Look Back At The U. S. Department Of Energy/'s Aquatic Species ...
National Renewable Energy Laboratory
NREL/TP-580-24190
A Look Back at the
U.S. Department of Energy’s
Aquatic Species Program:
Biodiesel from Algae
Close-Out Report
A Look Back at the U.S. Department of Energy’s Aquatic Species
Program—Biodiesel from Algae
July 1998
By
John Sheehan
Terri Dunahay
John Benemann
Paul Roessler
Prepared for:
U.S. Department of Energy’s
Office of Fuels Development
Prepared by: the
National Renewable Energy Laboratory
1617 Cole Boulevard
Golden, Colorado 80401-3393
A national laboratory of the U.S. Department of Energy
Operated by Midwest Research Institute
Under Contract No. DE-AC36-83CH10093
NREL/TP-580-24190
Executive Summary
From 1978 to 1996, the U.S. Department of Energy’s Office of Fuels Development funded a program to
develop renewable transportation fuels from algae. The main focus of the program, know as the Aquatic
Species Program (or ASP) was the production of biodiesel from high lipid-content algae grown in ponds,
utilizing waste CO2 from coal fired power plants. Over the almost two decades of this program,
tremendous advances were made in the science of manipulating the metabolism of algae and the
engineering of microalgae algae production systems. Technical highlights of the program are summarized
below:
Applied Biology
A unique collection of oil-producing microalgae.
The ASP studied a fairly specific aspect of algae—their ability to produce natural
oils. Researchers not only concerned themselves with finding algae that produced a
lot of oil, but also with algae that grow under severe conditions—extremes of
temperature, pH and salinity. At the outset of the program, no collections existed that
either emphasized or characterized algae in terms of these constraints. Early on,
researchers set out to build such a collection. Algae were collected from sites in the
west, the northwest and the southeastern regions of the continental U.S., as well as
Hawaii. At its peak, the collection contained over 3,000 strains of organisms. After
screening, isolation and characterization efforts, the collection was eventually
winnowed down to around 300 species, mostly green algae and diatoms. The
collection, now housed at the University of Hawaii, is still available to researchers.
This collection is an untapped resource, both in terms of the unique organisms
available and the mostly untapped genetic resource they represent. It is our sincere
hope that future researchers will make use of the collection not only as a source of
new products for energy production, but for many as yet undiscovered new products
and genes for industry and medicine.
Shedding light on the physiology and biochemistry of algae.
Prior to this program, little work had been done to improve oil production in algal
organisms. Much of the program’s research focused attention on the elusive “lipid
trigger.” (Lipids are another generic name for TAGs, the primary storage form of
natural oils.) This “trigger” refers to the observation that, under environmental stress,
many microalgae appeared to flip a switch to turn on production of TAGs. Nutrient
deficiency was the major factor studied. Our work with nitrogen-deficiency in algae
and silicon deficiency in diatoms did not turn up any overwhelming evidence in
support of this trigger theory. The common thread among the studies showing
increased oil production under stress seems to be the observed cessation of cell
division. While the rate of production of all cell components is lower under nutrient
starvation, oil production seems to remain higher, leading to an accumulation of oil in
the cells. The increased oil content of the algae does not to lead to increased overall
productivity of oil. In fact, overall rates of oil production are lower during periods of
nutrient deficiency. Higher levels of oil in the cells are more than offset by lower
rates of cell growth.
National Renewable Energy Laboratory
Breakthroughs in molecular biology and genetic engineering.
Plant biotechnology is a field that is only now coming into its own. Within the field of plant
biotechnology, algae research is one of the least trodden territories. The slower rate of advance in this field
makes each step forward in our research all the more remarkable. Our work on the molecular biology and
genetics of algae is thus marked with significant scientific discoveries. The program was the first to isolate
the enzyme Acetyl CoA Carboxylase (ACCase) from a diatom. This enzyme was found to catalyze a key
metabolic step in the synthesis of oils in algae. The gene that encodes for the production of ACCase was
eventually isolated and cloned. This was the first report of the cloning of the full sequence of the ACCase
gene in any photosynthetic organism. With this gene in hand, researchers went on to develop the first
successful transformation system for diatoms—the tools and genetic components for expressing a foreign
gene. The ACCase gene and the transformation system for diatoms have both been patented. In the
closing days of the program, researchers initiated the first experiments in metabolic engineering as a means
of increasing oil production. Researchers demonstrated an ability to make algae over-express the ACCase
gene, a major milestone for the research, with the hope that increasing the level of ACCase activity in the
cells would lead to higher oil production. These early experiments did not, however, demonstrate increased
oil production in the cells.
Algae Production Systems
Demonstration of Open Pond Systems for Mass Production of Microalgae.
Over the course of the program, efforts were made to establish the feasibility of large-scale algae
production in open ponds. In studies conducted in California, Hawaii and New Mexico, the ASP proved
the concept of long term, reliable production of algae. California and Hawaii served as early test bed sites.
Based on results from six years of tests run in parallel in California and Hawaii, 1,000 m2 pond systems
were built and tested in Roswell, New Mexico. The Roswell, New Mexico tests proved that outdoor ponds
could be run with extremely high efficiency of CO2 utilization. Careful control of pH and other physical
conditions for introducing CO2 into the ponds allowed greater than 90% utilization of injected CO2. The
Roswell test site successfully completed a full year of operation with reasonable control of the algal species
grown. Single day productivities reported over the course of one year were as high as 50 grams of algae
per square meter per day, a long-term target for the program. Attempts to achieve consistently high
productivities were hampered by low temperature conditions encountered at the site. The desert conditions
of New Mexico provided ample sunlight, but temperatures regularly reached low levels (especially at
night). If such locations are to be used in the future, some form of temperature control with enclosure of
the ponds may well be required.
The high cost of algae production remains an obstacle.
The cost analyses for large-scale microalgae production evolved from rather
superficial analyses in the 1970s to the much more detailed and sophisticated studies
conducted during the 1980s. A major conclusion from these analyses is that there is
little prospect for any alternatives to the open pond designs, given the low cost
requirements associated with fuel production. The factors that most influence cost
are biological, and not engineering-related. These analyses point to the need for
highly productive organisms capable of near-theoretical levels of conversion of
sunlight to biomass. Even with aggressive assumptions about biological
productivity, we project costs for biodiesel which are two times higher than current
petroleum diesel fuel costs.
A Look Back at the Aquatic Species Program—Executive Summary ii
National Renewable Energy Laboratory
Resource Availability
Land, water and CO2 resources can support substantial biodiesel production and CO2
savings.
The ASP regularly revisited the question of available resources for producing biodiesel from microalgae.
This is not a trivial effort. Such resource assessments require a combined evaluation of appropriate climate,
land and resource availability. These analyses indicate that significant potential land, water and CO2
resources exist to support this technology. Algal biodiesel could easily supply several “quads” of
biodiesel—substantially more than existing oilseed crops could provide. Microalgae systems use far less
water than traditional oilseed crops. Land is hardly a limitation. Two hundred thousand hectares (less than
0.1% of climatically suitable land areas in the U.S.) could produce one quad of fuel. Thus, though the
technology faces many R&D hurdles before it can be practicable, it is clear that resource limitations are not
an argument against the technology.
A Look Back at the Aquatic Species Program—Executive Summary iii
A Look Back at the U.S.
Department of Energy’s
Aquatic Species Program:
Biodiesel from Algae
Part I:
Program Summary
Background
Origins of the Program
This year marks the 20th anniversary of the National Renewable Energy Laboratory
(NREL). In 1978, the Carter Administration established what was then called the
Solar Energy Research Institute (SERI) in Golden, CO. This was a first-of-its kind
federal laboratory dedicated to the development of solar energy. The formation of
this lab came in response to the energy crises of the early and mid 1970s. At the
same time, the Carter Administration consolidated all federal energy activities under
the auspices of the newly established U.S. Department of Energy (DOE).
Among its various programs established to develop all forms of solar energy, DOE
initiated research on the use of plant life as a source of transportation fuels. Today,
this program—known as the Biofuels Program—is funded and managed by the
Office of Fuels Development (OFD) within the Office of Transportation
Technologies under the Assistant Secretary for Energy Efficiency and Renewable
Energy at DOE. The program has, over the years, focused on a broad range of
alternative fuels, including ethanol and methanol (alcohol fuel substitutes for
gasoline), biogas (methane derived from plant materials) and biodiesel (a natural oil-
derived diesel fuel substitute). The Aquatic Species Program (ASP) was just one
component of research within the Biofuels Program aimed at developing alternative
sources of natural oil for biodiesel production.
Close-out of the Program
The Aquatic Species Program (ASP) was a relatively small research effort intended
to look at the use of aquatic plants as sources of energy. While its history dates back
to 1978, much of the research from 1978 to 1982 was focused on using algae to
produce hydrogen. The program switched emphasis to other transportation fuels, in
particular biodiesel, beginning in the early 1980s. This report provides a summary of
the research activities carried out from 1980 to 1996, with an emphasis on algae for
biodiesel production.
In 1995, DOE made the difficult decision to eliminate funding for algae research
within the Biofuels Program. Under pressure to reduce budgets, the Department
chose a strategy of more narrowly focusing its limited resources in one or two key
areas, the largest of these being the development of bioethanol. The purpose of this
report is to bring closure to the Biofuels Program’s algae research. This report is a
summary and compilation of all the work done over the last 16 years of the program.
It includes work carried out by NREL researchers at our labs in Golden, as well as
subcontracted research and development activities conducted by private companies
and universities around the country. More importantly, this report should be seen not
as an ending, but as a beginning. When the time is right, we fully expect to see
renewed interest in algae as a source of fuels and other chemicals. The highlights
presented here should serve as a foundation for these future efforts.
A Look Back at the Aquatic Species Program—Program Summary
What is the technology?
Biological Concepts
Photosynthetic organisms include plants, algae and some photosynthetic bacteria.
Photosynthesis is the key to making solar energy available in useable forms for all
organic life in our environment. These organisms use energy from the sun to
combine water with carbon dioxide (CO2) to create biomass. While other elements of
the Biofuels Program have focused on terrestrial plants as sources of fuels, ASP was
concerned with photosynthetic organisms that grew in aquatic environments. These
include macroalgae, microalgae and emergents. Macroalgae, more commonly known
as “seaweed,” are fast growing marine and freshwater plants that can grow to
considerable size (up to 60m in length). Emergents are plants that grow partially
submerged in bogs and marshes. Microalgae are, as the name suggests, microscopic
photosynthetic organisms. Like macroalgae, these organisms are found in both
marine and freshwater environments. In the early days of the program, research was
done on all three types of aquatic species. As emphasis switched to production of
natural oils for biodiesel, microalgae became the exclusive focus of the research.
This is because microalgae generally produce more of the right kinds of natural oils
needed for biodiesel (see the discussion of fuel concepts presented later in this
overview).
In many ways, the study of microalgae is a relatively limited field of study. Algae
are not nearly as well understood as other organisms that have found a role in today’s
biotechnology industry. This is part of what makes our program so valuable. Much
of the work done over the past two decades represents genuine additions to the
scientific literature. The limited size of the scientific community involved in this
work also makes it more difficult, and sometimes slower, compared to the progress
seen with more conventional organisms. The study of microalgae represents an area
of high risk and high gains.
These photosynthetic organisms are far from monolithic. Biologists have categorized
microalgae in a variety of classes, mainly distinguished by their pigmentation, life
cycle and basic cellular structure. The four most important (at least in terms of
abundance) are:
• The diatoms (Bacillariophyceae). These algae dominate the
phytoplankton of the oceans, but are also found in fresh and
brackish water. Approximately 100,000 species are known to
exist. Diatoms contain polymerized silica (Si) in their cell walls.
All cells store carbon in a variety of forms. Diatoms store
carbon in the form of natural oils or as a polymer of
carbohydrates known as chyrsolaminarin.
• The green algae (Chlorophyceae). These are also quite
abundant, especially in freshwater. (Anyone who owns a
swimming pool is more than familiar with this class of algae).
They can occur as single cells or as colonies. Green algae are the
evolutionary progenitors of modern plants. The main storage
compound for green algae is starch, though oils can be produced
under certain conditions.
2 A Look Back at the Aquatic Species Program—Program Summary
• The blue-green algae (Cyanophyceae). Much closer to bacteria
in structure and organization, these algae play an important role
in fixing nitrogen from the atmosphere. There are approximately
2,000 known species found in a variety of habitats.
•
The golden algae (Chrysophyceae). This group of algae is
similar to the diatoms. They have more complex pigment
systems, and can appear yellow, brown or orange in color.
Approximately 1,000 species are known to exist, primarily in
freshwater systems. They are similar to diatoms in pigmentation
and biochemical composition. The golden algae produce natural
oils and carbohydrates as storage compounds.
The bulk of the organisms collected and studied in this program fall in the first two
classes—the diatoms and the green algae.
Microalgae are the most primitive form of plants. While the mechanism of
photosynthesis in microalgae is similar to that of higher plants, they are generally
more efficient converters of solar energy because of their simple cellular structure.
In addition, because the cells grow in aqueous suspension, they have more efficient
access to water, CO2, and other nutrients. For these reasons, microalgae are capable
of producing 30 times the amount oil per unit area of land, compared to terrestrial
oilseed crops.
Put quite simply, microalgae are remarkable and efficient biological factories capable of
taking a waste (zero-energy) form of carbon (CO2) and converting it into a high density
liquid form of energy (natural oil). This ability has been the foundation of the research
program funded by the Office Fuels Development.
Algae Production Concepts
Like many good ideas (and certainly many of the concepts that are now the basis for
renewable energy technology), the concept of using microalgae as a source of fuel is
older than most people realize. The idea of producing methane gas from algae was
proposed in the early 1950s1. These early researchers visualized a process in which
wastewater could be used as a medium and source of nutrients for algae production.
The concept found a new life with the energy crisis of the 1970s. DOE and its
predecessors funded work on this combined process for wastewater treatment and
energy production during the 1970s. This approach had the benefit of serving
multiple needs—both environmental and energy-related. It was seen as a way of
introducing this alternative energy source in a near-term timeframe.
In the 1980s, DOE’s program gradually shifted its focus to technologies that could
have large-scale impacts on national consumption of fossil energy. Much of DOE’s
publications from this period reflect a philosophy of energy research that might,
somewhat pejoratively, be called “the quads mentality.” A quad is a short-hand name
for the unit of energy often used by DOE to describe the amounts of energy that a
given technology might be able to displace. Quad is short for “quadrillion Btus”—a
unit of energy representing 1015 (1,000,000,000,000,000) Btus of energy. This
perspective led DOE to focus on the concept of immense algae farms.
A Look Back at the Aquatic Species Program—Program Summary 3
Such algae farms would be based on the use of open, shallow ponds in which some
source of waste CO2 could be efficiently bubbled into the ponds and captured by the
algae (see the figure below).
Water
Nutrients
Motorized
paddle
wheel
Algae
Waste CO2
The ponds are “raceway” designs, in which the algae, water and nutrients circulate
around a racetrack. Paddlewheels provide the flow. The algae are thus kept
suspended in water. Algae are circulated back up to the surface on a regular
frequency. The ponds are kept shallow because of the need to keep the algae
exposed to sunlight and the limited depth to which sunlight can penetrate the pond
water. The ponds are operated continuously; that is, water and nutrients are
constantly fed to the pond, while algae-containing water is removed at the other end.
Some kind of harvesting system is required to recover the algae, which contains
substantial amounts of natural oil.
The concept of an “algae farm” is illustrated on the next page. The size of these
ponds is measured in terms of surface area (as opposed to volume), since surface area
is so critical to capturing sunlight. Their productivity is measured in terms of
biomass produced per day per unit of available surface area. Even at levels of
productivity that would stretch the limits of an aggressive research and development
program, such systems will require acres of land. At such large sizes, it is more
appropriate to think of these operations on the scale of a farm.
There are quite a number of sources of waste CO2. Every operation that involves
combustion of fuel for energy is a potential source. The program targeted coal and
other fossil fuel-fired power plants as the main sources of CO2. Typical coal-fired
power plants emit flue gas from their stacks containing up to 13% CO2. This high
concentration of CO2 enhances transfer and uptake of CO2 in the ponds. The concept
of coupling a coal-fired power plant with an algae farm provides an elegant approach
to recycle of the CO2 from coal combustion into a useable liquid fuel.
4 A Look Back at the Aquatic Species Program—Program Summary
CO2
Recovery
System
Algae/Oil
Fuel
Recovery
Production
System
Other system designs are possible. The Japanese, French and German governments
have invested significant R&D dollars on novel closed bioreactor designs for algae
production. The main advantage of such closed systems is that they are not as
subject to contamination with whatever organism happens to be carried in the wind.
The Japanese have, for example, developed optical fiber-based reactor systems that
could dramatically reduce the amount of surface area required for algae production.
While breakthroughs in these types of systems may well occur, their costs are, for
now, prohibitive—especially for production of fuels. DOE’s program focused
primarily on open pond raceway systems because of their relative low cost.
The Aquatic Species Program envisioned vast arrays of algae ponds covering acres of
land analogous to traditional farming. Such large farms would be located adjacent to
power plants. The bubbling of flue gas from a power plant into these ponds provides a
system for recycling of waste CO2 from the burning of fossil fuels.
Fuel Production Concepts
The previous sections have alluded to a number of potential fuel products from algae.
The ASP considered three main options for fuel production:
• Production of methane gas via biological or thermal gasification.
• Production of ethanol via fermentation
A Look Back at the Aquatic Species Program—Program Summary 5
• Production of biodiesel
A fourth option is the direct combustion of the algal biomass for production of steam
or electricity. Because the Office of Fuels Development has a mandate to work on
transportation fuels, the ASP did not focus much attention on direct combustion. The
concept of algal biomass as a fuel extender in coal-fired power plants was evaluated
under a separate program funded by DOE’s Office of Fossil Fuels. The Japanese
have been the most aggressive in pursuing this application. They have sponsored
demonstrations of algae production and use at a Japanese power plant.
Algal biomass contains three main components:
• Carbohydrates
• Protein
• Natural Oils
The economics of fuel production from algae (or from any biomass, for that matter)
demands that we utilize all the biomass as efficiently as possible. To achieve this, the
three fuel production options listed previously can be used in a number of
combinations. The most simplistic approach is to produce methane gas, since the
both the biological and thermal processes involved are not very sensitive to what
form the biomass is in. Gasification is a somewhat brute force technology in the
sense that it involves the breakdown of any form of organic carbon into methane.
Ethanol production, by contrast, is most effective for conversion of the carbohydrate
fraction. Biodiesel production applies exclusively to the natural oil fraction. Some
combination of all three components can also be utilized as an animal feed. Process
design models developed under the program considered a combination of animal feed
production, biological gasification and biodiesel production.
The main product of interest in the ASP was biodiesel. In its most general sense,
biodiesel is any biomass-derived diesel fuel substitute. Today, biodiesel has come to
mean a very specific chemical modification of natural oils. Oilseed crops such as
rapeseed (in Europe) and soybean oil (in the U.S.) have been extensively evaluated as
sources of biodiesel. Biodiesel made from rapeseed oil is now a substantial
commercial enterprise in Europe. Commercialization of biodiesel in the U.S. is still
in its nascent stage.
The bulk of the natural oil made by oilseed crops is in the form of triacylglycerols
(TAGs). TAGs consist of three long chains of fatty acids attached to a glycerol
backbone. The algae species studied in this program can produce up to 60% of their
body weight in the form of TAGs. Thus, algae represent an alternative source of
biodiesel, one that does not compete with the existing oilseed market.
As a matter of historical interest, Rudolph Diesel first used peanut oil (which is
mostly in the form of TAGs) at the turn of the century to demonstrate his patented
diesel engine2. The rapid introduction of cheap petroleum quickly made petroleum
the preferred source of diesel fuel, so much so that today’s diesel engines do not
operate well when operated on unmodified TAGs. Natural oils, it turns out, are too
viscous to be used in modern diesel engines.
6 A Look Back at the Aquatic Species Program—Program Summary
In the 1980s, a chemical modification of natural oils was introduced that helped to
bring the viscosity of the oils within the range of current petroleum diesel3. By
reacting these TAGs with simple alcohols (a chemical reaction known as
“transesterification” already commonplace in the oleochemicals industry), we can
create a chemical compound known as an alkyl ester4, but which is known more
generically as biodiesel (see the figure below). Its properties are very close to those
of petroleum diesel fuel.
1 TAG
3 molecules of biodiesel
O
CH2
COOH
HC
HC
COOH
O
CH2
HC
COOH
O
CH2
+
3(CH2OH)
+
3 molecules of alcohol
HCOH
HCOH
HCOH
1 molecule of glycerol
Commercial experience with biodiesel has been very promising5. Biodiesel performs
as well as petroleum diesel, while reducing emissions of particulate matter, CO,
hydrocarbons and SOx. Emissions of NOx are, however, higher for biodiesel in many
engines. Biodiesel virtually eliminates the notorious black soot emissions associated
with diesel engines. Total particulate matter emissions are also much lower6,7,8.
Other environmental benefits of biodiesel include the fact that it is highly
biodegradable9 and that it appears to reduce emissions of air toxics and carcinogens
(relative to petroleum diesel)10. A proper discussion of biodiesel would require much
more space than can be accommodated here. Suffice it to say that, given many of its
environmental benefits and the emerging success of the fuel in Europe, biodiesel is a
very promising fuel product.
High oil-producing algae can be used to produce biodiesel, a chemically modified
natural oil that is emerging as an exciting new option for diesel engines. At the same
time, algae technology provides a means for recycling waste carbon from fossil fuel
combustion. Algal biodiesel is one of the only avenues available for high-volume re-use
of CO2 generated in power plants. It is a technology that marries the potential need for
carbon disposal in the electric utility industry with the need for clean-burning
alternatives to petroleum in the transportation sector.
Why microalgae technology?
There are a number of benefits that serve as driving forces for developing and
deploying algae technology. Some of these benefits have already been mentioned.
Four key areas are outlined here. The first two address national and international
issues that continue to grow in importance—energy security and climate change. The
A Look Back at the Aquatic Species Program—Program Summary 7
remaining areas address aspects of algae technology that differentiate it from other
technology options being pursued by DOE.
Energy Security
Energy security is the number one driving force behind DOE’s Biofuels Program.
The U.S. transportation sector is at the heart of this security issue. Cheap oil prices
during the 1980s and 1990s have driven foreign oil imports to all time highs. In
1996, imports reached an important milestone—imported oil consumption exceeded
domestic oil consumption. DOE’s Energy Information Administration paints a dismal
picture of our growing dependence on foreign oil. Consider these basic points11:
• Petroleum demand is increasing, especially due to new demand
from Asian markets.
• New demand for oil will come primarily from the Persian Gulf.
• As long as prices for petroleum remain low, we can expect our
imports to exceed 60% of our total consumption ten years from
now.
• U.S. domestic supplies will likewise remain low as long as prices
for petroleum remain low.
Not everyone shares this view of the future, or sees it as a reason for concern. The
American Petroleum Institute12 does not see foreign imports as a matter of national
security. Others have argued that the prediction of increasing Mideast oil
dependence worldwide is wrong. But the concern about our foreign oil addiction is
widely held by a broad range of political and commercial perspectives13.
While there may be uncertainty and even contention over when and if there is a
national security issue, there is one more piece to the puzzle that influences our
perspective on this issue. This is the fact that, quite simply, 98% of the transportation
sector in the U.S. relies on petroleum (mostly in the form of gasoline and diesel fuel).
The implication of this indisputable observation is that even minor hiccups in the
supply of oil could have crippling effects on our nation. This lends special
significance to the Biofuels Program as a means of diversifying the fuel base in our
transportation sector.
Our almost complete reliance on petroleum in transportation comes from the demand for
gasoline in passenger vehicles and the demand for diesel fuel in commerce. Bioethanol
made from terrestrial energy crops offers a future alternative to gasoline, biodiesel made
from algal oils could do the same for diesel fuel.
Climate Change
CO2 is recognized as the most important (at least in quantity) of the atmospheric
pollutants that contribute to the “greenhouse effect,” a term coined by the French
mathematician Fourier in the mid-1800s to describe the trapping of heat in the
Earth’s atmosphere by gases capable of absorbing radiation. By the end of the last
century, scientists were already speculating on the potential impacts of anthropogenic
8 A Look Back at the Aquatic Species Program—Program Summary
CO2. The watershed event that brought the question of global warming to the
forefront in the scientific community was the publication of Revelle’s data in 1957,
which quantified the geologically unprecedented build-up of atmospheric CO2 that
began with the advent of the industrial revolution. Revelle14 characterized the
potential risk of global climate change this way:
“Human beings are carrying out a large scale geophysical experiment of
a kind that could not have happened in the past nor be produced in the
future. Within a few centuries, we are returning to the atmosphere and
the oceans the concentrated organic carbon stored in sedimentary rocks
over hundreds of millions of years.”
Despite 40 years of research since Revelle first identified the potential risk of global
warming, the debate over the real impacts of the increased CO2 levels still rages. We
may never be able to scientifically predict the climatic effects of increasing carbon
dioxide levels due to the complexity of atmospheric and meteorological modeling.
Indeed, Revelle’s concise statement of the risks at play in global climate change
remains the best framing of the issue available for policy makers today. The question
we face as a nation is how much risk we are willing to take on an issue like this. That
debate has never properly taken place with the American public.
As Revelle’s statement implies, the burning of fossil fuels is the major source of the
current build up of atmospheric CO2. Thus, identifying alternatives to fossil fuels must be
a key strategy in reducing greenhouse gas emissions. While no one single fuel can
substitute for fossil fuels in an all of the energy sectors, we believe that biodiesel made
from algal oils is a fuel which can make a major contribution to the reduction of CO2
generated by power plants and commercial diesel engines.
The Synergy of Coal and Microalgae
Many of our fossil fuel reserves, but especially coal, are going to play significant
roles for years to come. On a worldwide basis, coal is, by far, the largest fossil energy
resource available. About one-fourth of the world’s coal reserves reside in the
United States. To put this in perspective, consider the fact that, at current rates of
consumption, coal reserves could last for over 200 years.
Regardless of how much faith you put in future fossil energy projections, it is clear
that coal will continue to play an important role in our energy future—especially
given the relatively large amounts of coal that we control within our own borders.
DOE’s Energy Information Administration estimates that electricity will become an
increasingly large contributor to future U.S. energy demand. How will this new
demand be met? Initially, low cost natural gas will grow in use. Inevitably, the
demand for electricity will have to be met by coal. Coal will remain the mainstay of
U.S. baseline electricity generation, accounting for half of electricity generation by
the year 2010.
The long term demand for coal brings with it a demand for technologies that can
mitigate the environmental problems associated with coal. While control
technologies will be used to reduce air pollutants associated with acid rain, no
technologies exist today which address the problem of greenhouse gas emissions.
Coal is the most carbon-intensive of the fossil fuels. In other words, for every Btu of
energy liberated by combustion, coal emits more CO2 than either petroleum or
A Look Back at the Aquatic Species Program—Program Summary 9
natural gas. As pressure to reduce carbon emissions grows, this will become an
increasingly acute problem for the U.S.
One measure of how serious this problem could be is the absurdity of some of the
proposals being developed for handling carbon emissions from power plants. The
preferred option offered by researchers at MIT is ocean disposal, despite the expense
and uncertainty of piping CO2 from power plants and injecting the CO2 in the
ocean15.
Commonsense suggests that recycling of carbon would be more efficacious than deep
ocean disposal. No one clearly understands the long-term effects of injecting large
amounts of CO2 into our oceans. Beyond these environmental concerns, such large-
scale disposal schemes represent an economic sinkhole. Huge amounts of capital and
operating dollars would be spent simply to dispose of carbon. While such Draconian
measures may ultimately be needed, it makes more sense to first re-use stationary
sources of carbon as much as possible. Algae technology is unique in its ability to
produce a useful, high-volume product from waste CO2.
Consumption of coal, an abundant domestic fuel source for electricity generation, will
continue to grow over the coming decades, both in the U.S. and abroad. Algae
technology can extend the useful energy we get from coal combustion and reduce carbon
emissions by recycling waste CO2 from power plants into clean-burning biodiesel. When
compared to the extreme measures proposed for disposing of power plant carbon
emissions, algal recycling of carbon simply makes sense.
Terrestrial versus Aquatic Biomass
Algae grow in aquatic environments. In that sense, algae technology will not
compete for the land already being eyed by proponents of other biomass-based fuel
technologies. Biomass power and bioethanol both compete for the same land and for
similar feedstocks—trees and grasses specifically grown for energy production.
More importantly, many of the algal species studied in this program can grow in
brackish water—that is, water that contains high levels of salt. This means that algae
technology will not put additional demand on freshwater supplies needed for
domestic, industrial and agricultural use.
The unique ability of algae to grow in saline water means that we can target areas of
the country in which saline groundwater supplies prevent any other useful application
of water or land resources. If we were to draw a map showing areas best suited for
energy crop production (based on climate and resource needs), we would see that
algae technology needs complement the needs of both agriculture and other biomass-
based energy technologies.
In a world of ever more limited natural resources, algae technology offers the
opportunity to utilize land and water resources that are, today, unsuited for any other
use. Land use needs for microalgae complement, rather than compete, with other
biomass-based fuel technologies.
10 A Look Back at the Aquatic Species Program—Program Summary
Technical Highlights of the Program
Applied Biology
A unique collection of oil-producing microalgae.
The ASP studied a fairly specific aspect of algae—their ability to produce natural
oils. Researchers not only concerned themselves with finding algae that produced a
lot of oil, but also with algae that grow under severe conditions—extremes of
temperature, pH and salinity. At the outset of the program, no collections existed that
either emphasized or characterized algae in terms of these constraints. Early on,
researchers set out to build such a collection. Algae were collected from sites in the
west, the northwest and the southeastern regions of the continental U.S., as well as
Hawaii. At its peak, the collection contained over 3,000 strains of organisms. After
screening, isolation and characterization efforts, the collection was eventually
winnowed down to around 300 species, mostly green algae and diatoms. The
collection, now housed at the University of Hawaii, is still available to researchers.
This collection is an untapped resource, both in terms of the unique organisms
available and the mostly untapped genetic resource they represent. It is our sincere
hope that future researchers will make use of the collection not only as a source of
new products for energy production, but for many as yet undiscovered new products
and genes for industry and medicine.
Shedding light on the physiology and biochemistry of algae.
Prior to this program, little work had been done to improve oil production in algal
organisms. Much of the program’s research focused attention on the elusive “lipid
trigger.” (Lipids are another generic name for TAGs, the primary storage form of
natural oils.) This “trigger” refers to the observation that, under environmental stress,
many microalgae appeared to flip a switch to turn on production of TAGs. Nutrient
deficiency was the major factor studied. Our work with nitrogen-deficiency in algae
and silicon deficiency in diatoms did not turn up any overwhelming evidence in
support of this trigger theory. The common thread among the studies showing
increased oil production under stress seems to be the observed cessation of cell
division. While the rate of production of all cell components is lower under nutrient
starvation, oil production seems to remain higher, leading to an accumulation of oil in
the cells. The increased oil content of the algae does not to lead to increased overall
productivity of oil. In fact, overall rates of oil production are lower during periods of
nutrient deficiency. Higher levels of oil in the cells are more than offset by lower
rates of cell growth.
Breakthroughs in molecular biology and genetic engineering.
Plant biotechnology is a field that is only now coming into its own. Within the field
of plant biotechnology, algae research is one of the least trodden territories. The
slower rate of advance in this field makes each step forward in our research all the
more remarkable. Our work on the molecular biology and genetics of algae is thus
marked with significant scientific discoveries. The program was the first to isolate
A Look Back at the Aquatic Species Program—Program Summary 11
the enzyme Acetyl CoA Carboxylase (ACCase) from a diatom. This enzyme was
found to catalyze a key metabolic step in the synthesis of oils in algae. The gene that
encodes for the production of ACCase was eventually isolated and cloned. This was
the first report of the cloning of the full sequence of the ACCase gene in any
photosynthetic organism. With this gene in hand, researchers went on to develop
the first successful transformation system for diatoms—the tools and genetic
components for expressing a foreign gene. The ACCase gene and the transformation
system for diatoms have both been patented. In the closing days of the program,
researchers initiated the first experiments in metabolic engineering as a means of
increasing oil production. Researchers demonstrated an ability to make algae over-
express the ACCase gene, a major milestone for the research, with the hope that
increasing the level of ACCase activity in the cells would lead to higher oil
production. These early experiments did not, however, demonstrate increased oil
production in the cells.
Algae Production Systems
Demonstration of Open Pond Systems for Mass Production of Microalgae.
Over the course of the program, efforts were made to establish the feasibility of
large-scale algae production in open ponds. In studies conducted in California,
Hawaii and New Mexico, the ASP proved the concept of long term, reliable
production of algae. California and Hawaii served as early test bed sites. Based on
results from six years of tests run in parallel in California and Hawaii, 1,000 m2 pond
systems were built and tested in Roswell, New Mexico. The Roswell, New Mexico
tests proved that outdoor ponds could be run with extremely high efficiency of CO2
utilization. Careful control of pH and other physical conditions for introducing CO2
into the ponds allowed greater than 90% utilization of injected CO2. The Roswell
test site successfully completed a full year of operation with reasonable control of the
algal species grown. Single day productivities reported over the course of one year
were as high as 50 grams of algae per square meter per day, a long-term target for the
program. Attempts to achieve consistently high productivities were hampered by low
temperature conditions encountered at the site. The desert conditions of New Mexico
provided ample sunlight, but temperatures regularly reached low levels (especially at
night). If such locations are to be used in the future, some form of temperature
control with enclosure of the ponds may well be required.
A disconnect between the lab and the field.
An important lesson from the outdoor testing of algae production systems is the
inability to maintain laboratory organisms in the field. Algal species that looked very
promising when tested in the laboratory were not robust under conditions
encountered in the field. In fact, the best approach for successful cultivation of a
consistent species of algae was to allow a contaminant native to the area to take over
the ponds.
The high cost of algae production remains an obstacle.
12 A Look Back at the Aquatic Species Program—Program Summary
The cost analyses for large-scale microalgae production evolved from rather
superficial analyses in the 1970s to the much more detailed and sophisticated studies
conducted during the 1980s. A major conclusion from these analyses is that there is
little prospect for any alternatives to the open pond designs, given the low cost
requirements associated with fuel production. The factors that most influence cost
are biological, and not engineering-related. These analyses point to the need for
highly productive organisms capable of near-theoretical levels of conversion of
sunlight to biomass. Even with aggressive assumptions about biological
productivity, we project costs for biodiesel which are two times higher than current
petroleum diesel fuel costs.
Resource Availability
Land, water and CO2 resources can support substantial biodiesel production and CO2
savings.
The ASP regularly revisited the question of available resources for producing
biodiesel from microalgae. This is not a trivial effort. Such resource assessments
require a combined evaluation of appropriate climate, land and resource availability.
These analyses indicate that significant potential land, water and CO2 resources exist
to support this technology. Algal biodiesel could easily supply several “quads” of
biodiesel—substantially more than existing oilseed crops could provide. Microalgae
systems use far less water than traditional oilseed crops. Land is hardly a limitation.
Two hundred thousand hectares (less than 0.1% of climatically suitable land areas in
the U.S.) could produce one quad of fuel. Thus, though the technology faces many
R&D hurdles before it can be practicable, it is clear that resource limitations are not
an argument against the technology.
A Brief Chronology of the Research Activities
Part II of this report details the specific research accomplishments of the program on
a year-to-year basis. In order to provide a consistent context and framework for
understanding this detail, we have attempted to outline the major activities of the
program as they unfolded over the course of the past two decades. The timeline on
the following page shows the major activities broken down into two main
categories—laboratory studies and outdoor testing/systems analysis. For the sake of
clarity and brevity, many of the research projects and findings from the program are
not presented here. Instead, only those findings that form a thread throughout the
work are highlighted. There were many other studies and findings of value in the
program. The reader is encouraged to review Part II of this report for a more
comprehensive discussion of the research.
Laboratory Studies
The research pathway in the lab can be broken down into three types of activities:
• Collection, screening and characterization of algae.
• Biochemical and physiological studies of lipid production
• Molecular biology and genetic engineering studies
A Look Back at the Aquatic Species Program—Program Summary 13
There is a logic to the sequence of these activities. Researchers first identified a need
to collect and identify algae that met minimal requirements for this technology.
Collection and screening occurred over a seven-year period from 1980 to 1987.
Once a substantial amount of information was available on the types of oil-producing
algae and their capabilities, the program began to switch its emphasis to
understanding the biochemistry and physiology of oil production in algae. A natural
next step was to use this information to identify approaches to genetically manipulate
the metabolism of algae to enhance oil production.
Algae collection efforts initially focused on shallow, inland saline habitats,
particularly in western Colorado, New Mexico and Utah. The reasoning behind
collecting strains from these habitats was that the strains would be adapted to at least
some of the environmental conditions expected in mass culture facilities located in
the southwestern U.S. (a region identified early on as a target for deployment of the
technology). Organisms isolated from shallow habitats were also expected to be
more tolerant to wide swings in temperature and salinity. In the meantime,
subcontractors were collecting organisms from the southeastern region of the U.S.
(Florida, Mississippi, and Alabama). By 1984, researchers in the program had
developed improved tools and techniques for collecting and screening organisms.
These included a modified rotary screening apparatus and statistically designed saline
media formulations that mimicked typical brackish water conditions in the southwest.
In 1985, a rapid screening test was in place for identifying high oil-producing algae.
In the last years of the collection effort, the focus switched to finding algae that were
tolerant to low temperature. This expanded the reach of the collection activities into
the northwest. By 1987, the algae collection contained over 3,000 species.
As the collection efforts began to wind down, it became apparent that no one single
species was going to be found that met all of the needs of the technology. As a
result, about midway through the collection efforts, the program began studies on the
biochemistry and physiology of oil production in algae in hopes of learning how to
improve the performance of existing organisms. A number of ASP subcontractors
struggled to identify the so-called “lipid trigger.” These studies confirmed
observations that deficiencies in nitrogen could lead to an increase in the level of oil
present in many species of algae. Observations of cellular structure also supported
the notion of a trigger that caused rapid build up of oil droplets in the cells during
periods of nitrogen depletion.
14 A Look Back at the Aquatic Species Program—Program Summary
Pre-1980
1980 1981 1982 1983 1984 1985 1986 1987 1988 1989 1990 1991 1992 1993 1994 1995 1996
Carribean, NM,CO,
AZ,CA,
UT, FL, HI, NE, AL
NV,NM,
CO, UT
TX,UT
Lab Studies
CO, AL,MI
UT, WA, CO, CA,
NV
Collection,
By 1987, over
Screening,
3,000 strains of
Characterization
algae had been
Artificial saline
collected.
media; Temp-
Future efforts
Salinity Gradient
Nile Red lipid
Screening
screening
Isolation and
Biochemistry, Physiology
characterization
of Lipid Production
of ACCase
N-deficiency
enzyme
lipid trigger Si-deficiency
lipid trigger Link between
Genetic Engineering
Si-deficiency
of Algae
and ACCase
Transient
expression of
Outdoor Culture Studies and Systems Analysis
1st successful
foreign gene in
genetic
algae using
transformation of
protoplasts
Algae Production in Wastewater
diatom
Treatment
<100 sq.m. Pond Studies (CA, HI)
1000 sq.m. Pond Study (NM)
Systems Analysis and Resource Assessment
In the end, however, the studies conducted both by NREL researchers and program
subcontractors concluded that no simple trigger for lipid production exists. Instead,
we found that environmental stresses like nitrogen depletion lead to inhibition of cell
division, without immediately slowing down oil production. It appeared that no
simple means existed for increasing oil production, without a penalty in overall
productivity due to a slowing down of cell growth. The use of nutrient depletion as a
means of inducing oil production may still have merit. Some experiments conducted
at NREL suggested that the kinetics of cell growth and lipid accumulation are very
subtle. With a better understanding of these kinetics, it may be possible to provide a
net increase in total oil productivity by carefully controlling the timing of nutrient
depletion and cell harvesting.
In 1986, researchers at NREL reported on the use of Si depletion as a way to increase
oil levels in diatoms. They found that when Si was used up, cell division slowed
down since Si is a component of the diatoms’ cell walls. In the diatom C. cryptica,
the rate of oil production remained constant once Si depletion occurred, while growth
rate of the cells dropped. Further studies identified two factors that seemed to be at
play in this species:
1. Si-depleted cells direct newly assimilated carbon more toward
lipid production and less toward carbohydrate production.
2. Si-depleted cells slowly convert non-lipid cell components to
lipids.
Diatoms store carbon in lipid form or in carbohydrate form. The results of these
experiments suggested that it might be possible to alter which route the cells used for
storage (see schematic below):
CO2
Photosynthesis
C
Lipid Synthesis
Carbohydrate
Synthesis
16 A Look Back at the Aquatic Species Program—Program Summary
Through the process of photosynthesis, algae cells assimilate carbon. There are
numerous metabolic pathways through which the carbon can go, resulting in
synthesis of whatever compounds are needed by the cell. These pathways consist of
sequences of enzymes, each of which catalyzes a specific reaction. Two possible
pathways for carbon are shown on the previous page. They represent the two storage
forms that carbon can take.
Researchers at NREL began to look for key enzymes in the lipid synthesis pathway.
These would be enzymes whose level of activity in the cell influences the rate at
which oils are formed. Think of these enzymes as valves or spigots controlling the
flow of carbon down the pathway. Higher enzyme activity leads to higher rates of oil
production. When algae cells increase the activity of active enzymes, they are
opening up the spigot to allow greater flow of carbon to oil production. Finding such
critical enzymes was key to understanding the mechanisms for controlling oil
production.
By 1988, researchers had shown that increases in the levels of the enzyme Acetyl
CoA Carboxylase (ACCase) correlated well with lipid accumulation during Si
depletion. They also showed that the increased levels correlated with increased
expression of the gene encoding for this enzyme. These findings led to a focus on
isolating the enzyme and cloning the gene responsible for its expression. By the end
of the program, not only had researchers successfully cloned the ACCase gene, but
they had also succeeded in developing the tools for expressing foreign genes in
diatoms.
In the 1990s, genetic engineering had become the main focus of the program. While
we have highlighted the successes of over-expressing ACCase in diatoms, other
approaches were also developed for foreign gene expression—in green algae as well
as in diatoms. Another interesting sideline in the research involved studies aimed at
identifying key enzymes involved in the synthesis of storage carbohydrates. Instead
of over-expressing these enzymes, researchers hoped to inactivate them. Returning
to our “spigot” analogy, this approach was like shutting off the flow of carbon to
carbohydrates, in the hopes that it would force carbon to flow down the lipid
synthesis pathway (again, see the schematic on the previous page). This work led to
the discovery of a unique multifunctional enzyme in the carbohydrate synthesis
pathway. This enzyme and its gene were both patented by NREL in 1996.
Outdoor Testing and Systems Analysis
The first work done in earnest by DOE on demonstration of algae technology for
energy production predates the Aquatic Species Program. In 1976, the Energy
Research and Development Administration (before it was folded into DOE) funded a
project at the University of California Berkeley’s Richmond Field Station to evaluate
a combined wastewater treatment/fuel production system based on microalgae. Over
the course of several years, the Richmond Field Station demonstrated techniques for
algae harvesting and for control of species growing in open ponds.
By the time the Aquatic Species Program took on microalgae research, emphasis had
already moved from wastewater treatment based systems to dedicated algae farm
operations. From 1980 to 1987, the program funded two parallel efforts to develop
large scale mass culture systems for microalgae. One effort was at the University of
California, and it was based on a so-called “High Rate Pond” (HRP) design. The
other effort was carried out at the University of Hawaii, where a patented “Algae
A Look Back at the Aquatic Species Program—Program Summary 17
Raceway Production System” (ARPS). Both designs utilized open raceway designs.
The HRP design was based on a shallow, mixed raceway concept developed at
Berkeley in 1963 and successfully applied in wastewater treatment operations in
California. The ARPS was really a variation on the same concept. Both efforts
carried out their test work in ponds of 100 square meters or less. They studied a
variety of fundamental operational issues, such as the effects of fluid flow patterns,
light intensity, dissolved oxygen levels, pH and algae harvesting methods.
At the conclusion of the smaller scale tests conducted in California and Hawaii, the
program engaged in a competitive bidding process to select a system design for scale
up of algae mass culture. The HRP design evaluated at UC Berkeley was selected for
scale-up. The “Outdoor Test Facility” (OTF) was designed and built at the site of an
abandoned water treatment plant in Roswell, New Mexico. From 1988 to 1990,
1,000 square meter ponds were successfully operated at Roswell. This project
demonstrated how to achieve very efficient (>90%) utilization of CO2 in large ponds.
The best results were obtained using native species of algae that naturally took over
in the ponds (as opposed to using laboratory cultures). The OTF also demonstrated
production of high levels of oil in algae using both nitrogen and silica depletion
strategies. While daily productivities did reach program target levels of 50 grams per
square per day, overall productivity was much lower (around 10 grams per square
meter per day) due to the number of cold temperature days encountered at this site.
Nevertheless, the project established the proof-of-concept for large scale open pond
operations. The facility was shut down in 1990, and has not been operated since.
A variety of other outdoor projects were funded over the course of the program,
including a three-year project on algal biodiesel production conducted in Israel. In
addition, research at the Georgia Institute of Technology was carried out in the late
1980s. This work consisted of a combination of experimental and computer
modeling work. This project resulted in the development of the APM (Algal Pond
Model), a computer modeling tool for predicting performance of outdoor pond
systems.
Two types of systems analysis were conducted frequently over the course of the
program—resource assessments and engineering design/cost analyses. The former
addresses the following important question: how much impact can algae technology
have on petroleum use within the limits of available resources? Engineering designs
provide some input to this question as well, since such designs tell us something
about the resource demands of the technology. These designs also tell us how much
the technology will cost.
As early as 1982, the program began to study the question of resource availability for
algae technology. Initial studies scoped out criteria and methodology that should be
used in the assessment. In 1985, a study done for Argonne National Lab produced
maps of the southwestern U.S. which showed suitable zones for algae production
based on climate, land and water availability. In 1990, estimates of available CO2
supplies were completed for the first time. These estimates suggested that that there
was enough waste CO2 available in the states where climate conditions were suitable
to support 2 to 7 quads of fuel production annually. The cost of the CO2 was
estimated to range anywhere from $9 to $90 per ton of CO2. This study did not
consider any regionally specific data, but drew its conclusions from overall data on
CO2 availability across a broad region. Also in 1990, a study was funded to assess
land and water availability for algae technology in New Mexico. This study took a
more regionally specific look at the resource question, but did so by sacrificing any
18 A Look Back at the Aquatic Species Program—Program Summary
consideration of available CO2 supplies. This last study sums up the difficulties
faced in these types of studies. The results obtained on resource availability are
either able to provide a complete, but general, perspective on resources or they are
more detailed in approach, but incomplete in the analysis of all resources.
Engineering design and cost studies have been done throughout the course of the
ASP, with ever increasing realism in the design assumptions and cost estimates. The
last set of cost estimates for the program was developed in 1995. These estimates
showed that algal biodiesel cost would range from $1.40 to $4.40 per gallon based on
current and long-term projections for the performance of the technology. Even with
assumptions of $50 per ton of CO2 as a carbon credit, the cost of biodiesel never
competes with the projected cost of petroleum diesel.
Program Funding History
Like all of the renewable fuels programs, the ASP has always been on a fiscal roller
coaster
Funding History for the Aquatic Species Program
3000
2500
2000
1500
$1000s per year
1000
500
0
1978
1980
1982
1984
1986
1988
1990
1992
1994
1996
In its heyday, this program leaped to levels of $2 to $2.75 million in annual funding.
In most cases, these peaks came in sudden bursts in which the funding level of the
program would double from one year to the next. After the boom years of 1984 and
1985, funding fell rapidly to its low of $250,000 in 1991. The last three years of the
program saw a steady level of $500,000 (not counting FY 1996, which were mostly
used to cover the cost of employee terminations). Ironically, these last three years
were among the most productive in the history of the program (given the
breakthroughs that occurred in genetic engineering). Though funding levels were
A Look Back at the Aquatic Species Program—Program Summary 19
relatively low, they were at least steady—providing a desperately needed stability for
the program. The years of higher spending are, for the most part, dominated by
costly demonstration work (the tests carried out in California, Hawaii and
culminating in New Mexico), engineering analysis and culture collection activities.
High Return for a Small Investment of DOE Funds
Aquatic Species
45000
Program
40000
Total Biof uels Program
35000
30000
25000
20000
$1000s per year
15000
10000
5000
0
1978
1980
1982
1984
1986
1988
1990
1992
1994
1996
The total cost of the Aquatic Species Program is $25.05 million over a twenty-year
period. Compared to the total spending under the Biofuels Program ($458 million
over the same period), this has not been a high cost research program. At its peak,
ASP accounted for 14% of the annual Biofuels budget; while, on average, it
represented only 5.5% of the total budget. Given that relatively small investment,
DOE has seen a tremendous return on its research dollars.
Future Directions
Put less emphasis on outdoor field demonstrations and more on basic biology
Much work remains to be done on a fundamental level to maximize the overall
productivity of algae mass culture systems. The bulk of this work is probably best
done in the laboratory. The results of this program’s demonstration activities have
proven the concept of outdoor open pond production of algae. While it is important
to continue a certain amount of field work, small scale studies and research on the
20 A Look Back at the Aquatic Species Program—Program Summary
basic biological issues are clearly more cost effective than large scale demonstration
studies.
