Microcalorimetric studies of the growth of sulfate-reducing bacteria: comparison of the growth parameters of some Desulfovibrio species.

We performed a comparative study of the growth energetics of some species of Desulfovibrio by measuring microcalorimetric and molar growth yield values. Lactate and pyruvate were used as energy sources for sulfate reduction. On lactate-sulfate media Desulfovibrio desulfuricans Norway, Desulfovibrio gigas, and Desulfovibrio africanus exhibited molar growth yields of 4.1 +/- 0.6, 3.7 +/- 1.7, and 1.8 +/- 0.1 g/mol, respectively, whereas on pyruvate-sulfate media the molar growth yields were higher (8.5 +/- 0.8, 7.7 +/- 1.6, and 3.5 +/- 0.5 g/mol, respectively). Thus, we found that D. africanus was the least efficient species in converting energy into cell material. The uncoupling of energy in this strain was obvious since its catabolic activities were high compared with those of the two other strains. The enthalpy changes associated with lactate and pyruvate metabolism were -49 +/- 0.7 and -70.2 +/- 6.0 jK/mol, respectively, for D. desulfuricans, -76.6 +/- 1.8 and -91.2 +/- 1.1 kJ/mol, respectively, for D. gigas, and -78.8 +/- 7.2 and -88.0 +/- 6.2 kJ/mol, respectively, for D. africanus. D. gigas and D. africanus produced only acetate, CO2 and hydrogen sulfide as metabolic end products. In addition to these normal end products, D. desulfuricans Norway produced a small amount of butanol. This butanol production was interpreted as reflecting a regulatory system of electron flow during the catabolism of both substrates. Such metabolism was comparable to that reported for D. vulgaris, which lost part of the reducing power of its energy sources through hydrogen evolution.     

Energetics of growth of a defined mixed culture of Desulfovibrio vulgaris and Methanosarcina barkeri: maintenance energy coefficient of the sulfate-reducing organism in the absence and presence of its partner

The maintenance energy coefficient of Desulfovibrio vulgaris was studied by using a chemostat, with Methanosarcina barkeri or sulfate as the electron acceptor; lithium lactate or sodium pyruvate served as the electron donor. The experiments showed that the growth energetics of D. vulgaris or M. barkeri were greatly affected by maintenance energy coefficients. When D. vulgaris grew on lactate or pyruvate medium with sulfate, these coefficients reached 4.40 and 2.80 mM g-1 h-1, respectively; on lactate medium in the presence of M. barkeri the same coefficient reached a value of 2.90 mM g-1 h-1. Results also showed that the increase of the value of the maintenance energy coefficient corresponded to a decrease of the biomass produced. D. vulgaris maximal growth yield values calculated by use of the Pirt equation were slightly higher with M. barkeri (maximal growth yield, 10 g/mol) than with sulfate (maximal growth yield, 7.5 g/mol). This finding could be interpreted by reference to the ATP-generating reactions involved in D. vulgaris growth in the presence of sulfate or M. barkeri.     

Physiological function of hydrogen metabolism during growth of sulfidogenic bacteria on organic substrates.

Desulfovibrio vulgaris Madison and Thermodesulfobacterium commune contained functionally distinct hydrogenase activities, one which exchanged 3H2 into 3H2O and was inhibited by carbon monoxide and a second activity which produced H2 in the presence of CO. Cell suspensions of D. vulgaris used either lactate, pyruvate, or CO as the electron donor for H2 production in the absence of sulfate. Both sulfidogenic species produced and consumed hydrogen as a trace gas during growth on lactate or pyruvate as electron donors and on thiosulfate or sulfate as electron acceptors. Higher initial levels of hydrogen were detected during growth on lactate-sulfate than on pyruvate-sulfate. D. vulgaris but not T. commune also produced and then consumed CO during growth on organic electron donors and sulfate or thiosulfate. High partial pressures of exogenous H2 inhibited growth and substrate consumption when D. vulgaris was cultured on pyruvate alone but not when it was metabolizing pyruvate plus sulfate or lactate plus sulfate. The data are discussed in relation to supporting two different models for the physiological function of H2 metabolism during growth of sulfidogenic bacteria on organic electron donors plus sulfate. A trace H2 transformation model is proposed for control of redox processes during growth on either pyruvate or lactate plus sulfate, and an obligate H2 cycling model is proposed for chemiosmotic energy coupling during growth on CO plus sulfate.     

