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Previous Article | Next Article 
Applied and Environmental Microbiology, August 1999, p. 3272-3278, Vol. 65, No. 8
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Low-Molecular-Weight Sulfonates, a Major Substrate
for Sulfate Reducers in Marine Microbial Mats
Pieter T.
Visscher,*
Rachel F.
Gritzer, and
Edward
R.
Leadbetter
Department of Marine Sciences, and Department
of Molecular and Cell Biology, University of Connecticut, Groton,
Connecticut 06340
Received 16 March 1999/Accepted 18 May 1999
 |
ABSTRACT |
Several low-molecular-weight sulfonates were added to microbial mat
slurries to investigate their effects on sulfate reduction. Instantaneous production of sulfide occurred after taurine and cysteate
were added to all of the microbial mats tested. The rates of production
in the presence of taurine and cysteate were 35 and 24 µM
HS
h1 in a stromatolite mat, 38 and 36 µM
HS h1 in a salt pond mat, and 27 and 18 µM HS h1 in a salt marsh mat,
respectively. The traditionally used substrates lactate and acetate
stimulated the rate of sulfide production 3 to 10 times more than
taurine and cysteate stimulated the rate of sulfide production in all
mats, but when ethanol, glycolate, and glutamate were added to
stromatolite mat slurries, the resulting increases were similar to the
increases observed with taurine and cysteate. Isethionate,
sulfosuccinate, and sulfobenzoate were tested only with the
stromatolite mat slurry, and these compounds had much smaller effects
on sulfide production. Addition of molybdate resulted in a greater
inhibitory effect on acetate and lactate utilization than on sulfonate
use, suggesting that different metabolic pathways were involved. In all
of the mats tested taurine and cysteate were present in the pore water
at nanomolar to micromolar concentrations. An enrichment culture from
the stromatolite mat was obtained on cysteate in a medium lacking
sulfate and incubated anaerobically. The rate of cysteate consumption
by this enrichment culture was 1.6 pmol cell1
h1. Compared to the results of slurry studies, this rate
suggests that organisms with properties similar to the properties of
this enrichment culture are a major constituent of the sulfidogenic population. In addition, taurine was consumed at some of highest dilutions obtained from most-probable-number enrichment cultures obtained from stromatolite samples. Based on our comparison of the
sulfide production rates found in various mats, low-molecular-weight sulfonates are important sources of C and S in these ecosystems.
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INTRODUCTION |
The sulfur cycle plays an important
role in the geomicrobiology of intertidal and coastal sediments
(35, 59). In addition to sulfate, sulfide, and various
inorganic intermediates, organosulfur compounds constitute an important
sulfur pool as well (5, 50). In the pelagic marine
environment, dimethyl sulfide and methanethiol are thought to be
biologically important species (38) because of their
presumed importance in climate feedback models (11). In
addition to these organosulfur compounds, organic polysulfides, which
result from chemical reactions of sulfide and low-molecular-weight organic molecules, probably constitute a significant reservoir, especially in the benthic environment (40, 46).
The sulfonates are a group of organosulfur compounds that are commonly
found in household and industrial wastewater. Linear alkyl sulfonates
are major constituents of detergents (58), and the
catabolism of these compounds has been well documented (6,
18) and includes carbon-sulfur bond breakage pathways (39,
44). Recently, it has been shown that a range of
low-molecular-weight sulfonates are present in the marine environment.
A variety of different marine sediments contain significant amounts of
sulfonates, which comprise 20 to 40% of the organosulfur pool
(65). Unfortunately, the composition of the sulfonate pool
was not characterized further in the study of Vairavamurthy et al.
(65). However, several sources of sulfonates have been
identified in the marine environment. Sulfonates can be the sole source
of sulfur or nitrogen for marine phytoplankton (7), and
sulfolipids can be major biomembrane constituents in microscopic algae
(56, 57), cyanobacteria (2, 26), diatoms
(1), and bacteria (24). Sulfonate-containing exopolymers may be present in benthic diatoms and support the gliding
motility of these organisms, like suggestions made for bacteria
(25). Taurine (2-aminoethanesulfonate) is present in marine
diatoms (34) and zooplankton (9), presumably as a major osmolyte. The intracellular concentration of these compounds typically exceeds 0.2 M, which could easily explain the occurrence of
sulfonates in the estuarine environment. Similarly, cysteate (alanine
3-sulfonate) is one of the oxidation products of cysteine residues in
proteins (55, 60), and isethionate
(2-hydroxyethanesulfonate) has been found in marine algae
(31). Clearly, many different sources contribute to the pool
of low-molecular-weight sulfonates in the marine environment.
Taurine is one of the several amino acids that are readily bioavailable
in marine sediments (48). In addition to being used for
anabolic purposes, amino acids are catabolized by microbes. Under
anoxic conditions, this occurs through fermentation and, more
importantly, sulfate reduction (8, 51, 61). The
nitrogen-containing sulfonates taurine and cysteate can also be
utilized by sulfate-reducing bacteria (SRB) (27, 28,
42-44). Previously, most workers have focused on using amino
acids as electron donors, but recently Lie et al. (44)
demonstrated that in an SRB strain isolated from a salt marsh both
carbon and the sulfur moiety are catabolized. After cleavage of the
carbon-sulfur bond, the sulfite that is presumably generated can be
reduced to sulfide. Other low-molecular-weight sulfonates, such as
isethionate, also support growth of a variety of SRB (41, 42,
44).
Microbial mats, including microbial mats associated with salt marshes
and modern marine stromatolites, are typically dominated by a
cyanobacterial community near the surface. High rates of primary
production are coupled to high rates of aerobic respiration. High rates
of aerobic respiration rapidly deplete O2, and the oxic-anoxic interface is therefore found at a depth of a few
millimeters (66). As a result, a significant part of the
carbon fixed by the cyanobacteria is oxidized by SRB (36,
68). The maximum sulfate reduction rates (SRR) that have been
reported for microbial mats are high and range from 126 to 270 µM
h1 in the salt pond mats of Guerrero Negro
(10) and from 13 to 26 µM h1 in temperate
intertidal mats (67). Although the level of local production
of organic carbon due to photosynthesis is generally high, sulfate
reduction is generally thought to be carbon limited (21).
