Bernhard, Joan M., Pieter T. Visscher, And Samuel S. Bowser ...

Limnol. Oceanogr., 48(2), 2003, 813–828
2003, by the American Society of Limnology and Oceanography, Inc.
Submillimeter life positions of bacteria, protists, and metazoans in laminated sediments
of the Santa Barbara Basin
Joan M. Bernhard1
Department of Environmental Health Sciences, Norman J. Arnold School of Public Health, University of South Carolina,
Columbia, South Carolina 29208
Pieter T. Visscher
Department of Marine Sciences, University of Connecticut, Groton, Connecticut 06340
Samuel S. Bowser
Wadsworth Center, New York State Department of Health, P.O. Box 509, Albany, New York 12201-0509
Abstract
To provide insights into the biogeochemistry of environments where steep chemical gradients place anaerobes,
microaerophiles, and aerobes in close proximity, it might be necessary to survey biotic distributions on scales that
are not possible using conventional ecological approaches. To overcome such limitations, we adapted sedimento-
logical and cell biological methods to examine the life positions of microbes within sediments. This ﬂuorescently
labeled embedded core (FLEC) technique was used to survey the submillimeter distributions of eukaryotic nanobiota
and meiofauna, plus co-occurring prokaryotes, inhabiting laminated sediments of the bathyal Santa Barbara Basin
(SBB; 34 13 N, 120 02 W). Although SBB sediments were vertically structured on the scale of millimeters (i.e.,
as laminae), and microelectrode proﬁles suggested vertically distinct oxygenated and anoxic-sulﬁdic layers, the
distributions of aerobes, microaerophiles, and sulﬁde-tolerant anaerobes were not concomitantly structured. Un-
precedented associations were observed among microorganisms. For example, relatively deep in the sediments,
where high sulﬁde concentrations were expected, ﬂagellates were intimately associated with Beggiatoa. Ciliates
were typically solitary, whereas ﬂagellates were often aggregated in nearly monomorphotypic swarms of
3
104
mm 3. Such aggregations could signiﬁcantly affect geochemical pore–water processes at scales
1
l. Our obser-
vations indicate that a mosaic of chemically heterogeneous microhabitats exist in both vertical and horizontal
dimensions, suggesting that biogeochemical processes in the SBB are more complex than predictions based on
standard biotic assessments and microelectrode proﬁling.
Conventional methods used to study nano-, meio- and
A high abundance and biovolume of eukaryotes reside in
macrofauna, such as sieving or density gradient extractions,
the surface centimeter of sediments from the Santa Barbara
obliterate the life positions of benthic organisms. Conse-
Basin (SBB), a deep-water locale with steep oxygen and
quently, little is known about their subcentimeter–scale spa-
sulﬁde gradients (Bernhard et al. 2000). A mix of aerobes,
tial distributions. This information is crucial to understand-
microaerophiles, and anaerobic sulﬁde-tolerant taxa com-
ing the biotic and physicochemical interactions in sediments,
prise the SBB consortium; the majority of these protists and
particularly those with steep pore–water gradients. Within
metazoans have associated prokaryotes. Here, we report the
the surface centimeter in such settings, for example, condi-
submillimeter life positions of these eukaryotes and larger
tions can range from oxygen depletion to anoxia, with or
prokaryotes in laminated SBB sediments, visualized using
without sulﬁde enrichment (e.g., Jørgensen 1988).
the life position approach described by Bernhard and Bowser
(1996) and augmented by hot-knife microtomy (McGee-
Russell et al. 1990). In contrast to previous reports (Bernhard
1 Corresponding author (joan.bernhard@sc.edu).
1996, 2000; Bernhard et al. 1997; Bernhard and Sen Gupta
Acknowledgments
1999; Pike et al. 2001), results presented here are for a com-
Assistance from the following is gratefully acknowledged: the
plete range of taxa. The objectives of this contribution are
captain and crew of the RV Robert Gordon Sproul, as well as the
to determine whether biotic distributions in SBB sediments
resident technicians of Scripps Institution of Oceanography; all
reﬂect microelectrode-based measurements of oxygen,
members of the 1998 and 1999 cruises’ scientiﬁc parties (especially
whether sedimentary laminations affect those distributions,
Ellen Braun-Howland, Dennis Bazylinski, Brad Dubbels, Annette
and how species composition and abundance change with
Dean, Shelly Hoeft, and Dan Rogers); Sam McGee-Russell for in-
respect to sediment depth, Beggiatoa density, and different
troducing us to hot knife microtomy, Sara Meyers, Brenda Famo-
physicochemical regimes.
laro, Kat Benson, and Adam Meyer for help in adapting the tech-
nique; Monterey Bay Aquarium Research Institute for bathymetric
data; Jim Barry for construction of the baseline map; Tom Chandler
Material and methods
for LSCM access, and two anonymous reviewers for their helpful
comments. This work was supported by NSF grants OCE 9417097
Site description—The SBB is a silled basin with a max-
and OCE 9711812 (to J.M.B.) and OPP 9725830 (to S.S.B.).
imum depth of
600 m and sill depth of
425 m (Fig. 1).
813

