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.     

Comparison of Energy and Growth Yields for Desulfitobacterium dehalogenans during Utilization of Chlorophenol and Various Traditional Electron Acceptors

Desulfitobacterium dehalogenans grew with formate as the electron donor and 3-chloro-4-hydroxyphenylacetate (3-Cl-4-OHPA) as the electron acceptor, yielding Y(X/formate), Y(X/2e), and Y(X/ATP) ranging from 3.2 to 11.3 g of biomass (dry weight)/mol, thus indicating that energy was conserved through reductive dechlorination. Pyruvate was utilized as the electron donor and acceptor, yielding stoichiometric amounts of acetate and lactate, respectively, and a Y(X/reduced acceptor) of 13.0 g of biomass (dry weight)/mol. The supplementation of pyruvate-containing medium with additional electron acceptors, such as 3-Cl-4-OHPA, nitrate, fumarate, or sulfite, caused pyruvate to be replaced as the electron acceptor and nearly doubled the Y(X/ATP) (Y(X/acetate formed)). A comparison of the yields for 3-Cl-4-OHPA with those for other traditional electron acceptors indicates that the dehalogenation reaction led to the formation of similar amounts of energy equivalents. The various electron acceptors were used concomitantly with 3-Cl-4-OHPA in nonacclimated cultures, but the utilization rates and amounts utilized differed.     

Pyruvate and lactate metabolism by Shewanella oneidensis MR-1 under fermentation, oxygen limitation, and fumarate respiration conditions

Shewanella oneidensis MR-1 is a facultative anaerobe that derives energy by coupling organic matter oxidation to the reduction of a wide range of electron acceptors. Here, we quantitatively assessed the lactate and pyruvate metabolism of MR-1 under three distinct conditions: electron acceptor-limited growth on lactate with O(2), lactate with fumarate, and pyruvate fermentation. The latter does not support growth but provides energy for cell survival. Using physiological and genetic approaches combined with flux balance analysis, we showed that the proportion of ATP produced by substrate-level phosphorylation varied from 33% to 72.5% of that needed for growth depending on the electron acceptor nature and availability. While being indispensable for growth, the respiration of fumarate does not contribute significantly to ATP generation and likely serves to remove formate, a product of pyruvate formate-lyase-catalyzed pyruvate disproportionation. Under both tested respiratory conditions, S. oneidensis MR-1 carried out incomplete substrate oxidation, whereby the tricarboxylic acid (TCA) cycle did not contribute significantly. Pyruvate dehydrogenase was not involved in lactate metabolism under conditions of O(2) limitation but was required for anaerobic growth, likely by supplying reducing equivalents for biosynthesis. The results suggest that pyruvate fermentation by S. oneidensis MR-1 cells represents a combination of substrate-level phosphorylation and respiration, where pyruvate serves as an electron donor and an electron acceptor. Pyruvate reduction to lactate at the expense of formate oxidation is catalyzed by a recently described new type of oxidative NAD(P)H-independent d-lactate dehydrogenase (Dld-II). The results further indicate that pyruvate reduction coupled to formate oxidation may be accompanied by the generation of proton motive force.     

Catabolic thiosulfate disproportionation and carbon dioxide reduction in strain DCB-1, a reductively dechlorinating anaerobe.

Strain DCB-1 is a strict anaerobe capable of reductive dehalogenation. We elucidated metabolic processes in DCB-1 which may be related to dehalogenation and which further characterize the organism physiologically. Sulfoxy anions and CO2 were used by DCB-1 as catabolic electron acceptors. With suitable electron donors, sulfate and thiosulfate were reduced to sulfide. Sulfate and thiosulfate supported growth with formate or hydrogen as the electron donor and thus are probably respiratory electron acceptors. Other electron donors supporting growth with sulfate were CO, lactate, pyruvate, butyrate, and 3-methoxybenzoate. Thiosulfate also supported growth without an additional electron donor, being disproportionated to sulfide and sulfate. In the absence of other electron acceptors, CO2 reduction to acetate plus cell material was coupled to pyruvate oxidation to acetate plus CO2. Pyruvate could not be fermented without an electron acceptor. Carbon monoxide dehydrogenase activity was found in whole cells, indicating that CO2 reduction probably occurred via the acetyl coenzyme A pathway. Autotrophic growth occurred on H2 plus thiosulfate or sulfate. Diazotrophic growth occurred, and whole cells had nitrogenase activity. On the basis of these physiological characteristics, DCB-1 is a thiosulfate-disproportionating bacterium unlike those previously described.     