Modern marine stromatolite mats have much lower primary production
rates than the rates that have been reported for other microbial mats
(10, 36, 69), and so carbon limitation of the SRB should be
more pronounced in the former mats.
The aims of this study were to investigate whether low-molecular-weight
sulfonates stimulate sulfate reduction in stromatolite mats and, if
they do, to establish the importance of these compounds as sources of
carbon and sulfur. Furthermore, by comparing the effects of these
sulfonates on SRR in a variety of mats, we assessed their importance in
biogeochemical cycling of carbon and sulfur.
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MATERIALS AND METHODS |
Site description and field measurements.
The microbial mats
associated with modern marine stromatolites which are found in the
Exumas (Bahamas) have been described in detail previously (54,
69). Briefly, these laminated lithified structures typically
contain a cyanobacterial community that is dominated by
Schizothrix spp. in a 0.5- to 1-mm-thick surface layer which
is lithified (layer 1). Under this is a layer which is a few
millimeters thick, is soft (unlithified), and contains less biomass
(layer 2) and then another lithified layer, which is several
millimeters thick and is associated with greater microbial biomass
(layer 3). Under this, soft and hard layers alternate. The other
microbial mats used in this study were Microcoleus-dominated systems; samples of these mats were obtained from salt ponds (salt contents, 72 and 96 ppt) in Guerrero Negro, Mexico (14), and an intertidal salt marsh in Stonington, Conn. The mats from the salt
marsh were similar to the mats found in intertidal areas of the North
Sea (66, 68).
The stromatolite mats, which were the mats that were investigated in
most detail, were collected in March and August 1998 from a depth of 50 to 75 cm (mean high water) and were processed immediately. Salt pond
samples were collected in December 1998 and were stored on ice in the
dark before experiments were performed. Salt marsh samples were
obtained in September 1998 and February 1999, stored on ice, and
processed within 1 h.
Porewater samples were collected in situ with 22-gauge needles or were
recovered from sectioned cores in the lab by centrifugation (15,000 × g). The samples were filtered (pore size,
0.2 µm) and frozen until the taurine and cysteate concentrations were
measured by high-performance liquid chromatography (see below).
Microelectrode measurements were obtained at each site, as described
previously (69), by using needle electrodes for
O2 and HS (Diamond General, Ann Arbor, Mich.;
Microscale Measurements, Haren, The Netherlands).
We determined the SRR in discrete layers (69). Samples were
separated into layers and kept under air (oxic layers) or an N2 atmosphere (anoxic layers). Triplicate samples of each
layer were incubated with 1 µCi of
35SO42 (carrier free; Amersham,
Chicago, Ill.) under oxic or anoxic conditions for 6 h. Zinc
acetate and freezing were used to stop microbial activity, and after
this SRR were determined by using the single-step reduction method
(20). Based on 24-h measurements of O2 and
HS concentrations (69), diel SRR were
calculated by assuming that 14 h of oxic conditions and 10 h
of anoxic conditions occurred in the top 1 mm, 10 h of oxic
conditions and 14 h of anoxic conditions occurred in the 1- to
3-mm layer, 8 h of oxic conditions and 16 h of anoxic
conditions occurred in the 3- to 5-mm layer, and 24 h of anoxic
conditions occurred in the 5- to 10-mm layer.
Most-probable-number (MPN) incubations of SRB were done in the same
layers used for sulfate reduction measurements by employing sterilized
seawater supplemented with a carbonate-buffered lactate-acetate medium
(29, 67). Final scores were obtained after 8 weeks, and
population sizes were calculated by using the method of De Man
(15).
Taurine and cysteate concentrations in porewater samples were
determined by high-performance liquid chromatography by using precolumn
derivatization with o-phthalaldehyde and fluorescent detection (49). The detection limit for these sulfonated
amino acids was 50 pM. The hydrogen sulfide concentrations in slurry and cell suspension experiments were determined with ion-specific needle electrodes or by the methylene blue method (63).
Laboratory experiments.
Slurry experiments were performed
with stromatolite samples by using either bulk sediments (upper 10 mm)
or material from layer 3. For mat samples obtained from the salt pond
(salt content, 72 ppt) and the salt marsh, the upper 40 mm of the mat
was used. Samples were homogenized under an N2 atmosphere,
and slurries were prepared by mixing the resulting homogenates with
equal volumes of filter-sterilized deoxygenated seawater collected from
the respective sampling sites. The slurries (10 to 40 ml) were
incubated in small, dark polyvinyl chloride vessels that were sealed
without a headspace. After substrate was added, sulfide production was monitored continuously over a 15- to 60-min period with a sulfide needle electrode. The slurries were stirred except when
HS readings were being obtained. The following substrates
were tested: acetate, lactate, taurine, and cysteate (all three mat
slurries); glutamate (salt marsh and stromatolite mat slurries); and
ethanol, glycolate, Schizothrix exopolymer (EPS),
isethionate, 2-sulfobenzoate, and sulfosuccinate (stromatolite mat
slurries). The initial substrate concentrations in the slurries ranged
from 2 to 15 µM (except for EPS, which was added at a concentration
of 1.67 µg ml1), and all substrate additions were
replicated at least twice. The effect of adding
MoO42 (final concentration, 20 mM) on the
sulfide production rates in stromatolite samples was determined in
slurries containing acetate, lactate, taurine, and cysteate. All slurry
experiments were carried out at the ambient temperature. Taurine use
was also tested in two or three of the highest positive MPN enrichment cultures obtained from layers 1 and 3.
