AEM
Home Help [Feedback] [For Subscribers] [Archive] [Search] [Contents]
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Supplemental material
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Habicht, K. S.
Right arrow Articles by Canfield, D. E.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Habicht, K. S.
Right arrow Articles by Canfield, D. E.
Agricola
Right arrow Articles by Habicht, K. S.
Right arrow Articles by Canfield, D. E.
Applied and Environmental Microbiology, July 2005, p. 3770-3777, Vol. 71, No. 7
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.7.3770-3777.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Effect of Low Sulfate Concentrations on Lactate Oxidation and Isotope Fractionation during Sulfate Reduction by Archaeoglobus fulgidus Strain Z{dagger}

Kirsten S. Habicht,* Lilian Salling, Bo Thamdrup, and Donald E. Canfield

Danish Center of Earth System Science, University of Southern Denmark, Institute of Biology, Campusvej 55, DK-5230 Odense M, Denmark

Received 27 August 2004/ Accepted 19 January 2005


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The effect of low substrate concentrations on the metabolic pathway and sulfur isotope fractionation during sulfate reduction was investigated for Archaeoglobus fulgidus strain Z. This archaeon was grown in a chemostat with sulfate concentrations between 0.3 mM and 14 mM at 80°C and with lactate as the limiting substrate. During sulfate reduction, lactate was oxidized to acetate, formate, and CO2. This is the first time that the production of formate has been reported for A. fulgidus. The stoichiometry of the catabolic reaction was strongly dependent on the sulfate concentration. At concentrations of more than 300 µM, 1 mol of sulfate was reduced during the consumption of 1 mol of lactate, whereas only 0.6 mol of sulfate was consumed per mol of lactate oxidized at a sulfate concentration of 300 µM. Furthermore, at low sulfate concentrations acetate was the main carbon product, in contrast to the CO2 produced at high concentrations. We suggest different pathways for lactate oxidation by A. fulgidus at high and low sulfate concentrations. At about 300 µM sulfate both the growth yield and the isotope fractionation were limited by sulfate, whereas the sulfate reduction rate was not limited by sulfate. We suggest that the cell channels more energy for sulfate uptake at sulfate concentrations below 300 to 400 µM than it does at higher concentrations. This could explain the shift in the metabolic pathway and the reduced growth yield and isotope fractionation at low sulfate levels.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The first life on Earth originated more than 3.8 billion years ago (24), possibly in high-temperature hydrothermal vent environments (31). Sulfate reduction had evolved by 3.5 billions years ago, as indicated by stable sulfur isotopes (26). The modern sulfate reducers that are most closely related to ancient sulfate-reducing hypothemophiles may be the species of Archaeoglobus, which is the only genus of sulfate reducers in the Archaea (28, 31). The stable sulfur isotope record is perhaps the best tool linking the present sulfur cycle with the past. Therefore, a study of the metabolic characteristics of Archaeoglobus spp., together with stable sulfur isotope fractionation studies, may shed light on early life.

Species of the hyperthermophilic genus Archaeoglobus have optimum growth temperatures of 83°C and today are found in various marine and terrestrial hydrothermal areas, including Vulcano, Italy; the Guayamas Basin, Mexico; the mid-Atlantic ridge; hot oil field water; and hot springs of Yellowstone National Park in the United States (23, 28, 32). Three sulfate-reducing Archaeoglobus species have been isolated, A. fulgidus, A. profundus, and A. lithotrophicus, whereas A. veneficus reduces only thiosulfate and sulfite (13). The best-studied sulfate-reducing Archaeoglobus is A. fulgidus strain VC-16, whose complete genome sequence is known (18). This strain oxidizes lactate completely to CO2 during sulfate reduction. It can also use pyruvate, formate, and oxaloacetate but not acetate as an electron donor. A. fulgidus grows chemoautotophically on H2 and CO2 with sulfate or thiosulfate as an electron acceptor (13, 28). In contrast to strain VC-16, lactate is oxidized incompletely to acetate by A. fulgidus strain Z (32). The complete oxidation of 1 mol lactate to 3 mol CO2 by A. fulgidus strain VC-16 goes through acetyl coenzyme A (acetyl-CoA) and the C1-CO dehydrogenase pathway, whereas the reaction pathway for incomplete oxidation by A. fulgidus strain Z is unknown (21, 32). In the complete absence of sulfate some sulfate reducers can ferment, but strains of Archaeglobus cannot. Instead, they can use thiosulfate and sulfite as electron acceptors (13).

Depending on the environment, natural populations of sulfate reducers may be limited in their energy metabolism by either electron donors or sulfate. In modern marine environments the sulfate concentration is high, around 28 mM, and the availability of organic substrates is generally the factor that limits sulfate reduction rates (17). In freshwater environments the sulfate concentrations are typically low, below about 500 µM, and sulfate might therefore limit sulfate reduction in these environments (14). Similar low sulfate concentrations have been inferred for the early archean ocean (11). The adaptation to low sulfate and electron donor concentrations by Archaeoblobus sp. was the focus of this study.

Microbial sulfate reduction can be identified in sedimentary sulfides deposited throughout Earth's history by measuring the isotope composition of sulfur species. Sulfate-reducing organisms, including Archaeoglobus spp., preferentially reduce 32S-sulfate compared to 34S-sulfate and produce sulfide enriched in 32S by 2 to 42{per thousand}, with fractionation of 17 to 26{per thousand} reported for A. fulgidus (4, 8). Sedimentary sulfides enriched in 32S are, therefore, considered to have microbial origins. Isotope fractionation is reduced as the sulfate concentration is reduced from about 2 mM, and below concentrations around 200 µM no fractionation has been observed during sulfate reduction (11, 12). Why low sulfate concentrations affect isotope fractionation so severely is not well understood.