Growth yields and energy generation by Campylobacter sputorum subspecies bubulus during growth in continuous culture with different hydrogen acceptors

Campylobacter sputorum subspecies bubulus was grown in continuous culture with excess of l-lactate or formate, and growth-limiting amounts of oxygen, fumarate, nitrate or nitrite. l-Lactate was oxidized to acetate, fumarate was reduced to succinate, and nitrate and nitrite were reduced to ammonia. The Y _lactate values (g dry weight bacteria/g mol lactate) for the respective hydrogen acceptors were much higher than the Y _formate values. Steady state cultures on formate and nitrite could only be obtained at a low dilution rate and low nitrite concentrations in the growth medium. In H⁺/2e measurements with lactate-grown cells proton ejections were observed with lactate or pyruvate as a hydrogen donor, and oxygen or hydrogen peroxide as a hydrogen acceptor. Proton ejection was also observed with pyruvate and nitrate. Proton ejection did not occur with lactate and nitrate, neither with lactate or pyruvate and fumarate or nitrite. With formate as a hydrogen donor acidification occurred with all hydrogen acceptors mentioned. It has been concluded that during growth on lactate and fumarate or nitrite substrate level phosphorylation at acetate formation is the sole ATP-generating system. Growth on formate and fumarate or nitrite is explained by a proton gradient generated as a result of oxidation of formate at the periplasmic side of the cytoplasmic membrane. With oxygen and nitrate additional ATP is formed by electron transport-linked phosphorylation. The low molar growth yields with formate are explained by the observation that formate-grown cells had a great permeability to protons.Abbreviations H⁺/2e value number of protons ejected per electron pair transported in the respiratory system - P/2e value mol of ATP formed per electron pair transported in the respiratory system - CCCP carbonyl cyanide m-chlorophenyl-hydrazone     

Microcalorimetric studies of the growth of sulfate-reducing bacteria: energetics of Desulfovibrio vulgaris growth.

The metabolism of Desulfovibrio vulgaris Hildenborough grown on medium containing lactate or pyruvate plus a high concentration of sulfate (36 mM) was studied. Molecular growth yields were 6.7 +/- 1.3 and 10.1 +/- 1.7 g/mol for lactate and pyruvate, respectively. Under conditions in which the energy source was the sole growth-limiting factor, we observed the formation of 0.5 mol of hydrogen per mol of lactate and 0.1 mol of hydrogen per mol of pyruvate. The determination of metabolic end products revealed that D. vulgaris produced, in addition to normal end products (acetic acid, carbon dioxide, hydrogen sulfide) and molecular hydrogen, 2 and 5% of ethanol per mol of lactate and pyruvate, respectively. Power-time curves of growth of D. vulgaris on lactate and pyruvate were obtained, by the microcalorimetric Tian-Calvet apparatus. The enthalpies (delta Hmet) associated with the oxidation of these substrates and calculated from growth thermograms were -36.36 +/- 5 and -70.22 +/- 3 kJ/mol of lactate and pyruvate, respectively. These experimental values were in agreement with the homologous values assessed from the theoretical equations of D. vulgaris metabolism of both lactate and pyruvate. The hydrogen production by this sulfate reducer constitutes an efficient regulatory system of electrons, from energy source through the pathway of sulfate reduction. This hydrogen value may thus facilitate interactions between this strain and other environmental microflora, especially metagenic bacteria.     

