Sulfur Isotope Enrichment during Maintenance Metabolism in the Thermophilic Sulfate-Reducing Bacterium Desulfotomaculum putei

Abstract

Values of Δ³⁴S (, where δ³⁴S_HS and indicate the differences in the isotopic compositions of the HS⁻ and SO₄²⁻ in the eluent, respectively) for many modern marine sediments are in the range of −55 to −75‰, much greater than the −2 to −46‰ ɛ³⁴S (kinetic isotope enrichment) values commonly observed for microbial sulfate reduction in laboratory batch culture and chemostat experiments. It has been proposed that at extremely low sulfate reduction rates under hypersulfidic conditions with a nonlimited supply of sulfate, isotopic enrichment in laboratory culture experiments should increase to the levels recorded in nature. We examined the effect of extremely low sulfate reduction rates and electron donor limitation on S isotope fractionation by culturing a thermophilic, sulfate-reducing bacterium, Desulfotomaculum putei, in a biomass-recycling culture vessel, or “retentostat.” The cell-specific rate of sulfate reduction and the specific growth rate decreased progressively from the exponential phase to the maintenance phase, yielding average maintenance coefficients of 10⁻¹⁶ to 10⁻¹⁸ mol of SO₄ cell⁻¹ h⁻¹ toward the end of the experiments. Overall S mass and isotopic balance were conserved during the experiment. The differences in the δ³⁴S values of the sulfate and sulfide eluting from the retentostat were significantly larger, attaining a maximum Δ³⁴S of −20.9‰, than the −9.7‰ observed during the batch culture experiment, but differences did not attain the values observed in marine sediments.

Dissimilatory SO₄²⁻ reduction is a geologically ancient, anaerobic, energy-yielding metabolic process during which SO₄²⁻-reducing bacteria (SRB) reduce SO₄²⁻ to H₂S while oxidizing organic molecules or H₂. SO₄²⁻ reduction is a dominant pathway for organic degradation in marine sediments (23) and in terrestrial subsurface settings where sulfur-bearing minerals dominate over Fe³⁺-bearing minerals. For example, at depths greater than 1.5 km below land surface in the fractured sedimentary and igneous rocks of the Witwatersrand Basin of South Africa, SO₄²⁻ reduction is the dominant electron-accepting process (3, 26, 46, 48, 61).

The enrichment of ³²S in biogenic sulfides, with respect to the parent SO₄²⁻, imparted by SRB, is traceable through the geologic record (10, 54). The magnitude of the Δ³⁴S (= δ³⁴S_pyrite − δ³⁴S_{barite/gypsum}, where δ³⁴S_pyrite and δ³⁴S_{barite/gypsum} are the isotopic compositions of pyrite and barite or gypsum) increases from −10‰ in the 3.47-billion-year-old North Pole deposits to −30‰ in late-Archaean deposits (55), to −75‰ in Neoproterozoic to modern sulfide-bearing marine sediments (13).

The kinetic isotopic enrichment, ɛ³⁴S, deduced from trends in the δ³⁴S values of SO₄²⁻ and HS⁻ in batch culture microbial SO₄²⁻ reduction experiments using the Rayleigh relationship, ranges from −2‰ to −46‰ (6, 7, 11, 17, 22, 27, 28, 30, 31, 38, 39). The variation in ɛ³⁴S values has been attributed to the SO₄²⁻ concentration, the type of electron donor and its concentration, the SO₄²⁻ reduction rate per cell (csSRR) (22), temperature, and species-specific isotope enrichment effects. In these laboratory experiments, doubling times are on the order of hours and csSRRs range from to 0.1 to 18 fmol cell⁻¹ h⁻¹ (7, 12, 17, 22, 30, 32, 39, 40).

Experiments performed during the 1960s found that the magnitude of ɛ³⁴S was inversely proportional to the csSRR for organic electron donors (16, 31, 38, 39) when SO₄²⁻ was not limiting. More-recent batch culture experiments on 3 psychrophilic (optimum growth temperature, <20°C) and mesophilic (optimum growth temperature, between 20°C and 45°C) SRB strains (7) and on 32 psychrophilic to thermophilic SRB strains (22), however, have failed to reproduce such a relationship. In 2001, Canfield (11) reported an inverse correlation between ɛ³⁴S and reduction rate using a flowthrough sediment column and demonstrated that ɛ³⁴S values of approximately −35 to −40‰ were produced when organic substrates added by way of amendment were limited with respect to SO₄²⁻. Because it was not possible to readily evaluate changes in biomass in the sediment column with changes in temperature or substrate provision rate, it was inferred that changes in ɛ³⁴S were related to changes in the csSRRs. More recently, Canfield et al. (12) observed a 6‰ variation in ɛ³⁴S values related to the temperature of the batch culture experiments relative to the optimum growth temperature. The few early experiments that were performed using H₂ as the electron donor yielded ɛ³⁴S values ranging from −3 to −19‰ (22, 38, 39), which appear to correlate with the csSRR (39). Hoek et al. (32) also found that the ɛ³⁴S values for the thermophilic SO₄²⁻ reducer Thermodesulfatator indicus increased from between −1.5‰ and −10‰ in batch cultures with high H₂ concentrations to between −24‰ and −37‰ in batch cultures grown under H₂ limitation with respect to SO₄²⁻. Detmers et al. (22) found that the average ɛ³⁴S of SRB that oxidize their organic carbon electron donor completely to CO₂ averaged −25‰, versus −9.5‰ for SRB that release acetate during their oxidation of their organic carbon electron donor. Detmers et al. (22) speculated that the greater free energy yield per mole of SO₄²⁻ from incomplete carbon oxidation relative to that for complete carbon oxidation promotes complete SO₄²⁻ reduction and hinders isotopic enrichment due to isotopic exchange of the intracellular sulfur species pools.