An enrichment culture was obtained from a piece of the stromatolite mat
(NS8) (69) for which the SRR and MPN were also determined. The inoculum was incubated in an anaerobic basal salt medium
(71) from which SO42 was omitted
and to which cysteate (10 mM) was added as the sole electron donor and
acceptor. Incubation was carried out in Balch tubes under an
N2-CO2 (80:20) headspace. The enrichment
culture was transferred approximately 20 times and served as the basis for cell suspension experiments.
Cell suspension experiments were carried out with cells in the early
stationary phase. A culture was harvested by centrifugation (15,000 × g, 4°C), washed twice, and resuspended in
medium without substrate reduced with 18 µM titanous chloride. Cells
were incubated in serum bottles sealed with butyl rubber stoppers lined
with Teflon. At zero time, each cell suspension received 18, 52, or 154 µM cysteate. Samples were withdrawn every 0.33 to 1 h through the stoppers by using sterile glass syringes, and sulfide and cysteate
concentrations were measured. The cell densities in these experiments
were determined by acridine orange epifluorescence microscopy
(30).
The chemicals used were standard reagent grade. Taurine, cysteic acid,
and isethionic acid were purchased from Sigma Chemical Co., St. Louis,
Mo.; sulfosuccinate was obtained from Fluka, Milwaukee, Wis.; and
2-sulfobenzoic acid was acquired from Aldrich, Milwaukee, Wis.
| |
RESULTS |
Field studies. (i) Stromatolite mats.
The highest SRR were
associated with the lithified layer at a depth of 3 to 5 mm (layer 3),
for which a diel SRR of 32 nmol cm3 h1 was
calculated (Fig. 1). The organic
compound-rich cyanobacterial surface layer (layer 1) also had a
significant SRR (14 nmol cm3 h1) despite
the high concentrations of oxygen that were present in that layer
during the day (Fig. 1). During the night, however, the depth of
O2 penetration was less than 0.6 mm; at this depth HS was detected, suggesting that SRB played an important
role in carbon oxidation in the surface layer. The vertical
distribution of SRB determined by the MPN method (Fig.
2) showed that the largest population was
in layer 3 at a depth of 3 to 5 mm (2 × 106 cells
cm3), which coincided with the layer that had the highest
SRR. Interestingly, the surface layer (layer 1) also contained a
significant viable population of SRB (6 × 104 cells
cm3), although it was much smaller than the populations
found in deeper layers. However, the cell-specific SRR were much higher at the surface at a depth of 3 to 5 mm. This could have been due to
underestimation of the SRB population at the surface due to selectivity
of the enumeration medium or, alternatively, to overestimation of the
SRR at a depth of 0 to 1 mm. The selectivity of the MPN technique has
been well documented (22, 37), and therefore the values
determined for populations of SRB probably are underestimates. However,
modifying the medium to mimic natural growth conditions, including
using a carbonate-buffered medium that contained site water, greatly
improved the sensitivity (37).
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FIG. 1.
(A and B) Depth profiles for the SRR (A) and the oxygen
and sulfide concentrations (B) in a modern marine stromatolite mat
(NS8) (69) in Highborne Cay, Bahamas. (C) Approximate
locations of layers 1 to 4. Open symbols, values obtained at night
(3:30 a.m.); solid symbols, values obtained during the day (12:30
p.m.); squares, O2 concentrations; circles, sulfide
concentrations. Measurements were obtained in August 1998. Error bars
indicate standard deviations (n = 3).
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FIG. 2.
Viable population of SRB as assessed by the MPN method
in the presence of lactate plus acetate for the stromatolite mat
described in Fig. 1. Error bars indicate the 95% confidence
intervals.
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Taurine and cysteate were detected in porewater samples obtained from a
depth of ca. 8 to 10 mm (Table 1); the
concentrations of these two compounds were 0.55 and 0.07 µM,
respectively.
(ii) Salt pond mats.
The presence of sulfide, as determined
with needle electrodes (data not shown), confirmed that an active SRB
population was present, as reported previously (10).
Cysteate was detected only in the lower-salinity mat (72 ppt) at a
concentration of 0.08 µM, but the taurine concentrations were much
higher and increased with increasing salinity; the taurine
concentrations were 0.87 and 2.34 µM in the mats containing 72 and 96 ppt of salt, respectively (Table 1).
(iii) Salt marsh mats.
The MPN analysis of SRB from salt marsh
mats (data not shown) revealed that the population increased with depth
in the upper 25 mm of the mat. At a depth of 15 to 25 mm, the
population size was 2.3 × 107 cells cm of
sediment3. Porewater samples contained the highest
concentrations of both taurine (1.61 µM) and cysteate (0.28 µM) in
the surface layer (0 to 10 mm). The cysteate concentration was below
the detection limit (50 pM) in deeper layers. The taurine
concentrations ranged from 0.2 to 1.6 µM and were similar to the
concentrations in stromatolite and salt pond mats (Table 1). The depth
profile of taurine indicated that consumption of this sulfonate
occurred at a depth of 20 to 30 mm.
Laboratory experiments. (i) Sulfide production in slurries.
The depth profiles for O2 and HS
concentrations, MPN measurements, and/or SRR measurements suggested
that very active populations of SRB were present in the upper parts of
all of the mats tested. Therefore, the upper 10 mm of the stromatolite
mat and the upper 40 mm of the salt pond and salt marsh mats were used
to obtain slurries in which SRB activity was high. Addition of organic
carbon increased the SRR, measured as HS production, in
all mat samples (Table 2). Sulfide was
produced without a lag in stromatolite slurries, but the salt pond and salt marsh slurries required preincubation for 10 and 60 min, respectively. An experiment in which sulfide was added to autoclaved slurry indicated that a chemical reaction of Fe2+ with
HS occurred. This buffering capacity of the slurry for
sulfide may explain the observed lag. In all three mats,
HS production was stimulated the most when acetate and
lactate were added. The rates were 15 to 20 times higher than the
endogenous production rates when acetate was added and 10 to 15 times
higher when lactate was added to the slurries. The rates increased
slightly more in salt pond and salt marsh slurries than in stromatolite slurries. Comparable rates were obtained for the stromatolite slurries
when slurries containing the top 10 mm and layer 3 were used (data not
shown). Addition of taurine and addition of cysteate also stimulated
HS production in all of the slurries (Table 2); the rate
of HS production increased 2- to 4.5-fold in the presence
of taurine and 1.5- to 3.5-fold when cysteate was added. In both cases,
sulfide was produced without a lag. The relative increases caused by
these substrates were higher in stromatolite and salt pond slurries than in salt marsh samples. Isethionate, 2-sulfobenzoate, and sulfosuccinate were tested only with stromatolite slurries, in which
the increases in HS production were small (Table 2).