Carbon monoxide cycling by Desulfovibrio vulgaris Hildenborough

Sulfate-reducing bacteria, like Desulfovibrio vulgaris Hildenborough, use the reduction of sulfate as a sink for electrons liberated in oxidation reactions of organic substrates. The rate of the latter exceeds that of sulfate reduction at the onset of growth, causing a temporary accumulation of hydrogen and other fermentation products (the hydrogen or fermentation burst). In addition to hydrogen, D. vulgaris was found to produce significant amounts of carbon monoxide during the fermentation burst. With excess sulfate, the hyd mutant (lacking periplasmic Fe-only hydrogenase) and hmc mutant (lacking the membrane-bound, electron-transporting Hmc complex) strains produced increased amounts of hydrogen from lactate and formate compared to wild-type D. vulgaris during the fermentation burst. Both hydrogen and CO were produced from pyruvate, with the hyd mutant producing the largest transient amounts of CO. When grown with lactate and excess sulfate, the hyd mutant also exhibited a temporary pause in sulfate reduction at the start of stationary phase, resulting in production of 600 ppm of headspace hydrogen and 6,000 ppm of CO, which disappeared when sulfate reduction resumed. Cultures with an excess of the organic electron donor showed production of large amounts of hydrogen, but no CO, from lactate. Pyruvate fermentation was diverse, with the hmc mutant producing 75,000 ppm of hydrogen, the hyd mutant producing 4,000 ppm of CO, and the wild-type strain producing no significant amount of either as a fermentation end product. The wild type was most active in transient production of an organic acid intermediate, tentatively identified as fumarate, indicating increased formation of organic fermentation end products in the wild-type strain. These results suggest that alternative routes for pyruvate fermentation resulting in production of hydrogen or CO exist in D. vulgaris. The CO produced can be reoxidized through a CO dehydrogenase, the presence of which is indicated in the genome sequence.     

Energetics of Growth of a Defined Mixed Culture of Desulfovibrio vulgaris and Methanosarcina barkeri: Interspecies Hydrogen Transfer in Batch and Continuous Cultures

Interspecies hydrogen transfer was studied in Desulfovibrio vulgaris-Methanosarcina barkeri mixed cultures. Experiments were performed under batch and continuous growth culture conditions. Lactate or pyruvate was used as an energy source. In batch culture and after 30 days of simultaneous incubation, these organisms were found to yield 1.5 mol of methane and 1.5 mol of carbon dioxide per mol of lactate fermented. When M. barkeri served as the hydrogen acceptor, growth yields of D. vulgaris were higher compared with those obtained on pyruvate without any electron acceptor other than protons. In continuous culture, all of the carbon derived from the oxidation of lactate was recovered as methane and carbon dioxide, provided the dilution rate was minimal. Increasing the dilution rate induced a gradual accumulation of acetate, causing acetate metabolism to cease at above μ = 0.05 h⁻¹. Under these conditions all of the methane produced originated from carbon dioxide. The growth yields of D. vulgaris were measured when sulfate or M. barkeri was the electron acceptor. Two key observations resulted from the present study. First, although sulfate was substituted by M. barkeri, metabolism of D. vulgaris was only slightly modified. The coculture-fermented lactate produced equimolar quantities of carbon dioxide and methane. Second, acetogenesis and methane formation from acetate were completely separable.     