Take Advantage of Plant Biotechnology
We have only scratched the surface in the area of genetic engineering for algae. With
the advances occurring in this field today, any future effort on modifying algae to
increase natural oil production and overall productivity are likely to proceed rapidly.
The genetic engineering tools established in the program serve as a strong foundation
for further genetic enhancements of algae.
Start with what works in the field
Select strains that work well at the specific site where the technology is to be used.
These native strains are the most likely to be successful. Then, focus on optimizing
the production of these native strains and use them as starting points for genetic
engineering work.
Maximize photosynthetic efficiency.
Not enough is understood about what the theoretical limits of solar energy conversion
are. Recent advances in our understanding of photosynthetic mechanisms at a
molecular level, in conjunction with the advances being made in genetic engineering
tools for plant systems, offer exciting opportunities for constructing algae which do
not suffer the limitations of light saturation photoinhibition.
Set realistic expectations for the technology
Projections for future costs of petroleum are a moving target. DOE expects
petroleum costs to remain relatively flat over the next 20 years. Expecting algal
biodiesel to compete with such cheap petroleum prices is unrealistic. Without some
mechanism for monetizing its environmental benefits (such as carbon taxes), algal
biodiesel is not going to get off the ground.
Look for near term, intermediate technology deployment opportunities such as
wastewater treatment.
Excessive focus on long term energy displacement goals will slow down
development of the technology. A more balanced approach is needed in which more
near term opportunities can be used to launch the technology in the commercial
arena. Several such opportunities exist. Wastewater treatment is a prime example.
The economics of algae technology are much more favorable when it is used as a
waste treatment process and as a source of fuel. This harks back to the early days of
DOE’s research.
A Look Back at the Aquatic Species Program—Program Summary 21
Footnotes
1 Meier, R.L. (1955) “Biological Cycles in the Transformation of Solar Energy into Useful Fuels.” In
Solar Energy Research (Daniels, F.; Duffie, J.A.; eds), Madison University Wisconsin Press, pp. 179-
183.
2 Peterson, C. L. (1986) “Vegetable Oil as a Diesel Fuel: Status and Research Priorities,” Transactions
of the ASAE, pp 1413-1422. American Society of Agricultural Engineers, St. Joseph, MO.
3 Bruwer, J.; van D. Boshoff, B.; du Plessis, L.; Fuls, J.; Hawkins, C,; van der Walt, A.; Engelbrecht, A.,
(1980)“Sunflower Seed Oil As an Extender for Diesel Fuel in Agricultural Tractors,” presented at the
1980 Symposium of the South African Institute of Agricultural Engineers.
4 Markley, K. (1961) “Chapter 9: Esters and Esterfication,” in Fatty Acids: Their Chemistry, Properties,
Production and Uses Part 2, 2nd Edition (Markley, K.; ed.). Interscience Publications, New York.
5 European engine manufacturers have had very positive experience using rapeseed oil-derived
biodiesel. In the U.S., engine manufacturers have expressed tentative support for blends of soy-derived
biodiesel of up to 20%. See Alternative Fuels Committee of the Engine Manufacturers Association
(1995) Biodiesel Fuels and Their Use in Diesel Engine Applications Engine Manufacturers’
Association, Chicago, IL.
6 Graboski, M.; McCormick, R. (1994) Final Report: Emissions from Biodiesel Blends and Neat
Biodiesel from a 1991 Model Series 60 Engine Operating at High Altitude. Colorado Institute for High
Altitude Fuels and Engine Research. Subcontractor’s report to National Renewable Energy Laboratory,
Golden,CO.
7 FEV Engine Technology, Inc. (1994) Emissions and Performance Characteristics of the Navistar
T444E DI Diesel Engine Fueled with Blends of Biodiesel and Low Sulfur Diesel Fuel: Phase I final
Report. Contractor’s report to the National Biodiesel Board, Jefferson City, MO.
8 Fosseen Manufacturing and Development, Ltd. (1994) Emissions and Performance Characteristics of
the Navistar T444E DI Diesel Engine Fueled with Blends of Biodiesel and Low Sulfur Diesel Fuel:
Phase I final Report. Contractor’s report to National Biodiesel Board, Jefferson City, MO.
9 Peterson, C.; Reece, D. (1994) “Toxicology, Biodegradability and Environmental Benefits of
Biodiesel,” in Biodiesel '94 (Nelson, R.; Swanson. D.; Farrell, J.;eds). Western Regional Biomass
Energy Program, Golden, CO.
10 Sharpe, Chris, Southwest Research Institute (1998). Presentation on speciated emissions presented at
the Biodiesel Environmental Workshop.
11 Annual Energy Outlook 1996 with Projections to 2015. U.S. Department of Energy, Energy
Information Administration, DOE/EIA-0383(96), Washington, D.C. 1996.
12 Reinventing Energy: Making the Right Choices. The American Petroleum Institute, Washington, DC.
1996.
13 See Romm, J. The Atlantic Monthly, April 1996, pp 57-74.
14 Revelle, R.; Suess, H. Tellus, 9/1, pp 18-21, 1957.
15 Herzog, H., et al (1993) The Capture, Utilization and Disposal of Carbon Dioxide from Fossil
FuelPower Plants. Report to the U.S. Department of Energy DOE/ER-30194.
22 A Look Back at the Aquatic Species Program—Program Summary
A Look Back at the U.S.
Department of Energy’s
Aquatic Species Program:
Biodiesel from Algae
Part II:
Technical Review
National Renewable Energy Laboratory
Table of Contents
I.
INTRODUCTION............................................................................................................................ 1
II.
LABORATORY STUDIES ............................................................................................................. 5
II.A.
COLLECTION, SCREENING, AND CHARACTERIZATION OF MICROALGAE
5
II.A.1.
Collection, Screening, and Characterization of Microalgae by SERI In-House Researchers.......5
II.A.1.a. Introduction ...................................................................................................................................5
II.A.1.b. Collection and Screening Activities - 1983...................................................................................8
II.A.1.c. Collection and Screening Activities - 1984...................................................................................8
II.A.1.d. Collection and Screening Activities - 1985.................................................................................17
II.A.1.e. Collection and Screening Activities - 1986 and 1987 .................................................................19
II.A.1.f. Development of a Rapid Screening Procedure for Growth and Lipid Content of Microalgae ....21
II.A.1.g. Statistical Analysis of Multivariate Effects on Microalgal Growth and Lipid Content ..............27
II.A.1.h. Detailed Analyses of Microalgal Lipids......................................................................................28
II.A.2.
Collection, Screening, and Characterization of Microalgae: Research by SERI
Subcontractors.............................................................................................................................32
II.A.2.a. Introduction .................................................................................................................................32
II.A.2.b. Yields, Photosynthetic Efficiencies, and Proximate Chemical Composition of Dense
Cultures of Marine Microalgae....................................................................................................33
II.A.2.c. Selection of High-Yielding Microalgae from Desert Saline Environments ................................36
II.A.2.d. Screening and Characterizing Oleaginous Microalgal Species from the Southeastern United
States ...........................................................................................................................................40
II.A.2.e. Collection of High Energy Strains of Saline Microalgae from Southwestern States ..................43
II.A.2.f. Collection of High Energy Yielding Strains of Saline Microalgae from the Hawaiian Islands ..45
II.A.2.g. Characterization of Hydrocarbon Producing Strains of Microalgae ...........................................46
II.A.2.h. Collection of High Energy Yielding Strains of Saline Microalgae from South Florida..............48
II.A.2.i. Collection and Selection of High Energy Thermophilic Strains of Microalgae ..........................50
II.A.3.
The SERI Microalgae Culture Collection....................................................................................50
II.A.3.a. History of SERI Microalgae Culture Collection .........................................................................51
II.A.3.b. Current status of the SERI/NREL Microalgae Culture Collection..............................................55
II.A.4.
Collection and Screening of Microalgae—Conclusions and Recommendations.........................64
II.B.
MICROALGAL STRAIN IMPROVEMENT
67
II.B.1.
Physiology, Biochemistry, and Molecular Biology of Lipid Production: Work by SERI
Subcontractors.............................................................................................................................67
II.B.1.a. Introduction .................................................................................................................................67
II.B.1.b. Chrysophycean Lipids: Effects of Induction Strategy in the Quantity and Types of Lipids .......68
II.B.1.c. Genetic Variation in High Energy Yielding Microalgae .............................................................70
II.B.1.d. Ultrastructure Evaluation of Lipid Producing Microalgae ..........................................................75
II.B.1.e. Improvement of Microalgal Lipid Production by Flow Cytometry.............................................78
II.B.1.f. Biochemical Elucidation of Neutral Lipid Synthesis in Microalgae ...........................................81
II.B.1.g. Biochemical Elucidation of Neutral Lipid Synthesis in Microalgae ...........................................83
II.B.1.h. Transformation and Somatic Cell Genetics for the Improvement of Energy Production in
Microalgae...................................................................................................................................87
II.B.2.
Physiology, Biochemistry, and Molecular Biology of Lipid Production: NREL In-House
Researchers .................................................................................................................................95
II.B.2.a. Introduction .................................................................................................................................95
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II.B.2.b. Lipid Accumulation Induced by Nitrogen Limitation .................................................................96
II.B.2.c. Studies on Photosynthetic Efficiency in Oleaginous Algae ........................................................97
II.B.2.d. Lipid Accumulation in Silicon-Deficient Diatoms......................................................................98
II.B.2.e. Isolation and Characterization of Acetyl-CoA Carboxylase from C. cryptica ..........................102
II.B.2.f. Cloning of the Acetyl-CoA Carboxylase Gene from C. cryptica ..............................................105
II.B.2.g. Biochemistry of Lipid Synthesis in Nannochloropsis ...............................................................108
II.B.2.h. Biochemistry and Molecular Biology of Chrysolaminarin Synthesis .......................................108
II.B.3
Manipulation of Lipid Production in Microalgae via Genetic Engineering..............................113
II.B.3.a. Introduction ...............................................................................................................................113
II.B.3.b. Mutagenesis and Selection ........................................................................................................114
II.B.3.c. Development of a Genetic Transformation System for Microalgae ..........................................116
II.B.3.d. Attempts to Manipulate Microalgal Lipid Composition via Genetic Engineering....................137
II.B.3.e. The Effect of Different Promoters on Expression of Luciferase in Cyclotella..........................139
II.B.4. Microalgal Strain Improvement – Conclusions and Recommendations .............................................142
III. OUTDOOR STUDIES AND SYSTEMS ANALYSIS............................................................... 145
III.A.
PROJECTS FUNDED BY ERDA/DOE 1976-1979
145
III.A.1.
Introduction...............................................................................................................................145
III.A.2.
Species Control in Large-Scale Algal Biomass Production ......................................................147
III.A.3
An Integrated System for the Conversion of Solar Energy with Sewage-Grown Microalgae ...152
III.A.4.
Large-Scale Freshwater Microalgal Biomass Production for Fuel and Fertilizer ...................156
III.A.5.
Other Microalgae Projects During the ERDA/DOE Period .....................................................161
III.B. THE ASP MICROALGAL MASS CULTURE
162
III.B.1. Introduction .......................................................................................................................................162
III.B.2.
The ARPS Project in Hawaii, 1980-1987 ..................................................................................165
III.B.2.a. Hawaii ARPS Project Initiation, 1980-1981 .............................................................................165
III.B.2 b. Second Year of the Hawaii ARPS Project, 1981-1982 .............................................................166
III.B.2.c. Third Year of the Hawaii ARPS Project, 1982-1983 ................................................................169
III.B.2.d. Fourth Year of the Hawaii ARPS Project, 1983-1984...............................................................170
III.B.2.e. Fifth Year of the ARPS Project, 1984-1985..............................................................................172
III.B.2.f. Sixth Year of the Hawaii ARPS Project, 1985-1986.................................................................172
III.B.2.g. Seventh Year of the Hawaii ARPS Project, 1986-1987 ............................................................173
III.B.2.h. Hawaii ARPS Project, Conclusions...........................................................................................174
III.B.3.
High Rate Pond (HRP) Operations in California, 1981-1986 ..................................................176
III.B.3.a. HRP Design and Construction Phase, 1981 ..............................................................................176
III.B.3.b. HRP Operations in California, Oct-Nov. 1982..........................................................................179
III.B.3.c. Continuing California HRP Pond Operations, 1983-1984 ........................................................179
III.B.3.d. Completion of the California HRP Project, 1985-1986.............................................................185
III.B.4.
The Israeli Microalgae Biodiesel Production Project...............................................................190
III.B.5.
Design and Operation of a Microalgae Outdoor Test Facility (OTF) in New Mexico .............193
III.B.5.a. Facility Design and Construction. .............................................................................................193
III.B.5.b. First Year OTF Experiments .....................................................................................................195
III.B.3.c. Full OTF System Operations.....................................................................................................195
III.B.5.d. Conclusions ...............................................................................................................................198
III.B.6.
The Effects of Environmental Fluctuation on Laboratory Cultures ..........................................199
III.B.6.a. Species Control and Productivity ..............................................................................................199
III.B.6.b. The Algal Pond Growth Model. ................................................................................................202
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III.B.6.c. Microalgae Competition under Fluctuating Conditions in the Laboratory................................205
III.B.6.d. Lipid Productivity of Microalgae ..............................................................................................205
III.B.6.e. Competition Studies with Continuous and Semicontinuous Cultures .......................................208
III.C.
RESOURCE ANALYSES
211
III.C.1.
Introduction...............................................................................................................................211
III.C.2.
The Battelle Columbus 1982 Resource Assessment Report.......................................................212
III.C.3.
The 1982 Argonne Study of CO2 Availability ............................................................................212
III.C.4.
The 1985 SERI Resource Evaluation Report.............................................................................213
III.C.5.
The 1990 SERI Study on CO2 Sources.......................................................................................215
III.C.6.
The 1990 SERI Study of Water Resources in New Mexico ........................................................217
III.C.7.
Conclusions ...............................................................................................................................219
III.D.
ENGINEERING SYSTEMS AND COST ANALYSES
219
III.D.1.
Introduction...............................................................................................................................219
III.D.2.
The Algal Pond Subsystem of the “Photosynthesis Energy Factory” .......................................220
III.D.3.
Cost Analysis of Microalgae Biomass Systems..........................................................................221
III.D.4.
Cost Analysis of Aquatic Biomass Systems................................................................................224
III.D.5.
Microalgae as a Source of Liquid Fuels....................................................................................225
III.D.6.
Fuels from Microalgae Technology Status, Potential and Research Requirements..................229
III.D.7.
Design and Analysis of Microalgae Open Pond Systems ..........................................................233
III.D.8.
Systems and Economic Analysis of Microalgae Ponds for Conversion of CO2 to Biomass ......237
III.D.9.
NREL Studies of Flue Gas CO2 Utilization by Microalgae.......................................................241
III.D.10.
Conclusions. ..............................................................................................................................245
IV. CONCLUSIONS AND RECOMMENDATIONS ..................................................................... 248
IV.A.
MICROALGAL STRAIN IMPROVEMENT
248
IV.A.1.
Conclusions ...............................................................................................................................248
IV.A.2.
R & D Recommendations ..........................................................................................................250
IV.A.2.a. General Considerations .............................................................................................................250
IV.A.2.b. Maximum Efficiency of Photosynthesis....................................................................................250
IV.A.2.c. Overcoming Light Saturation, Photooxidation, and Other Limitations.....................................252
IV.A.2.d. Microalgal Strains for Mass Culture: Source and Genetic Improvements ................................253
IV.B.
MICROALGAL MASS CULTURE
255
IV.B.1.
Conclusions ...............................................................................................................................255
IV.B.1.a. Cost and Productivity Goals ......................................................................................................255
IV.B.1.b. Higher Value Byproducts and Coproducts ................................................................................256
IV.B.1.c. The Japanese R&D Program for Microalgae CO2 Utilization...................................................257
IV.B.1.d. Resource Projections and Microalgae Biodiesel R&D..............................................................259
IV.B.1.e. Summary of Major Conclusions from the ASP Microalgal Mass Culture Work ......................260
IV.B.2.
R & D Recomendations .............................................................................................................260
IV.B.2 a. Biodiesel Production and Algal Mass Culture for Wastewater Treatment ................................260
IV.B.3.
Conclusions ...............................................................................................................................261
V.
BIBLIOGRAPHY ........................................................................................................................ 263
V.A.
SERI/NREL/DOE REPORTS AND PUBLICATIONS
263
V.B.
ADDITIONAL REFERENCES
293
V.C.
PATENTS
294
A Look Back at the Aquatic Species Program—Technical Review
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National Renewable Energy Laboratory
I.
Introduction
Photosynthetic organisms, including plants, algae, and some photosynthetic bacteria, efficiently
utilize the energy from the sun to convert water and CO2 from the air into biomass. The Aquatic
Species Program (ASP) at SERI1 was initiated as a long term, basic research effort to produce
renewable fuels and chemicals from biomass. It emphasized the use of photosynthetic organisms
from aquatic environments, expecially species that grow in environments unsuitable for crop
production. Early in the program, macroalgae, microalgae, and emergents were investigated for
their ability to make lipids (as a feedstock for liquid fuel or chemical production) or
carbohydrates (for fermentation into ethanol or anaerobic digestion for methane production).
Macroalgae (seaweeds) are fast-growing marine or freshwater plants that can reach considerable
size; for example, the giant brown kelp can grow a meter in 1 day and as long as 60 m.
Emergents are plants such as cattails or rushes that grow partially submerged in bogs or marshes.
Macroalgae and emergents were found to produce small amounts of lipid, which function mainly
as structural components of the cell membranes, and produce carbohydrate for use as their
primary energy storage compound. In contrast, many microalgae, (microscopic, photosynthetic
organisms that live in saline or freshwater environments), produce lipids as the primary storage
molecule. By the early 1980s, the decision was made to focus ASP research efforts on the use of
microalgal lipids for the production of fuels and other energy products. The studies on the
growth and chemical composition of macroalgae and emergents will not be discussed in this
report. However, interested readers are referred to reports by subcontractors J.D. Ryther, Harbor
Branch Foundation, Florida (seaweeds), and D. Pratt, from the University of Minnesota, St. Paul
(emergents) listed in the Bibliography.
Microalgae, like higher plants, produce storage lipids in the form of triacyglycerols (TAGs).
Although TAGs could be used to produce of a wide variety of chemicals, work at SERI focused
on the production of fatty acid methyl esters (FAMEs), which can be used as a substitute for
fossil-derived diesel fuel. This fuel, known as biodiesel, can be synthesized from TAGs via a
simple transesterification reaction in the presence of acid or base and methanol. Biodiesel can be
used in unmodified diesel engines, and has advantages over conventional diesel fuel in that it is
renewable, biodegradable, and produces less SOX and particulate emissions when burned. The
technology is available to produce biodiesel from TAGs, and there are growing biodiesel
industries both in the United States and Europe that use soybean or rapeseed oil as the biodiesel
feedstock. However, the potential market for biodiesel far surpasses the availability of plant oils
not designated for other markets. Thus, there was significant interest in the development of
microalgal lipids for biodiesel production.
Microalgae exhibit properties that make them well suited for use in a commercial-scale biodiesel
production facility. Many species exhibit rapid growth and high productivity, and many microalgal
species can be induced to accumulate substantial quantities of lipids, often greater than 60% of
1 The Solar Energy Research Institute (SERI) became the National Renewable Energy Laboratory (NREL)
in 1990. In this report, the laboratory may be referred to as either SERI or NREL, depending on the time
period during which the work being described was performed.
A Look Back at the Aquatic Species Program—Technical Review
National Renewable Energy Laboratory
their biomass. Microalgae can also grow in saline waters that are not suitable for agricultural
irrigation or consumption by humans or animals. The growth requirements are very simple,
primarily carbon dioxide (CO2) and water, although the growth rates can be accelerated by
sufficient aeration and the addition of nutrients. A brief overview of the characteristics of the
major microalgal classes can be found in Section II.A.2.
A major undertaking by ASP researchers in the early stages of the program was to identify
candidate microalgal species that exhibited characteristics desirable for a commercial production
strain. Resource analyses carried out by SERI (discussed in Section III.C.) indicated that the
desert regions of the southwestern United States were attractive areas in which to locate
microalgal-based biofuel production facilities. This, in part, dictated the required strain
characteristics. These characteristics included the ability of the strains to grow rapidly and have
high lipid productivity when growing under high light intensity, high temperature, and in saline
waters indigenous to the area in which the commercial production facility is located. In addition,
because it is not possible to control the weather in the area of the ponds, the best strains should
have good productivity under fluctuating light intensity, temperature, and salinity.
A multi-faceted effort was carried out to:
• isolate microalgae from a variety of saline habitats (including oceans, lakes, ponds,
and various ephemeral water bodies),
• screen those isolates for the ability to grow under a variety of conditions,
• analyze the biochemical components of the strains (especially with respect to lipids),
and
• determine the effects of environmental variables on the growth and lipid composition
of selected strains.
This effort involved in-house researchers and subcontractors from academia, industry, and other
government laboratories. Section II.A.1. documents the efforts of SERI in-house researchers in
the area of microalgal strain isolation and screening. It also describes the methodologies
developed and employed during the isolation, screening, and characterization phases of the work.
Section II.A.2. describes parallel efforts conducted by SERI subcontractors. An account of the
history and current status of the NREL Microalgae Culture Collection is presented in Section
II.A.3.
Although the collection and screening efforts produced a number of viable candidate strains, no
one algal strain was identified that exhibited the optimal properties of rapid growth and high
constitutive lipid production. Many microalgae can be induced to accumulate lipids under
conditions of nutrient deprivation. If this process could be understood, it might be possible to
manipulate either the culture conditions, or to manipulate the organisms themselves, to increase
lipid accumulation in a particular strain. Therefore, studies were initiated both at SERI and by
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National Renewable Energy Laboratory
ASP subcontractors to study the biochemistry and physiology of lipid production in oleaginous
(oil-producing) microalgae. Work performed by several ASP subcontractors was designed to
understand the mechanism of lipid accumulation. In particular, these researchers tried to
determine whether there is a specific “lipid trigger” that is induced by factors such as nitrogen
(N) starvation. Subcontractors also studied ultrastructural changes induced in microalgae during
lipid accumulation. They also initiated efforts to produce improved algae strains by looking for
genetic variability between algal isolates, attempting to use flow cytometry to screen for
naturally-occurring high lipid individuals, and exploring algal viruses as potential genetic
vectors. The work performed by ASP subcontractors is described in Section II.B.1.
Although some of the efforts of the in-house SERI researchers were also directed toward
understanding the lipid trigger induced by N starvation, they showed that silica (Si) depletion in
diatoms also induced lipid accumulation. Unlike N, Si is not a major component of cellular
molecules, therefore it was thought that the Si effect on lipid production might be less complex
than the N effect, and thus easier to understand. This initiated a major research effort at SERI to
understand the biochemistry and molecular biology of lipid accumulation in Si-depleted diatoms.
This work led to the isolation and characterization of several enzymes involved in lipid and
carbohydrate synthesis pathways, as well as the cloning of the genes that code for these enzymes.
One goal was to genetically manipulate these genes in order to optimize lipid accumulation in
the algae. Therefore, reseach was performed simultaneously to develop a genetic transformation
system for oleaginous microalgal strains. The successful development of a method to genetically
engineer diatoms was used in attempts to manipulate microalgal lipid levels by overexpressing or
down-regulating key genes in the lipid or carbohydrate synthetic pathways. Unfortunately,
program funding was discontinued before these experiments could be carried out beyond the
prelimilary stages.
Cost-effective production of biodiesel requires not only the development of microalgal strains
with optimal properties of growth and lipid production, but also an optimized pond design and a
clear understanding of the available resources (land, water, power, etc.) required. Section III
reviews the R&D on outdoor microalgae mass culture for production of biodiesel, as well as
supporting engineering, economic and resource analyses, carried out and supported by ASP
during the 1980s and early 1990s. It also covers work supported by DOE and its predecessor
agency, the Energy Research and Development Administration (ERDA), during the 1970s and
some recent work on utilization of CO2 from power plant flue gases.
From 1976 to 1979, researchers at the University of California-Berkeley used shallow, paddle
wheel mixed, raceway-type (high-rate) ponds to demonstrate a process for the simultaneous
treatment of wastewater and production of energy (specifically methane). Starting in 1980, the
ASP supported outdoor microalgal cultivation projects in Hawaii and California, using fresh and
seawater supplies, respectively, in conjunction with agricultural fertilizers and CO2. The two
projects differed in the types of algae cultivated and the design of the mass culture system, with
the project in California continuing to develop the high-rate pond design, and the Hawaii project
studying an (initially) enclosed and intensively mixed system. From 1987 to 1990, an “Outdoor
Test Facility” was designed, constructed and operated in Roswell, New Mexico, including two
A Look Back at the Aquatic Species Program—Technical Review
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National Renewable Energy Laboratory
1,000 m2 high-rate ponds. This last project represented the culmination of ASP R&D in
large-scale algal mass culture R&D. These studies are described in Section III.A. Some
supporting laboratory studies and development of an “Algal Pond Model” (APM) are also
reviewed at the end of that section. The conclusion from these extensive outdoor mass culture
studies was that the use of microalgae for the low-cost production of biodiesel is technically
feasible, but still requires considerable long-term R&D to achieve the high productivities
required.
Section III.B. reviews the resource assessments, for water, land, CO2, etc., carried out by the
ASP, primarily for the southwestern United States. These studies demonstrated the potential
availability of large brackish and saline water resources suitable for microalgae mass cultures,
large land and CO2 resources. They suggest that the potential production of microalgae-derived
biodiesel may represent more than 10% of U.S. transportation fuels, although full resource
exploitation would be significantly constrained in practice. Several engineering and economic
cost analyses were also supported by DOE and the ASP, and these are reviewed in Section III.C.,
including recent work by the ASP and DOE on power plant flue gas utilization for greenhouse
gas (CO2) mitigation.
The overall conclusion of these studies was that in principle and practice large-scale microalgae
production is not limited by design, engineering, or net energy considerations and could be
economically competitive with other renewable energy sources. However, long-term R&D
would be required to actually achieve the very high productivities and other assumptions made in
such cost analyses. Section III.D. provides recommendations for future research that could make
this technology commercially feasible.
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National Renewable Energy Laboratory
II.
Laboratory Studies
II.A.
Collection, Screening, and Characterization of Microalgae
II.A.1.
Collection, Screening, and Characterization of Microalgae by SERI
In-House Researchers
II.A.1.a. Introduction
This chapter describes the research performed at SERI in the area of microalgal collection and
screening. In addition to performing actual research in this area, SERI personnel were
responsible for coordinating the efforts of the many subcontractors performing similar activities,
and for standardizing certain procedures and analyses. These efforts ultimately resulted in the
development of the SERI Microalgal Culture Collection, which is more fully described in
Chapter II.A.3.
Brief review of algal taxonomic groups and characteristics:
For the purposes of this report, microalgae are defined as microscopic organisms that can grow
via photosynthesis. Many microalgae grow quite rapidly, and are considerably more productive
than land plants and macroalgae (seaweed). Microalgae reproduction occurs primarily by
vegetative (asexual) cell division, although sexual reproduction can occur in many species under
appropriate growth conditions.
There are several main groups of microalgae, which differ primarily in pigment composition,
biochemical constituents, ultrastructure, and life cycle. Five groups were of primary importance
to the ASP: diatoms (Class Bacillariophyceae), green algae (Class Chlorophyceae), golden-
brown algae (Class Chrysophyceae), prymnesiophytes (Class Prymnesiophyceae), and the
eustigmatophytes (Class Eustigmatophyceae). The blue-green algae, or cyanobacteria (Class
Cyanophyceae), were also represented in some of the collections. A brief description of these
algal groups follows.
• Diatoms. Diatoms are among the most common and widely distributed groups
of algae in existence; about 100,000 species are known. This group tends to
dominate the phytoplankton of the oceans, but is commonly found in fresh- and
brackish-water habitats as well. The cells are golden-brown because of the
presence of high levels of fucoxanthin, a photosynthetic accessory pigment.
Several other xanthophylls are present at lower levels, as well as β-carotene,
chlorophyll a and chlorophyll c. The main storage compounds of diatoms are
lipids (TAGs) and a β-1,3-linked carbohydrate known as chrysolaminarin. A
distinguishing feature of diatoms is the presence of a cell wall that contains
substantial quantities of polymerized Si. This has implications for media costs
in a commercial production facility, because silicate is a relatively expensive
chemical. On the other hand, Si deficiency is known to promote storage lipid
A Look Back at the Aquatic Species Program—Technical Review
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National Renewable Energy Laboratory
accumulation in diatoms, and thus could provide a controllable means to induce
lipid synthesis in a two-stage production process. Another characteristic of
diatoms that distinguishes them from most other algal groups is that they are
diploid (having two copies of each chromosome) during vegetative growth;
most algae are haploid (with one copy of each chromosome) except for brief
periods when the cells are reproducing sexually. The main ramification of this
from a strain development perspective is that it makes producing improved
strains via classical mutagenesis and selection/screening substantially more
difficult. As a consequence, diatom strain development programs must rely
heavily on genetic engineering approaches.
• Green Algae. Green algae, often referred to as chlorophytes, are also abundant;
approximately 8,000 species are estimated to be in existence. This group has
chlorophyll a and chlorophyll b. These algae use starch as their primary storage
component. However, N-deficiency promotes the accumulation of lipids in
certain species. Green algae are the evolutionary progenitors of higher plants,
and, as such, have received more attention than other groups of algae. A
member of this group, Chlamydomonas reinhardtii (and closely related species)
has been studied very extensively, in part because of its ability to control sexual
reproduction, thus allowing detailed genetic analysis. Indeed, Chlamydomonas
was the first alga to be genetically transformed. However, it does not
accumulate lipids, and thus was not considered for use in the ASP. Another
common genus that has been studied fairly extensively is Chlorella.
• Golden-Brown Algae. This group of algae, commonly referred to as
chrysophytes, is similar to diatoms with respect to pigments and biochemical
composition. Approximately 1,000 species are known, which are found
primarily in freshwater habitats. Lipids and chrysolaminarin are considered to
be the major carbon storage form in this group. Some chysophytes have lightly
silicified cell walls.
• Prymnesiophytes. This group of algae, also known as the haptophytes, consists
of approximately 500 species. They are primarily marine organisms, and can
account for a substantial proportion of the primary productivity of tropical
oceans. As with the diatoms and chrysophytes, fucoxanthin imparts a brown
color to the cells, and lipids and chrysolaminarin are the major storage products.
This group includes the coccolithophorids, which are distinguished by
calcareous scales surrounding the cell wall.
• Eustigmatophytes. This group represents an important component of the
“picoplankton”, which are very small cells (2-4 µm in diameter). The genus
Nannochloropsis is one of the few marine species in this class, and is common
in the world’s oceans. Chlorophyll a is the only chlorophyll present in the cells,
although several xanthophylls serve as accessory photosynthetic pigments.
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National Renewable Energy Laboratory
• Cyanobacteria. This group is prokaryotic, and therefore very different from all
other groups of microalgae. They contain no nucleus, no chloroplasts, and have
a different gene structure. There are approximately 2,000 species of
cyanobacteria, which occur in many habitats. Although this group is
distinguished by having members that can assimilate atmospheric N (thus
eliminating the need to provide fixed N to the cells), no member of this class
produces significant quantities of storage lipid; therefore, this group was not
deemed useful to the ASP.
Collection and Screening of Microalgae: Programmatic Rationale
The in-house collection effort was focused on collecting strains from inland saline habitats,
particularly in Colorado, New Mexico, and Utah. The reasoning behind collecting strains from
these habitats was that the strains would be adapted to at least some of the environmental
conditions in mass culture facilities in the southwestern United States (i.e., high light intensity
and high temperatures). They would also be well suited for growth in the saline waters available
for use in such facilities. In addition, many of the aquatic habitats in this region are shallow, and
therefore subject to large variations in temperature and salinity; thus, the strains collected in this
region might be expected to better withstand the fluctuations that would occur in a commercial
production pond. Cyanobacteria, chrysophytes, and diatoms often dominate inland saline
habitats. The latter were of particular interest to the program because of their propensity to
accumulate lipids. There had never been a large-scale effort to collect strains with this
combination of characteristics; therefore, they were not available from culture collections.
The stated objectives2 of the SERI culture collection and screening effort were to:
• Assemble and maintain a set of viable mono-specific algal cultures stored under
conditions best suited to the maintenance of their original physiological and
biochemical characteristics.
• Develop storage techniques that will help maintain the genetic variability and
physiological adaptability of the species.
• Collect single species cultures of microalgae from the arid regions of Colorado,
Utah, and New Mexico for product and performance screening.
• Develop media which are suitable for their growth.
• Evaluate each species for its temperature and salinity tolerances, and quantify
growth rates and proximate chemical composition for each species over the
range of tolerated conditions.
2 Taken from the Proceedings of the April 1984 Aquatic Species Program Principal Investigators’ Meeting.
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National Renewable Energy Laboratory
Each objective was met during the course of research within the ASP. The following pages
describe in detail the major findings of the work conducted by SERI researchers.
II.A.1.b.
Collection and Screening Activities - 1983
The first collecting trips made by SERI researchers took place in the fall of 1983. Five saline hot
springs in western Colorado were selected for sampling because of their abundant diatom
populations, and because a variety of water types was represented. Water samples were used to
inoculate natural collection site water that had been enriched with N (ammonium and nitrate) and
phosphate (P) and then filter sterilized. Water samples were also taken for subsequent chemical
analyses. The temperature and conductivity of the site water were determined at the time of
collection. Conductivity ranged from 1.9 mmhos•cm-2 at South Canyon Spring to 85.0
mmhos•cm-2 (nearly three times the conductivity of seawater) at Piceance Spring. Water
temperature at the time of collection ranged from 11º to 46ºC.
In the laboratory, researchers tried to isolate the dominant diatoms from the enriched water
samples. Cyanobacteria and other contaminants were removed primarily with agar plating.
Approximately 125 unialgal diatom strains were isolated. The predominant genera found were
Achnanthes, Amphora, Caloneis, Camphylodiscus, Cymbella, Entomoneis, Gyrosigma, Melosira,
Navicula, Nitzschia, Pleurosigma, and Surirella.
A standardized lipid analysis protocol was not yet in place to screen these strains. However,
many algal strains were known to accumulate lipids under conditions of nutrient stress.
Microscopic analysis of cells grown under N-deficient conditions revealed lipid droplets in
several of the strains, particularly in Amphora and Cymbella.
In addition to yielding several promising algal strains, this initial collection trip was useful for
identifying areas for improving the collection and screening protocols. Some of these
improvements were implemented for the 1984-collecting season, and are described in the next
section.
Publications:
Barclay, W.R. (1984) “Microalgal technology and research at SERI: Species collection and
characterization.” Aquatic Species Program Review: Proceedings of the April 1984 Principal
Investigators’ Meeting, Solar Energy Research Institute, Golden, Colorado, SERI/CP-231-2341;
pp. 152-159.
II.A.1.c.
Collection and Screening Activities - 1984
The screening and characterization protocols used by SERI researchers were refined for the 1984
collecting season. Included in these refinements was the development of a modified “rotary
screening apparatus”, a standard type of motorized culture mixing wheel for 16x150-mm culture
tubes. The rotating wheel was constructed of Plexiglas to allow better light exposure (see Figure
II.A.1). The wheel was typically illuminated with a high-intensity tungsten stage lamp, and
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could be placed inside a box behind a CuSO4-water heat filter for temperature control. The
Plexiglas wheel allowed all the cultures to receive equal illumination. Another technological
advance used a temperature-salinity gradient table to characterize the thermal and salinity
preferences and tolerances of the isolates.
Development of artificial saline media.
One of the most significant contributions made by SERI researchers during 1984 was the
development of media that mimicked the saline water in shallow aquifers in the southwestern
United States. This was an important undertaking because it allowed algal strains to be screened
for growth in the types of water that would likely be available in an outdoor mass culture facility.
To identify the major water types available in the southwestern United States, state and federal
reports that described the chemical characteristics of water from 85 saline wells in New Mexico
were studied. The data were statistically analyzed to identify the relationships between the
various ionic constituents. (Data from wells deeper than 83 m was not used in this analysis,
because the cost of pumping water from those depths was prohibitive.) R-mode factor analysis
indicated that two factors were largely responsible for the differences between the waters
examined (Barclay et al. 1988). The first factor, monovalent ion concentration, was responsible
for 40% of the variance; the second factor, divalent ion concentration, for 30%. A plot of these
factors against each other clearly delineated two primary water types, referred to as “Type I” and
“Type II”. Type I waters were characterized by a low monovalent-to-divalent ion ratio (average
value = 0.4), whereas Type II waters had a higher level of monovalent ions (monovalent-to-
divalent ion ratio of 9.4). The major ions present in Type I water were Na+, Cl-, Mg2+, and Ca2+.
The major ions of Type II water were Na+, Cl-, SO 2-
-
4 , and HCO3 . Type II water is consequently
termed a “sodium bicarbonate class” of water. Approximately three-fourths of the saline well
waters were of the Type II variety, and one-fourth could be characterized as Type I.
The survey indicated that both types of water exhibited a range of conductivities; the researchers
believed that the higher-conductivity waters resulted from evaporation of the lower conductivity
waters. In addition, they recognized that the conductivity of the water in an outdoor production
pond would increase with time because of the high rates of evaporation in the southwestern
United States (as high as 1 cm•day-1). Therefore, artificial media that covered a wide range of
conductivities had to be developed. To this end, an experiment was conducted in which media
that contained the salts typically present in low-conductivity Type I and Type II waters were
allowed to evaporate with stirring at 35ºC. Samples were removed at various times and filtered.
The ions still dissolved in the waters were quantified using an inductively coupled plasma
spectrometer and a high-performance liquid chromatograph. In this manner, media formulations
were derived at SERI that covered a range of conductivities (from 10 to 70 mmho•cm-1) for both
media types. The media most commonly used were designated SERI Type I/10, Type I/25, Type
I/55, Type I/70, Type II/10, Type II/25, Type II/55, and Type II/70, in which the number
following the slash indicates the specific conductivity of the medium. The compositions of these
media are given in Figure II.A.2.
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National Renewable Energy Laboratory
In order to assess whether these media formulations accurately reflected the types of water in
desert region surface waters, samples of the water at numerous algal collection sites in the
southwestern United States were chemically analyzed. The relative compositions of the anionic
and cationic constituents were then plotted on separate trilinear plots, which allowed a graphical
representation of the various water samples relative to SERI Type I and Type II media (Figure
II.A.3). This analysis indicated that Type I water has higher proportions of Mg2+ and Ca2+ than
most surface waters examined, whereas Type II water was fairly representative of the sampled
waters with respect to these cations. On the other hand, natural surface waters often had an anion
composition similar to both SERI Type I and Type II media. The researchers concluded that
these artificial media would serve well as standardized media for testing newly acquired strains,
thereby allowing all ASP researchers (both in-house personnel and subcontractors) to screen
strains for growth potential in waters similar to those that would be available for commercial
production.
Collection activities.
Collecting trips made by SERI researchers in 1984 focused on shallow saline habitats, including
ephemeral ponds, playas, and springs in the arid regions of Colorado and Utah. After collection,
the water and sediment samples were kept under cool, dark conditions for 1 to 3 days until they
could be further treated in the laboratory. The pH, temperature, conductivity, redox potential,
and alkalinity of the collection site waters were determined, and water samples were taken for
subsequent ion analysis. In the laboratory, the samples were enriched with 300 µM urea, 30 µM
PO4, 36 µM Na2SiO3, 3 µM NaFeEDTA, trace metals (5 mL/L PII stock, see Figure II.A.2), and
vitamins. The enrichment tubes were then placed in the rotary screening apparatus (maintained
at 25ºC or 30ºC) and illuminated at ~400 µE•m-2•s-1. Over a 5-day period, the illumination
provided by the stage lamp was gradually increased to 1,000 µE•m-2•s-1. The predominant strains
present in the tubes were isolated as unialgal cultures by agar plating or by serial dilution in
liquid media.
The isolated strains were then tested for their ability to grow in incubators at 25ºC at 150-200
µE•m-2•s-1 in the standard media types described above. and in artificial seawater (termed “Rila
Salts ASW,” using Rila Marine Mix, an artificial sea salt mixture produced by Rila Products,
Teaneck, NJ. The strains that grew well in at least one of these media were further characterized
with respect to growth on a temperature-salinity gradient table at a light intensity of 200
µE•m-2•s-1. Thirty combinations of temperature (10º to 35ºC) and salinity (10 to 70 mmho•cm-1)
were included in this analysis, representing the ranges that might be expected in actual outdoor
production systems. Once again, the cultures were enriched with nutrients to maximize growth
rates. The cultures used to inoculate the test cultures were preconditioned by growth in the
media at 17° and 27ºC. The optical density at 750 nm (OD750) of the cultures was measured
twice daily for 5 days, and the growth rates were calculated from the increase in culture density
during the exponential phase of growth. A refinement of this method was to measure the growth
rates in semicontinuous cultures, wherein the cultures were periodically diluted by half with fresh
medium; this method provided more reproducible results than the batch mode experiments.
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Figure II.A.3 gives an example of the type of growth data generated by the use of temperature-
salinity gradient tables. The contour lines in the plot are interpolations indicating where a
particular combination of temperature and salinity would result in a given growth rate. Many
such plots were generated for various strains, and are shown in the culture collection catalogs and
ASP annual reports.
Approximately 300 strains were collected from the 1984 trips to Utah and Colorado. Of these,
only 15 grew well at temperatures ≥30ºC and conductivities greater than 5 mmho•cm-1. Nine
were diatoms, including the genera Amphora, Cymbella, Amphipleura, Chaetoceros, Nitzschia,
Hantzschia, and Diploneis. Several chlorophytes (Chlorella, Scenedesmus, Ankistrodesmus, and
Chlorococcum) were also identified as promising strains, along with one chrysophyte
(Boekelovia).
Two strains isolated as a result of the 1984 collecting effort (Ankistrodesmus sp. and Boekelovia
sp.) were characterized in greater detail using the temperature-salinity matrix described earlier.
Boekelovia exhibited a wide range of temperature and salinity tolerance, and grew faster than one
doubling•day-1 from 10 to 70 mmho•cm-1 conductivity and from 10º to 32ºC, exhibiting maximal
growth of 3.5 doublings•day-1 in Type II/25 medium. Reasonable growth rates were also
achieved in SERI Type I and ASW-Rila salts media (as many as 1.73 and 2.6 doublings•day-1,
respectively). Ankistrodesmus was also able to grow well in a wide range of salinities and
temperatures, with maximal growth rates occurring in Type II/25 medium (3.0 doublings•day-1).
Boekelovia and Ankistrodesmus were also examined with regard to their lipid accumulation
potential. Two-liter cultures were grown in media that contained high (600 µM) and low (300
µM) urea concentrations at a light intensity of 200 µE•m-2•s-1. Half of each culture was harvested
2 days after the low-N culture entered stationary phase to determine the lipid content of N-
sufficient cells and cells that were just entering N-deficient growth. After 10 days of N-limited
growth, the remainder of the low-N culture was harvested. Lipids were extracted via a
modification of the method of Bligh and Dyer (1959) and lipid mass was determined
gravimetrically. The lipid content of Boekelovia was 27% of the organic mass in N-sufficient
cells, increasing to 42% and 59% after 2 days and 10 days of N-deficiency, respectively. There
was less effect of N starvation on the lipid content of Ankistrodesmus; the lipid content only
increased from 23% in N-sufficient cells to 29% in cells that were N-deficient for 10 days.
In conclusion, research at SERI in 1984 led to the development of artificial media that mimicked
the saline groundwater typically found in the desert regions of the southwestern United States.
This allowed the strains isolated during collecting trips at various ionic concentrations to be
systematically screened and provided standardized media that could be used in different
laboratories performing ASP-sponsored research. Numerous strains were characterized with
respect to growth at several temperatures and salinities using these new media.
A Look Back at the Aquatic Species Program—Technical Review
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National Renewable Energy Laboratory
Publications:
Barclay, W.; Johansen, J.; Chelf, P.; Nagle, N.; Roessler, R.; Lemke, P. (1986) “Microalgae
Culture Collection 1986-1987.” Solar Energy Research Institute, Golden, Colorado, SERI/SP-
232-3079, 147 pp.
Barclay, B.; Nagle, N.; Terry, K. (1987) “Screening microalgae for biomass production potential:
Protocol modification and evaluation.” FY 1986 Aquatic Species Program Annual Report, Solar
Energy Research Institute, Golden, Colorado, SERI/CP-231-3071; pp. 23-40.
Barclay, B.; Nagle, N.; Terry, K.; Roessler, P. (1985) “Collecting and screening microalgae from
shallow, inland saline habitats.” Aquatic Species Program Review: Proceedings of the March
1985 Principal Investigators’ Meeting, Solar Energy Research Institute, Golden, Colorado,
SERI/CP-231-2700; pp. 52-68.
Barclay, W.R.; Nagle, N.J.; Terry, K.L.; Ellingson, S.B.; Sommerfeld, M.R. (1988)
“Characterization of saline groundwater resource quality for aquatic biomass production: A
statistically-based approach.” Wat. Res. 22:373-379.
Sommerfeld, M.R.; Ellingson, S.B. (1987) “Collection of high energy yielding strains of saline
microalgae from southwestern states.” FY 1986 Aquatic Species Program Annual Report, Solar
Energy Research Institute, Golden, Colorado, SERI/CP-231-3071; pp. 53-66.
Additional References:
Bligh, E.G.; Dyer, D.J. (1959) “A rapid method for total lipid extraction and purification.” Can.
J. Biochem. Physiol. 37:911-917.
Siver, P. (1983) “A new thermal gradient device for culturing algae.” British J. Phycol. 18:159-
163.
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Figure II.A.1. Rotary screening apparatus used for microalgal screening.
A Look Back at the Aquatic Species Program—Technical Review
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National Renewable Energy Laboratory
Figure II.A.2. Formulations for SERI Type I and Type II artificial inland saline waters.
Recipes for the preparation of Type I and Type II media at five different salinities, expressed as
conductivity of the final solution. Formulas for these media were developed by statistical
analysis of saline groundwater data for the state of New Mexico. For each salt, necessary
additions in mg/L are listed. (Source: Barclay et al. 1986).
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Figure II.A.3. Trilinear plots showing the ionic constitutents of various water samples
relative to SERI Type I and SERI Type II artificial saline media. (Source: Sommerfeld and
Ellingson 1987.)
A Look Back at the Aquatic Species Program—Technical Review
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National Renewable Energy Laboratory
Figure II.A.4. Growth contour plots. Examples of growth contour plots generated from
data obtained by the use of a temperature-gradient table. The contour lines represent
interpolated values indicating where a particular combination of temperature and salinity
would result in a given growth rate. The data shown, given as doublings•day) represent the
exponential growth of Monoraphidium sp. (S/MONOR-2) in semicontinuous culture. Each
point represents the mean of at least five separate daily growth rate determinations. (Source:
Barclay et al. 1987).
A: Type I inland saline water
B: Type II inland saline water
C: Seawater
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II.A.1.d.
Collection and Screening Activities - 1985
In 1985, the strain enrichment procedure utilizing the rotary screening apparatus described
previously was modified to include incubation of samples in SERI Type I and Type II media (25
and 55 mmho•cm-1 conductivity) and in artificial seawater, in addition to the original site water.
The cultures that exhibited substantial algal growth were further treated to isolate the
predominant strains as unialgal (clonal) isolates. These strains were then tested for growth using
the temperature-salinity matrix described earlier.
Collection activities.
Collection efforts by SERI researchers in 1985 again focused on shallow inland saline habitats.
This time collecting trips were also made to New Mexico and Nebraska, in addition to Colorado
and Utah. Eighty-six sites were sampled during the year, 53 of which were sampled in the
spring. From these 53 sites, 17 promising strains were isolated. An analysis was conducted
comparing the results of the new protocol with those that would have resulted from the protocol
used in prior years. This analysis indicated that the revised protocol was in fact superior to the
older protocol. For example, only six of the 17 strains selected via the new protocol would also
have been selected using the old protocol. Only three of the 17 strains grew best in the artificial
medium type that most closely resembled the collection site water; in fact, only six strains were
even considered to grow well in the collection site water relative to growth in at least one of the
artificial medium. This analysis clearly indicated the value of performing the initial screening
and enrichment in a variety of relevant media. The results suggest that the shallow saline
environments sampled probably contain a large number of species whose metabolism is arrested
at any given time. In other words, the water quality of such sites varies greatly, depending on
precipitation and evaporation, so probably only a few of the many species present are actively
growing at any given time. This also may explain the wide range of salinities and temperatures
tolerated by many of these strains.
Growth rates.
Six promising strains were analyzed in SERI Type I, Type II, and ASW (Rila) using the
temperature-salinity gradient described previously. These included the diatoms Chaetoceros
muelleri (CHAET14), Navicula (NAVIC1), Cyclotella (CYCLO2), Amphora (AMPHO1 and
AMPHO2), and the chlorophyte Monoraphidium minutum (MONOR2). (NAVIC1 and
CYCLO2 were actually collected from the Florida keys; the remaining strains were collected in
Colorado and Utah.) All strains exhibited rapid growth over a wide range of conductivities in at
least two media types. Furthermore, all strains exhibited temperature optima of 30ºC or higher.