In this study we grew A. fulgidus strain Z in a chemostat at 80°C under lactate-limited conditions and at sulfate concentrations between 0.3 mM and 14 mM. During our experiments we monitored the stoichiometry of lactate oxidation during sulfate reduction, and our data provide new insights into the incomplete lactate oxidation pathway. Furthermore, we compare the kinetics of sulfate uptake to the extent of isotope fractionation, the growth yield, and the sulfate reduction rate during sulfate reduction by A. fulgidus strain Z.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Bacterial strain.
We used a culture of A. fulgidus kindly donated by Karl O. Stetter, University of Regensburg, Germany, which is also available as strain Z, DSMZ 4139, from the Deutsche Sammlung von Mikroorganismen und Zellkulturen, Braunschweig, Germany (http://www.dsmz.de).

Culture medium.
The composition of the culture medium was as follows: KCl, 0.34 g liter–1; MgCl2 · 6H2O, 4.0 g liter–1; NH4Cl, 0.25 g liter–1; CaCl2 · 2H2O, 0.14 g liter–1; K2HPO4, 0.14 g liter–1; NaCl, 18.00 g liter–1; NaHCO3, 5.00 g liter–1; yeast extract, 0.25 g liter–1; lactate, 5 mM; and Fe(NH4)2(SO4)2 · 7H2O, 2 mg liter–1. The trace element solution used (10 ml liter–1) is described in detail in the Deutsche Sammlung von Mikroorganismen und Zellkulturen description of the medium for A. fulgidus (http://www.dsmz.de/media/med399.htm). Sulfate was added as MgSO4 to final concentrations between 3 mM and 19 mM (input concentrations). The medium was autoclaved and then cooled to room temperature while it was gassed with N2-CO2 (80:20). After cooling, sodium bicarbonate was added, which yielded a final pH of 7.0.

Chemostat setup.
The organism was grown in a chemostat (continuous culture; total volume, 1 liter) containing 0.7 liter of medium. The reactor of the chemostat was made of glass, and the lid was constructed of autoclavable and sulfide-resistant plastic (polyacetal [POM]), with connections consisting of Swagelok fittings (perfluoroalcoxy [PFA]). The medium was delivered with a peristaltic pump (Ole Dich, Denmark) from an N2-CO2 (80:20)-pressurized 5-liter reservoir bottle at dilution rates varying between 0.189 day–1 and 0.624 day–1. The chemostat was placed on a heating plate with magnetic stirring connected to a thermostat (IKA Labortechnik) that maintained the temperature of the culture at 80 ± 1°C. The pH was controlled with a pH-stat (JENCO) connected to a pH electrode (Metler-Toledo model 405-DPAS-SC-K8S/120) and was kept at 7.0 ± 0.1 by titration with 0.5 N HCl. The headspace of the chemostat was gassed with N2-CO2 (80:20) at a flow rate of 20 ml min–1 to keep the culture anoxic. Lactate was the only electron donor, and it was supplied in limiting amounts. Excess sulfate was present, and the concentrations in the reactor were between 0.3 mM and 15 mM. Each experiment was performed until a steady state was reached, which was generally after four total volume turnovers.

Samples were withdrawn from a sample port in the lid to measure the following: (i) concentrations and {delta}34S values of sulfate, (ii) concentrations of lactate, acetate, formate, and (iii) cell numbers. For the sulfate concentration measurements 1 ml of sample was fixed in 1 ml of 20% zinc acetate (ZnAc) to prevent further cell metabolism and to fix sulfide as zinc sulfide (ZnS). For isotope analysis of sulfate sulfur, between 20 ml and 100 ml of sample (depending on the sulfate concentration) was fixed in 10 ml of 20% ZnAc and stored frozen until further analysis. For fatty acid analyses, samples were filter sterilized through 0.22-µm filters and stored frozen. For cell counting, 0.9 ml of a sample was fixed in 0.1 ml of glutaraldehyde (20%).

The sulfide produced during sulfate reduction was carried from the chemostat with the N2-CO2 purge gas and trapped in 60 ml of 2% ZnAc. The gas traps were changed periodically (every 0.5 to 3 days) and used to measure the concentration and {delta}34S of sulfide.

Analytical procedure.
Cell numbers were determined using a phase-contrast microscope equipped with a UV lamp (Leica) and a Neubauer grid (0.0025 mm2 by 0.1 mm) after staining with 4',6'-diamidino-2-phenylindole (DAPI) (standard deviation [SD], ±5%).

The sulfate concentration was measured with a Dionex autosuppressed ion chromatograph equipped with a conductivity detector (Column Ionpac AS4S-SC 4 mm; SD, ±1%). The carrier was 1.5 mM HCO3–-CO32–, and the flow rate was 2 ml min–1. Sulfide was measured spectrophotometrically at 670 nm by the methylene blue method (SD, ±1%) (5). Fatty acids were analyzed with an autosuppressed ion chromatograph (SYKAM) by using an Aminex HPX-87H column (300 by 7.8 mm; Bio-Rad catalog no. 125-0140) and UV detection at 210 nm. The carrier was 5 mM sulfuric acid, and the flow rate was 0.6 ml min–1 (SD, ±1%). The detection limits for lactate, formate, and acetate were 10 µM, 20 µM, and 50 µM, respectively.

Sulfur isotope compositions were determined with BaSO4 and Ag2S. Barium sulfate was precipitated from the chemostat solution after addition of 1 M BaCl2 at about pH 4. Sulfide was converted to Ag2S after filtration of ZnS from the sulfide traps, distillation of the ZnS with 6 N HCl, and collection of the H2S gas produced as Ag2S in a 0.1 M AgNO3 trap. Dry BaSO4 (300 µg to 400 µg) or Ag2S (200 µg to 300 µg) was weighed in tin cups containing a 10-fold excess of V2O5. Sulfur isotope compositions were determined at the Center for Environmental Research Leipzig-Halle, Halle/Saale, Germany, by using SO2 gas produced from the combustion of either BaSO4 or Ag2S in a Carlo Erba elemental analyzer connected to a gas source mass spectrometer. The data are reported below as per mille differences relative to the Vienna Canyon Diablo troilite standard (SD, ±0.5{per thousand}).