None of these experiments, however, have yielded ɛ³⁴S factors capable of producing the Δ³⁴S values of −55 to −75‰ observed in the geological record from ∼1.0 billion years ago to today. Various schemes have been hypothesized, and observations that involve either the disproportionation of S₂O₃²⁻ (36), the disproportionation of S⁰ produced by oxidation of either H₂S or S₂O₃²⁻ (15), or the disproportionation of SO₃²⁻ (29) have been made. Attribution of the increasing Δ³⁴S values recorded for Achaean to Neoproterozoic sediments to the increasing role of H₂S oxidative pathways makes sense in the context of increasing O₂ concentrations in the atmosphere (14) but is not consistent with the lack of significant fractionation observed during oxidative reactions (29). To explain the Δ³⁴S values of −55 to −77‰ reported to occur in interstitial pore waters from 100- to 300-m-deep, hypersulfidic ocean sediments (51, 64, 67), where the presence of a S-oxidative cycle is unlikely, an alternative, elaborate model of the SO₄²⁻-reducing pathway has been proposed by Brunner and Bernasconi (9). This model attributes the large Δ³⁴S values to a multistep, reversible reduction of SO₃²⁻ to HS⁻ involving S₃O₆²⁻ and S₂O₃²⁻ (20, 25, 41, 42, 52, 66). The conditions under which the maximum ɛ³⁴S values might be expressed are a combination of elevated HS⁻ concentrations, electron donor limitations, nonlimiting SO₄²⁻ concentrations, and a very low csSRR. The csSRR for subsurface environments has been estimated from biogeochemical-flux modeling to be 10⁻⁶ to 10⁻⁷ fmol cell⁻¹ h⁻¹ (23), with a corresponding cell turnover rate greater than 1,000 years (37).

Batch and chemostat culture systems, despite low growth rates, cannot completely attain a state of zero growth with constant substrate provision and therefore do not accurately reflect the in situ nutritional states of microbes in many natural settings. Retentostats, or recycling fermentor vessels, recycle 100% of biomass to the culturing vessel, allowing experimenters to culture microbial cells to a large biomass with a constant nutrient supply rate until the substrate supply rate itself becomes the growth-limiting factor and cells enter a resting state in which their specific growth rate approaches zero and they carry on maintenance metabolism (1, 47, 53, 58, 59, 62, 63). Utilizing this approach, Colwell et al. (18) were able to obtain a cell-specific respiration rate of 7 × 10⁻⁴ fmol of CH₄ cell⁻¹ h⁻¹ for a mesophilic marine methanogen, a rate that is comparable to that estimated for methanogenic communities in deep marine sediments off the coast of Peru (49).

In this study, the conditions that Brunner and Bernasconi (9) hypothesized would lead to the large Δ³⁴S values seen in nature were recreated in the laboratory by limiting the electron donor supply rate with respect to the SO₄²⁻ supply rate in a retentostat vessel. The S isotopic enrichment by a resting culture of Desulfotomaculum putei at an extremely low csSRR was compared to that of a batch culture experiment to determine whether the ɛ³⁴S values produced under the former conditions approach the Δ³⁴S seen in nature.

MATERIALS AND METHODS

Cultivation of D. putei.

D. putei strain TH15 represents an appropriate model subsurface SRB because it is the closest culturable phylogenetic relative to the 16S rRNA clone sequences belonging to the SRB that are prevalent in South African subsurface sediments (26, 44, 46), and it was isolated from an ∼2.7-km depth in the Taylorsville Triassic Basin, VA (45). D. putei is a strictly anaerobic, SO₄²⁻- and S₂O₃²⁻-reducing thermophile that grows on lactate and H₂ (45). D. putei was grown in a minimal salts medium of NH₄Cl (18.7 mM), MgCl₂·6H₂O (5 mM), CaCl₂·2H₂O (2.7 mM), and 0.1% Resazurin (2 ml liter⁻¹) with N₂-CO₂ (70:30) headspace gas. This medium had been passed through an O₂ scrubber; autoclaved; amended with sterile, anoxic solutions of NaHCO₃ (10 mM final concentration), K₂HPO₄·3H₂O (35 μM final concentration), peptones (0.2% final concentration), lactic acid (20 mM final concentration), Na₂SO₄ (20 mM final concentration), 10× ATCC vitamin solution (10 ml liter⁻¹), ATCC trace mineral solution (15 ml liter⁻¹), and Na₂S to remove trace O₂ (1 mM final concentration); and titrated with 3 M NaOH to a final pH of 7.8.

Batch culture cultivation.

D. putei was grown in batch culture for 72 h at 65°C and sampled for changes in biomass, aqueous geochemistry, and sulfur isotope enrichment. The lactate and SO₄²⁻ concentrations were 4 and 20 mM, respectively, consistent with that of the retentostat experiments. Changes in cell concentration and morphology were determined by staining 100-μl aliquots of cells with 5 nM Sytox-13 green fluorescent dye and analyzing them using flow cytometry (FACScan; Becton-Dickinson). The cell concentration and size were calibrated to 1-μm fluorescent microspheres at known concentrations (Molecular Probes, Eugene, OR). The uncertainty for enumeration was determined by triplicate analyses, and the 95% error of confidence was typically ±6%.