Characterization of Desulfitobacterium chlororespirans sp. nov., which grows by coupling the oxidation of lactate to the reductive dechlorination of 3-chloro-4-hydroxybenzoate.

Strain Co23, an anaerobic spore-forming microorganism, was enriched and isolated from a compost soil on the basis of its ability to grow with 2,3-dichlorophenol (DCP) as its electron acceptor, ortho chlorines were removed from polysubstituted phenols but not from monohalophenols. Growth by chlororespiration was indicated by a growth yield of 3.24 g of cells per mol of reducing equivalents (as 2[H]) from lactate oxidation to acetate in the presence of 3-chloro-4-hydroxybenzoate but no growth in the absence of the halogenated electron acceptor. Other indicators of chlororespiration were the fraction of electrons from the electron donor used for dechlorination (0.67) and the H2 threshold concentration of < 1.0 ppm. Additional electron donors utilized for reductive dehalogenation were pyruvate, formate, butyrate, crotonate, and H2. Pyruvate supported homoacetogenic growth in the absence of an electron acceptor. Strain Co23 also used sulfite, thiosulfate, and sulfur as electron acceptors for growth, but it did not use sulfate, nitrate or fumarate. The temperature optimum for growth was 37 degrees C; however, the rates of dechlorination were optimum at 45 degrees C and activity persisted to temperatures as high as 55 degrees C. The 16S rRNA sequence was determined, and strain Co23 was found to be related to Desulfitobacterium dehalogenans JW/IU DC1 and Desulfitobacterium strain PCE1, with sequence similarities of 97.2 and 96.8%, respectively. The phylogenetic and physiological properties exhibited by strain Co23 place it into a new species designated Desulfitobacterium chlororespirans.     

Pyruvate Fermentation by Oenococcus oeni and Leuconostoc mesenteroides and Role of Pyruvate Dehydrogenase in Anaerobic Fermentation

The heterofermentative lactic acid bacteria Oenococcus oeni and Leuconostoc mesenteroides are able to grow by fermentation of pyruvate as the carbon source (2 pyruvate → 1 lactate + 1 acetate + 1 CO₂). The growth yields amount to 4.0 and 5.3 g (dry weight)/mol of pyruvate, respectively, suggesting formation of 0.5 mol ATP/mol pyruvate. Pyruvate is oxidatively decarboxylated by pyruvate dehydrogenase to acetyl coenzyme A, which is then converted to acetate, yielding 1 mol of ATP. For NADH reoxidation, one further pyruvate molecule is reduced to lactate. The enzymes of the pathway were present after growth on pyruvate, and genome analysis showed the presence of the corresponding structural genes. The bacteria contain, in addition, pyruvate oxidase activity which is induced under microoxic conditions. Other homo- or heterofermentative lactic acid bacteria showed only low pyruvate fermentation activity.     

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.     

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.     

Pyruvate fermentation by Oenococcus oeni and Leuconostoc mesenteroides and role of pyruvate dehydrogenase in anaerobic fermentation

The heterofermentative lactic acid bacteria Oenococcus oeni and Leuconostoc mesenteroides are able to grow by fermentation of pyruvate as the carbon source (2 pyruvate --> 1 lactate + 1 acetate + 1 CO(2)). The growth yields amount to 4.0 and 5.3 g (dry weight)/mol of pyruvate, respectively, suggesting formation of 0.5 mol ATP/mol pyruvate. Pyruvate is oxidatively decarboxylated by pyruvate dehydrogenase to acetyl coenzyme A, which is then converted to acetate, yielding 1 mol of ATP. For NADH reoxidation, one further pyruvate molecule is reduced to lactate. The enzymes of the pathway were present after growth on pyruvate, and genome analysis showed the presence of the corresponding structural genes. The bacteria contain, in addition, pyruvate oxidase activity which is induced under microoxic conditions. Other homo- or heterofermentative lactic acid bacteria showed only low pyruvate fermentation activity.     