Consumption rate.
As A. fulgidus was grown in a continues culture, the rates of consumption (q) (µmol time–1) of sulfate and lactate during sulfate reduction and the rates of production (p) of acetate and formate were calculated as follows:

(1)

(2)
where Sin is the concentration of the substrate (lactate or sulfate) entering the reactor, Sout is the concentration of the substrate leaving the reactor, Pin is the concentration of the product (acetate or formate) entering the reactor, Pout is the concentration of the product (acetate or formate) leaving the reactor, D is the dilution rate (time–1) of the medium in the reactor, and V is the volume (cm3) of the medium in the chemostat (700 cm3 in this study). The sulfate reduction rate (µmol time–1) was also calculated from the rate of sulfide flushed from the chemostat into the ZnAc traps, as follows (data not shown):

(3)
where [H2S]trap is the concentration of sulfide trapped as ZnS, v is the volume of the trap, and t is the time during which sulfide was flushed into the trap. The volume-based sulfate reduction rate (SRR) (µmol cm–3 time–1) was calculated from the rate of consumption of sulfate (qsulfate) calculated from equation 1 as follows:

(4)

The specific sulfate reduction rate (spSRR) (fmol cell–1 day–1) was calculated as follows:

(5)
where N is the cell number (cells ml–1).

We constrained the electron balance by testing if the electrons donated by lactate could account for the electrons accepted by sulfate (eight electrons per sulfate molecule). During lactate oxidation a maximum of 12 electrons per molecule can be donated from the organic matter to sulfate if all of the carbon is oxidized to CO2. The maximum was not observed in our experiment as both acetate and formate were formed during incomplete oxidation and 8 of the 12 electrons remained in acetate and 2 remained in formate. Assuming that all lactate carbon is converted to acetate, formate, or CO2, this gives the following electron balance:

(6)
where qsulfate and qlactate are the rates of consumption of sulfate and lactate, respectively, calculated from equation 1, and pacetate and pformate are the rates of production of acetate and formate calculated from equation 2.

Growth yield.
The growth yield (Y) expressed the amount of cell material produced during the utilization of 1 mol substrate (lactate or sulfate):

(7)
To calculate Y, we assumed that A. fulgidus has a spherical cell diameter of 1 µm, a cell density of 1 g cm–3, and a cell dry weight of 30% and that 40% of the dry weight is organic carbon. These values yield a cell dry weight fraction of 1.56 x 10–13 g cell–1 and a carbon content of 5.2 fmol C cell–1.

Sulfur isotope fractionation.
In a chemostat, which is a well-mixed open system, fractionation can be calculated by determining the isotope difference between sulfide [{delta}34H2S(out)] and sulfate [{delta}34SO42–(out)] (2), as follows:

(8)


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Lactate oxidation.
The lactate concentration in the culture solution was always below the detection limit for the lactate analysis (<10 µM), which implies that A. fulgidus grew under lactate-limited conditions. Up to 3.6 mM acetate and up to 6.1 mM formate were formed as products. To determine the stoichiometry of carbon metabolism during sulfate reduction with lactate, we calculated the rates of consumption of sulfate and lactate and the rates of production of sulfide, acetate, and formate (Table 1). The stoichiometries were calculated for five different sulfate concentrations (experiments I to V) ranging from 0.3 mM to 14 mM. At each sulfate concentration two to four measurements were taken over a 1- to 8-day period after steady state was established. The dilution rates were 0.288 day–1 in experiments I, II, and V, 0.624 day–1 in experiment III, and 0.189 day–1 in experiment IV. For the mass balance calculation we included only data with at least 80% electron balance (Table 1). The reason for electron imbalance in some cases is not known. It is known that methane can be produced by A. fulgidus during sulfate reduction, which could result in a deficit of electrons in the carbon budget (28). There was, however, no clear trend in the electron imbalance.


View this table:
[in this window]
[in a new window]
  		TABLE 1. Mass balance calculations for lactate oxidation during sulfate reduction by A. fulgidus growing in a chemostat

 
At sulfate concentrations between 0.6 mM and 14 mM (experiments I to IV) the stoichiometry of lactate oxidation during sulfate reduction by A. fulgidus was always the same within the experimental error (Table 2). For each 1 mol of lactate oxidized, 1 mol of sulfate was reduced and 0.25 mol of acetate, 1 mol of sulfide, and 1 mol of formate were produced. At a sulfate concentration of 0.3 mM (experiment V), the stoichiometry was much different from the stoichiometry for higher sulfate concentrations (Table 2). Thus, the cells reduced only 0.6 mol of sulfate per mol of lactate oxidized and produced 1.3 mol of acetate, 0.5 mol of formate, and 0.6 mol of sulfide. These results indicate that in A. fulgidus the metabolic pathway changed at low levels of sulfate.


View this table:
[in this window]
[in a new window]
  		TABLE 2. Stoichiometry of lactate and sulfate utilization by A. fulgidusa

 
Sulfate reduction rate and growth yield.
The volume-based sulfate reduction rates at steady state (last measurement) by A. fulgidus ranged from 0.78 µmol · cm–3 · day–1 to 2.82 µmol · cm–3 · day–1, and the cell-specific rates ranged from 18 fmol · cell–1 · day–1 to 36 fmol · cell–1 · day–1 (Fig. 1). The highest specific rates were measured at lower sulfate concentrations. However, lactate limited sulfate reduction at all sulfate concentrations.