Maximal growth rates of these strains, along with the optimal temperature, conductivity, and
media type determined in these experiments are shown in Table II.A.1. (Higher growth rates
were determined for some of these strains in subsequent experiments; see results presented in
Barclay et al. [1987]). Temperature-salinity growth contours are provided for these strains in the
1986 ASP Annual Report (Barclay et al. 1986).
A Look Back at the Aquatic Species Program—Technical Review
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National Renewable Energy Laboratory
Table II.A.1. Growth characteristics of various microalgal strains collected in 1985.
Strain
Maximum
Optimal
Optimal
Optimal Medium Type
Growth Rate
Temperature
Conductivity
(dependent on temperature
(doublings•day-1)
(ºC)
(mmho•cm-1)
and conductivity used)
AMPHO1 1.7
30
10-25
Type
I,
ASW
AMPHO2
2.48
30-35
40-70
Type I, Type II
CHAET14
2.87
35
25-70
Type II, ASW
CYCLO2
1.63
30-35
10-70
Type I, ASW
MONOR2
2.84
25-30
25
Type I, II, ASW
NAVIC1
2.77
30
10-40
Type I, Type II
Experiments were also conducted in an attempt to identify the chemical components of SERI
Type I and Type II media most important for controlling the growth of the various algal strains.
Bicarbonate and divalent cation concentrations were found to be important determinants in
controlling the growth of Boekelovia sp. (BOEKE1) and Monoraphidium (MONOR2). The
growth rate of MONOR2 increased by more than five-fold as the bicarbonate concentration of
Type II/25 medium was increased from 2 to 30 mM, and the growth of BOEKE1 by
approximately 60% over this range. These results make sense, since media enriched in
bicarbonate would have more dissolved carbon available for photosynthesis. An unexpected
finding was that there was a decrease of nearly 50% in the growth rate of BOEKE1 as the
divalent cation concentration increased from 5 mM to 95 mM (in Type I/10 medium containing
altered amounts of calcium and magnesium). The effects of magnesium and calcium
concentration on the growth of MONOR2 were less pronounced. These results indicate that
matching the chosen strain for a particular production site to the type of water available for mass
cultivation will be important.
Lipid content.
The lipid contents of several strains were determined for cultures in exponential growth phase
and for cultures that were N-limited for 7 days or Si-limited for 2 days. In general, nutrient
deficiency led to an increase in the lipid content of the cells, but this was not always the case.
The highest lipid content occurred with NAVIC1, which increased from 22% in exponential
phase cells to 49% in Si-deficient cells and to 58% in N-deficient cells. For the green alga
MONOR2, the lipid content increased from 22% in exponentially growing cells to 52% for cells
that had been N-starved for 7 days. CHAET14 also exhibited a large increase in lipid content in
response to Si and N deficiency, increasing from 19% to 39% and 38%, respectively. A more
modest increase occurred for nutrient-deficient AMPHO1 cells, whereas the lipid content of
CYCLO2 was similar in exponential phase and nutrient-deficient cells, and actually decreased in
AMPHO2 as a result of nutrient deficiency.
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These results suggested that high lipid content was indeed achievable in many strains by
manipulating the nutrient levels in the growth media. However, these experiments did not
provide information on actual lipid productivity in the cultures, which is the more important
factor for developing a commercially viable biodiesel production process. This lack of lipid
productivity data also occurred with most of the ASP subcontractors involved in strain screening
and characterization, but was understandable because the process for maximizing lipid yields
from microalgae grown in mass culture never was optimized. Therefore, there was no basis for
designing experiments to estimate lipid productivity potential.
Publications:
Barclay, B.; Nagle, N.; Terry, K. (1986) “Screening microalgae for biomass production potential:
protocol modification and evaluation.” FY 1986 Aquatic Species Program Annual Report, Solar
Energy Research Institute, Golden, Colorado, SERI/SP-231-3071, pp. 22-40.
Barclay, W.R.; Terry, K.L.; Nagle, N.J.; Weissman, J.C.; Goebel, R.P. (1987) “Potential of new
strains of marine and inland saline-adapted microalgae for aquaculture.” J. World. Aquaculture
Soc. 18:218-228.
II.A.1.e.
Collection and Screening Activities - 1986 and 1987
SERI in-house algal strain collection and screening efforts during 1986-1987 were focused in
three separate areas. First, detailed characterization of previously collected strains continued.
Second, because the desert southwest sites targeted for biodiesel production facilities can be quite
cool during the winter, a new effort to collect strains from cold-water sites was initiated. Finally,
a strategy was developed and implemented to reduce the number of strains that had accumulated
as a result of in-house and subcontracted research efforts, which allowed researchers to focus on
the most promising strains.
Strain characterization.
Eight additional strains collected previously from warm-water sites that grew well during the
initial screening procedures were characterized with respect to temperature and salinity
tolerances, growth rates, and lipid content under various conditions. These strains were
Chaetoceros muelleri (strains CHAET6, CHAET9, CHAET10, CHAET15, and CHAET39),
Cyclotella cryptica (CYCLO4), Pleurochrysis carterae (PLEUR1), and Thalassiosira weissflogii
(THALA2). Each strain was grown in a variety of temperature-salinity combinations by the use
of a temperature-salinity gradient table. The maximal growth rate achieved under these
conditions occurred with CHAET9, which exhibited a growth rate of 4.0 doublings•day-1. The
remaining strains all had maximum growth rates that exceeded 1.4 doublings•day-1, and several
grew at rates exceeding 2.5 doublings•day-1 (i.e., CHAET6, CHAET10, and CHAET39). All had
an optimal temperature of 30°C or higher, except for PLEUR1 and THALA2, which had optimal
temperatures of 25°C and 28°C, respectively. Most of the strains grew well in a wide range of
salinities (e.g., five of the eight strains exhibited a growth rate greater than one doubling•day-1 at
A Look Back at the Aquatic Species Program—Technical Review
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National Renewable Energy Laboratory
conductivities between 10 and 70 mmho•cm-1). With respect to the effect of water type on
growth, CHAET39, CYCLO4, and PLEUR1 grew best on SERI Type I medium. On the other
hand, CHAET6, CHAET9, and CHAET10 grew best in SERI Type II medium, but also exhibited
good growth on Type I medium and artificial seawater. CHAET15 and THALA2 achieved
maximal growth rates on artificial seawater, and, along with PLEUR1, grew very poorly on Type
II medium. These results again highlight the need to have a variety of algal strains available for
the specific water resources that would be available for mass culture in various locations.
The lipid contents of these 10 strains were also determined for exponentially growing cells, as
well as for cells that were grown under nutrient-limited conditions. Nitrogen deficiency led to an
increase in the lipid contents of CHAET6, CHAET9, CHAET10, CHAET15, CHAET39, and
PLEUR1. The mean lipid content of these strains increased from 11.2% (of the total organic
mass) in nutrient-sufficient cells to 22.7% after 4 days of N deficiency. Silicon deficiency led to
an increase in the lipid content of all strains (although in some cases the increase was small and
probably not statistically significant). The mean lipid content of the eight strains increased from
12.2% in nutrient-sufficient cells to 23.4% in Si-deficient cells. A few strains were poor lipid
producers, such as CHAET6, CYCLO4, and PLEUR1, which did not produce more than 20%
lipid under any growth conditions.
Cold water strain collection efforts.
Most microalgal collection efforts carried out under the auspices of the ASP before 1987 focused
on sites that were expected to naturally experience high temperatures; indeed, one subcontractor,
Keith Cooksey (Montana State University) specifically searched for thermophilic strains isolated
from hot springs. This was because the temperatures of production ponds in the southwestern
United States during the prime growing season were expected to reach high levels; thus the
production strains would have to thrive under such conditions. However, temperatures in this
region are quite cool for several months of the year and can drop to below freezing at night.
Consequently, an effort was initiated by SERI researchers to collect, screen, and characterize
strains from cold-water habitats.
Four collecting trips were made between October 1986 and March 1987 to various inland saline
water sites in Utah and eastern Washington, and to the coastal lagoon waters in southern
California. Water samples were enriched with N, P, trace metals, and vitamins; artificial media
were not used in the initial selection protocol for these experiments. The rotary screening
apparatus was maintained at 15°C for the duration of the screening process by including a copper
cooling coil inside the screening chamber. The cultures were incubated for 5-10 days, which is
longer than for warm-water strains because of the slower growth at the cooler temperature. This
procedure created a problem, however, in that many more strains survived the selection process
than when 30°C was used as the selection temperature. As a consequence, separating strains
from each other and identifying which were the best for further characterization were more
difficult.
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An interesting finding from the cold water strain collection project was that many species that
predominate after the enrichment procedure were the same as the warm water species selected in
previous collection efforts. The genera and species that were commonly found in both the cold
water and warm water screening projects were C. muelleri, Amphora coffeiformis, Cyclotella,
Navicula, and Nitzschia. However, some ochromonids and green coccoid algae were also
isolated from the cold water collection effort; these types of alga were less commonly isolated
during the warm-water selection procedures. Additional work would be needed to characterize
these strains with respect to lipid production potential. Future work should look at the fatty acid
profiles of oil found in the cold-water strains. Such cold-water organisms often contain high
levels of polyunsaturated fatty acids, which would perform poorly as a feedstock for biodiesel
because of their low oxidative stability and tendency to polymerize during combustion
(Harrington et al. 1986).
Publications:
Johansen, J.R.; Doucette, G.J.; Barclay, W.R.; Bull, J.D. (1988) “The morphology and ecology of
Pleurochrysis carterae var dentata nov. (Prymnesiophyceae), a new coccolithophorid from an
inland saline pond in New Mexico, USA.” Phycologica 27:78-88.
Johansen, J.; Lemke, P.; Barclay, W.; Nagle, N. (1987) “Collection, screening, and
characterization of lipid producing microalgae: Progress during Fiscal Year 1987.” FY 1987
Aquatic Species Program Annual Report, Solar Energy Research Institute, Golden, Colorado,
SERI/SP-231-3206, pp. 27-42.
Johansen, J.R.; Theriot, E. (1987) “The relationship between valve diameter and number of
central fultoportulae in Thalassiosira weissflogii (Bacillariophyceae).” J. Phycol. 23:663-665.
Tadros, M.G.; Johansen, J.R. (1988) “Physiological characterization of six lipid-producing
diatoms from the southeastern United States.” J. Phycol. 24:445-452.
Additional References:
Harrington, K.J. (1986) Biomass 9:1-17.
II.A.1.f.
Development of a Rapid Screening Procedure for Growth and Lipid Content
of Microalgae
By 1987, SERI researchers and subcontractors had collected approximately 3,000 algal strains.
Most of these strains had not been well characterized, especially with respect to lipid production
capabilities. As a consequence, work commenced on the development of a simple screening
procedure to estimate the lipid contents of cells to determine which strains had the best potential
as biofuel production organisms. Ideally, the procedure should be simple and reproducible so
that it could be used as a standard method in numerous laboratories. The researchers hoped that
such a screening tool would allow the size of the strain collection to be reduced to a manageable
number (~200) representing the most promising strains.
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National Renewable Energy Laboratory
Development of a rapid screen for lipid content.
In an attempt to develop a reproducible, easy-to-use screening procedure to identify algal strains
with high lipid contents, Dr. Keith Cooksey (an ASP subcontractor at Montana State University)
suggested that investigators explore the possibility of using the lipophilic dye Nile Red (9-
diethylamino-5H-benzo{a}phenoxazine-5-one) to stain cells. Nile Red was first isolated from
Nile Blue by Greenspan et al. (1985), who showed that Nile Red will fluoresce in a nonpolar
environment and could serve as a probe to detect nonpolar lipids in cells. Nile Red permeates all
structures within a cell, but the characteristic yellow fluorescence (approximately 575 nm) only
occurs when the dye is in a nonpolar environment, primarily neutral storage lipid droplets. Earlier
work within the ASP by Dr. Steve Lien had shown the utility of Nile Blue in microscopically
assessing the lipid content of algal cells (Lien 1981). The active ingredient in these Nile Blue
preparations may in fact have been Nile Red. Parallel efforts to develop a Nile Red staining
procedure were carried out by SERI researchers and ASP subcontractors, notably Drs. Cooksey
and Sommerfeld.
Cooksey et al. (1987) used the diatom Amphora coffeiformis to optimize the Nile Red staining
procedure. The dye was dissolved in acetone and used at a concentration of 1 mg/mL of cell
suspension. In this species, the fluorescence of the dye in live stained cells was stable for only 2-
7 minutes; fluorescence measurements had to be completed rapidly to ensure consistent results.
The kinetics of fluorescence in stained cells varied in different species, presumably due to
differences in the permeability of cell walls to the stain, and differences in how the lipid is stored
in the cells, i.e., as large or small droplets. Fixing the stained cells with formaldehyde or ethanol
preserved the Nile Red fluorescence for 2 hours, but cells that were chemically fixed before Nile
Red staining did not exhibit the characteristic yellow fluorescence. When Nile Red fluorescence
was measured in algal cultures over time, the fluorescence increased as the culture became N
deficient. The fluorescence level was linearly correlated with an increase in the total lipid
content, determined gravimetrically, in a growing culture of algal cells. Fractionation of the lipids
by silicic acid column chromatography demonstrated that the increase in lipid was due primarily
to an increase in neutral lipids rather than in the polar lipids or glycolipids, which are found
primarily in cell membranes.
Additional development of the Nile Red screening procedure occurred at SERI and at Milt
Sommerfeld’s laboratory at Arizona State University. The resultant protocol involved taking a
fixed volume of a diluted algal culture (typically 4 mL), adding 0.04 mL of a Nile Red solution
(0.1 mg/mL in acetone), and determining the fluorescence after 5 min using a fluorometer
equipped with the appropriate excitation and emission filters.
Although the use of Nile Red allowed various microalgae to be rapidly screened for neutral lipid
accumulation, interspecies comparisons may be subject to misinterpretation because of the
species-specific staining differences described earlier. Nonetheless, before Nile Red was used,
quantitating lipids from cells was very time consuming. It required the extraction of lipid from a
large number of cells using organic solvents, evaporation of the solvent, and determination of the
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National Renewable Energy Laboratory
amount of lipid by weighing the dried extract. Consequently, the use of Nile Red as a rapid
screening procedure can still have substantial value.
Screening for growth in high conductivity media.
The estimation of lipid content using a simple procedure such as the Nile Red assay is clearly an
important component of a rapid screening procedure for identifying promising strains, but an
equally important component is a means to identify strains that grow rapidly under the expected
culture conditions. Reports detailing the amount and types of saline groundwater available in the
southwestern United States, along with data concerning the high rates of evaporation in this
region, indicated that tolerance of algal strains to high conductivity (higher than 50 mmho•cm-1)
could be important. Therefore, an additional component of the secondary screening procedure
developed to reduce the number of strains being maintained by ASP researchers. Algae were
tested for the ability to grow at high conductivity (55 mmho•cm-1, both Type I and Type II
media), high temperature (30ºC), and high light intensity (average of 200 µE•m-2•s-1, 12 h
light:12 h dark cycle) in cultures that were continually agitated via aeration. To prevent osmotic
shock, strains were adapted to higher conductivities via a stepwise transfer into media with
increasing conductivity at 2-day intervals. Tubes were used that could be placed directly in a
spectrophotometer (i.e., 25 mm diameter, 50 mL volume), allowing the culture density to be
measured without removing a sample. The tubes also held enough medium to allow samples to
be taken for Nile Red lipid analysis (both for N-sufficient and N-deficient cells), and for ash-free
dry mass determinations. The tubes were placed in a rack and illuminated by fluorescent lamps
from below for the screening procedure. Optical density measurements were taken twice daily
for 4 days during exponential growth to determine growth rates. Samples were removed for Nile
Red fluorometric analysis during exponential growth and after 2 days (Arizona State University)
or 4 days (SERI) of N deficiency.
This newly developed rapid screening protocol was subsequently used both in Milt Sommerfeld’s
laboratory and at SERI to screen many microalgal isolates. Keith Cooksey’s laboratory also
examined numerous strains using this procedure. Sommerfeld’s laboratory examined
approximately 800 strains that had been collected over the previous 2 years of the subcontract.
Only 102 of these strains survived transfer into media having a conductivity of 55 mmho•cm-1.
Of these strains, 40 grew in both Type I/55 and Type II/55 media, 42 grew only in Type I/55
medium, and 19 grew only in Type II/55 medium. The 10 fastest-growing strains, along with
their preferred media, are shown in Table II.A.2.
A Look Back at the Aquatic Species Program—Technical Review
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National Renewable Energy Laboratory
Table II.A.2 Fastest growing strains from Arizona State University collection.
Strain
Genus
Class
Growth Rate
Medium
(doublings•day-1)
OSCIL2
Oscillatoria
Cyanophyceae
4.23
I/55
OSCIL3
Oscillatoria
Cyanophyceae
3.50
I/55
CHLOC4
Chlorococcum/
Chlorophyceae
3.47
I/55
Eremosphaera
SYNEC5
Synechococcus
Cyanophyceae
3.25
II/55
ASU0735
Oscillatoria
Cyanophyceae
3.06
I/55
AMPHO46
Amphora
Bacillariophyceae
2.81
I/55
NANNO13
Nannochloris
Chlorophyceae
2.78
I/55
POLYC1
Synechococcus
Cyanophyceae
2.73
I/55
CHLOR23
Chlorella
Chlorophyceae
2.66
I/55
SYNEC3
Synechococcus
Cyanophyceae
2.51
II/55
The lipid production potential of these strains was evaluated by the use of the fluorometric Nile
Red assay. In Type I/55 medium, 49 strains had a higher apparent lipid content after 2 days of
N-deficient growth, whereas 13 strains had the same or lower apparent lipid levels in response to
N deficiency. In Type II/55 medium, 42 strains had higher lipid levels, and 7 strains had lower
or unchanged lipid levels as a consequence of N deficiency. Of note was that the mean lipid
level in cells grown in Type II/55 medium was nearly twice that of Type I/55-grown cells.
The strains exhibiting the highest Nile Red fluorescence levels are shown in Table II.A.3. All of
these strains are diatoms, confirming the propensity of this group to accumulate lipids.
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Table II.A.3. Strains from the Arizona State University collection having the highest
Nile Red fluorescence.
Strain
Genus
Class
Triolein
Triolein
equivalents
equivalents
(mg•L-1)
(mg•L-1)
Exponential
N-deficient
growth
growth
NITZS54
Nitzschia
Bacillariophyceae
8
1003
NITZS53
Nitzschia
Bacillariophyceae
17
934
NITZS55
Nitzschia
Bacillariophyceae
37
908
ASU3004
Amphora
Bacillariophyceae
9
593
NAVIC36
Nitzschia
Bacillariophyceae
61
579
AMPHO45 Amphora
Bacillariophyceae
39
308
FRAGI2
Fragilaria
Bacillariophyceae
6
304
AMPHO27 Amphora
Bacillariophyceae
38
235
NITZS52
Nitzschia
Bacillariophyceae
24
234
These researchers also ranked strains according to the estimated lipid productivity of rapidly
growing cells, based on the calculated growth rates and estimated lipid contents of exponential
phase cells. The top strains resulting from this analysis are shown in Table II.A.4. However, the
optimal strategy for maximizing lipid yield in actual mass culture facilities may require an
“induction” step (i.e., manipulation of the culture environment, possibly involving nutrient
deficiency). The ranking of strains would obviously be very different in that case.
A Look Back at the Aquatic Species Program—Technical Review
25
National Renewable Energy Laboratory
Table II.A.4. Strains in the Arizona State University collection with the highest
apparent lipid productivity during exponential growth, based on Nile Red staining.
Strain
Genus
Class
Triolein equivalents
(mg•L-1•day-1)
AMPHO27
Amphora
Bacillariophyceae
345
CHLOC4
Eremosphaera/
Chlorophyceae
117
Chlorococcum
SYNEC5
Synechococcus
Cyanophyceae
86
AMPHO46
Amphora
Bacillariophyceae
71
SYNEC4
Synechococcus
Cyanophyceae
64
AMPHO45
Amphora
Bacillariophyceae
63
NITZS55
Nitzschia
Bacillariophyceae
48
OOCYS9
Oocystis
Chlorophyceae
46
NITZS52
Nitzschia
Bacillariophyceae
45
SERI researchers also started to evaluate various strains by the rapid screening procedure. Initial
work focused on 25 partially characterized strains. These strains were analyzed for growth and
Nile Red fluorescence in exponentially growing cultures and in cultures grown under N-deficient
conditions for 4 days. The results of the SERI and Sommerfeld laboratories cannot be compared
directly, because Nile Red units are expressed differently and the time duration of N deficiency
was not the same. The best strains of the 25 tested (based on the highest Nile Red fluorescence
normalized to ash-free dry weight (AFDW) and rapid exponential growth) were determined to be
CHAET9 (muelleri), NAVIC2 (Navicula saprophila), and NITZS12 (Nitzschia pusilla).
Twenty-eight strains of Chaetoceros were also examined using this screening protocol. The best
strains indentified were CHAET21, CHAET22, CHAET23, and CHAET25 (all muelleri). All
but the latter strain were isolated from various regions of the Great Salt Lake in Utah.
The departure of Dr. Bill Barclay and Dr. Jeff Johansen from the ASP, along with a greater
emphasis on genetic improvement of strains, marked the end of the in-house collection and
screening work. As a consequence, many of the 3,000 strains collected by ASP researchers
during the course of this research effort were never analyzed via this rapid screening protocol.
Nonetheless, enough strains had been analyzed at SERI and at the laboratories of various
subcontractors to obtain a substantial number of promising strains. The next step was to
determine their ability to grow in actual outdoor mass culture ponds. This work is described in
Section III of this report.
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Publications:
Berglund, D.; Cooksey, B.; Cooksey, K.E.; Priscu, L.R. (1987) “Collection and screening of
microalgae for lipid production: possible use of a flow cytometer for lipid analysis.” FY 1986
Aquatic Species Program Annual Report, Solar Energy Research Institute, Golden, Colorado,
SERI/SP-231-3071, pp. 41-52.
Cooksey, K.E.; Guckert, J.B.; Williams, S.A.; Collis, P.R. (1987) “Fluorometric determination of
the neutral lipid content of microalgal cells using Nile Red”, J. Microbiol. Methods 6:333-345.
Johansen, J.; Lemke, P.; Barclay, W.; Nagle, N. (1987) “Collection, screening, and
characterization of lipid producing microalgae: Progress during Fiscal Year 1987.” FY 1987
Aquatic Species Program Annual Report, Solar Energy Research Institute, Golden, Colorado,
SERI/SP-231-3206, pp. 27-42.
Lien, S. (1981) In Proc. Brighton Int. Conf. On Energy from Biomass (Palz, W.; Chartier, P.; and
Hall, D.O., eds.), Applied Science Publishers, London, pp. 697-702.
Sommerfeld, M.; Ellingson, S.; Tyler, P. (1987) “Screening microalgae isolated from the
southwest for growth potential and lipid yield.” FY 1987 Aquatic Species Program Annual
Report, Solar Energy Research Institute, Golden, Colorado, SERI/SP-231-3206, pp. 43-57.
Additional References:
Greenspan, P.; Mayer, E.P.; Fowler, S.D. (1985) “Nile Red: A selective fluorescent stain for
intracellular lipid droplets.” J. Cell Biol 100:965-973.
Greenspan, P.; Fowler, S.D. (1985) J. Lipid Res. 26:787
II.A.1.g.
Statistical Analysis of Multivariate Effects on Microalgal Growth and Lipid
Content
As discussed earlier, environmental variables (particularly nutrient status) can have great effects
on growth and the quantity and quality of lipids in microalgae. To determine the effects of
several environmental variables alone and in combination on the growth and lipid contents of
microalgae, a multivariate, fractional factorial design experiment was carried out with two
promising diatoms, Navicula saprophila (NAVIC1) and C. muelleri (CHAET9). For these
experiments, cells were grown in modified SERI Type II/25 medium in which the alkalinity was
adjusted by adding sodium carbonate and sodium bicarbonate and the conductivity was adjusted
by adding sodium chloride. Cultures were grown on the temperature gradient table described
previously at 200 µE•m-2•s-1. The following variables were tested in the multivariate analysis:
conductivity (20 to 80 mmho•cm-1), temperature (17° to 32ºC), N (urea) concentration (0 to 144
mg•L-1), sodium silicate concentration (0 to 500 mg·L-1), and alkalinity (8.8 to 88 meq•L-1). In
these experiments, growth was measured by changes in AFDW and lipid was measured by the
use of the Nile Red fluorometric assay.
A Look Back at the Aquatic Species Program—Technical Review
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National Renewable Energy Laboratory
The results indicated that the N content and conductivity of the medium were the most important
variables affecting lipid content (Nile Red fluorescence) of both NAVIC1 and CHAET9. As N
levels and conductivity increased, the amount of neutral lipid per mg of AFDW decreased. The
interaction of N and conductivity was an important determinant of lipid content as well. Silicon
level and alkalinity were more important factors in determining the lipid content for CHAET9
than for NAVIC1. N concentration was by far the most important factor in determining final cell
mass for NAVIC1, and was a major factor for cell mass yield in CHAET9 (along with the
interaction of conductivity and alkalinity, which had a large negative impact on growth).
Alkalinity was a major factor for growth of both NAVIC1 and CHAET9. However, these
experiments did not determine actual growth rates, but only the final cell yields; thus, how actual
cell division rates compared with each other is not known.
These experiments indicate the importance of examining the interactions of environmental
variables in determining the effects on growth and lipid production. However, the models
generated by these kinds of experiments are specific for the strains being studied, and the results
cannot necessarily be used to predict the effects of these variables on other strains. Furthermore,
for such models to be truly predictive of growth and lipid production in an actual mass culture,
much more sophiticated (and realistic) experimental setups would be required.
Publications:
Chelf, P. (1990) “Environmental control of lipid and biomass production in two diatom species.”
J. Appl. Phycol. 2:121-129.
II.A.1.h.
Detailed Analyses of Microalgal Lipids
In addition to the in-house research being conducted in the area of strain collection and
screening, there was an effort by Dr. Thomas Tornabene and others to characterize various
strains via detailed lipid compositional analyses. Dr. Tornabene’s laboratory at SERI (and later
at the Georgia Institute of Technology) served as the focal point for the analysis of lipids in algal
samples supplied by various researchers in the ASP. This section will describe the results of
these analyses, and will provide details about the analytical methods used, as these methods were
the most comprehensive used in the program. An early report by Tornabene et al. (1980)
described the lipids that were present in the halophilic alga Dunaliella that had been isolated
from the Great Salt Lake in Utah. The cells were grown to late logarithmic phase, harvested, and
extracted with chloroform/methanol via the method of Bligh and Dyer (1959). Additional
extraction by acetate buffer, followed by refluxing with an alkaline methanol/water mixture was
then performed, followed by partitioning of lipids into petroleum ether. The extracted lipids
were fractionated on the basis of polarity using silicic acid columns via differential elution with
hexane, benzene, chloroform, acetone, and methanol. In this procedure, the lipids are eluted as
follows:
1. hexane:
acyclic
hydrocarbons
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2. benzene: cyclic hydrocarbons, polyunsaturated acyclic hydrocarbons, sterols,
and xanthophylls
3. chloroform: mono-, di- and triacylglycerols, free fatty acids, and phaeophytin a
4. acetone: glycolipids, carotenoids, and chlorophyll a and b; and
5. methanol: phospholipids and chlorophyll c.
The various lipid classes were further analyzed via Si gel thin layer chromatography (both one-
and two-dimensional), wherein lipids were detected via the use of iodine vapors (and
autoradiography in the case of 14C-labeled lipids). In addition, lipids containing amino groups
were detected via the ninhydrin reagent, and phospholipids were detected by the use of
molybdate/H2SO4. Fatty acids were analyzed via gas chromatography using either flame
ionization or mass spectroscopic detection after being converted to their methyl ester derivatives
in the presence of methanolic HCl. The head groups of the polar lipids were identified via gas
chromatography after being converted to alditol acetates. These and related methods were
described by Tornabene et al. (1982).
These analyses indicated that lipids comprised 45%-55% of the total organic mass of Dunaliella
cells. Based on the distribution of 14C after labeling the cells with 14C-bicarbonate, neutral lipids
accounted for 58.5% of the lipid mass, whereas phospholipids and galactolipids were 22.9% and
10.9% of the lipid mass, respectively. Isoprenoid hydrocarbons (including β-carotene) and
aliphatic hydrocarbons (in which the major components were tentatively identified as straight-
chain and methyl-branched C17 and C19 hydrocarbons with various degrees of unsaturation)
represented 7.0% and 5.2% of the lipids, respectively. The major fatty acids present were
palmitic (20.6%), linolenic (12.5%), linoleic (10.7%) and palmitoleic (7.8%), but no attempt was
made to ascertain whether any of these fatty acids predominated a particular lipid class. The high
hydrocarbon content of this alga is rather atypical of most of the strains characterized in the ASP.
These types of hydrocarbons would probably require catalytic conversion into a usable fuel
source, which would perhaps limit their utility as a production organism.
A detailed analysis of the lipids present in the green alga Neochloris oleoabundans was also
carried out by Tornabene (who was later to hold a position at the Georgia Institute of
Technology), along with G. Holzer (Colorado School of Mines), S. Lien and N. Burris (SERI)
(Tornabene et al. 1983). The strain used in this study was obtained from the University of Texas
Algal Culture Collection, and reportedly contained substantial quantities of lipid when grown
under N-deficient conditions. (However, this is a freshwater strain). Exponentially growing cells
were transferred into a low-N medium, and after 5 to 7 days of growth in stirred cultures that
were bubbled with 1% CO2 in air, the cells were harvested and the lipids were extracted.
Analytical methods were similar to those described earlier, and included the use of pyrrolidine-
acetic acid/mass spectrometry to determine the position of double bonds in the fatty acids. These
analyses indicated that 35%-54% of the cellular dry weight was in the form of lipids in N-
deficient cells. Neutral lipids accounted for more than 80% of the total lipids, and were
A Look Back at the Aquatic Species Program—Technical Review
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National Renewable Energy Laboratory
predominantly in the form of TAGs. Small amounts of straight-chain hydrocarbons and sterols
were also found (one sterol was identified as a ∆7 sterol, but low quantities of material made
identification of the sterols difficult). A number of polar lipids were also quantified, but all polar
lipids combined accounted for less than 10% of the lipid mass. The fatty acids that comprised
the TAGs were present in the following proportions: 36% oleic (18:1 ∆9), 15% palmitic (16:0),
11% stearic (18:0), 8.4% iso-17:0 (an unusual fatty acid for microalgae), and 7.4% linoleic (18:2
∆9,12). Other saturated and monounsaturated fatty acids were present in TAGs, but represented
less than 5% each of the total fatty acids present. The high proportion of saturated and
monounsaturated fatty acids in this alga is considered optimal from a fuel quality standpoint, in
that fuel polymerization during combustion would be substantially less than what would occur
with polyunsaturated fatty acid-derived fuel (Harrington, 1986).
Additional research carried out in Tornabene’s laboratory (Ben-Amotz et al. 1985) examined the
lipid composition of 7 algal species. Some were from existing culture collections and others
were isolated by ASP researchers. The lipid contents of these strains were determined under
conditions of N sufficiency, after 10 days of N deficiency, and under different salinity levels.
Botryocooccus braunii has received considerable interest as a fuel production organism in other
laboratories because of its high lipid content. This study confirmed the high lipid levels (55% of
the organic mass for N-deficient cells). Most of this lipid was in the form of hydrocarbons,
including C29 to C34 aliphatic hydrocarbons and a variety of branched and unsaturated
isoprenoids. Glycerolipids were less abundant than the hydrocarbons, and were composed
primarily of 16:0 and various C18 fatty acids. These data, coupled with the fact that this species
grows very slowly (one doubling per 72 hours), indicated that Botryococcus would not function
well as a feedstock for lipid-based fuel production.
The other species examined in this study were the chlorophytes Ankistrodesmus, Dunaliella, and
Nannochloris, the diatom Nitzschia, and the chrysophyte Isochrysis. N deficiency led to an
increase in the lipid content of Ankistrodesmus (from 24.5% to 40.3%), Isochrysis (from 7.1% to
26.0%), and Nannochloris (from 20.8% to 35.5%), but resulted in a decrease in the lipid content
of Dunaliella (from 25.3% to 9.2%). Elevating the NaCl concentration of the medium had little
effect on the lipid content of Botryococcus cells, but caused a slight decrease in the lipid content
of Dunaliella salina (from 25.3% to 18.5% with an increase in [NaCl] from 0.5 to 2 M).
Conversely, the lipid content of Isochrysis increased from 7.1% to 15.3% as the NaCl increased
from 0.5 to 1 M. These results once again highlight the impact of culture conditions on the
quantities of lipids present. However, as stated before, the most important characteristic of a
lipid production strain is the overall lipid productivity for a given amount of time, which was not
examined in this study.
The polar lipid composition of the strains examined in this study were typical of photosynthetic
microalgae, and included phosphatidylcholine, phosphatidylinositol, phosphatidylethanolamine,
phosphatidylglycerol, monogalactosyldiacylglycerol, and digalactosyldiacylglycerol.
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Table II.A.5 indicates the major fatty acids (those at levels exceeding 5% of the total) present in
these strains, both under N-sufficient and N-deficient growth conditions.
In conclusion, the work carried out by Tornabene’s laboratory provided a detailed
characterization of the lipids present in a variety of microalgae. No general conclusions could be
made from the work except that the lipid composition of various microalgal strains can differ
quite substantially. Because the nature of the lipids can have a large impact on the quality of the
fuel product, characterizing the potential production strains is important to ensure that deleterious
lipids (e.g., highly polyunsaturated fatty acids in the case of biodiesel fuel) are not present at high
levels.
Table II.A.5. Major fatty acids of various microalgae. (Fatty acids in bold are present
at levels of 15% or higher)
Strain
Nitrogen-sufficient cells
Nitrogen-deficient cells
Ankistrodesmus
16:0, 16:4, 18:1, 18:3
16:0, 18:1, 18:3
Botryococcus braunii
16:0, 18:1, 18:2, 18:3
16:0, 18:1, 18:3, 20:5
Dunaliella bardawil
not determined
12:0, 14:0/14:1, 16:0, 18:1, 18:2,
18:3
Dunaliella salina
14:0/14:1, 16:0, 16:3, 16:4, 16:0, 16:3, 18:1, 18:2, 18:3
18:2, 18:3
Isochrysis sp.
14:0/14:1, 16:0, 16:1, 18:1, 14:0/14:1, 18:1, 18:2, 18:3, 18:4,
18:3, 18:4, 22:6
22:6
Nannochloris sp.
14:0/14:1, 16:0, 16:1, 16:2, not determined
16:3, 20:5
Nitzschia sp.
14:0/14:1, 16:0, 16:1, 16:2, not determined
16:3, 20:6
Publications:
Ben-Amotz, A.; Tornabene, T.G. (1983) “Chemical profile of algae with emphasis on lipids of
microalgae.” Aquatic Species Program Review: Proceedings of the March 1983 Principal
Investigators’ Meeting, Solar Energy Research Institute, Golden, Colorado, SERI/CP-231-1946,
pp. 123-134.
Ben-Amotz, A.; Tornabene, T.G.; Thomas, W.H. (1985) “Chemical profiles of selected species
of microalgae with emphasis on lipids.” J. Phycol. 21:72-81.
Tornabene, T.G.; Holzer, G.; Peterson, S.L. (1980) “Lipid profile of the halophilic alga,
Dunaliella salina.” Biochem. Biophys. Res. Comm. 96:1349-1356.
A Look Back at the Aquatic Species Program—Technical Review
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National Renewable Energy Laboratory
Tornabene, T.G.; Ben-Amotz, A.; Hubbard, J.S. (1982) “Isolation, analysis and identification of
lipids.” SERI ASP Report prepared under contract XK-2-02149-01.
Tornabene, T.G.; Holzer, G.; Lien, S.; Burris, N. (1983) “Lipid composition of the nitrogen
starved green alga Neochloris oleoabundans.” Enzyme Microb. Technol. 5:435-440.
Tornabene, T.G. (1984) “Chemical profile of microalgae with emphasis on lipids.” Aquatic
Species Program Review: Proceedings of the April 1984 Principal Investigators’ Meeting, Solar
Energy Research Institute, Golden, Colorado, SERI/CP-231-2341, pp. 64-78.
Tornabene, T.G.; Benemann, J.R. (1985) “Chemical profiles on microalgae with emphasis on
lipids.” Aquatic Species Program Review: Proceedings of the March 1985 Principal
Investigators’ Meeting, Solar Energy Research Institute, Golden, Colorado, SERI/CP-231-2700,
pp. 83-99.
Additional References:
Harrington, K.J. (1986) Biomass 9:1-17.
II.A.2.
Collection, Screening, and Characterization of Microalgae: Research
by SERI Subcontractors
II.A.2.a. Introduction
Included in this section are summaries of the research conducted by various subcontractors
within the ASP who contributed to the collection, screening, and characterization of microalgal
strains for potential use in biofuel production facilities. Initially, a variety of strain isolation and
screening procedures were carried out by the various research groups, as there was no established
protocol. This lack of uniformity in the screening protocols made comparing the results from one
laboratory with those of another difficult, and meant that the criteria for selecting the best strains
differed between the laboratories. At the same time, however, this arrangement provided the
opportunity for new ideas regarding collecting and screening to be pursued, thereby allowing
individual creativity in a manner that might be beneficial to the entire program.
Several subcontractors participated in the strain collection and screening effort. Dr. Bill Thomas
and colleagues (Scripps Institution of Oceanography) collected a large number of strains from the
desert regions of eastern California and western Nevada. Additional microalgal strains from
desert waters in Arizona, New Mexico, California, Nevada, Utah, and Texas were obtained
through the efforts of Dr. Milt Sommerfeld’s laboratory at Arizona State University. Dr.
Mahasin Tadros (Alabama A&M University) collected strains habitats in the southeastern United
States (Alabama, Mississippi, and Florida). Additional strains from the Florida Keys and
Everglades were collected by John Rhyther (Harbor Branch Foundation); Richard York (Hawaii
Institute of Marine Biology) isolated a number of strains from the Hawaiian islands and
surrounding waters. Certain environmental niches were focused on as well; Dr. Keith Cooksey
(Montana State University) isolated several strains from thermal springs in Yellowstone National
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Park; Dr. Ralph Lewin (Scripps Institution of Oceanography) focused on picopleustonic algae
(“floating” algae at the air-sea interface). The results of these efforts are described below.
II.A.2.b.
Yields, Photosynthetic Efficiencies, and Proximate Chemical Composition
of Dense Cultures of Marine Microalgae
Subcontractor:
University of California, San Diego
Principal Investigator:
William H. Thomas
Period of Performance:
1980 - 1983
Subcontract Number:
XK-0-9111-1
Work carried out under this subcontract represented one of the first attempts by an ASP
subcontractor to characterize the productivity and lipid yields of various microalgae. Six algal
strains (B. braunii, Dunaliella primolecta, Isochrysis sp., Monallanthus salina, Phaeodactylum
tricornutum, and Tetraselmis sueica) were obtained from existing culture collections and
analyzed with respect to lipid, protein, and carbohydrate content under various growth
conditions. For these experiments, all cultures except for B. braunii were grown in natural
seawater that was enriched with N, P, and trace metals. B. braunii was grown in an artificial
seawater medium. Initial experiments to determine productivities of these species were
performed using batch cultures in 9-L serum bottles. Of the strains tested, the highest growth
rates were observed with P. tricornutum (Thomas strain) and M. salina.
Additional experiments were performed in plexiglas vessels that were 5 cm thick, 39 cm deep,
and 24 cm wide (surface area ~940 cm2). The cultures were illuminated from the side with a
2,000-watt tungsten-halide lamp, which was placed behind a water/CuSO4 thermal filter. In
these experiments, the cultures were typically maintained for 40 to 90 days. In the early stages of
an experiment, the cultures were maintained in a batch mode, and then converted to a continuous
or semi-continuous dilution mode. Various culture parameters (including light intensity, dilution
rate, and N status) were manipulated during the course of these experiments to determine their
effects on the productivities and proximate chemical composition of the strains. The results of
these experiments with each species tested are discussed below. These experiments are difficult
to compare because the experiments were all carried out slightly differently (i.e., different light
intensities, different culturing methods [batch, semi-continuous, and continuous], different means
of obtaining N-deficient cultures, and inconsistent use of a CuSO4 heat filter, which resulted in
differences in light quality and culture temperature). Nonetheless, the general conclusions of this
study are of interest.
P. tricornutum (Thomas strain):
This strain has been used for several past studies, and was concomitantly being tested in outdoor
mass culture by another subcontractor (the University of Hawaii; principal investigator Dr.
Edward Laws; discussed in Section III). Therefore, this strain was subjected to more extensive
testing than the other strains in this subcontract. In one experiment reported for this strain, the
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effects of light intensity on productivity were determined in batch cultures (i.e., in the Plexiglas
culture apparatus described earlier without culture replacement and dilution). The maximum
productivity observed for this strain (21 to 22 g dry weight•m-2•d-1)3 was observed at a total daily
illumination of 63-95 kcal (representing approximately 40%-60% of full sunlight in southern
California during the summer). This value was slightly higher than the productivity observed
with a total daily illumination of 70% full sunlight (17.1 g dry weight•m-2•d-1). Productivities
under N-limiting, continuous growth mode conditions were between 7 and 11 g dry
weight•m-2•d-1. Likewise, productivities under N-sufficient, continuous growth mode conditions
were reduced relative to batch cultures.
In addition to measuring overall productivities, the levels of protein, carbohydrate, lipid, and ash
were determined for cells grown under the various conditions described earlier. Illumination of
the cultures from 40% to 70% of full sunlight did not have a large impact on the cellular
composition. Growth of P. tricornutum cells under N-deficient conditions resulted in a reduction
of the protein content from 55% (in N-sufficient cells) to 25% of the cellular dry weight.
Carbohydrate content increased from 10.5% to 15.1%, and the mean lipid content increased from
19.8% to 22.2%, although these differences in carbohydrate and lipid contents did not appear to
be statistically significant. At one stage of the experiment, however, a time course of N
deficiency led to a consistent rise in lipid content from 19.9% to 30.8% over the course of 7 days.
The actual rate of lipid production did not increase, however, because the overall productivity of
the cultures was reduced under N-deficient growth.
D. primolecta:
The maximum productivity observed for this species (12.0 g dry weight•m-2•d-1 ) occurred during
continuous culture at 60% full sunlight under N-sufficient conditions. Doubling the light
intensity lowered the productivity to 6.1 g dry weight•m-2•d-1. The chemical composition of N-
sufficient cells (as an average percentage of total cell dry weight) was 64.2% protein, 12.6%
carbohydrate, and 23.1% lipid. After 7 days of growth under N-deficient conditions, the
composition was 26.8% protein, 59.7% carbohydrate, and 13.7% lipid. Therefore, this alga
accumulates carbohydrates rather than lipids in response to nutrient deficiency, limiting its
usefulness as a lipid production strain.
M. salina:
This alga reportedly contained high levels of lipids when grown under N-deficient conditions.
The highest productivity (13.9 g dry weight•m-2•d-1) was observed under N-sufficient conditions
at a light intensity of 50% full sunlight, although detailed experiments with regards to the effects
of light intensity on productivity were not conducted. There was little difference in the lipid
3Reporting of productivities in g dry weight·m-2 ·d-1 derives from the goal of mass culturing the algae in
shallow open ponds. The objective would be to maximize biomass produced per area of pond. However, it
is often difficult to compare results between experiments when the data are reported in this manner, as
factors such as culture depth and vessel design would significantly affect productivity of the cultures.
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content of cells grown under N-sufficient and N-deficient conditions (20.7% and 22.1%,
respectively).
T. sueica:
The highest productivity observed for this strain was 19.1 g dry weight•m-2•d-1, which occurred
in N-sufficient batch cultures grown under a light intensity of 60% full sunlight. N deficiency
resulted in a large increase in carbohydrate content (from a mean value of 10.7% to a mean value
of 47.1%). On the other hand, protein content was reduced substantially (from 67.6% to 28.3%),
and the lipid content decreased from 23.1% to 14.6% in response to N deficiency.
Isochrysis sp.(Tahitian strain T-ISO):
This strain is commonly used as a feed organism in aquaculture production systems. A
productivity of 11.5 g dry weight•m-2•d-1 was typical for batch cultures of this species, which was
approximately 33% higher than the value recorded during semi-continuous growth (dilution of
0.15 L/d). Productivity was lowered during N-deficient growth to 5.5-7.6 g dry weight•m-2•d-1.
This strain accumulated carbohydrate in response to N deficiency (from a mean value of 23.1%
to 56.9%). Lipid content also increased slightly (from 28.5% to 33.4%), whereas protein content
was reduced from 44.9% to 27.3%. The higher lipid content of N-deficient cells did not translate
to higher lipid productivities, however, because of the lower overall productivity of the stressed
cultures.
B. braunii:
Some very limited experiments were conducted with this species, which is known to accumulate
hydrocarbons. A culture grown under a light intensity of 60% full sunlight had a productivity of
only 3.4 g dry weight•m-2•d-1. The lipid content of these cells was 29% of the cellular dry
weight; the N status of the cells was not reported, but it is assumed that the cells were grown
under N-sufficient conditions.
Overall Conclusions
Of the species examined, P. tricornutum and T. sueica had the highest overall productivities.
These species also had the highest lipid productivities, which were 4.34 and 4.47 g lipid•m-2•d-1,
respectively. For both species, the maximal productivities were obtained in batch cultures, as
opposed to semi-continuous or continuous cultures. Although the lipid contents of cells were
often higher in response to N deficiency, the lipid productivities of all species tested were
invariably lower under N deficiency because of an overall reduction in the culture growth rates.
For the species tested under continuous or semi-continuous growth conditions, lipid
productivities were reduced from 14% to 45% of the values measured for N-sufficient cultures.
A Look Back at the Aquatic Species Program—Technical Review
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National Renewable Energy Laboratory
The results also pointed to the importance of identifying strains that are not photoinhibited at
light intensities that would occur in outdoor ponds. Finally, this work highlighted the fact that
some microalgae accumulate carbohydrates during nutrient-deficient growth; such strains are
clearly not acceptable for use as a feedstock for lipid-based fuel production.
Publications:
Thomas, W.H.; Seibert, D.L.R.; Alden, M.; Neori, A. (1981) “Effects of light intensity and
nitrogen deficiency on yield, proximate composition, and photosynthetic efficiency of
Phaeodactylum.” Proceedings of the Subcontractors’ Review Meeting—Aquatic Species
Program, Solar Energy Research Institute, Golden, Colorado, SERI/CP-624-1228, pp. 33-58.
Thomas, W.H.; Seibert, D.L.R.; Alden, M.; Neori, A.; Eldridge, P. (1984a) “Yields,
photosynthetic efficiency, and proximate composition of dense marine microalgal cultures. I.
Introduction and Phaeodactylum tricornutum experiments.” Biomass 5:181-209.
Thomas, W.H.; Seibert, D.L.R.; Alden, M.; Neori, A.; Eldridge, P. (1984b) “Yields,
photosynthetic efficiency, and proximate composition of dense marine microalgal cultures. II.
Dunaliella primolecta and Tetraselmis suecica experiments.” Biomass 5:211-225.
(Also see references listed in the following section.)
II.A.2.c.
Selection of High-Yielding Microalgae from Desert Saline Environments
Subcontractor:
University of California, San Diego
Principal Investigator:
William H. Thomas
Period of Performance:
1983 - 1986
Subcontract Number:
XK-2-02170-0-01
The work carried out under this subcontract represented one of the first efforts to collect
microalgae from inland saline habitats and to screen those strains for rapid growth rates and lipid
content. Collecting trips were made to eastern California and western Nevada, and initial
culturing efforts were conducted at the Sierra Nevada Aquatic Research Laboratory near
Mammoth Lakes, California, and the Desert Studies Center (Zzyzx Springs), which is near
Baker, California. Various saline waters and soils were sampled during these collecting trips.
The collection sites included Pyramid Lake, Black Lake, Owens Lake, Walker Lake, Saline
Valley, Zzyzx Springs, Armagosa River, Sperry River, Harper Lake, and Salt Creek. The water
samples were enriched with N, P, Si, and trace metals, then incubated under natural conditions.
The algae that grew up were isolated by the use of micropipettes. Soil samples were placed in
“Zzyzx medium” before algal isolation (see Thomas et al. [1986] for media compositions).
Diatoms, green algae, and cyanobacteria were the dominant types of algae isolated using these
procedures. Of the 100 strains isolated, 42 were grown under standardized conditions in various
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artificial media that were designed to mimic the water from which the strains were originally
obtained. The pH of these various media formulations was typically high because of the
presence of high levels of carbonate and bicarbonate, and the total dissolved solids ranged from
approximately 1.5 g•L-1 to over 260 g•L-1. The growth of the cultures was visually scored, and
nine of the fastest growing strains were further analyzed with respect to growth under scaled-up
conditions.