Glutamate added to stromatolite and salt marsh slurries and ethanol and
EPS added to stromatolite slurries stimulated HS
production to the same extent that taurine and cysteate stimulated HS production (Table 2). Also, in the stromatolite mat
slurries, additions of substrates at concentrations ranging from 2 to
15 µM resulted in increased rates of HS production as
the substrate concentrations increased. Therefore, these observations
were used to determine the kinetic effects of substrates on SRR.
Saturation curves for acetate, taurine, and cysteate were linearized by
using single-reciprocal (Eadie-Hofstee) plots (19). Kinetic
indices describe the rate-limiting activity of the entire SRB community
rather than a single enzyme system. The Vmax and
apparent half-saturation constant (Km) were 170 µM h1 and 5.8 µM for acetate (data not shown), 56 µM h1 and 8.8 µM for taurine, and 33 µM
h1 and 7.2 µM for cysteate, respectively (Fig.
3). In the presence of
MoO42, HS production was
inhibited, albeit not completely; after acetate, lactate, taurine, and
cysteate were added, the rates of HS production decreased
82, 87, 43, and 21%, respectively.
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TABLE 2.
SRR in microbial mat slurries prepared from marine
stromatolites (Highborne Cay, Bahamas), a salt pond (Guerrero Negro,
Mexico), and a salt marsh (Stonington, Conn.) after electron donors
were addeda
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FIG. 3.
Eadie-Hofstee single-reciprocal plot of cysteate
(squares) and taurine (circles) consumption kinetics. Solid symbols,
cell suspension data; open symbols data from slurry experiments. All
measurements were obtained with stromatolite samples (the same samples
used for the experiments whose results are shown in Fig. 1 and 2). The
slope is defined by  Km.
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(ii) Cysteate utilization.
Suspensions of cells from the
enrichment culture consumed cysteate immediately when it was added as
the sole electron donor. The use of cysteate coincided with production
of HS (Fig. 4); 87 to 93%
of the sulfonate sulfur was recovered as HS. All of the
substrate was depleted within hours in cell suspensions that contained
3.5 × 107 to 5.5 × 107 cells
ml1. The maximum specific rate of cysteate consumption
was 1.6 pmol cell1 h1. The three cysteate
concentrations tested (18, 52, and 154 µM) were used in the
single-reciprocal (Eadie-Hofstee) plot (Fig. 3). The
Vmax and apparent Km for
cysteate utilization were 76 µM h1 and 4.8 µM,
respectively.
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FIG. 4.
Time course of cysteate consumption and sulfide
production in a cell suspension from an enrichment culture from a
Bahamian stromatolite. Solid symbols, cysteate; open symbols, sulfide;
circles, control (chemical destruction of cysteate when 184 µM
cysteate was added); squares and triangles, microbial consumption of
154 and 52 µM cysteate, respectively. The results obtained when 18 µM cysteate was added are not shown.
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(iii) Taurine utilization.
Utilization of taurine was tested
with the highest positive dilutions of the MPN series of layer 3; no
consumption was detected in the 107 dilution, but two of
three 106 dilution preparations consumed taurine when it
was added at a final concentration of 100 µM to medium containing 20 mM sulfate. One of two 104 dilutions tested from layer 1 also consumed taurine. This implied that at least 10% of the total SRB
population in the stromatolite mat could utilize taurine.
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DISCUSSION |
Taurine and cysteate are two of the low-molecular-weight
sulfonates which are rapidly degraded when they are added to anoxic microbial mat slurries that are producing HS
concomitantly. All microbial mats investigated in this study had high
SRR. The use of low-molecular-weight sulfonates observed in the slurry
experiments was confirmed with an enrichment culture obtained from the
stromatolite mat grown with cysteate. This corroborated previous
reports that both taurine and cysteate are enrichment substrates that
are used by aerobic (23, 39, 64) and anaerobic bacteria
(12, 16), including SRB (41, 43, 44). The
enrichment culture rapidly converted cysteate stoichiometrically to
HS in the absence of sulfate and thus must have depended
on C-S bond cleavage. Some, but not all, of the MPN enrichment
cultures, which were selected by using acetate and lactate as electron
donors, were also capable of taurine utilization.
Low-molecular-weight sulfonates are dominant organosulfur species in
terrestrial and marine environments (3, 48, 65). Taurine and
cysteate (at nanomolar to micromolar concentrations) were detected in
the porewater of a variety of microbial mats, and the concentrations of
taurine were higher when the salinity was greater. This finding
supports the hypothesis that taurine may be used as an osmolyte by a
variety of marine organisms (9, 13, 33, 34, 47, 53). The
observed pool sizes of taurine and cysteate may have been small, but
the rapid increase in HS production under anoxic
conditions suggests that the in situ turnover rates were high. Tight
coupling of production and consumption of organic carbon is typical for
microbial mats (66). Traditional substrates, such as
lactate, acetate, and ethanol, clearly stimulated SRR more than the
sulfonates tested stimulated SRR. However, in stromatolite slurries the
rates observed when glutamate, glycolate, and EPS were added were the
same as the rates observed when taurine and cysteate were added in this
system. Interestingly and surprisingly, Schizothrix EPS
stimulated sulfate reduction more than it stimulated aerobic
respiration (69). Although the composition of this EPS is
not known, it is conceivable that sulfonated residues are part of the
polymer matrix. The sulfur moiety can be a significant part (up to 14%
of the total dry weight) of cyanobacterial EPS (17) and is
believed to be associated with sulfate esters. However, due to the use
of rigorous digestion techniques in certain assays, sulfonates could
account for part of the EPS sulfur as well.