Aqueous geochemistry (SO₄²⁻, S₂O₃²⁻, acetate, and lactate) samples were collected in 1.5-ml microcentrifuge tubes and were analyzed by ion chromatography-mass spectrometry (Dionex 320; Dionex, Sunnyvale, CA; Finnigan MSQ; Thermo Scientific). The detection limits were 1 μM for SO₄²⁻, acetate, and lactate and 5 μM for S₂O₃²⁻. The 95% probability envelopes were ±5% for SO₄²⁻, acetate, and lactate and ±10% for S₂O₃²⁻. Sulfide samples were collected in 1.5-ml microcentrifuge tubes, and the sulfide was immediately quantified by UV spectroscopy by following the method of Cord-Ruwisch (19), which has a detection limit of 0.01 mM and a 95% probability envelope of ±5%.

Cell-specific rates of SO₄²⁻ reduction (csSRRs in mol of SO₄ cell⁻¹ h⁻¹) were calculated for the exponential phase using the changes in the SO₄²⁻ levels and cell concentrations between time points 1 and 2 (T₁ and T₂, respectively) [SO₄^{2−₍₁₎} and SO₄^{2−₍₂₎} and X₁ and X₂, respectively] according to the following equation (22):

(1)

The standard deviation of the csSRR was determined from duplicate measurements and was equal to ∼25%.

Retentostat cultivation.

All retentostat experiments were performed within an incubator that was housed in an anaerobic glove bag (Coy Laboratory Products, Inc., Grass Lake, MI) filled with N₂-CO₂ (70:30). The retentostat experiments were performed using a 1-liter culture vessel with a constant rate of fresh medium supplied at a rate of 22 ml h⁻¹ for runs 1 and 2 and of 10 ml h⁻¹ for run 3. The medium contained 4 mM lactate and 26 mM SO₄²⁻ for run 1 and the same lactate concentration and 20 mM SO₄²⁻ for runs 2 and 3. The other medium constituents were the same as described above. The vessel was inoculated with 10 ml of batch culture.

A Pellican XVG50 tangential-flow filter (Millipore Inc., CA) with a maximum pore size of 0.2 μm was used to recycle cells back into the culture vessel while allowing effluent medium to be removed at the same rate that fresh medium was being added (Fig. 1). The medium was constantly stirred with a magnetic stir bar. Samples were collected simultaneously with inoculation for time zero measurements, and the retentostat was then sampled three times every 24 h. The retentostat vessel fluid volume, which fluctuated due to slight variations in the pump rates (Fig. 1), was recorded to correct for the calculation of substrate supply rates. The maximum vessel volume fluctuation was never more than 5% of the total volume.

FIG. 1.

Retentostat experimental design. A, B, and C are peristaltic pumps where the fluid pump rate in ml h⁻¹ for A is equal to the fluid pump rate for C. The tangential flow filter (TFF) separates biomass from effluent and delivers cells back into the culture vessel. Black dots above the stir rod represent bacterial cells. δ³⁴S_CdS represents the isotopic composition of the cadmium sulfide precipitate, and δ³⁴S_SO4 represents the isotopic composition of the sulfate.

Biomass samples were collected in 1.5-ml microcentrifuge tubes from an in-line sampling port on the recycling biomass loop and were fixed immediately in a 2.2% glutaraldehyde solution. Cell concentration and morphology for each time point was determined by flow cytometry as described above.

Aqueous geochemistry (SO₄²⁻, S₂O₃²⁻, acetate, and lactate) samples were collected in 1.5-ml microcentrifuge tubes from the effluent port (Fig. 1) and immediately frozen at −80°C until analysis by ion chromatography-mass spectrometry, as described above. Sulfide samples were collected in 1.5-ml microcentrifuge tubes and were immediately quantified as described above. The pH of the effluent was measured at each time point using pH paper in two ranges, pHs 5 to 10 (Colorphast intermediate range; accuracy, 0.3 to 0.5; EMD Chemicals) and pHs 6.5 to 10 (Colorphast narrow range; accuracy, 0.2 to 0.3; EMD Chemicals). The dissolved inorganic carbon was measured using a LI6252 apparatus (LICOR Biosciences, Lincoln, NE). The H₂ concentration in the glove bag headspace was measured by gas chromatographic analyses (Kappa5; Trace Analytical, Menlo Park, CA) on gas samples collected in serum vials before the exponential growth phase and during the maintenance metabolism phase of run 3.

The csSRR for each time point (T_i) of the experiment (csSSR_i) was calculated using the following formula:

(2)

where VI is the volume flow rate for the incoming fresh medium (liters h⁻¹), VO_i is the volume flow rate of the outgoing medium for sample i (liters h⁻¹), [SO₄²⁻_i] is the SO₄²⁻ concentration (mol liter⁻¹) for sample i, [SO₄²⁻₍₀₎] is the SO₄²⁻ concentration at time zero, and X_i is the cell concentration (cells liter⁻¹) for sample i.

In order to test the effects of various csSRRs on the ɛ³⁴S, the initial cell concentration was increased from 4.4 × 10¹⁰ cells liter⁻¹ for run 1 to 1.5 × 10¹¹ cells liter⁻¹ for run 2 and to 4.7 × 10¹² cells liter⁻¹ for run 3. To determine if temperature affected the ɛ³⁴S, the temperature of run 3 was reduced from 65 to 60°C at 640 h and then from 60 to 55°C at 692 h. The temperature was reduced from 55 to 50°C at 840 h, but the fresh medium had become contaminated at this point and the experiment was terminated.

Sulfur isotope methods.