For larger scale cultures, 6 L of medium that was enriched with N (nitrate, urea, or ammonium),
phosphate, trace metals, and vitamins were placed in 9-L serum bottles, and the cultures were
illuminated with fluorescent bulbs at a light intensity of 18% full sunlight. To enhance growth,
the cultures were bubbled with 1% CO2 in air, and more nutrients were added as the cell density
of the cultures increased. The strains tested in this manner included Nitzschia, Ankistrodesmus,
Nannochloris, Oocystis (two strains), Chlorella (three strains), and Selenastrum. The estimated
productivities ranged from 8.8 g dry weight•m-2•d-1 for for Nitzschia S-16 (NITZS14) grown in
the presence of urea to 45.8 g dry weight•m-2•d-1 for Oocystis pusilla 32-1, which was also grown
with urea as the N source. (These productivity values were considered overestimates in that there
was incidental side lighting of the flasks under the incubation conditions.) Also the productivity
values did not always correlate with final biomass yield values, indicating that growth saturation
was reached at different culture densities for the various strains. The maximum biomass yield
was obtained with O. pusilla 32-1 (2.29 g dry weight•L-1). The results of these experiments
indicated that certain strains had a clear preference for either urea or nitrate as the N source.
Because urea is significantly less expensive than nitrate, these results have economic
implications with respect to algal mass culture. However, the results regarding a preference for a
particular N source were not always reproducible.
Additional experiments were carried out to assess the combined effect of temperature and salinity
on the growth of several of the isolates. A thermal gradient table was used for these experiments
in which the incubation temperature at six stations on the table varied between 11°C and 35°C.
Salinities of the media were varied in five increments along the other axis (as much as twice that
of the natural waters), leading to a total of 30 different combinations of temperature and salinity.
Growth was determined via optical density measurements, and contour lines were drawn upon a
matrix chart of the various temperature/salinity combinations. This approach was later used by
other subcontractors and SERI in-house researchers to determine the optimal growth
characteristics of numerous promising algal strains. Results from this analysis were reported for
eight different strains. In general, the strains grew better at higher salinities, indicating their
halophilic nature, and had temperature optima for growth between 20°C and 30°C. Of the strains
tested, the Mono Lake isolate NITZS1 had the highest temperature optimum (between 30 and
36°C).
The effect of light intensity on the growth of Ankistrodesmus falcatus 91-1 (ANKIS1, from
Pyramid Lake) and O. pusilla 32-1 (from Walker Lake) was determined in 0.83-L cultures that
were placed at varying distances from a tungsten lamp source. Neutral density filters were
4NREL Microalgae Culture Collection strain designations are provided when relevant.
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National Renewable Energy Laboratory
placed between the light source and the cultures. This arrangement provided between 30% and
70% of full sunlight. For ANKIS1, maximum productivity (21.9 g dry weight•m-2•d-1) was
attained when the cells were subjected to 50% full sunlight. At 30% full sunlight, the
productivity fell off to 14.7 g dry weight•m-2•d-1 (because of light limitation), and at 70% full
sunlight, the productivity was reduced to 19.0 g dry weight•m-2•d-1 (most likely because of
photoinhibition). For O. pusilla, a maximum productivity of 25.8 g dry weight•m-2•d-1 was also
attained at 50% full sunlight, whereas productivity at 30% and 70% full sunlight was 18.7 and
23.2 g dry weight•m-2•d-1, respectively. These productivity values are believed to be more
accurate than those reported in the preceding section because light was able to enter the culture
vessels only from one side. The relative high densities of these cultures (>1 g dry weight•L-1)
permitted the cells to tolerate higher light intensities than would be possible in less dense
cultures, because of the self-shading of the cells.
Experiments were also conducted to determine the effects of varying the culture vessel width
(i.e., culture depth) on overall productivities. ANKIS1 was grown in containers that were 5, 10,
and 15 cm wide. The containers were illuminated with a tungsten lamp at 50% full sunlight, and
were bubbled vigorously with 1% CO2 in air. The results of these experiments indicated that
growth rate, when expressed as g dry weight•L-1•d-1, was highest in the 5-cm thick culture and
lowest in the 15-cm thick culture. However, when productivities were expressed as g dry
weight•m-2•d-1, which takes into account the actual surface area that is illuminated, the thicker
cultures were more productive. For example, the volumetric productivities over 10 days were
0.72, 0.35 and 0.31 g dry weight•L-1•d-1) for the 5-, 10-, and 15-cm thick cultures, respectively,
whereas the corresponding areal productivities for these cultures were 41.1, 40.2, and 52.7 g dry
weight•m-2•d-1. Because the economic constraints regarding an actual algal biodiesel production
facility dictate the use of open pond systems, the areal productivity values are the more important
consideration, although less water (and consequently less water handling) is required when
dealing with more dense cultures. The productivities reported for these experiments may be
overestimates of what these strains could achieve in outdoor mass culture because of the
optimized mixing and aeration regime.
In the final year of this subcontract, additional collecting trips were taken to gather more
microalgal strains. These strains, along with some that had been collected during earlier trips,
were screened more rigorously than before. In this revised selection process, the strains were
subjected to higher light intensities and higher temperatures, and the abilities of the strains to
grow in “SERI standard media5“ were investigated. This selection procedure resulted in the
isolation of 41 additional strains. Initial screening of strains involved incubating the isolates at
25°C and 30°C under 40% full sunlight (provided by a 2000-W tungsten-halide lamp) in the
SERI standard medium that most closely resembled the water from which they were originally
isolated. Twelve of the strains that grew best under these conditions were then tested under the
same temperature and light conditions in an early version of standard SERI media (Type I and
Type II at low, medium, and high salinities; see media compositions in Thomas et al. [1985]).
5The development of the SERI standard media is discussed in Chapter II.A.1. The compositions of these
media are given in Table II.A.1.
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The results indicated that most of the strains had a definite preference for a particular medium
type and level of salinity. The results also indicated some inconsistencies in the growth rates of
cells grown in the two experiments. For example, Chlorella BL-6 (CHLOR2) grew very well in
the preliminary experiments in Type II/low salinity medium (2.48 doublings•d-1), but grew much
more poorly when grown in all five SERI media (including Type II/low salinity) in the second set
of experiments. Conversely, Chlamydomonas HL-9 grew much more quickly in the second set
of experiments than in the first set. The reasons for these discrepancies are unclear, as the culture
conditions were essentially the same, and underscore the need to perform replicate experiments.
Several marine microalgae were also tested for the ability to grow in SERI standard media.
Phaeodactylum tricornutum, Chaetoceros gracilis, and Platymonas all grew well (>1.25
doublings•d-1) in at least one SERI medium. Isochrysis T-ISO was unable to grow in any SERI
medium, however.
The combined effects of temperature and salinity on the growth rates of eight of these newly
collected strains were determined by the use of a temperature-salinity gradient table. In general,
the strains grew best in the range of salinity that was similar to that of the water from which they
were originally isolated. The optimal temperature for growth was generally in the 25°C to 35°C
range, although one Chlorella strain from Salt Creek grew well at 40°C. Based on the results of
these experiments, two strains were selected for analysis of growth characteristics in larger scale
(12 L) cultures at 50%-70% full sunlight. CHLOR2 achieved a productivity of 55.5 g dry
weight•m-2•d-1 under these conditions, and Nannochloris MO-2A had a productivity of 31.9 g dry
weight•m-2•d-1.
This subcontract represented one of the first efforts in the ASP to collect and screen microalgal
strains to identify suitable biofuel production strains. As a consequence, many of the screening
and characterization protocols were still being developed; therefore, there is a substantial lack of
uniformity in the testing of the various strains isolated. Nonetheless, a number of promising
strains were isolated during the course of this research, and several methods were developed that
helped establish standard screening protocols used by other ASP researchers.
Publications:
Thomas, W.H.; Gaines, S.R. (1982) “Algae from the arid southwestern United States: an
annotated bibliography.” Report for Subcontract XK-2-0270-01. Solar Energy Research
Institute, Golden, Colorado, October 1982.
Thomas, W.H.; Seibert, D.L.R.; Alden, M.; Eldridge, P.; Neori, A.; Gaines, S. (1983b)
“Selection of high-yielding microalgae from desert saline environments.” Aquatic Species
Program Review: Proceedings of the March 1983 Principal Investigators’ Meeting, Solar Energy
Research Institute, Golden, Colorado, SERI/CP-231-1946, pp. 97-122.
Thomas, W.H. (1983b) “Microalgae from desert saline waters as potential biomass producers.”
Progress in Solar Energy 6:143-145.
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National Renewable Energy Laboratory
Thomas, W.H.; Seibert, D.L.R.; Alden, M.; Eldridge, P. (1984c) “Cultural requirements, yields,
and light utilization efficiencies of some desert saline microalgae.” Aquatic Species Program
Review: Proceedings of the April 1984 Principal Investigators’ Meeting, Solar Energy Research
Institute, Golden, Colorado, SERI/CP-231-2341, pp. 7-63.
Thomas, W.H.; Tornabene, T.G.; Weissman, J. (1984d) “Screening for lipid yielding microalgae:
Activities for 1983.” Final Subcontract Report. Solar Energy Research Institute, Golden,
Colorado, SERI/STR-231-2207.
Thomas, W.H.; Seibert, D.L.R.; Alden, M.; Eldridge, P. (1985) “Selection of desert saline
microalgae for high yields at elevated temperatures and light intensities and in SERI Standard
artificial media.” Aquatic Species Program Review: Proceedings of the March 1985 Principal
Investigators’ Meeting, Solar Energy Research Institute, Golden, Colorado SERI/CP-231-2700,
pp. 5-27.
Thomas, W.H.; Seibert, D.L.R.; Alden, M.; Eldridge, P. (1986) “Cultural requirements, yields
and light utilization efficiencies of some desert saline microalgae.” Nova Hedwigia 83:60-69.
II.A.2.d.
Screening and Characterizing Oleaginous Microalgal Species from the
Southeastern United States
Subcontractor: Alabama
A&M
University
Principal Investigator:
Mahasin Tadros
Period of Performance:
1983 -
Subcontract Number:
XK-3-03-50-1
The goal of this subcontract was to isolate and characterize strains of microalgae from the
southeastern United States that have attributes desirable for a biodiesel production strain. During
the first year of this work, field trips were made to several sites in Alabama to collect microalgal
strains from a variety of habitats. Freshwater and brackish water strains were collected from
rivers, lakes, estuaries, and ponds, and marine strains were collected from the waters surrounding
Dauphin Island in the Gulf of Mexico. Collected samples were inoculated into various artificial
media, including Bold’s Basal Medium, Chu no. 10, and “f/2” (Barclay et al. 1986). Artificial
sea salts were used in place of seawater for the saltwater media. For initial strain selection, the
cultures were incubated at 29-30°C with shaking at a light intensity of 100 to 125 µE•m-2•s-1
provided by cool white fluorescent bulbs with a 14 h:10 h light:dark cycle. The fastest growing
strains were isolated via micropipetting or by spreading samples on agar plates. In these
preliminary experiments, the marine strains exhibiting the fastest growth were Cyclotella DI-35
(CYCLO1), Hantzschia DI-160 (NITZS2), and Chlorococcum DI-34. The freshwater strains
exhibiting the fastest growth rates were Chlorella MB-31, Scenedesmus TR-84, Ankistrodesmus
TR-87, and Nitzschia TR-114.
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CYCLO1, Nitzschia TR-114, and Scenedesmus TR-84 were selected for more detailed growth
analyses under various combinations of temperature, salinity, and light intensity. A temperature
gradient table was employed for these experiments that was similar in design to the tables used
by Dr. William Thomas (discussed earlier) and SERI researchers for screening purposes. Growth
of standing cultures was determined by measuring final cell densities after 12 days of incubation.
CYCLO1 achieved maximum cell density at a temperature of 30°C, a light intensity of 100
µE•m-2•s-1, and a salinity of 15 ppt (parts per thousand). Growth was nearly as good at a light
intensity of 200 µE•m-2•s-1 and a temperature of 35°C, and substantial growth occurred at a
salinity of 32 ppt. Growth did not occur at 15° to 20°C. Nitzschia TR-114 achieved maximal
cell density at 30°C, 15 ppt salinity, and 100 µE•m-2•s-1; growth was severely inhibited at 0 and
45 ppt salinity. Growth was similar at 100 and 200 µE•m-2•s-1 for this strain, although the higher
light intensity seemed to increase the thermal tolerance of the cells. The freshwater strain
Scenedesmus TR-84 grew best at 25°C, and grew increasingly slower as the salt concentration of
the medium was increased.
The lipid contents of several strains isolated during the initial collecting trips were determined.
For 14-day-old cultures that were reportedly N-limited (although no evidence is provided to
support this), the lipid contents (as a percentage of the organic mass) were as follows: CYCLO1,
42.1%; Nitzschia TR-114, 28.1%; Chlorella MB-31, 28.6%-32.4%; Scenedesmus TR-84, 44.7%;
Ankistrodesmus TR-87, 28.1%; and Hantzschia DI-160 (NITZS2), 66%.
Additional strains were collected the next year from intertidal waters near Biloxi, Mississippi and
St. Joseph Bay, Florida. Preliminary screening experiments indicated that five strains (all of
which were diatoms) had the best growth rates and lipid accumulation potential: Navicula
acceptata (two strains, NAVIC6 and NAVIC8), N. saprophila (NAVIC7), Nitzschia dissipata
(NITZS13), and Amphiprora hyalina (ENTOM3). These strains and CYCLO1 were grown semi-
continuously in media with six different salinities at 25°, 30°, and 35°C. Cells were grown at
light intensities of 80 µE•m-2•s-1 and 160 µE•m-2•s-1 (approximately 4% and 8% of full sunlight,
respectively). The media were produced by adding various quantities of artificial sea salts to
“f/2” medium; the resulting conductivities were <1, 10, 20, 35, 45, and 60 mmho•cm-1. (Note:
seawater is typically 35-45 mmho•cm-1.) All strains exhibited more rapid growth under 160
µE•m-2•s-1 illumination than at 80 µE•m-2•s-1; and even higher growth rates might well have been
obtained at light intensities greater than 160 µE•m-2•s-1.
ENTOM3 grew best (2.0 to 2.3 doublings•d-1) at 30°C in media with conductivities of 20-60
mmho•cm-1. Growth was better with urea or nitrate as the N source rather than with ammonium.
The lipid content of nutrient-sufficient cells was 22.1% of the organic mass, and increased to
37.1% and 30.2% under Si-deficient and N-deficient conditions, respectively.
CYCLO1 achieved the highest growth rates (2.8 to 3.0 doublings•d-1) at 35°C between 10 and 35
mmho·cm-1. Cells grew best with nitrate as a N source, followed by ammonium and then urea.
The highest lipid content was observed in N-deficient cells (42.1%), but was also elevated in Si-
deficient cells (38.6%) relative to nutrient-sufficient cells (13.2%).
A Look Back at the Aquatic Species Program—Technical Review
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National Renewable Energy Laboratory
NAVIC8 grew most rapidly (3.8 doublings•d-1) at 35°C and 45 mmho•cm-1. Nitrate and
ammonium were more suitable N sources than urea. Lipid contents of 21.8%, 48.5%, and 32.4%
were observed for cells grown under nutrient-sufficient, Si-deficient, and N-deficient conditions,
respectively.
During the final year of this subcontract, additional promising strains were isolated. Included in
this group was Navicula BB-324 (NAVIC9), which had a growth rate exceeding 2.5
doublings•d-1 at 30°C in artificial seawater and SERI Types I/10, I/25, I/40, II/10, II/25, II/40,
and II/55 media. Navicula SB-304 (NAVIC8) also exhibited excellent growth (1.5-3.0
doublings•d-1) in each medium. These two strains had Si starvation-induced lipid contents of
42.5% and 47.2%, respectively. Other notable strains were Nitzschia SB-307 (NITZS13), which
had a maximal growth rate of 2.5 doublings•d-1 and a lipid content of 45%-47% under nutrient-
stressed conditions. Amphiprora BB-333 (ENTOM3), Chaetoceros BB-330 (CHAET66), and
Cylindrotheca AB-204 also grew rapidly (2.3-6.0 doublings•d-1), with stress-induced lipid
contents ranging from 16.5%-37.1%.
In conclusion, many promising strains were isolated as a result of this subcontract. The nutrient
status of the cells again was played an important role in lipid accumulation. Furthermore, the
nature of the N source included in the medium had a substantial impact on growth of the cultures.
Several of these strains were further tested in outdoor mass culture, as described in Section III.
Publications:
Barclay, W.; Johansen, J.; Chelf, P.; Nagle, N.; Roessler, R.; Lemke, P. (1986) “Microalgae
Culture Collection 1986-1987.” Solar Energy Research Institute, Golden, Colorado, SERI/SP-
232-3079, 147 pp.
Tadros, M.G. (1985) “Screening and characterizing oleaginous microalgal species from the
southeastern United States.” Aquatic Species Program Review: Proceedings of the March 1985
Principal Investigators’ Meeting, Solar Energy Research Institute, Golden, Colorado, SERI/CP-
231-2700, pp. 28-42.
Tadros, M.G. (1987a) “Screening and characterizing oleaginous microalgal species from the
southeastern United States.” FY 1986 Aquatic Species Program Annual Report, Solar Energy
Research Institute, Golden, Colorado, SERI/SP-231-3071, pp. 67-89.
Tadros, M.G. (1987b) “Conclusion of the warm-water algae collection and screening efforts
conducted in the southeastern United States.” FY 1987 Aquatic Species Program Annual Report,
Solar Energy Research Institute, SERI/SP-231-3206, pp. 58-74.
Tadros, M.G.; Johansen, J.R. (1988) “Physiological characterization of six lipid-producing
diatoms from the southeastern United States.” J. Phycol. 24:445-452.
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II.A.2.e.
Collection of High Energy Strains of Saline Microalgae from Southwestern
States
Subcontractor:
Arizona State University
Principal Investigator:
Milton Sommerfeld
Period of Performance:
1985 - 1987
Subcontract Number:
N/A
The objectives of this subcontract were to collect microalgal strains from a variety of locations in
the desert regions of the Southwestern United States and to screen them for their ability to grow
under conditions in a commercial microalgal biodiesel facility. Studies were also conducted to
optimize a fluorometric procedure for estimating cellular lipid content, and to use this method to
screen some of the strains.
Collecting trips took place between April 1985 and June 1986. Water samples containing
microalgae were collected from 125 sites in Arizona, California, Nevada, New Mexico, Texas,
and Utah. Some samples were taken from saline surface waters in the regions of Arizona and
New Mexico that were deemed suitable for microalgal mass culture, based in part on the
availability of large quantities of saline groundwater (Lansford et al. 1987). Researchers
believed that strains from these areas would be well adapted to the indigenous waters available
for mass culture. These areas included the Palo Verde Irrigation District in Arizona, and the
Pecos River Basin, the Crow Flats area, and the Tularosa Basin in New Mexico. The
temperature, pH, specific conductance, and water depth were recorded at each collection site.
Most of the waters sampled had a specific conductance exceeding 2 mmho•cm-1. Temperatures
ranged from 18°C to 45°C (mean = 26.9°C), pH ranged from 6.1 to 10.2 (mean = 8.0), and
specific conductance ranged from 0.45 mmho•cm-1 to 474 mmho•cm-1 (mean = 22.7 mmho•cm-1).
Planktonic algae were the primary type of alga collected, although neustonic and benthic forms
were also collected when algal growth in such habitats was clearly visible. From these samples,
more than 1,700 strains of microalgae were obtained. From these strains, approximately 700
unialgal cultures were established. Initial strain isolations were performed by streaking out
samples onto 1.5% agar plates containing seawater, sterilized collection site water, or SERI Type
I or Type II medium having a conductivity similar to that of the collection site water. In some
cases, an enrichment step was performed before streaking out the cells, wherein the samples were
placed in tubes on a rotary agitation wheel in liquid media at a light intensity of 1500 µE•m-2•s-1
(~75% of full sunlight) using a 12h:12h light:dark cycle.
Of the 700 unialgal cultures, 120 were identified taxonomically; 24 genera were represented in
this group, including 60 chlorophytes, 40 diatoms, and 20 cyanophytes. The most common
genera were Dunaliella, Chlorococcum, Chlorosarcina, Amphora, Nitzschia, Navicula,
Oscillatoria, and Chroococcus. Initial screening typically involved visual assessment of growth
at 25°C at 200 µE•m-2•s-1 in tubes containing SERI Type I/40 and Type II/40 media. Strains that
grew the most rapidly were subjected to further characterization, including analysis of the effects
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of temperature, salinity, light intensity, and N source on growth rates. Cultures were grown in
several different media (SERI Types I and II media at various conductivities, and artificial
seawater) at 30°C and 500 µE•m-2•s-1 using a 12h:12h light:dark cycle on a rotary screening
apparatus. Thirty-one diatoms were tested under these conditions, and 11 exhibited growth rates
exceeding one doubling•d-1. The highest growth rate observed for a diatom under these
conditions was 1.96 doublings•d-1 for Amphora ASU0308. Of the 50 chlorophyte species tested,
17 strains exhibited growth rates exceeding one doubling•d-1; the highest growth rate (2.58
doublings•d-1) was observed for a strain of Dunaliella (ASU0038). Of the strains that were tested
for growth in all seven standard media, 80% of the cultures grew in the low salinity SERI Type I
and Type II media and more than 50% of the strains grew in seawater. The highest growth rates
were typically observed in SERI Type I/10 and Type II/10 media and seawater, although the
mean growth rates of all strains combined at the highest salinities (70 mmho•cm-1) were 60% to
80% of the mean growth rates obtained at the lower salinities. Most of the strains were isolated
from waters with specific conductances below 40 mmho•cm-1, which may explain the lower
growth rates in the media having higher salinities. A few strains, however, grew quite well in the
higher salinity media. Amphora ASU0032 (AMPHO27), Synechococcus ASU0071 (CHLOC5),
also referred to by the subcontractor as Chroococcus, and Navicula ASU0267 were the only
strains that had a growth rate that exceeded one doubling•d-1 in Type II/70 medium. Certain
strains had high growth rates in both Type I/70 and Type II/70 media; included in this group
were the strains mentioned earlier along with Synechococcus ASU0075 (CHROC2) and
Dunaliella ASU0038. Some strains were clearly euryhaline (i.e., able to tolerate a wide range of
salinities), and could grow in all media tested (e.g., CHLOC5 had a growth rate that exceeded
one doubling•d-1 in each of the seven media tested). Other strains were stenohaline, and grew
much better at one particular salinity. Certain strains showed no real preference for SERI Type I
versus SERI Type II medium, despite the very different ionic composition of these media types.
Other strains exhibited a clear preference for one media type over the other (e.g., Dunaliella
ASU0038 grew much better in SERI Type I medium or seawater than in SERI Type II medium).
Twenty-eight of the strains tested had growth rates exceeding one doubling•d-1 in at least one
media type, three strains had growth rates that exceeded two doublings•d-1, and one strain (either
Eremosphaera or Chlorococcum ASU0132 [CHLOC6] or ASU0048 [CHLOC4]) had a growth
rate higher than three doublings•d-1.
The proximate chemical compositions of 11 isolates were also determined in this study. Total
lipid was determined by the Bligh-Dyer procedure (Bligh and Dyer 1959). Protein was
determined by the heated biuret-Folin assay, and total carbohydrate was estimated by the phenol-
sulfuric acid method (see Sommerfeld et al. [1987b] for details). Of the newly isolated strains
tested, Franceia ASU0146 (FRANC1) had the highest lipid content under normal growth
conditions (26.5% of the AFDW). The strains were not evaluated under nutrient-deficient
conditions, which often increases the lipid content of microalgae. Because analyzing the lipid
content of all the strains that had been isolated by this type of procedure would be very difficult,
the strains were examined by the use of lipophilic dyes. The dyes Nile Blue A, Sudan Black B,
Oil Red O, and Nile Red were used in conjunction with fluorescence microscopy to check for oil
droplets within the cells. Inconsistent results were obtained when using the first three stains, but
Nile Red appeared to give more reliable results. The stained cells were visually scored for the
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presence of fluorescing lipid droplets. Additional work was carried out to develop a Nile Red
staining procedure that could (in theory) provide a quantitative measure of lipid content by the
use of a fluorometer. This latter work was discussed in Section II.A.1. along with the results of
similar efforts carried out by SERI researchers and other subcontractors.
This subcontract was somewhat unusual in that many of the strains were collected from the
actual areas in which the commercial microalgal biodiesel facilities could be. As a consequence,
many of the strains that were isolated had characteristics that could make them good candidates
for production strains. Additional results from this subcontract are presented in Section II.A.1.
Publications:
Sommerfeld, M.R.; Ellingson, S.B. (1987a) “Collection of high energy yielding strains of saline
microalgae from southwestern states.” FY 1986 Aquatic Species Program Annual Report, Solar
Energy Research Institute, Golden, Colorado, SERI/SP-231-3071, pp. 53-66.
Sommerfeld, M.; Ellingson, S.; Tyler, P. (1987b) “Screening microalgae isolated from the
southwest for growth potential and lipid yield.” FY 1987 Aquatic Species Program Annual
Report, Solar Energy Research Institute, Golden, Colorado, SERI/SP-231-3206, pp. 43-57.
Additional References:
Lansford, R.R.; Hernandez, J.W.; Enis, P.J.(1987) “Evaluation of available saline water resources
in New Mexico for the production of micoralgae.” FY 1986 Aquatic Species Program Annual
Report, Solar Energy Research Institute, Golden, Colorado, SERI/SP-231-3071, pp. 227-248.
II.A.2.f.
Collection of High Energy Yielding Strains of Saline Microalgae from the
Hawaiian Islands
Subcontractor:
Hawaii Institute of Marine Biology
Principal Investigator:
Richard H. York, Jr.
Period of Performance:
1985
Subcontract Number:
N/A
Microalgae were collected from a variety of sites in the Hawaiian islands, including ocean sites
and inland saline habitats. The conductivity, dissolved oxygen content, pH, and temperature of
each site was recorded. Individual cells were isolated via micropipetting and placed into glass
tubes or fluorohalocarbon plastic bags containing either the original sample water, offshore
seawater, SERI Type I medium, or SERI Type II medium. The plastic bags, which transmit the
full visible solar spectrum, were placed in full sunlight without temperature control. This
treatment was therefore believed to provide a good selection for strains that would be able to
thrive under outdoor mass culture conditions. The glass tubes were incubated at 25°-26°C at 40
µE•m-2•s-1 under a 16h:8h light:dark regime; these conditions were less stressful than the outdoor
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National Renewable Energy Laboratory
conditions, and therefore led to the recovery of less hardy strains. A large-scale outdoor
enrichment culture was also prepared by pumping 1700 L of enriched seawater into a 5.5 m
diameter open tank, then strains arising in this culture were isolated.
As a result of these procedures, 100 of the most rapidly growing strains were selected and
maintained for further analysis. This group included members of the Chlorophyceae,
Cyanophyceae, Bacillariophyceae, and Pyrrophyceae. Two strains, Chaetoceros SH 9-1
(CHAET38) and Cyclotella 14-89 (THALA6), were grown in outdoor cultures consisting of cells
in 1-L fluorohalocarbon plastic bags. The highest growth rates measured for these strains were
2.12 doublings•d-1 for CHAET38 and 1.43 doublings•d-1 for THALA6. These growth rates were
reported to correspond to 31 and 33 g dry weight•m-2•d-1, respectively, although how these values
were derived is not clear.
Publications:
York, Jr., R.H. (1987) “Collection of high energy yielding strains of saline microalgae from the
Hawaiian Islands.” FY 1986 Aquatic Species Program Annual Report, Solar Energy Research
Institute, Golden, Colorado, SERI/SP-231-3071, pp. 90-104.
II.A.2.g.
Characterization of Hydrocarbon Producing Strains of Microalgae
Subcontractor:
Scripps Institution of Oceanography
Principal Investigator:
Ralph A. Lewin
Period of Performance:
1985 - 1986
Subcontract Number:
N/A
This subcontract focused on the collection and characterization of picopleustonic algae, which
are defined as algae (including the prokaryotic cyanophytes) that are very small (1-5 µm) and
that live on the surface of the water. In February 1985, water samples were taken from various
sites in the Caribbean Sea, including sites near the U.S. Virgin Islands (St. John, St. Thomas, and
St. Croix), Tortola, Puerto Rico, Curaçao, Panama, and the Florida Keys. 130 samples (250 mL
each) were collected and filtered through a 3-8-µm filter to remove larger cells. Smaller cells
were collected on a 0.45 µm nitrocellulose filter, which was rolled up and placed in the original
sampling water that had passed through the filter. These samples were placed under natural
lighting at 20°C to 25°C until transported to the laboratory. The filters were then transferred into
a tube of sterile enriched seawater (containing additional N and other nutrients) and incubated at
25°C under continuous illumination from a fluorescent lamp at 30 µE•m-2•s-1. In an attempt to
stimulate lipid accumulation via nutrient deficiency, a portion of each culture was transferred
after 4 weeks of growth to a fresh tube of unenriched seawater and then allowed to grow under
the same conditions. After 4 more weeks, a film of cells was often observed floating on the
surface of the cultures. Small samples of these cells were transferred to fresh enriched seawater.
After incubation for an additional 2 weeks, the cells in these cultures were microscopically
examined, and cultures that were dominated by diatoms, cyanophytes, and flagellates were
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discarded, leaving approximately 60 cultures of small (1-5-µm) green cells. Unialgal cultures
were established from these cells by isolating colonies on agar plates. Of these purified cultures,
there were 14 isolates of Stichococcus, 21 isolates of Nannochloris, four strains of Chlorella, and
several representatives of other genera. Stichococcus, Nannochloris, and Chlorella are all
chlorophytes. Because cyanophytes typically do not accumulate lipids, they were eliminated
from further study in this subcontract. The researchers anticipated that isolating strains in this
manner would enrich for lipid-accumulating microalgae.
The isolated strains were tested for the ability to grow in freshwater; all the Stichococcus and
Chlorella strains grew well in freshwater, suggesting perhaps a brackish water origin for these
strains. Only six of the 21 Nannochloris strains could grow on non-marine media.
To quantitatively determine lipid content in the isolated strains, 1-L cultures were grown for 3
weeks in enriched seawater under continuous illumination at 30 µE•m-2•s-1. The cultures were
bubbled with 0.5% CO2 in air. Cells were harvested, frozen and lyophilized, and then extracted
three times with a chloroform/methanol mixture (2:1 v/v). After the solvents evaporated, the
lipid mass was determined gravimetrically and normalized to the cellular AFDW. The 13
Stichococcus strains had lipid contents ranging from 9% to 59% of the AFDW, with an average
of 33%. The lipid contents of the 21 Nannochloris strains ranged from 6% to 63%, with an
average of 31%. Data were not presented for the lipid contents of the four Chlorella strains,
although three strains of the eustigmatophyte Nannochloropsis that were isolated from Qingdao,
China were examined; for this genus, the lipid content ranged from 31% to 68%, with an average
of 46%. These reported lipid contents may be slight overestimates, in that there was apparently
no attempt to remove somewhat polar materials that may have also been extracted via the use of
an aqueous washing step.
Some preliminary experiments were also conducted during the course of this subcontract
regarding the growth of the eustigmatophyte Nannochloropsis (strain Nanno-Q, one of the
Qingdao strains). This strain is euryhaline, and is able to grow in seawater as well as brackish
water with one-tenth the salinity of seawater. The cells grew to a higher final yield with nitrate
as the N source than with ammonium, and the lipid content rose substantially when the N source
was initially supplied at levels below 200 µM (as determined by the percentage of cells that were
floating due to elevated lipid levels). A number of Nannochloropsis strains that had been
obtained primarily from the Culture Collection of Marine Phytoplankton at Bigelow Laboratory
(West Boothbay Harbor, Maine) were analyzed with respect to maximum cell yields after 4
weeks of growth at different temperatures. Most of the strains had temperature optima at or
below 25°C, although one strain that had been collected near Long Island, New York had a
temperature optimum of 33°C.
This subcontract examined a group of microalgae that had not received much attention in the
ASP until that point. The small size of picopleustonic algae could hinder harvesting efficiency in
a mass culture facility, which would have a negative impact on the economics of biodiesel
production. However, if the cells could be made to consistently float due to high lipid levels, this
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property might facilitate harvesting. Outdoor testing of the most promising strains would help to
evaluate this group of microalgae.
Publications:
Lewin, R.A. (1985) “Production of hydrocarbons by micro-algae; isolation and characterization
of new and potentially useful algal strains.” Aquatic Species Program Review: Proceedings of the
March 1985 Principal Investigators’ Meeting, Solar Energy Research Institute, Golden,
Colorado, SERI/CP-231-2700, pp. 43-51.
Lewin, R.A.; Burrascano, C; Cheng, L. (1987) “Some picopleuston algae from the Caribbean
region.” FY 1986 Aquatic Species Program Annual Report, Solar Energy Research Institute,
Golden, Colorado, SERI/SP-231-3071, pp. 105-121.
II.A.2.h.
Collection of High Energy Yielding Strains of Saline Microalgae from South
Florida
Subcontractor:
Harbor Branch Foundation
Principal Investigator:
John H. Ryther
Period of Performance:
1985 - 1986
Subcontract Number:
N/A
The goal of the work performed under this subcontract was to collect and screen microalgal
species from southern Florida. It emphasized collecting chromophytic algae (e.g., diatoms,
chrysophytes, and prymnesiophytes), because this group of algae was known to often accumulate
lipids. Collection trips were made in June and September 1985, and in February 1986 to the
Florida Keys and the Everglades. Samples were taken from 123 sites, including various
mangrove swamps, salt flats, canals, ditches, and shallow ponds. The basic physicochemical
characteristics of the collection site waters were determined. The mean temperature was 29°-
30°C both for sites in the Florida Keys and the Everglades. The mean conductivity of the water
from the Keys (35.6 mmho•cm-1) was somewhat higher than that of the Everglades (25.7
mmho•cm-1), whereas the pH values were similar (~8). To select for the fastest growing
microalgal strains in the water samples, the original samples were enriched with nitrate, trace
metals, and vitamins, and incubated under continuous light (880 µE•m-2•s-1, or 45% of full
sunlight) at 30°C. The strains that became dominant in the cultures were isolated into unialgal
cultures via micropipetting, serial dilution, and spreading onto agar plates. As a consequence of
these experiments, 61 unialgal cultures were produced.
Preliminary evaluation of the growth of these strains in various media was performed in test
tubes containing enriched seawater, SERI Type I/25, Type I/40, Type II/25, and Type II/40
media. The test tubes were incubated at 30°C under constant illumination at 300 µE•m-2•s-1.
Growth rates were determined by measuring the OD750 every day for 5 days, and the final culture
density was measured after 10 days. One hundred ten strains (including some strains already in
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the Harbor Branch algal collection) were screened in this manner. In general, the strains that
were newly isolated under the selection scheme outlined above grew more rapidly than the
culture collection strains. Members of the Prymnesiophyceae, particularly coccolithophorids and
ochromonads, tended to grow well in most media types, but the dinoflagellates isolated via these
procedures did not grow well in the SERI standard media. Most species grew better in Type II
medium than in Type I medium, although there were certainly exceptions to this. The highest
growth rate (3.26 doublings•d-1) was observed with a strain of Hymenomonas HB152
(HYMEN3) in Type II/25 medium. Seven strains had growth rates that exceeded 2 doublings•d-1
in at least one media type; included in this group were Dunaliella HB37 (DUNAL2),
Nannochloris HB44 (NANNO2), a yellow green unicell HB54 (UNKNO4), Chlorella HB82
(CHLOR7) and HB87, Pyramimonas HB133 (PYRAM2), and HYMEN3. Nine strains had
growth rates of at least one doubling•d-1 in all five media (including Chlorella HB84 (CHLOR8)
and HB97 (CHLOR9), Nannochloris HB85 (NANO3), and all the strains mentioned earlier in
this paragraph except for HYMEN3).
Several of the most promising strains were examined in more detail; they were grown in a matrix
of five different salinities (8-60 mmho•cm-1) at five different temperatures (15°-35°C) by the use
of a temperature-salinity gradient table, as described in previous sections. An artificial seawater
medium (ASP-2) diluted with varying amounts of distilled water was used for these experiments.
The cultures were exposed to constant illumination at 180 µE•m-2•s-1. Each of the four strains
tested (Tetraselmis HB47 [TETRA4], PYRAM2, UNKNO4, and an olive green unicell HB154
[UNKNO5]) exhibited excellent growth over a wide range of conditions. All these strains had a
growth rate greater than one doubling•d-1 between 8 and 60 mmho•cm-1 and between 20° and
35°C. UNKNO4 had growth rates higher than 1.5 doublings•d-1 between 15 and 60 mmho•cm-1
and between 20° and 35°C.
A visual assessment of the lipid contents of the most rapidly growing strains was conducted by
staining the cells with Nile Red, the stained cells were examined using fluorescence microscopy.
Based on this assessment (which was not carried out with nutrient-starved cells), TETRA5 and
UNKNO4 had the highest estimated lipid contents. TETRA4, HYMEN2, PYRAM2, and
UNKNO5 also appeared to accumulate substantial quantities of lipid.
Publications:
Rhyther, J.H.; Carlson, R.D.; Pendoley, P.D.; Jensen, P.R. (1987) “Collection and
characterization of saline microalgae from South Florida.” FY 1986 Aquatic Species Program
Annual Report, Solar Energy Research Institute, Golden, Colorado, SERI/SP-231-3071, pp. 122-
136.
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National Renewable Energy Laboratory
II.A.2.i.
Collection and Selection of High Energy Thermophilic Strains of Microalgae
Subcontractor:
Montana State University
Principal Investigator:
Keith E. Cooksey
Period of Performance:
3/86 - 4/87
Subcontract Number:
XK-4-04136-04
The goal of this research was to develop a technique for rapidly screening microalgae for high
lipid content, and to use this method to select microalgae with potential for liquid fuel
production. Dr. Cooksey’s laboratory initiated the development of the Nile Red lipid staining
procedure, which is fully described in Section II.A.1.f. The Nile Red staining procedure was used
to screen for high lipid strains of microalgae, first using cultures collected mainly from Florida
and maintained at Montana State University, and in cultures containing diatoms freshly isolated
from hot springs in Yellowstone National Park. Because algae to be used in outdoor mass
culture in the desert southwest would be subject to high temperatures, the Florida strains, isolated
at 28°C, were first tested for growth and lipid production at 35°C. Although some strains
produced fairly high levels of lipid, most grew poorly. Some diatom strains were then isolated
from the hot springs, based on the premise that they would be more likely to tolerate extremes of
temperature and pH variation. In these cultures, Nile Red was used to screen the initial sample
for lipid-producing strains. These cells were then cultured, made unialgal and axenic, and tested
for growth rate and lipid production. The strains tested showed growth rates of 0.5 to 2
doublings/d and lipid contents of 9%-54%, similar to the properties of oil-producing algae
isolated by other methods.
Publications:
Berglund, D.; Cooksey, B.; Cooksey, K.E.; Priscu, L.R. (1987) “Collection and screening of
microalgae for lipid production: Possible use of a flow cytometer for lipid analysis.” FY 1986
Aquatic Species Program Annual Report, Solar Energy Research Institute, Golden, Colorado,
SERI/SP-231-3071, pp. 41-52.
Cooksey, K.E. (1987) “Collection and screening of microalgae for lipid production.” Final
Subcontract Report to the Solar Energy Research Institute, Solar Energy Research Institute,
Golden, Colorado, May 1987, 42 pp.
Cooksey, K.E.; Guckert, J.B.; Williams, S.A.; Collis, P.R. (1987) “Fluorometric determination of
the neutral lipid content of microalgal cells using Nile Red,” Journal of Microbiological Methods
6:333-345.
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II.A.3.
The SERI Microalgae Culture Collection
II.A.3.a.
History of SERI Microalgae Culture Collection
The SERI Microalgae Culture Collection was first established in 1984 by Dr. Bill Barclay to
provide a central repository for strains that were believed to have potential as biomass fuel
production organisms. The intent was to provide documented and partially characterized
microalgal strains to researchers interested in conducting biofuels research or in developing algal
mass culture technologies. The publicly available collection was described in a series of Culture
Collection Catalogs published between 1984 and 1987. It was initially limited to strains that had
been characterized quite extensively with respect to growth properties and chemical composition,
and that were believed to hold the most promise. These catalogs contain a wealth of information
for many of these strains, often including photomicrographs, proximate chemical compositions,
lipid contents of cells grown under various environmental conditions, growth characteristics in
different media types and different temperatures, and the results of small-scale outdoor
production pond trials. Furthermore, media compositions are provided in these catalogs.
The original 1984-1985 Microalgae Culture Collection Catalog listed the following criteria for
selection of strains to be placed in the cataloged public collection (in descending order of
importance):
• Energy yield (growth rate x energy content)
• Type of fuel products available from biomass (hydrocarbon, diesel, alcohol,
methanol)
• Environmental tolerance range (temperature, salinity, pH)
• Performance in mass culture (highly competitive, predator resistant)
• Media supplementation requirements (addition of vitamins, trace minerals)
• Amount of culture and composition data available on the clone or strain
• Budget for the culture collection
Although conceptually sound, these criteria carried with them the requirement to characterize the
strains fairly extensively before a decision could be made as to whether they should be included
in the collection. This detailed characterization became increasingly difficult as the number of
strains available increased. As a consequence, many strains were maintained that were not
officially documented in the catalogs.
From the inception of the culture collection until the late 1980s, strains in the collection were
provided free of charge to anyone who requested them, with the hope that the research conducted
(and published) using these strains would increase the overall understanding of these organisms.
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National Renewable Energy Laboratory
Many laboratories took advantage of this. In the first year after the publication of the SERI
Microalgae Culture Collection Catalog, more than 100 cultures were shipped to various groups
studying biofuels production, natural product discovery, aquaculture, and the general physiology,
biochemistry, and molecular biology of microalgae. In the ensuing years, hundreds of additional
cultures were provided to researchers free of charge. Then, in the early 1990s, concerns were
raised by the SERI Legal Department that the cultures should be considered as valuable
intellectual property, and a moratorium was placed on providing cultures to outside parties; this
moratorium persisted until the ASP were eliminated in late 1995.
The SERI Microalgae Culture Collection consists almost exclusively of eukaryotic, single-celled
microalgae. Included in the collection are members of several algal classes, with a predominance
of chlorophytes (green algae) and diatoms. These two groups tended to dominate under the high
temperature/high light screening and selection regimes used to identify good production strain
candidates. Of the strains present in the final culture catalog produced (published in 1987,
including an addendum), 26% were chlorophytes, 60% were diatoms, 8% were chrysophytes, and
6% were eustigmatophytes. The following pages list the strains described in each of the three
culture collection catalogs published during the course of the ASP.
The first culture collection catalog (1984-1985) listed 11 strains, which included five
chlorophytes, four diatoms, one chrysophyte, and one eustigmatophyte. Some of these strains
were obtained from other culture collections, including the University of Texas algal culture
collection. The strains listed in the original 1984-1985catalog were as follows:
Table II.A.6. Microalgal strains listed in the first SERI Culture Collection Catalog,
1984-1985.
Species
Original SERI
Final SERI
Strain Alias
Collector
Strain
Strain
Designation
Designation
Ankistrodesmus falcatus
S/ANKIS-1
ANKIS1
Pyramid Lake 91-
W. Thomas
Botryococcus braunii
S/BOTRY-1
BOTRY1
UTEX #572
---
Chaetoceros gracilis
S/CHAET-1
CHAET1
CHGRA
R. York
Chlorella sp.
S/CHLOR-1
CHLOR1
S01
S. Lien
Isochrysis galbana
S/ISOCH-1
ISOCH1
Tahitian T-ISO
J.-L.
Nannochlropsis salina
S/NANNO-1
NANNP1
GSBSTICHO
J. Rhyther
Nitzschia sp.
S/NITZS-1
NITZS1
Mono Lake
D.
Oocystis pusilla
S/OOCYS-1
OOCYS1
Walker Lake
W. Thomas
Phaeodactylum
S/PHAEO-1
PHAEO1
TFX-1
---
Phaeodactylum
S/PHAEO-2
PHAEO2
BB
W. Thomas
Platymonas sp.
S/PLATY-1
TETRA1
---
E. Laws
(later Tetraselmis suecia)
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The next edition of the SERI Microalgae Culture Collection Catalog (1985-1986) included the
following additional strains:
Table II.A.7. Microalgal strains added to the SERI Culture Collection Catalog, 1985-
1986.
Species
Original SERI
Final SERI
Strain Alias
Collector
Strain
Strain
Designation
Designation
Amphora coffeiformis S/AMPHO-1
AMPHO1 ---
W.
Barclay
Boekelovia hooglandii
S/BOEKE-1
BOEKE1
---
W. Barclay
Chaetoceros sp.
S/CHAET-2
CHAET14
SS-14
W. Thomas
Chlorella ellipsoidea
S/CHLOR-2
CHLOR2
BL-6
W. Thomas
Chlorella sp.
S/CHLOR-3
CHLOR3
SC-2
W. Thomas
Cyclotella cryptica
S/CYCLO-1
CYCLO1
DI-35
M. Tadros
Monoraphidium sp.
S/MONOR-1
MONOR1
Mom’s Ranch
W. Barclay
Monoraphidium sp.
S/MONOR-2
MONOR2
---
W. Barclay
Nannochloropsis sp.
S/NANNO-2
NANNP2
Nanno-Q
R. Lewin
Nitzschia dissipata
S/NITZS-2
NITZS2
DI-160
M. Tadros
The 1986-1987 SERI Microalgae Culture Collection Catalog (including an addendum) added 29
more strains, bringing the total number of strains in the collection to 50. The 1986-1987 catalog
included the following additional strains:
Table II.A.8. Microalgal strains added to the SERI Culture Collection Catalog, 1986-
1987.
Species
Final SERI
Strain Alias
Collector
Strain
Designation
Amphiprora hyalina ENTOM3 BB-333
M.
Tadros
Amphora sp.
AMPHO27
MLS-1, ASU 0032
M. Sommerfeld
Amphora sp.
AMPHO28
GR-2, ASU 3001
M. Sommerfeld
Chaetoceros muelleri
CHAET6
NM-6
W. Barclay
Chaetoceros muelleri
CHAET9
UT-147
S. Rushforth
Chaetoceros muelleri
CHAET10
S/CHAET-4, UT-27
S. Rushforth
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Chaetoceros muelleri
CHAET15
49-1A
W. Thomas
Chaetoceros muelleri
CHAET38
SH9-1
R. York
Chaetoceros muelleri
CHAET58
---
J. Johansen
Chaetoceros muelleri
CHAET61
---
S. Rushforth
Chaetoceros muelleri
CHAET63
---
S. Rushforth
Cyclotella cryptica
CYCLO2
F-1
P. Roessler
Cyclotella cryptica
CYCLO4
UT-65
W. Barclay
Ellipsoidon sp.
ELLIP1
70-01
R. Lewin
Franceia sp.
FRANC1
LCC-1, ASU 0146
M. Sommerfeld
Nannochloris sp.
NANNO2
HB44
R. Carlson
Nannochloris sp.
NANNO12
120-01
R. Lewin
Navicula saprophila
NAVIC1
F-2
P. Roessler
Navicula acceptata
NAVIC6
SB-264
M. Tadros
Navicula sp.
NAVIC7
BB 260
M. Tadros
Navicula acceptata
NAVIC8
SB-304
M. Tadros
Navicula saprophila
NAVIC24
---
J. Johansen
Nitzschia dissipata
NITZS13
SB-307
M. Tadros
Nitschia communis
NITZS28
---
J. Johansen
Pleurochrysis carterae
PLEUR1
---
W. Barclay
Tetraselmis sp.
TETRA4
HB47
R. Carlson
Thalassiosira weissflogii
THALA2
CO-F15
W. Barclay
Thalassiosira weissflogii
THALA6
SH14-89
R. York
Unidentified Prasinophyte
GREEN3
---
J. Johansen
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II.A.3.b.
Current status of the SERI/NREL Microalgae Culture Collection
Of the strains included in the most recent Culture Collection Catalog (the 1986-1987 edition,
including the addendum), 37 are still viable. In addition, approximately 260 additional strains
are part of the collection, but were never characterized well enough to be included in the catalog.