Hydrogen metabolism in Desulfovibrio desulfuricans strain New Jersey (NCIMB 8313)--comparative study with D. vulgaris and D. gigas species

This article aims to study hydrogen production/consumption in Desulfovibrio (D.) desulfuricans strain New Jersey, a sulfate reducer isolated from a medium undergoing active biocorrosion and to compare its hydrogen metabolism with two other Desulfovibrio species, D. gigas and D. vulgaris Hildenborough. Hydrogen production was followed during the growth of these three bacterial species under different growth conditions: no limitation of sulfate and lactate, sulfate limitation, lactate limitation, pyruvate/sulfate medium and in the presence of molybdate. Hydrogen production/consumption by D. desulfuricans shows a behavior similar to that of D. gigas but a different one from that of D. vulgaris, which produces higher quantities of hydrogen on lactate/sulfate medium. The three species are able to increase the hydrogen production when the sulfate became limiting. Moreover, in a pyruvate/sulfate medium hydrogen production was lower than on lactate/sulfate medium. Hydrogen production by D. desulfuricans in presence of molybdate is extremely high. Hydrogenases are key enzymes on production/consumption of hydrogen in sulfate reducing organisms. The specific activity, number and cellular localization of hydrogenases vary within the three Desulfovibrio species used in this work, which could explain the differences observed on hydrogen utilization.     

CO metabolism of Desulfovibrio vulgaris strain Madison: physiological function in the absence or presence of exogeneous substrates

Abstract Trace amounts of carbon monoxide were produced and subsequently consumed during the growth of Desulfovibrio vulgaris on organic electron donors. D. vulgaris also utilized carbon monoxide as the sole electron donor for growth and sulfate reduction. Growth of D. vulgaris on CO, H₂ or organic electron donors was inhibited at ≥4.5% CO in the culture headspace. At lower CO concentrations, hydrogen was produced as a consequence of CO consumption and consumed when the CO partial pressure was decreased. The rate of CO consumption was ten-fold higher in D. vulgaris grown on either CO, lactate or pyruvate than when cells were grown on H₂ as electron donor. The physiological function of CO metabolism and a CO-dependent hydrogen cycle in D. vulgaris is discussed.     

Energy yield of respiration on chloroaromatic compounds in Desulfitobacterium dehalogenans

The amount of energy that can be conserved via halorespiration by Desulfitobacterium dehalogenans JW/IU-DC1 was determined by comparison of the growth yields of cells grown with 3-chloro-4-hydroxyphenyl acetate (Cl-OHPA) and different electron donors. Cultures that were grown with lactate, pyruvate, formate, or hydrogen as an electron donor and Cl-OHPA as an electron acceptor yielded 3.1, 6.6, 1.6, and 1.6 g (dry weight) per mol of reduction equivalents, respectively. Fermentative growth on pyruvate yielded 14 g (dry weight) per mol of pyruvate oxidized. Pyruvate was not fermented stoichiometrically to acetate and lactate, but an excess of acetate was produced. Experiments with 13C-labeled bicarbonate showed that during pyruvate fermentation, approximately 9% of the acetate was formed from the reduction of CO2. Comparison of the growth yields suggests that 1 mol of ATP is produced per mol of acetate produced by substrate-level phosphorylation and that there is no contribution of electron transport phosphorylation when D. dehalogenans grows on lactate plus Cl-OHPA or pyruvate plus Cl-OHPA. Furthermore, the growth yields indicate that approximately 1/3 mol of ATP is conserved per mol of Cl-OHPA reduced in cultures grown in formate plus Cl-OHPA and hydrogen plus Cl-OHPA. Because neither formate nor hydrogen nor Cl-OHPA supports substrate-level phosphorylation, energy must be conserved through the establishment of a proton motive force. Pyruvate ferredoxin oxidoreductase, lactate dehydrogenase, formate dehydrogenase, and hydrogenase were localized by in vitro assays with membrane-impermeable electron acceptors and donors. The orientation of chlorophenol-reductive dehalogenase in the cytoplasmic membrane, however, could not be determined. A model is proposed, which may explain the topology analyses as well as the results obtained in the yield study.     