814
Bernhard et al.
Fig. 1.
Map of Santa Barbara Basin showing primary sampling site (*).
In the basin, bottom-water oxygen concentration rarely ex-
frame and rigged to close when the corer tripped. Oxygen
ceeds 5
mol L 1 ( 0.1 ml L 1) and is typically
2
mol
concentrations of Niskin samples were determined using the
L 1 (e.g., Reimers et al. 1996; Kuwabara et al. 1999). Sed-
microwinkler method (Broenkow and Cline 1969). As soon
iments in the central portion of the basin are laminated be-
as possible (i.e., within 5–10 min), after the recovery of an
cause [O ] is too low to support bioturbating macrofauna.
undisturbed box core, it was subsampled with syringe cores
2
These varved sediments have been the focus of many paleo-
(12 cc, 1.5 cm diameter), which were taken into a refriger-
ceanographic studies assessing climate change on annual,
ated (5 C) van and processed for life position studies as de-
decadal, and millennial time scales (e.g., Behl and Kennett
scribed below. For some box cores, a 7.5-cm-diameter core
1996; Kennett et al. 2000).
was also taken for proﬁling of oxygen, hydrogen sulﬁde, or
SBB sediments have a high pore–water content ( 93–
both using either single or combined needle electrodes
98% in surface 0.5 cm; Reimers et al. 1990) and high or-
(Visscher et al. 1991). The pH was measured using a needle
ganic carbon content ( 5–8% in surface 0.5 cm; Reimers et
electrode (Diamond General) or a Ross pH probe (Thermo
al. 1990). Sediment composition varies between the two
Orion). In the refrigerated van, the core was transferred to a
laminae types: dark laminae contain very ﬁne grained ter-
glove bag and microelectrode readings were taken every
rigenous detritus (mostly silt and clay particles), whereas
0.2–0.4 mm; calibration followed Visscher and coworkers
lighter ones consist mostly of biosilica (e.g., diatom frus-
(1991, 2002). Sulﬁde concentrations were calculated from
tules; Grimm et al. 1996). Mean grain size of SBB sediments
interpolation of pH 7.5, 8.0, and 8.5 calibration curves using
is about 3
m, and the contribution of sand is very low
the speciﬁc in situ pH. Detection limits were
1
mol L 1
( 1% in laminated sediments; Fleischer 1972).
for oxygen,
5
mol L 1 for sulﬁde, and
0.1 pH.
Sample collection and microelectrode proﬁling—Sedi-
FLEC method—The life position method, which is de-
ments were collected from the central portion of the SBB
scribed in Bernhard and Bowser (1996), combines the well-
(Fig. 1;
590 m water depth;
34 13.5 N, 120 01.9 W) us-
established epoxy-embedding core technique of Frankel
ing a Soutar box corer. Bottom-water samples were taken
(1970) and the application of ﬂuorogenic probes to visualize
with a 2-liter Niskin bottle, which was attached to the corer
live cells. We introduce here the use of hot-knife microtomy

Benthic microbiota in situ
815
(McGee-Russell et al. 1990) to section polymerized cores,
Results
allowing serial sectioning at
0.1 mm without sample loss
(as opposed to conventional mineralogical methods of saw-
Representative FLEC results are presented for two box
ing and subsequent polishing). The combined use of the life
cores that were collected 7 months apart (19 February and
position approach and hot-knife microtomy is hereby denot-
22 September 1999) from the same site in the central SBB.
ed the ﬂuorescently labeled embedded core (FLEC) method.
The sediment surface changed visibly between sampling
Fluorogenic probes are nonﬂuorescent compounds that
events, as evidenced by photographs of the box core surfaces
yield a ﬂuorescent product that accumulates intracellularly
before subsampling (Fig. 2A,B). In February, all four box
after enzymatic hydrolysis (see Bernhard et al. 1995 and
cores collected from the site showed that the sediment sur-
references therein). The ﬂuorogenic probe utilized in this
face was an orange-brown color. In the box core from which
study, Cell Tracker Green CMFDA
(CTG, Molecular
the February FLEC core was taken, three individuals of the
Probes) is dependent on nonspeciﬁc esterase activity. In the
epifaunal gastropod Astrys permodesta were observed, along
refrigerated van, each syringe core was injected with CTG
with a few cohesive boli of white material (Fig. 2A). Prelim-
to a ﬁnal concentration of 1
mol L 1, recapped to prevent
inary 16S rRNA data indicate that the boli were the ﬁla-
aeration, and incubated for
4–6 h. Cores were then ﬁxed
mentous, sulﬁde-oxidizing bacterium Beggiatoa (B. Dub-
in 3% glutaraldehyde/0.1 mol L 1 cacodylate buffer (pH
bels, D. Bazylinski, and J. Bernhard unpubl. data). In
7.2), perfused three times in buffer, and secured for transport
September, all ﬁve box cores collected from the site had a
with a layer of alginic acid ( 0.5%), which gelled in place
well-developed mat of Beggiatoa veiling the sediment sur-
on the sediment surface after the addition of CaCl . In the
face (Fig. 2B). Bottom-water [O ] also changed between the
2
2
laboratory, the alginate was dissolved with multiple incu-
two sampling events, with relatively high concentrations in
bations in 50 mmol L 1 ethylenediaminetetraacetic acid
February (2.4
mol L 1), but nearly undetectable levels in
(EDTA). Subcores were then dehydrated using an ascending
September (0.1
mol L 1). FLEC sections oriented perpen-
series of ethanol and ultimately incubated for
2 weeks in
dicular to the sediment surface showed that laminations were
100% ethanol. A ﬁnal 2- to 4-week incubation in acetone
present in both cores (Fig. 2C,D). The laminae are not as
preceded inﬁltration with Spurrs’ low-viscosity epoxy resin.
obvious in Fig. 2C because the FLEC section is thicker than
The inﬁltrated cores were polymerized at 70 C.
that shown in Fig. 2D.
For sectioning, the top
1.5 cm of a polymerized core
was mounted on a brass stub made to ﬁt a custom-modiﬁed
Biotic distributions revealed by FLEC—FLEC results
Sorvall MT1 microtome. The core was oriented either ver-
show complex biotic distribution patterns, wherein organ-
tically or horizontally, depending on the desired orientation
isms with different energy metabolisms existed along the
of serial sections (i.e., down-core slices or sections oriented
same horizon and aerobes commonly occurred deeper than
perpendicular to the sediment surface). The two cores de-
anaerobes (Table 1). The observations indicate that SBB
tailed here were sectioned vertically, permitting down-core
pore–water conditions are similarly complex. Over 1,000 eu-
observations of each section. Two additional cores were ex-
karyotes, including metazoans, were observed throughout
amined in less detail: one was sectioned perpendicular to the
the examined sections, which extended to a depth of 11 mm.
sediment–water interface and the other was sectioned verti-
Eukaryotic abundances do, however, decline down-core
cally. Because SBB sediments rarely have terrigenous par-
from maxima near the sediment–water interface (Table 1),
ticles
63
m (Fleischer 1972), the sediments are easily
which is consistent with results obtained from traditional ap-
sectioned with hot-knife microtomy (which is ineffective
proaches. Ciliates and ﬂagellates were not common deeper
with sandy deposits; Bernhard and Bowser pers. obs.). Sec-
than
4–5 mm unless they were associated with Beggiatoa
tions were mounted between two coverslips with epoxy resin
(Table 1; see below).
and polymerized. Sections and specimens located within 1–
Visual inspection of FLEC material conﬁrms few Beggia-
2 mm of the core edge were rejected from analysis because,
toa ﬁlaments at the sediment surface in February 1999 when
during subcoring, downward smearing could have occurred
the bottom-water oxygen concentration was relatively high
at the periphery. Preliminary observations were made with
(2.4
mol L 1); ﬁlaments were more abundant below the
an Olympus SZX12 stereomicroscope equipped with epiﬂu-
sediment surface (Fig. 3). In a representative area, a speci-
orescence optics; more detailed studies used an Olympus
men of Nonionella stella resided near the sediment surface
Fluoview Personal Confocal Microscope System. Although
(0.3 mm below the sediment–water interface; Fig. 4A), al-
this laser scanning confocal microscope (LSCM) was
though this foraminiferan also dwelled consistently in the
equipped with both Ar and Kr lasers, images were obtained
subsurface (Fig. 8E). Along a horizon located 0.5 mm below
using only the ﬂuorescein channel (i.e., 488 nm excitation,
the sediment–water interface, ciliates of one morphotype
520 nm emission) in most cases. The depth of an organism
were encountered (Figs. 3, 4A). In counts reported in Table
below the sediment–water interface was measured by super-
1, this morphotype was only observed twice at depths be-
imposing a stage micrometer (10 mm in 0.1-mm divisions)
tween 1 and 2 mm, whereas 20 individuals occurred in the
on the area in question and viewing with the ﬂuorescence-
surface millimeter; none were seen deeper or in examined
equipped Olympus SZX12. To visually assess sediment lam-
material collected in September 1999.
inations, digital ﬂash photographs of FLEC sections were
In this same section, at a depth of
2.1 mm below the
imported into Photoshop
v. 4.0 as grayscale image ﬁles,
sediment–water interface, a high density ( 200 pl 1) of var-
and pixel percent saturation values were recorded at intervals
ious ﬁlamentous bacterial types occurred (Fig. 3). Closer in-
of 1 mm down-core.
spection revealed nanoliter-scale spatial aggregations, with