View larger version (12K):
[in this window]
[in a new window]
  		FIG. 1. Effects of sulfate concentrations on the specific rate of sulfate reduction (spSRR) by A. fulgidus grown in a chemostat at 80°C with lactate as the limiting substrate. The dilution rates were 0.189 day–1 at a sulfate concentration of 0.6 mM, 0.288 day–1 at sulfate concentrations of 0.3 mM, 3.1 mM, and 14.5 mM, and 0.624 day–1 at a sulfate concentration of 1.5 mM. Data available as supplemental material.

 
The growth yield for A. fulgidus was between 0.8 and 4.0 g (dry weight) cells per mol sulfate (Fig. 2A). The growth yield depended on the sulfate concentration; low growth yields were obtained at low sulfate concentrations, and high growth yields were obtained at high sulfate concentrations. About 3 to 11% of the lactate consumed by A. fulgidus during sulfate reduction was assimilated as carbon into the cell material, and less carbon was incorporated at low sulfate concentrations (Fig. 2B).



View larger version (17K):
[in this window]
[in a new window]
  		FIG. 2. (A) Effects of sulfate concentrations on the growth yield during sulfate reduction by A. fulgidus grown under lactate-limiting conditions. The model line assumes that there is a Michaelis-Menten dependence between the sulfate concentration and the growth yield. The data in parentheses were not included when the model line was drawn. Km-Y, Km for the growth yield; Ymax, maximum growth yield. (B) Effects of sulfate on the carbon fraction assimilated into the cell. Data available as supplemental material.

 
Sulfur isotope fractionation at different sulfate concentrations.
The {delta}34S of sulfide was depleted by 14{per thousand} to 26{per thousand} compared to sulfate during sulfate reduction by A. fulgidus at 80°C (Fig. 3). The highest fractionations were measured at high sulfate concentrations (>2 mM), whereas generally low fractionations were associated with low sulfate concentrations (≤2 mM). At the lowest sulfate concentration, 0.3 mM, the {delta}34S of sulfide was depleted by 17{per thousand} compared to sulfate.



View larger version (20K):
[in this window]
[in a new window]
  		FIG. 3. Effects of sulfate concentrations on fractionation during sulfate reduction by A. fulgidus grown under lactate-limiting conditions. The model line assumes that there is a Michaelis-Menten correlation between the sulfate concentration and fractionation. The data in parentheses were not included when the model line was drawn. Data available as supplemental material.

 
There was no relationship between fractionation and the sulfate reduction rate (Fig. 4), whereas there was a clear correlation between fractionation and sulfate at concentrations below 2 mM (Fig. 3). This indicates that at concentrations below about 2 mM, sulfate controlled the extent of fractionation, whereas the sulfate reduction rate did not.



View larger version (13K):
[in this window]
[in a new window]
  		FIG. 4. Sulfur isotope fractionation during sulfate reduction by A. fulgidus grown in a chemostat with different sulfate reduction rates. (A) Volume-based sulfate reduction rates (SRR). (B) Sulfate reduction rates per cell (spSRR). Symbols: {blacktriangleup}, rates measured at sulfate concentrations less than 2 mM; {triangleup}, rates measured at concentrations greater than 2 mM. Data available as supplemental material.

 

   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Lactate oxidation.
A. fulgidus strain Z, which was used in this study, was originally isolated from coastal anoxic hydrothermal sediments close to mount Vulcanell on Vulcano Island, Italy (32). This strain was described as an organism that oxidizes lactate incompletely to acetate and CO2. This is in contrast to the first strain of A. fulgidus isolated, strain VC-16, which oxidized lactate completely to CO2 (28). Studies of the enzymes and coenzymes in A. fulgidus strain VC-16 suggested that the complete oxidation of lactate to 3CO2 goes through acetyl-CoA and the C1-CO dehydrogenase pathway (21).

Similar to Zellner et al. (32), we observed incomplete oxidation of lactate to acetate and CO2 by A. fulgidus strain Z during sulfate reduction, but we observed that formate is an important metabolic product, a new finding. Our stoichiometric analysis for ≥0.6 mM sulfate showed that for each 1 mol of lactate consumed, 1 mol of sulfate was reduced, producing approximately 0.25 mol of acetate, 1 mol of formate, and 1 mol of sulfide. Using batch cultures, Zellner and coworkers (32) observed production of 0.24 mol of acetate and consumption of 0.98 mol of sulfate per mol of lactate oxidized, similar to our observations. However, these workers did not analyze formate and assumed that the remaining carbon from lactate was converted to CO2. They therefore suggested that the oxidation of lactate involved two major steps. First, lactate was incompletely oxidized to acetate and CO2. Then more than 65% of the acetate was completely oxidized to CO2, as follows:

(9)

(10)

(11)
The stoichiometries of these reactions, however, do not correspond to our data, which showed that significant amounts of formate were formed (Table 2).

Instead, we suggest that formate is produced together with acetyl-CoA during pyruvate degradation catalyzed by the enzyme pyruvate formate-lyase (PFL) (Fig. 5). Pyruvate is a well-known product during lactate degradation, and enterobacteria, lactobacilli, and some clostridia can degrade pyruvate to formate by using PFL under strict anoxic conditions (25). Genome sequences revealed that genes for PFL enzymes are present in A. fulgidus strain VC-16, and it has therefore been suggested that PFL-like enzymes also are present in Archaeoglobus spp. (18, 25). The formation of formate during pyruvate degradation could explain the measured stoichiometries in our study.



View larger version (16K):
[in this window]
[in a new window]
  		FIG. 5. Proposed pathway for the oxidation of lactate during sulfate reduction to sulfide, acetate, formate, and CO2 in A. fulgidus strain Z at high sulfate concentrations (≥0.6 mM) and low sulfate concentrations (≤0.3 mM). The numbers indicate the moles that are produced or reduced during the oxidation of 1 mol of lactate.