Gene expression analysis of energy metabolism mutants of Desulfovibrio vulgaris Hildenborough indicates an important role for alcohol dehydrogenase

Comparison of the proteomes of the wild-type and Fe-only hydrogenase mutant strains of Desulfovibrio vulgaris Hildenborough, grown in lactate-sulfate (LS) medium, indicated the near absence of open reading frame 2977 (ORF2977)-coded alcohol dehydrogenase in the hyd mutant. Hybridization of labeled cDNA to a macroarray of 145 PCR-amplified D. vulgaris genes encoding proteins active in energy metabolism indicated that the adh gene was among the most highly expressed in wild-type cells grown in LS medium. Relative to the wild type, expression of the adh gene was strongly downregulated in the hyd mutant, in agreement with the proteomic data. Expression was upregulated in ethanol-grown wild-type cells. An adh mutant was constructed and found to be incapable of growth in media in which ethanol was both the carbon source and electron donor for sulfate reduction or was only the carbon source, with hydrogen serving as electron donor. The hyd mutant also grew poorly on ethanol, in agreement with its low level of adh gene expression. The adh mutant grew to a lower final cell density on LS medium than the wild type. These results, as well as the high level of expression of adh in wild-type cells on media in which lactate, pyruvate, formate, or hydrogen served as the sole electron donor for sulfate reduction, indicate that ORF2977 Adh contributes to the energy metabolism of D. vulgaris under a wide variety of metabolic conditions. A hydrogen cycling mechanism is proposed in which protons and electrons originating from cytoplasmic ethanol oxidation by ORF2977 Adh are converted to hydrogen or hydrogen equivalents, possibly by a putative H(2)-heterodisulfide oxidoreductase complex, which is then oxidized by periplasmic Fe-only hydrogenase to generate a proton gradient.     

Reduction of the amount of periplasmic hydrogenase in Desulfovibrio vulgaris (Hildenborough) with antisense RNA: direct evidence for an important role of this hydrogenase in lactate metabolism.

To establish the function of the periplasmic Fe-only hydrogenase in the anaerobic sulfate reducer Desulfovibrio vulgaris (Hildenborough), derivatives with a reduced content of this enzyme were constructed by introduction of a plasmid that directs the synthesis of antisense RNA complementary to hydrogenase mRNA. It was demonstrated that the antisense RNA technique allowed specific suppression of the synthesis of this hydrogenase in D. vulgaris by decreasing the amount of hydrogenase mRNA but did not result in the complete elimination of the enzyme, as is usual with most conventional mutagenesis techniques. The hydrogenase content in these antisense RNA-producing D. vulgaris clones was two- to threefold lower than in the parental strain when the strains were grown in batch cultures with lactate as a substrate and sulfate as a terminal electron acceptor. Under these conditions, several differences in growth parameters were measured between the hydrogenase-suppressed clones and wild-type D. vulgaris: growth rates of the clones decreased two- to threefold, and at excess lactate, growth yields were reduced by 20%. Furthermore, the amount of hydrogen measured in the culture headspaces was reduced three- to fivefold for the clones. These observations indicate that this hydrogenase has an important function during growth on lactate and is involved in hydrogen production from protons and electrons originating from at least one of the two oxidation reactions in the conversion of lactate to acetate. The implications for the energy metabolism of D. vulgaris are discussed.     

Energy Yield of Respiration on Chloroaromatic Compounds in Desulfitobacterium dehalogenans

The amount of energy that can be conserved via halorespiration by Desulfitobacterium dehalogenans JW/IU-DC1 was determined by comparison of the growth yields of cells grown with 3-chloro-4-hydroxyphenyl acetate (Cl-OHPA) and different electron donors. Cultures that were grown with lactate, pyruvate, formate, or hydrogen as an electron donor and Cl-OHPA as an electron acceptor yielded 3.1, 6.6, 1.6, and 1.6 g (dry weight) per mol of reduction equivalents, respectively. Fermentative growth on pyruvate yielded 14 g (dry weight) per mol of pyruvate oxidized. Pyruvate was not fermented stoichiometrically to acetate and lactate, but an excess of acetate was produced. Experiments with ¹³C-labeled bicarbonate showed that during pyruvate fermentation, approximately 9% of the acetate was formed from the reduction of CO₂. Comparison of the growth yields suggests that 1 mol of ATP is produced per mol of acetate produced by substrate-level phosphorylation and that there is no contribution of electron transport phosphorylation when D. dehalogenans grows on lactate plus Cl-OHPA or pyruvate plus Cl-OHPA. Furthermore, the growth yields indicate that approximately 1/3 mol of ATP is conserved per mol of Cl-OHPA reduced in cultures grown in formate plus Cl-OHPA and hydrogen plus Cl-OHPA. Because neither formate nor hydrogen nor Cl-OHPA supports substrate-level phosphorylation, energy must be conserved through the establishment of a proton motive force. Pyruvate ferredoxin oxidoreductase, lactate dehydrogenase, formate dehydrogenase, and hydrogenase were localized by in vitro assays with membrane-impermeable electron acceptors and donors. The orientation of chlorophenol-reductive dehalogenase in the cytoplasmic membrane, however, could not be determined. A model is proposed, which may explain the topology analyses as well as the results obtained in the yield study.     