816
Bernhard et al.
Fig. 2.
(A, B) Photographs of box core surfaces collected in (A) February and (B) September 1999. The white tufts (magenta arrows) are
small aggregations of Beggiatoa. Four individuals of the epifaunal gastropod A. permodesta (black arrows) can also be seen. Surface of box
core collected in September has abundant Beggiatoa (white material, not labeled). For scale, gastropods are
1 cm long. (C, D) Photographs
of hot-knife sections from box cores shown in panels A, B. (C) Box core shown in panel A (February); (D) box core shown in panel B
(September). The boxed area is shown in detail using LSCM in Figs. 4, 6; selected areas are shown at higher magniﬁcation in Figs. 5, 7.
Beggiatoa in some areas and nonvacuolate ﬁlamentous bac-
interface), a transverse section of a nematode was encoun-
terial morphotypes in an adjacent zone. Nonvacuolate ﬁla-
tered (Figs. 3, 4D). Because adult SBB nematodes are typ-
mentous bacteria were densely packed in a prolate spheroid
ically
2 mm in length, their entire body is rarely intact
shape similar to that of a fecal pellet (Fig. 4B). A dense
within one FLEC section. Even deeper below the sediment
accumulation of small ( 6–10
m) ﬂagellates dwelled
surface (5.5 mm), a ciliate morphologically similar to Par-
among bacterial ﬁlaments at a depth of
2.3 mm (Fig. 4C).
ablepharisma occurred (Figs. 3, 4E). Members of Parable-
The teeming ﬂagellates were mainly of one morphotype,
pharisma are obligate anaerobes (Fenchel 1996b), and con-
which is an undescribed euglenoid with epibionts (Bernhard
geners in the SBB harbor ectosymbiotic bacteria (Bernhard
et al. 2000). A single foraminifer occurred in this swarm,
et al. 2000). At a sediment depth of 7.5 mm, two thick ﬁl-
and a ciliate was situated at the edge of the aggregation.
aments of Beggiatoa were entwined (Fig. 3). Detailed in-
Deeper down-core ( 2.8 mm below the sediment–water
spection showed that 13 ﬂagellates, all of one morphotype,