 
Another potential source of formate during lactate oxidation is production in the C1-CO dehydrogenase pathway. Sulfate-reducing bacteria employing the C1-CO dehydrogenase pathway can form formate from formyltetrahydropterin during the oxidation of acetyl-CoA (23, 30). However, A. fulgidus strain VC-16 is known to use a modified C1-CO dehydrogenase pathway, which does not produce formate (23). Furthermore, a 1:1 stoichiometric relationship between lactate and formate, as measured in this study, does not result if formate is produced during the oxidation of acetyl-CoA. It is therefore less likely that formate is produced in the C1-CO dehydrogenase pathway by A. fulgidus strain Z.

Based on the stoichiometries measured at 0.6 to 14 mM sulfate, including the formation of formate and the genomic sequence observations, we suggest that the incomplete oxidation of lactate by A. fulgidus strain Z involves the following set of reactions (Fig. 5 and Table 3):

(12)

(13)

(14)

(15)
The {Delta}G0' is –69.3 kJ/mol lactate.


View this table:
[in this window]
[in a new window]
  		TABLE 3. Effect of sulfate on {Delta}G during sulfate reduction by A. fulgidus strain Z grown in a chemostat with lactate at limiting concentrations

 
The overall stoichiometry is explained by an initial oxidation of lactate to pyruvate, as previously suggested for A. fulgidus strain VC-16 by Möller-Zinkhan and Thauer (21). Pyruvate might then be dissimilated to formate and acetyl-CoA in a reaction catalyzed by PFL. This result would be the first indication that a PFL-like enzyme is active in A. fulgidus. In contrast, during the complete oxidation of lactate by A. fulgidus strain VC-16, pyruvate is oxidized to CO2 and acetyl-CoA by the enzyme pyruvate ferredoxin oxidoreductase (19, 29). The gene for pyruvate ferredoxin oxidoreductase has also been identified in A. fulgidus strain VC-16 (18). About 75% of the acetyl-CoA produced during pyruvate fermentation might then be completely oxidized to CO2 through the C1-CO dehydrogenase pathway (21), whereas the rest is converted to acetate, in a reaction probably catalyzed by the ADP-forming acetyl-CoA synthetase (19). Definitive proof that the suggested enzymes are indeed present in A. fulgidus strain Z requires their detection in crude extracts.

Lactate oxidation at low sulfate concentrations.
When A. fulgidus was grown at a sulfate concentration of 0.3 mM, the stoichiometry of the sulfate reduction reaction changed markedly (Table 2). At the low sulfate concentration, 0.6 mol of sulfate was reduced for each 1 mol of lactate oxidized, in contrast with the results obtained with high sulfate concentrations, where 1 mol of sulfate was reduced per mol of lactate. Also, three times more acetate was produced at low sulfate concentrations, whereas only one-half as much formate was formed. The reaction stoichiometry at a low sulfate concentration can be explained by following oxidation pathways (Fig. 5 and Table 3):

(16)

(17)

(18)

(19)
The {Delta}G0' is –53.8 kJ/mol lactate.

From the overall stoichiometry we inferred that only 1 mol HCO3– was produced per mol lactate, compared to 1.5 mol HCO3– at high sulfate concentrations (Table 2). This means that less carbon was completely oxidized at low sulfate concentrations and that a larger portion was converted to acetate. The only way that this stoichiometry could fit the reactions was by assuming that formate is oxidized by A. fulgidus strain Z. Both A. fulgidus strain Z and strain VC-16 can grow on formate in the presence of thiosulfate, but only A. fulgidus strain VC-16 has previously been shown to grow with formate and sulfate (32). It is possible that A. fulgidus strain Z oxidizes formate only at low levels of sulfate. When there is no sulfate present in the medium, A. fulgidus stops growing (32).

An alternative explanation for our observations is that at low sulfate concentrations A. fulgidus could express a pyruvate ferredoxin oxidoreductase-type enzyme, which oxidizes pyruvate to CO2 and acetyl-CoA. Thus, less formate would be produced compared to the level measured at high sulfate concentrations. The stoichiometry for this oxidation pathway, however, does not fit the measured data as too much acetate would be produced.

Free energy calculations showed that the cells did not gain energy by changing the metabolic pathway at low sulfate concentrations (Table 3). At a sulfate concentration of 300 µM the {Delta}G is –37.0 kJ/mol lactate for the reaction stoichiometry at high sulfate concentrations (equation 15) (Table 3) compared to the –30.1 kJ/mol lactate calculated from the reaction stoichiometry measured at low sulfate concentrations (equation 19) (Table 3). Instead, the shift in carbon metabolism at low levels of sulfate indicates that A. fulgidus is affected by the supply of sulfate to the cell at sulfate concentrations below 600 µM.

Growth yield.
The maximum growth yield during lactate oxidation by A. fulgidus strain Z was 2.8 g (dry weight) cells formed per mol sulfate (Fig. 2A). This value is lower than the values obtained for other sulfate reducers oxidizing various organic substrates or H2, where growth yields between 4 and 17 g are typically found (23). The relatively low growth yield found for A. fulgidus in this study might have resulted from the lactate-limiting growth conditions in the chemostat. At sulfate concentrations below 1.3 mM the growth yield decreased with decreasing sulfate concentration, and only 1.4 g (dry weight) cells per mol sulfate was formed with 300 µM sulfate. We used a Michaelis-Menten relationship to describe the correlation between growth yield and sulfate concentration, which yielded a Km for the growth yield of 0.40 mM sulfate and a maximum growth yield of 2.95 g (dry weight) cells per mol sulfate. This observation also demonstrates that the growth yield is limited by the sulfate supply at sulfate concentrations below 600 µM.

