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Syntrophic growth of sulfate-reducing bacteria and colorless sulfur bacteria during oxygen limitation

Frank P. van den Ende , Jutta Meier , Hans van Gemerden
DOI: http://dx.doi.org/10.1111/j.1574-6941.1997.tb00392.x 65-80 First published online: 1 May 1997

Abstract

Stable co-cultures of the sulfate-reducing bacterium Desulfovibrio desulfuricans PA2805 and the colorless sulfur bacterium Thiobacillus thioparus T5 were obtained in continuous cultures supplied with limiting amounts of lactate and oxygen while sulfate was present in excess. Neither species could grow in pure culture under these conditions. Desulfovibrio could grow only when the oxygen concentration was kept low by Thiobacillus. Zerovalent sulfur (S0) produced by Thiobacillus was preferred over sulfate as electron acceptor by Desulfovibrio, but the affinity for S0 seemed to be rather low. This substrate was more efficiently used when sulfide was present suggesting that S0 is preferably used in the form of polysulfides. Through the use of S0 as electron acceptor the sulfide production per lactate by Desulfovibrio was four times higher than with sulfate as acceptor. Thiobacillus produces less sulfate and more S0 when the amount of sulfide available per oxygen increases. The elevated sulfide production by Desulfovibrio thus resulted in an increase of the S0 production by Thiobacillus, again leading to a further increase of the sulfide production. This positive feedback mechanism stabilizes the syntrophic association. The yield on lactate of Desulfovibrio was doubled in the mixed culture compared with growth on lactate and sulfate in pure culture. This yield increase was attributed to the use of zerovalent sulfur instead of sulfate as electron acceptor. Both organisms were thus shown to benefit from a syntrophic interaction in which lactate was oxidized with oxygen, with a rapid cycling of sulfide and zerovalent sulfur serving the transfer of reducing equivalents between the species. These observations shed some light on the occurrence of colorless sulfur bacteria and sulfate-reducing bacteria at the same depth horizons in microbial mats.

Keywords
  • Thiobacillus
  • Desulfovibrio
  • Oxygen limitation
  • Sulfide oxidation
  • Sulfate reduction

1 Introduction

Coexistence of sulfate-reducing bacteria and colorless sulfur bacteria has been reported to occur in various habitats, e.g., marine sediments [1], microbial mats [2], hydrothermal vents [3] and biofilms in waste water treatment systems [4]. Aerobic colorless sulfur bacteria require the availability of both reduced sulfur compounds and oxygen, and optimum conditions obviously occur at oxygen-sulfide interfaces. Although sulfate-reducing bacteria are anaerobes that prefer anoxic conditions highest population densities in a microbial mat were found around the oxygen sulfide interface [2]. This is not surprising because in these ecosystems the primary food source is organic matter derived from organisms in the oxic zone. Considering the avoidance of oxygen and positioning as close as possible to the food source, optimum conditions are expected at the oxygen sulfide interface. The depth of oxygen penetration however is not static. Especially in microbial mats and intertidal sediments the activities of oxygenic phototrophs cause a light dependent up and downward movement of the oxygen sulfide interface [57]. Organisms living at the delicate balance between maximum availability of food and avoidance of oxygen are expected to face regular contact with oxygen.

Many sulfate-reducing bacteria tolerate the periodic exposure to oxygen [811]. High numbers of viable sulfate-reducing bacteria have been found in oxic zones of marine sediments and microbial mats [12, 13, 2]. Fine scale sulfate reduction rate measurements have shown the continuation of sulfate reduction during oxic periods in microbial mats [1416, 2]. On the other hand Cypionka and coworkers showed that oxygen can be used as electron acceptor by many strains of sulfate-reducing bacteria and is even preferred over sulfur compounds [1720]. Although aerobic respiration did not seem to support prolonged growth, respiration rates and affinity for oxygen resembled those of aerobes. There may therefore be a direct competition for oxygen between sulfate-reducing and colorless sulfur bacteria.

Under oxygen limitation colorless sulfur bacteria oxidize sulfide partially to zerovalent sulfur and thiosulfate [2123]. These compounds can be used by sulfate-reducing bacteria as electron acceptor in the reduction of hydrogen and organic compounds [2426]. However, zerovalent sulfur and thiosulfate may also be disproportionated or even oxidized with oxygen [27, 28, 18]. Due to the versatility of sulfate-reducing bacteria interactions with colorless sulfur bacteria could be argued to be competitive just as well as commensalistic.

The aim of the present study was to investigate the interactions between a sulfate-reducing and a colorless sulfur bacterium under conditions as they occur at the oxygen sulfide interface in marine systems. To this end both pure and mixed cultures were studied. Lactate was supplied as model organic substrate and the availability of oxygen was varied. In all experiments sulfate was present in excess.

2 Materials and methods

2.1 Bacterial strains

The marine sulfate-reducing bacterium Desulfovibrio desulfuricans strain PA2805, isolated from intertidal sediment in the Basin d'Arcachon, was kindly provided by Prof. P. Caumette. The colorless sulfur bacterium Thiobacillus thioparus strain T5 was originally isolated from a marine microbial mat [29].

2.2 Culture medium

The composition of the mineral medium used was as follows (in g·l−1): NaCl (25.0), MgSO4·7H2O (4.9), Na2CO3 anh. (4.0), CaCl2·2H2O (0.225), NH4Cl (0.2), KCl (0.2), MgCl2·6H2O (0.2), KH2PO4 anh. (0.02), FeSO4·7H2O (0.001) and trace element solution SL12B (1 ml·l−1) [30]. Filter-sterilized vitamin solution (1 ml·l−1) and lactic acid (see Table 1) were added after the autoclaved medium had cooled. A pinch of dithionite was added before inoculation when the medium was used for batch cultures. The vitamin solution contained (in g·l−1): p-aminobenzoic acid (0.1), riboflavin (0.1), thiamin (0.2), nicotinic acid (0.2), pyridoxamin (0.5) pantothenic acid (0.1), cyanocobalamin (0.1) and biotin (0.02).