Sulfur isotope samples were collected by adding 1 ml of 2 M CdCl₂ solution to the 10 ml of medium in each tube during the course of the batch culture experiment. An uninoculated medium tube served as the negative control and was processed in the same manner to obtain the initial SO₄²⁻ isotopic composition. Sulfur isotope samples were collected for the retentostat experiments by collecting 45 ml of eluent in sterile 120-ml, O₂-free serum vials preloaded with 5 ml of a 2 M CdCl₂ solution, capped with butyl rubber stoppers, and frozen at −20°C until processing, as previously described (8, 22). In the presence of H₂S, CdS was precipitated and recovered on quartz filters, and the filters were dried in an oven overnight at 60°C. The filtrate solution was acidified to about pH 3 and heated to 80°C. Drops of a saturated BaCl₂ solution were added to the filtrate until there was no visible whitening due to suspended BaSO₄ crystals. The BaSO₄ precipitate was washed and centrifuged three times using distilled, deionized water from a Millipore gradient Mili-Q water filtration system with a QuantumEX and Q-Gard-2 dual-filtration system.

Sulfur isotope ratios for BaSO₄ and CdS were determined using an EA1110 elemental analyzer coupled to a Finnigan Mat 252 isotope ratio mass spectrometer via a ConFlo II split interface (57). Isotope data are reported as the δ³⁴S per mille deviation from the Vienna Cañon Diablo Troilite (VCDT) calculated by using equation 3:

(3)

where r_sample is equal to ³⁴S_sample/³²S_{sample i} and r_VCDT is equal to 0.0441638 (24).

Each set of 12 samples was bracketed by three working standards ranging in isotopic composition from −32.5‰ to +38.7‰ based on calibration to the International Atomic Energy Agency (IAEA) S-1 (−0.30 ‰), IAEA S-3 (−32.5‰), and NBS-127 (+20.3 ‰) external standards. Corrections were assumed to be linear over the entire range of isotopic compositions, and analytical reproducibility was better than ±0.2‰ for standards and analytical duplicates.

The ɛ³⁴S during exponential-phase growth in batch culture was calculated using the following standard Rayleigh model for closed systems:

(4)

where δ³⁴S_product is the isotopic composition in the integrated HS⁻ pool, δ³⁴S_init is the isotopic composition in the initial SO₄²⁻ pool, and f is the fraction of SO₄²⁻ remaining at a given time. The δ³⁴S_init was 9.97‰.

The ɛ³⁴S during retentostat operation can be modeled as a steady-state system (50) using the following equation:

(5)

where and δ³⁴S_HS are the isotopic compositions of the SO₄²⁻ and HS⁻ in the eluent (Fig. 1).

The overall S isotope mass balance was verified by comparing the total S isotopic composition of the eluent to that of the SO₄²⁻ in the fresh medium supply by using the following relationship:

(6)

The δ³⁴S value of the SO₄²⁻ in the fresh medium supply was determined at the initiation of each experiment and was −0.41‰ for run 1, 0.40‰ for run 2, and 5.44‰ for run 3. These calculations also take into account the HS⁻ present in the inoculum (∼10 mM in 10 ml) and in the 1-liter culture vessel at the beginning of each experiment (∼0.256 mM HS⁻) and in the 3 liters of fresh medium supply in the case of run 2. The δ³⁴S value of the HS⁻ in the medium supply was determined at the initiation of each experiment and was 8.74‰ for run 1, 4.94‰ for run 2, and 5.94‰ for run 3.

Geochemical modeling.

The retentostat experimental data were simulated using the React module of Geochemist's Workbench standard version 7.04 (5) by using the “flush” command and by following the dual-Monod reaction model of Jin and Bethke (34, 35):

(7)

where the reaction rate, k, is in mol kg of H₂O⁻¹ s⁻¹, V_max is the maximum cellular rate for SO₄²⁻ reduction in mol mg of biomass⁻¹ s⁻¹, and X is the cell concentration in mg of biomass kg of H₂O⁻¹. F_A is the factor controlled by the electron-accepting reaction, F_T is the thermodynamic-potential factor, and F_D is the kinetic parameter controlled by the electron-donating reaction and is defined by the equation

(8)

where m_D^PD and m_D+^PD+ are the concentrations of the electron donor reactant and product, respectively. Similarly, the electron-accepting reaction is controlled by the kinetic parameter F_A, defined by

(9)

where m_A^PA and m_A−^PA are the concentrations of the reactants of the electron-accepting reactant and product, respectively. PA and PD are assumed to be 1 for all reactions and PA− and PD+ are assumed to be 0 for all reactions. K_D and K_A are in molar units and are equivalent to the half-saturation constants K_m and K_s. The K_D value was constrained by matching the initial rate of lactate depletion. The K_A value of 10⁻⁵ M reported for SRB by Ingvorsen and Jorgensen (33) was assumed, but because of the high SO₄²⁻ concentrations used in these experiments, the K_A had no effect on the outcome of the simulations.

F_T is defined as

(10)

where R is the real gas constant 8.314472 J K⁻¹ mol⁻¹, T is the temperature in kelvins, x is the average stoichiometric number (34) or the ratio of the free-energy change of the overall reaction to the sum of the free energy changes for each elementary step, and Γ is the net thermodynamic driving force of the reaction defined by equation 11:

(11)

where ΔG is the free-energy change of the redox reaction and ΔG_p is the free energy for the phosphorylation reaction ADP + P → ATP (35). ΔG_p can vary from about −40 to −70 kJ mol⁻¹, depending upon the temperature, pH, and concentrations of ADP and ATP in the cell (43), and more likely ranges from −50 to −88 kJ mol⁻¹ if a thermodynamic efficiency of 80% is assumed. n is the number of moles of ATP generated per mole of reaction mixture. Values of four-thirds and 0.5 were utilized for n and x, respectively, according to the method of Jin and Bethke (35), but the ΔG_p was constrained by matching the lactate concentration in the maintenance phase of the retentostat experiments, i.e., the threshold concentration of the lactate.