Growth with hydrogen,and further physiological characteristics of Desulfotomaculum species

Growth of Desulfotomaculum orientis, D. ruminis, D. nigrificans and the Desulfotomaculum strains TEP, TWC and TWP, that were newly isolated with sulfate and fatty acids, was studied using defined mineral media. Four of these strains grew with hydrogen plus sulfate as the only energy source. Under these conditions the growth yield of D. orientis in batch culture was 7.5 g cell dry mass per mol sulfate reduced. Growth on methanol with growth yields of about 6 g cell dry mass per mol sulfate was obtained with D. orientis and strain TEP. All strains tested grew slowly with formate as electron donor. Fatty acids from propionate to palmitate were utilized by the strains TEP, TWC and TWP. D. orientis and the strains TEP and TWC were able to utilize the methoxyl groups of trimethoxybenzoate for growth. D. orientis was found to grow chemoautotrophically with hydrogen, carbon dioxide and sulfate; during growth with C₁-compounds no additional organic carbon source was required. Furthermore, D. orientis was able to grow slowly in sulfate-free medium with formate, methanol, ethanol lactate, pyruvate or trimethoxybenzoate. Under these conditions acetate was excreted, indicating the function of carbon dioxide as electron acceptor in a homoacetogenic process. A growth-promoting effect of pyrophosphate added to the medium of Desulfotomaculum species was not observed. The results show a high catabolic and anabolic versatility among Desulfotomaculum species, and indicate that electron transport to sulfate can be the sole energy conserving process in this genus.     

Sulfate-Dependent Interspecies H(2) Transfer between Methanosarcina barkeri and Desulfovibrio vulgaris during Coculture Metabolism of Acetate or Methanol

We compared the metabolism of methanol and acetate when Methanosarcina barkeri was grown in the presence and absence of Desulfovibrio vulgaris. The sulfate reducer was not able to utilize methanol or acetate as the electron donor for energy metabolism in pure culture, but was able to grow in coculture. Pure cultures of M. barkeri produced up to 10 mumol of H(2) per liter in the culture headspace during growth on acetate or methanol. In coculture with D. vulgaris, the gaseous H(2) concentration was 相似文献   

Desulfobulbus mediterraneus sp. nov., a sulfate-reducing bacterium growing on mono- and disaccharides

A new sulfate-reducing bacterium, strain 86FS1, was isolated from a deep-sea sediment in the western Mediterranean Sea with sodium lactate as electron and carbon source. Cells were ovoid, gram-negative and motile. Strain 86FS1 contained b- and c-type cytochromes. The organism was able to utilize propionate, pyruvate, lactate, succinate, fumarate, malate, alanine, primary alcohols (C(2)-C(5)), and mono- and disaccharides (glucose, fructose, galactose, ribose, sucrose, cellobiose, lactose) as electron donors for the reduction of sulfate, sulfite or thiosulfate. The major products of carbon metabolism were acetate and CO(2), with exception of n-butanol and n-pentanol, which were oxidized only to the corresponding fatty acids. The growth yield with sulfate and glucose or lactate was 8.3 and 15 g dry mass, respectively, per mol sulfate. The temperature limits for growth were 10 degrees C and 30 degrees C with an optimum at 25 degrees C. Growth was observed at salinities ranging from 10 to 70 g NaCl l(-1). Sulfide concentrations above 4 mmol l(-1) inhibited growth. The fatty acid pattern of strain 86FS1 resembled that of Desulfobulbus propionicus with n-14:0, n-16:1omega7, n-16:1 omega5, n-17:1 omega6 and n-18:1 omega7 as dominant fatty acids. On the basis of its phylogenetic position and its phenotypic properties, strain 86FS1 affiliates with the genus Desulfobulbus and is described as a new species, Desulfobulbus mediterraneus sp. nov.     