Similarly, the carbon fraction assimilated into the cell decreased with decreasing sulfate concentration. At sulfate concentrations of >0.6 mM up to 9% of the lactate consumed during sulfate reduction was assimilated as carbon into the cell material (Fig. 2B). This fraction is similar to the fraction found in other sulfate reducers, which assimilate about 9 to 11% carbon per mol substrate (23). However, at sulfate concentrations below 0.6 mM, only about 1% carbon was assimilated. In contrast, it seemed that the specific rate of sulfate reduction increased at low sulfate concentrations (Fig. 1). This indicates that at low sulfate concentrations the cells used less of the energy gained from the metabolism for growth and more of the energy for maintaining the cell activities.

Overall, the energy gained during sulfate reduction is used for four major activities in the cell, (i) maintenance of cellular components, (ii) carbon metabolism and growth, (iii) transport in and out of cell, and (iv) sulfate reduction, in which ATP activates sulfate to form adenosine-5'-phosphosulfate (APS), which is reduced to sulfide. In the discussion below we assume that the energy used to maintain the cell components is the same at both high and low sulfate concentrations. The growth yield data show that less energy was used for growth at low sulfate concentrations (Fig. 2). This can be explained in part by the reduced energy yield at low sulfate concentrations. However, an increased energy demand to transport sulfate into the cell (a shift of energy from growth to uptake of sulfate) can explain the reduced growth yields at low sulfate concentrations and probably also the higher specific rates of sulfate reduction.

For several sulfate-reducing organisms it has been shown that sulfate is transported into the cell together with two protons or sodium ions (6). However, at sulfate-limiting concentrations sulfate accumulates in the cell by an electrogenic mechanism using three protons or sodium ions per sulfate. The increased energy requirement for sulfate transport can be estimated by assuming that three protons are consumed per ATP formed by ATPase (6). An electro-neutral uptake using two protons would correspond to consumption of 2/3 ATP per sulfate transported into the cell, whereas an electrogenic uptake using three protons corresponds to consumption of 1 ATP. The cell therefore uses 1/3 ATP more per sulfate at low sulfate concentrations than at high sulfate concentrations. This is costly for the cell in view of the low energy yield obtained during sulfate reduction and in view of the fact that about –44 kJ is required for production of 1 mol ATP (20).

The increased energy demand for sulfate uptake might be the reason for the shift in the metabolic pathway at 300 µM sulfate, in which less sulfate is reduced per mol lactate than in the metabolic pathway used at high sulfate concentrations. During sulfate reduction, the activation of sulfate is associated with consumption of 2 ATP/SO42– (22, 23). Thus, when the cell needs about 1/3 ATP more for sulfate uptake at 300 µM sulfate, it can partially compensate for this loss by shifting its metabolic pathway (from equation 15 to equation 19) and thereby use less sulfate (3/8 mol less sulfate and 3/4 mol less ATP) per mol of lactate during lactate oxidation. Furthermore, the cell uses less energy for growth and increases the specific sulfate reduction rate. Thus, the cell gains enough energy to maintain the cell activities, although the sulfate concentration is low.

Sulfur isotope fractionation at different sulfate concentrations.
A preliminary analysis of the isotope data was reported by Habicht et al. (11), who pointed out that isotope fractionation during sulfate reduction by A. fulgidus is independent of sulfate at concentrations above 2 mM (Fig. 3). Under these conditions A. fulgidus expressed a maximum isotope fractionation of 26{per thousand}. This fractionation value is similar to values obtained for bacterial sulfate reducers growing at high sulfate concentrations. Overall, sulfate reducers in pure cultures express fractionations ranging from 2 to 46{per thousand} (average, 18{per thousand}) (8, 12), and for natural sulfate-reducing populations the values range from 16 to 43{per thousand} (average, 28{per thousand}) (1, 3, 9, 10).

The fractionation during sulfate reduction by A. fulgidus decreased as the sulfate concentration dropped below 2 mM (Fig. 3). At a sulfate concentration of 300 µM the fractionation was only about one-half the maximum fractionation (26{per thousand}). At this concentration, A. fulgidus likely is limited by the sulfate supply, as demonstrated by the stoichiometric calculations. We previously found that natural populations of sulfate reducers from both marine and freshwater sediments express fractionations of up to 32{per thousand} at sulfate concentrations down to 200 µM (11). At sulfate concentrations of less than 50 µM, however, the average fractionation for the natural population was not more than 0.7{per thousand} ± 5.2{per thousand}. Thus, for A. fulgidus and for natural populations of sulfate reducers, sulfate limits isotope fractionation at concentrations around 50 to 300 µM.

Similar to the growth yield, it is possible to express the correlation between fractionation and sulfate by use of a modified Michaelis-Menten equation:

(20)
where {varepsilon} ({per thousand}) is the fractionation at sulfate concentration S (mM), {varepsilon}max is the maximum isotope fractionation, and Km-frac (mM) is the half-saturation constant expressing the sulfate concentration at one-half of the maximum isotope fractionation. Using linear regression of the correlation between 1/fractionation and 1/sulfate, we obtained a maximum fractionation of 25.5{per thousand} and a Km-frac of 363 µM (Fig. 3). Using equation 20 it is also possible to calculate the limiting sulfate concentration for the isotope fractionation (<5{per thousand}), which for A. fulgidus is 88 µM. This value is in the concentration range found for natural populations of sulfate-reducing bacteria (11).

The Km values for both the growth yield and the isotope fractionation (Km-frac) were about 400 µM. In contrast, the Km for the sulfate reduction rate probably is less than 300 µM as there was no decrease in the sulfate reduction rate at sulfate concentrations down to 300 µM (Fig. 1). The exact Km for the sulfate reduction rate for A. fulgidus is not known. For comparison, Km values for cultured sulfate-reducing strains are between 3 and 68 µM for freshwater strains and between 77 and 200 µM for marine strains (7, 15, 16, 22, 27).