All of these strains are in the process of being transferred to the University of Hawaii, where they
will be maintained within the Center for Marine Biotechnology. The university intends to again
make these strains available to the research community. A complete list of the strains still being
maintained is included below:
Table II.A.9. Current list of microalgal strains in the SERI Culture Collection
Strain
Species Class
ACHNA1
Achnanthes orientalis Bacillariophyceae
ACHNA2
Achnanthes orientalis Bacillariophyceae
AMPHO1
Amphora coffeiformis Bacillariophyceae
AMPHO2
Amphora coffeiformis Bacillariophyceae
AMPHO3
Amphora coffeiformis Bacillariophyceae
AMPHO5
Amphora coffeiformis Bacillariophyceae
AMPHO6
Amphora coffeiformis Bacillariophyceae
AMPHO7
Amphora delicatissima capitata Bacillariophyceae
AMPHO8
Amphora coffeiformis punctata Bacillariophyceae
AMPHO10
Amphora delicatissima Bacillariophyceae
AMPHO11
Amphora coffeiformis Bacillariophyceae
AMPHO12
Amphora coffeiformis punctata Bacillariophyceae
AMPHO13
Amphora coffeiformis punctata Bacillariophyceae
AMPHO14
Amphora coffeiformis Bacillariophyceae
AMPHO18
Amphora coffeiformis Bacillariophyceae
AMPHO21
Amphora coffeiformis linea Bacillariophyceae
AMPHO22
Amphora coffeiformis Bacillariophyceae
AMPHO23
Amphora delicatissima Bacillariophyceae
AMPHO24
Amphora coffeiformis Bacillariophyceae
AMPHO25
Amphora sp. Bacillariophyceae
AMPHO26
Amphora coffeiformis tenuis Bacillariophyceae
AMPHO28
Amphora coffeiformis Bacillariophyceae
AMPHO29
Amphora coffeiformis Bacillariophyceae
AMPHO30
Amphora coffeiformis Bacillariophyceae
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Strain
Species Class
AMPHO31
Amphora coffeiformis Bacillariophyceae
AMPHO32
Amphora coffeiformis Bacillariophyceae
AMPHO33
Amphora delicatissima Bacillariophyceae
AMPHO34
Amphora coffeiformis Bacillariophyceae
AMPHO35
Amphora coffeiformis taylori Bacillariophyceae
AMPHO36
Amphora coffeiformis taylori Bacillariophyceae
AMPHO37
Amphora coffeiformis linea Bacillariophyceae
AMPHO38
Amphora delicatissima Bacillariophyceae
AMPHO40
Amphora delicatissima Bacillariophyceae
AMPHO46
Amphora sp. Bacillariophyceae
ANKIS1
Ankistrodesmus falcatus Chlorophyceae
BOROD2
Borodinella sp. Chlorophyceae
BOTRY1
Botryococcus braunii Chlorophyceae
CARB
--- ---
CHAET1
Chaetoceros muelleri Bacillariophyceae
CHAET3
Chaetoceros gracilis Bacillariophyceae
CHAET5
Chaetoceros muelleri Bacillariophyceae
CHAET6
Chaetoceros muelleri Bacillariophyceae
CHAET7
Chaetoceros muelleri Bacillariophyceae
CHAET9
Chaetoceros muelleri Bacillariophyceae
CHAET10
Chaetoceros muelleri Bacillariophyceae
CHAET11
Chaetoceros muelleri Bacillariophyceae
CHAET14
Chaetoceros muelleri Bacillariophyceae
CHAET15
Chaetoceros muelleri Bacillariophyceae
CHAET17
Chaetoceros muelleri Bacillariophyceae
CHAET18
Chaetoceros muelleri Bacillariophyceae
CHAET19
Chaetoceros muelleri Bacillariophyceae
CHAET20
Chaetoceros muelleri Bacillariophyceae
CHAET21
Chaetoceros muelleri Bacillariophyceae
CHAET22
Chaetoceros muelleri Bacillariophyceae
CHAET23
Chaetoceros muelleri Bacillariophyceae
CHAET24
Chaetoceros muelleri Bacillariophyceae
CHAET30
Chaetoceros sp. Bacillariophyceae
CHAET39
Chaetoceros muelleri Bacillariophyceae
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Strain
Species Class
CHAET40
Chaetoceros muelleri Bacillariophyceae
CHAET41
Chaetoceros muelleri Bacillariophyceae
CHAET43
Chaetoceros muelleri Bacillariophyceae
CHAET44
Chaetoceros muelleri Bacillariophyceae
CHAET45
Chaetoceros muelleri Bacillariophyceae
CHAET46
Chaetoceros muelleri Bacillariophyceae
CHAET47
Chaetoceros muelleri Bacillariophyceae
CHAET48
Chaetoceros muelleri Bacillariophyceae
CHAET50
Chaetoceros muelleri Bacillariophyceae
CHAET51
Chaetoceros muelleri Bacillariophyceae
CHAET54
Chaetoceros muelleri subsalsum Bacillariophyceae
CHAET55
Chaetoceros muelleri subsalsum Bacillariophyceae
CHAET57
Chaetoceros muelleri-trans Bacillariophyceae
CHAET58
Chaetoceros muelleri muelleri Bacillariophyceae
CHAET59
Chaetoceros muelleri subsalsum Bacillariophyceae
CHAET60
Chaetoceros muelleri subsalsum Bacillariophyceae
CHAET62
Chaetoceros muelleri subsalsum Bacillariophyceae
CHAET64
Chaetoceros muelleri subsalsum Bacillariophyceae
CHAET66
Chaetoceros muelleri Bacillariophyceae
CHAET67
Chaetoceros sp. Bacillariophyceae
CHAET68
Chaetoceros sp. Bacillariophyceae
CHAET69
Chaetoceros sp. Bacillariophyceae
CHAET73
Chaetoceros sp. Bacillariophyceae
CHAET75
Chaetoceros sp. Bacillariophyceae
CHAET76
Chaetoceros sp. Bacillariophyceae
CHAET78
Chaetoceros sp. Bacillariophyceae
CHLOC1
Chlorococcum sp. Chlorophyceae
CHLOC2
Chlorococcum sp. Chlorophyceae
CHLOC3
Chlorococcum sp. Chlorophyceae
CHLOC6
Chlorococcum sp. Chlorophyceae
CHLOC7
Chlorococcum sp. Chlorophyceae
CHLOC8
Chlorococcum sp. Chlorophyceae
CHLOC10
Chlorococcum sp. Chlorophyceae
CHLOC11
Chlorococcum sp. Chlorophyceae
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Strain
Species Class
CHLOR1
Chlorella sp. Chlorophyceae
CHLOR2
Chlorella ellipsoidea Chlorophyceae
CHLOR3
Chlorella sp. Chlorophyceae
CHLOR5
Chlorella sp. Chlorophyceae
CHLOR6
Chlorella sp. Chlorophyceae
CHLOR8
Chlorella sp. Chlorophyceae
CHLOR9
Chlorella sp. Chlorophyceae
CHLOR10
Chlorella sp. Chlorophyceae
CHLOR11
Chlorella sp. Chlorophyceae
CHLOR12
Chlorella sp. Chlorophyceae
CHLOR13
Chlorella sp. Chlorophyceae
CHLOR14
Chlorella salina Chlorophyceae
CHLOR15
Chlorella salina Chlorophyceae
CHLOR16
Chlorella sp. Chlorophyceae
CHLOR17
Chlorella sp. Chlorophyceae
CHLOR18
Chlorella sp. Chlorophyceae
CHLOR20
Chlorella sp. Chlorophyceae
CHLOR24
Chlorella sp. Chlorophyceae
CHLOR27
Chlorella sp. Chlorophyceae
CHOOC3
(Chlorococcales) Chlamydophyceae
CHOOC4
(Chlorococcales) Chlamydophyceae
CHOOC5
(Chlorococcales) Chlamydophyceae
CHOOC7
(Chlorococcales) Chlamydophyceae
CHOOC8
(Chlorococcales) Chlamydophyceae
CHOOC10
(Chlorococcales) Chlamydophyceae
CHOOC13
(Chlorococcales) Chlamydophyceae
CHOOC14
(Chlorococcales) Chlamydophyceae
CHOOC16
(Chlorococcales) Chlamydophyceae
CHROO1
Chroomonas sp. Bacillariophyceae
CHRYS1
Chrysosphaera sp. Bacillariophyceae
CHRYS2
Sarcinoid chrysophyte
Bacillariophyceae
CRICO1
Cricosphaera sp. Bacillariophyceae
CRYPT1
Cryptomonas sp. Bacillariophyceae
CYCLO1
Cyclotella cryptica Bacillariophyceae
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Strain
Species Class
CYCLO6
Cyclotella cryptica Bacillariophyceae
CYCLO8
Cyclotella meneghiniana Bacillariophyceae
CYCLO9
Cyclotella meneghiniana Bacillariophyceae
CYCLO10
Cyclotella sp. Bacillariophyceae
CYCLO11
Cyclotella cryptica Bacillariophyceae
DIATO1
Navicula sp. nov
Bacillariophyceae
DUNAL1
Dunaliella sp. Bacillariophyceae
DUNAL2
Dunaliella sp. Bacillariophyceae
ELLIP1
Ellipsoidon sp. Eustigmatophyceae
ENTOM1
Amphiprora hyalina Bacillariophyceae
EUSTI1
(Eustigmatophyte) Eustigmatophyceae
EUSTI2
(Eustigmatophyte) Eustigmatophyceae
EUSTI3
(Eustigmatophyte) Eustigmatophyceae
EUSTI5
(Eustigmatophyte) Eustigmatophyceae
EUSTI6
(Eustigmatophyte) Eustigmatophyceae
EUSTI7
(Eustigmatophyte) Eustigmatophyceae
EUSTI8
(Eustigmatophyte) Eustigmatophyceae
EUSTI9
(Eustigmatophyte) Eustigmatophyceae
EUSTI10
(Eustigmatophyte) Eustigmatophyceae
EUSTI11
(Eustigmatophyte) Eustigmatophyceae
EUSTI12
(Eustigmatophyte) Eustigmatophyceae
EUSTI13
(Eustigmatophyte) Eustigmatophyceae
EUSTI14
(Eustigmatophyte) Eustigmatophyceae
EUSTI15
(Eustigmatophyte) Eustigmatophyceae
EUSTI16
(Eustigmatophyte) Eustigmatophyceae
EUSTI17
(Eustigmatophyte) Eustigmatophyceae
EUSTI18
(Eustigmatophyte) Eustigmatophyceae
EUSTI19
(Eustigmatophyte) Eustigmatophyceae
EUSTI20
(Eustigmatophyte) Eustigmatophyceae
EUSTI21
(Eustigmatophyte) Eustigmatophyceae
EUSTI22
(Eustigmatophyte) Eustigmatophyceae
EUSTI23
(Eustigmatophyte) Eustigmatophyceae
FLAGE1
Flagellate ---
FLAGE2
Unknown flagellate
---
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Strain
Species Class
FLAGE3
Pleurochrysis sp. ---
FLAGE6
Green Flagellate
---
FRANC1
Franceia sp. Chlorophyceae
GLECA2
Gleocapsa sp. Cyanophyceae
GLOEO1
Gloeothamnion sp. ---
GREEN1
Green unicell
---
GREEN2
Green unicell
---
GREEN4
Green coccoid
---
GREEN6
Green coccoid
---
GREEN7
Green coccoid
---
GREEN8
Unknown green coccoid
---
GREEN9
Unknown green coccoid
---
GREEN10
Unknown green coccoid
---
GREEN11
Unknown green coccoid
---
HYMEN2
Hymenomonas sp. Prymnesiophyceae
ISOCH1
Isochrysis aff. galbana Prymnesiophyceae
MONOR1
Monoraphidium sp. Chlorophyceae
MONOR2
Monoraphidium sp. Chlorophyceae
MONOR3
Monoraphidium sp. Chlorophyceae
MONOR4
Monoraphidium minutum Chlorophyceae
NANNO2
Nannochloris sp. Chlorophyceae
NANNO3
Nannochloris sp. Chlorophyceae
NANNO5
Nannochloris sp. Chlorophyceae
NANNO7
Nannochloris sp. Chlorophyceae
NANNO8
Nannochloris sp. Chlorophyceae
NANNO9
Nannochloris sp. Chlorophyceae
NANNO10
Nannochloris sp. Chlorophyceae
NANNO12
Nannochloris sp. Chlorophyceae
NANNO13
Nannochloris sp. Chlorophyceae
NANNP1
Nannochloropsis salina Eustigmatophyceae
NANNP2
Nannochloropsis sp. Eustigmatophyceae
NAVIC1
Navicula saprophila Bacillariophyceae
NAVIC2
Navicula saprophila Bacillariophyceae
NAVIC3
Navicula saprophila Bacillariophyceae
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Strain
Species Class
NAVIC5
Navicula saprophila Bacillariophyceae
NAVIC7
Navicula saprophila Bacillariophyceae
NAVIC9
Navicula pseudotenelloides Bacillariophyceae
NAVIC10
Navicula biskanterae Bacillariophyceae
NAVIC12
Navicula acceptata Bacillariophyceae
NAVIC13
Navicula saprophila Bacillariophyceae
NAVIC14
Navicula pseudotenelloides Bacillariophyceae
NAVIC15
Navicula pseudotenelloides Bacillariophyceae
NAVIC16
Navicula pseudotenelloides Bacillariophyceae
NAVIC17
Navicula pseudotenelloides Bacillariophyceae
NAVIC20
Navicula pseudotenelloides Bacillariophyceae
NAVIC21
Navicula pseudotenelloides Bacillariophyceae
NAVIC22
Navicula saprophila Bacillariophyceae
NAVIC23
Navicula saprophila Bacillariophyceae
NAVIC24
Navicula saprophila Bacillariophyceae
NAVIC26
Navicula saprophila Bacillariophyceae
NAVIC28
Navicula saprophila Bacillariophyceae
NAVIC31
Navicula acceptata Bacillariophyceae
NAVIC32
Navicula acceptata Bacillariophyceae
NAVIC33
Navicula pseudotenelloides Bacillariophyceae
NAVIC35
Navicula acceptata Bacillariophyceae
NEPHC1
Nephrochloris sp. ---
NEPHR1
Nephroselmis sp. ---
NITZS1
Nitzschia pusilla monoensis Bacillariophyceae
NITZS3
Nitzschia pusilla elliptica Bacillariophyceae
NITZS4
Nitzschia alexandrina Bacillariophyceae
NITZS5
Nitzschia quadrangula Bacillariophyceae
NITZS6
Nitzschia pusilla monoensis Bacillariophyceae
NITZS7
Nitzschia quadrangula Bacillariophyceae
NITZS9
Nitzschia inconspicua Bacillariophyceae
NITZS10
Nitzschia microcephala Bacillariophyceae
NITZS12
Nitzschia pusilla Bacillariophyceae
NITZS13
Nitzschia dissipata Bacillariophyceae
NITZS14
Nitzschia communis Bacillariophyceae
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Strain
Species Class
NITZS17
Nitzschia hantzschiana Bacillariophyceae
NITZS19
Nitzschia inconspicua Bacillariophyceae
NITZS20
Nitzschia inconspicua Bacillariophyceae
NITZS23
Nitzschia intermedia Bacillariophyceae
NITZS24
Nitzschia hantzschiana Bacillariophyceae
NITZS30
Nitzschia hantzschiana Bacillariophyceae
NITZS35
Nitzschia inconspicua Bacillariophyceae
NITZS37
Nitzschia pusilla Bacillariophyceae
NITZS38
Nitzschia pusilla Bacillariophyceae
NITZS39
Nitzschia pusilla Bacillariophyceae
NITZS40
Nitzschia pusilla Bacillariophyceae
NITZS43
Nitzschia pusilla Bacillariophyceae
NITZS44
Nitzschia frustulum Bacillariophyceae
NITZS45
Nitzschia inconspicua Bacillariophyceae
NITZS49
Nitzschia sp. Bacillariophyceae
OCHRO2
Ochromonas sp. Chrysophyceae
OOCYS1
Oocystis pusilla Chlorophyceae
OOCYS3
Oocystis parva Chlorophyceae
OOCYS5
Oocystis sp. Chlorophyceae
OOCYS9
Oocystis sp. Chlorophyceae
OOCYS11
Oocystis sp. Chlorophyceae
OOCYS14
Oocystis sp. Chlorophyceae
OSCIL1
Oscillatoria limnetica Cyanophyceae
OSCIL3
Oscillatoria subbrevis Cyanophyceae
OSCIL8
Oscillatoria sp. Cyanophyceae
OSCIL9
Oscillatoria sp. Cyanophyceae
OSCIL10
Oscillatoria sp. Cyanophyceae
OSCIL11
Oscillatoria sp. Cyanophyceae
OSCIL12
Oscillatoria sp. Cyanophyceae
OSCIL13
Oscillatoria sp. Cyanophyceae
OSCIL14
Oscillatoria sp. Cyanophyceae
OSCIL15
Oscillatoria sp. Cyanophyceae
PAVLO2
Pavlova sp. Chrysophyceae
PHAEO1
Phaeodactylum tricornutum Bacillariophyceae
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Strain
Species Class
PHAEO2
Phaeodactylum tricornutum Bacillariophyceae
PLATY1
Platymonas sp. Chlorophyceae
PLEUR1
Pleurochrysis dentata Prymnesiophyceae
PLEUR4
Pleurochrysis dentata Prymnesiophyceae
PLEUR5
Pleurochrysis sp. Prymnesiophyceae
PLEUR6
Pleurochrysis sp. Prymnesiophyceae
PRYMN2
(Prymnesiophyte) Prymnesiophyceae
PSEUD1
Pseudoanabaena sp. Cyanophyceae
PSEUD4
--- ---
PYRAM2
Pyramimonas sp. Prasinophyceae
STICH1
Stichococcus sp. Chlorophyceae
STICH2
Stichococcus sp. Chlorophyceae
SYNEC1
Synechococcus sp. Cyanophyceae
SYNEC3
Synechococcus sp. Cyanophyceae
SYNEC5
Synechococcus sp. Cyanophyceae
TETRA1
Tetraselmis suecica Prasinophyceae
TETRA2
Tetraselmis sp. Prasinophyceae
TETRA3
Tetraselmis sp. Prasinophyceae
TETRA4
Tetraselmis sp. Prasinophyceae
TETRA5
Tetraselmis sp. Prasinophyceae
TETRA6
Tetraselmis sp. Prasinophyceae
TETRA7
Tetraselmis sp. Prasinophyceae
TETRA8
Tetraselmis sp. Prasinophyceae
TETRA9
Tetraselmis sp. Prasinophyceae
TETRA11
Tetraselmis sp. Prasinophyceae
THALA6
Thalassiosira weissflogii Bacillariophyceae
THALA7
Thalassiosira weissflogii Bacillariophyceae
THALA14
Thalassiosira weissflogii Bacillariophyceae
THALA15
Thalassiosira weissflogii Bacillariophyceae
THALA16
Thalassiosira weissflogii Bacillariophyceae
UNKNO1
--- ---
UNKNO5
Unknown olive-green unicell
UNKNO6
Unknown coccolithophorid
Prymnesiophyceae
UNKNO8
Unknown coccolithophorid
Prymnesiophyceae
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Strain
Species Class
UNKNO10
Nitzschia sp. Bacillariophyceae
UNKNO24
--- ---
UNKNO36
--- ---
UNKNO52
--- Cyanophyceae
UNKNO58
--- ---
VW291
--- ---
Publications:
Microalgae Culture Collection 1984-1985. Solar Energy Research Institute, SERI/SP-231-2486;
59 pp.
Microalgae Culture Collection 1985-1986. Solar Energy Research Institute, SERI/SP-232-2863,
97 pp.
Barclay, W.; Johansen, J.; Chelf, P.; Nagle, N.; Roessler, R.; Lemke, P. (1986) “Microalgae
Culture Collection 1986-1987.” Solar Energy Research Institute, SERI/SP-232-3079, 149 pp.
Johansen, J.; Lemke, P.; Nagle, N.; Chelf, P.; Roessler, R.; Galloway, R.; Toon, S. (1987)
“Addendum to Microalgae Culture Collection 1986-1987.” Solar Energy Research Institute,
SERI/SP-232-3079a, 23 pp.
II.A.4.
Collection and Screening of Microalgae—Conclusions and
Recommendations
The collection, screening, and characterization of microalgal strains represent a major endeavor
of ASP researchers during the 1980s. More than 3,000 algal strains were collected from sites
within the continental U.S. (Figure II.A.5.) and Hawaii or obtained from other culture
collections. This was the first major collection of microalgae that emphasized strains suitable for
cultivation in saline waters at high (or variable) temperatures, and with the potential for oil
production. The establishment of the SERI Culture Collection as a genetic resource was a major
accomplishment of the ASP; unfortunately a large proportion of the collection was lost due to
funding cutbacks. However, the approximately 300 strains remaining in the collection will be
transferred to the University of Hawaii, and should be available to interested researchers.
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Figure II.A.5. Microalgae Collection Sites Within the Continental U.S.
The collection and screening effort resulted in a large number of strains that had many
characteristics deemed important for a biodiesel production organism. In reviewing the many
procedures used by ASP researchers, however, it is clear that a more consistent screening
protocol might have yielded results that could be compared more meaningfully. Although these
types of standard protocols were being developed near the end of the collection and screening
effort, they were not consistently used. Furthermore, because an optimized microalgal-based
biofuel production process was never fully developed, the screening protocols could not be based
on an actual process. Therefore, whether the screening criteria used in the ASP were accurate
predictors of good performance in a biodiesel production facility is not really known. For
example, lipid productivity over a given amount of time is one of the most important factors in a
production process. There were no clear guidelines as to whether lipid productivity in an outdoor
pond was better in a continuous, steady-state process or in a multistage process involving
substantial culture manipulation (e.g., nutrient level control, “ripening” tanks). This information
is critical, and has a profound impact on the type of screening that should be conducted.
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In addition to the need for a better understanding of the most economically feasible commercial
biodiesel production process, additional information about the true constraints with regard to
lipid properties (e.g., fatty acid chain length, degree of unsaturation, polar lipid constituents, etc.)
there is a need for better information on the impact of lipid composition on fuel quality. The
lipophilic dye Nile Red was used as a screening tool to rapidly assess the lipid contents of
isolates, but in retrospect this technique probably does not provide the level of detail regarding
lipid quality that may be necessary. Indeed, the variability in the ability of various strains to take
up this dye is a major problem that must be recognized. With the rapid advances that have been
made in recent years in automated high performance liquid chromatography and detection, this
technique seems readily adaptable for use as a powerful screening tool.
For future screening endeavors, we recommend that an effort be made to naturally select strains
at the locations that would likely be commercial microalgal production sites. In this manner, the
algae would be exposed to the prevailing environmental conditions, particularly the indigenous
waters. In small open-air vessels, the medium would be “inoculated” with a variety of
indigenous strains, and a process of natural selection would occur such that the most competitive
strains would dominate the cultures after a short while. Of course, the disadvantage of this
method is that the dominant strains may not be good lipid producers. For this reason, genetically
manipulating the dominant strains by classical or recombinant means may be necessary, such that
they remain competitive and yet make acceptable amounts of lipid. Whether such manipulations
can be made awaits further experimentation.
One thing that was clear from the collection and screening effort was that diatoms and green
algae would most likely be well represented in a “natural selection” screen as described in the
preceding paragraph. Therefore, future efforts should probably focus on developing
sophisticated genetic engineering tools focused on these groups. Such tools could be transferable
to many different species within these groups; such transfer would be facilitated by the fact that
powerful methods for generating genetic sequence information are becoming routine.
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II.B.
Microalgal Strain Improvement
II.B.1.
Physiology, Biochemistry, and Molecular Biology of Lipid
Production: Work by SERI Subcontractors
II.B.1.a. Introduction
Eukaryotic algae, like all photosynthetic organisms, efficiently convert solar energy into biomass.
The algal research program at SERI was designed as a long-term basic research effort to adapt or
use photosynthesis and related metabolic pathways to produce renewable fuels and chemicals.
Research at SERI under the Aquatic Species Program (Biodiesel) has focused on ways to
increase the yield of oil from microalgae for cost-effective liquid fuel production. Initially, a
large component of the research performed both by subcontractors and by SERI researchers was
the collection of microalgal strains from saline environments in the desert southwest of the
United States (a region targeted as a feasible location for large-scale microalgal culture), marine
environments, and established culture collections. These organisms were then screened and
numerous species were identified as candidates for biodiesel production; this research was
described in Sections II.A. and II.B. of this report. However, no one species was identified that
displayed the ideal combination of rapid growth, environmental tolerance, and high lipid
production. Subsequent research efforts were directed toward understanding the biochemistry
and physiology of lipid production in oleaginous microalgal strains, with the idea of using strain
improvement technologies (breeding, cell fusion, genetic engineering, mutagenesis and selection)
to develop algal strains with optimized traits for biodiesel production.
Early in the research program it became obvious the maximal lipid accumulation in the algae
usually occurred in cells that were undergoing physiological stresses, such as nutrient deprivation
or other conditions that inhibited cell division. Unfortunately, these conditions are the opposite of
those that promote maximum biomass production. Thus, the conditions required for inexpensive
biodiesel production, high productivity and high lipid content, appeared to be mutually exclusive.
To overcome this problem, research efforts were focused on understanding the biochemistry and
physiology of lipid accumulation, with emphasis on understanding the “lipid trigger”, a
mechanism that could induce production of large quantities of lipid under nutrient deprivation.
In addition, research was directed toward understanding genetic variation within microalgal
populations and to develop methods to screen for high-lipid subpopulations within algal cultures.
The knowledge of the biochemistry and physiology of lipid synthesis, combined with basic
studies on microalgal molecular biology, was used in the later years of the project in attempts to
use genetic engineering to develop microalgal strains with optimal properties of growth and lipid
production.
Part II.B.2. of this report describes work by ASP subcontractors to understand the biochemistry
and physiology of lipid accumulation in microalgae, including ultrastructural studies, the
development of methods for screening for high lipid strains, and attempts to understand the
biochemical lipid trigger. The research performed by SERI/NREL subcontractors on the
physiology, biochemistry, and molecular biology of lipid production in oleaginous microalgae
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took place during the second half of the 1980s and is presented here, roughly chronologically,
according to the work performed by the individual subcontractors.
II.B.1.b.
Chrysophycean Lipids: Effects of Induction Strategy in the Quantity and
Types of Lipids
Subcontractor:
Selma
University
Principal Investigator:
Shobha Sriharan
Period of Performance: 9/85 - 5/88
Subcontract Number:
XK-5-05104-1
The purpose of the research performed by Dr. Sriharan and coworkers was to study the effects of
nutrient deprivation and temperature on growth and lipid production in microalgae with potential
for liquid fuel production. All the reports generated by this laboratory during the subcontract
describe a virtually identical set of experiments performed on several species of microalgae. The
benefit of this approach is that the productivity data can be compared between the species.
However, the flaws in experimental design and reporting were carried through all experiments
and reports.
The basic design for these experiments was to grow the algal cells in batch cultures in media that
contained either “sufficient” or “deficient” levels of N or Si, and to test for algal growth rate,
productivity, and lipid production. Cultures were grown at 20°C and at 30°C to test for the effect
of temperature on growth and lipid induction. Exponentially growing cells were inoculated into
fresh media containing high or low levels of Si or N. Growth was monitored by measuring the
optical density of the culture, and the growth rate was reported as the number of cell doublings
per day. The cells were harvested and processed to determine lipid content (reported as a
percentage of the AFDW), and fatty acid composition under the various growth conditions.
The organisms studied were all diatoms, except for the chlorophyte M. minutum (which the
authors initially reported to be a diatom, but they corrected this error in a later report). The
diatoms tested were Chaetoceros SS-14, C. muelleri var subsalsum, Navicula saprophila,
obtained from SERI, Cyclotella DI-35, Cyclotella cryptica Reimann, Lewin, and Guillard, and
Hantzchia DI-60, obtained from M. Tadros at Alabama A&M University. All the organisms
tested grew more rapidly at 30°C (versus 20°C) and in nutrient-sufficient media. A decrease in
the total AFDW was reported for all strains grown in nutrient-deficient media compared to
nutrient-sufficient cultures, and this was accompanied by an increase in the percentage of the
AFDW made up of lipids.
The most dramatic increases in the lipid content of the cultures were seen under N-deficient
conditions in cells grown at 30°C. In C. cryptica, the total lipids, as a percentage of AFDW,
increased from 15% to 44%. In Hantzschia, lipids increased from 29% to 53%, and in Navicula
saprophila, lipids increased from 26% to 44%. In all cases, the increase in total lipids was due to
increases in both the neutral lipid and polar lipid fractions. In several cases, the ratio of neutral
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lipids to polar lipids increased significantly in the nutrient-stressed cells (i.e., in Hantzschia and
C. muelleri grown in Si-deficient media, and in Navicula grown in N-deficient media). Dr.
Sriharan also presented data comparing the fatty acid profiles of lipids from diatoms grown under
nutrient-sufficient and nutrient-deficient conditions. Although the data was incomplete, it
indicated that changes in the fatty acid composition (lipid quality) did occur in nutrient-stressed
cells, suggesting that nutrient deprivation can affect the lipid biosynthetic pathways.
In all cases nutrient-deficiency resulted in a decreased rate of cell growth and a decrease in total
cell productivity. Therefore, an increase in lipid as a percentage of cell mass may not be
economically advantageous for liquid fuel production from mass-cultured algae if the conditions
that induce lipid accumulation also result in a significant drop in total biomass, and thus in total
lipid produced. Although this was not discussed by Dr. Sriharan, the total effect of nutrient
limitation on lipid content of the algal cultures could be estimated by multiplying the total
biomass (AFDW) produced by the percentage of the AFDW attributable to lipid under nutrient-
sufficient or nutrient-deficient conditions. In general, these calculations demonstrated an
increase in lipid content of the cultures induced by nutrient stress, in the range of a 20% to 30%
increase in total lipid.
The results reported here clearly suggest that algal productivity is increased under nutrient-
sufficient conditions and at elevated temperatures. However, it is difficult to determine the
validity of the data presented regarding nutrient-deprivation as a lipid trigger. Growth curves
were not presented for the organisms studied. Therefore, it cannot be determined if the low
nutrient levels limited growth throughout the period of the experiment, or whether the nutrients
became depleted and the lipid effects correlated with a decrease in cell division, as reported
elsewhere. It is also not clear at what point in the cell cycle the cells were harvested for
determination of AFDW and lipid content, and how to compare the data for nutrient-sufficient
and nutrient-deficient cells. In several experiments, the authors reported that the cultures were
only harvested when lipid droplets were seen in the cells, although this would seem to bias
experiments designed to test nutrient effects on lipid production. All in all, it is difficult to
determine whether the experiments were badly performed, or just poorly reported.
In summary, the data presented by Dr. Sriharan was difficult to interpret for these reasons;
however, several general conclusions can be made. Diatoms seem to be promising candidates for
neutral lipid production. Many species produce constitutively high levels of lipid, and the level
of lipid as a percentage of biomass can be increased by growing the cells under nutrient-limited
conditions (the data presented here suggests that N-limitation may be more effective than Si-
limitation). In addition, Dr. Sriharan’s results suggest that nutrient-limitation may alter the lipid
biosynthetic pathways in diatoms to increase lipid production and possibly affect lipid
composition.
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Publications:
Sriharan, S.; Bagga, D. (1987a) “Effects of induction strategies on Chaetoceros (SS-14), growth
with emphasis on lipids.” FY 1986 Aquatic Species Program Annual Report, Solar Energy
Research Institute, Golden, Colorado, SERI/SP-231-3071, pp. 273-284.
Sriharan, S.; Bagga, D. (1987b) “Influence of nitrogen and temperature on lipid production in
microalgae (Hantzschia DI-60).” Energy from Biomass and Wastes X, Conference Proceedings,
(Klas, D.L., ed.), Institute of Gas Technology, Chicago, pp. 1689-1190.
Sriharan, S.; Bagga, D.; Sriharan, T.; Das, M. (1987) “Effect of nutrients and temperature on
lipid production and fatty acid composition in Monoraphidium minutum and Cyclotella DI-35.”
FY 1987 Aquatic Species Program Annual Report, Solar Energy Research Institute, Golden,
Colorado, SERI/SP-231-3206, pp. 108-126.
Sriharan, S.; Bagga, D.; Sriharan, T.P. (1988) “Algal lipids: Effect of induction steategies on the
quantity and types of lipids produced.” Final Technical Report, Solar Energy Research Institute,
Golden, Colorado, 16 pp.
Sriharan, S.; Sriharan, T.P.; Bagga, D. (1989) “Lipids and fatty acids of diatom Chaetoceros
muelleri var subsalsum and the control of their production by environmental factors.” Energy
from Biomass and Wastes XII, Conference Proceedings, (Klas, D.L., ed.), Institute of Gas
Technology, Chicago, 10 pp.
Sriharan, S.; Bagga, D.; Nawaz, M. (1991) “The effects of nutrients and temperature on biomass,
growth, lipid production, and fatty acid composition of Cyclotella cryptica Reimann, Lewin, and
Guillard.” Appl. Biochem. Biotech. 28/29:317-326.
Sriharan, S.; Bagga, D.; Sriharan, T.P. (1989) “Environmental control of lipids and fatty acid
production in the diatom Navicula saprophila.” Appl. Biochem. Biotech. 20/21:281-291.
Sriharan, S.; Bagga, D.; Sriharan, T.P. (1990) “Effects of nutrients and temperature on lipid and
fatty acid production in the diatom Hantzshia DI-60.” Appl. Biochem. Biotech. 24/25:309-316.
II.B.1.c.
Genetic Variation in High Energy Yielding Microalgae
Subcontractor:
City College of the City University of New York
Principal Investigator:
Jane C. Gallagher
Period of Performance:
3/86 - 12/87
Subcontract Numbers:
ZK-4-04136-5; ZK 4-04-136-04
The purpose of these studies was to investigate the intragenetic variability (e.g., between various
isolates of a single species) in microalgae with potential for high lipid production. The rationale
for this work with respect to the ASP is that variability within and between species of microalgae
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has implications for algal collection strategies, for strain selection for high lipid producers, and
for genetic manipulation of microalgae by classical breeding or genetic engineering.
Historically, microalgae have been classified based on morphological similarities. Previous
studies by Dr. Gallagher and others (see Gallagher 1986) suggested significant physiological
variability between isolates of a single species. In these studies, various isolates of a species
grown under identical conditions (to control for environmentally induced changes in gene
expression) often showed significant differences in characteristics such as nutrient uptake,
growth rates, and pigment content. These results indicated that there may be inherent genetic
differences between the individual strains. The studies by Dr. Gallagher compared
electrophoretic banding patterns of specific proteins to obtain quantitative estimates of the
genetic differences between isolates of two genera of oil-producing microalgae.
The organisms studied were A. coffeiformis (class Bacilliarophyceae) and Nannochloropsis spp.
(class Eustigmatophyceae). The basic approach was to streak the isolated algae onto agar plates,
then to pick single colonies and restreak the cells to ensure that each strain was unialgal. The
isolates were propagated under identical growth conditions to minimize differences caused by
environmentally induced changes in gene expression. Each strain was examined using light
microscopy (LM) and scanning electron microscopy (SEM) to look for morphological
differences and to confirm species identity. Crude protein extracts from each strain were
separated by polyacrylamide gel electrophoresis. The gel was then stained to detect several
specific enzymes, including phosphoglucose isomerase, hypoxanthine dehydrogenase, α-
ketoglutarate dehydrogenase, malate dehydrogenase, α-hydroxybutarate dehydrogenase, and
tetrazolium oxidase. (Dr. Gallagher also tried unsuccessfully to stain for several other enzymes.
Poor staining may be a consequence of the location of these enzymes within cellular membranes
in microalgae.) An extract from the diatom Skeletonema costatum (clone SKEL) was run on
each gel to serve as an internal standard, and the migration pattern for each enzyme was reported
as the ratio of the migration distance for the unknown Amphora or Nannochloropsis enzyme to
the migration of the known enzyme from Skeletonema. An example of this type of experiment is
shown in Figure III.B.1. This method allowed for detection of very small differences in the
migration patterns of the various forms of the enzymes. These differences could result from
subtle variations in protein charge or conformation due to one or several amino acid changes.
Isolates that showed two bands for a specific enzyme were assumed to be heterozygous at that
allele.
For the studies of Amphora, Dr. Gallagher obtained 47 isolates, 32 of which were isolated from a
salt marsh in Woods Hole, Massachusetts, on the same day in August 1985. Another six strains
had been isolated from the same site during the summers of 1979 or 1980 and maintained in
culture, and five strains were obtained from laboratory cultures maintained by other investigators.
It is unclear from Dr. Gallagher's reports how many of the 47 Amphora isolates were tested as
described earlier.
All strains that were subsequently analyzed for enzyme banding patterns were first examined by
LM and SEM. Microscopy confirmed that all the strains were A. coffeiformis, although some
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variation was observed in the morphology of the frustule between strains, for example, in the
presence or absence of costae (rib-like protrusions) or in the shape or number of punctae (holes).
These changes were assumed to be due to genetic differences between the strains, as unialgal
clones maintained in culture for 6-7 years did not show variations in frustule morphology
between individuals. Genetic similarity was calculated based on the electrophoretic banding
patterns using the statistical methods of Nei (1972). The zymograms indicated significant
variation between isolates of the same species, even between strains isolated from the same site
on the same day. These differences were not correlated with the extent of morphological
variation, and some morphologically identical strains showed differences in the protein banding
patterns.
For Nannochloropsis, 115 strains were obtained, all from culture collections. The electrophoretic
banding patterns also indicated significant genetic diversity between strains, even between
samples isolated from the same location. However, the zymogram data for Nannochloropsis was
limited due to the high percentage of “null” alleles (no staining of some enzymes) in some
isolates. It was unclear whether this was caused by undetermined genetic differences between the
isolates (and between Amphora and Nannochloropsis), or due to difficulties in extraction of the
proteins from Nannochloropsis. More data would be needed to fully analyze the genetic
differences between isolates of this genus.
What are the implications of this research for the Aquatic Species Program? The significant
amount of genetic diversity between individuals of a species, even when isolated from very
similar locations, suggests that researchers involved in collecting microalgal strains as potential
lipid producers should obtain more than one isolate from each site. In fact, these results suggest
that it may be adequate to sample fewer sites to obtain a sufficiently varied collection of
microalgal strains.
In a previous study (discussed in Gallagher 1985), Dr. Gallagher described experiments
performed on isolates of the diatom S. costatum similar to those described here. The data
suggested significantly less genetic variation between isolates of Skeletonema, even between
strains isolated from different locations, than was seen between Amphora strains isolated from
the same environment. This difference was attributed to the fact that Amphora is an attached,
benthic organism that produces amoeboid gametes, whereas Skeletonema is planktonic, and
produces swimming sperm. These “lifestyle” differences would result is lower potential for gene
flow between Amphora populations, although the presence of heterozygotes indicates
interbreeding among Amphora at localized sites. These observations suggest that benthic
organisms may have greater genetic diversity than planktonic forms.
Based on the data in this study, Dr. Gallagher also concluded that breeding or genetic
engineering of microalgae may be more successful using morphologically similar phenotypes, as
her results suggest less diversity at the protein level between identical morphotypes. However,
genetic engineering research during the past 15 years in other organisms indicates that cells can
often express genes from very different species, so these differences between strains probably
will not affect the expression of genes transferred between these similar organisms.
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While working under the SERI subcontract, Dr. Gallagher also participated in a study that
provided evidence that the carotenoid violaxanthin functions as a major light harvesting pigment
in Nannochloropsis (Owens et al. 1987). Carotenoids generally are considered accessory
pigments in photosynthetic organisms, involved primarily in photoprotection, fluorescence
quenching, and light harvesting. Nannochloropsis is a member of the class Eustigmatophyceae,
which are unusual in that they can contain violaxanthin as up to 60% of their total pigments.
These authors used room temperature fluorescence excitation and emission data to provide the
first evidence that violaxanthin can function in photosynthetic light harvesting.
Understanding the fundamental processes involved in microalgal photosynthesis is important to
the ASP since light-driven photosynthesis results in the production of chemical reductants that
drive the synthetic dark reactions; lipids are storage products that can be produced from excess
photosynthate. One possible implication is that carotenoids absorb at different wavelengths than
chlorophyll, absorbing green light that penetrates into the water column. This feature could be
beneficial for mass culture of organisms, allowing denser cultures to grow in a deep raceway.
Publications:
Gallagher, J.C. (1986). “Population genetics of microalgae.” In Algal Biomass Technologies: An
Interdisciplinary Perspective (Barclay, W.; McIntosh, R., eds.), Beihefte zur Nova Hedwigia,
Heft 83, Gebrüder Borntraeger, Berlin-Stugart, pp. 6-14.
Gallagher, J. C. (1987a). “Patterns of genetic diversity in three genera of oil-producing
microalgae” (abstr.), FY 1987 Aquatic Species Program Annual Report, (Johnson, D.A.; Sprague,
S., eds.), Solar Energy Research Institute, Golden, Colorado, SERI/SP-231-3206, p. 209.
Gallagher, J.C. (1987b). “Genetic variation in oil-producing microalgae.” FY 1986 Aquatic
Species Program Annual Report, (Johnson, D.A., ed.), Solar Energy Research Institute, Golden,
Colorado, SERI/SP-231-3071, p.331-336.
Owens, T.G.; Gallagher, J.C.; Alberte, R.S. (1987) “Photosynthetic light-harvesting function of
violaxanthin in Nannochloropsis spp. (Eustigmatophyceae).” J. Phycol. 23:79-85.
Additional References:
Nei, M. (1972) Amer. Natur. 106:283.
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Figure II.B.1. Drawing of representative gels showing the banding patterns of various
enzymes from clones of Amphora coffeiformis.
All bands are graphed as a ratio of the distance travelled by protein bands in Amphora to the
distance travelled by stand bands in Skeletonema costatum. PGI: phosphoglucose isomerase;
XDH: hypoxanthine dehydrogenase; TO: tetrazolium oxidase; ADH: analine dehydrogenase.
(Source: Gallagher 1987).
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II.B.1.d. Ultrastructure
Evaluation of Lipid Producing Microalgae
Subcontractor:
Oak Ridge National Laboratory, Oak Ridge, Tennessee
Principal Investigator:
Jean A. Solomon
Period of Performance:
10/84 - 11/86
Subcontract Number:
N/A
The goal of this project was to gain further understanding of the physiology of lipid accumulation
in microalgae by examination of the ultrastructure of cells containing high levels of storage
lipids. The questions that Dr. Solomon addressed were:
1. Where does the lipid accumulate within the cells; and
2. What other ultrastructual changes are seen in microalgae induced to accumulate
lipid?
Three oleaginous microalgal strains were used in this study, Ankistrodesmus fulcatus (SERI
strain ANKIS1; class Chlorophyceae), Isochrysis aff. glabana (ISOCH1, class
Prymnesiophyceae), and Nannochloropsis salina (NANNO1, class Eustigmatophyceae).
Ultrastructural changes were monitored by transmission electron microscopy (TEM). In this
technique, cells are chemically fixed and embedded in a plastic resin. The resin is then cut into
thin sections (70-100nm), stained with heavy metals, and viewed in an electron microscope. The
first step was to develop adequate fixation and embedding techniques for the algal species to be
studied. This is often problematic for microalgae, presumably due to the chemical and physical
properties of the algal cell wall, which can act as barriers to penetration of the fixatives or resin.
Dr. Solomon tested five fixation protocols (see Solomon 1985, p.74, Table 1), all variations of
standard methods of fixation using glutaraldehyde and osmium tetroxide, dehydration with an
organic solvent, embedding of the cells in an acrylic resin, and poststaining of the sections with
uranyl acetate and lead citrate. Initially, Dr. Solomon reported that the best fixation of
Ankistrodesmis and Isochrysis was achieved by exposing the cells briefly to glutaraldehyde and
osmium simultaneously, followed by dehydration in acetone and embedding in Spurrs resin.
However, a later report stated that Ankistrodesmis was better preserved by exposing the cells
sequentially to glutaraldehyde and then osmium (in cacodylate buffer supplemented with sucrose
as an osmoticum). Also, Araldite/Embed12 resin was used, as it appeared to provide better
penetration into the cells. For Isochrysis, the initial protocol was also modified by adding
sucrose. Fixation of Nannochloropsis was poor with any method used; the scaley cell wall of
this organism seemed to provide a significant barrier to adequate penetration of fixatives and
resins.
Nitrogen deprivation was used to trigger the production of lipids in the cells. The cells were
grown in N-replete medium, then collected by centrifugation and resuspended in growth medium
without added N. Samples were fixed immediately and at regular intervals during the following
13 days, and thin sections were cut and examined for ultrastructual changes by TEM.
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As expected, N deprivation resulted in the accumulation of lipid within the cells of all three
microalgal species. The lipid appeared primarily as droplets within the cytoplasm, not within the
chloroplast or other cellular organelles. The lipid droplets often appeared adjacent to a
mitochondrion. In Ankistrodesmus, N-deficiency also produced an increased number of starch
granules within the chloroplasts, and resulted in the formation of unusual membrane structures
consisting of packed, concentric layers of double membranes within the cytoplasm. Whether
these unusual structures were the site of excess lipid accumulation, or were structural artifacts of
the fixation process, was unclear.
It is difficult to conclude much more about ultrastructural changes that might have been induced
in these cells following N deprivation. The sample size was very small. One hundred-nm thick
sections may represent less than 1/100th of the volume of a microalgal cell. In addition, only a
few cells within a population can be examined easily by this technique. Finally, there is a high
likelihood that the chemical fixation methods used in the study can create artifacts that are not
related to actual cell structure. However, these studies supported the observation that significant
levels of storage lipids can accumulate in the cytoplasm of microalgal cells exposed to N
deficiency. Dr. Solomon's microscopic observations in Ankistrodesmus also suggested the
presence of a discrete lipid trigger mechanism within each cell, as lipid did not appear to
accumulate gradually within all cells of a population after N deprivation. Instead, individual
cells appeared to accumulate large amounts of lipid during a 1-2 day period. This result was
supported by the flow cytometric data also performed in Dr. Solomon's laboratory, which is
described below.
Publications:
Solomon, J.A.; Hand, R.E.; Mann, R.C. (1986b) “Ultrastructural and Flow Cytometric Analyses
of Lipid Accumulation in Microalgae: A Subcontract Report.” Solar Energy Research Institute,
Golden, Colorado, SERI/STR-231-3089.
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Figure II.B.2. Electron micrographs of nitrogen-sufficient (top) and nitrogen-deficient
(bottom) cells of Nannochlorposis salina.
Note the accumulation of large lipid droplets (L) in the cytoplasm in the nitrogen-deficient cells.
The lipid often appeared adjacent to a mitochondrion (M). N: nucleus. C: chloroplast. Scale
bars = 0.5 µm. The numbers in the lower left corner of each figure are from the original
publication (Solomon et al.1986b).
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II.B.1.e.
Improvement of Microalgal Lipid Production by Flow Cytometry
Subcontractor:
Oak Ridge National Laboratory, Oak Ridge, Tennessee
Principal Investigator:
Jean A. Solomon
Period of Performance:
12/86 - 11/87
Subcontract Number:
DK-4-04142-01
The purpose of this project was to determine if flow cytometry could be used to select for
subpopulations of high lipid-producing algae within an algal culture. Flow cytometry is a
method that measures the light scattered or emitted by particles as they pass through a laser
beam. Scattered light is believed to reflect the size, shape, and refractive properties of cells. Dr.
Solomon initially used exponentially growing and nutrient-stressed cells of the chrysophycean
alga Boekelovia to demonstrate that the extent of right-angle scatter, which indicates changes in
internal cell morphology, can be correlated to the lipid content of microalgal cells.
In subsequent studies, a lipid-specific fluorescent dye, Nile Red (see work by Dr. Cooksey,
described in Section II.A.1.f.), was used to stain intracellular lipids. Nile Red is excited at a
wavelength of 488 nm, and emits yellow-green light at 520-580 nm. In contrast, chlorophyll
autofluorescence can be measured at wavelengths greater than 630 nm. Therefore, in contrast to
the scattered light data mentioned above, flow cytometric analyses of cells stained with Nile Red
would be more specific for changes in lipid content. Preliminary experiments in which cells of
Boekelovia were stained with Nile Red demonstrated that increased yellow green fluorescence
could be correlated with increased numbers of lipid droplets in the cells, suggesting that this
method could work to screen for cells with high lipid contents.
Of the three microalgal species analyzed by TEM (Section II.B.1.d.), only Isochrysis was found
to be appropriate for flow cytometric analysis. The cells of this strain are small and spherical, the
optimal shape for flow cytometric analysis, and take up Nile Red well. In contrast,
Ankistrodesmus cells are long and thin (40 nm x 4 nm). Nannochloropsis did not take up the
Nile Red dye, possibly because of cell wall properties that also prevented good chemical fixation
for microsopy.
In the initial experiments, cells were screened for lipid content based on Nile Red fluorescence
alone. Several improvements to this procedure were implemented during the course of the study.
First, efforts were made to optimize the Nile Red staining protocol. The best staining was
achieved using a concentration of 1 mg Nile Red in 1 mL of cell suspension. The solvent for the
Nile Red stain was changed from heptane to acetone, due to interfering fluorescence from
undissolved heptane droplets. Finally, the researchers found that the fluorescence signal from
Nile Red is unstable and decays rapidly. However, the fluorescence level stabilizes after about
45 minutes, so all readings were taken at least 45 minutes after staining the cells with Nile Red.
Another important change was to measure the chlorophyll autofluorescence as well as Nile Red
fluorescence. This ensured that only viable cells containing lipid and intact chloroplasts would
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be analyzed. In addition, the amount of chlorophyll is an indication of cell size. Cell sorting
based on the ratio of chlorophyll fluorescence to Nile Red fluorescence would normalize the
results to account for differences in cell size and age and allow detection of individual cells with
unusually high lipid levels resulting from natural genetic variation. A decrease in the ratio of
chlorophyll to Nile Red fluorescence would indicate lipid accumulation.
In one set of experiments, Isochrysis cultures were stressed by transferring the cells into N-
deficient media, then screened for lipid content using flow cytometry, either by lipid content
alone (Nile Red fluorescence) or by monitoring the chlorophyll to Nile Red fluorescence ratios.
The daughter cells containing high or low levels of lipid were recultured in N-replete medium for
1 week or 1 month, then subjected again to N deprivation, and resorted. The lipid content of the
daughter population was compared to that of the parent cells. These experiments produced
inconsistent results. In some cases, the population of daughter cells selected for their high lipid
content showed a wider range of lipid contents than the parent cells; other sorts produced
daughters without significant differences in lipid content from the parents. One interesting
observation was the bimodal distribution of cells in all populations subjected to N stress. Cells
fell into two classes with low or high chlorophyll-to-lipid ratios. This again supports the author's
theory, discussed in the section on ultrastructural analysis of lipid accumulation, that cells
respond as individuals to a lipid trigger, rather than gradually increasing the lipid content of the
entire culture.