The compositions of the SRB populations in sediments change with depth
(37, 67). The changes are determined in part by the
available substrates (21). At the surface of a mat the
substrates are directly derived from cyanobacteria (e.g., glycolate),
but at depth they are produced by fermentative organisms (which produce ethanol, lactate, etc.). Similarly, different populations of SRB are
expected when different mats are compared, depending on physicochemical parameters, such as temperature, light, and substrate availability. The
SRR in the stromatolite mat is 10 to 20 times lower than the SRR
reported for salt pond mats (10). Despite this difference in
SRR, the salt pond slurries had only slightly higher HS
production rates than the stromatolite slurries had when traditional substrates were added and similar rates when taurine and cysteate were
added. A possible explanation for this is that the SRB in the
stromatolite mats are more severely substrate limited, perhaps due to
diffusion barriers (lithified layers) that are present (32).
Alternatively, the stromatolite mats may contain different types of
SRB, which, given the very distinct physicochemical parameters, would
not be too surprising. Interestingly, despite the pronounced differences in SRR, SRB population sizes, etc., all mat slurries consumed low-molecular-weight sulfonates.
Molybdate is a specific inhibitor of sulfate reduction (52),
and its effect on SRB in slurries when acetate and lactate were added
was as expected (82 and 87% inhibition, respectively). However, the
inhibitory effect of MoO42 was less
pronounced when a sulfonate was added; HS production with
taurine was inhibited 43%, and HS production with
cysteate was inhibited 21%. A similar effect was noticed by Lie et al.
(45) when pure cultures of a SRB were amended with
MoO42 and either isethionate or cysteate.
Molybdate competes with sulfate for active sites of ATP sulfurylase. If
indeed C-S cleavage of sulfonates results in
SO3 instead of
SO42, as suggested previously (39, 41,
43, 44), production of APS is not required, and thus, no
inhibitory effects of MoO42 are expected. The
fact that some inhibition was observed could be attributed to the
presence of both SO42 and ATP sulfurylase in
the slurry, which should have resulted in some ATP depletion when
MoO42 was added (62) and, perhaps,
a lower rate of overall HS production. Alternatively, as
mentioned above, a significant number of novel S-reducing organisms,
which are less sensitive to MoO42, could be
present in the stromatolite mat. Clearly, the true inhibitory effect of
molybdate needs to be reevaluated, so the use of molybdate in
environmental studies does not lead to misinterpretations, as has been
suggested previously (45, 52, 67).
The use of low-molecular-weight sulfonates in slurries and cell
suspensions revealed remarkably similar substrate affinities (defined
as the initial slope of the v-s graph, or
Vmax/Km) (Fig. 5). This indicates that organisms similar
to the enrichment culture organisms consume sulfonate in situ. When the
maximum rate observed in the slurry is divided by the highest MPN
dilution of SRB that used taurine, the theoretical cell-specific
consumption rates for taurine and cysteate are 35 and 24 pmol
cell1 h1, respectively. Compared to the
maximum cell-specific rate observed in the cell suspension (1.6 pmol
cell1 h1), these rates are 1 order of
magnitude higher, which is not atypical for slurry experiments
(8). We should emphasize that the size of the
taurine-utilizing fraction of SRB used for this calculation is only a
rough approximation. Clearly, the size of the population of
sulfidogenic organisms that use low-molecular-weight sulfonates needs
to be assessed more carefully. Lie et al. (44) observed that
H2 utilization by Desulfovibrio desulfuricans
IC1 occurs at a lower H2 concentration (3 ppmv) in the
presence of isethionate as the electron acceptor than in the presence
of either sulfate or sulfite (H2 thresholds, 15 and 8 ppmv,
respectively). Similar mechanisms could contribute to higher rates of
HS production in slurries in which the presence of
H2 is conceivable.
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FIG. 5.
Michaelis-Menten kinetics for taurine utilization
(slurry) and cysteate utilization (slurry and cell suspension). The
kinetic indices Vmax and
Km were 56 h1 and 8.8 µM, 33 h1 and 7.2 µM, and 76 h1 and 15.8 µM
for taurine, for cysteate in slurries, and for cysteate in a cell
suspension, respectively. The corresponding affinities
(Vmax/Km) were 6.36, 4.58, and 4.80 h1 µM1, respectively. The
Vmax and Km for acetate
in slurry experiments (data not shown) were 170 h1 and
5.8 µM, respectively, while the affinity was 29.31 h1
µM1.
|
|
Amino acids are important substrates for bacteria in marine sediments,
and several amino acids support SRB growth (8, 27). The
importance of sulfonate-containing amino acids for SRB metabolism seems
clear, especially when the HS production rates on taurine
and cysteate are compared with the HS production rates on
glutamate. In addition to anaerobic consumption of sulfonates, aerobic
degradation was observed as well; in stromatolite slurries, a rapid
change in the oxygen concentration over time (d[O2]/dt) was observed, and the
rates were approximately one-third the rates observed when acetate or
lactate was added (69, 70). Similarly, methanesulfonate,
another low-molecular-weight sulfonate and a chemical oxidation product
of dimethyl sulfide, was also degraded aerobically by marine
methylotrophs (4).
We concluded that low-molecular-weight sulfonates contribute
significantly to the substrates that support SRB growth in microbial mats. The fact that the relative stimulation of the SRR was much greater in stromatolites could indicate either that the substrate limitation in these mats was greater or that a substantially different community of SRB was present or both. The observation that relatively high concentrations of O2 are present in the layers which
exhibit high SRR for at least part of a diel cycle (69) and
the observation that MoO42 has a limited
inhibitory effect indicate that a more detailed study of the
physiological diversity and the molecular diversity of SRB is necessary.
| |
ACKNOWLEDGMENTS |
This study was supported by NSF grant OCE 9619314 and by NASA
collaborative agreement NCC2-1067.