Benthic microbiota in situ
817
Table 1. Observed occurrences of individuals in FLEC sections presented by depth and major taxa. Data represent compilations from
observations of two sides per FLEC section for one section per time point. Values of sediment reﬂectance are averaged for the depth range
indicated; higher values indicate darker sediments. Beggiatoa is ranked from maximal (***) to moderate (**) to low (*) abundance. Bold
denotes presence of at least one known anaerobe; italic denotes inclusion of at least one known aerobe; bold italic denotes presence of at
least one known aerobe and one known anaerobe; normal font denotes all specimens of unknown energy metabolism. If the same specimen
was visible in more than one surface, it was only recorded once. Metazoan counts are conservative to minimize size sampling bias; for
example, the same nematode can appear multiple times in a given section.
No. individuals
Depth
Sediment reﬂectance
(mm)
(% saturation)
Beggiatoa
Foraminifera
Flagellates
Ciliates
Metazoa
Feb 99 (O
2.4
mol L 1)
2
0–1
57
*
64
64
31
1
1–2
70
*
19
156
12
2
2–3
67
***
17
500†‡
5
1
3–4
59
**
8
16
3
1
4–5
68
*
7
7
4
1
5–6
77
**
4
0
5
1
6–7
85
*
2
2
0
2
7–8
72
***
8
13†
0
1
8–9
51
***
7
0
0
1
9–10
65
*
1
1
0
3
10–11
53
*
0
0
0
3
Sep 99 (O
0.1
mol L 1)
2
0–1
73
**
11
23
4
3
1–2
79
***
6
18†§
6
3
2–3
80
***
0
3
5†
3
3–4
68
***
0
5
2
1
4–5
69
*
1
0
0
2
5–6
86
*
0
0
0
3
6–7
98
**
1
1
1
0
7–8
77
*
1
0
1
0
8–9
71
*
0
0
0
1
9–10
65
**
0
0
0
2
10–11
68
*
0
0
0
1
† Some or all were associated with Beggiatoa or other ﬁlamentous bacteria.
‡ Swarm, estimated count.
§ Some or all were associated with metazoans.
were closely associated with the Beggiatoa (Fig. 4F). Living
et al. 1995; Simpson et al. 1996/1997), were oriented toward
foraminifera were quite common to a depth of
3 mm in
a bolus of thin, ﬁlamentous bacteria located just below the
this section (n
100), but they also existed deeper in the
nematode (Fig. 6B). Vacuolate and nonvacuolate ﬁlamentous
sediments (n
37, 3–10 mm; Table 1).
bacteria of various diameters appeared enriched from
1.2–
In September 1999, when bottom-water oxygen was near-
4.4 mm down-core (Fig. 5. Three conspeciﬁc ciliates were
ly undetectable (0.1
mol L 1), Beggiatoa ﬁlaments pene-
found within this cluster of bacterial ﬁlaments (Fig. 6C).
trated through the sediments and extended into the overlying
At a sediment depth of
3.5–4.0 mm, a horizon (i.e., a
water column (Figs. 5, 6A), which is a life habit thought to
lamina) of phytodetritus was inﬁltrated by bundled Beggia-
indicate oxygen limitation (e.g., Møller et al. 1985) or nitrate
toa (Figs. 5, 6D). When this area was examined with dual-
chemotaxis (Huettel et al. 1996). The ﬁlaments typically
channel LSCM, which can be used to view photosynthetic
emerged from the sediments in clusters rather than as single
pigment autoﬂuorescence in addition to the signal from the
ﬁlaments. Thus, the Beggiatoa life mode shown in the pho-
CTG, it was clear that the pigments were well preserved
tograph of the box core is preserved in the FLEC samples
(data not shown). In this layer, individual plastids, as well
(compare Figs. 2B, 6A). In general, protists were less abun-
as chains of resting spores of the diatom Chaetoceros, oc-
dant and occurred shallower in the sediment column in Sep-
curred in high densities. At a depth of
7 mm, bundles of
tember 1999 compared to February 1999 (Table 1). At a
entwined, vacuolated ﬁlamentous bacteria occurred (Figs. 5,
depth of 1.2 mm below the sediment–water interface, a ﬂa-
6E). At 7.1 mm depth, a lone ciliate, morphologically similar
gellate (morphologically similar to the aerobic, nonsym-
to Litonotus, was encountered (Figs. 5, 6F).
biont-bearing Notosolenus ostium) occurred in close prox-
Beggiatoa ﬁlament widths were typically
10
m in di-
imity to a nematode that was preserved among Beggiatoa
ameter (Figs. 4B, 6A–C, 7F), although larger Beggiatoa ﬁl-
ﬁlaments (Figs. 5, 6B). Three individuals of a different ﬂa-
aments were also noted ( 20–25
m diameter). The ob-
gellate morphotype, morphologically similar to the ectosym-
served range in Beggiatoa ﬁlament diameter is not surprising
biont-bearing anaerobe Postgaardi mariagerensis (Fenchel
because widths reportedly vary by species between 1–2
m

818
Bernhard et al.
Fig. 3.
LSCM montage of area demarcated in Fig. 2C. The depth scale is in millimeters; the right column is a continuation of the left
column. Scale bar
200
m.

Benthic microbiota in situ
819
Fig. 4.
LSCM images of selected areas in Fig. 3. (A) The foraminifer N. stella and four ciliates, possibly of the genus Pleuronema.
Note the horizontal distribution of the ciliates; three additional specimens can be seen along the same horizon in Fig. 3. (B) Aggregation
of various ﬁlamentous bacteria. Note the ovoid shape delimited by thin nonvacuolate ﬁlaments in the lower left. Also present is the anterior
of a ciliate, C. (C) Swarm of euglenoid ﬂagellates; ciliate, C; and a portion of a foraminifer, F. (D) Portion of a nematode showing the
pharynx. (E) A ciliate, possibly of the genus Parablepharisma. Note the oral ciliature and macronucleus. (F) Entwined ﬁlaments of Beggiatoa
with 13 associated ﬂagellates of one morphotype. Number of images compiled/distance between images ( m): A
90/0.5; B
80/0.5; C
83/0.8; D
70/0.5; E
63/0.4; F
166/0.5. Scale bars: A, B, F
100
m; C
200
m; D, E
50
m.

820
Bernhard et al.
Fig. 5.
LSCM montage of area demarcated in Fig. 2D. The depth scale is in millimeters; the right column is a continuation of the left
column. Scale bar
200
m.