These results suggested that flow cytometry might be used to select for populations of high lipid
algae if more was understood about the relationship of the physiological state of parent cells to
lipid accumulation. Analysis of the growth of Isochrysis in N-replete media showed several
phases, including a period of exponential growth that declined to a stationary phase. After about
2 weeks, the nutrients were depleted and the cells entered a stressed phase. A series of
experiments was performed in which cells in various growth phases were stained with Nile Red
and sorted based on lipid content alone (no chlorophyll measurements). The sorts on cells in
exponential phase were usually not successful, but if the parent cells were in stationary phase or
from very old cultures (stressed), the mean lipid content of the daughter population was about
20% higher than that of the parental cells. These results suggested that successful screening for
high-lipid cells using flow cytometry was related to the cell cycle. The exponential cultures
contained cells in all stages of cell growth and division. Cells that were preparing to divide
would be larger and would be selected as high-lipid, so that the set of high lipid cells selected
would actually contain only the largest and oldest cells, rather than high lipid genetic variants.
Stationary or stressed populations are not actively dividing, so the cells are more uniform in size
and sorting of the cells for high lipid should be more likely to identify true high lipid variants
within the population.
The experiments using Isochrysis cultures in various growth stages as the parental population
were repeated to test these assumptions. Cells were sorted based either on lipid levels alone (Nile
Red fluorescence only) or using the ratio of chlorophyll to Nile Red fluorescence to control for
cell size. The results presented in the available reports support the hypotheses. Sorting cells
based solely on lipid content produced high-lipid daughter populations if the parent population
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was in a stationary or stressed growth phase. Exponential cultures produced variable results. If
the parental cells were sorted based on ratios of chlorophyll-to-lipid fluorescence, high-lipid
populations could be produced from exponentially growing parent cultures.
These conclusions were based on very limited data. Only a few experiments were performed. In
addition, several daughter cultures did not grow up after the sorting process, and failure of a
growth chamber resulted in the loss of some cultures. The data were written up in only two
technical reports to SERI, and it was difficult to determine the exact protocol used for each
sorting experiment. However, the results of these studies are intriquing, and suggest that flow
cytometry might be a viable method for screening for high lipid genetic variants within (or
between) strains of oleaginous microalgae. The procedure would be limited to strains in which
the cells are small and spherical. Dr. Solomon's data suggest the best results would be achieved
by using stationary or stressed cells as the parent population. In addition, cells should be selected
based on a low chlorophyll-to-Nile Red fluorescence ratio, which would indicate high-lipid
levels with respect to cell size. However, it is interesting that high-lipid daughter strains were not
produced in the experiments in which exponentially growing cells were transferred directly to N-
deficient media; yet, cells allowed to gradually deplete their N supply to induce the stressed
condition could be used successfully in a flow cytometric screen. This suggests either that lipid
accumulation occurs by different mechanisms under these two conditions, or, more likely, that
the stressed cells from very old cultures had all entered a similar metabolic state so that size and
lipid contents would be more indicative of genetic differences.
Publications:
Solomon, J.A (1985) “Ultrastructure evaluation of lipid accumulation in microalgae.” Aquatic
Species Program Review: Proceedings of the March 1985 Principal Investigators’ Meeting,
Solar Energy Research Institute, Golden, Colorado, SERI/CP-231-2700, pp. 71-82.
Solomon, J.A. (1987) “Flow cytometry techniques for species improvement.” FY 1986 Aquatic
Species Program: Annual Report (abstr.), Solar Energy Research Institute, Golden, Colorado,
SERI/SP-231-3071, p. 252.
Solomon, J.A.; Hand, R.E.; Mann, R.C. (1986a) “Ultrastructural and flow cytometric analyses of
lipid accumulation in microalgae.” Annual Report, Oak Ridge National Laboratory, Oak Ridge,
Tennessee, ORNL/M-258, 50 pp.
Solomon, J.A.; Hand, R.E.; Mann, R.C. (1986b) “Ultrastructural and Flow Cytometric Analyses
of Lipid Accumulation in Microalgae: A Subcontract Report.” Solar Energy Research Institute,
Golden, Colorado, SERI/STR-231-3089.
Solomon, J.A.; Palumbo, A.V. (1987) “Improvement of microalgal strains for lipid production.”
FY 1987 Aquatic Species Program: Annual report, Solar Energy Research Institute, Golden,
Colorado, SERI/SP-231-3206.
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II.B.1.f.
Biochemical Elucidation of Neutral Lipid Synthesis in Microalgae
Subcontractor:
Montana State University
Principal Investigator:
Keith E. Cooksey
Period of Performance:
1/86 - 10/87
Subcontract Numbers:
XK-5-05073-1; XK-6-05073-1
The goal of this research was to understand the biochemistry of lipid accumulation in microalgae,
in particular, to provide information on the biochemical triggers that induce lipid synthesis. The
Nile Red fluorescence technique developed by Dr. Cooksey's laboratory and at SERI (described
above in Sections II.A.1.f. and III.B.1.e.) was used to study lipid accumulation in microalgae in
response to N or Si depletion. Nile Red fluorescence was used to monitor the lipid levels in
batch cultures of Chlorella over time. As the N became depleted, lipids accumulated in the
cultures, predominately as triglycerides. The triglyceride levels began to increase before N was
totally depleted from the medium. Microscopic examination showed that individual cells within
the population began to accumulate lipid at different times, similar to results obtained by Dr.
Solomon in Isochrysis. Dr. Cooksey concluded that lipid accumulation begins as the cells enter
stationary phase and cell division ceases; the timing of this event would be different for
individual cells within a population.
Dr. Cooksey's laboratory next performed a complex series of experiments designed to correlate
media factors, (i.e., nitrate concentrations, pH, and carbon availability), with lipid accumulation
in CHLOR-1. Cell growth and lipid accumulation were monitored in batch cultures, with cells
grown in unbuffered Bold's medium, or media buffered at pH 7, 9, or 10. In unbuffered Bold's
medium, the initial pH was 7, and increased to pH 8 by day 6, and up to pH 9.5 by day 9. The
cells grew in all conditions, with the best growth at pH 9. Under all growth conditions, the level
of nitrate in the media decreased, but never fell below 25% of the initial levels.
Accumulation of neutral lipids was monitored by Nile Red fluorescence. There was no lipid
accumulation in cultures maintained at or below pH 9. However, in buffered medium with a pH≥
10, or in unbuffered medium that experienced an increase in pH during the growth period, the
cultures generally showed a significant increase in lipid levels that was accompanied by a
decrease in cellular growth rates.
Nutrient limitation, generally nitrate or silica, can trigger lipid accumulation in microalgae.
Nutrient deprivation can cause a decrease in cell division, which presumably results in
“targeting” of excess fixed carbon into storage lipids. The data obtained by Dr. Cooksey's
laboratory suggested that a shift in pH, which has been correlated with decreased rates of cell
division, could also trigger lipid accumulation. These data suggested that nutrient limitation
might not directly affect biochemical pathways to enhance lipid synthesis; rather, lipid
accumulation may be an indirect consequence of inhibition of a stage in the cell cycle. In other
photosynthetic systems studied, the data indicated that cells synthesize triglycerides in the light
and utilize these lipids as energy stores in the dark and during cell division. If division were
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blocked, the rate of neutral lipid utilization would be slower than the rate of synthesis, so
triglycerides would accumulate in the cells. To help test this hypothesis, Dr. Cooksey used light
microscopy to examine cells grown in media with different pH ranges. Cultures grown at pH 7-9
consisted almost entirely of small, single cells. However, at pH 10 and higher, a large proportion
of the cells was in the form of autosporangial complexes, i.e., their nucleii had divided, but the
autospores had not separated. The specific effect of increased pH on cell division is not clear,
although some evidence suggests that increased pH can lead to increased flexibility of the
autospore wall, preventing individual cells from breaking free. Alternatively, increased pH could
affect precipitation of media components, indirectly affecting the cell cycle.
Although the data presented here suggest that nutrient deprivation or increased pH may affect
lipid levels simply as a consequence of decreased cell division, additional research by Dr.
Cooksey's laboratory suggested that treatments that increase lipid accumulation may also affect
the biochemistry of lipid biosynthesis. Analysis of the lipid classes present in the cells at the end
of the 10-day growth period showed accumulation of triglycerides in cells at high pH, with a
decrease in glycolipids and polar lipids. The nonpolar storage lipids predominantly contain 16-
and 18-carbon saturated or monounsaturated fatty acids (16:0 and 18:1), which are considered
“precursor” fatty acids in lipid biosynthesis. The polar lipids and glycolipids usually contain a
higher proportion of polyunsaturated fatty acids. However, analysis of the fatty acid composition
of the storage lipids showed that at higher pH, more of these precursor lipids were seen in the
polar lipids and glycolipids. This suggests a switch in the lipid synthesis patterns that results in
less desaturation of the fatty acids esterified to the polar lipids.
In summary, the finding that an increase in pH can also lead to lipid accumulation in cells before
N is depleted suggested a method to uncouple lipid accumulation from nutrient deprivation, and
provided another method to study the biochemistry of lipid accumulation in microalgae. The
data from Dr. Cooksey's laboratory supported the premise that lipid triggers such as nutrient
deprivation or pH increase affect lipid accumulation in microalgae by similar mechanisms, i.e.,
inhibition of cell division, leading to decreased utilization of storage lipid while new synthesis of
lipid continues. However, he also proposed that different stresses may affect different stages of
the cell cycle. As there is evidence that the different lipid classes (neutral lipids versus polar
lipids) may be synthesized at different times during the cell cycle, this could affect the quality
and the quantity of the lipids synthesized. For example, pH stress appears to block release of
autospores (after DNA replication); N deprivation could have multiple effects on the
photosynthetic machinery or on a number of biochemical pathways in the cell that could directly
or indirectly affect lipid synthesis. R. Thomas, a graduate student working with Dr. Cooksey,
found that treating the cells with monofluoroacetate (MFA) also decreased cell growth and
caused neutral lipid accumulation. MFA inhibits the TCA cycle, presumably preventing TCA
oxidation of fatty acids and thus increasing the pool of acetyl CoA for synthesis of new fatty
acids. (Thomas suggested that MFA could be used as a trigger for lipid accumulation in algal
ponds, with the caveat that MFA is toxic to all living systems).
Dr. Cooksey concluded that to understand the biochemistry of neutral lipid accumulation in
microalgae, it would be necessary to understand cellular cycles of lipid synthesis and utilization
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that are coupled to cell growth and division. It will also be important to consider not only factors
that affect synthesis of storage lipid, but also to understand the metabolic shifts that result in
production of membrane lipids or storage lipids.
Publications:
Cooksey, K.E. (1987) “Collection and screening of microalgae for lipid production.” Final
Subcontract Report, Solar Energy Research Institute, Golden, Colorado, May 1987, 42 pp.
Cooksey, K.E.; Guckert, J.B.; Thomas, R. (1989) “Triglyceride accumulation and the cell cycle
in microalgae.” Aquatic Species Program Annual Report, Solar Energy Research Institute,
Golden, Colorado, pp. 139-158.
Cooksey, K.E.; Guckert, J.B.; Williams, S.A.; Collis, P.R.(1987) Fluorometric determination of
the neutral lipid content of microalgal cells using Nile Red, J. of Microbiol. Methods 6:333-345.
Cooksey, K.E.; Williams, S.A.; Collis, P.R. (1987) “Nile Red, a fluorophore useful in assessing
the relative lipid content of single cells,” In The Metabolism, Structure and Function of Plant
Lipids, (Stumpf, P.K; Williams, S.A.; Callis, R.P., eds.), Plenum Press, N.Y., pp. 645-647.
Guckert, J.B.; Cooksey, K.E.; Jackson, L.L. (1987) “Lipid solvent systems are not equivalent for
analysis of lipid classes in the microeukaryotic green alga, Chlorella.” Unpublished manuscript.
Guckert, J.B.; Cooksey, K.E. (1990) “Triglyceride accumulation and fatty aid profile changes in
Chlorella (Chlorophyta) during high pH-induced cell cycle inhibition.” J. Phycol. 26:72-79.
Thomas, R.M. (1990) “Triglyceride accumulation and the cell cycle in Chlorella.” Masters
Thesis, Montana State University, July 1990.
II.B.1.g.
Biochemical Elucidation of Neutral Lipid Synthesis in Microalgae
Subcontractor: University
of
Nebraska
Principal Investigator:
Steven D. Schwartzbach
Period of Performance:
3/87 - 3/88
Subcontract Number:
XK-5-05073-3
The goal of the research performed by Dr. Schwartzbach and coworkers was to understand the
biochemistry and physiology of lipid accumulation in microalgae, in particular the biochemical
responses to N deficiency as a trigger for lipid accumulation. Lipid biosynthesis is dependent on
the availability of fixed carbon and the activity of enzymes involved in lipid synthesis. These
experiments were directed at understanding how these processes are affected by N limitation in
the algal cells.
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The first set of experiments analyzed lipid synthesis in the eustigmatophytes Nannochloropsis
salina and Nanno Q, two oleaginous strains from the SERI Culture Collection. Similar results
were obtained for the two strains. The basic protocol was to innoculate the algal cells into media
containing either non-limiting levels or low levels of nitrogen (0.1 mM NaNO3), and to monitor
cell growth, chlorophyll content, and the lipid levels per cell and per culture volume. In cultures
containing low N, cell division ceased after 50-60 hours, and the cells entered stationary phase as
the N was depleted. In contrast, cells grown with sufficient N continued to divide. In the N-
replete cultures, the lipid content of the individual cells remained constant, and there was a
steady increase in the amount of lipid per mL of culture as the cell number increased. In contrast,
the N-deficient culture showed a significant increase in the level of lipid per cell. However, the
lipid content of the culture per mL (or percentage of the AFDW composed of lipid) did not
change. In N. salina, lipid made up 26%-32% of the AFDW, and in Nanno Q, lipid was 23%-
24% of the AFDW in cultures grown under N-replete and N-depleted conditions. These results
indicate that N depletion causes the cells to stop dividing, while lipid synthesis continues.
However, there is no net increase in lipid synthesis, and the trigger in these cells does not change
the activity of enzymes involved in lipid biosynthesis. One caveat to these studies is that Dr.
Schwartzbach measured only total lipid produced in the cells, including polar membrane lipids
and nonpolar storage lipids; it is unclear from these studies and those described later whether N
deficiency could differentially affect accumulation of the nonpolar lipids in these algae.
Another result from the studies on Nannochloropsis was that the level of chlorophyll in the cells
declined rapidly in N depleted cells. Thus, N depletion would also presumably decrease
photosynthetic efficiency and the availability of fixed carbon. The next set of experiments was
designed to separate the effects of reduced photosynthetic efficiency from direct effects of N
limitation on biosynthetic enzyme activities. To accomplish this, a series of experiments was
performed using the eukaryotic green alga Euglena gracilis var. bacillaris Cori. Euglena can
grow heterotrophically using ethanol as the sole carbon source. The growth of cells in the
presence of externally supplied carbon (ethanol) should not be limited by decreased
photosynthesis, so the rate of lipid synthesis would be solely limited by lipid biosynthetic
capabilities.
Euglena is unique compared to most algae of interest to the ASP as potential producers of
biodiesel. Euglena produces both lipid (primarily in form of the wax ester myristyl-miristate)
and carbohydrate (the major product is paramylum, a β-1,3-glucan) as storage products. Using
Euglena, a complicated series of experiments was conducted comparing the growth, lipid and
carbohydrate content, and chlorophyll levels in algae under photosynthetic and heterotrophic
growth conditions, as well as under aerobic and anaerobic conditions (Coleman et al. 1988b).
Basically, cells were grown to N deficiency, then resuspended in fresh media containing either
sufficient or limiting amounts of N. The new media also did, or did not, contain ethanol as a
carbon source, and the cells were grown in the dark or in light.
As was seen with the Nannochloropsis strains, cell growth under N deficient conditions caused
an increase in the levels of storage products (in this case, lipid plus carbohydrate) per cell.
However, there was no net increase in total lipid/carbohydrate when measured as a percentage of
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dry cell weight. This was true in cells grown autotrophically or heterotrophically. Nitrogen
depletion caused the cells to stop dividing, but the storage products continued to accumulate in
the cells at the same rate as in non-nitrogen limited cells. In addition, the proportion of
carbohydrate and lipid was unchanged, thus there did not appear to be a N trigger effect, either
directly or indirectly via carbon limitation, on the enzymes of the lipid or carbohydrate synthetic
pathways. One caveat to this result was that in very old cultures, (i.e., 12 days after transfer of
the cells to N-deficient media), the lipid as a percentage of the dry cell weight increased in all
cultures. However, this was accompanied by a decrease in the total cell mass, and the lipids are
apparently more stable than other cell components.
Growth of Euglena under N-deficient conditions resulted in loss of chlorophyll, as seen for
Nannochloropsis. Dr. Schwartzbach also used two-dimensional gel electrophoresis to monitor
changes in the levels of chloroplast and mitochondrial proteins under N-deficient conditions
(Coleman et al., 1988a). Under photosynthetic growth conditions (high light), exposure of the
cells to N-deficient conditions resulted in a decrease in the levels of 37 proteins identified as
components of the chloroplast. Under low light conditions, there was little change in the
population of chloroplast proteins. The degradation of the chloroplasts under low N conditions
was presumably due to photooxidation of chlorophyll, accompanied by degradation of newly
synthesized photosynthetic membrane proteins that could not assemble properly into the unstable
chloroplast. Synthesis of chlorophyll requires N to form δ-aminolevulinic acid, a chlorophyll
precursor. Although photooxidation of chlorophyll occurs constantly in the light, synthesis of
new chlorophyll molecules also occurs to replace the degraded molecules. However, if N levels
are depleted, new chlorophyll cannot be produced, and photosynthetic efficiency decreases. This
result is important with regard to biodiesel production. It suggests that there would be limitations
on the amount of lipid that could be produced in outdoor ponds using N limitation as a trigger for
lipid accumulation even if carbon was not limiting (i.e., for cells grown in outdoor ponds).
One process that affected the biosynthetic pathways in Euglena and resulted in an increase in the
total lipid in the cultures was cell growth under anaerobic conditions with ethanol as a carbon
source (lipids increased from 5-10% to 45% of the AFDW). Growth via anaerobiosis caused the
activation of the oxygen-sensitive pyruvate dehydrogenase in the mitochondria. This led to
increased levels of acetyl CoA in the mitochondria, which activates the mitochondrial fatty acid
synthesis pathways. However, the increased flow of carbon to lipid synthetic pathways was
accompanied by degradation of non-lipid components under anaerobic condition, including
paramylum, the main storage carbohydrate, which resulted in a decrease in total cell mass. Dr.
Schwartzbach estimated that if the anaerobic cells had increased in cell mass to the same extent
as cells grown aerobically, the lipids would only compose 15% of the dry weight.
This observation that anaerobiosis could result in increased lipid yields by actually affecting the
lipid biosynthetic pathway suggested that lipid synthesis could be increased by increasing the
levels of the lipid precursors acetyl CoA and malonyl CoA. Little is known about lipid synthesis
in algae, but data from other organisms suggested that pyruvate dehydrogenase and acetyl CoA
carboxylase could function as regulatory enzymes in algal lipid synthesis. Understanding the
biochemical factors that limit production of the lipid precursors could lead to biochemical or
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genetic engineering strategies to increase the activity of these enzymes that could produce an
organism with the ability to produce very high lipid levels. To this end, Dr. Schwartzbach
initiated a project to isolate and characterize these enzymes from several algae, including
Euglena, N. salina, Nanno Q, and Monoraphidium 2 (Smith and Schwartzbach 1988). They
reported some very preliminary information on protein extraction techniques and assay
techniques for these enzymes. This work was a precursor to a major effort at SERI/NREL in the
late 1980s through the end of project in 1996 to identify key enzymes in the algal biosynthetic
pathways and to increase lipid levels by manipulating these pathways through genetic
engineering (see Sections II.B.2. and II.B.3.).
In summary, although Euglena is not typical of the oleaginous microalgae targeted as potential
biodiesel producers by the ASP, the data from Dr. Schwartzbach's laboratory point out the
importanance of understanding the biochemical mechanisms by which algae accumulate lipids.
For Euglena and the Nannochloropsis strains described here, N deprivation does not seem to
function as an actual trigger to induce biosynthesis of lipid. Rather, it acts as a block to cell
division. Lipid synthesis continues by normal pathways, and lipid levels increase per cell, with
no net accumulation in the culture. This result confirms the conclusions of Cooksey and
coworkers. In addition, N deficiency also affects other cell processes, such as photosynthetic
efficiency, which could affect lipid accumulation as the availability of fixed carbon is decreased.
Publications:
Coleman, L.W.; Rosen, B.H.; Schwartzbach, S.D. (1987a) “Environmental control of lipid
accumulation in Nannochloropsis salina, Nanno Q and Euglena.” FY 1987 Aquatic Species
Program Annual Report (Johnson, D.A.; Sprague, S., eds.), Solar Energy Research Institute,
Golden, Colorado, SERI/SP-231-3206, pp. 190-206.
Coleman, L.W.; Rosen, B.H.; Schwartzbach, S.D. (1987b) “Biochemistry of neutral lipid
synthesis in microalgae.” FY 1986 Aquatic Species Program Annual Report (Johnson, D.A., ed.),
Solar Energy Research Institute, Golden, Colorado, SERI/SP-231-3071, p. 255.
Coleman, L.W.; Rosen, B.H.; Schwartzbach, S.D. (1988a) “Preferential loss of chloroplast
proteins in nitrogen deficient Euglena.” Plant Cell Physiol 29:1007-101. (Note: a preprint of this
article was also submitted as a SERI Report, 47pp.)
Coleman, L.W.; Rosen, B.H.; Schwartzbach, S.D. (1988b) “Environmental control of
carbohydrate and lipid synthesis in Euglena.” Plant Cell Physiol. 29:423-432.
Smith, C.W.; Schwartzbach, S.D. (1988) “Preliminary characterization of pyruvate
dehydrogenase and acetyl-CoA synthetase.” Manuscript submitted as a report to SERI, 6 pp.
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II.B.1.h.
Transformation and Somatic Cell Genetics for the Improvement of Energy
Production in Microalgae
Subcontractor:
University of Nebraska; Oregon State University
Principal Investigator:
Russel H. Meints
Period of Performance:
1/86 - 12/89
Subcontract Number:
XK-5-05073-2
By the mid-1980s, SERI researchers became convinced that optimal lipid production in
microalgae could be achieved only by genetic manipulation of the cells to produce the desired
traits. The overall goal of the research performed by Dr. Meints for SERI was to develop an
algal virus system as a tool for the genetic manipulation of microalgae with potential for liquid
fuel production. Viral vector systems had been used successfully to transfer DNA into other cell
types. Dr. Meints' laboratory was preeminent in the field of algal viruses. The work performed
from 1986 to 1989 was based on earlier studies from this laboratory on isolating and
characterizing a unique group of viruses from symbiotic algae. Funding from SERI contributed
to ongoing research on the algal viral system by Dr. Meints; much of this work was done in
collaboration with Dr. James Van Etten, in the Department of Plant Pathology at the University
of Nebraska. The list of publications at the end of this section includes articles produced before
and after the period of the SERI subcontract. This information was included to give interested
readers an overview of the work performed on this topic.
Background:
In the early 1980s, Dr. Meints and Dr. Van Etten were studying Chlorella-like green algae that
live in a symbiotic relationship within cells of the protozoan Hydra viridis. They found that the
algal cells could be excised from the hydra, which could exist free of the symbiont if given
proper nutrients. However, it was not possible to culture the algae free of the hydra host. Further
study demonstrated that when the algal cells were isolated from the host, virus particles rapidly
began to multiply within the algae, resulting in lysis of the algal population within 24 hours.
Ultrastructual and biochemical studies on this algal virus system produced the following results:
• The virus consisted of a large (approximately 190-nm), polygonal particle,
containing 30 to 40 polypeptides, the most abundant of which was a 46 kDa
glycoprotein, presumably associated with the viral capsid.
• The virus genome consists of about 130 kbp of double-stranded DNA.
• New virus particles were assembled in the cytoplasm of the algal cells and
released upon lysis of the algal cell wall.
This virus, called HVCV (for Hydra viridis Chlorella virus), was one of the few viruses
described in eukaryotic algae. HVCV might play a role in initiating or maintaining the symbiotic
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relationship between the alga and its hydra host, possibly by altering the algal cell wall.
Subsequent studies identified viruses in four other strains of Hydra obtained from commercial
sources. The viruses fell into two classes, based on particle size, bouyant density, and DNA
restriction patterns (HVCV-1, HVCV-2). In addition, a similar virus was isolated from symbiotic
Chlorella from Paramecium bursaria (PBCV-1).
To facilitate the study of these viruses, it was desirable to identify an algal strain that could be
cultured free of the hydra or Paramecium host, which was susceptible to infection by the virus.
This would allow production of large quantities of the virus and the study of viral replication and
development. Sixteen strains of culturable Chlorella, which had been isolated from invertebrates
such as Paramecium, Hydra, Stentor, and sponges, plus two free-living strains, were obtained.
Attempts were made to infect these Chlorella strains with all the virus strains described earlier.
None of the HVCV viruses (from Chlorella-Hydra hosts) were able to infect any Chlorella strain
tested. However, two culturable Chlorella strains originally isolated from Paramecium
(Chlorella strains N1a and NC64) were infected with PBCV-1 (the P. bursaia Chlorella virus).
The infection led to lysis of the algal cells and production of large amounts of infectious viral
progeny. This result led to the development of a plaque assay system for the algal viruses,
similar to a bacteriophage assay on bacterial lawns. The availability of this system, which caused
synchronous infection of the algal cells and the production of large quantities of viral particles,
allowed the researchers to characterize the virus biochemically. It also allowed researchers to
study the regulation of viral gene expression and the effects of viral replication on algal
physiology and gene expression. A large number of publications resulted from this research (see
below). Several of the most interesting and possibly relevant findings are summarized here.
• The virus particles attach at one vertex of their polygonal capsid to receptor sites
on the algal cell wall. A lytic enzyme produced by the virus degrades the wall
at this site, and the viral DNA is released into the cell. Living algal cells are not
required for virus attachment and wall degradation (viruses can attach to and
degrade isolated wall fragments), but living cells are necessary for release of the
viral DNA. Complete viral capsids are assembled in viral assembly sites within
the cytoplasm and subsequently filled with DNA. Virus particles are released
through holes produced at discrete locations in the algal cell wall.
• The plaque assay system was used to screen for other virues that infect algae.
Viruses that infect Chlorella strains N1a or NC64A were found to be very
common in nature. The viruses all had similar features, including a large,
polygonal capsid and dsDNA; however, some viruses were distinguishable
based on plaque size, reactivity to anti-PBCV-1 antisera, variations in the DNA
restriction patterns and the extent of nucleotide modification.
• The viruses are highly infectious and grow rapidly within the algal cells. Algal
growth is inhibited rapidly following viral infection. Synthesis of host DNA
and RNA is shut down, and the organellar and genomic DNA is degraded. Viral
gene expression entails transcription of early and late genes, and may include
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expression of overlapping genes or transcription of genes from both DNA
strands. Analysis of DNA from some viral isolates showed that the viral DNA
is modified to varying extents, primarily in the methylation of adenine and
cytosine residues. The data suggested that the virus produces a unique
restriction enzyme that is specific for non-methylated sequences for degradation
of the host DNA. The virus also produces a corresponding methyltransferase,
which recognizes the same sequence as the restriction endonuclease. The
methyltransferase methylates newly synthesized viral DNA, protecting it from
degradation in the next round of infection.
Dr. Meints received funding from SERI from 1986 through 1989. The overall goal of the SERI-
funded research was to use the algal virus system to develop methods for genetically
manipulating microalgae with potential for liquid fuel production. The research from Dr. Meints'
laboratory is reviewed below with respect to the specific goals of the project.
Isolation and characterization of natural hosts for the algal viruses.
Water samples isolated from various sites were screened for the presence of algal viruses using
the plaque assay. If viruses were not detected initially, an enrichment protocol was used in which
a few algal cells were added to the water sample; after 48-72 hours the algal and cell debris were
removed by centrifugation and the sample was reassayed for the presence of virus. Using these
procedures, more than 50 individual algal viral isolates were identified. Although the viruses
were all large polygonal particles with dsDNA genomes, analysis of the viral DNA by digestion
with restriction endonucleases showed there were at least 15 different types of virus found. Sites
that contained virus were further analyzed for the natural algal hosts; however, none were
identified. It is unclear why the natural hosts could not be found. Dr. Meints proposed either
that the viruses were propagated or maintained by some unknown mechanism, or, more likely,
that the natural host was present in the sites tested at very low concentrations. This is possible in
that each virus that infects an algal cell could produce 350 new virus particles, and that up to 100
algal cells can exist within a single Paramecium. Based on the density of viral particles isolated
from the various sites, a single Paramecium with algal symbionts could theoretically sustain a
virus population in 350 liters of water. Because a natural alga host for the viruses was not found,
the goal of characterizing the viral host was dropped, and further screening efforts were
discontinued.
In a separate, but related, series of experiments, Dr. Meints received 250 water samples collected
by Dr. Ralph Lewin of the Scripps Institute in La Jolla, California. These were also screened for
the presence of algal viruses, but viral particles were not found in any of Dr. Lewin's samples.
Screening of the SERI algal collection for infection with the Chlorella viruses.
Dr. Meints received a number of algal strains from Dr. Bill Barclay at SERI that were believed to
have potential for biodiesel production. These included Chlorella 501 and A. falcatus (A record
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of the precise number and identity of the strains sent to Dr. Meints could not be found). Growth
conditions for these strains were optimized, and the algae were then tested for infection by the
algal viruses. None of the SERI strains were susceptible to infection by any virus isolate tested.
This project was discontinued in early 1987.
Use of the lytic enzyme(s) produced by the virus for degradation of the host cell wall for the
production of microalgal protoplasts.
Infection of Chlorella N1a or NC64A by the algal viruses resulted in rapid lysis of the algal cells.
Dr. Meints' laboratory developed methods to isolate the lytic enzymes and to use the lysin
preparation to produce algal protoplasts (cells without cell walls). The protoplasts could be used
in studies of somatic cell fusion (genetic improvement by fusion of two individuals with useful
traits such as pH tolerance and high lipid production). Alternatively, the protoplasts could be
used as targets in a genetic transformation system in which DNA plasmids are taken up directly
into cells without walls.
A crude lysin preparation was produced by infecting Chlorella N1a with PBCV-1. After several
lytic cycles (approximately 24 hours), the sample was centrifuged to remove cell debris and
virus. Initially, the supernatant from this preparation was used directly to produce protoplasts
from Chlorella N1a cells. Alternatively, lysin activity was precipitated from the supernatant with
65% ammonium sulfate. Cells were exposed to lysin in the presence of 1 M sorbitol as an
osmoticum. (One interesting result from the protoplast studies is that algal strains exhibited
significant differences in their sensitivities to osmotica commonly used for higher plant cells; i.e.
mannitol, but not sorbitol, was toxic to Chlorella N1a. The sensitivity of individual algal strains
to different osmotica will need to be determined empirically.) The cell wall of algal cells
exposed to lysin was rapidly degraded over the entire cell surface, as judged by electron
microscopy and staining of the cells with calcofluor white, which stains plant cell walls. The
lysin preparation could be purified further by exposing the crude sample to an affinity matrix
composed of algal cell wall fragments. Lysin activity was eluted by salt washes. Exposure of
Chlorella N1a cells to this lysin sample resulted in degradation of the algal wall at specific sites;
when the osmoticum was reduced to half strength, the alga protoplast was released through
discrete holes in the wall. This result suggested the presence of more than one enzymatic activity
in the crude lysin preparation.
Somewhat surprisingly, the protoplasts did not lyse when transferred to water. However,
exposure of lysin-treated cells, but not untreated cells, to low concentrations of detergent caused
the release of chlorophyll. The amounts of chlorophyll released from lysin-treated cells was used
as a measure of the extent of protoplast formation in a cell sample. The protoplasts were viable,
as judged by staining with fluorescein diacetate. Unfortunately, although some regeneration of
the cell wall occurred, the lysin-treated cells never formed new colonies. Attempts to use the
viral-lysin to produce protoplasts from other microalgal strains met with little success. A
manuscript was prepared and submitted to SERI that described the progress made on the use of
lysin to produce algal protoplasts, but the article was never published in a technical journal.
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Characterization of the chloroplast and mitochondrial genomes of microalgal strains.
The goal of this research was to characterize the organellar genomes of Chlorella and other
microalgae. As organellar DNA is thought to be highly conserved evolutionarily, the idea was to
use similarities or differences between chloroplast or mitochondrial DNA as a measure of the
taxomonic relatedness of algal strains. This information could be useful for experiments
involving somatic cell fusion or gene transfer, as these procedures would likely have a higher
chance of success between more closely related strains. Studies of the organellar genomes could
also lead to the identification of promoters or replication origins that could be used to develop
vectors for algal transformation. Due to the lack of significant progress on the first three goals,
Dr. Meints concentrated the efforts of his laboratory on this project for the last 2 years of the
subcontract.
The first step was to develop methods for isolation of chloroplast and mitochondrial DNA from
Chlorella N1a. Based on protocols used for higher plants, Dr. Meints exploited the differences in
the C/G content between chloroplast DNA and nuclear DNA to separate the two genomes using
density gradient centrifugation. Chloroplast DNA was identified by hybridization with
heterologous chloroplast DNA markers. The chloroplast genome of Chlorella N1a was found to
be circular, containing approximately 120 kbp of DNA. A restriction map of the chloroplast
genome was produced and several genes were localized on the map by hybridization with
chloroplast gene sequences from maize. Most chloroplast genomes contain two inverted repeats,
each of which contains a copy of the 23S, 16S, and 5S ribosomal RNA genes. These repeats are
flanked by a short and long single copy DNA region. Although Dr. Meints initially reported that
Chlorella N1a chloroplast DNA contained this inverted repeat structure (Meints 1987), a
subsequent article reported that the chloroplast genome of Chlorella N1a contains only a single
copy of the ribosomal RNA gene region (Schuster et al. 1990b). This result was confirmed by
Dr. Meints via a recent personal communication. Although most other green algae, including
other chlorellans, contain chloroplast DNA similar to that commonly seen in most higher plants,
i.e., containing two inverted repeats, this unusual chloroplast structure has been seen in two
legumes (peas, broad beans), conifers, some red algae, and in at least one other green alga,
Codium.
Restriction analysis of chloroplast DNA from several exsymbiont and free-living strains of
Chlorella showed variations between the strains that indicate genetic divergence and that suggest
gene transfer and cell fusion between these species may be problematic. The results suggest that
chloroplast DNA structure may be a useful taxonomic parameter, but more study is needed
before definite conclusions on algal taxonomy or cell-cell compatability based on chloroplast
DNA structure can be made.
Isolating mitochondrial DNA from Chlorella N1a was technically problematic, and the
mitochondrial genome isolated was first presumed to be a plasmid. Unlike some species in
which mitochondrial DNA has a G/C content similar to that of nuclear DNA, in Chlorella N1a,
the mitochondrial DNA had a low G/C content similar to that of chloroplast DNA, and the two
genomes banded very closely on the density gradients. As with the chloroplast DNA,
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heterologous probes were used to identify the mitochondrial DNA and to localize specific
mitochondrial genes on the restriction map. The gene organization in the Chlorella N1a
mitochondrial DNA was similar to that in higher plants, and distinct from the organization of
mitochondrial genes in animals and fungi. It has been proposed that mitochondria in plants and
green algae originated from a separate endosymbiotic event as compared to animals and fungi.
This is supported by Dr. Meints’ results.
Although not included in the original Statement of Work, Dr. Meints also reported under this task
other related research efforts in his laboratory toward the development of a genetic
transformation system for microalgae. Libraries were prepared from Chlorella N1a nuclear
DNA and DNA from the algal virus. The goal of this project was to identify DNA sequences
that could be used to develop transforming vectors, such as origins of replication, regulatory
regions for gene expression, or algal genes to use in selectable marker systems. A library of the
viral DNA was prepared in a lambda vector, which allowed for the sequencing of the viral
genome and studies of viral gene stucture and expression. This work led to several significant
discoveries that were published after SERI funding stopped, including the cloning of the major
viral capsid protein (Graves and Meints, 1992), and the identification of a viral gene promoter
that also functioned in higher plants (Mitra and Higgins, 1994).
Dr. Meints’ laboratory also made several attempts to produce a library of Chlorella nuclear
DNA, with little success. This appeared to be due to modification (probably methylation) of the
algal DNA that resulted in degradation of the DNA by the bacterial host used for library
construction. Several ways around this problem were proposed, including the use of a yeast
cloning system or the use of a bacterial host that did not contain the enzymes for degradation of
methylated DNA. A cDNA library was produced successfully before the end of the SERI-funded
research efforts.
Dr. Meints and his coworkers and collaborators produced a large quantity of data during the 4
years of SERI-funded research and during the following years. They made significant
contributions to the study of the biology, biochemistry, and molecular biology of a eukaryotic
algal virus, and to the biology and molecular biology of the algal hosts, particularly with respect
to the algal organellar genomes. Unfortunately, because of the specificity of the virus/algal
interactions, the results obtained were not directly applicable to the development of a
transformation system for the oleaginous algal strains of interest to NREL. The research also
generated some valuable technical information, regarding toxicity of microalgae to common
osmotica, construction of genomic DNA libraries, and organellar genome isolation, which could
be useful for further studies of algal molecular biology and the development of genetic
engineering techniques. The studies of the algal virus also resulted in the identification of a new
restriction endonuclease (Jin et al. 1994) and a new adenine methyltransferase (Stefan et al.
1991), as well as a viral promoter sequence that can function in plants (Mitra and Higgens 1994).
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Publications:
Anberg, S.M.; Meints, R.H. (1991) “Nucleotide sequence of the genes for ribulose-1,5-
bisphosphate carboxylase/oxygenase large subunit and ribosomal protein S145 from a Chlorella-
like alga.” J. Phycol. 27:753-758.
Graves, M.V.; Meints, R.H. (1992) “Characterization of the major capsid protein and cloning of
its gene from algal virus PBCV-1.” Virology 188:198-207.
Graves, M.V.; Meints, R.H. (1992) “Characterization of the gene encoding the most abundant in
vitro translation product from a virus infected Chlorella-like alga.” Gene 113:149-155.
Ivey, R.G.; Henry, E.C.; Meints, R.H. (1996) “A Feldmania algal virus has two genome size
classes.” Virology 220:267-.
Jin, A.; Zhang, Y.; Van Etten, J.L. (1994) “New restriction endonuclease CviRI cleaves DNA at
TG/CA sequences.” Nuc. Acids. Res. 22:3928-.
Krueger, S.K.; Ivey, R.G.; Meints, R.H. (1996) “A brown algal virus genome contains a ring zinc
finger motif.” Virology 219:307-.
Lee, A. M.; Ivey, R.G.; Meints, R.H. (1995) “Characterization of a repetitive DNA element in a
brown algal virus.” Virology 212:474-.
McCluskey, K.; Graves, M.V.; Mills, D.; Meints, R.H. (1992) “Replication of Chlorella virus
PBCV-1 and host karyotype determination studies with pulse-field gel elecrtrophoresis.” J.
Phycol. 28:846-850.
Meints, R. (1987) “Studies of the chloroplast genome of exsymbiotic and free-living Chlorella.”
FY 1987 Aquatic Species Program Annual Report, Solar Energy Research Institute, SERI/SP-
231-3206, pp. 210-228.
Meints, R.H.; Burbank, D.E.; Van Etten, J.L.; Lamport, D.T.A. (1988) “Properties of the
Chlorella receptor for the PBCV-1 virus.” Virology 164:15-21.
Meints, R.H.; Lee, K.; Burbank, D.E.; Van Etten, J.L. (1984) “Infection of a Chlorella-like alga
with the virus PBCV-1: Ultrastructural studies.” Virology 138:341-346.
Meints, R.H.; Lee, K.; Van Etten, J.L. (1986) “Assembly site of the virus PBCV-1 in
Chlorella-like alga: Ultrastructural studies.” Virology 154:240-245.
Meints, R.H.; Kennedy, J.; Ziegelbein, M.; Amberg, S.; Van Etten, J.L. (1988) “Production of
protoplasts from Chlorella-like algae.” Unpublished manuscript; submitted to SERI as a
research report.
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Meints, R.H.; Schuster, A.M.; Van Etten, J.L. (1985) “Chlorella viruses.” Plant Mol. Biol.
Reports 3:180-187.
Meints, R.H.; Van Etten, J.L. (1991) “Eukaryotic Algae.” In Microbioal Ecology: Principles,
Methods and Applications, (Levin, M.A.; Seidler, R.J.; Rogul, M., eds.), McGraw-Hill, Inc. New
York, pp. 883-888.
Meints, R.H.; Van Etten, J.L.; Kuczmarski, D.; Ang, B. (1981) “Viral infection of the symbiotic
Chlorella-like alga present in Hydra viridis.” Virology 113:698-703.
Reisser, W.; Burbank, D.E.; Meints, S.M.; Meints, R.H.; Becker, B.; Van Etten, J.L. (1988) “A
comparison of viruses infecting two different Chlorella-like green algae.” Virology 167:143-
149.
Schuster, A.M.; Burbank, D.E.; Meister, B.; Skrdla, M.; Meints, R.H.; Hattman, S.; Swinton, D.;
Van Etten, J.L. (1986) “Characterization of viruses infecting a eukayotic Chlorella-like green
alga.” Virology 150:170-177.
Schuster, J.A.; Graves, M.; Korth, K.; Ziegelbein, M.; Brumbaugh, J.; Grone, D.; Meints, R.H.
(1990a) “Transcription and sequence studies of a 4.3 kbp fragment from a dsDNA eukaryotic
alga virus.” Virology 176:515-523.
Schuster, A.M.; Waddle, J.A.; Korth, K.; Meints, R.H. (1990b) “The chloroplast genome of an
exsymbiotic Chlorella-like alga.” Plant Mol. Biol. 14:859-862.
Skrdla, M.P.; Burbank, D.E.; Xia, T.; Meints, R.H.; Van Etten, J.L. (1984) “Structural proteins
and lipids in a virus, PBCV-1, which replicates in a Chlorella-like virus.” Virology 135:308-315.
Stefan, C.; Xia, Y.; Van Etten, J.L. (1991) “Molecular cloning and characterization of the gene
encoding the adenine methyltransferase M.Cviri from Chlorella virus XZ-6E” Nuc. Acids Res.
19:307-.
Van Etten, J.L.; Burbank, D.E.; Joshi, J.; Meints R.H. (1984) “DNA synthesis in a Chlorella-like
alga following infection with the virus PBCV-1.” Virology 134:443-449.
Van Etten, J.L.; Burbank, D.E.; Kuczmarski, D.; Meints, R.H. (1983) “Virus infection of a
culturable Chlorella-like algae and development of a plaque assay.” Science 219:994-996.
Van Etten, J.L.; Burbank, D.E.; Meints, R.H (1986) “Replication of PBCV-1 in ultra-violet
irradiated Chlorella.” Intervirology 26:115-120.
Van Etten, J.L.; Burbank, D.E.; Schuster, A.M.; Meints, R.H. (1985) “Lytic viruses infecting a
Chlorella-like alga.” Virology 140:135-143.
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Van Etten, J.L.; Burbank, D.E.; Xia, Y.; Meints, R.H. (1983) “Growth cycle of a virus, PBCV-1,
that infects Chlorella-like algae.” Virology 126:117-125.
Van Etten, J.L.; Meints, R.H. (1991) “Viruses and virus-like particles of eukaryotic algae.”
Microbiological Reviews 55:586-620.
Van Etten, J.L.; Lane, L.; Meints, R.H. (1991) “Unicellular plants also have large dsDNA
viruses.” Seminars in Virology 2:71-77.
Van Etten, J.L.; Meints, R.H.; Burbank, D.E.; Kuczmarski, D.; Cuppels, D.A.; Lane, L.C. (1981)
“Isolation and characterization of a virus from the intracellular green alga symbiotic with Hydra
viridis.” Virology 113:704-111.
Van Etten, J.L.; Meints, R.H.; Kuczmarski, D.; Burbank, D.E.; Lee, K. (1982) “Viruses of
symbiotic Chlorella-like algae isolated from Paramecium bursaria and Hydra viridis.” Proc.
Nat'l. Acad. Sci. 79:3867.
Van Etten, J.L.; Schuster, A.M.; Meints, R.H. (1988) “Viruses of eukaryotic Chlorella-like
algae.” In Viruses of Fungi and Simple Eukaryotes, Mycology Series vol. 7 (Koltin, Y.;
Leibowitz, M.J., eds.), Marcel Dekker, Inc., New York, pp. 411-428.
Van Etten, J.L.; Xia, Y.; Meints, R.H. (1987) “Viruses of a Chlorella-like green alga.” In Plant-
Microbe Interactions II, (Kosuge, T.; Nester, E.W., eds.), Macmillan Publishing Co., pp 307-325.
Van Etten, J.L.; Xia, Y.; Narva, K.E.; Meints, R.H. (1987) “Chlorella Algal Viruses.” In
Extrachromasomal Elements in Lower Eukaryotes (Fink, G.; et al., eds.), Plenum Press, New
York, pp. 337-347.
Waddle, J.A.; Schuster, A.M.; Lee, K.; Meints, R.H. (1989) “The mitochondrial genome of the
exsymbiotic Chlorella-like green alga N1a.” Aquatic Species Program Annual Report,
September 1989, Solar Energy Research Institute, Golden, Colorado, SERI/SP-231-3579, pp. 91-
118.
Waddle, J.A.; Schuster, A.M.; Lee, K.; Meints, R.H. (1990) “The mitochondrial genome of an
exsymbiotic Chlorella-like green alga.” Plant Mol. Biol. 14:187-195
II.B.2.
Physiology, Biochemistry, and Molecular Biology of Lipid
Production: NREL In-House Researchers
II.B.2.a. Introduction
During the first few years of the ASP, in-house research efforts in the area of lipid biosynthesis
focused on understanding the lipid trigger and the effects of N starvation on lipid synthesis and
photosynthetic efficiency. It was also shown that lipid accumulation can be induced in diatoms
by Si starvation, a major component of the diatom cell wall. During the second half of the 1980s
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and early 1990s, a major component of the research at SERI/NREL was the study of lipid
biosynthesis in the oleaginous diatom C. cryptica. This was primarily the work of Paul Roessler,
who identified a key enzyme involved in lipid accumulation and isolated and characterized both
the protein and the gene for the enzyme acetyl-CoA carboxylase from Cyclotella. Other research
efforts at NREL examined related biosynthetic pathways, including synthesis of chrysolaminarin,
a storage carbohydrate, and lipid processing reactions such as fatty acid desaturation. The basic
biochemistry and molecular biology research formed the basis of the efforts to manipulate
microalgal lipids by genetic engineering, which will be described in Section II.B.3.
One aspect of the algal research at SERI was the possibility of producing hydrogen by
microalgae, for use as a gaseous fuel. During photosynthetic electron transport, electrons from
reduced ferredoxin can be transferred to hydrogen ions to produce H2. This reaction is catalyzed
by the enzyme hydrogenase. Unfortunately, hydrogenase is inhibited by molecular oxygen, a by-
product of the photosynthetic reaction, making the practical application of this process difficult.
There was a significant research effort at SERI in the early 1980s, primarily by Dr. Steve Lien
and Dr. Paul Roessler, to understand the biochemistry of hydrogen production by microalgae.
The work on hydrogenase was funded by the Hydrogen Program at DOE, not the ASP, and will
not be included in this report. However, studies on hydrogen production by microalgae are
ongoing at NREL (SERI) in the Center for Basic Sciences, and interested readers should contact
Dr. Michael Seibert for more information.
II.B.2.b.
Lipid Accumulation Induced by Nitrogen Limitation
As a result of the algal screening efforts by SERI subcontractors and in-house researchers,
several algal species were identified as good candidates for biodiesel production during the early
1980s. This was facilitated by the development of a cytochemical staining technique for
intracellular lipids that allowed researchers to visualize storage lipid droplets in algal cells (see
Section II.A.1.f. and Lien 1981a). Two of the most promising candidates were the green alga N.
oleoabundans, which showed a high lipid content and rapid growth, and a Chlorella strain
(CHLS01) isolated from a local site.
First, a sensitive method to monitor nitrate levels in liquid cultures using ion chromatography
was developed to study the effects of N limitation on lipid accumulation in these organisms.
Algal growth, lipid content, and chlorophyll a content were measured in batch cultures of N.
oleoabundans and CHLS01. Cell division and chlorophyll accumulation occurred rapidly in the
cultures as long as N was present. When N was depleted, cell division stopped, although
biomass accumulation continued for several days. The major portion of the new biomass was
composed of lipids and storage oils. N depletion resulted in a rapid decrease in the level of
chlorophyll a in the cultures, suggesting that the cells might metabolize chlorophyll during
periods of nitrogen stress. There was also an increase in the ratio of carotenoid to chlorophyll
and a significant decrease in the complexity of the intracellular membranes in N-starved cells.