Alan Decho kindly provided Schizothrix slime. The technical
assistance of and fruitful discussions with Shelley Hoeft, Tonna-Marie Surgeon, Pamela Reid, Brad Bebout, John Thompson, and Tom Lie are much appreciated.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Marine Sciences, University of Connecticut, 1084 Shennecossett Road, Groton, CT 06340. Phone: (860) 405-9159. Fax: (860) 405-9153. E-mail:
pieter.visscher{at}uconn.edu.
This is RIBS contribution number 03.
| |
REFERENCES |
| 1.
|
Anderson, R.,
M. Kates, and B. E. Volcani.
1978.
Identification of the sulfolipids in the non-photosynthetic diatom Nitzschia alba (Algae).
Biochim. Biophys. Acta
528:89-106.
|
| 2.
|
Archer, S. D.,
K. A. McDonald, and A. P. Jackman.
1997.
Effect of light irradiance on the production of sulfolipids from Anabaena 7120 in a fed-batch photobioreactor.
Appl. Biochem. Biotechnol.
67:139-152.
|
| 3.
|
Autry, A. R., and J. W. Fitzgerald.
1990.
Sulfonate S: a major form of forest soil organic sulfur.
Biol. Fertil. Soils
10:50-56.
|
| 4.
|
Baker, S. C.,
D. P. Kelly, and J. C. Murrell.
1991.
Microbial degradation of methanesulphonic acid: a missing link in the biogeochemical sulphur cycle.
Nature
350:627-628.
|
| 5.
|
Bates, T. E., and R. Carpenter.
1979.
Organo-sulfur compounds in sediments in the Puget Sound.
Geochim. Cosmochim. Acta
43:1209-1221.
|
| 6.
|
Berna, J. L.,
A. Moreno, and J. Ferrer.
1991.
The behavior of LAS in the environment.
J. Chem. Technol. Biotechnol.
50:387-398.
|
| 7.
|
Biedlingmaier, S.,
H.-P. Körst, and A. Schmidt.
1986.
Utilization of sulfonic acids as the only sulfur source for growth of photosynthetic organisms.
Planta
169:518-523.
|
| 8.
|
Burdige, D. J.
1989.
The effect of sediment slurrying on microbial processes, and the role of amino acids as substrates for sulfate reduction in anoxic sediments.
Biogeochemistry
8:1-23.
|
| 9.
|
Burton, R. S., and M. W. Feldman.
1982.
Changes in free amino acid concentrations during osmotic response in the intertidal copepod Tigriopus californicus.
Comp. Biochem. Physiol.
73:441-445.
|
| 10.
|
Canfield, D. E., and D. J. DesMarais.
1991.
Aerobic sulfate reduction in microbial mats.
Science
251:1471-1473.
|
| 11.
|
Charlson, R. J.,
J. E. Lovelock,
M. O. Andreae, and S. G. Warren.
1987.
Oceanic phytoplankton, atmospheric sulfur, cloud albedo and climate.
Nature
326:655-661.
|
| 12.
|
Chien, C.-C.,
E. R. Leadbetter, and W. Godchaux, III.
1995.
Sulfonate-sulfur can be assimilated for fermentative growth.
FEMS Microbiol. Lett.
129:189-194.
|
| 13.
|
Costa, C. J.,
S. K. Pierce, and M. K. Warren.
1980.
The intracellular mechanism of salinity tolerance in polychaetes: volume regulation by isolated Glycera dibranchiata red coelomocytes.
Biol. Bull.
159:626-638.
|
| 14.
|
D'Amelio, E. D.,
Y. Cohen, and D. J. DesMarais.
1989.
Comparative functional ultrastructure of two hypersaline submerged cyanobacterial mats: Guerrero Negro, Baja California Sur, and Solar Lake, Sinai, Egypt, p. 97-113.
In
Y. Cohen, and E. Rosenberg (ed.), Microbial mats: physiological ecology of benthic microbial communities. American Society for Microbiology, Washington, D.C.
|
| 15.
|
De Man, J.
1975.
The probability of most probable numbers.
Eur. J. Appl. Microbiol.
1:67-78.
|
| 16.
|
Denger, K.,
H. Laue, and A. M. Cook.
1997.
Anaerobic taurine oxidation: a novel reaction by nitrate-reducing Alcaligenes sp.
Microbiology
143:1919-1924.
|
| 17.
|
De Philippis, R., and M. Vincenzini.
1998.
Exocellular polysaccharides from cyanobacteria and their possible applications.
FEMS Microbiol. Rev.
22:151-175.
|
| 18.
|
Federle, T. W., and R. M. Ventullo.
1990.
Mineralization of surfactants by the microbiota of submerged plant detritus.
Appl. Environ. Microbiol.
56:333-339.
|
| 19.
|
Fersht, A.
1985.
Enzyme structure and mechanism. W. H.
Freeman & Co., New York, N.Y.
|
| 20.
|
Fossing, H., and B. B. Jørgensen.
1989.
Measurement of bacterial sulfate reduction in sediments: evaluation of a single-step chromium reduction method.
Biogeochemistry
8:205-222.
|
| 21.
|
Fründ, C., and Y. Cohen.
1992.
Diurnal cycles of sulfate reduction under oxic conditions in microbial mats.
Appl. Environ. Microbiol.
58:70-77.
|
| 22.
|
Gibson, G. R.,
R. J. Parkes, and R. A. Herbert.
1987.
Evaluation of viable counting procedures for the enumeration of sulfate-reducing bacteria in estuarine sediments.
J. Microbiol. Methods
7:201-210.
|
| 23.
|
Giehl, T. J.,
M. W. Qodronfleh, and B. J. Wilkinson.
1987.
Transport, nutritional and metabolic studies of taurine in staphylococci.