Deletion of the hmc operon of Desulfovibrio vulgaris subsp. vulgaris Hildenborough hampers hydrogen metabolism and low-redox-potential niche establishment

The hmc operon of Desulfovibrio vulgaris subsp. vulgaris Hildenborough encodes a transmembrane redox protein complex (the Hmc complex) that has been proposed to catalyze electron transport linking periplasmic hydrogen oxidation to cytoplasmic sulfate reduction. We have replaced a 5-kb DNA fragment containing most of the hmc operon by the cat gene. The resulting chloramphenicol-resistant mutant D. vulgaris H801 grows normally when lactate or pyruvate serve as electron donors for sulfate reduction. Growth with hydrogen as electron donor for sulfate reduction (acetate and CO2 as the carbon source) is impaired. These results confirm the importance of the Hmc complex in electron transport from hydrogen to sulfate. Mutant H801 is also deficient in low-redox-potential niche establishment. On plates, colony development takes 14 days longer than colony development of the wild-type strain, when the cells use hydrogen as the electron donor. This result suggests that, in addition to transmembrane electron transport from hydrogen to sulfate, the redox reactions catalyzed by the Hmc complex are crucial in establishment of the required low-redox-potential niche that allows single cells to grow into colonies.     

Physiological characteristics and growth behavior of single and double hydrogenase mutants of Desulfovibrio fructosovorans

The presence of one periplasmic [NiFe] hydrogenase, one periplasmic [Fe] hydrogenase, and one cytoplasmic NADP-reducing hydrogenase has been previously established in Desulfovibrio fructosovorans. In the present work, marker-exchange mutagenesis was performed to determine the function of the tetrameric NADP-reducing hydrogenase encoded by the hndA, B, C, and D genes. The mutations performed were not lethal to the cells, although the H₂-dependent NADP reduction was completely abolished. The double-mutated DM4 (ΔhynABC, ΔhndD) strain was still able to grow on hydrogen plus sulfate as the sole energy source. The growth may have occurred under these culture conditions because of the presence of the remaining [Fe] hydrogenase. The cells grew differently on various substrates depending on whether fructose, lactate, or pyruvate was used in the presence of sulfate. The (hnd mutant growth rates were 25–70% lower than those of the wild-type strain, although the molar growth yield remained unchanged. By contrast, mutants devoid of both [NiFe] hydrogenase and NADP-reducing hydrogenase had 24-38% lower growth yields and showed a corresponding drop in the growth rates. We concluded that each of the three hydrogenases may contribute to the energy supply in D. fructosovorans and that the loss of one enzyme might be compensated for by another. However, the loss of two hydrogenases affected the phosphorylation accompanying the metabolism of fructose, lactate, and pyruvate. Received: 17 September 1996 / Accepted: 5 November 1996     

Effects of Deletion of Genes Encoding Fe-Only Hydrogenase of Desulfovibrio vulgaris Hildenborough on Hydrogen and Lactate Metabolism

The physiological properties of a hyd mutant of Desulfovibrio vulgaris Hildenborough, lacking periplasmic Fe-only hydrogenase, have been compared with those of the wild-type strain. Fe-only hydrogenase is the main hydrogenase of D. vulgaris Hildenborough, which also has periplasmic NiFe- and NiFeSe-hydrogenases. The hyd mutant grew less well than the wild-type strain in media with sulfate as the electron acceptor and H(2) as the sole electron donor, especially at a high sulfate concentration. Although the hyd mutation had little effect on growth with lactate as the electron donor for sulfate reduction when H(2) was also present, growth in lactate- and sulfate-containing media lacking H(2) was less efficient. The hyd mutant produced, transiently, significant amounts of H(2) under these conditions, which were eventually all used for sulfate reduction. The results do not confirm the essential role proposed elsewhere for Fe-only hydrogenase as a hydrogen-producing enzyme in lactate metabolism (W. A. M. van den Berg, W. M. A. M. van Dongen, and C. Veeger, J. Bacteriol. 173:3688-3694, 1991). This role is more likely played by a membrane-bound, cytoplasmic Ech-hydrogenase homolog, which is indicated by the D. vulgaris genome sequence. The physiological role of periplasmic Fe-only hydrogenase is hydrogen uptake, both when hydrogen is and when lactate is the electron donor for sulfate reduction.     