Utilization of aminoaromatic acids by a methanogenic enrichment culture and by a novel<Emphasis Type="Italic"> Citrobacter freundii</Emphasis> strain

Following incubation of mesophilic methanogenic floccular sludge from a lab-scale upflow anaerobic sludge bed reactor used to treat cattle manure wastewater, a stable 5-aminosalicylate-degrading enrichment culture was obtained. Subsequently, a Citrobacter freundii strain, WA1, was isolated from the 5-aminosalicylate-degrading methanogenic consortium. The methanogenic enrichment culture degraded 5-aminosalicylate completely to CH₄, CO₂ and NH₄ ⁺, while C. freundii strain WA1 reduced 5-aminosalicylate with simultaneous deamination to 2-hydroxybenzyl alcohol during anaerobic growth with electron donors such as pyruvate, glucose or serine. When grown on pyruvate, C. freundii WA1 converted 3-aminobenzoate to benzyl alcohol and also reduced benzaldehyde to benzyl alcohol. Pyruvate was fermented to acetate, CO₂, H₂ and small amounts of lactate, succinate and formate. Less lactate (30%) was produced from pyruvate when C. freundii WA1 grew with 5-aminosalicylate as co-substrate.     

Complete oxidation of catechol by the strictly anaerobic sulfate-reducing Desulfobacterium catecholicum sp. nov.

From an anaerobic enrichment culture with vanillate as substrate, a catechol-degrading lemon-shaped nonsporing sulfate-reducing bacterium, strain NZva20, was isolated in pure culture. Growth occurred in defined, bicarbonate-buffered, sulfide-reduced freshwater medium with catechol as sole electron donor and carbon source. Catechol was completely oxidized to CO₂ with an average growth yield of 31 g cell dry mass per mol of catechol, corresponding to 9.5 g cell dry mass per mol of sulfate reduced. Further substrates utilized as electron donors and carbon sources were resorcinol, hydroquinone, benzoate and several other aromatic compounds, hydrogen plus carbon dioxide, formate, lactate, pyruvate, alcohols including methanol, dicarboxylic acids, acetate, propionate and higher fatty acids up to 18 carbon atoms. Instead of sulfate, sulfite, thiosulfate, dithionite or nitrate served as electron acceptors. Nitrate was reduced to ammonium. Strain NZva20 is the first bacterium in which the complete oxidation of organic substrates is linked to the ammonification of nitrate. Elemental sulfur was not utilized as electron acceptor. In the absence of an electron acceptor slow growth occurred on pyruvate or fumarate. The G+C content of the DNA of strain NZva20 was 52.4 mol%. Cytochromes were present. Desulfoviridin could not be detected. Strain NZva20 is described as type strain of a new species, Desulfobacterium catecholicum sp. nov.Affectionately dedicated to Professor Ralph S. Wolfe on the occassion of his 65th birthday     

Pyruvate formate lyase and acetate kinase are essential for anaerobic growth of Escherichia coli on xylose

During anaerobic growth of bacteria, organic intermediates of metabolism, such as pyruvate or its derivatives, serve as electron acceptors to maintain the overall redox balance. Under these conditions, the ATP needed for cell growth is derived from substrate-level phosphorylation. In Escherichia coli, conversion of glucose to pyruvate yields 2 net ATPs, while metabolism of a pentose, such as xylose, to pyruvate only yields 0.67 net ATP per xylose due to the need for one (each) ATP for xylose transport and xylulose phosphorylation. During fermentative growth, E. coli produces equimolar amounts of acetate and ethanol from two pyruvates, and these reactions generate one additional ATP from two pyruvates (one hexose equivalent) while still maintaining the overall redox balance. Conversion of xylose to acetate and ethanol increases the net ATP yield from 0.67 to 1.5 per xylose. An E. coli pfl mutant lacking pyruvate formate lyase cannot convert pyruvate to acetyl coenzyme A, the required precursor for acetate and ethanol production, and could not produce this additional ATP. E. coli pfl mutants failed to grow under anaerobic conditions in xylose minimal medium without any negative effect on their survival or aerobic growth. An ackA mutant, lacking the ability to generate ATP from acetyl phosphate, also failed to grow in xylose minimal medium under anaerobic conditions, confirming the need for the ATP produced by acetate kinase for anaerobic growth on xylose. Since arabinose transport by AraE, the low-affinity, high-capacity, arabinose/H+ symport, conserves the ATP expended in pentose transport by the ABC transporter, both pfl and ackA mutants grew anaerobically with arabinose. AraE-based xylose transport, achieved after constitutively expressing araE, also supported the growth of the pfl mutant in xylose minimal medium. These results suggest that a net ATP yield of 0.67 per pentose is only enough to provide for maintenance energy but not enough to support growth of E. coli in minimal medium. Thus, pyruvate formate lyase and acetate kinase are essential for anaerobic growth of E. coli on xylose due to energetic constraints.     