These last three observations indicated that the photophysiology of the cells was affected,
suggesting that the lipid trigger could also directly or indirectly alter photosynthetic efficiency in
the treated cells (discussed in more detail below).
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II.B.2.c.
Studies on Photosynthetic Efficiency in Oleaginous Algae
SERI researchers Lien and Roessler (1986) tried a somewhat different approach to understand
the processes affecting lipid accumulation (Lien and Roessler 1986). A recently published
technical evaluation (Hill et al. 1984) identified two major requirements for economic feasibility
of biodiesel production:
1. Photosynthetic efficiency (which can simply be thought of as the percentage of
incident radiation that is converted into biomass) needs to be 18%, and
2. Algal biomass needs to consist of 60% lipid.
Because very high lipid production is usually correlated with stress conditions (nutrient
deprivation) that result in decreased photosynthetic efficiency and decreased growth, the two
conditions of high lipid and high productivity seemed to be mutually exclusive. To overcome
this technical hurdle, Lien and Roessler initiated a study to help understand the effects of
nitrogen deprivation and lipid accumulation on photosynthetic efficiency.
Three strains of oleaginous algae were used in this study: Chlorella CHLSO1, Ankistrodesmus
sp., and a newly isolated chrysophyte strain Chryso/F-1. The cells were grown in batch culture
and monitored for nitrate concentration, light levels in the culture, chlorophyll concentration, and
yield of cell mass and lipid (including total, neutral, and polar lipids). Maximum energy
efficiency occurred as the culture approached N depletion. At this point, the culture showed a
maximum density of photosynthetic pigments (before chlorophyll degradation and after N
depletion), but the light energy reaching the cells was decreased due to the higher culture density.
Thus, photosynthetic efficiency (biomass produced per light energy input) was maximized and
the individual cells suffered less photooxidative damage due to lower light exposure. After the N
in the culture was depleted, cell mass continued to increase for a time, eventually leveling off.
All cultures experienced a two- to three-fold increase in total lipid, primarily as non-polar lipid.
The photosynthetic efficiency decreased over the duration of the batch culture. However, in the
early stages after the N was depleted, the cultures showed a decrease in energy efficiency with
respect to total cell mass (AFDW) and with respect to the non lipid cell components, while
photosynthetic efficiency remained constant or increased slightly with respect to lipid
accumulation. In addition, N deprivation caused an increase in the efficiency of neutral, storage
lipid production and suppressed the efficiency of polar structural lipid production.
These studies provided interesting preliminary data on the energetics of cell mass and lipid
accumulation in algae. Follow-up experiments were proposed, including investigations of the
relationship between initial N concentration and photosynthetic efficiency and lipid production
after N depletion, and studying the effects of N resupply after depletion to attempt to extend the
period of lipid production. These experiments were not continued; however, the results
described earlier suggest that understanding the timing or kinetics of lipid accumlation in
microalgae will be essential to maximize lipid production in a mass culture facility. If N
starvation is used to trigger lipid accumulation, the data suggest that maximal photosynthetic
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efficiency with respect to lipid production (and probably the best time for harvesting lipid-
producing cells), occurs just after the N is depleted from the cultures.
Another set of experiments directed at optimizating photosynthetic efficiency in algal ponds was
performed by SERI researcher Dr. Ken Terry. Previous studies had indicated that algal cells
grown under high-intensity flashing light can use that light energy more efficiently than cells
grown under the same intensity under constant illumination. The evidence suggests that an algal
cell can integrate absorbed light energy such that the photosynthetic efficiency achieved under
intermittent light conditions is similar to that attained under constant light of the same average
intensity. This flashing light, or photomodulation, effect can be mimicked in vertically-mixed
algal ponds, as cells circulate to the surface and back down to the lower levels in the pond where
they receive minimal light. Thus, the photosynthetic efficiency of algal cells grown in ponds
may be increased in high light by using mixing strategies that optimize this photomodulation
effect.
In order to better understand the effects of intermittent light on photosynthetic efficiency of
microalgal cultures, Dr. Terry set up a system to measure photosynthetic rates and oxygen
evolution in laboratory cultures of Chlorella pyrenoidosa and Phaeodactylum tricorutum under
flashing light conditions. Intermittent light conditions were simulated by placing sectored disks
in front of a light source, and using this to illuminate exponentially growing cultures that had
been placed in an oxygen electrode chamber. Photosynthesis was then measured under varying
light/dark ratios (generated by changing the configuration of the disk) and light intensities. The
data generated were used to calculate the percent “integration” of the incident light by the algal
cultures. More rapid flashing led to greater integration, although lower flash frequencies
produced higher levels of integration as the percentage of time the cells spent in the light
decreased. Although these data were preliminary, they supported the proposal that
photosynthetic efficiency in microalgal ponds could be enhanced by optimized vertical mixing
strategies. However, increased photosynthetic efficiency might be compromised by increased
losses to respiration as the cells spend increased time away from the surface, and the energy costs
to achieve optimal mixing could be prohibitive.
Although Dr. Terry proposed follow-up studies using modulated light regimes that more closely
mimic those seen in algal ponds, little further research on understanding photosynthetic
efficiency in algal cultures was performed at SERI. Instead, the emphasis of the in-house
research shifted to understanding the biochemistry and molecular biology of lipid accumulation.
II.B.2.d.
Lipid Accumulation in Silicon-Deficient Diatoms
A note added to a chapter of the 1986 Annual Report (Lien and Roessler 1986) described
preliminary data on the use of Si deficiency to trigger lipid accumulation in diatoms. Silicon is
major component of diatom cell walls. Similar to the lipid trigger effect produced by N-
deficiency, Si depletion also results in a decrease in cell growth and often is accompanied by an
accumulation of lipid within the cells. However, Si (unlike N) is not a component of other
cellular macromolecules (enzymes, membranes) or cell structures such as the photosynthetic
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apparatus. Therefore, any changes in cellular biochemistry and lipid accumulation induced by Si
deficiency might be more easily interpreted than changes induced by N starvation. This work
initiated a series of experiments by Paul Roessler during the late 1980s and early 1990s on the
biochemistry and molecular biology of lipid accumulation in Si-deficient diatoms.
The first set of experiments compared the effects of Si deficiency on lipid accumulation and cell
physiology in several species of diatoms, including C. cryptica T13L, Thalassiosira pseudonana,
and Cylindrotheca fusiformis. Exponentially growing cultures were transferred to media that
contained either excess Si or limited levels of Si so that the media became Si deficient while the
cells were still growing exponentially. Cell growth, chlorophyll a content, AFDW, lipid, and
photosynthetic capacity were monitored under both conditions. In all three species, cell division
decreased as soon as the Si was depleted in the media. However the species responded
differently with respect to other physiological parameters. In C. cryptica, chlorophyll a synthesis
was almost completely inhibited after 12 hrs in Si-depleted media; C. fusiformis showed little
change in chlorophyll a synthesis after 72 hrs. T. pseudonana exhibited an intermediate effect,
with some decrease in chlorophyll a synthesis noted after 36 hours without Si. The effect on
photosynthetic capacity, measured as O2 evolution, also varied between the three species. In C.
fusiformis and C. cryptica, photosynthetic capacity decreased 33% and 58%, respectively, after
12 hours; T. pseudonana showed a steady decline in photosynthetic capability following Si-
depletion. (However, photosynthetic capacity decreased in Si-replete cultures as well during the
72 hours time course of the experiment, presumably due to the increased ratio of antenna
chlorophyll molecules versus reaction center molecules in the self-shaded, dense cultures).
The three species were also analyzed for accumulation of total biomass and lipid (Figure II.B.3.).
In C. fusiformis, biomass accumulation (measured as AFDW) for the duration of the experiment
was similar in cultures with or without sufficient Si, although lipids made up a higher percentage
of the AFDW in the Si-deficient cultures (26% versus 21% in Si-replete cells). In T.
pseudonana, synthesis of cell mass and lipid was not affected until 36 hours after Si depletion.
At this point, biomass and lipid accumulation rates decreased; however, there was little
difference in the percentage of total lipid in the cells with or without Si at the end of the 72 hours
experimental period. The situation with C. cryptica was very different. Twelve hours following
Si depletion, there was a 38% decrease in the growth rate of these cells compared to the Si-
replete culture. However, lipid synthesis continued at the same rate in the Si-deficient cells as in
the Si-replete cells, resulting in a significant increase in the lipid content of the Si-starved cells.
Interestingly, after these initial changes, the Si deficient cultures of C. cryptica showed little gain
in total AFDW or lipid during the remaining 72 hours of the experiment.
In order to determine if Si deprivation affected the composition of the lipids produced, the lipids
were extracted and analyzed for the percentage of polar versus neutral lipids present. In all three
species, the Si-deficient cultures showed a significant increase in the level of neutral lipids,
primarily TAGs. For example, the percentage of neutral lipids in Si-deficient cultures of C.
cryptica was 64%, compared to 32% in Si-replete cultures. In C. fusiformis, the percentage of
neutral lipids increased from 17%-20% to 57% in Si-deprived cultures.
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Based on these studies, C. cryptica was identified as the best candidate for further studies on the
biochemistry of lipid accumulation. To determine the effects of Si deficiency on the synthesis of
the cell components, the levels of protein, carbohydrate, and lipid were examined at various
times after Si was depleted in the cultures. During the first 12 hours, protein and carbohydrate
synthesis decreased. Lipid accumulation continued at a rate similar to that of the Si-replete
cultures. This resulted in an increase in lipid content of the Si deficient cells from 19% to 27%.
This observation was confirmed in subsequent studies that followed the incorporation of newly
assimilated carbon (as H14CO -3) into the various cell components. Si depletion resulted in a net
decrease in the rate of photosynthesis and carbon assimilation, but the individual cell fractions
were affected differently. For example, the rate of 14C accumulation into lipids decreased by
48% in the first 4 hours of Si-deprivation; the uptake of 14C into chrysolaminarin, the major
carbohydrate storage product in diatoms, decreased 84%. Therefore, the increase in lipid content
of Si-deficient cells was not due to an increase in the rate of lipid synthesis, but to a relative
decrease in the rate of synthesis of protein and carbohydrate.
Pulse-chase experiments were performed to test whether Si deficiency also caused the conversion
of non-lipid cellular components into lipids. In these experiments, Si-replete cells were labeled
with H14CO -3 for 1 hour, then transferred into Si-deficient media without labeled bicarbonate.
The amount of labeled carbon in the lipid fraction was determined at various times following
transfer to Si-free media. This experiment showed that carbon was slowly redistributed from the
nonlipid components of the cells into lipid under Si-deficient conditions, but not under Si-replete
conditions. Therefore, the accumulation of lipids in diatoms in response to Si-deficiency is
apparently due to two factors:
1. An increase in the proportion (but not the net amount) of newly assimilated
carbon that is incorporated into lipids, resulting from a disproportionate
decrease in the rate of lipid synthesis versus carbohydrate synthesis, and
2. A slow conversion of nonlipid cell material into lipids.
Fractionation of the lipids produced demonstrated that Si deprivation resulted in an increase in
the proportion of total lipid as neutral lipids, primarily TAGs, from 43% to 63% after only 4
hours of Si deficiency. Analysis of the fatty acid composition of the accumulated lipids also
showed changes induced by Si starvation. In Si-deficient cells, there was an increase in the
proportions of mono- and unsaturated fatty acids (16:1, palmitoleic acid; 16:0, palmitic acid; and
14:0, myristic acid), and a reduction in the proportions of the three major polyunsaturated fatty
acids, (16:3, 20:5, and 22:6). These results are consistent with the finding that the predominant
fatty acids found in triacylglycerol storage lipids in C. cryptica are 16:1, 16:0, and 14:1. These
shorter, more highly saturated fatty acids are also the most desirable substrates for conversion
into fatty acid methyl esters (biodiesel), as they would be less likely to polymerize during
combustion and “gum up” an engine.
Although Si depletion causes all diatoms tested to stop dividing, species responded differently
with repsect to continued accumulation of biomass and lipid. C. cryptica showed a rapid
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response to Si-depletion, with a decrease in growth accompanied by a significant increase in the
proportion of the biomass as lipid within 12 hours (the response to N starvation was usually
much slower, as the cells could utilize internal N stores). This result again emphasizes the need
to understand the kinetics of lipid accumulation in individual species under specific conditions
for cost-effective lipid production in the ponds.
Figure II.B.3. Changes in lipid mass, ash-free dry mass, and lipid content in Si-deficient
cultures of three diatoms.
A. C. fusiformis B. C. cryptica C. T. pseudonana.
Symbols: ( ) Si-deficient cultures; ( ) Si-replete cultures.
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II.B.2.e.
Isolation and Characterization of Acetyl-CoA Carboxylase from C. cryptica
To better understand the processes involved in lipid accumulation in microalgae, and to identify
potential molecular targets for genetic manipulation, studies were initiated to examine the effects
of Si deficiency on the enzymatic pathways involved in lipid and carbohydrate synthesis in C.
cryptica. One possibility is that the increased levels of storage lipid in cells exposed to Si
starvation could result from shifts in the relative activities of one or more enzymes in the lipid
biosynthesis pathway. Acetyl-coenzyme A (acetyl-CoA) is known to be the immediate precursor
of fatty acid synthesis, but the source of this compound varies in different organisms. For
example, in mammalian cells, acetyl-CoA used in cytosolic fatty acid synthesis is produced from
citrate via the action of ATP citrate lyase. In plants, acetyl-CoA can be produced in the
chloroplasts from pyruvate, catalyzed by pyruvate dehydrogenase. Alternatively, acetyl-CoA
could be produced by the mitochondrial pyruvate dehydrogenase. In this case, the acetyl-CoA
(which cannot diffuse across the organellar membranes) would be broken down to acetate and
free CoA by acetyl-CoA hydrolase. Acetate would diffuse to the chloroplast and become
incorporated into acetyl-CoA by the action of acetyl-CoA synthetase. Once acetyl-CoA is
produced, it is then used as a substrate by acetyl-CoA carboxylase (ACCase) to produce malonyl
CoA. Malonyl-CoA is a substrate for fatty acid synthase and this reaction is considered to be the
first committed step in fatty acid synthesis.
These pathways had not previously been well-characterized in diatoms. To better understand the
lipid synthesis pathways, Roessler first looked for the presence of these enzymes in extracts of C.
cryptica, but found no citrate lyase activity. However, acetyl-CoA hydrolase, acetyl-CoA
synthetase, and ACCase activity were all present. Enzyme activities were studied in Si-replete
and Si-deficient cells (Figure II.B.4). The level of acetyl-CoA synthetase activity was similar
under both conditions; however, the level of ACCase activity was two fold higher in Si-deficient
cells after 4 hours, and four fold higher after 15 hours. Based on subsequent studies using
protein synthesis inhibitors, the increased specific activity of the ACCase was believed to result
from an increase in expression of the ACCase gene (Roessler 1988a; 1988c).
ACCase is a biotin-containing enzyme that catalyses the carboxylation of acetyl-CoA to form
malonyl-CoA. This reaction entails two partial reactions: the carboxylation of biotin, followed
by the transfer of the carboxyl group from biotin to acetyl-CoA. In bacteria, the enzyme is
composed of four non-identical subunits. However, in eukaryotes, biotin binding, biotin
carboxylation, and carboxyl-transfer all occur on a single large multifunctional protein; the
functional ACCase occurs as a multimer of this polypeptide. ACCase had previously been
shown to play a key regulatory role in the rates of fatty acid synthesis in both animal and plant
systems. A project was initiated to isolate and characterize ACCase from C. cryptica to clarify
the role of this enzyme in lipid accumulation induced by Si starvation, and to compare the
microalgal enzyme with those isolated from plants, animals, yeast, and bacteria.
The enzyme was purified from C. cryptica by a combination of (NH4)2SO4 precipitation, gel
filtration chromatography, and affinity chromatography based on the affinity of biotin to avidin.
Consistent with ACCase enzymes isolated from other eukaryotes, C. cryptica ACCase was found
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to consist of a homo-tetramer of 185 kDa subunits. The activity of the enzyme was assayed by
the incorporation of 14C bicarbonate into malonyl-CoA, and other factors were identified that
affect the stability and activity of the enzyme. As seen for other ACCases, the enzyme required a
slightly alkaline pH for optimum activity (pH 8.2), although the enzyme was most stable when
stored at pH 6.5. The enzyme was also stabilized by sulfhydryl reductants (i.e., dithiothreitol),
citrate, NaCl, and KCl; divalent metal cations (Mg2+ or Mn2+) were required for activity. A
number of cellular metabolites were also tested for their affects on ACCase activity. The enzyme
was inhibited by products of the ACCase reaction, including malonyl-CoA, ADP, and NaH2PO4,
and also by palmitoyl-CoA, but it was not affected by various glycolytic or photosynthetic
intermediates or by free CoA. Two herbicides that inhibit ACCases from monocot plants were
also had little or no effect on C. cryptica ACCase. Thus, the ACCase from this diatom was
found to be similar to higher plant ACCase enzymes in that it is composed of multiple, identical,
multifunctional subunits. In addition, the Kms for the ACCase substrates (acetyl-CoA, MgATP,
and bicarbonate) in C. cryptica were similar to those found in plant ACCase enzymes (Roessler
1989; 1990).
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Figure II.B.4. Activity of various enzymes in Si-replete and Si-deficient C. cryptica.
A. (Top) - Activities of several enzymes in Si-replete and Si-deficient C. cryptica cells. There is
no significant difference in the activities of UDPglucose pyrophosphorylase, acetyl-CoA
synthetase, or citrate synthase in the cells under the two conditions. However, there is a relative
increase in acetyl-CoA carboxylase activity, and a decrease in chrysolaminarin synthase activity
in Si-deficient cells.
B (Bottom) - Graph showing the activity of ACCase in C. cryptica cells. Exponential-phase cells
were transferred into Si-free media at 0 hr. At 6 hr, (arrow), the culture was split and 1.8 mM
Na2SiO3 was added to one culture. ( ) Si-deficient cells; ( ) Si-replete cells.
(Source: Roessler 1988a).
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II.B.2.f.
Cloning of the Acetyl-CoA Carboxylase Gene from C. cryptica
Work on the key role of ACCase in lipid biosynthesis in other plant and animal systems,
suggested that this enzyme might be a viable target for genetic manipulation in order to increase
lipid production in microalgae. This notion was further supported by the work at SERI that
showed changes in ACCase activity in Si-starved C. cryptica cells.
The next step was to isolate the ACCase gene from a microalgal species. Although the ACCase
gene had been isolated from yeast, rats, and the bacteria E. coli, the gene had not previously been
isolated from any photosynthetic organism. In 1990 and 1991, Dr. Roessler took a sabbatical
from SERI to work with Dr. John Ohlrogge at Michigan State University. Dr. Ohlrogge studies
lipid biosynthetic pathways in higher plants. This work was partially funded by a Plant Biology
Postdoctoral Fellowship to Paul Roessler from the National Science Foundation. The goal of this
collaboration was to clone and characterize the ACCase gene from C. cryptica. To accomplish
this task, the purified ACCase protein was first cleaved with cyanogen bromide (CNBr); the
peptides generated were separated by SDS-PAGE, purified, and several of these peptides were
analyzed to determine their amino acid sequence. (This work was done in collaboration with
Calgene, a plant biotechnology company in Davis, California.)
The amino acid sequences were used to design degenerate oligonucleotide primers that were used
in a polymerase chain reaction (PCR) to amplify an ACCase gene fragment from C. cryptica’s
total DNA. A 32P-labeled RNA transcript was produced from the ACCase DNA and used to
screen a genomic library of C. cryptica DNA. A 14 kb cloned fragment that hybridized to the
ACCase probe was cleaved into smaller fragments that were subcloned, sequenced, and analyzed
for the presence of open reading frames (ORFs) and non coding intron sequences. This analysis
showed that the ACCase gene from C. cryptica contains approximately 6.3 kbp of coding
sequence, separated by a 447 bp intron close to the 5' end, and a 73 bp intron just upstream from
the biotin binding site. The protein predicted by this nucleotide sequence would contain 2,089
amino acids and have a molecular weight of 230 kDa. This is somewhat larger than the
molecular weight of 185 kDa estimated by SDS-PAGE, discussed earlier. This discrepancy
could be accounted for by inaccuracies inherent in using SDS-PAGE to estimate protein size,
particularly for large proteins, and the probability of a signal sequence on the ACCase enzyme
for targeting the protein to the chloroplast. Post-translational cleavage of the signal would result
in a mature protein smaller than predicted from the primary DNA sequence.
The deduced amino acid sequence of the ACCase from C. cryptica was compared with known
sequences from yeast and rat (Figure II.B.5). The algal sequence showed approximately 50%
identity with other sequences in the biotin carboxylase domain (at the amino terminus of the
protein) and in the carboxyl transferase domain (at the carboxyl terminus of the sequence).
However, the central portion of enzyme showed only about 30% identity with the yeast and rat
enzymes, with most of the similarity in this region occurring in the biotin binding domain. This
suggests that the central region of the protein probably functions primarily as a linker or spacer
that moves the carboxylated biotin residue closer to the carboxyl transferase domain. The
isolation of the ACCase gene from C. cryptica was an important step for the ASP; significantly
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in that this was the first time a full-length sequence for an ACCase gene had been isolated from a
photosynthetic organism. NREL was granted a patent on this gene in 1996, and there has been
interest from at least one major plant biotechnology company in using this gene to manipulate
oils and lipids in higher plants (Roessler and Ohlrogge 1993; Roessler et al. 1994).
The availability of the purified ACCase protein and the cloned ACCase gene allowed NREL
researchers to study the effects of Si deficiency on ACCase gene expression. Southern blots, in
which a fragment of the cloned ACCase gene was used as a probe to analyze C. cryptica DNA,
indicated that there is probably only a single copy of the ACCase gene in C. cryptica. ACCase
gene fragments were used to monitor mRNA levels in Si-deficient cells using the ribonuclease
protection assay (RPA). ACCase mRNA levels increased 2.5-fold between 2 and 6 hours after
the beginning of Si-deprivation as compared to Si-replete cells, but then decreased to the control
level after 23 hours. Thus, Si concentration appears to affect ACCase gene expression at the
level of gene transcription, possibly as a result of increased promoter activity and/or by altering
the rates of mRNA degradation. ACCase activity was also measured in cell lysates from Si-
starved cultures; enzyme activity increased steadily over 23 hours to a final level 4.5-fold higher
than that of Si-replete cultures. The increased level of ACCase activity was correlated with an
increase in the amount of ACCase protein, as determined by Western blotting using anti-ACCase
polyclonal antibodies. Although Si deficiency caused the levels of ACCase mRNA and protein
to increase, the kinetics of the two processes were different.
These results supported the hypothesis that diatoms could respond to Si deprivation by altering
the activity of enzymes involved in lipid biosynthesis to partition more fixed carbon into storage
lipids. If the activity of ACCase could be increased using mutation or genetic manipulation, it
might be possible to produce a strain with constitutively high levels of TAG synthesis. This was
a major premise of the genetic engineering experiments discussed in Section II.B.3.
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Figure II.B.5. Schematic diagram indicating regions of similarity among the primary
sequences of ACCase from C. cryptica, rat, and yeast. Regions (≥20 amino acids) that are
present in all three sequences and that exhibit statistically significant homology (p<10-6) are
indicated by thickened areas.
(Source: Roessler and Ohlrogge 1993).
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II.B.2.g.
Biochemistry of Lipid Synthesis in Nannochloropsis
From 1992 to 1995, Dr. Jane Schneider worked at NREL with Dr. Roessler on a project funded
by the United States–Israel Binational Agricultural Research and Development Fund. The
research was performed in collaboration with Dr. Assaf Sukenik and other scientists at the Israel
Oceanographic and Limnological Institute in Haifa. The goal of the research was to understand
the biochemistry of lipid synthesis in the eustigmatophyte Nannochloropsis sp., particularly with
respect to fatty acid desaturation pathways. There has been a significant amount of research on
lipid synthesis pathways in higher plants, and the pathways hve been assumed to be similar in
lipogenic algae. However, unlike plants, nutrient deprivation produces major effects on the
quantity and quality of lipids in algae; so there are likely to be significant differences in the
biochemical pathways. In addition, like many algae, Nannochloropsis contains a high proportion
of long fatty acids (i.e., C-20, C-22) with a high degree of unsaturation (20:5). These very long
chain-polyunsaturated fatty acids (VLC-PUFAs) are important in aquaculture applications as
they improve the nutritional quality of feed for fish and shellfish, and have nutritional and
pharmaceutical applications for humans. Understanding the details of the biochemistry of lipid
accumulation in microalgae could help researchers develop strategies for genetic manipulation of
lipid synthesis pathways to affect not only the quantity but also the quality (chain length, degree
of desaturation) of lipids produced for optimal biodiesel performance.
In one set of experiments, pulse-chase radiolabeling was used to study de novo synthesis of lipids
in Nannochloropsis. Exponentially growing cells under low light were fed 14C-bicarbonate or
acetate for 1 hour. The cells were then washed and allowed to grow in unlabeled medium. At
various time points, cells were removed and lipids extracted. The substrates resulted in a
different distribution of labeled carbon in the lipids and fatty acids. The work demonstrated the
probable existence of two pools of malonyl-CoA used as substrates for fatty acid synthesis, and
resulted in a new model for the sites of desaturation of fatty acids and the identification of a new
acyltransferase activity in this organism. In another set of experiments, Nannochloropsis cells
were mutagenized using UV light and screened for unusual fatty acid profiles using gas
chromatography. This work resulted in the isolation of a mutant deficient in 20:5 fatty acids,
probably due to a mutation affecting a desaturase enzyme that utilizes 20:4 fatty acids as
substrate.
These experiments will not be described in detail here, primarily because the funding for this
research did not come from DOE. In addition, it would require a lengthy discussion of the details
of fatty acid synthesis and processing for the reader to understand the relevance of the findings.
Readers interested in the details of this research are referred to the three publications that resulted
from this research (Schneider and Roessler 1994; Schneider et al. 1995; Schneider and Roessler
1995).
II.B.2.h.
Biochemistry and Molecular Biology of Chrysolaminarin Synthesis
Another strategy that has been proposed to increase the proportion of lipid in algal cells is to
limit the flow of newly assimilated carbon into other cellular pathways. Many diatoms, including
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C. cryptica, can produce a significant amount of a storage carbohydrate called chrysolaminarin, a
β-(1→3)-linked glucan. Although some data were available on the chemistry of this compound,
the biochemical pathways involved in the synthesis of chrysolaminarin were not known. The
synthesis of most storage polysaccharides involves the condensation of nucleoside diphosphate
sugars; for example, starch is formed in plants from ADPglucose, and UDPglucose is used to
form sucrose in plants and glycogen in mammalian cells. These reactions are catalyzed by
nucleoside diphosphate sugar pyrophosphorylases, such as UDPglucose pyrophosphorylase
(UGPase), which catalyzes the following reaction:
glucose-1-phosphate + UTP → UDPglucose + PPi
Roessler first looked for nucleoside diphosphate sugars pyrophosphorylases in cell-free extracts
of C. cryptica, and identified significant amounts of UGPase activity. The enzyme activity was
characterized to optimize in vitro assay conditions. The enzyme was activated in the presence of
Mn2+ and Mg2+ but was not affected by 3-phosphoglycerate or inorganic phosphate; these
chemicals are known to affect the activity of ADPglucose pyrophosphorylase in higher plants.
Incubation of cell-free extracts with UDP[14C]glucose resulted in the incorporation of the labeled
carbon into a β-(1→3)-glucan polymer, presumably chrysolaminarin, supporting the role of
UGPase in chrysolaminarin synthesis in diatoms. Subsequent studies identified a second
enzyme, UDPglucose:β-(1→3)-glucan-β-glucosyltransferase (also known as chrysolaminarin
synthase), which catalyzes the synthesis of glucan using UDPglucose as substrate. The specific
activity of both enzymes was examined in C. cryptica cells under Si-replete and Si-depleted
conditions. The activity of UDPglucose pyrophosphorylase was similar under both conditions;
however, the activity of chrysolaminarin synthase decreased by 31% in Si-deficient cells,
suggesting that the partitioning of newly assimilated carbon into lipid may be partly due to
decreased synthesis or inhibition of the chrysolaminarin synthase enzyme (Roessler 1987;
1988a).
Further research on UGPase in C. cryptica was put on hiatus for several years while the emphasis
was on ACCase (discussed earlier) and on the development of genetic engineering protocols for
microalgae (discussed in Section II.B.3.). However, the development of a successful genetic
transformation system for C. cryptica, as well as advances in techniques that allow the down-
regulation of particular genes (i.e., antisense RNA, ribozymes) generated a renewed interest in
UGPase. NREL researcher Eric Jarvis spent 6 months working at Ribozyme Pharmaceuticals,
Inc., a biotechnology company in Boulder, Colorado, learning about these new methods.
Antisense RNA is a method in which a cell is transformed with a synthetic gene that produces an
RNA sequence complimentary to a specific messenger RNA (mRNA). Although the exact
mechanism is not clear, the antisense RNA prevents translation from its complimentary mRNA,
effectively lowering the level of that particular protein in the cell. Ribozymes are also RNA
molecules produced by synthetic genes that can bind to, and cleave, very specific RNA
sequences. Ribozymes can be designed to degrade specific mRNA molecules, effectively
decreasing expression of a specific gene.
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In C. cryptica, chrysolaminarin can make up 20%-30% of the cell dry weight, and thus
chrysolaminarin synthesis pathways presumably compete for newly fixed carbon with the
pathways for lipid biosynthesis. Dr. Jarvis and Dr. Roessler proposed that inhibiting
chrysolaminarin production by inhibiting one or more genes in the carbohydrate synthesis
pathway could result in the flow of more carbon into lipid production. Based on the earlier
studies on chrysolaminarin synthesis, Dr. Jarvis initiated an effort to isolate the UGPase gene
from C. cryptica DNA. A fragment of the C. cryptica UGPase gene was first produced by the
PCR using degenerate oligonucleotide primers based on conserved sequences from known
UGPase genes from potato, human, yeast, and Dictyostelium. This fragment was cloned and
sequenced; the derived amino acid sequence showed 37% identity with the corresponding
sequence from potato UGPase, confirming that a C. cryptica UGPase gene fragment had been
cloned. The cloned PCR product was then used as a probe to isolate a genomic DNA clone
containing the entire C. cryptica UGPase gene from a lambda library. One clone contained a
DNA segment with a single long open reading frame, the 5' end of which showed homology to
known UGPase genes. Surprisingly, the 3' end of this DNA showed homology to known genes
coding for the enzyme phosphoglucomutase (PGMase). In chrysolaminarin synthesis, PGMase
catalyzes the following reaction:
glucose-6-phosphate → glucose-1-phosphate
The glucose-1-P produced in this reaction is the substrate for UGPase in the production of
UDPglucose, an immediate precursor of chrysolaminarin, as described earlier. Although
PGMase and UGPase are thought to catalyze successive steps in the chrysolaminarin
biosynthesis pathway, this was the first report of a naturally occurring fusion of these two genes
in any organism. The C. cryptica UGPase/PGMase gene, designated upp1, contained 3,640 bps,
including 3 introns, and coded for a protein composed of 1,056 amino acids, with a molecular
weight of 114.4 kd.
To confirm that the protein coded for by upp1 actually catalyzes both the UGPase and PGMase
reactions, the protein was isolated from extracts of C. cryptica by sequential column
chromatography (ion exchange, hydroxylapatite, and gel filtration). The two enzyme activities
co-eluted throughout the purification procedure, and all fractions containing UGPase/PGMase
activity contained a 114 kd protein as determined by SDS-PAGE. These results supported the
presence of both enzyme activities in C. cryptica on a single multifunctional protein. A patent
submitted by NREL on this unique gene was allowed in October 1996. The research at NREL
involving attempts to manipulate upp1 gene expression to affect carbon partitioning in C.
cryptica will be discussed in Section II.B.3. of this report.
Publications:
Bergeron, P.W., Corder, R.E., Hill, A.M., Lindsey, H.; Lowenstein, M.Z. (1983) SERI Biomass
Program Annual Technical Report: 1982. Solar Energy Research Institute, Golden, Colorado,
SERI/TR-231-1918, 83 pp.
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Corder, R.E.; Hill, A.M.; Lindsey, H.; Lowenstein, M.Z.; McIntosh, R.P. (1984) SERI Biomass
Program FY 1983 Annual Report, Solar Energy Research Institute, Golden, Colorado, SERI/TR-
231-2159, 107 pp.
Hill, A.; Feinberg, A.; McIntosh, R.; Neenan, B.; Terry, K. (1984) “Fuels from microalgae:
Technical status, potential, and research issues.” Solar Energy Research Institute Report,
SERI/SP-231-2550.
Jarvis, E.E.; Dunahay, T.G.; Brown, L.M. (1992) “DNA nucleoside composition and methylation
in several species of microalgae.” J. Phycol. 28:356-362.
Lien, S. (1981a) “Photobiological production of fuels by microalgae.” In Energy from Biomass:
Proceedings of the First E.C. Conference (Palz, W.; Chartier, P.; Hall, D.O., eds.), Applied
Science Publishers, London, pp. 697-702.
Lien, S. (1981b) “Algal research at SERI: A basic research on photobiological production of
fuels and chemicals in microalgae.” Proceedings of the July 1981 Subcontractors' Review
Meeting, Aquatic Species Program, Solar Energy Research Institute, Golden, Colorado,
SERI/CP-624-1228, pp. 59-65.
Lien, S. (1982) “Studies on the production and accumulation of oil and lipids by microalgae.” In
Proceedings of the SERI Biomass Program Principal Investigators' Review Meeting, Aquatic
Species Program Reports, June 23-25, 1982, Solar Energy Research Institute, Golden, Colorado,
SERI/CP-231-1808, pp. 45-54.
Lien, S.; Roessler, P. (1986) “The energetics of biomass and lipid production by lipogenic
microalgae under nitrogen deprivation.” Aquatic Species Program Review: Proceedings of the
March 1985 Principal Investigators’ Meeting, Solar Energy Research Institute, Golden,
Colorado, SERI/CP-231-2700, pp. 100-117.
Lien, S.; Spencer, K.G. (1983) “Algal oil production and lipid metabolism research.” Aquatic
Species Program Review: Proceedings of the March 1983 Principal Investigators' Meeting, Solar
Energy Research Institute, Golden, Colorado, SERI/CP-231-1946, pp. 3-18.
Ohlrogge, J.; Jaworski, J.; Post-Beittenmiller, D.; Roughan, G.; Roessler, P.; Nakahira, K. (1993)
“Regulation of flux through the fatty acid biosynthesis pathway.” In Biochemistry and
Molecular Biology of Membrane and Storage Lipids of Plants (Murata, N.; Somerville, C.R.,
eds.), American Society of Plant Physiologists, Rockville, Maryland, pp. 102-112.
Raymond, L.P. (1983a) “Aquatic biomass as a source of fuels and chemicals.” Conference
report: First U.S.-China Conference on Energy, Resources, and Environment. Beijing, People’s
Republic of China, Nov. 7-12, 1982. Solar Energy Research Institute, Golden, Colorado,
SERI/TP-231-1699, 8 pp.
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Roessler, P.G. (1987a) “Biochemical aspects of lipid accumulation in silicon-deficient diatoms.”
FY 1986 Aquatic Species program Annual Report, Solar Energy Research Institute, Golden,
Colorado, SERI/SP-231-3071.
Roessler, P.G. (1987c) “Lipid accumulation in silicon-deficient diatoms.” In The Metabolism,
Structure, and Function of Plant Lipids, (Stumpf, P.K.; Mudd, M.B.; Nes, W.D., eds.), Plenum,
Publishing Corp., pp. 649-651.
Roessler, P.G. (1987d) “UDPglucose pyrophosphorylase activity in the diatom Cyclotella
cryptica. Pathway of crysolaminarin biosynthesis.” J. Phycol. 23:494-498.
Roessler, P.G. (1988a) “Changes in the activities of various lipid and carbohydrate biosynthetic
enzymes in the diatom Cyclotella cryptica in response to silicon deficiency.” Arch. Biochem.
Biophys. 267:521-528.
Roessler, P.G. (1988b) “Characteristics of abrupt size reduction in Synedra ulna
(Bacillariophyceae).” Phycologia 27:294-297.
Roessler, P.G. (1988c) “Effects of silicon deficiency on lipid composition and metabolism in the
diatom Cyclotella cryptica.” J. Phycol. 24:394-400.
Roessler, P.G. (1989) “Purification and characterization of acetyl-CoA carboxylase from the
diatom Cyclotella cryptica.” Aquatic Species Program Annual Report, Solar Energy Research
Institute, (Bollmeier, W.S.; Sprague, S., eds.), SERI/SP-231-3579, pp. 121-129.
Roessler, P.G. (1990) “Purification and characterization of acetyl-CoA carboxylase from the
diatom Cyclotella cryptica.” Plant. Physiol. 92:73-78.
Roessler, P.G.; Bleibaum, J.L.; Thompson, G.A.; Ohlrogge, J.B. (1994) “Characteristics of the
gene that encodes acetyl-CoA carboxylase in the diatom Cyclotella cryptica.” In Recombinant
DNA Technology II (Bajpai, K.; Prokop, A., eds.), Ann. N.Y. Acad. Sci. 721:250-256.
Roessler, P.G.; Ohlrogge, J.B. (1993) “Cloning and characterization of the gene that encodes
acetyl-coenzyme A carboxylase in the alga Cyclotella cryptica.” J. Biol. Chem. 268:19254-
19259.
Schneider, J.C.; Roessler, P. (1994) “Radiolabeling studies of lipids and fatty acids in
Nannochloropsis (Eustigmatophyceae), an oleaginous marine alga.” J. Phycol. 30:594-598.
Schneider, J.C.; Livine, A.; Sukenik, A.; Roessler P.G. (1995) “A mutant of Nannochloropsis
deficient in eicosapentaenoic acid production.” Phytochem. 40:807-814.
Schneider, J.C.; Roessler, P.G. (1995) “A novel acyltransferase activity in an oleaginous alga.” In
Plant Lipid Metabolism (Kader, L.C.; Mazliak, P., eds.), Kluwer Academic Publishers, the
Netherlands, pp. 105-107.
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Terry, K.L. (1984a) “Microalgal technology research at SERI: Modulated light photobiology.”
Aquatic Species Program Review: Proceedings of the April 1984 Principal Investigators’
Meeting, Solar Energy Research Institute, Golden, Colorado, SERI/CP-231-2341, pp. 160-169.
Tornabene, T.G.; Holzer, G.; Lien, S.; Burris, N. (1983) “Lipid composition of the nitrogen
starved green alga Neochloris oleoabundans.” Enzyme Microb. Technol. 5:435-440.
Walsh, R.G. (1984) “Microalgal technology research at SERI: The biochemistry of lipid trigger
mechanisms in microalgae—alteration of lipid metabolism during transition from rapid growth to
rapid lipid accumulation.” Aquatic Species Program Review: Proceedings of the April 1984
Principal Investigators Meeting, Solar Energy Research Institute, Golden, Colorado, SERI/CP-
231-2341, pp. 170-183.
Patents:
Roessler, P.G; Ohlrogge, J.B., “Gene Encoding Acetyl-Coenzyme A Carboxylase”, U.S. Patent
5,559,220, issued 9/24/96.
Jarvis, E.E.; Roessler, P.G. “Isolated-Gene Encoding an Enzyme with UDP-Glucose
Pyrophosphorylase Activities from Cyclotella cryptica.” Allowed 10/24/96.
II.B.3
Manipulation of Lipid Production in Microalgae via Genetic
Engineering
II.B.3.a. Introduction
The overall goal of the ASP was to cost-effectively produce biodiesel fuel from microalgal lipids.
The early laboratory efforts focused on the characterization of microalgae with regard to traits
deemed desirable for mass culture and fuel production, i.e., rapid growth, tolerance to
environmental fluxes, and high production of TAG storage lipids. Although numerous promising
organisms were identified, no individual strain demonstrated rapid growth with constitutively
high lipid production. Although high lipid levels could be induced in many strains by starving
the cells for an essential nutrient such as N or Si, the increase in lipid was accompanied by a
decrease in cell division and total productivity.
During the late 1980s and 1990s, the direction of the laboratory research efforts at NREL shifted
to the study of the biochemical pathways involved in lipid synthesis, with the goal of identifying
targets for genetic manipulation. As discussed earlier, the desirable traits for biodiesel
production (high productivity and high lipid content) were found to be mutually exclusive
conditions in the organisms studied. Therefore, it was decided to use mutagenesis or genetic
engineering to manipulate the algal biosynthetic pathways to produce algal strains with
constitutively high lipid levels. Another possibility would be to engineer an organism in which
lipid synthesis could be regulated by inducing or repressing key genes. Very little was known
about the molecular biology of oleaginous microalgae and the genetic regulation of lipid
biosynthesis pathways, so a concentrated research effort in this direction was deemed critical to
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the success of the biodiesel project. Another reason for the shift to research on genetic
manipulation of algae was more practical. Funding levels for ASP decreased during this period
from the high funding levels in the mid-1980s that had allowed large numbers of subcontractors
and the development of the Outdoor Test Facility in Roswell, New Mexico (Section II.B.5.).
Laboratory experiments emphasizing biochemistry, molecular biology, and genetic engineering
could be performed with a limited budget and few personnel.
This section of the report will describe the in-house research efforts at NREL to develop high
lipid algae by genetically manipulating selected oleaginous strains. Microalgae generally
reproduce asexually by simple fission. Many strains can also produce sexually, but the
conditions required to induce algal sexual reproduction in the laboratory are not known for most
species. Thus, genetic manipulation by classical “breeding” was not an option for the algae. The
approaches explored at NREL were (1) mutagenesis and selection; and (2) genetic engineering.
The information summarized in this report was taken from annual reports, scientific publications
and meeting reports. No annual reports were generated by the ASP after 1993, and no quarterly
reports after September 1995, so some of the most recent information presented was derived
from the personal experience of Terri Dunahay and discussions with former coworkers (Paul
Roessler and Eric Jarvis).
II.B.3.b. Mutagenesis
and
Selection
Work by SERI/NREL subcontractors in the early 1980s supported the idea that there is
significant genetic variation within algal populations (i.e., Gallagher, Section II.B.1.c.).
Therefore, one possible method for producing high lipid algal strains would be selection of
natural genetic variants with desired traits, such as high lipid levels or increased tolerance to high
salinity or temperature. The limiting factor to this approach has always been the difficulty of
selecting for individuals exhibiting a desired trait among a large population of cells. The use of
lipophilic dyes such as Nile Blue or Nile Red, coupled with flow cytometry, showed some
potential for isolation of high lipid strains of microalgae (see work by Solomon and Cooksey,
Sections II.B.1.e and f). It was not clear, however, that the variations detected in subpopulations
of cells were the result of genetic variations that would be passed on to progeny.
An alternative approach is to induce genetic variation in a population of cells using mutagenesis.
Again, the ability to select for the desired trait is a limiting factor, but the production of large
numbers of mutants by artificial means is a proven method for generating organisms with
heritable traits, often the result of a mutation within a single gene. As a prelude to the initiation
of mutagenesis and selection experiments with oleaginous microalgae, NREL researcher Ruth
Galloway performed a series of experiments designed to understand the factors required to
produce mutants in microalgae. These included media requirements for growth, the ability to
form colonies on agar plates, sensitivity to herbicides and other growth inhibitors, and the
sensitivity of algal strains to mutagens such as UV light or fluorodeoxyuridine (Galloway 1990).
Nine algal strains from the SERI Culture Collection were tested, including organisms from three
classes: the chlorophyceae (M. minutum MONOR1 and MONOR2), the eustigmatophyceae
(Nannochloropsis (NANNP1 and NANNP2), and the bacilliarophyceae (C. cryptica T13L, C.
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mulleri CHAET9, Amphora AMPHO17, Nitzschia pusilla NITSC12, and N. saprophila
NAVIC1).
Growth of each strain was evaluated qualitatively after spotting the cultures onto media
containing nitrogen or carbon sources, or after growing the cells under phototrophic,
mixotrophic, or heterotrophic conditions. Cell growth was also evaluated in the presence of a
large number of growth inhibitors including various antibiotics and herbicides. Although there
was some variability between the algal strains, several generalizations could be made. Most
strains could use either NO -
+
3 or NH4 as a nitrogen source. Mixotrophic growth on various
carbon sources was more variable, and only AMPHO17, MONOR2, and CYCLOT13L were able
to grow heterotrophically, using glucose as a carbon source. The ability to grow
heterotrophically would be important for the isolation of photosynthetic mutants.
Predictably, antibiotics that inhibit bacterial cell wall synthesis such as ampicillin and
carbenicillin did not inhibit the growth of the algal strains. Antibiotics that inhibit bacterial
protein synthesis by binding to the 30S ribosome showed variation in their effects on algal
growth. For example, all strains tested grew well on kanamycin and neomycin and showed no
growth on erythromycin; while the growth response differed for the strains on spectinomycin and
streptomycin. Whether this result was due to differences in 30S (organellar) ribosomal structure
between the algal strains, to differences in uptake of the antibiotics by the individual strains, or to
other factors that affect sensitivity, is unclear. All strains showed sensitivity to photosynthesis
inhibitors diuron, metronidazol, and atrazine, and to the herbicide glyphosate (“RoundUp”),
which affects the shikimic acid pathway. However, sensitivity varied between the strains to
compounds that affect the enzyme acetolactate synthase and to chemicals that inhibit microtubule
synthesis. (The details of these growth experiments can be found in Tables 2, 3, 4, 5, and 7 of
Galloway 1990). Many of the growth inhibitors used in this study affect specific proteins in the
target organism, and many of these proteins have been well characterized in a number of
systems. Isolating the corresponding gene from an algal mutant using heterologous gene probes
to characterize the mutation and/or to use the mutant gene as a selectable marker for
transformation studies should be relatively easy.
Attempts were also made to generate mutants in the algal strains by exposing the cells to UV
light or to fluorodeoxyuridine, followed by plating the cultures on toxic levels of various growth
inhibitors. Using UV mutagenesis, streptomycin-resistant mutants were obtained in MONOR2,
as well as glyphosate-resistant mutants in both strains of Nannochloropsis, sulfometuron methyl-
resistant mutants in NANNP1, and atrazine-diuron-resistant mutants in NAVIC1. In addition,
tunicamycin-resistant mutants of NAVIC1 were produced following treatment with
fluorodeoxyuridine. Mutants were not obtained for the other diatoms, CYCLOT13L, CHAET9,
or NITZS12, whether this was due to poor colony formation by these strains, inefficacy of the
mutagen, or inappropriately high levels of the selective agent is not known. One interesting point
was that the green algal strains, Monoraphidium and Nannochloropsis, produced mutants with
traits thought to be due to recessive nuclear gene mutations (i.e., glyphosate resistance or
photosynthesis mutants). On the other hand, in Navicula, the only diatom in which mutants were
generated, the types of mutations produced were indicative of dominant mutations, i.e.,
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atrazine/diuron resistance (resulting from a chloroplast gene mutation) or resistance to
tunicamycin, an inhibitor of n-glycosylation. These results indicate that Monoraphidium and
Nannochloropsis are probably haploid; the diatoms are diploid. Design of strategies for
generation of algal mutants will have to consider the ploidy of the target organism. For example,
generating nitrate reductase-deficient mutants for use in a genetic transformation system using
homologous selectable markers (described in detail later) should be relatively simple in haploid
strains, but would be much more difficult in diploids. For the diatoms, a better approach would
be to utilize a dominant gene as a selectable marker, such as a mutant form of the enzyme
acetolactate synthase (discussed later), or a heterologous gene such as the neomycin
phosphotransferase II (NPTII). The latter gene confers drug resistance by inactivating antibiotics
such as kanamycin or geneticin (G418).
In summary, the research performed by Dr. Galloway demonstrated the potential to produce algal
mutants with a wide variety of phenotypes, particularly in the green algae, using simple
mutagenesis and selection techniques. It would be important to first optimize and understand the
growth conditions for the target strains. The conditions to be used for selection (inhibitor
specificities and concentrations) should be determined for each strain. However, the generation
of mutants will probably be more useful as a tool in developing selectable marker systems, rather
than as a method to directly produce high lipid algal strains, primarily because there is no simple
way to screen for high-lipid phenotypes. The use of mutagenesis to develop of homologous
selectable marker systems for algal transformation will be discussed in detail later.
Mutagenesis and selection was used successfully in another study at NREL to generate mutants
in one aspect of lipid synthesis, fatty acid desaturation (Schneider et al. 1995, described in
Section II.B.2.g.). In this experiment, UV mutagenized cells of Nannochloropsis were allowed
to form colonies, then grown in small-scale liquid cultures. Lipids were extracted from each
sample and analyzed by gas chromatography for any significant alteration in the proportion of
fatty acids. This project resulted in the identification of a mutant lacking in 20:5 fatty acids,
apparently due to a mutation in a 20:4 desaturase. In this case, a simple screen was used to look
for changes in a quantitative trait. This result suggests that, with the right method to screen for
mutants with the desired properties, mutagenesis could result in microalgae with altered lipid
compositions. However, this project was very labor intensive, with hundreds of colonies
screened to identify a single mutant.
II.B.3.c.