Overall, isotope fractionation occurs as sulfate is reduced in a number of enzymatic steps to sulfide. Theoretically, a maximum fractionation of 45 to 50{per thousand} can occur during sulfate reduction (2). However, such large fractionations can occur only when all the reversible steps during sulfate reduction, including the uptake of sulfate into the cell (step 1), the activation of sulfate with ATP to form APS (step 2), and the reduction of APS to SO32– (step 3), are near exchange equilibrium. Thus, the irreversible reduction of SO32– to H2S (step 4) is the rate-limiting step and probably the main step that determines the final fractionation (2). At limiting sulfate concentrations less sulfate is exchanged back out the cell and the equilibrium is shifted toward sulfate uptake. Possible active sulfate uptake probably further reduces the amount of sulfate exchanged back out the cell and thereby enhances the reduction in the isotope fractionation but not necessarily the specific sulfate reduction rate.

Therefore, when the normal sulfate uptake mechanism in A. fulgidus becomes limited by sulfate at concentration between 0.6 mM and 1.3 mM, the cell probably actively starts to pump sulfate into the cell. This reduces the free exchange of sulfate in and out of the cell and thus also the isotope fractionation during sulfate reduction. The energy used for the active sulfate uptake reduces the growth yield, and to maintain energy for growth the specific sulfate reduction rate increases. At sulfate concentrations below 0.6 mM the cell shifts the metabolic pathway to a reaction that uses less sulfate per mole of organic substrate than is used at high sulfate concentrations. Only at very low sulfate concentrations (below 300 µM) does the sulfate reduction rate decrease with decreasing sulfate concentrations.


   ACKNOWLEDGMENTS
 
This work was supported by a European Commission Marie Currie fellowship and by the Danish National Research Foundation (Danmarks Grundforskningsfond).

We thank Kjeld Ingversen and three anonymous reviewers for discussions concerning the manuscript and Peter Søholt for technical assistance. Theo Hansen from the University of Groningen, The Netherlands, is thanked for introducing the chemostat techniques, and Karl O. Stetter from the University of Regensburg, Germany, is thanked for the donation of an A. fulgidus culture.


   FOOTNOTES
 
* Corresponding author. Mailing address: Danish Center of Earth System Science, University of Southern Denmark, Institute of Biology, Campusvej 55, DK-5230 Odense M, Denmark. Phone: (45) 65502743. Fax: (45) 65930457. E-mail: khabicht{at}biology.sdu.dk. Back

{dagger} Supplemental material for this article may be found at http://aem.asm.org/. Back