The cell concentration is given by the following relationship:

(12)

where dX/dt is the change in cell concentration with time, i.e., the growth rate, Y is the yield in mg of biomass mol of reaction mixture⁻¹, k is the reaction rate in mol of reaction mixture kg of H₂O⁻¹ s⁻¹, and D is the death rate in s⁻¹. Cell counts were converted to mg of biomass by assuming a cellular mass of 1.56 × 10⁻¹³ g cell⁻¹ (30). Y and V_max were determined by simultaneously fitting the rate of SO₄⁻² reduction and the cell concentrations during the growth phase. D was determined by the trend in cell concentrations during the maintenance phase. Kinetic enrichment of S isotopes was simulated by introducing ³⁴S species into the thermodynamic database, defining a ³⁴S microbial reaction, and then adjusting the V_max, D, and K_A for the ³⁴S microbial reaction to fit the data.

TEM protocol.

Transmission electron microscope (TEM) observations of the samples were performed subsequent to pelleting of cell suspensions and fixation with 2.5% glutaraldehyde in 200 mM Na cacodylate (pH 7.2) for 2 h on ice. The pellets were then washed three times with 100 mM Na cacodylate buffer (pH 7.2) and left in the buffer overnight at 4°C. The pellets were then fixed with 2% Os tetroxide in Na Veronal buffer (pH 7.2) for 1 h on ice, followed by three rinses with the Na Veronal buffer and three rinses with 50 mM Na maleate buffer (pH 5.2). The pellets were stained with 0.5% uranyl acetate in the Na maleate buffer overnight at room temperature and then rinsed three times with 50 mM Na maleate buffer. The pellets were then sequentially dehydrated with 30%, 50%, 70%, and 95% ethanol and twice with 100% ethanol, followed by immersion in propylene oxide for 30 min, a propylene oxide-resin (1:1) mixture overnight, a propylene oxide-resin (1:2) mixture for 24 h, and straight resin overnight. The pellets were then placed in Beem capsules and polymerized at 60°C overnight. Unstained 70-nm sections were obtained using a diamond knife on a Leica UC6 ultramicrotome and observed at 80 kV on a Zeiss 912AB TEM equipped with an Omega energy filter (at Princeton University) and on a Philips 420 TEM operating at 80 kV (at the University of Western Ontario). Micrographs were captured using a digital camera from Advanced Microscopy Techniques and saved as TIFF files onto a Dell personal computer (at Princeton University) and using a Hamamatsu digital camera and HP computer (at the University of Western Ontario).

Lipid analyses.

Biomass samples for membrane lipid analyses were shipped to the University of Tennessee on dry ice and stored at −80°C until analyzed. The total lipids were extracted using methanol-chloroform-buffer (2:1:0.8 [vol/vol/vol]) and subsequently fractionated on a silicic acid column with only the polar lipids then transesterified into phospholipid fatty acid (PLFA) methyl esters (65). The PLFA methyl esters were separated, quantified, and identified by gas chromatography-mass spectrometry (65).

RESULTS

Batch culture experiment.

During the batch culture experiments, 6.6 mM SO₄²⁻ and 5.4 mM lactate were consumed over the 72-h period, with the production of 5.8 mM acetate. S₂O₃²⁻ was not detected. The csSRR for exponential-phase growth of D. putei was (0.9 ± 0.4) × 10⁻¹⁵ mol cell⁻¹ h⁻¹, with a Y of 5,180 mg mol of lactate⁻¹, and an ɛ³⁴S of −9.7‰ ± 0.4‰ was measured.

Retentostat experiment.

The pH and temperature remained at 7.8 ± 0.5 and 65°C, respectively, with no fluctuations throughout the duration of the experiments, except in run 3, where the temperature was purposely decreased in two steps to 55°C toward the end. During all three runs, the medium in the retentostat became progressively darker. During run 1, the SO₄²⁻ concentrations decreased from 25 mM to ∼19 mM and the lactate concentrations decreased from 4.0 mM to below the detection level (Fig. 2) by the end of exponential-phase growth, when an inflection occurs in the model growth curve (Fig. 3). SO₄²⁻ concentrations decreased from 20 mM to ∼14 mM and HS⁻ concentrations increased from 0.1 mM to a maximum of 6 mM in runs 2 and 3 (Fig. 2) by the end of exponential-phase growth (Fig. 3). S₂O₃²⁻ concentrations increased from <6 μM to 0.3 ± 0.1 mM in a manner synchronistic with HS⁻ production (Fig. 2). Lactate concentrations decreased from 4 mM to 4.8 ± 1.7 μM (Fig. 2) by the end of the exponential-phase growth (∼30 h in Fig. 3) of run 2 and from 4 mM to 77 ± 44 μM (Fig. 2) by the end of the exponential-phase growth (∼92 h in Fig. 3) of run 3 and remained at these low concentrations for the remainder of these experiments. Acetate increased from below 0.2 μM to 9 ± 2 mM by the end of the exponential-phase growth of run 2 and increased to 8 ± 2 mM by the end of the exponential-phase growth of run 3; it remained at these concentrations for the remainder of these experiments. No formate was detected. The HCO₃⁻ varied from 8 mM to 13 mM. The decrease in SO₄²⁻ and lactate concentrations and the increase in HS⁻ concentrations during the course of each run were consistent with the following reaction stoichiometry:

(13)

The declines in SO₄²⁻ concentrations for runs 1, 2, and 3 (Fig. 2) were best fitted with specific V_max reaction rates of 8.7 × 10⁻¹⁰, 1.7 × 10⁻¹⁰, and 5.3 × 10⁻¹² mol, respectively, of SO₄²⁻ mg of biomass⁻¹ s⁻¹. Geochemical modeling indicated that the pH should remain at 7.8, as it is buffered by the precipitation of dolomite and the 30% CO₂ in the headspace, which is consistent with the observed pH and explains the darkening of the medium. The average threshold lactate concentration was fit with a ΔG_p value of −63 ± 1 kJ (mol of SO₄²⁻)⁻¹ for run 2 and of −75 ± 1 kJ (mol of SO₄²⁻)⁻¹ for run 3.