View this table:
1

List of symbols used

[H]= reducing equivalent
Th=Thiobacillus thioparus T5
Dv=Desulfovibrio desulfuricans PA2805
N= cell density (cells·l−1)
pThC,pDvC= protein content per cell
vThP= rate of protein synthesis of Thiobacillus (mg·l−1·h−1)
vThO2= oxygen consumption rate of Thiobacillus (mmol·l−1·h−1)
vDvO2= oxygen consumption rate of Desulfovibrio (mmol·l−1·h−1)
vtotalO2= measured total oxygen consumption rate (mmol·l−1·h−1), equals supply rate when oxygen is limiting.
v[H]= rate of release of reducing equivalents from lactate oxidation (mmol·l−1·h−1)
v[H]→S0= flux of reducing equivalents to S0 (mmol·l−1·h−1)
vTh[H]= rate of transfer of reducing equivalents to Thiobacillus (mmol·l−1·h−1)
vDv[H]→S= rate of transfer of reducing equivalents to sulfur compounds by Desulfovibrio (mmol·l−1·h−1)
vDv[H]→O2= rate of transfer of reducing equivalents to oxygen by Desulfovibrio (mmol·l−1·h−1)
[H]x= mmol H per mmol compound x
bDv, bTh= proportion of [H] used for biosynthesis
= steady-state concentration
Yx= yield (mg protein mg−1 substrate x)
D= dilution rate (h−1)

2.3 Culture set-up

The organisms were grown in continuous culture at a constant dilution rate of 0.037 h−1. The medium was pumped from two reservoir bottles at equal rates. One bottle contained a double strength solution of carbonate and lactate at pH 11, the other bottle a double strength solution of the other constituents at pH 4.5. The pH in the culture was kept at 7.8±0.05 by titration with 1M HCl. Temperature was maintained at 250C. Lactate was the sole electron donor supplied, while sulfate and oxygen were supplied as electron acceptors. Sulfate was present in excess, whereas oxygen was supplied in limiting amounts. The culture volume was 1 l, with a headspace of 0.5 l. The culture was gassed with an adjustable water-saturated air/nitrogen mixture at a rate of 0.8 l h−1. The oxygen supply rate, and thus the degree of oxygen limitation, was controlled by changing the air content in the gassing mixture. The oxygen concentration in the culture fluid and in the out-flowing gas were monitored continuously with an oxygen electrode (Ingold, Switzerland). Samples for gas-chromatographic oxygen determination were taken from sample ports in the gas in- and outlets.

2.4 Analytical procedures

Lactate and acetate were assayed gas-chromatographically [31]. Oxygen and carbon dioxide were analyzed with a gas-chromatograph equipped with a katharometer. Oxygen uptake rates were calculated from the gas flow rate (determined accurately with film-flow pipettes) and the difference between the oxygen concentration in the in- and out-flowing gas. Gas-volumes were corrected for temperature, pressure, oxygen uptake and carbon dioxide and sulfide purged from the culture.

Sulfide was measured colorimetrically using the methyleneblue method of Pachmayr [32]. Sulfate was measured colorimetrically as chloranilate [33]. Zerovalent sulfur (S0) was pelleted by centrifugation and quantified spectrophotometrically in methanol extracts [34]. Thiosulfate, tetrathionate and polysulfides were assayed colorimetrically after cyanolysis of 0.2 μm filtered samples [35, 36].

Protein was assayed according to Lowry et al. [37] on cell pellets solubilized in 1 M NaOH, after extraction of zerovalent sulfur with methanol. Bovine serum albumin was used as a standard. Reserve sugars were measured as total hexose with the anthrone reagent [38]. PHB was determined gas-chromatographically after Braunegg et al. [39]. Cell numbers were counted by epifluorescence microscopy after staining with DAPI [40].The proportion of Thiobacillus and Desulfovibrio cells was determined separately with a bright-field phase-constrast microscope. Growth in batch cultures of Desulfovibrio was monitored as optical density at 660 nm.

2.5 Calculations

2.5.1 Oxygen consumption

(a) Estimation of the rate of oxygen uptake of Desulfovibrio by quantification of the path of the oxygen supplied.

Using the parameters as described in Table 1 the rate of protein synthesis of Thiobacillus can be calculated from its cell density and growth rate: Embedded Image with μ=D in steady state. This value can be used to calculate the oxygen consumption rate of Thiobacillus: Embedded Image

The rate of oxygen consumption by Desulfovibrio then can be deduced from: Embedded Image

The corresponding rate of transfer of reducing equivalents [H] from lactate to oxygen by Desulfovibrio follows: Embedded Image

(b) Estimation of the rate of oxygen uptake of Desulfovibrio by quantifying the path of reducing equivalents ([H]) released during lactate oxidation:

The rate at which [H] are released during lactate oxidation by Desulfovibrio equals: Embedded Image

The number of [H] released per lactate depends on the amount of pyruvate used for biosynthetic purposes: Embedded Image

Since the medium supplied contained only sulfate as sulfur source, S0 must have been formed through sulfate reduction. The corresponding flow of [H] amounts to: Embedded Image

The rate of transfer of reducing equivalents to Thiobacillus is calculated from the oxygen consumption (Eqn. 2), adding [H] used for CO2 fixation Embedded Image

Both [H] used by Thiobacillus and [H] ending up in S0 have derived from the reduction of sulfate or other sulfur compounds. Thus the transfer of [H] by Desulfovibrio to sulfur compounds can be quantified: Embedded Image the rate of transfer of [H] by Desulfovibrio to oxygen then follows: Embedded Image

Taking into account that 4 reducing equivalents are needed to reduce molecular oxygen, the oxygen consumption by Desulfovibrio can be calculated.