   REFERENCES
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Brüchert, V., C. Knoblauch, and B. B. Jorgensen. 2001. Controls on stable sulfur isotope fractionation during bacterial sulfate reduction in Arctic sediments. Geochim. Cosmochim. Acta 65:763-776.[CrossRef]
  2. Canfield, D. E. 2001. Biogeochemistry of sulfur isotopes, p. 607-636. In J. W. Yalley and D. R. Cole (ed.), Reviews in mineralogy & geochemistry. Stable isotope geochemistry, vol. 43. The Mineralogical Society of America, Washington, DC.
  3. Canfield, D. E. 2001. Isotope fractionation by natural populations of sulfate-reducing bacteria. Geochim. Cosmochim. Acta 65:1117-1124.
  4. Canfield, D. E., K. S. Habicht, and B. Thamdrup. 2000. The archean sulfur cycle and the early history of atmospheric oxygen. Science 288:658-661.[Abstract/Free Full Text]
  5. Cline, J. D. 1969. Spectrophotometric determination of hydrogen sulfide in natural waters. Limnol. Oceanogr. 14:454-458.
  6. Cypionka, H. 1995. Solute transport and cell energetics, p. 151-184. In L. L. Barton (ed.), Sulfate-reducing bacteria, vol. 8. Plenum Press, New York, NY.
  7. Dalsgaard, T., and F. Bak. 1994. Nitrate reduction in a sulfate-reducing bacterium, Desulfovibrio desulfuricans, isolated from rice paddy soil: sulfide inhibition, kinetics, and regulation. Appl. Environ. Microbiol. 60:291-297.[Abstract/Free Full Text]
  8. Detmers, J., V. Brüchert, K. S. Habicht, and J. Kuever. 2001. Diversity of sulfur isotope fractionations by sulfate-reducing prokaryotes. Appl. Environ. Microbiol. 67:888-894.[Abstract/Free Full Text]
  9. Habicht, K. H., and D. E. Canfield. 1997. Sulfur isotope fractionation during bacterial sulfate reduction in organic rich sediments. Geochim. Cosmochim. Acta 61:5351-5361.
  10. Habicht, K. S., and D. E. Canfield. 2001. Isotope fractionation by sulfate-reducing natural populations and the isotope composition of sulfide in marine sediments. Geology 29:555-558.[Abstract/Free Full Text]
  11. Habicht, K. S., M. Gade, B. Thamdrup, P. Berg, and D. E. Canfield. 2002. Calibration of sulfate levels in the archean ocean. Science 298:2372-2374.[Abstract/Free Full Text]
  12. Harrison, A. G., and H. G. Thode. 1958. Mechanism of the bacterial reduction of sulphate from isotope fractionation studies. Trans. Faraday Soc. 54:84-92.[CrossRef]
  13. Hartzell, P., and D. Reed. 12 May 2003, posting date. The genus Archaeoglobus. In The prokaryotes. An evolving electronic resource for the microbiology community. [Online.] Springer-Verlag, Berlin, Germany. http://141.150.157.117:8080/prokPUB/chaphtm/COMPLETE.htm.
  14. Holmer, M., and P. Storkholm. 2001. Sulphate reduction and sulphur cycling in lake sediments: a review. Freshwater Biol. 46:431-451.[CrossRef]
  15. Ingvorsen, K., and B. B. Jørgensen. 1984. Kinetics of sulfate uptake by freshwater and marine species of Desulfovibrio. Arch. Microbiol. 139:61-66.[CrossRef]
  16. Ingvorsen, K., J. G. Zeikus, and T. D. Brock. 1981. Dynamics of bacterial sulfate reduction in a eutrophic lake. Appl. Environ. Microbiol. 42:1029-1036.[Abstract/Free Full Text]
  17. Jørgensen, B. B. 1982. Mineralization af organic matter in the sea bed—the role of sulphate reduction. Nature 296:643-645.
  18. Klenk, H. P., R. A. Clayton, J. F. Tomb, O. White, K. E. Nelson, K. A. Ketchum, R. J. Dodson, M. Gwinn, E. K. Hickey, J. D. Peterson, D. L. Richardson, A. R. Kerlavage, D. E. Graham, N. C. Kyrpides, R. D. Fleischmann, J. Quackenbush, N. H. Lee, G. G. Sutton, S. Gill, E. F. Kirkness, B. A. Dougherty, K. McKenney, M. D. Adams, B. Loftus, S. Peterson, C. I. Reich, L. K. McNeil, J. H. Badger, A. Glodek, L. X. Zhou, R. Overbeek, J. D. Gocayne, J. F. Weidman, L. McDonald, T. Utterback, M. D. Cotton, T. Spriggs, P. Artiach, B. P. Kaine, S. M. Sykes, P. W. Sadow, K. P. Dandrea, C. Bowman, C. Fujii, S. A. Garland, T. M. Mason, G. J. Olsen, C. M. Fraser, H. O. Smith, C. R. Woese, and J. C. Venter. 1997. The complete genome sequence of the hyperthermophilic, sulphate-reducing archaeon Archaeoglobus fulgidus. Nature 390:364-370.[CrossRef][Medline]
  19. Labes, A., and P. Schonheit. 2001. Sugar utilization in the hyperthermophilic, sulfate-reducing archaeon Archaeoglobus fulgidus strain 7324: starch degradation to acetate and CO2 via a modified Embden-Meyerhof pathway and acetyl-CoA synthetase (ADP-forming). Arch. Microbiol. 176:329-338.[CrossRef][Medline]
  20. Lengeler, J. D., G. Drews, and H. G. Schlegel. 1999. Biology of the prokaryotes. Blackwell Science, Stuttgart, Germany.
  21. Möller-Zinkhan, D., and R. K. Thauer. 1990. Anaerobic lactate oxidation to 3 CO2 by Archaeoglobus fulgidus via the carbon monoxide dehydrogenase pathway: demonstration of the acetyle-CoA carbon-carbon cleavage reaction in cell extracts. Arch. Microbiol. 153:215-218.[CrossRef]
  22. Nethe-Jaenchen, R., and R. K. Thauer. 1984. Growth yields and saturation constant of Desulfovibrio vulgaris in chemostat culture. Arch. Microbiol. 137:236-240.[CrossRef]
  23. Rabus, R., T. Hansen, and F. Widdel. 8 September 2000, posting date. Dissimilatory sulfate- and sulfur- reducing prokaryotes. In The prokaryotes. An evolving electronic resource for the microbiology community. [Online.] Springer-Verlag, Berlin, Germany. http://141.150.157.117:8080/prokPUB/chaphtm/COMPLETE.htm.
  24. Rosing, M. T. 1999. C-13-depleted carbon microparticles in >3700-Ma sea-floor sedimentary rocks from west Greenland. Science 283:674-676.[Abstract/Free Full Text]
  25. Sawers, G., and G. Watson. 1998. A glycyl radical solution: oxygen-dependent interconversion of pyruvate formate-lyase. Mol. Microbiol. 29:945-954.[CrossRef][Medline]
  26. Shen, Y., R. Buick, and D. E. Canfield. 2001. Isotopic evidence for microbial sulphate reduction in the early Archean era. Nature 410:77-81.[CrossRef][Medline]
  27. Sonne-Hansen, J., P. Westermann, and B. K. Ahring. 1999. Kinetics of sulfate and hydrogen uptake by the thermophilic sulfate-reducing bacteria Thermodesulfobacterium sp. strain ASP and Thermodesulfovibrio sp. strain R1Ha3. Appl. Environ. Microbiol. 65:1304-1307.[Abstract/Free Full Text]
  28. Stetter, K. O., G. Lauerer, M. Thomm, and A. Neuner. 1987. Isolation of extremely thermophilic sulfate reducers: evidence for a novel branch of Archaebacteria. Science 236:822-824.[Abstract/Free Full Text]
  29. Thauer, R. K., and J. Kunow. 1995. Sulfate-reducing archaea, p. 33-48. In L. L. Barton (ed.), Sulfate-reducing bacteria. Plenum Press, New York, NY.
  30. Thauer, R. K., D. Möller-Zinkhan, and A. M. Spormann. 1989. Biochemistry of acetate catabolism in anaerobic chemotrophic bacteria. Annu. Rev. Microbiol. 43:43-67.[Medline]
  31. Woese, C. R. 1987. Bacterial evolution. Microbiol. Rev. 51:221-271.[Free Full Text]
  32. Zellner, G., E. Stackerbrandt, H. Kneifel, P. Messner, U. B. Sleytr, E. C. de Macario, H.-P. Zabel, K. O. Stetter, and J. Winter. 1989. Isolation and characterization of a thermohpilic, sulfate reducing archaebacterium, Archaeoglobus fulgidus strain Z. Syst. Appl. Microbiol. 11:151-160.

Applied and Environmental Microbiology, July 2005, p. 3770-3777, Vol. 71, No. 7
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.7.3770-3777.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Supplemental material
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Habicht, K. S.
Right arrow Articles by Canfield, D. E.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Habicht, K. S.
Right arrow Articles by Canfield, D. E.
Agricola
Right arrow Articles by Habicht, K. S.
Right arrow Articles by Canfield, D. E.

Home Help [Feedback] [For Subscribers] [Archive] [Search] [Contents]
J. Bacteriol. Microbiol. Mol. Biol. Rev. Eukaryot. Cell All ASM Journals