J. Gen. Microbiol.
133:849-856.
|
| 24.
|
Godchaux, W., III, and E. R. Leadbetter.
1983.
Unusual sulfonolipids are characteristic of Cytophaga-Flexibacter group.
J. Bacteriol.
153:1238-1246.
|
| 25.
|
Godchaux, W., III, and E. R. Leadbetter.
1984.
Sulfonolipids of gliding bacteria: structures of the N-acylaminosulfonates.
J. Biol. Chem.
259:621-623.
|
| 26.
|
Guler, S.,
A. Seeliger,
H. Hartel,
G. Renger, and C. Benning.
1996.
A null mutant of Synechococcus sp. PCC7942 deficient in the sulfolipid sulfoquinovosyl dialkylglycerol.
J. Biol. Chem.
271:7501-7507.
|
| 27.
|
Hansen, L. S.,
M. Holmer, and T. H. Blackburn.
1993.
Mineralization of organic nitrogen and carbon (fish food) added to anoxic sediment in microcosms: role of sulphate reduction.
Mar. Ecol. Prog. Ser.
102:199-204.
|
| 28.
|
Hansen, L. S., and T. H. Blackburn.
1995.
Amino acid degradation by sulfate-reducing bacteria.
Limnol. Oceanogr.
40:502-510.
|
| 29.
|
Hines, M. E.,
P. T. Visscher, and R. Devereux.
1996.
Sulfur cycling, p. 324-334.
In
S. Y. Newell, et al. (ed.), Manual of environmental microbiology. Aquatic environments. American Society for Microbiology, Washington, D.C.
|
| 30.
|
Hobbie, J. E.,
R. J. Daley, and S. Jasper.
1977.
Use of Nuclepore filters for counting bacteria by fluoerescence microscopy.
Appl. Environ. Microbiol.
33:1225-1228.
|
| 31.
|
Holst, P. B.,
S. E. Nielsen,
U. Anthoni,
K. S. Bisht,
C. Christophersen,
S. Gupta,
V. S. Parmar,
P. H. Nielsen,
D. B. Sahoo, and A. Singh.
1994.
Isethionate in certain red algae.
J. Appl. Phycol.
6:443-446.
|
| 32.
|
Huettel, M.,
W. Ziebis, and S. Foster.
1996.
Flow-induced uptake of particulate matter in permeable sediments.
Limnol. Oceanogr.
41:309-322.
|
| 33.
|
Huxtable, R. J.
1992.
Physiological aspects of taurine.
Physiol. Rev.
72:101-163.
|
| 34.
|
Jackson, A. E.,
S. W. Ayer, and M. V. Laycock.
1992.
The effect of salinity on growth and amino acid composition in the marine diatom Nitzschia pungens.
Can. J. Bot.
70:2198-2201.
|
| 35.
|
Jørgensen, B. B.
1982.
Mineralization of organic matter in the sea bed the role of sulphate reduction.
Nature
296:643-645.
|
| 36.
|
Jørgensen, B. B., and Y. Cohen.
1977.
Solar Lake (Sinai). 5. The sulfur cycle of the benthic cyanobacterial mats.
Limnol. Oceanogr.
22:657-666.
|
| 37.
|
Jørgensen, B. B., and F. Bak.
1991.
Pathways and microbiology of thiosulfate transformations and sulfate reduction in a marine sediment (Kattegat, Denmark).
Appl. Environ. Microbiol.
57:847-856.
|
| 38.
|
Kiene, R. P.
1993.
Microbial sources and sinks for methylated sulfur compounds in the marine environment, p. 15-33.
In
J. C. Murrell, and D. P. Kelly (ed.), Microbial growth on C1 compounds. Intercept Ltd., Andover, United Kingdom.
|
| 39.
|
Kondo, H.,
H. Niki,
S. Takahashi, and M. Ishimoto.
1977.
Enzymatic oxidation of isethionate to sulfoacetaldehyde in bacterial extract.
J. Biochem.
81:1911-1916.
|
| 40.
|
LaLonde, R. T.,
L. M. Ferrara, and M. P. Hayes.
1987.
Low-temperature, polysulfide reactions of conjugated ene carbonyls: a reaction model of S-heterocycles.
Org. Geochem.
11:563-571.
|
| 41.
|
Laue, H.,
K. Denger, and A. M. Cook.
1997.
Taurine reduction in anaerobic respiration of Bilophila wadsworthia RZATAU.
Appl. Environ. Microbiol.
63:2016-2021.
|
| 42.
|
Laue, H.,
K. Denger, and A. M. Cook.
1997.
Fermentation of cysteate by a sulfate-reducing bacterium.
Arch. Microbiol.
168:210-214.
|
| 43.
|
Lie, T. J.,
T. Pitta,
E. R. Leadbetter,
W. Godchaux III, and J. R. Leadbetter.
1996.
Sulfonates: novel electron acceptors in anaerobic respiration.
Arch. Microbiol.
166:204-210.
|
| 44.
|
Lie, T. J.,
J. R. Leadbetter, and E. R. Leadbetter.
1998.
Metabolism of sulfonic acids and other organosulfur compounds by sulfate reducing bacteria.
Geomicrobiol. J.
15:135-149.
|
| 45.
| Lie, T. J., W. Godchaux III, and E. R. Leadbetter. Sulfidigenesis from sulfonates: sulfonates as terminal
electron acceptors for growth of sulfite-reducing bacteria
(Desulfitobacterium spp.) and sulfate-reducing bacteria.
Submitted for publication.
|
| 46.
|
Luther, G. W., III,
T. M. Church,
J. R. Scudlark, and M. Cosman.
1986.
Inorganic and organic sulfur cycling in salt-marsh porewaters.
Science
232:746-749.
|
| 47.
|
Manahan, D. T., and S. Nourizadeh.
1988.
Requirements for specific amino acids and proteins by oyster larvae (Crassostrea gigas).
J. Shellfish Res.
8:322-323.
|
| 48.
|
Mayer, L. M.,
L. L. Schick,
T. Sawyer,
C. J. Plante,
P. A. Jumars, and R. L. Self.
1985.