2.5.2 Production of zerovalent sulfur and thiosulfate

Growth and conversions of sulfur in pure cultures of Thiobacillus subjected to oxygen limitation with sulfide as substrate are known from previous experiments [22]. Oxygen limitation occurred when the ratio of the rates at which oxygen and sulfide were supplied (O2/HS) was below ∼1.6. At lower ratios there was not enough oxygen to oxidize all sulfide completely to sulfate. Under these circumstances Thiobacillus, instead of leaving sulfide behind, oxidized part of the sulfide to S0 and thiosulfate. The more severe the oxygen limitation the more S0 and thiosulfate were formed until a maximum was reached at O2/HS∼0.4, when 82% of the supplied sulfide was oxidized to S0 and 18% to thiosulfate, whereas no sulfate was formed. When the O2/HS ratio was lower than ∼0.4 sulfide accumulated.

These values cannot be used directly for the mixed cultures because the relation between the O2/HS ratio and the oxidation products formed depends on the yield, which in turn is dependent on the growth rate among other things. A generally applicable description can be derived as follows. According to the stoichiometry, 2 O2/HS is needed for the complete oxidation of sulfide to sulfate. Oxidation of sulfide to sulfur requires 0.5 O2/HS and the oxidation of sulfide to thiosulfate 1 O2/HS. Oxidation of 82% of HS to S0 and 18% of HS to thiosulfate, as observed in Thiobacillus cultures, requires a ratio O2/HS=(0.82×0.5 O2/HS)+(0.18·1O2/HS)=0.59. In growing cultures the response to the O2/HS ratio depends on the amount of reducing equivalents used for biosynthesis. Full oxidation of sulfide to sulfate occurs at O2/HS=2·(1−bTh), and to S0 and thiosulfate at O2/HS=0.59·(1−bTh). The production rates of S0 by Thiobacillus can then be calculated: Embedded Image Embedded Image Embedded Image Embedded Image

Assuming bTh=0.15 these equations convert to: Embedded Image Embedded Image Embedded Image Embedded Image

A completely analogous set of equations can be used to calculate thiosulfate production rate by substituting 0.5·0.18·1/0.59=0.15 for 1.39 in Eqns. 11 and 12.

2.6 Conversion factors (see Table 1 for symbols)

[pThC]: A pThC value of 21 fg protein cell−1 was obtained in pure continuous cultures of Thiobacillus at a comparable growth rate [41].

[pDvC]: a value of 60 fg protein cell–1 was used. This was the average (range 51–70) of three steady states of pure cultures of Desulfovibrio with varying SR−lactate.

[YThO2]: When growing on sulfide YThO2 has been shown to be independent of the degree of oxygen limitation [22]. For the present organism YThO2 amounted 1.92 mg protein per mmol oxygen consumed when growing at D=0.1 h−1[22], whereas YThO2 was 1.62 at D=0.05 h−1[41]. The yield thus decreases with decreasing growth rate, which is in accordance with observations of Kuenen [42]. Since the growth rate in the current experiments was 0.038 h−1, YThO2 will have been lower than 1.62. Consequently a specific oxygen consumption (1/YThO2) of 0.62 mmol O2·mg−1 protein should thus be regarded a minimum estimate. Embedded Image Embedded Image

3 Results

3.1 Pure cultures of Desulfovibrio desulfuricans PA2805

3.1 Batch cultures

Growth was followed via optical density in two sets (five replicates each) of screw-capped tubes completely filled with a medium containing 13.8 mM lactate and excess sulfate (20.2 mM). The pH, which may have a profound effect on growth rate [26], was kept constant at 7.8 using a 20 mM carbonate buffer. Temperature was maintained at 30°C. After lactate depletion acetate amounted to 12.9 mM, indicating that 6.5% of lactate supplied was used for biosynthesis. The sulfide concentration was 6.6 mM. Only very low concentrations of zerovalent sulfur (0.040 mM S), tetrathionate (0.036 mM) and polysulfide (0.014 mM S) were found, however, their abiotic formation during sample handling can not be excluded. Growth was exponential (μmax=0.072 h−1) and did not slow down until the stationary phase was reached. There was no significant difference between tubes. Apparently growth was not inhibited by sulfide at concentrations <6.6 mM. The protein yield was 3.68 mg·mmol−1 lactate. During the exponential growth on lactate, reserve sugar concentrations were extremely low (41 μg sugar·mg−1 protein) and PHB was absent.

3.1.2 Continuous cultures

In lactate-limited continuous cultures in steady state operated at D=0.039 h−1, the lactate concentration was below the limit of detection (<0.05 mM). With SR−lactate being 15.6 mM, the concentration of acetate amounted to 13.0 mM. This discrepancy can be explained by biosynthesis, but removal of some acetate due to gassing cannot be excluded. For this reason acetate data have not been used for calculations. The steady state protein concentration was 42.7 mg·l−1 was formed, which corresponds to a yield of 2.73 g·mol−1 lactate. The sulfate concentration in the in-flowing medium was 13.4 mM, whereas the steady state concentration was 7.8 mM, indicating the formation of 6.5 mM sulfide. The actual concentration of sulfide was lower (4.23 mM), because part of the sulfide (35%) was removed due to continuous gassing of the culture. These data were obtained using nitrogen gas completely devoid of oxygen. Addition of oxygen at rates >0.15 mmol l−1 h−1 (equivalent to 0.29 mmol O2/mmol lactate) resulted in detectable oxygen concentrations in the culture fluid, the consequence being cessation of growth, followed by wash-out of the culture. Lower oxygen supply rates were not tested. When prior to complete wash-out the gassing mixture was switched back to oxygen-free nitrogen, growth and sulfide production resumed.

3.2 Mixed cultures of Desulfovibrio and Thiobacillus

Co-cultures (D=0.039 h−1, SR−lactate 13.5 mM, see Table 2) were obtained by mixing steady state chemostat cultures of both organisms. Addition of oxygen at rates up to 0.36 mmol·l−1·h−1 (i.e., a molar oxygen/lactate ratio of 0.71) resulted in stable co-cultures in which the oxygen concentration was below the detection limit (<2 μM). These observations indicate that oxygen was effectively removed by Thiobacillus, thus allowing Desulfovibrio to grow and produce sulfide. When the oxygen supply rate was increased to 0.41 mmol·l−1·h−1 (0.81 O2/lactate) oxygen became detectable, resulting in complete wash-out of both organisms.