A unified model describing the role of hydrogen in the growth of desulfovibrio vulgaris under different environmental conditions

A unified model for the growth of Desulfovibrio vulgaris under different environmental conditions is presented. The model assumes the existence of two electron transport mechanisms functioning simultaneously. One mechanism results in the evolution and consumption of hydrogen, as in the hydrogen-cycling model. The second mechanism assumes a direct transport of electrons from the donor to the acceptor, without the participation of H2. A combination of kinetic and thermodynamic conditions control the flow of electrons through each pathway. The model was calibrated using batch experiments with D. vulgaris grown on lactate, in the presence and absence of sulfate, and was verified using additional batch experiments under different conditions. The model captured the general trends of consumption of substrates and accumulation of products, including the transient accumulation and consumption of H2. Furthermore, the model estimated that 48% of the electrons transported from lactate to sulfate involved H2 production, indicating that hydrogen cycling is a fundamental process in D. vulgaris. The presence of simultaneous electron transport mechanisms might provide D. vulgaris with important ecological advantages, because it facilitates a rapid response to changes in environmental conditions. This model increases our ability to study the microbial ecology of anaerobic environments and the role of Desulfovibrio species in a variety of environments. Copyright 1998 John Wiley & Sons, Inc.     

Growth of Methanosarcina barkeri (Fusaro) under nonmethanogenic conditions by the fermentation of pyruvate to acetate: ATP synthesis via the mechanism of substrate level phosphorylation.

A mutant of Methanosarcina barkeri (Fusaro) is able to grow on pyruvate as the sole carbon and energy source. During growth, pyruvate is converted to CH4 and CO2, and about 1.5 mol of ATP per mol of CH4 is formed (A.-K. Bock, A. Prieger-Kraft, and P. Schönheit, Arch. Microbiol. 161:33-46, 1994). The pyruvate-utilizing mutant of M. barkeri could also grow on pyruvate when methanogenesis was completely inhibited by bromoethanesulfonate (BES). The mutant grew on pyruvate (80 mM) in the presence of 2 mM BES with a doubling time of about 30 h up to cell densities of about 400 mg (dry weight) of cells per liter. During growth on pyruvate, the major fermentation products were acetate and CO2 (about 0.9 mol each per mol of pyruvate). Small amounts of acetoin, acetolactate, alanine, leucine, isoleucine, and valine were also detected. CH4 was not formed. The molar growth yield (Yacetate) was about 9 g of cells (dry weight) per mol of acetate, indicating an ATP yield of about 1 mol/mol of acetate formed. Growth on pyruvate in the presence of BES was limited; after six to eight generations, the doubling times increased and the final cell densities decreased. After 9 to 11 generations, growth stopped completely. In the presence of BES, suspensions of pyruvate-grown cells fermented pyruvate to acetate, CO2, and H2. CH4 was not formed. Conversion of pyruvate to acetate, in the complete absence of methanogenesis, was coupled to ATP synthesis. Dicyclohexylcarbodiimide, an inhibitor of H(+)-translocating ATP synthase, did not inhibit ATP formation. In the presence of dicyclohexylcarbodiimide, stoichiometries of up to 0.9 mol of ATP per mol of acetate were observed. The uncoupler arsenate completely inhibited ATP synthesis, while the rates of acetate, CO2, and H2 formation were stimulated up to fourfold. Cell extracts of M. barkeri grown on pyruvate under nonmethenogenic conditions contained pyruvate: ferredoxin oxidoreductase (0.5 U/mg), phosphate acetyltransferase (12 U/mg), and acetate kinase (12 U/mg). From these data it is concluded that ATP was synthesized by substrate level phosphorylation during growth of the M. barkeri mutant on pyruvate in the absence of methanogenesis. This is the first report of growth of a methanogen under nonmethanogenic conditions at the expense of a fermentative energy metabolism.