Benthic microbiota in situ
821
and 40
m (e.g., Larkin and Strohl 1983; Teske et al. 1999).
Because the bottom-water values determined from Niskin sam-
The Beggiatoa in the examined material were typically seen
ples were 0.2
mol L 1 for February 1998 and 0.1
mol L 1
as discrete populations of uniform ﬁlament widths. Thicker
for September 1999, oxygen values shown in proﬁles for over-
ﬁlaments (i.e.,
20
m) generally occurred deeper in the
lying water and at the sediment–water interface in Fig. 8 are
cores (e.g., Figs. 4F, 6D,F). Some of these thicker ﬁlaments
likely to be higher than in situ values. Contamination from
were bundled (Fig. 6D,F), suggesting that they were Thio-
atmospheric oxygen could have occurred during box core sub-
ploca rather than Beggiatoa.
sampling prior to sediment proﬁling, even though precautions
were taken during microelectrode measurements (i.e., imme-
Observations from other cores—Several observations are
diate subcoring and proﬁling in a capped core barrel). Assum-
worth noting from other sections of the same FLEC cores
ing that the time elapsed between box core recovery and se-
shown in Figs. 3–6, as well as two additional FLEC cores of
curing the subcore for proﬁling was
15 min, and the diffusion
SBB sediments. In at least three cases, Beggiatoa inhabited
time t
[z2/2D] (where z is the diffusional distance and mo-
microburrows, which were presumably made by nematodes or
lecular diffusion coefﬁcient D
2
10 5 cm2 s 1), O could
2
other metazoans (e.g., Fig. 7A; Pike et al. 2001). In two ob-
have diffused downward an additional 2 mm. We could there-
served instances, eukaryotes also took advantage of the in-
fore argue that, in situ, O penetrated only to
2 mm depth in
2
creased pore space (and presumably a more favorable geo-
September 1999. Importantly, FLEC cores, from which life-
chemical microenvironment) from such microbioturbation (e.g.,
position observations were made, were separate from the cores
Fig. 7A). Empty foraminiferal shells also provided space for
proﬁled and were not subject to such atmospheric exposure.
protists. For example, an empty shell of Chilostomella ovoidea
harbored at least six ﬂagellates of two morphotypes as well as
Discussion
ﬁlamentous bacteria (Fig. 7B). It is unknown whether these
ﬂagellates were consuming organic remains of the foraminifer
or were merely occupying pore space. As noted, ﬂagellate
The FLEC method: Caveats and comments—Previously,
swarms were not uncommon (Fig. 4C). A higher magniﬁcation
ﬁne-scale biotic distributions have been correlated with pore–
view of one swarm is shown in Fig. 7C, where the estimated
water chemistry by manipulating organism abundances and
ﬂagellate density was 3.1
104 mm 3.
measuring millimeter-scale pore–water solute concentrations
At a depth of 3.3–3.9 mm, a specimen of the gastrotrich
(e.g., Aller and Aller 1992) as well as microelectrode proﬁling
Urodasys anorektoxys was observed (Fig. 7D). The body
of ﬁeld samples paired with small-volume sediment sampling
extension of this specimen indicates lack of trauma during
(0.2–1.0 cm 2; e.g., Wetzel et al. 1995; Fenchel and Bernard
FLEC processing; isolated U. anorektoxys retract when ﬁxed
1996). Although these approaches provide useful information,
for conventional microscopy.
they do not assess actual life positions. Previous work noted
Besides being the most abundant foraminifer when oxy-
subsurface foraminiferal positions in sediment cores embedded
gen was nearly undetectable (e.g., Fig. 5; see also Bernhard
with epoxy resin (Frankel 1970). Bright-ﬁeld imaging of non-
and Reimers 1991; Bernhard et al. 1997), 21 N. stella oc-
vitally stained specimens severely limits the sensitivity and util-
curred in subsurface sediments (1–11 mm in February 1999
ity of this original approach. A similar impregnation technique
FLEC section). Four individuals were observed at
7.5 mm
was used to survey the potential food resources for deposit
sediment depth in a different portion of the February 1999
feeders at a ﬁne scale (Watling 1988), but there was no attempt
FLEC section; two are shown in Fig. 7E. The vacuolar char-
to identify meiofaunal, nanobiotic, or prokaryotic constituents.
acter of typical foraminiferal cytoplasm can be seen in Fig.
Here, we merge core embedding and microelectrode measure-
7E (see also Fig. 4A).
ments to show submillimeter life positions of ﬂuorescently la-
Occasionally, numerous prokaryotic and eukaryotic taxa
beled individuals.
of differing energy metabolisms occurred within a small vol-
Before our ﬁndings can be discussed, however, two points
ume. For example, in 9 nl, the fringe of a Beggiatoa bolus
regarding the FLEC method need to be examined. First, the
contained the anaerobic ciliate Metopus verrucosus, two
positive identiﬁcation of microorganisms in FLEC sections
small nematodes, and four ﬂagellates of two morphotypes
was sometimes hampered (e.g., by specimen opacity or ob-
(Fig. 7F). One of the ﬂagellate morphotypes observed in this
struction by mineral grains and detritus). Also, entire indi-
association was an anaerobe that harbors ectobionts (P. mar-
viduals were not always contained within a section. There-
iagerensis; Fenchel et al. 1995; Simpson et al. 1996/1997),
fore, some systematic identiﬁcations are tentative. Second,
but the other is known to lack symbionts (Sphenomonas sp.;
it is possible that particulates can be transported down-core
Bernhard et al. 2000) and is a presumptive aerobe, like its
by ﬂuid percolation through the sediment matrix during pro-
congeners (Lee and Patterson 2000).
cessing. If this occurred, however, one would expect to ob-
serve size sorting, with microbiota nearer the sediment–wa-
Microelectrode proﬁles—Matched oxygen and sulﬁde pro-
ter interface and nanobiota deeper in the sediment column.
ﬁles are not available for either box core for which detailed
Because nanobiota were imaged throughout the core, we re-
FLEC results are presented. An O proﬁle is presented for Sep-
ject this possibility. Furthermore, if down-core transport oc-
2
tember 1999, and matched proﬁles are presented for February
curred, we might also expect elongated specimens to exhibit
1998 (proﬁle data not available for February 1999; Fig. 8).
a particular orientation in response to ﬂuid motion (i.e., ob-
Data shows that sulﬁde can reach considerable concentrations
long specimens streamlined in the direction of ﬂuid ﬂow).
(i.e., 45
mol L 1; Fig. 8A) between 0.5–1.0 cm. Oxygen was
No such aligned orientation was seen in our material. For
undetectable at
4 mm depth in September 1999 (Fig. 8B).
these reasons, and because submillimeter scale resolution of