Bioavaialble amino acids in sediments: a biomimetic kinetic-based approach.
Limnol. Oceanogr.
40:511-520.
|
| 49.
|
Mopper, K., and D. Delmas.
1984.
Trace determination of biological thiols by liquid chromatography and precolumn fluorometric labeling with o-phtalaldehyde.
Anal. Chem.
56:2557-2560.
|
| 50.
|
Mopper, K., and B. F. Taylor.
1986.
Biogeochemical cycling of sulfur: thiols in coastal marine sediments, p. 323-339.
In
M. L. Sohn (ed.), Organic marine geochemistry. American Chemical Society, Washington, D.C.
|
| 51.
|
Nanninga, H. J., and J. C. Gottschal.
1985.
Amino acid fermentation and hydrogen transfer in mixed cultures.
FEMS Microbiol. Ecol.
31:261-269.
|
| 52.
|
Oremland, R. S., and D. G. Capone.
1988.
Use of "specific" inhibitors in biogeochemistry and microbial ecology.
Adv. Microb. Ecol.
10:285-383.
|
| 53.
|
Pierce, S. K.,
L. M. Rowand-Faux, and S. M. O'Brien.
1992.
Different salinity mechanism in Atlantic and Chesapeake Bay conspecific oysters: glycine betaine and amino acid pool variations.
Mar. Biol.
113:115-117.
|
| 54.
|
Reid, R. P.,
I. G. Macintyre,
R. S. Steineck,
K. M. Brown, and T. E. Miller.
1995.
Stromatolites in the Exuma Cays: uncommonly common.
Facies
33:1-18.
|
| 55.
|
Roy, A. B., and P. A. Trudinger.
1970.
The biochemistry of inorganic compounds of sulphur.
Cambridge University Press, Cambridge, United Kingdom.
|
| 56.
|
Saidha, T., and J. A. Schiff.
1989.
The role of mitichondria in sulfolipid biosynthesis by Euglena chloroplasts.
Biochim. Biophys. Acta
1001:263-273.
|
| 57.
|
Sandman, G., and P. Boger.
1982.
Formation and degradation of photosynthetic membranes determined by 35S (sulfur isotope) labeled sulfolipid (Scenedesmus acutus, algae).
Plant Sci. Lett.
24:347-352.
|
| 58.
|
Schmidt, W. W.,
W. Lillienthal,
K. H. Raney, and S. T. Dubey.
1994.
A novel dianionic surfactant from the reaction of C14-alkenylsuccinic acid with sodium isethionate.
J. Am. Oil Chem. Soc.
71:695-703.
|
| 59.
|
Sørensen, J., and B. B. Jørgensen.
1987.
Early diagenesis in sediments from Danish coastal waters: microbial activity and Mn-Fe-S geochemistry.
Geochim. Cosmochim. Acta
51:1583-1590.
|
| 60.
|
Spindle, M.,
R. Stadler, and H. Tanner.
1984.
Amino acid analysis of feedstuffs: determination of methionine and cysteine after oxidation with performic acid and hydrolysis.
J. Agric. Food Chem.
32:1366-1371.
|
| 61.
|
Stams, A. J. M.,
T. A. Hansen, and G. W. Skyring.
1985.
Utilization of amino acids as energy substrates by two marine Desulfovibrio strains.
FEMS Microbiol. Ecol.
31:11-15.
|
| 62.
|
Taylor, B. F., and R. S. Oremland.
1979.
Depletion of adenosine triphosphate in Desulfovibrio by oxyanions of group VI elements.
Curr. Microbiol.
3:101-103.
|
| 63.
|
Trüper, H. G., and H. G. Schlegel.
1964.
Sulphur metabolism in Thiorhodaceae. I. Quantitative measurements of growing cells of Chromatium okenii.
Antonie Leeuwenhoek J. Microbiol. Serol.
30:225-238.
|
| 64.
|
Uria-Nickelsen, M. R.,
E. R. Leadbetter, and W. Godchaux, III.
1993.
Sulphonate utilization by enteric bacteria.
J. Gen. Microbiol.
139:203-208.
|
| 65.
|
Vairavamurthy, A.,
W. Zhou,
T. Eglinton, and B. Manowitz.
1994.
Sulfonates: a novel class of organic sulfur compounds in marine sediments.
Geochim. Cosmochim. Acta
58:4681-4687.
|
| 66.
|
Van Gemerden, H.
1993.
Microbial mats: a joint venture.
Mar. Geol.
113:3-25.
|
| 67.
|
Visscher, P. T.,
R. A. Prins, and H. van Gemerden.
1992.
Rates of sulfate reduction and thiosulfate consumption in a marine microbial mat.
FEMS Microbiol. Ecol.
86:383-394.
|
| 68.
|
Visscher, P. T., and H. van Gemerden.
1993.
Sulfur cycling in laminated marine ecosystems, p. 672-693.
In
R. S. Oremland (ed.), Biogeochemistry of global change: radiatively active trace gases. Chapman and Hall, New York, N.Y.
|
| 69.
|
Visscher, P. T.,
R. P. Reid,
B. M. Bebout,
S. E. Hoeft,
I. G. Macintyre, and J. A. Thompson, Jr.
1998.
Formation of lithified micritic laminae in modern marine stromatolites (Bahamas): the role of sulfur cycling.
Am. Minerol.
83:1482-1493.
|
| 70.
| Visscher, P. T. 1998. Unpublished data.
|
| 71.
|
Widdel, F., and F. Bak.
1992.
Gram-negative mesophilic sulfate-reducing bacteria, p. 3353-3378.
In
A. Balows, H. G. Trüper, M. Dworkin, W. Harder, and K.-H. Schleifer (ed.), The prokaryotes. A handbook on the biology of bacteria: ecophysiology, isolation, identification, applications, 2nd ed. Springer-Verlag, New York, N.Y.
|
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Abstract
Introduction