FIG. 2.

Concentrations of SO₄²⁻, HS⁻, and S₂O₃²⁻ versus time in retentostat runs 1, 2, and 3, respectively. Sulfide data for run 1 are not shown. Modeled reaction rates for the equation lactate + 1.5 SO₄²⁻ → 1.5 HS⁻ + 3 HCO₃⁻ + 0.5 H⁺ are shown for runs 1 (solid line), 2 (solid line), and 3 (sulfate, dashed line; sulfide, dashed and dotted line). ±2 S.D., 95% error in confidence error bar for SO₄²⁻ (95% error in confidence error bars for HS⁻ and S₂O₃²⁻ are equal to or less than the size of their symbols). 60°C and 55°C, beginnings of temperature intervals for run 3.

FIG. 3.

Cellular concentration versus time during retentostat runs 1, 2, and 3. Modeled growth curves shown for runs 1 (solid line), 2 (dashed line), and 3 (solid line). The 95% error in confidence error bars are smaller than the symbols. 60°C and 55°C, beginnings of temperature intervals for run 3.

As has been observed in other retentostat experiments, cell concentrations increased from the inoculum concentration through an exponential phase of growth until the substrate feed became limiting and the growth rate declined and, in the case of run 1, approached zero (Fig. 3). The geochemically modeled curves simulate the overall trend in declining growth rates (Fig. 3). A maximum cell concentration of 2 × 10⁹ cells ml⁻¹ was observed for run 1 by 408 h, 8 × 10⁹ cells ml⁻¹ was observed for run 2 by 171 h, and 3 × 10¹⁰ cells ml⁻¹ was observed for run 3 by 450 h (Fig. 3). Run 2 may have been terminated prior to attaining maximum cell concentration and zero growth rate. Toward the end of run 3 when the temperature was reduced by a total of 10°C in two 5°C increments, the cell concentration attained 4 × 10¹⁰ cells ml⁻¹. The lowest csSRR values attained at the end of the runs were 0.10 ± 0.02, 0.016 ± 0.012, and 0.0017 ± 0.0003 fmol of SO₄²⁻ cell⁻¹ h⁻¹.

In runs 1 and 2, the cell concentrations declined briefly at the end of exponential growth (Fig. 3), indicating that some cell death process was occurring in these experiments, and this was seen by TEM. TEM observations also confirmed that dividing cells were more abundant in the samples collected prior to the onset of maintenance, were detected by the presence of lysed cells, indicated a reduction in cell volume from 4.9 to 0.5 μm³, and revealed the formation of endospores (Fig. 4). Spores comprised ∼10% of the total cell counts, and the csSRR values were corrected for this nonactive portion of the population.

FIG. 4.

(A) Transmission electron micrograph of D. putei cells at the end of exponential-phase growth (at 192 h) showing large healthy cells, one dividing cell, and a few smaller-diameter and denser cells. (B) Transmission electron micrograph of D. putei cells during maintenance metabolism (at 640 h) showing more smaller-diameter and denser cells, one larger cell, a couple of spores with spore coats, and remnants of cell walls and membranes.

Lipid analyses.

PLFA results were obtained for samples from the end of the exponential phase through the maintenance phase of runs 2 and 3 (Fig. 5). The total PLFA ranged from 286 to 2,470 pmol ml⁻¹. These concentrations compared to the cell counts correspond to a cell number (pmol of PLFA⁻¹) ratio of 2.7 × 10⁷ with a ±1 S.D. of +3.3 × 10⁷ and −1.6 × 10⁷. This value is ∼1,000 times the 2.5 × 10⁴ normally used for conversion of PLFA concentrations into cell concentrations (4). The PLFA composition was dominated by normal saturates (33 to 57%) and terminally branched saturates (29 to 52%), with the monoenoics comprising 3 to 11%, the branched monoenoics comprising 4 to 17%, and the one cyclic fatty acid present, 17:0 cyc, comprising only 0 to 3%. The relative proportions of the major fatty acid groups present in the retentostat experiment match those reported for D. putei (45), but the normal saturate abundance was somewhat higher and the monoenoics lower than those reported by Liu et al. (45). At the beginnings of runs 2 and 3, the proportions of normal saturates were greater than those of the terminally branched saturates, but further into the maintenance phase the terminally branch saturates became proportionally greater than the normal saturates, with the crossover occurring at ∼150 h for run 2 and ∼400 h for run 3 (Fig. 5). Toward the end of run 3, the proportions of terminally branched saturates decreased and of normal saturates increased as the temperature decreased from 65 to 55°C.

FIG. 5.

PLFA classes for runs 2 and 3 compared to the PLFA composition of D. putei (TH15) grown in batch culture (45) (solid symbols). NSATS, normal saturates; TBSATS, terminally branched saturates; BRMONO, branched monoenoics; MONO, monoenoics; CYCLO, cyclic fatty acid.

S isotope analyses.

The S mass balances for all aqueous sulfur species (SO₄²⁻, HS⁻, and S₂O₃²⁻) in runs 2 and 3 remained within 7% and 10%, respectively, of the initial 20 mM SO₄²⁻ pool. The S mass balance for run 1 could not be confirmed due to inaccurate HS⁻ measurements. The S isotope mass balance for runs 2 and 3 as determined using equation 6 were ∼1‰ ± 1‰ greater than the S isotopic composition of the supply medium. Assuming that the isotopic composition of S₂O₃²⁻, which was not determined, accounts for this small isotope imbalance, its δ³⁴S values ranged from −55 to −100‰ and from −20 and −60‰ for runs 2 and 3, respectively.