Physiological characterization of strain DCB-1, a unique dehalogenating sulfidogenic bacterium

Strain DCB-1 is an obligately anaerobic bacterium which carries out the reductive dehalogenation of halobenzoates and was previously known to grow only on pyruvate plus 20% ruminal fluid. When various electron acceptors were supplied, thiosulfate and sulfite were found to stimulate growth. Sulfide was produced from thiosulfate. Cytochrome c and desulfoviridin were detected. The mol% G+C was 49 (at the thermal denaturation temperature). Of 55 carbon sources tested, only pyruvate supported growth as the sole carbon source in mineral medium. Lactate, acetate, L- and D-malate, glycerol, and L- and D-arabinose stimulated growth when supplemented with 10% ruminal fluid and 20 mM thiosulfate. In mineral medium, pyruvate was converted to acetate and lactate, with small amounts of succinate and fumarate accumulating transiently. During growth with thiosulfate, all of these products accumulated transiently. Addition of excess hydrogen to pyruvate-grown cultures resulted in diversion of carbon to formate, lactate, and butyrate, which caused a decrease in cell yield. We conclude that strain DCB-1 is a new type of sulfidogenic bacterium.     

Physiological characterization of strain DCB-1, a unique dehalogenating sulfidogenic bacterium.

Strain DCB-1 is an obligately anaerobic bacterium which carries out the reductive dehalogenation of halobenzoates and was previously known to grow only on pyruvate plus 20% ruminal fluid. When various electron acceptors were supplied, thiosulfate and sulfite were found to stimulate growth. Sulfide was produced from thiosulfate. Cytochrome c and desulfoviridin were detected. The mol% G+C was 49 (at the thermal denaturation temperature). Of 55 carbon sources tested, only pyruvate supported growth as the sole carbon source in mineral medium. Lactate, acetate, L- and D-malate, glycerol, and L- and D-arabinose stimulated growth when supplemented with 10% ruminal fluid and 20 mM thiosulfate. In mineral medium, pyruvate was converted to acetate and lactate, with small amounts of succinate and fumarate accumulating transiently. During growth with thiosulfate, all of these products accumulated transiently. Addition of excess hydrogen to pyruvate-grown cultures resulted in diversion of carbon to formate, lactate, and butyrate, which caused a decrease in cell yield. We conclude that strain DCB-1 is a new type of sulfidogenic bacterium.     

A Desulfitobacterium strain isolated from human feces that does not dechlorinate chloroethenes or chlorophenols

An anaerobic bacterium, strain DP7, was isolated from human feces in mineral medium with formate and 0.02% yeast extract as energy and carbon source. This rod-shaped motile bacterium used pyruvate, lactate, formate, hydrogen, butyrate, and ethanol as electron donor for sulfite reduction. Other electron acceptors such as thiosulfate, nitrate and fumarate stimulated growth in the presence of 0.02% yeast extract and formate. Acetate was the only product during fermentative growth on pyruvate. Six mol of pyruvate were fermented to 7 mol of acetate. 13C-NMR labeling experiments showed homoacetogenic 13C-CO2 incorporation into acetate. The pH and temperature optimum of fermentative growth on pyruvate was 7.4 and 37 degrees C, respectively. The growth rate under these conditions was approximately 0.10 h(-1). Strain DP7 was identified as a new strain of Desulfitobacterium frappieri on the basis of 16S rRNA sequence analysis (99% similarity) and DNA-DNA hybridization (reassociation value of 83%) with Desulfitobacterium frappieri TCE1. In contrast to described Desulfitobacterium strains, the newly isolated strain has not been isolated from a polluted environment and did not use chloroethenes or chlorophenols as electron acceptor.     