822
Bernhard et al.
Fig. 6.
LSCM images of selected areas in Fig. 5. (A) The sediment–water interface, with Beggiatoa ﬁlaments extending from the
sediments into the overlying water. Also shown is a portion of a ciliate, C, of unknown identity. (B) Flagellates (*) and a portion of
nematode among bacterial ﬁlaments. Note the single ﬂagellate located near the nematode cuticle and the three ﬂagellates associated with
the bolus of thin bacterial ﬁlaments. (C) Three ciliates of a single morphotype among various ﬁlamentous bacteria. (D) Dense accumulation
of algal remains penetrated by Beggiatoa or Thioploca ﬁlaments. Note the empty centric diatom frustule, D; Chaetoceros chain (open
arrow); and ciliate, C. (E) Thick strands of entwined ﬁlamentous bacteria (Beggiatoa or Thioploca); each bundle is composed of about

Benthic microbiota in situ
823
SBB laminations is preserved using other epoxy impregna-
species when oxygen is nearly undetectable (Fig. 5), suggest
tion methods (e.g., Grimm et al. 1996), down-core displace-
that this species avoids higher [O ] while seeking higher
2
ment of organisms during FLEC processing does not appear
[H S]. Its possession of sequestered chloroplasts (Bernhard
2
to be a signiﬁcant concern.
and Bowser 1999; Grzymski et al. 2002) and peroxisome–
endoplasmic reticulum complexes (Bernhard 1996) could
Taxon-speciﬁc distributions and associations—The prox-
provide the cellular machinery required for surviving dy-
imate intra- and interspeciﬁc associations revealed with the
soxic/sulﬁdic conditions.
FLEC method are, to our knowledge, unprecedented. In gen-
In addition to the coupled interactions between symbionts
eral, ciliates occurred as solitary individuals that were not
and their hosts, loose but complex associations of free-living
associated with other organisms (Figs. 4A,E, 6A,D,F, 7D;
prokaryotes and eukaryotes were observed in these laminat-
Table 1), whereas ﬂagellates were typically encountered in
ed SBB sediments (e.g., Figs. 6B, 7F). In Fig. 7F, metazoans
groups (Fig. 7B) or even swarms (Figs. 4C, 7C; Table 1).
occurred in close proximity to known anaerobes (the ciliate
Flagellate aggregates were commonly afﬁliated with pro-
M. verrucosus, the ﬂagellate P. mariagerensis) and chemo-
karyotes or other eukaryotes (Figs. 4F, 6B). Exceptions to
lithoautotrophs (the sulﬁde oxidizer Beggiatoa). Thus, it is
these generalities exist; for example, where a ciliate was ob-
possible that this particular microzone, which was
9 nl in
served among Beggiatoa ﬁlaments (Fig. 7F; Table 1). The
volume, was either (1) anoxic and the aerobic specimens
near absence of ciliates and ﬂagellates
5 mm (Table 1) was
were transients, perhaps attracted to food, or (2) oxygenated
probably because of the decreased pore space in the more
enough for aerobes to respire but depleted enough for an-
compacted subsurface sediments.
aerobes to avoid oxygen toxicity. Because microaerophilic
It might be somewhat surprising that the majority of in-
ciliates can move as fast as
900
m s 1 when exposed to
dividuals in ﬂagellate swarms were of a single morphotype.
suboptimal conditions (Fenchel and Bernard 1996)—and
It is unclear whether such swarms were clonal aggregates
would, therefore, easily travel through the area shown in Fig.
that had not dispersed or congregations of individuals that
7F in less than a second—we conclude that the specimens
were seeking a particular microenvironment. All of the iden-
depicted in that image were not transients.
tiﬁable ﬂagellates in these swarms harbor ectobionts (i.e.,
Although a variety of afﬁliations among taxa was ob-
Euglenoid sp., Calkinsia aureus; Bernhard et al. 2000), sug-
served, we can only speculate on the nature of these asso-
gesting that the hosts are anaerobes. If this is the case, the
ciations. For example, it is possible that the nematode and
observation of a swarm at 0.2 mm depth (Fig. 7C) indicates
ﬂagellates observed in close association with the sulﬁde-ox-
an anoxic microzone at a depth normally expected to be
idizing Beggiatoa gained access to a microhabitat devoid of
aerated (albeit weakly). Regardless of the energy metabolism
potentially toxic hydrogen sulﬁde (Figs. 4F, 7A). Although
of the ﬂagellates, these aggregations must be hotspots of
the identity of the ﬂagellates in Fig. 4F is uncertain, it is
biological activity. Considering the total density of ﬂagel-
clear that the Beggiatoa provide some physical or chemical
lates observed ( 4
105 cm 3; Bernhard et al. 2000), as
advantage to that particular morphotype in that microenvi-
well as an estimated swarm density calculated from Fig. 7C,
ronment. The ﬂagellates in Fig. 6B also could have been
it is possible that there are 13–14 ﬂagellate swarms cm 3 in
beneﬁting from a microzone of low sulﬁde, or merely in-
the top cm of SBB sediments. The effect of such swarms on
gesting the thin ﬁlamentous bacteria comprising the bolus.
benthic and geochemical processes (e.g., carbon cycling, sol-
The FLEC method of examining spatial relationships among
ute transport) could be signiﬁcant.
organisms at the submillimeter scale provides a foundation
The restriction of one ciliate morphotype along a speciﬁc
for future experimental investigations to begin addressing
sedimentary horizon (e.g., Figs. 3, 4A) suggests that this
such questions.
morphospecies has strict geochemical or sedimentological
requirements. Given that the microfabric does not appear
Temporal differences—Quantitative comparisons between
different from that at other depths (Fig. 2C), we infer that
replicate cores collected at different times are beyond the
geochemistry drives the distribution of this morphotype. If
scope of this paper, but striking differences between the two
these individuals belong to Pleuronema, which is a micro-
cores examined in detail provide a new understanding of
aerophilic genus (e.g., Fenchel 1996b; Fenchel and Bernard
changes that occur in the SBB eukaryotic community as bot-
1996) that lacks symbionts in SBB (Bernhard et al. 2000;
tom-water oxygen concentrations vary. It is clear that pro-
reported as Frontonia sp.), we might conclude that both an
tists penetrated deeper into sediments and were more abun-
avoidance of sulﬁde and a dependence on oxygen drive this
dant when bottom-water [O ] was higher (Table 1). This
2
species’ distribution pattern. In contrast, the observations of
view discounts the smallest of the ﬂagellates (e.g., Euglenoid
the foraminifer N. stella, which commonly lives consider-
sp. of Bernhard et al. 2000), because their bodies are gen-
ably below the sediment–water interface during more aerated
erally
10
m in length and are thus difﬁcult to identify
times (e.g., Fig. 7E) and is the most common foraminiferal
with conﬁdence in FLEC images, unless they occur en mas-
←
three ﬁlaments. Note that the ciliate shown in panel F appears in the lower right corner. (F) Contorted ciliate, possibly of the genus
Litonotus. Inset shows same specimen from a different angle (i.e., in a tilted rendering from compiled optical sections), providing conﬁr-
mation of ciliate’s sigmoidal shape at the time of ﬁxation. Number of images compiled/distance between images ( m): A
108/0.8; B
89/0.9; C
100/0.8; D
97/0.9; E
121/0.8; F
187/0.3. Scale bars: A, C, D, E
200
m; B
100
m; F
50
m.