The ɛ³⁴S values ranged from 12 to −15‰ during the course of run 1, with the most negative ɛ³⁴S recorded at 408 h (Fig. 6). The ɛ³⁴S values ranged from 5 to −21‰ during the course of run 2, with the most negative ɛ³⁴S recorded at 95 h (Fig. 6). The ɛ³⁴S values ranged from 1 to −18‰ during the course of run 3, with the most negative ɛ³⁴S recorded at 192 h (Fig. 6). In the cases of runs 2 and 3, the most negative ɛ³⁴S occurred just after the exponential phase and before the maintenance phase, and as the cells entered the state of maintenance metabolism, the ɛ³⁴S steadily declined. This was most readily apparent in run 2, but run 3 exhibited an extension of the same trend. As the temperature dropped from 65 to 60°C, the ɛ³⁴S changed from −15 to −5‰. As the temperature dropped from 60 to 55°C, the ɛ³⁴S changed from −5 to −10‰.

FIG. 6.

ɛ³⁴S was equal to δ³⁴S_HS minus versus time during retentostat runs 1, 2, and 3. One standard deviation is 0.4‰, which is smaller than the symbol. Modeled enrichment curves are shown for runs 1 (small solid squares), 2 (small gray squares), and 3 (small open squares). The csSRR values are in fmol of SO₄²⁻ cell⁻¹ h⁻¹. 60°C and 55°C, beginnings of temperature intervals for run 3.

DISCUSSION

The retentostat cellular concentration profiles and substrate consumption curves correspond well with those found in previously published retentostat studies (47, 53, 58, 59, 62, 63), with the exception that distinct linear regions in the cellular concentration profiles were not readily discernible (Fig. 3). The average calculated maintenance coefficients during the final stages of runs 1, 2, and 3 were 4.7 × 10⁻¹⁷, 1.2 × 10⁻¹⁷, and 1.2 × 10⁻¹⁸ mol of lactate cell⁻¹ h⁻¹, respectively. These values are comparable to values reported for other non-SRB retentostat experiments run under conditions of electron donor limitation (18, 47, 62, 63). The 75-kJ (mol of SO₄²⁻)⁻¹ ΔG_p values are consistent with the net production of 1 mol of ATP per mol of SO₄²⁻ but are less than the 23- and 35-kJ (mol of SO₄²⁻)⁻¹ ΔG_p values reported by Sonne-Hansen et al. (56) for H₂-oxidizing, thermophilic SRB. The 75 kJ (mol of SO₄²⁻)⁻¹ translates into a maintenance energy dissipation rate of ∼0.1 kJ C mol of biomass⁻¹ h⁻¹, which is ∼1,000 times less than values reported by Tijhuis et al. (60), which were based upon chemostat experiments.

Lactate oxidation during maintenance metabolism.

The 8 to 9 mM acetate concentrations observed during the maintenance phases of each experiment indicate that 16 to 18 mmol of organic carbon was produced for every 12 mmol of carbon from the 4 mmol of lactate added to the retentostat vessel. Simultaneously, 48 mmol of electrons is required to explain the reduction of 6 mmol of SO₄²⁻ to HS⁻ for every 4 mmol of lactate added to the retenotstat vessel. This observation suggests that complete oxidation of lactate with concomitant SO₄²⁻ reduction occurred during the maintenance phase of D. putei. Batch culture experiments revealed the same reaction stoichiometry for SO₄²⁻ reduction and a much smaller, but still discernible, excess acetate production, 4.4 mM of acetate produced per 4 mM of lactate consumed. For their batch culture experiments, Liu et al. (45) reported the incomplete conversion of lactate to acetate, but because the amount of SO₄²⁻ reduced requires the complete oxidation of lactate, other potential origins for the acetate production were examined.

First, because the retentostat was being operated in an anaerobic glove bag, some residual H₂ from the normal 10% H₂-90% N₂ headspace may have survived multiple purges by the 30% CO₂-70% N₂ gas. This H₂ could then enter the retentostat through the supply medium and be utilized by D. putei for acetogenic pathways (4H₂ + 2HCO₃⁻ + H⁺ → CH₃COO⁻ + 4H₂O), thereby adding to the measured acetate pool and ultimately affecting the interpretation of the ɛ³⁴S data due to mixed electron donor availability. This hypothesis seems unlikely, however, because of the following: (i) analyses of the glove bag headspace before exponential phase and during maintenance metabolism phase yielded 20 parts per million by volume H₂ at both time points, indicating that H₂ existed in the glove bag but had remained unconsumed throughout the course of the experiment; (ii) even if all of this H₂ were consumed to produce acetate, given that the volume of the glove bag is ∼30 mol of gas, less than 1 mmol of acetate could be produced compared to the ∼52 mmol of acetate produced during run 3; (iii) at 65°C only submicromolar concentrations of the H₂ would be dissolved in the medium, and this would be insufficient to account for the millimolar concentrations of acetate recorded; and (iv) excess acetate was observed in the batch culture where no H₂ was added or detected.

Second, peptones in the fresh medium supply (see Materials and Methods) may have provided an alternate source of organic C to the system. Assuming that the 0.2% (by weight) peptones in the supply medium contained 40% organic C by weight (2) and that 100% of it was bioavailable and could subsequently contribute to acetate production via fermentation, as has been reported for other bacterial strains (50), then ∼33 mM could potentially result from this source compared to the 8 to 10 mM acetate concentrations observed. If true, this would mean that our calculated maintenance coefficients are minimum estimates.