Development of a Genetic Transformation System for Microalgae
Introduction:
During the past 2 decades, manipulation of organisms via genetic engineering has become
routine in a number of animal, bacterial, fungal, and plant systems. However, before the research
was done at NREL, very little work in this area had been done with microalgae. In fact, the only
species for which there was a reproducible transformation system was the single-celled,
flagellated green alga C. reinhardtii, which is studied extensively in laboratories as a model
photosynthetic cell. The focus of the research in the ASP during the early 1990s was to develop
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genetic transformation methods for microalgae with potential for biodiesel production. Based on
the collection and screening efforts of the 1980s, this approach was considered to have the
highest potential to produce organisms with high constitutive lipid levels, and to use genetic
manipulation to understand the molecular regulation of lipid synthesis in the oleaginous algae.
Studies on the biochemistry and molecular biology of lipid production in C. cryptica had
identified acetyl-CoA carboxylase as a key regulatory enzyme in lipid synthesis (Section
II.B.2.e.). One initial goal was to introduce additional copies of this gene into C. cryptica with
the hope of increasing the activity of the enzyme and the flux of fixed carbon into lipid.
Several projects will be discussed in the following section of this report that were directed
towards the development and use of genetic transformation systems in oleaginous microalgae.
The initial approach was to use use available promoters and marker genes that were reported to
function in other eukaryotic systems. Various methods were also tried to get DNA into the cell,
initially focusing on enzymatically removing the cell wall or perturbating the cell membrane
using electroporation. Unsuccessful experiments represented a “Catch 22” scenario, as negative
results could mean either the DNA was not getting into the cells, or the DNA entered but could
not be expressed at detectable levels. Subsequent experiments were designed to increase the
understanding of the processes involved in DNA uptake and expression and to increase the
probability of obtaining transformants by developing methods for detecting rare transformation
events within a population of cells.
The projects that will be discussed here include a basic study on the DNA composition of
microalgal strains, with implications for the choice of reporter or marker genes used to monitor
gene expression in transgenic algae. Other aspects of the research that will be discussed include:
• the use of the luciferase gene to monitor DNA uptake and expression in
Chlorella protoplasts,
• attempts to develop heterologous and homologous genetic markers for algal
transformation,
• the development of methods to introduce DNA into algal cells through the cell
wall, and
• the successful development of a stable genetic transformation system for
diatoms.
Once the methods were available to obtain genetic transformants, efforts were made to use the
transformation system to manipulate lipid content in the algae by overexpressing or
downregulating key genes. In addition, the transformation system was used to introduce a
reporter gene under the control of various regulatory sequences, to better understand the
regulation of gene expression in microalgae.
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Analysis of Microalgal DNA Composition:
Several oleaginous microalgal strains had been identified as potential candidates for biodiesel
fuel production. These organisms became the target of genetic engineering efforts to manipulate
the lipid biosynthetic pathways. Before the work on genetic transformation of algae at NREL,
very little information was available on the molecular biology of these organisms. One of the
first steps was to develop techniques to isolate and purify DNA from these organisms. A
desirable protocol would disrupt the cell wall using methods gentle enough to prevent shearing of
the genomic DNA. This was not trivial for some species, such as Monoraphidium, which has a
very resistant wall that contains sporopollenin. A method that worked for most species tested
(described in Jarvis et al. 1992) was developed based on a protocol used to isolate yeast DNA
(Hoffman and Winston 1987). The cells were suspended in buffer that contained 2% Triton X-
100 and 1% SDS, then added to a tube that contained glass beads and an equal volume of
phenol:cholorform:isoamyl alcohol (PCI). The cells were agitated for 1 minute using a vortex
mixer. The DNA in the aqueous phase was purified by re-extraction with PCI, ethanol
precipitation, and treated with RNase A. For some species, the DNA had to be purified further
by using precipitation with hexadecyltrimethylammonium bromide (CTAB; Murray and
Thompson 1980) to remove contaminating carbohydrates or by purifying the DNA on CsCl
gradients. This procedure produced DNA that digested well with many common restriction
endonucleases, but even highly purified DNA would not digest well with all restriction enzymes.
NREL researcher Eric Jarvis theorized that poor digestion of the DNA by some enzymes could
be attributable to characteristics of the DNA. All DNA is composed of four nucleosides;
deoxycytidine, deoxyguanosine, deoxythymidine, and deoxyadenosine, (abbreviated dC, dG, dT,
dA); in double stranded DNA, dC is always paired with dG, and dT with dA. The percentage of
each nucleoside (often represented as %GC) is variable between species. Restriction enzymes
cut DNA at specific nucleotide sequences, generally recognizing 4-6 bp motifs. Therefore, the
frequency of cutting by a particular enzyme will be affected by the total nucleotide composition
of the DNA (i.e., an enzyme that recognizes CCGG would cut infrequently in an organism with a
low %GC). The GC content is also reflected in the codon usage by each organism, as DNA with
a high GC content would show a bias toward codons ending with G or C in the variable third
position. DNA can also contain unusual modified nucleosides, including 5-
hydroxymethyldeoxycytidine (hm5dC) and 5-hydroxymethyldeoxyuridine (hm5dU), although the
biological significance is unclear. Another common modification is the presence of methylated
nucleosides, in particular 5-methyldeoxycytidine (m5dC) and 6-methyldeoxyadenosine (m6dA).
The degree of methylation has been associated with levels of gene expression. In addition, some
microorganisms use DNA methylation as a defense mechanism, in that methylated DNA
sequences are often not recognized by endonucleases from invading pathogens. Although the
presence of methylated nucleosides is characteristic for some species, the degree of methylation
can vary on a short time scale with changing environmental conditions. In contrast, the %GC
and presence of modified nucleosides are characteristic for a particular organism. These
characteristics only on an evolutionary time scale.
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DNA was isolated from microalgae strains, including 10 species from 5 classes. The nucleoside
composition was analyzed by reverse-phase HPLC and by digestion with restriction
endonucleases. The results of the HPLC analysis are summarized in Table I.B.4-1. Although the
diatoms showed a GC content typical for most eukaryotes (42%-48% GC), the GC content of the
green algae (excepting Stichococcus) was significantly higher. In particular, Monoraphidium
DNA contains 71% GC. The table also shows the presence of m5dC in the algal DNA. All
species tested contained some level of this modified base, although once again Monoraphidium
stands out with approximately 11% m5dC. The only other unusual feature was the presence of
12% hm5dU in the dinoflagellate C. cohnii (data not shown); dinoflagellates were not considered
to be good candidates for biodiesel fuel production, so this observation was not explored further.
These data provided a good background for developing genetic transformation systems for these
organisms. As mentioned above, the GC content of an organism can be reflected in the codon
usage, suggesting that an organism with a high GC content such as Monoraphidium may not
successfully express heterologous marker genes. This was found to be true for the green alga
Chlamydomonas; successful transformation of this organism was achieved only by the use of
homologous selectable markers (discussed in more detail later). Also, GC content should be
considered when designing synthetic DNA probes based on protein sequences, i.e., for isolation
of algal genes by PCR. In addition, DNA methylation can affect the ability to construct DNA
libraries and to clone algal DNA, and may require the use of bacterial host strains that are
insensitive to DNA methylation.
Table II.B.1. DNA Nucleoside Composition of Several Microalgal Strains (Modified
from Dunahay, et al, 1992, p .333 and Jarvis et al, 1992)
Algal species
M5dC 5GC
Chlorophyceae
Chlamydomonas reinhardtii
0.16 61.6
Chlorella ellipsoidea
1.48 51.6
Monoraphidium minutum
11.2 70.9
Bacillariophyceae
Cyclotella cryptica
1.95 43.2
Navicula saprophila
0.20 46.2
Nitzschia pusilla
0.78 45.4
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Phaeodactylum tricornutum
0.14 48.0
Charophyceae
Stichococcus sp.
0.30
44.8
Prasinophyceae
Tetraselmis suecica
3.32 57.5
Dinophyceae
Crypthecodinium cohnii
1.54 43.7
Transient Expression of Luciferase in Chlorella ellipsoidea:
The first step in transformating any organism is getting the foreign DNA inside the cell. For
organisms with a cell wall, methods must be devised to either remove or permeabilize the wall,
or to get DNA into the cell through the intact wall. Bacterial cell walls do not seem to represent
a significant barrier to DNA uptake, and can be induced to take up foreign DNA simply by being
washed in low osmotic medium and glycerol, followed by a brief heat shock. Cell walls can be
removed enzymatically from yeast cells to form spheroplasts, or from plant cells to form
protoplasts. These wall-less cells can be induced to take up DNA by chemically permeabilizing
the cell membrane with polyethylene glycol and/or calcium. Alternatively, DNA can enter yeast
spheroplasts or plant protoplasts via electroporation, a method in which a rapid, high voltage
electric pulse is used to produce transient pores in a cell membrane.
Based on their research backgrounds, NREL researchers tended to view microalgae as either
single cell plants, or pigmented yeasts. In either case, the initial tendency was to try to produce
wall-less algal cells as targets for transformation. There had previously been some reports of
protoplast production in green microalgae of the genus Chlorella (Braun and Aach 1975;
Berliner 1977). NREL researcher Eric Jarvis decided to attempt to introduce foreign DNA into
Chlorella protoplasts, with the eventual goal of adapting these protocols for other algal strains
with biodiesel production potential.
The production of a stably transformed line of cells involves several steps, including introducing
the foreign DNA into the target cell, expressing the foreign gene, stabilizating (replicating) the
new DNA by the host cell, and survival and proliferation of the genetically altered cells.
Transient expression assays can be used to monitor and optimize just the first two of these
processes, i.e., DNA entry and expressing a foreign gene in a population of cells, and thus can be
useful intermediate steps in developing genetic transformation systems. Transient assays usually
involve the introduction of a gene that codes for an enzyme detectable by a simple biochemical
assay (often referred to as a reporter gene). Dr. Jarvis decided to use one such gene, the firefly
luciferase gene, to monitor the entry and expression of foreign DNA into Chlorella protoplasts.
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The alga used for these studies was C. ellipsoidea (strain CCAP 211/1a, obtained from the
Culture Collection of Algae and Protozoa, Freshwater Biological Association, United Kingdom).
Protoplasts were produced using a protocol adapted from Global and Aach (1985). The cells
were grown to early stationary phase, then incubated overnight in 10 mg/mL Cellulysin, a crude
commercial preparation of the cellulose-degrading enzyme cellulase. Protoplast production was
monitored by sonication of the treated cells in water; generally about 80% of the cells were
disrupted by this treatment and were considered to be protoplasts. A plasmid containing the
luciferase gene driven by plant regulatory sequences was introduced in the protoplasts by mixing
the cells with the plasmid DNA for 30 minutes in the presence of 50 mM CaCl2 and 13%
polyethylene glycol (mw 4000). The cells were washed and incubated in a regeneration medium
overnight. The cells were then harvested and luciferase activity was monitored in crude protein
extracts. Luciferase catalyzes the oxidation of luciferin with the production of a photon of light
via the following reaction:
luciferase, Mg2+, O2
LUCIFERIN + ATP -----------------------> OXYLUCIFERIN + AMP + CO2 + hv
The light produced can be monitored using a scintillation counter or a luminometer.
The results of these experiments are shown in Figure II.B.6. Luciferase activity was detectable in
protoplasts treated with the luciferase plasmid, but not in protoplasts that had not been exposed to
plasmid or to polyethylene glycol. Intact cells did not take up the DNA. There was a significant
decrease in luciferase expression when carrier DNA was left out of the transformation reaction
(“carrier DNA” is usually sheared genomic DNA from calf thymus or salmon sperm that is added
to reduce the effects of cellular nucleases on the added plasmid DNA). Monitoring of the
luciferase activity over time showed that the activity was maximal at about 24 hours after
exposing the protoplasts to the plasmid; expression decreased over time and was virtually
undetectable after 80-100 hours. Unfortunately, attempts to regenerate the protoplasts into viable
walled cells were unsuccessful.
These results were important as they demonstrated the first successful steps in developing a
genetic transformation system for microalga, including the production of viable protoplasts, the
introduction of DNA into the protoplasts, and the expression of a foreign gene by the algal cells.
This last point was very significant, as homologous genes were required to achieve
transformation in another green alga (Chlamydomonas). The dogma in the field was that
heterologous gene expression in green algae would likely be unsuccessful due to codon biases
resulting from high GC contents. The work resulted in a publication (Jarvis and Brown 1991),
and was the basis for later studies in which the luciferase gene was used to monitor promoter
activities in Cyclotella (discussed later). However, attempts to adapt this procedure to algal
strains with significance to the biodiesel project were unsuccessful. The composition of
microalgal cell walls is highly variable between species and even between isolates of the same
species. Some unsuccessful efforts were made to determine the enzymatic conditions for wall
degradation for several oleaginous algal strains. However, the conclusion, in the words of the
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project manager at the time, was that this was “an endless pit of fruitless endeavor”, and the
decision was made to explore other methods of introducing DNA into microalgal cells. In
addition, although low levels of luciferase expression were acheived in Chlorella, the decision
was made to pursue the development of selectable marker systems that would allow the isolation
of very rare individual transformants within a population of microalgal cells. This will be
discussed in the following section.
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Figure II.B.6. Transient expression of firefly luciferase in Chlorella ellipsoidea.
A. (Top) - Histogram showing luciferase expression in protoplasts of C. ellipsoidea.
Expression of the luciferase gene is expressed in relative light units (RLU), which are the net
photons counted during a 5-min period. See text for explanation.
B. (Bottom) - Kinetics of luciferase expression in C. ellipsoidea protoplasts. Each symbol
represents the result of a single assay. Control cultures were grown in the dark (▲) or light
(∆). Duplicate cultures of plasmid-treated protoplasts were also grown in either the dark (■)
or light (□).
(Source: Jarvis and Brown 1991).
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Development of Homologous Selectable Markers for Monoraphidium and Cyclotella:
Transient expression assays can be useful for the rapid assessment of DNA uptake and
expression by cells as demonstrated by the expression of luciferase in Chlorella protoplasts,
described earlier. However, attempts to produce similar results in other algal strains were
unsuccessful. The problem with an experiment that produces no signal is that it is impossible to
know if this is because the DNA did not get into the cell, or if the DNA entered the cell but was
not expressed at detectable levels. In the latter case, poor expression could result from
degradation of the foreign DNA, inappropriate regulatory signals, or differences in the codon
usage.
One of the most promising organisms with regard to high lipid production and tolerance to
environmental fluxes was the green alga M. minutum (strain MONOR2). However, MONOR2
DNA was shown to be highly unusual in GC content and degree of methylation. As mentioned
elsewhere in this report, successful transformation of the green alga C. reinhardtii, which also
has an elevated GC content, required the use of homologous selectable markers. The literature
suggested that this unusual GC content would inhibit the expression of foreign genes, such as
bacterial antibiotic resistance genes that had been used successfully as transformation markers in
plant and mammalian systems. Based on this information, it was decided to attempt to develop
homologous selectable markers for transforming MONOR2 and other strains with programmatic
importance. Use of a selectable marker, in contrast to a transient expression assay, would allow
the identification of very rare transformation events. Under the appropriate selection conditions,
one transformed cell can be detected in a very large population of nontransformed cells, whereas
in transient assays, a significant number of cells in a population must be expressing the foreign
gene in order to detect the new enzymatic activity. The use of a homologous gene as a marker
would greatly increase the chance for successful expression of the introduced gene, as there
would be no problems associated with codon bias or foreign regulatory sequences. Although
some success was achieved toward the development of a homologous selectable marker system,
the emphasis of the research at NREL was shifted after the successful development of a
transformation system for diatoms that used a chimeric selectable marker. A significant effort
was put into the development of homologous markers, particularly for non-diatom species, from
1989 to 1994, so it is relevant here to summarize the progress made in this area.
The general protocol for developing a homologous selectable transformation system involves
several steps. First, a mutation is created or identified in a specific gene. The gene should be
essential for growth under “normal” conditions; however, the mutated strains will grow under
modified growth conditions. This will allow for positive selection of transformed cells. Then the
corresponding wild-type gene is isolated and inserted into a plasmid vector. The wild-type gene
is introduced into the mutant cells, and transformants are detected by the ability to grow under
the normal, defined growth conditions. In contrast to the transient assay described earlier, use of
a selectable marker involves not only DNA entry and expression, but also stabilization of the new
DNA in the cell and viability and growth of the newly transformed cells. Genes with good
potential for use as selectable markers should not only code for a protein essential for growth
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under defined conditions, but should also produce a protein that can be detected by a simple
enzymatic assay. In addition, the use of a gene that has been well characterized in other systems
will help isolate the gene from the species of interest and simplify the development of enzyme
assays and growth conditions for isolating mutants and transformed cells.
Two genes that meet these criteria were targeted for the development of homologous selectable
markers for MONOR2 and for C. cryptica T13L. One codes for the enzyme nitrate reductase
(NR). NR had been used successfully to transform Chlamydomonas (Kindle et al. 1989) and
several species of fungi (Daboussi et al. 1989) and methods were available to isolate NR mutants
and selection of transformed strains. In addition, there was some interest at NREL in the role of
nitrogen uptake and utilization in lipid accumulation, and isolating the wild-type NR gene would
permit further investigation of these questions.
NR mutants can be isolated based on their resistance to chlorate. Cells with functional NR will
take up chlorate along with nitrate and reduce the chlorate to the toxic compound chlorite.
Therefore, cells with a mutation in the NR gene will be unable to grow using nitrate as the sole N
source, but will be able to grow in the presence of chlorate, as long as urea or ammonium is
added as an alternative N source. Using this scheme, several putative NR mutants grew from
non-mutagenized cells of MONOR2 and C. cryptica T13L. Biochemical assays suggested that at
least two of the MONOR2 mutants contained defects within the NR structural gene.
The next step was to isolate the wild-type gene from MONOR2 for complementation of the NR-
minus mutants. A partial cDNA clone of NR from Chlorella vulgaris was obtained from Dr.
Andrew Cannons (University of Southern Florida). Southern blot analysis indicated that the
Chlorella DNA sequence showed significant homology to a sequence in MONOR2 genomic
DNA. Degenerate primers for use in the PCR were designed based on conserved regions in the
NR genes from three green algal species and several higher plants. A 700-bp PCR product was
generated using MONOR2 genomic DNA as a template and confirmed to represent a fragment of
the NR gene by sequence analysis. A MONOR2 genomic DNA library was constructed in a
lambda phage vector. Although the library appeared to be representative of the algal genome in
that it contained approximately 300,000 separate clones of about 20,000 bp each, repeated
screening of the library with the NR gene fragment failed to produce any positive results. Two
additional libraries were constructed, but again, screening with the MONOR2 NR sequence did
not result in the isolation of a genomic NR sequence. It was concluded that the libraries were
probably incomplete; i.e., they did not contain DNA representative of the total algal genome,
possibly because of problems associated with the unusual composition of the MONOR2 DNA.
This project was put on hold when successful transformation was achieved in C. cryptica, and
had not been pursued further when the project was terminated in 1996.
A gene that encodes the enzyme orotidine-5'-phosphate decarboxylase (OPDase) was also
targeted for use as a selectable transformation marker. OPDase is a key enzyme in the synthesis
of pyrimidines. Organisms with defects in the OPDase gene will only grow if pyrimidines such
as uracil are added to the growth medium. OPDase mutants can be selected by growing cells in
the presence of the drug 5-fluoroorotic acid (FOA); OPDase converts FOA into a compound that
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is toxic to the cells. Therefore, OPDase mutants would grow in the presence of FOA and require
uracil; wild-type cells (or mutants transformed with the wild-type OPDase gene) would be
susceptible to FOA and would require added uracil in the growth media. NREL researcher Eric
Jarvis attempted to develop the OPDase system as a selectable marker for MONOR2. Cells were
mutagenized by exposure to UV light, then grown in the presence of uracil and FOA. Putative
OPDase mutants were identified as FOA-resistant colonies. Based on growth studies and
spectrophotometric measurements of OPDase activity, one isolate of MONOR2 (3180a-1) was
identified as a probable OPDase mutant for use as a host strain in the transformation system.
The next step, as for NR, was isolate the wild-type OPDase gene from MONOR2. OPDase had
previously been isolated from several species and demonstrated significant sequence
conservation between genes from different organisms. Dr. Jarvis made a number of attempts to
isolate the OPDase gene from MONOR2 via PCR, using degenerate primers based on conserved
OPDase gene sequences. Several PCR products were generated using this approach, but
sequence analysis of the cloned DNA fragments resulted in no clones with homology to the
OPDase gene. Why this approach did not work for OPDase is unclear, as this same PCR
technique had been used to isolate a fragment of NR. A second approach, in which a MONOR2
genomic DNA library was screened for OPDase sequences using heterologous probes, was also
unsuccessful.
By 1994, a transformation system had been developed for the diatoms using a chimeric gene as a
selectable marker (discussed in the following section); however, there was still interest in
producing a selectable marker system that would work for high lipid (although genetically
recalcitrant) green algal strains, such as MONOR2. Work began on developing a new selectable
marker system that used a mutated version of the acetolactate synthase (ALS) gene as a
selectable marker. ALS is an enzyme involved in the synthesis of branched-chain amino acid
such as leucine and valine. In plants, this enzyme is inhibited by sulfonylurea and imidazolinone
herbicides. Previous work at NREL by Galloway (1990) showed that many microalgae are also
sensitive to these herbicides. Eric Jarvis repeated these experiments for MONOR2 and
demonstrated that these cells are sensitive to low levels of the sulfonylurea herbicides
chlorsulfuron and sulfometural methyl. The approach was to isolate the wild-type gene for ALS
from MONOR2, and then to produce a gene that encodes a herbicide-resistant form of the
enzyme by site-directed mutagenesis. Degenerate primers were produced based on known ALS
sequences and used, this time successfully, to isolate an ALS gene fragment from MONOR2
DNA. This sequence was used to screen the MONOR2 DNA libraries for a full-length ALS
sequence, but once again, the screening efforts were unsuccessful.
The feeling among the NREL researchers was that the use of a homologous selectable marker
system would still be the best approach for developing genetic transformation systems for some
organisms, in particular, those with unusual DNA compositions, and for haploid organisms for
which generation of mutants should be relatively straightforward. Despite the promise of M.
minutum as a high lipid producer, it may have not been the best organism for these studies
because of its highly unusual DNA properties and “tough” cell wall that complicated biochemical
extractions and assays. Some of the cloning problems seen with this organism might have been
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solved if time had permitted the generation of a cDNA library, or a new genomic DNA library
using bacterial host strains optimized for use with highly modified or high GC DNA.
Transformation of Chlamydomonas reinhardtii Using Silicon Carbide Whiskers:
Based on the frustrating efforts to produce viable protoplasts from microalgae discussed earlier,
efforts were initated to develop other methods for introducing DNA into microalgal cells through
the intact algal cell walls. At the time this research was going on, the only microalga for which
there was a reproducible transformation system was C. reinhardtii. Early efforts to transform
this organism were facilitated by the availability of wall-less cells, either genetic mutants (cw-
15), or cells whose walls were degraded using autolysin, a species-specific cell wall-degrading
enzyme produced during mating by C. reinhardtii gametes. High-frequency nuclear
transformation was accomplished by agitating these wall-less cells in the presence of plasmid
DNA, glass beads, and polyethylene glycol (Kindle 1990). This method was reported to work for
walled cells, but at a very low frequency. DNA could also be introduced into walled cells of
Chlamydomonas and into higher plant cells using microprojectile bombardment, or biolistics;
however, this technique requires very expensive, specialized equipment. (This technique will be
described in detail “Development of a Genetic Transformation System for the Diatoms Cyclotella
and Navicula.”)
During the early 1990s, several reports demonstrated the feasibility of using silicon carbide
whiskers (SiC) to mediate the entry of DNA into intact plant cells (Kaeppler et al. 1990; Asano et
al. 1991). NREL researcher Terri Dunahay decided to try this approach to introduce DNA into
intact algal cells. As reliable selectable markers were not yet available for any oleaginous
microalgal strain, she decided to use Chlamydomonas as a model system. A strain of C
reinhardtii that contains a defect in the gene for nitrate reductase (CC2453 nit1-305 mt-) was
obtained from the Chlamydomonas Genetics Center at Duke University, Durham, North
Carolina. These cells cannot use nitrate as a N source, but grow well in the presence of ammonia
or urea. Kindle (1990) had shown previously that NR-deficient cells could be transformed with
the Chlamydomonas wild-type gene for NR; transformed cells expressing the added DNA could
be detected by their ability to grow on nitrate as the sole N source. A plasmid containing the
wild-type NR gene from Chlamydomonas was obtained from Dr. P. Lefebvre at the University of
Minnesota, St. Paul, Minnesota. A protocol for SiC-mediated transformation was developed
based on the glass bead transformation protocol of Kindle (1990). Exponentially growing cells
were washed once in NH +
4 -free medium, then suspended in the same medium with plasmid
DNA, sterilzed SiC whiskers, and polyethylene glycol (mw 8,000) to a final concentration of
4.5%-5.0% w/v. The samples were agitated using a vortex mixer for periods from 30 seconds to
10 minutes, then diluted into NH +
4 -free medium containing 0.6% agar (top agar) and plated onto
agar plates that contained the same medium. Transformed colonies (containing a funtional NR
gene) appeared in 1-2 weeks.
Attempts to transform walled cells of Chlamydomonas using SiC were made in parallel with
glass bead-mediated transformation to compare the two procedures. The results of a typical
experiment are shown in Figure II.B.7. The number of transformants obtained using SiC varied
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between experiments, but generally were in the range of 10-100 per 107 cells, comparable to
transformation efficiency obtained with glass beads. Probably the most significant finding was
the difference in cell viability after being agitated with either glass beads or SiC fibers. The
viability of the cultures was greater than 80% even after agitation with SiC fibers for 10 minutes;
only 10% of the cells survived agitation with glass beads for 60 seconds. The fact that SiC-
mediated transformation appears to be a more “gentle” protocol than glass bead treatment may be
important when adapting the transformation procedure to other species that may have different
wall properties. This work resulted in two publications (Dunahay 1993; Dunahay et al. 1997).
The second paper was a collaboration with Dr. Jonathan Jarvik at Carnegie Mellon University.
Dr. Jarvik's laboratory adopted and refined the SiC protocol and now uses it routinely to generate
transformants in Chlamydomonas strains with intact walls. After the initial development of the
SiC protocol, there was some work at NREL to adapt this procedure for other algal strains of
interest to the biodiesel project. Initially, no genetic markers for these strains were available;
however, the viability of Monoraphidium and Cyclotella were tested following agitation with
SiC; both strains showed high survival rates after extended agitation with SiC. However, the
successful development of a transformation system for Cyclotella using biolistics (discussed
later) precluded further work on SiC-mediated algal transformation. A few attempts were made
to generate transformants of Cyclotella or Navicula using SiC once a selectable marker system
was developed. Only one transformant was generated in one experiment. The silica frustule of
the diatoms likely acts as a significant barrier to penetration by SiC fibers. SiC would probably
work better for introducting DNA into non-diatom cells such as Monoraphidium; these cells are
very small and may not be a good target for biolistics, but might be readily pierced by SiC fibers.
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Figure II.B.7. Cell survival and transformation efficiency of intact C. reinhardtii following
vortex mixing with SiC fibers or glass beads. (Source: Dunahay 1993.)
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Development of a Genetic Transformation System for the Diatoms Cyclotella and Navicula:
Successful genetic transformation of microalgal strains with demonstrated potential for biodiesel
fuel production was finally accomplished in 1994. Two factors that were critical in the
development of the transformation system were:
• the cloning of the acetyl-CoA carboxylase gene from C. cryptica, and thus the
availability of ACCase regulatory sequences to drive expression of a foreign
gene in the diatoms, and
• the purchase by NREL of a microprojectile accelerator (also known as a particle
gun) that can efficiently deliver DNA-coated gold or tungsten beads into walled
cells.
Except for the transient expression of luciferase in Chlorella protoplasts, all previous attempts at
NREL to transform microalgae had been unsuccessful. Whether the problem was the inability to
deliver foreign DNA into the cells through the algal cell wall, or inefficient expression of the
foreign gene, is not clear.
As discussed in the previous section of this report, a significant amount of work went into
developing homologous selectable markers for microalgae, primarily for Monoraphidium.
However, there were some attempts, mainly with diatoms, to use a heterologous antibiotic
resistance gene as a selectable marker. The GC content of bacteria and diatoms are relatively
similar; thus, codon bias should not prevent expression of a bacterial gene in the diatoms. The
antibiotic kanamycin and its more potent analog G418, have been used extensively for genetic
transformation in higher plants. These antibiotics function by binding to 30S ribosomes and
inhibiting protein synthesis. Resistance to kanamycin or G418 can be induced in cells by
expressing the bacterial gene neomycin phosphotransferase (nptII). This enzyme phosphorylates
the antibiotic, preventing binding to the ribosome. Previous work at NREL (Galloway 1990)
demonstrated that some algal strains are sensitive to kanamycin, suggesting that the kanamycin-
G418/nptII system might be the basis of a successful transformation system for microalgae.
Further testing showed that most of the algal strains were sensitive to low concentrations of
G418; however, the conditions for complete inhibition of cell growth had to be determined
empirically for each strain. The required concentration of the antibiotic depended both on the
osmoticum of the plating medium and on the plating density of the cells. For example, C.
cryptica T13L grows well on both 10% and 50% ASW. When 2 x 106 cells of T13L were plated
on 50% ASW agar plates, the cells were resistant to 50 µg⋅mL-1 G418. The same number of cells
plated onto 10% ASW plus 50 µg⋅mL-1 G418 showed no growth, yet 3 x 107 cells produced a
confluent lawn of colonies under the same conditions.
Early attempts to use the nptII gene as a selectable marker used a plasmid construct that had been
used successfully for transformation in higher plants. This plasmid, pCaMVNeo, was obtained
from Dr. Michael Fromm at the USDA Plant Gene Expression Center, Albany California.
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pCaMVNeo contains the nptII gene driven by the cauliflower mosaic virus 35S ribosomal gene
promoter (CaMV35S). Attempts were made to introduce pCaMVNeo into C. cryptica CYCLO1
by electroporation, and later into C. ellipsoidea or CYCLO1 by agitating the cells with glass
beads or SiC fibers. No G418-resistant colonies were generated by these methods.
After the acetyl-CoA carboxylase (acc1) gene was cloned from C. cryptica T13L, NREL
researcher Paul Roessler decided to try to use the 5'- and 3'-regulatory regions from this gene to
drive expression of nptII in T13L. A plasmid (pACCNPT10) was constructed that contained a
chimeric gene consisting of the coding region of the nptII gene flanked by 445 bp of the acc1 5'
region (the putative promoter) and 275 bp of acc1 coding region following the nptII stop codon,
followed by the acc1 3' noncoding regions (the putative transcriptional terminator). To increase
the chance of encompassing the entire acc1 promoter, a second plasmid, pACCNPT5.1, was
constructed that contained 819 bp of upstream sequence. In addition, all but 13 bp of the acc1
coding region was removed from the 5' end of chimeric gene. Details of the plasmid
constructions can be found in Dunahay et al. (1995), and plasmid maps are shown in Figure
II.B.8.
DNA entry into the algal cells was accomplished using the DuPont/Bio-Rad PDS/1000He
microprojectile accelerator. The process, called biolistics, had been used successfully for
introducting DNA into walled cells of higher plants, fungi, bacteria, and Chlamydomonas. In this
procedure, plasmid DNA is precipitated onto small tungsten or gold particles and accelerated into
cells using a burst of helium pressure. Early versions of this device used a gun powder charge to
accelerate the particles. Because of prohibitive costs and restrictive licensing agreements, a
homemade version of the particle gun was designed and built at NREL. No transformants were
generated using this device, but as these experiments were performed before the acc1-nptII
chimeric plasmids where constructed, whether the device actually functioned as planned is
unclear. Ultimately, a commerical microprojectile accelerator was purchased. This device was
optimized for very simple operation and used helium pressure to propel the DNA-coated
particles. These properties resulted in greater reproducibility between shots and decreased
toxicity caused by gases generated during the explosive charge.
There was some initial skepticism on the part of at least one NREL researcher as to whether
microprojectile bombardment would work to introduce DNA into diatoms through the Si
frustule. However, the diatoms were transformed using the particle gun and the chimeric vectors
in the first try. This turned out to be a simple and reproducible procedure (Figure II.B.9.). For
each transformation, algal cells were harvested and spread in an approximate monolayer in the
center of an agar plate containing growth medium and 50 µg⋅mL-1 ampicillin to inhibit bacterial
growth. The plates were allowed to dry for 2 hours before bombardment. Just before
bombardment, plasmid DNA was precipitated onto 0.5-1.0 µm tungsten particles, which were
then propelled into the cells using the microprojectile accelerator. The exact parameters used are
described in Dunahay et al. (1995). The cells were incubated for 2 days under nonselective
conditions to allow the cells to recover and express the nptII gene. The cells were then washed
from the orignal plates and replated onto agar that contained the appropriate concentration of
G418. G418-resistant colonies appeared in 7-10 days. These putative transformants were picked
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from the plates and tested for continued resistance to G418. The presence of the foreign gene was
tested by hybridizing the algal DNA with an nptII gene probe (Southern analysis). The cells were
tested for the presence of the and for the NPTII protein by probing with an NPTII-specific
antibody (Western blotting), Figures II.B.10 and II.B.11.
Both the pACCNPT10 and pACCNPT5.1 plasmids worked well to generate transformants in two
strains of C. cryptica (T13L and CYCLO1), as well as in the diatom N. saprophila (NAVIC1).
These two species belong to different orders (C. cryptica is a centric diatom, Order Centrales; N.
saprophila is a pennate diatom, Order Pennales). Southern analysis indicated that the plasmid
DNA was not replicating independently in the cells but had integrated into the host genome,
presumably into the nuclear DNA. The chimeric gene integrated into one or more independent
sites, often in form of tandem repeats. The nptII DNA remained stably integrated into the host
genome for more than 1 year, even when the cells were grown under nonselective conditions.
The successful development of a genetic transformation system for the diatoms was a major
achievement for the ASP. This was the first report of genetic transformation of any diatom
species, and one of the few reports in which a heterologous gene was used as a selectable marker
for stable nuclear transformation of an alga. The use of algal regulatory sequences to drive
expression of the bacterial gene in diatoms apparently was a key factor in the successful
development of a transformation protocol for these organisms. When the pCaMVNeo plasmid
was introduced into diatoms via particle bombardment, no G418-resistant transformants were
generated. However, when another plasmid that contains the CaMV35S promoter and the firefly
luciferase gene were introduced into the diatoms by cotransformation with pACCNPT5.1, a
number of transformants selected based on their resistance to G418 also expressed significant
luciferase activity. This result suggests that even though microalgae can in some cases recognize
and use foreign promoter sequences, homologous promoters may be necessary to drive
expression of foreign selectable markers at levels high enough to overcome the selective
pressure. The research that resulted in the development of a genetic tranformation system for
diatoms resulted in a publication (Dunahay et al. 1995) that was a finalist for the Provasoli
Award for best publication in the Journal of Phycology for that year. In addition, a patent
describing this technology was applied for and issued in August 1997. Diatoms represent a very
large proportion of the world's biomass, and are responsible for nearly one-fourth of the net
primary production. However, little is known about the biochemistry and molecular biology of
these organisms. The availability of a genetic transformation system for diatoms could have a
major impact on increasing the understanding of the basic biology of these organisms and should
promote their use in biotechnological applications in addition to the intended goal of lipid
production. The following section will describe the initial attempts to use the genetic
transformation protocol to manipulate levels of storage lipids in C. cryptica.
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Figure II.B.8. Plasmid maps of pACCNPT10, pACCNPT5.1, pACC1
A. (Top) - Maps of plasmids containing the neomycin phosphotranferase gene (nptII) flanked
by regulatory regions from the acetyl-CoA carboxylase gene from C. cryptica. Both plasmids
worked well as expression vectors in the diatoms C. cryptica and Navicula saprophila.
B. (Bottom) - Map of plasmid pACC1, containing the full-length genomic sequence of the
acetyl-CoA carboxylase gene (acc1) from C. cryptica.
- nptII gene sequence;
- acc1 coding sequence;
- acc1 regulatory sequences
(Source: Dunahay et al. 1995; Roessler and Ohlrogge 1993).
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Figure II.B.9. Simplified schematic showing the protocol for transformation of diatoms by
microprojectile bombardment (“gene gun”).
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Figure II.B.10. Southern blot showing the presence of the nptII gene in transformed cells of
C. cryptica T13L.
Cells of C. cryptica T13L were transformed with pACCNPT5.1 via particle bombardment as
described in the text. DNA from wild-type cells (wt) or G418-resistant strains (lanes a-f) were
digested with HindIII and hybridized to a digoxigenin-labeled nptII sequence. The lane
designated “5.1” contains HindIII-digested pACCNPT5.1as a control. The sizes of DNA
fragments included as markers are indicated to the right. (Source: Dunahay et al. 1995).
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Figure II.B.11. Western blot, showing the presence of the 30-kDa NPTII protein in
transformed cells of C. cryptica T13L and N. saprophila.
In this example, C. cryptica and N. saprophila were transformed with pACCNPT10 and
pACCNPT5.1, respectively. Crude cell extracts were separated on SDS-polyacrylamide gels,
blotted onto a nitrocellulose filter, and NPTII protein was detected using anti-NPTII primary
antibodies and alkaline phosphatase-conjugated goat anti-rabbit IgG secondary antibodies. The
polyclonal antiNPTII antibody also recognizes a band of approximately 80 kDa in C. cryptica;
however, the 30 kDa NPTII protein is seen only in the G418-resistant transformants. (Source:
Dunahay et al. 1995).
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II.B.3.d.
Attempts to Manipulate Microalgal Lipid Composition via Genetic
Engineering
The overall goal of the studies on the biochemistry and molecular biology of lipid synthesis in
microalgae was to increase the understanding of the lipid biosynthetic pathways and to identify
enzymes that influence the rate of lipid accumulation and lipid quality. This information would
be used to genetically manipulate the biosynthetic pathways for improvement in lipid production
rates and to manipulate the nature of the lipids produced (i.e., the degree of fatty acid saturation
and chain length) to optimize the production of biodiesel.
The development of a genetic transformation system for diatoms allowed NREL researchers to
begin testing ways to manipulate microalgal biochemical pathways. The first target enzyme was
ACCase. Previous studies at NREL had shown that increased lipid production in diatoms
induced by Si starvation was accompanied by an increase in the activity of the ACCase enzyme.
Therefore, it was logical to ask whether the activity of the enzyme could be increased in the cells
by adding additional copies of the gene encoding ACCase (acc1), and, if so, would increased
activity of the protein stimulate the production of lipids in the algal cells?
A full-length copy of the C. cryptica acc1 gene had been cloned and characterized at NREL (see
Section II.B.2.f). The plasmid containing this sequence was designated pACC1 (Figure II.B.8).
Before the algal transformation system, attempts were made to express the algal gene in a
bacterial system to ensure that the cloned gene encoded a functional ACCase enzyme and to test
for the effects of overexpression. For expression of the C. cryptica acc1 gene in E. coli, the
introns were removed and the 5' terminus was replaced with the 5' end of the E. coli β-
galactosidase gene, which included the inducible promoter region. This fusion gene was
introduced into E. coli. The transformed cells were analyzed for the production of algal ACCase
protein by probing blots of (Sodium Dodecyl Sulfate, SDS) polyacrylamide gels with an anti-
ACCase antibody or with avidin conjugated to alkaline phosphatase. (Avidin binds to the biotin
moity in the functional ACCase protein.) The bacterial cells produced full-length algal ACCase,
as well as a large number of shorter polypeptides recognized by the anti-ACCase antibody.
Introducting the gene into other E. coli strains deficient in protease activity also produced these
shortened peptides; therefore, they were presumed to be the result of truncated transcription or
translation. The full-length ACCase protein was properly biotinylated in the transformed
bacteria, but not as efficiently as in the E. coli native biotin-binding ACCase subunit. No effects
were observed on lipid biosynthesis in the transformed E. coli strain. Attempts were also made
to introduce the C. cryptica acc1 gene into yeast, as expression in a eukaryotic system would
more likely mimic the effects in algae, but these experiments were unsuccessful.
The next step was to introduce additional copies of the acc1 gene in diatoms, with the goal of
increasing the activity of the ACCase enzyme and then assaying the effects of ACCase
overexpression on lipid accumulation. The plasmid containing the full-length acc1 gene
(pACC1) does not contain a selectable marker for transformation. Studies in other laboratories
showed that nonselectable plasmids can be introduced into cells via cotransformation with a
plasmid containing a selectable marker gene such as nptII. Although the exact mechanism for
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this phenomenon is not clear, it is believed that during a given transformation procedure, a
particular subpopulation of the cells becomes “transformation competent”. These cells may then
take up multiple copies of DNA molecules present in the reaction. Introduction of pACC1 into
the diatoms was mediated by microprojectile bombardment as described in a previous section,
but with pACCNPT5.1 and pACC1 precipitated onto the tungsten beads in equimolar amounts.
Transformed cells were selected based on their induced resistance to G418 and then screened for
additional copies of the acc1 wild-type gene using PCR and Southern analysis. Between 20%
and 80% of the G418-resistant colonies contained acc1 sequences in the cotransformation
experiments. To facilitate the selection of transformants containing extra copies of the acc1
gene, a plasmid was also constructed that contained both acc1 and nptII, designated pACCNPT4;
transformants generated using pACCNPT4 and selected for G418-resistance almost always
contained the acc1 gene as well.
Transformed cells containing additional C. cryptica acc1 gene sequences were isolated in C.
cryptica T13L, C. cryptica CYCLO1, and N. saprophila NAVIC1. Southern analysis indicated
that the foreign DNA inserted into host genome, often in one or more random sites, and often in
the form of tandem repeats. Several strains that contained one or more full-length sequences of
the inserted acc1 gene were analyzed further to test for ACCase overexpression. The CYCLO
T13L transformants showed two to three fold higher ACCase activity than wild-type cells, and
there was a corresponding increase in acc1 gene transcript (mRNA) levels. However,
preliminary analyses of the lipid composition of the cultures overexpressing acc1 did not indicate
a detectable increase in lipid levels. These results suggest that the lipid biosynthesis pathways
may be subject to feedback inhibition, so that increased activity of the ACCase enzyme is
compensated for by other pathways within the cells. It was hoped that expression of C. cryptica
T13L acc1 gene in other algal strains might overcome this inhibition. Numerous N. saprophila
transformants were generated that contained full-length copies of the C. cryptica acc1 gene;
although acc1 mRNA was detected using the RPA, the recombinant ACCase protein was not
detected in any of the N. saprophila strains tested. Whether this result was due to inefficient
translation of the mRNA, or degradation of the foreign protein due to improper biotinylation or
targeting, is not known. Transformants were also generated in a second strain of C. cryptica,
CYCLO1, but the program was discontinued before these strains could be analyzed fully.
NREL researcher Eric Jarvis took another approach to genetically manipulating algal pathways
for increased lipid production. Previous research had resulted in the cloning and characterization
of the upp1 gene from C. cryptica (described in Section II.B.2.h.). This gene codes for a fusion
protein containing the activities for UDPglucose pyrophosphorylase and phosphoglucomutase,
two key enzymes in the production of chrysolaminarin. It was postulated that decreasing
expression of the upp1 gene could result in a decrease in the proportion of newly assimilated
carbon into the carbohydrate synthesis pathways, and consequently increase the flow of carbon to
lipids.
Two techniques that are becoming widely used for gene inactivation are ribozymes and antisense
RNA. Dr. Jarvis spent 6 months working at Ribozyme Pharmaceuticals, Inc., a biotechnology
company in Boulder, Colorado, learning about these new methods. Antisense RNA is a method
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in which a cell is transformed with a synthetic gene that produces an RNA sequence
complimentary to a specific mRNA. Although the exact mechanism is not clear, the antisense
RNA prevents translation from its complimentary mRNA, effectively lowering the level of that
particular protein in the cell. Ribozymes are also RNA molecules produced by synthetic genes
that can bind to, and cleave, very specific RNA sequences. Ribozymes can be designed to
degrade specific mRNA molecules, effectively decreasing expression of a specific gene.
Several ribozymes sequences designed to cleave upp1 RNA were constructed based on computer
predictions of the secondary structure of the target RNA. The ribozyme constructs were shown
to specifically cleave the target RNA in vitro. The ribozyme sequences were then inserted into
the pACCNPT10 vector in the untranslated acc1 sequence between the nptII stop codon and the
acc1 termination sequence (see Figure II.B.8). C. cryptica T13L was transformed with these
vectors as described earlier and transformants were selected based on acquired resistance to
G418. Extracts were made of the transformed strains and analyzed for UGPase activity.
Unfortunately, insertion of the ribozyme sequences did not result in detectable decreases in
UGPase expression. Although these initial experiments were unsuccessful, gene inactivation
technologies acquired during this project seemed a promising approach for manipulation of algal
lipid synthesis pathways. At the time project funding was terminated, work was in progress to
continue with the ribozyme experiments and to test antisense RNA constructs as an additional
method for inactivating algal pathways.
II.B.3.e.
The Effect of Different Promoters on Expression of Luciferase in Cyclotella
Little is known about the regulation of gene expression in diatoms, partly because genetic
transformation was not possible in this group of algae before NREL’s transformation system was
developed. The availability of this transformation system now allows the study of the roles of
regulatory DNA sequences in gene expression. As a first step toward a better understanding of
gene transcription in diatoms, NREL researchers Paul Roessler and Steve Milstrey used the
firefly luciferase reporter gene (discussed earlier), to study the level of gene expression as
controlled by various DNA regulatory sequences from the diatom C. cryptica and other
organisms. They also used this system to try to define the regions of the ACCase gene promoter
involved in the Si-depletion response.
Various plasmids were constructed in which different combinations of 5’ regulatory DNA
sequences (promoters) and 3’ regulatory DNA sequences (terminators) were linked to the firefly
luciferase gene (luc). The regulatory sequences used in this study included both the ACCase
promoter and the UDP-glucose pyrophosphorylase/phosphoglucomutase promoters from C.
cryptica. Also tested were the simian virus 40 (SV40) promoter, which drives high levels of
gene expression in mammalian cells, and the cauliflower mosaic virus 35S RNA promoter
(CaMV35S), which is a strong constitutive promoter in plants. These plasmids were introduced
into C. cryptica via cotransformation with the selectable marker plasmid pACCNPT5.1 as
described earlier. Approximately half of the transformed strains produced in this manner
contained the luc gene, as determined by PCR analysis. Based on past results, it is expected that
the plasmid DNA was integrated into the genome of the cells. Luciferase activities in randomly
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chosen transformants (eight from each plasmid type) were determined by the use of a
luminometer.
As expected, the promoter regions of both C. cryptica genes drove luciferase expression in the
transformed C. cryptica cell at high levels. Less predictable was the finding that the SV40
mammalian promoter also drove luciferase expression in C. cryptica at relatively high levels
(although lower than seen using the homologous promoters), but the CaMV35S promoter was
much less effective. In most of the constructs used in this study, the 3' terminator regulatory
region was from the C. cryptica aac1 gene. Replacement of this sequence with the SV40
terminator did not affect the levels of luciferase expression driven by the acc1 promoter,
indicating that the source of the terminator sequence used may not be a critical determinant of
gene transcription efficiency.
Previous results at NREL indicated that Si deficiency may affect the expression of the acc1 gene
in C. cryptica (see Section II.B.2.d.). To try to identify regions of the acc1 promoter that might
be responsive to Si levels, three plasmids that contained varying lengths of the acc1 promoter
region (900, 445, and 396 bp, respectively) fused to the luc gene were used to transform C.
cryptica. Under Si replete conditions, the average luciferase activities of transformants
containing these plasmids were very similar. Furthermore, the luciferase activity increased to the
same extent (approximately twofold) 6 hours after transfer into Si-free medium. This suggests
that the Si-responsive elements are either within the shortest (396-bp) promoter region tested or
in a separate area of the genome.
These results indicate that the firefly luciferase gene can be expressed in recombinant C. cryptica
cells, to provide a sensitive reporter system for analyzing gene expression and promoter function
in diatoms. This and similar systems will likely be extremely useful for gaining a better
understanding of the molecular biology of this important group of organisms.
Publications:
Brown, L.M.; Dunahay, T.G.; Jarvis, E.E. (1990) Applications of genetics to microalgae
production. Dev. Industrial Microbiology, vol. 31 (J. Industrial Microbiol., Suppl. No.5), pp.
271-274.
Dunahay, T.G. (1993) “Transformation of Chlamydomonas reinhardtii with silicon carbide
whiskers.” Biotechniques 15:452-460.
Dunahay, T.G.; Adler, S.A.; Jarvik, J.W. (1997) “Transformation of microalgae using silicon
carbide whiskers.” In Methods in Molecular Biology, vol. 62: Recombinant Gene Expression
Protocols (Tuan, R., ed.), Humana Press, Totowa, New Jersey, pp. 503-509.
Dunahay, T.G.; Jarvis, E.E.; Dais, S.S.; Roessler, P.G. (1996) “Manipulation of microalgal lipid
production using genetic engineering.” Appl. Biochem. Biotechol. 57/58:223-231.
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Dunahay, T.G.; Jarvis, E.E.; Roessler, P.G. (1995) “Genetic transformation of the diatoms
Cyclotella cryptica and Navicula saprophila.” J. Phycol. 31:1004-1012.
Dunahay, T.G.; Jarvis, E.E.; Zeiler, K.G.; Roessler, P.G.; Brown, L.M. (1992) “Genetic
engineering of microalgae for fuel production.” Appl. Biochem. Biotechnol<