824
Bernhard et al.
Fig. 7.
LSCM images of various SBB FLEC cores. (A) Two microburrows inhabited by Beggiatoa as well as a ﬂagellate (*) and small
nematode, N. Note some other ﬂagellates outside the boli. Core collected in February 1996. (B) Shell remnant of the benthic foraminifer
C. ovoidea containing at least six ﬂagellates of two types as well as bacterial ﬁlaments. Specimens occurred in a different section of core
shown in Figs. 4, 5. (C) Swarm of euglenoid ﬂagellates, including at least one specimen of C. aureus (*), occurring
0.2 mm below

Benthic microbiota in situ
825
Fig. 8.
Proﬁles of oxygen and hydrogen sulﬁde in sediments from the SBB sampling site. (A)
Core collected in February 1998 when bottom-water [O ]
0.2
mol L 1 (arrow; see text). Note
2
the apparent nonoverlap of oxygen and sulﬁde. (B) Core collected in September 1999 when bottom-
water [O ]
0.1
mol L 1 (arrow; core collected 1 d after that shown in Figs. 2, 6, 7). Sulﬁde
2
data not available.
se. In contrast, an obvious change in metazoan abundance
clear-cut laminations at the ‘‘macro’’ or visible scale, there
and depth distribution was not observed. This could be be-
were no comparable clear-cut boundaries in terms of eu-
cause metazoans inhabit SBB sediments to depths of at least
karyotic types, abundances, or locations (Table 1). Metazo-
3 cm (Todaro et al. 2000; Mu¨ller et al. 2001), so differences
ans, which were mostly nematodes, occurred to the maxi-
in maximum penetration are likely to be seen deeper than
mum depth examined (11 mm; Table 1). Using a
11 mm. Longer scale (seasonal to annual or decadal) tem-
sedimentation rate of
1.0 cm yr 1 in the SBB (before com-
poral variability of bottom-water [O ] in the SBB probably
paction, Reimers et al. 1990), the age of the sediments where
2
causes the complex array of microhabitats to be dynamic,
these metazoans were observed is estimated to be
13
thereby introducing the potential for additional postdeposi-
months. Furthermore, metazoan presence to 3 cm (Todaro et
tional alterations to the sediment record, as discussed in the
al. 2000; Mu¨ller et al. 2001) suggests that sediments are
following section.
exposed to migrational activities for years before being bur-
ied. As noted by Pike and coworkers (2001), signiﬁcant blur-
Distributions related to physicochemical parameters—
ring of the stratigraphic record by interlaminal transport via
Possible effects on laminae: Although the primary factors in
microbioturbation is possible. Additionally, foraminiferal
varve formation are a debated topic (cf. Reimers et al. 1990;
migration cannot be discounted as a signiﬁcant factor af-
Thunell et al. 1995), it is generally agreed that laminated
fecting the sedimentary record: migration rates of
1.2–1.5
sediments such as those in the SBB are postdepositionally
mm h 1 have been observed in bathyal species (Gross 2000).
pristine in terms of physical disturbance. The observations
Given that foraminiferal migration is mediated by pseudo-
presented here cast a new light on this perspective. Despite
podial networks that ramify sediments (reviewed in Travis
←
sediment surface. A few other morphotypes of unknown identity are also present. From another core collected in September 1999 from a
shallower water depth (576 m; [O ]
0.3
mol L 1). (D) The gastrotrich U. anorektoxys at 3.3–3.9 mm depth in another section of the
2
February 1999 core that is also shown in Figs. 4, 5. Also present is a ciliate, C, possibly Parablepharisma sp. (E) Two foraminifers (N.
stella) at 7.6 mm depth in another section of the February 1999 core shown in Figs. 4, 5. (F) The ciliate M. verrucosus, M; four ﬂagellates
(*); and two small nematodes, N, at the periphery of a Beggiatoa bolus. Collected from 590 m water depth in February 1996 ([O ]
0.8
2
mol L 1). Number of images compiled/distance between images ( m): A
82/1.0; B
20/1.5; C
88/0.4; D
22/1.5; E
151/0.6;
F
23/0.9. Scale bars: A, C, E
100
m; B, D
200
m; F
50
m.