Third, as cells are nutritionally deprived, they tend to shrink in order to maximize their surface area-to-volume ratio for increased transmembrane transport of nutrients, as well as for conservation of energy by limiting production of potentially superfluous macromolecules required for growth and cell division. Self-cannibalization could occur at this time, depleting intracellular carbon stores and once again producing acetate by direct substrate level phosphorylation. TEM observations revealed that cell size reduction did occur, with an increasing proportion of cells (Fig. 4B) being smaller than the cells of more normal size seen in the growth phase (Fig. 4A). Results of PLFA analyses are compatible with a change in membrane composition reflecting the change from vegetative cells (normal saturates dominate) to resting cells in maintenance metabolism (terminally branched saturates dominate) (Fig. 5).

Whether the last two processes have an effect on the ɛ³⁴S results is uncertain, as the electron balance indicated that SO₄²⁻ reduction occurred primarily by the complete oxidation of lactate.

S isotopic enrichment during maintenance metabolism.

The S mass balance results indicate that the S species were conserved for the duration of the retentostat run, and the ∼1‰ ± 1‰ S isotope mass imbalance that was based only on the δ³⁴S of SO₄²⁻ and HS⁻ suggests that the δ³⁴S of the S₂O₃²⁻ was more negative than that of the HS⁻. Theoretically, H₂S comprises only 4.5% of the total sulfide at pH 7.8 and 65°C, and the theoretical equilibrium is −1.1‰ and −1.2‰ for 65° and 55°C, respectively. The δ³⁴S of the total sulfide, HS⁻ + H₂S, would be only 0.05 to 0.06‰ more negative than the values measured for HS⁻, and this falls within the error of measurement.

The lowest csSRRs attained in our retentostat experiments are substantially lower than the lowest csSRRs reported in the literature for batch culture experiments. The lowest reported csSRR for a batch culture experiment was 2 × 10⁻¹⁷ mol cell⁻¹ h⁻¹ from a psychrophilic SRB strain incubated at −2°C (7), and this rate is 10 times higher than that of run 3. The lowest reported csSRR for a thermophilic batch culture experiment was 3 × 10⁻¹⁶ mol cell⁻¹ h⁻¹ for Desulfotomaculum geothermicum (22), which is 100 times higher than that of run 3. The lowest csSRR attained in our experiments, 1.7 × 10⁻⁸ mol of SO₄ cell⁻¹ h⁻¹, is still 3 orders of magnitude higher than the in situ planktonic csSRR inferred from geochemical estimation (∼4 × 10⁻²¹ mol of SO₄ cell⁻¹ h⁻¹) for the deep terrestrial subsurface of the Witwatersrand Basin (44) but is of the same magnitude as the csSRR measured by ³⁵S microautoradiography for core-bound SRB (≥8 × 10⁻¹⁷ mol of SO₄ cell⁻¹ h⁻¹) from the same environment (21).

The maximum ɛ³⁴S attained during retentostat cultivation, −20.9‰ in run 2, is much greater than that for the batch culture performed with the same temperature, pH, and lactate/SO₄²⁻ ratio, −9.7‰. The ɛ³⁴S trends for all three experiments are similar, becoming increasingly negative during the exponential phase, attaining a peak negative value, which is then followed by a slow retreat from this peak negative value during the maintenance stage (Fig. 6). The decrease in ɛ³⁴S during the exponential phase represents the combined effect of flushing and replacement of the initial HS⁻ by microbially respired HS⁻. The decline in ɛ³⁴S values during the maintenance stage (Fig. 6) cannot be attributed to changes in the membrane transport rates or HS⁻ production rates as a function of temperature as outlined in the Canfield et al. (12) model for SO₄²⁻ reduction, because the retentostat runs were isothermal in this region. The TEM data indicate that the cell surface-to-volume ratio increases from the end of the exponential stage to the maintenance stage. If vegetative cells express an inherently more negative ɛ³⁴S than their resting cell counterparts, then one possible explanation for the slow decline in the magnitude of ɛ³⁴S during the maintenance stage is the decrease in the number of vegetative cells and the increase in the number of resting cells. In order for the resting cells to yield a lesser ɛ³⁴S than their vegetative progenitors, they would have to be more efficient in the conversion of intracellular SO₄²⁻ to HS⁻ rather than less efficient. The observed decrease and subsequent increase in the ɛ³⁴S as the temperature was decreased at the end of run 3, on the other hand, could conceivably be related to temperature-dependent changes in the membrane transport and SO₄²⁻ reduction rates as espoused by Canfield et al. (12) for their model.

The ɛ³⁴S values during the maintenance stage did not exhibit any correlation the with S₂O₃²⁻ or HS⁻ concentrations. Comparing the results from all three experiments, the ɛ³⁴S values during the maintenance stage also did not exhibit any correlation with the csSRR across 3 orders of magnitude (Fig. 6). This was surprising given that the SO₄²⁻ reduction pathway is the cumulative expression of multiple reductive and potentially reversible steps, each with its own ɛ³⁴S (9, 12). By performing the retentostat experiments under electron donor-limited conditions and at a very low csSRR, we appear to have enhanced the reverse flow of S species in the cells as witnessed by the presence of S₂O₃²⁻ in the medium, and the inferred δ³⁴S of the S₂O₃²⁻ is enriched in ³²S by 20 to 100‰. These observations are compatible with the hypothesis of Brunner and Bernasconi (9) that the final reduction of SO₃²⁻ to HS⁻ is indeed a multistep process for D. putei. Although the retentostat experiments yielded greater ɛ³⁴S values than did the batch culture, the final HS⁻ product was still 40 to 50‰ less enriched in ³²S than the 70‰ enhancement predicted by Brunner and Bernasconi (9). Further retentostat studies of SRB should be performed at lower temperatures to attain rates comparable to that in nature to fully test this theory.