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2

Data obtained from steady states of mixed cultures of Desulfovibrio and Thiobacillus at various oxygen supply rates

A. Culture conditions, oxygen uptake rates, acetate and protein concentrations, and cell densities
O2 in gas(%)O2 uptake(mmol·l−1·h−1)D(h−1)[SR-lactate](mM)[Acetate](mM)[Protein](mg·l−1)Desulfovibrio cell density(N·l−1)Thiobacillus cell density(N·l−1)
000.03915.613.042.76.1·10110
3.10.1510.03813.613.344.26.9·10111.7·1011
4.20.2590.03713.512.655.38.2·10113.7·1011
5.00.2960.03713.511.681.212.4·10115.0·1011
5.60.3640.03813.511.074.5a 8.6·1011a 5.2·1011
a‘near’ steady state.
B. Sulfur balance
O2 in gas(%)[SR-sulfate](mM)[SO42−](mM)[S4O62−](mM)[S2O32−](mM)[S0](mmol S·l−1)[Sx2−](mmol S·l−1)[S2−](mM)S-recovery(% of [SR])
019.913.40.070.040.050.014.2390
3.119.614.70.040.080.130.041.8386
4.219.715.20.010.050.790.010.6986
5.019.716.2n.d.n.d.3.30n.d.n.d.99
5.619.716.7n.d.n.d.3.05n.d.n.d.100
n.d., not detectable.
C. Redox balance
O2 in gas(%)a [H] from lactate oxidationa [H] in oxygen reductiona [H] in sulfur compoundsRedox balance(%)
S4O62−S2O32−S0Sx2−b S2−
063010005285
3.15416111039103
4.25428005029118
5.0543200200096
5.65438001800105
  • a [H], reducing equivalents (mmol·l−1).

  • b Sum of sulfide in culture-fluid and sulfide escaping with gas (estimated from sulfur balance).

With oxygen supply rates increasing from 0 to 0.3 mmol·l−1·h−1 (0.6 O2/lactate) the concentration of sulfide in the culture gradually decreased to zero, indicating that either the rate of sulfide oxidation by Thiobacillus increased or the rate of sulfide production by Desulfovibrio decreased. In the presence of sulfide, trace amounts of tetrathionate, thiosulfate and polysulfides were detected. At oxygen supply rates between 0.3 and 0.4 mmol·l−1·h−1 (0.6–0.8 O2/lactate) sulfide or other reduced forms of sulfur, with the exception of S0 which accumulated to approximately 3 mM S, could not be detected. In the absence of sulfide, the recovery of sulfur was close to 100% (Table 2).

Protein concentrations increased with increasing oxygen supply rates. The protein assay does not allow discrimination between Thiobacillus and Desulfovibrio, however, cell numbers of both Thiobacillus and Desulfovibrio increased (Table 1). Calculated protein values of Thiobacillus and Desulfovibrio are shown in Fig. 1, the sum of these agrees well with the measured total protein content (also included in Fig. 1). Reserve sugar concentrations were negligible (maximum 11 μg·mg−1 protein), while PHB was not formed at all.

1

Steady-state concentrations of sulfide, oxygen and total protein in mixed cultures of Desulfovibrio and Thiobacillus supplied with lactate and excess sulfate as a function of oxygen supply rate. Protein of individual species as calculated from counted cell numbers and a conversion factor for cellular protein content are shown as well.

NeitherThiobacillus nor Desulfovibrio can grow in pure culture under the conditions applied. Thiobacillus cannot grow on lactate as electron donor but needs reduced sulfur compounds to this end, which are formed by Desulfovibrio. Desulfovibrio, in turn, cannot grow with detectable oxygen concentrations in the culture, and depends on oxygen consumption by Thiobacillus. The mutual dependence of Desulfovibrio and Thiobacillus in these cultures is thus evident. Possible interactions are shown in Fig. 2.

2

Interactions of Desulfovibrio and Thiobacillus in oxygen limited mixed cultures. Only lactate, oxygen and sulfate are supplied externally. Width of arrows indicates relative importance of path. Dashed arrows indicate insignificant processes.

Desulfovibrio can grow by oxidizing lactate to acetate coupled to the reduction of sulfate to sulfide, as it does in the pure culture. Incomplete sulfate reduction resulting in thiosulfate formation, which has been reported to occur in certain sulfate reducers under electron donor limitation [43, 44], can be ruled out in the present experiments. Firstly, in pure cultures of Desulfovibrio thiosulfate concentrations measured were marginal and possibly originated from abiotic reactions during sample handling. Secondly, Thiobacillus T5 prefers sulfide over thiosulfate as electron donor when both are present (M.T.J. van der Meer, unpublished results). Sulfide is thus the sole electron donor used by Thiobacillus.

Thiobacillus can oxidize sulfide to sulfate. However, when oxygen is limiting Thiobacillus does not oxidize sulfide completely to sulfate but forms less oxidized sulfur compounds like thiosulfate and zero-valent sulfur [22]]. This zerovalent sulfur is probably excreted as soluble long chain polythionates, polysulfides or polysulfane thionic acids, which are in chemical equilibrium with zerovalent sulfur globules [45, 46, 41]. With zerovalent sulfur and thiosulfate produced by Thiobacillus, and sulfate and oxygen supplied in the feed there are four potential electron acceptors available to Desulfovibrio. To determine which of these is/are actually used by Desulfovibrio is crucial in understanding the processes in mixed cultures. This cannot be inferred directly from the measurements of the different compounds. In the case of oxygen the consumption observed may be attributed to both Thiobacillus and Desulfovibrio, whereas the use of sulfur compounds as electron acceptor is obscured because production and consumption of the various sulfur compounds by Thiobacillus and Desulfovibrio, respectively, may occur simultaneously.