Biochemical route and control of butyrate synthesis in Butyribacterium methylotrophicum

 Butyribacterium methylotrophicum produced more butyrate when grown on lactate than when grown on glucose, and only acetate was detected during growth on pyruvate. Higher levels of NADH were found in butyrate-producing than in acetate-producing cells. The addition of neutral red, an electron-flow modulator, to cells growing on pyruvate altered the carbon and electron flow from acetate plus H₂ synthesis to butyrate synthesis. Enzymatic analysis suggested that pyruvate was produced from glucose via an Embden-Meyerhof-Parnas pathway. Pyruvate was further metabolized to butyryl-CoA via, β-hydroxybutyryl-CoA and butyryl-CoA dehydrogenases. Lactate dehydrogenase, unlike butyryl-CoA dehydrogenase, was inducible and detected only in lactate-grown cells. Both of these dehydrogenases utilized 2,6-dichloroindophenol and other artificial electron acceptors but not NAD(P). Ferredoxin–NAD oxidoreductase levels were highest in lactate and lowest in pyruvate-grown cells. Cells contained both a ferredoxin–neutral-red reductase activity and a neutral-red–NAD reductase activity that coupled electron flow to butyrate synthesis. These results showed that butyrate synthesis by B. methylotrophicum was regulated by the carbon source and was dependent on the cellular NADH/NAD ratios, and the levels and direction of ferredoxin- and NAD-linked oxidoreductases. Received: 3 August 1995/Received revision: 31 October 1995/Accepted: 10 November 1995     

End-product induced metabolic shifts in <Emphasis Type="Italic">Clostridium thermocellum</Emphasis> ATCC 27405

When attempting to increase yields of desirable end-products during fermentation, there is the possibility that increased concentrations of one product redirects metabolism towards the synthesis of less desired products. Changes in growth, final end-product concentrations, and activities of enzymes involved in pyruvate catabolism and fermentative end-product formation were studied in Clostridium thermocellum in response to the addition of individual end-products (H₂, acetate, ethanol, formate, and lactate) to the growth medium. These were added to the growth medium at concentrations ten times greater than those found at the end of growth in cultures grown under carbon-limited conditions using cellobiose (1.1 g l⁻¹) as model soluble substrate. Although growth rate and final cell biomass decreased significantly with the addition of all end-products, addition of individual end-products had less pronounced effects on growth. Metabolic shifts, represented by changes in final end-product concentrations, were observed; H₂ and acetate yields increased in the presence of exogenous ethanol and lactate, while ethanol yields increased in the presence of exogenous hydrogen (H₂), acetate, and lactate. Late exponential phase enzyme activity data of enzymes involved in pyruvate catabolism and end-product formation revealed no changes in enzyme levels greater than 2-fold in response to the presence of any given end-product, with the exception of pyruvate:formate lyase (PFL), ferredoxin-dependent hydrogenase (Fd-H₂ase), and pyruvate:ferredoxin oxidoreductase (PFO): PFL and Fd-H₂ase activities increased 2-fold in the presence of ethanol, while PFO activity decreased by 57% in the presence of sodium formate. Changes in enzyme levels did not necessarily correlate with changes in final end-product yields, suggesting that changes in final end-product yields may be governed by thermodynamic considerations rather than levels of enzyme expressed under the conditions tested. We demonstrate that bacterial metabolism may be manipulated in order to selectively improve desired product yields.     

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.