826
Bernhard et al.
Table 2. Prokaryotic and symbiont-bearing eukaryotic taxa observed in FLEC images and the geochemical process(es) that they could
affect.
Taxon
Geochemical process
Reference
Prokaryote
Beggiatoa
Sulﬁde oxidation
Larkin and Strohl 1983
Eukaryote
Foraminifera
Nonionella stella
Nitrate reduction(?)
Grzymski et al. 2002
Buliminella tenuata
Unknown
Flagellates
Calkinsia aureus
Unknown
Lackey 1960
Postgaardi mariagerensis
Sulfate reduction(?), hydro-
Simpson et al. 1996/1997
gen production(?)
Euglenoid sp.
Unknown
Ciliates
Metopus verrucosus
Methanogenesis, hydrogen
Esteban et al. 1995; Fenchel and Ramsing 1992
production, sulfate reduc-
Fenchel and Finlay 1992
tion(?)
Parablepharisma sp.
Unknown
Fenchel and Ramsing 1992
Metazoa
Desmodora masira
Sulﬁde oxidation
Dubbels et al. unpubl. data
Xenonerilla bactericola
Sulﬁde reduction
Dubbels et al. unpubl. data
and Bowser 1991), it is likely that such activity affects sed-
thanogens and hydrogen producers (Table 2). It is important to
imentary microfabric.
stress that insights into the biogeochemistry of sediments can
be made only if the metabolic requirements and tolerance
Issues regarding pore–water chemistry: Another axiom re-
thresholds are known for the observed organism(s). Unfortu-
garding laminated sediments is that geochemical conditions and
nately, these thresholds and requirements are known for very
processes are consistent along any given lamina. Because mi-
few deep-water nanobiota and protistan meiofauna.
croelectrode data indicate that oxygen and sulﬁde do not co-
How is this complex mosaic of microhabitats formed and
exist within the SBB surface sediments (Fig. 8), we might ex-
maintained in the laminated SBB sediments? Chemosensory
pect aerobes to be concentrated within the surface 1–2 mm and
behavior most likely plays an important role in the distribu-
anaerobic thiobios (sulﬁde-tolerant taxa) to occur deeper than
tions of the more motile fauna, as suggested by Fenchel
6 mm, with the microaerophiles in between. FLEC obser-
(1996a) for shallow-water sediments. FLEC data indicate,
vations indicate, however, that this vertical zonation does not
however, that microorganism distributions and activities are
occur (Table 1). For example, the aerobic ciliate Litonotus was
also important in structuring chemical heterogeneity and, thus,
observed at a depth of 7.1 mm, which is far below the depth
in dictating community structure in these laminated sedi-
at which oxygen was detectable (Figs. 8B, 6F). On the other
ments. For example, aggregations of the sulﬁde-oxidizing
hand, individuals of the anaerobic ciliate genus Parablephar-
Beggiatoa commonly harbored eukaryotes (Figs. 4F, 7A), pre-
isma were observed at depths shallower than known aerobes
sumably creating a sulﬁde-free zone that promoted survival
(foraminifera; Fig. 3). Horizontally, variability in distributions
of these eukaryotes. Additionally, it has been shown that cer-
was also observed. For example, ﬂagellate swarms, which must
tain ciliates and bacteria affect solute transport, thereby alter-
be hotspots of carbon cycling, are limited in extent to a few
ing their immediate environments (Fenchel and Glud 1998;
cubic millimeters. In addition, boli of Beggiatoa, which are
Glud and Fenchel 1999). Certain metazoan meiofauna from
presumably zones of increased sulﬁde oxidation (Table 2 and
other shallow-water environments also increase solute trans-
references therein) existed in microburrows, which occurred
port (Aller and Aller 1992). Given the high densities of cili-
patchily at any given sediment depth. The observed patchy
ates and meiofaunal metazoans in SBB (Bernhard et al. 2000),
distributions are even more relevant to pore–water chemistry
as well as the high water content of its sediments ( 93–98%,
and redox processes when one considers the metabolic capa-
Reimers et al. 1990), it is expected that pore–water exchange
bilities of the symbionts associated with SBB eukaryotes. For
by both diffusion and microorganism-mediated solute trans-
example, a number of SBB eukaryotes could have sulfate-re-
port in surﬁcial SBB sediments is considerable.
ducing symbionts, which could produce microzones of sulﬁde
Our ﬁndings demonstrate that SBB biotic distributions are
enrichment. Other SBB eukaryotes might have sulfur-oxidizing
horizontally and vertically complex, suggesting that the lami-
symbionts (e.g., the nematode Desmodora masira, B. Dubbels,
nated sediments of the SBB comprise submillimeter hetero-
D. Bazylinski, J. Bernhard unpubl. data), which could locally
geneous chemical regimes ranging from aerated to suboxic to
deplete [H S] if the host were inactive for lengthy periods.
sulﬁdic environments. It is known that overlapping oxic, sub-
2
Furthermore, the symbionts of some taxa are known to be me-
oxic, and anoxic diagenetic reactions occur in the SBB, at least

Benthic microbiota in situ
827
on the centimeter scale (Reimers et al. 1996). The observed
otic distributions and inferred chemical regimes. Thus, phys-
submillimeter-scale heterogeneity would be unexpected if the
icochemical conditions along any given lamina should not
only available data were microelectrode proﬁles, but any given
be expected to be consistent and, given the likely activity of
proﬁle represents the net sum of production minus consumption
the SBB inhabitants, these laminated sediments should not
and diffusion. Thus, proﬁles do not necessarily reﬂect micro-
be considered postdepositionally pristine. Although pore–
scale environmental parameters experienced by the inhabitants.
water geochemistry is an important force in structuring bi-
For example, although undetectable by microelectrodes in the
otic distributions, we infer from our observations that the
top
3 mm in February 1998 (Fig. 8A), sulﬁde likely existed
distributions of the microorganisms modify the localized
in the surface few millimeters, being produced in microzones
geochemical environment. The dynamics of the SBB benthic
that contained numerous sulfate-reducing bacteria. Indeed, sul-
community, and the further effect of this community on the
fate reduction rates in SBB surface sediments are high (Rei-
laminated sediments, can only be addressed, however, with
mers et al. 1996), although it should be noted that these rate
time-resolved observations, preferably executed in situ.
data were obtained from the top 2 cm rather than at the mil-
limeter scale. Additionally, Kuwabara and coworkers (1999)
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