3.3 Oxygen consumption

Circumstantial evidence based on data collected in a co-culture supplied with 0.3 mmol O2·l−1·h−1 indicates that oxygen is not a quantitatively important electron acceptor for Desulfovibrio. This can de deduced in two different ways: (a) by determining the fate of oxygen, and (b) by determining the fate of reducing equivalents released during lactate oxidation.

(a) The amount of oxygen available for Desulfovibrio (vDvO2) can be determined by subtracting the rate of oxygen consumption by Thiobacillus (vThO2) from the measured total oxygen consumption rate (vtotalO2). Since the concentrations of both sulfide and oxygen remained below the detection limit (<1 μM) oxygen consumption by abiotic sulfide oxidation was considered negligible. The total flux of oxygen was 0.30 mmol O2·l−1·h−1. The oxygen requirement of Thiobacillus (vThO2) can be estimated from its biomass. Substituting the steady-state values of NTh, μ, and vtotalO2 in Eqns. 1, 2 and 3 it follows that vThP=0.39 mg protein·l−1·h−1 and vThO2≥0.24 mmol O2·l−1·h−1. Consequently the rate of aerobic respiration by Desulfovibrio (vDvO2) is estimated to amount maximally 0.06 mmol O2·l−1·h−1. Because four reducing equivalents are needed to reduce one O2 the flow of reducing equivalents (vDv[H]→O2) involved amounts at most to 0.24 mmol [H]·l−1·h−1.

(b). Data from the same steady state can also be used to calculate the rate at which reducing equivalents, derived from lactate oxidation, must have been transferred to a sulfur compound as electron acceptor in order to be able to explain both the observed Thiobacillus biomass and the amount of zerovalent sulfur. This can be stepwise illustrated as follows:

(1) The steady state S0 concentration was 3.30 mmol·l−1 (Table 2). Since the medium supplied contained only sulfate as sulfur source, the zerovalent sulfur must have originated through sulfate reduction. S0 is not an intermediate in the process of dissimilatory sulfate reduction and thus must have been formed from the oxidation of sulfide. Each S0 therefore represents a net flow of 6 [H] from lactate to sulfate. To obtain a S0 formation rate of 0.12 mmol·l−1·h−1 (3.30 mmol·l−1·0.037 h−1) the corresponding net flow of electrons from lactate must have been 0.72 mmol [H]·l−1·h−1 (6·0.12).

(2) As calculated above a consumption of ≥0.24 mmol O2·l−1·h−1 is needed to explain Thiobacillus biomass. For the chemical reduction of this amount of oxygen a flow of ≥0.96 mmol [H]·l−1·h−1 (4 [H] O2−1≥0.24 mmol O2·l−1·h−1) from a reduced sulfur compound is required. Taking into account that ∼10% of the reducing equivalents consumed by Thiobacillus is used for biosynthetic purposes, the total flow of electrons from lactate, via sulfide to Thiobacillus will have been ≥1.07 mmol [H]·l−1·h−1 (10/9·0.96).

(3) Adding this to the rate of 0.73 mmol electrons·l−1·h−1 required for S0 formation yields a flux of 1.79 mmol [H]·l−1·h−1 that is present in or has passed through reduced sulfur compounds, and thus cannot have been directly transferred to oxygen by Desulfovibrio.

(4) The total flow of electrons derived from the oxidation of lactate to acetate is estimated at 1.90 mmol l−1·h−1 (13.5 mmol lactate·l−1·0.037 h−1·4 electrons·lactate−1 minus 5% used by Desulfovibrio for biosynthetic purposes). Thus maximally 0.11 mmol electrons·l−1·h−1 (1.90–1.79) may have been used by Desulfovibrio using oxygen as acceptor.

There is good agreement between the two different ways of calculation. According to calculation (a) at most 0.24 mmol [H]·l−1·h−1 was carried over to oxygen by Desulfovibrio, which is ≤13% of the total flow of reducing equivalents from lactate oxidation. According to calculation (b) ≤0.11 mmol [H]·l−1·h−1 or ≤6% of the total flow of reducing equivalents was transferred to oxygen. Thus, with maximally about 10% of the lactate being aerobically respired by Desulfovibrio, oxygen appears to be at best a marginal electron acceptor for Desulfovibrio in the mixed cultures. This implies that either the affinity for oxygen of Desulfovibrio is lower than that of Thiobacillus or that Desulfovibrio prefers other electron acceptors present over oxygen.

3.4 Sulfur cycling

In previous experiments, pure cultures of Thiobacillus, grown in continuous culture on sulfide, have been subjected to oxygen limitation. The proportions in which S0, thiosulfate and sulfate are formed by Thiobacillus were shown to depend on the amount of oxygen available per sulfide supplied (i.e., the O2/HS ratio)[22]. The same response is expected in the mixed cultures since Thiobacillus was likewise subjected to oxygen limitation while growing on sulfide. In order to calculate the rates at which the various sulfur compounds are formed by Thiobacillus, the O2/HS ratio experienced by Thiobacillus needs to be known (see Section 2.5.2). The oxygen flux was measured directly (Table 2), but the sulfide flux has to be calculated. Taking into account that Desulfovibrio was lactate limited the rate of H2S production can be estimated from the lactate consumption rate, provided that the electron acceptor used is known.

In the mixed cultures virtually all oxygen was consumed by Thiobacillus. Assuming that Desulfovibrio in mixed culture performs as in pure culture without oxygen, i.e., using sulfate as electron acceptor, the sulfide production rate is expected to be constant and independent of the oxygen supply rate. Thiobacillus then experiences an increasing O2/HS ratio as the oxygen supply rate increases. The expected steady state concentrations of sulfur compounds have been calculated as described in the methods section (Fig. 3 top panel). At oxygen supplies lower than 0.23±0.02 O2/lactate not all sulfide formed by Desulfovibrio can be used by Thiobacillus. A maximum production of 0.38±0.01 S0/lactate and 0.05±0.01 thiosulfate/lactate is expected to occur when 0.23±0.02 O2 is supplied per lactate. These amounts gradually decrease with increasing oxygen supply until all sulfide is oxidized to sulfate at O2/lactate is 0.79±0.07.

3

Expected and observed amounts of sulfide, S0 and thiosulfate (mmol S/mmol lactate supplied) in mixed cultures of Desulfovibrio and Thiobacillus on lactate plus sulfate as a function of the oxygen supply. Lactate and oxygen are growth-limiting for Desulfovibrio and Thiobacillus, respectively; sulfate is supplied in excess. Band width of expected values represents possible variations in use of reducing equivalents for biosynthetic purposes. Upper panel: amounts expected when sulfate is the preferred electron acceptor for Desulfovibrio. Middle panel: observed concentrations. Lower panel: comparison of the sulfide concentration observed with the concentration expected when sulfate is the preferred electron acceptor and the concentration expected when S0 and thiosulfate formed by Thiobacillus are preferred by Desulfovibrio as electron acceptor over sulfate.

A comparison of the expected concentrations (Fig. 3 top panel) with measured values (Fig. 3 middle panel) shows large differences. In the first place, not all sulfide was consumed by Thiobacillus when O2/lactate >0.23±0.02. There even was sulfide present when the oxygen supply was more than twice as high. Secondly, at O2/lactate =0.3 the expected accumulation of S0 and thiosulfate was not observed. The possibility that these products were not formed appears unlikely on the basis of pure culture studies. Whenever measurable amounts of sulfide were present in oxygen limited steady states of Thiobacillus no sulfate was formed at all, and S0 and thiosulfate were the sole products of sulfide oxidation [22]. The second option is that S0 and thiosulfate were used by Desulfovibrio and for that reason did not accumulate.

The ability to use S0 and thiosulfate, either as acceptor or as substrate for disproportionation (Fig. 4), has not been studied for the Desulfovibrio strain used here but is widespread in sulfate-reducing bacteria [24, 47, 44]. Consumption of S0 and thiosulfate by Desulfovibrio not only explains the absence of accumulation of these compounds in the culture with an oxygen supply of 0.3 O2/lactate, but can also explain the presence of sulfide at relatively high oxygen supplies. This can be illustrated on the basis of data from a balanced mixed culture with an oxygen supply of 0.26 mmol·l−1·h−1 (=0.52 O2/lactate). Under these conditions a concentration of 0.69 mM HS (0.078 mmol/mmol lactate) was measured, which shows that Thiobacillus was unable to oxidize all sulfide. Since pure culture studies have demonstrated that Thiobacillus will oxidize all sulfide unless the O2/HS ratio is lower than 0.50 (see Section 2.5.2), it can be inferred that the sulfide production must have been higher than Embedded Image This is not feasible with sulfate as acceptor and acetate as end product. The maximum attainable sulfide production from the oxidation of lactate with sulfate is 0.5 HS−/lactate and, considering biosynthetic needs in the growing culture (Eqn. 6), rather 0.45–0.48 HS/lactate is expected. There must therefore have been alternative sources of sulfide. The partially oxidized sulfur compounds produced by Thiobacillus are the only alternative electron acceptor available.

4

Possible pathways of sulfur transformation in mixed cultures of Desulfovibrio and Thiobacillus supplied with lactate, sulfate and oxygen.

The possible ways of sulfide formation are shown in Fig. 4. These include disproportionation of S0 and thiosulfate, and the use of S0 and thiosulfate as electron acceptor. If S0 and thiosulfate are disproportionated these constitute an extra source of sulfide in addition to the reduction of sulfate. If S0 and thiosulfate replace sulfate as electron acceptor more sulfide is formed per lactate. Taking into account biosynthetic needs, 0.90–0.95 H2S/lactate can be formed with thiosulfate as acceptor, and 1.8–1.9 H2S/lactate with S0. With a mixture of S0 and thiosulfate as produced by Thiobacillus, 1.6–1.7 H2S can be formed per lactate.

Steady state sulfide concentrations in the cultures with oxygen supply rates of 0.29 and 0.52 O2/lactate were 1.83 and 0.69 mM respectively. Since disproportionation of S0 to sulfide and sulfate (option 3C in Fig. 4) is only then energetically favorable when the sulfide concentration is 10−5 M [28] this option can be excluded. Disproportionation of S0 to sulfide and sulfite is energetically even less favorable. However, subsequent reduction of the sulfite formed might pull the reaction (option 3A in Fig. 4). The net reaction in this case would be the same as in direct reduction of S0(option 3B in Fig. 4). It is not possible to distinguish between these two options in the present experiments. Disproportionation of thiosulfate yields less sulfide than the use as electron acceptor. However, since production of thiosulfate is quantitatively marginal compared to S0, it is not possible to distinguish whether it is used as acceptor or is disproportionated.

A calculation was made of the concentrations of sulfur compounds that are expected in the culture fluid when S0 and thiosulfate are used as electron acceptor by Desulfovibrio and are preferred over sulfate. Since S0 and thiosulfate are not supplied in the feed, the amounts of these compounds available as e-acceptor to Desulfovibrio are restricted by their formation by Thiobacillus. When S0 and thiosulfate are used by Desulfovibrio more H2S is formed per lactate than when only sulfate is used. As a consequence of the increased sulfide production the O2/HS ratio decreases, inciting Thiobacillus to make more S0 and thiosulfate and less sulfate, which will in turn lead to a higher sulfide production by Desulfovibrio. This positive feedback will go on until the O2/HS ratio decreases to a level where Thiobacillus cannot oxidize all sulfide because of shortage of oxygen. At that point a further increase of the sulfide production will not lead to a higher S0 and thiosulfate production rate. When supplies of lactate and oxygen are stoichiometrically balanced (O2/lactate=±0.8) a rapid cycling of sulfide, S0 and thiosulfate may take care of the transfer of reducing equivalents from Desulfovibrio to Thiobacillus, with neither of these compounds accumulating to measurable amounts. At lower oxygen supply rates the extent of this loop is limited by the oxygen supply rate. Only reducing equivalents that cannot be transferred to oxygen will be used in sulfate reduction. The resulting sulfide cannot be oxidized by Thiobacillus and remains in the culture. Fig. 3 (lower panel) shows the sulfide concentration expected in the culture fluid when all S0 and thiosulfate formed by Thiobacillus is used as electron acceptor by Desulfovibrio (sulfur and thiosulfate preferred over sulfate). The experimentally observed sulfide concentrations are not much lower than the calculated values. Whereas all S0 was consumed at low oxygen supplies an accumulation of S0was observed in the cultures with high oxygen supply rates. The S0 concentration was higher than expected based on the use of sulfate alone as electron acceptor for Desulfovibrio (compare Fig. 3 upper and middle panel). As reasoned above, this means that the O2/HS ratio was lower, and thus the sulfide production rate higher than attainable with sulfate as acceptor. This can only be achieved when S0 is used as electron acceptor. Apparently S0 is less efficiently used at high than at low O2 supplies. Possibly the presence of sulfide in the culture at low oxygen supplies facilitates the use of S0. S0 in the presence of sulfide is at equilibrium with polysulfides.

4 Discussion

4.1 Yield of Desulfovibrio

In the mixed cultures a two-fold increase of the cell yield of Desulfovibrio was observed with increasing oxygen supply. Major factors determining the yield of sulfate-reducing bacteria are: (1) the specific growth rate, (2) the toxicity of sulfide and (3) the nature of the electron acceptor [26, 9, 48]. The relevance of these factors for the present study is discussed below.

(1) In anoxic continuous culture (D=0.039 h−1, steady state) Desulfovibrio formed 2.73 mg protein per mmol lactate oxidized, whereas in batch culture (μ=0.072 h−1) a yield of 3.68 mg protein ·mmol−1 lactate was attained. A similar increase of the yield with increasing growth rate was observed in Desulfovibrio vulgaris Marburg and was attributed to high maintenance requirements [26]. From the present data a tentative Ymax of 6.7 g protein·mol−1 lactate and maintenance coefficient (m) of 8.4 mmol lactate·g protein−1·h−1 can be calculated for Desulfovibrio desulfuricans PA2805 growing on lactate plus sulfate. To avoid growth rate related effects due to high maintenance requirements care was taken that all mixed culture data were obtained from steady states at the same dilution rate. The observed yield increase of Desulfovibrio in mixed cultures with oxygen therefore cannot be attributed to growth rate differences.

(2) Toxicity of sulfide has been reported to be an important factor affecting cell yield in cultures of Desulfovibrio desulfuricans strain NCIB 8301. Upon partial removal of sulfide by gassing with oxygen-free nitrogen the yield of this strain more than doubled [48]. In the present mixed cultures the increase of cell yields of Desulfovibrio desulfuricans strain PA2805 with increasing oxygen supply coincided with decreasing sulfide concentrations (Fig. 1). However, batch cultures showed that there is no inhibitory effect of sulfide on growth with sulfide concentrations up to 6.6 mM. Since sulfide concentrations in the mixed cultures did not exceed this value (Table 2) an inhibitory effect of sulfide can be excluded as explanation for the observed yield differences.

(3) With respect to the utilization of different electron acceptors the following considerations are of relevance. Various sulfate-reducing bacteria, including the three strains of Desulfovibrio desulfuricans screened so far, are able to oxidize lactate with oxygen as e-acceptor [44]. The ΔG0 (pH 8) of the oxidation of lactate to acetate with O2 is −483 kJ·mol lactate−1. With sulfate the corresponding figure is −82 kJ·mol lactate−1 (calculated after [49]). In theory, the energy gain using oxygen thus is about six times higher. However, in none of the sulfate-reducing bacteria studied to date the use of oxygen instead of sulfate as electron acceptor resulted in a yield increase of the extent that would be expected based on free energy calculations [1719, 44, 50].

As shown in Section 3.3, above, in a steady state with an oxygen supply of 0.296 mmol O2·l−1·h−1 (0.59 O2/lactate) at least 90–95% of the reducing equivalents derived from lactate oxidation were transferred to oxidized sulfur compounds. The yield of Desulfovibrio in this steady state was calculated to be 5.52 mg protein·mmol−1 lactate. Assuming that the molar yield resulting from the transfer of electrons to oxidized sulfur compounds is equal to the molar yield obtained in the steady state without oxygen supply (2.73 mg protein mmol−1 lactate), only 2.5 to 2.6 mg·mmol−1 ((0.9–0.95)·2.73 mg·mmol−1) of the observed 5.52 mg protein mmol−1 lactate can be explained from the reduction of oxidized sulfur compounds. If the remaining 2.9–3.0 mg protein per mmol lactate were to be explained from the proportion of reducing equivalents transferred to oxygen (<5 to 10%) a yield of 30–58 mg protein per mmol lactate with oxygen as electron acceptor (3.0/0.1 to 2.9/0.05) is required. This means that an estimated 11–21-fold higher yield with oxygen compared to sulfate as electron acceptor is necessary to explain the observed yield. This is highly unlikely to be the case and it thus appears that the (limited) use of oxygen by Desulfovibrio cannot explain the yield doubling observed.

A more likely explanation for the observed yield increase with increasing oxygen supply is the shift from sulfate to S0 and thiosulfate as electron acceptor. Free-energy changes for lactate oxidation to acetate and carbondioxide under standard conditions (ΔG0) at pH 8 are not much different whether sulfate (ΔG0=−82 kJ·(mol lactate)−1), thiosulfate (ΔG0=−96 kJ·(mol lactate)−1) or zerovalent sulfur (ΔG0=−76 kJ·(mol lactate)−1) are used as e-acceptor. Considering these ΔG0 values it is anticipated that the ATP yield is equal as well. Hence free energy change alone cannot account for the observed yield increase upon change from sulfate to S0 and thiosulfate as electron acceptor. With sulfate concentrations generally several orders of magnitude higher than thiosulfate or S0 concentrations rather the opposite is expected. Data on the yield of sulfate-reducing bacteria have recently been summarized by Hansen [51]. For various Desulfovibrio species, growing on lactate plus sulfate, reported yields ranged from 3.0 to 7.8 g dry-weight (mol lactate)−1 [51, 52]. Applying a conversion factor of 7–8 g dry-weight·(mol ATP)−1[51] a gain of ∼0.7 (0.4–1.1) mol ATP·(mol lactate)−1 can be calculated. However, when sulfate is used as acceptor 2 ATP per sulfate have to be expended on sulfate activation, which comes down to 0.9–1 ATP per lactate. This expenditure is not included in the (net−) ATP yield figures based on dry-weight. The net ATP yield is thus at most half the gross ATP yield when sulfate is used as electron acceptor. An energy-expensive activation as in the case of sulfate is not necessary when thiosulfate or S0 are used, although reversed electron transport may be required [44]. Assuming an equal gross ATP yield for lactate oxidation with sulfate, zerovalent sulfur and thiosulfate, as suggested by the equal ΔG0, this would mean that the ATP spent in APS formation when sulfate is used as electron acceptor is available for other purposes when S0 or thiosulfate are used. As a consequence the net yield is expected to be much closer to the gross yield. In Desulfovibrio vulgaris strain Marburg, Badziong and Thauer [26] observed a tripling of Ymax upon change from hydrogen plus sulfate to hydrogen plus thiosulfate. Applying a protein to dry weight ratio of 0.5 and a dry weight to ATP conversion of 0.13 [51] a net ATP yield of ∼0.7 mol ATP·(mol lactate)−1 (2.73×2×0.13) can be estimated for growth with sulfate as e-acceptor. With an additional 0.9 mol ATP·(mol lactate)−1 available when S0 or thiosulfate are used as e-acceptor, a doubling of the yield is possible. This gain is large enough to explain the observed yield increment.

4.2 Preference of electron acceptor

Sulfate-reducing bacteria show a diversity with respect to the preference of electron acceptor. Pure cultures studied so far have revealed a preference of oxygen over sulfate [44, 20]. However, field measurements showing sulfate reduction in the presence of oxygen in microbial mats [15, 16, 2, 53] require the existence of sulfate-reducing bacteria that prefer sulfate over oxygen. Rapid reduction of thiosulfate in the sulfate-rich marine sediments [13, 2, 53], as well as inhibition of sulfate reduction after thiosulfate addition in freshwater sediment slurries [54] suggest the existence of sulfate-reducing bacteria that prefer thiosulfate over sulfate. This study shows that Desulfovibrio desulfuricans PA2805 prefers polysulfane-sulfur compounds, such as formed by thiobacilli, over sulfate. The existence of sulfate-reducing bacteria that prefer S0 over sulfate when both are present is of profound ecological importance. In anoxic marine sediments S0 is the main product of (abiotic) sulfide oxidation by iron and manganese oxides, and is an important sulfur sink. Sulfate-reducing bacteria that prefer S0 over sulfate will be able to exploit this source and thus influence net sulfur deposition rates. This activity is not detected with the standard methods used for sulfate reduction rate measurements.

As pointed out by Cypionka [44], the initial step in thiosulfate reduction, although energetically less expensive than in sulfate, still requires electrons of a quite low redox potential (E0[S2O32−/HS+HSO3]=−0.40 V) and thus reversed electron transport is required in the case of lactate as electron donor (E0[lactate/pyruvate]=−0.19 V). Zerovalent sulfur is an easier electron acceptor (E0[S0/HS]=−0.27 V) for that matter. Zerovalent sulfur is not part of the proposed pathways of sulfate reduction. Nevertheless growth of sulfate-reducing bacteria with S0 as acceptor has been widely observed. Recently Fuseler and Cypionka [55] showed that a disproportionation of S0 to sulfide and sulfite was part of the path of sulfide oxidation in Desulfobulbus propionicus. Such a step could be a connection between zerovalent sulfur and the sulfate reduction pathway (3A in Fig. 4). The type of zerovalent sulfur seems to be an important variable. Sulfur flower was, e.g., not used as e-acceptor by a D. desulfuricans studied by Postgate [24] whereas colloidal sulfur was used although at a low rate. The apparent difference in the affinity for S0 from Thiobacillus depending on the presence or absence of sulfide as observed in the present experiments also points at the importance of the nature of the zerovalent sulfur. Since the soluble sulfur precursors of sulfur globules formed by thiobacilli are metastable compounds their use will be difficult to establish experimentally in pure cultures. In Wollinella succinogenes the use of S0 and polythionates is thought to depend on the chemical reaction with sulfide to polysulfide, the latter being the actual substrate used [56]. Future experiments should reveal both the form and path of sulfur in Desulfovibrio.

The experimental conditions applied were designed to represent the situation at the oxygen sulfide interface in marine microbial mats. Both organisms were shown to benefit from a syntrophic interaction in which lactate was oxidized with oxygen, with a rapid cycling of sulfide and zerovalent sulfur serving the transfer of reducing equivalents between the species. These observations help explain the occurrence of colorless sulfur bacteria and sulfate-reducing bacteria at the same depth horizons in microbial mats.

Acknowledgements

The authors wish to thank M.T.J. van der Meer for providing unpublished data, Prof. Dr. H. Cypionka for critical comments on the manuscript, and Prof. Dr. P. Caumette for providing the sulfate-reducing bacterium.

Footnotes

  • 1 Department of Aquatic Ecotoxicology, University of Amsterdam, Kruislaan 320, 1098 SM Amsterdam, The Netherlands.

  • 2 Institut für Gewässerökologie, Neuglobsow, Germany

  • Dedicated to the memory of Prof. Dr. R.A. Prins.

References

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