Formation of formate and hydrogen, and flux of reducing equivalents and carbon in Ruminococcus flavefaciens Fd-1

A pathway for conversion of the metabolic intermediate phosphoenolpyruvate (PEP) and the formation of acetate, succinate, formate, and H₂ in the anaerobic cellulolytic bacterium Ruminococcus flavefaciens FD-1 was constructed on the basis of enzyme activities detected in extracts of cells grown in cellulose- or cellobiose-limited continuous culture. PEP was converted to acetate and CO₂ (via pyruvate kinase, pyruvate dehydrogenase, and acetate kinase) or carboxylated to form succinate (via PEP carboxykinase, malate dehydrogenase, fumarase, and fumarate reductase). Lactate was not formed even during rapid growth (batch culture, µ = 0.35/h). H₂ was formed by a hydrogenase rather than by cleavage of formate, and ¹³C-NMR and¹⁴ C-exchange reaction data indicated that formate was produced by CO₂ reduction, not by a cleavage of pyruvate. The distribution of PEP into the acetate and succinate pathways was not affected by changing extracellular pH and growth rates within the normal growth range. However, increasing growth rate from 0.017/h to 0.244/h resulted in a shift toward formate production, presumably at the presence of H₂. This shift suggested that reducing equivalents could be balanced through formate or H₂ production without affecting the yields of the major carbon-containing fermentation endproducts.     

Anaerobic degradation of sorbic acid by sulfate-reducing and fermenting bacteria: pentanone-2 and isopentanone-2 as byproducts

Strictly anaerobic bacteria were enriched and isolated from freshwater sediment sources in the presence and absence of sulfate with sorbic acid as sole source of carbon and energy. Strain WoSo1, a Gram-negative vibrioid sulfate-reducing bacterium which was assigned to the species Desulfoarculus (formerly Desulfovibrio) baarsii oxidized sorbic acid completely to CO₂ with concomitant stoichiometric reduction of sulfate to sulfide. This strain also oxidized a wide variety of fatty acids and other organic compounds. A Gram-negative rod-shaped fermenting bacterium, strain AmSo1, fermented sorbic acid stoichiometrically to about equal amounts of acetate and butyrate. At concentrations higher than 10 mM, sorbic acid fermentation led to the production of pentanone-2 and isopentanone-2 (3-methyl-2-butanone) as byproducts. Strain AmSo1 fermented also crotonate and 3-hydroxybutyrate to acetate and butyrate, and hexoses to acetate, ethanol, hydrogen, and formate. The guanine-plus-cytosine content of the DNA was 41.8±1.0 mol%. Sorbic acid at concentrations higher than 5 mM inhibited growth of this strain while strain WoSo1 tolerated sorbic acid up to 10 mM concentration.     

Influence of Metronidazole, CO, CO2, and Methanogens on the Fermentative Metabolism of the Anaerobic Fungus Neocallimastix sp. Strain L2

The effects of metronidazole, CO, methanogens, and CO₂ on the fermentation of glucose by the anaerobic fungus Neocallimastix sp. strain L2 were investigated. Both metronidazole and CO caused a shift in the fermentation products from predominantly H₂, acetate, and formate to lactate as the major product and caused a lower glucose consumption rate and cell protein yield. An increased lactate dehydrogenase activity and a decreased hydrogenase activity were observed in cells grown under both culture conditions. In metronidazole-grown cells, the amount of hydrogenase protein was decreased compared with the amount in cells grown in the absence of metronidazole. When Neocallimastix sp. strain L2 was cocultured with the methanogenic bacterium Methanobrevibacter smithii, the fermentation pattern changed in the opposite direction: H₂ and acetate production increased at the expense of the electron sink products lactate, succinate, and ethanol. A concomitant decrease in the enzyme activities leading to these electron sink products was observed, as well as an increase in the glucose consumption rate and cell protein yield, compared with those of pure cultures of the fungus. Low levels of CO₂ in the gas phase resulted in increased H₂ and lactate formation and decreased production of formate, acetate, succinate, and ethanol, a decreased glucose consumption rate and cell protein yield, and a decrease in most of the hydrogenosomal enzyme activities. None of the tested culture conditions resulted in changed quantities of hydrogenosomal proteins. The results indicate that manipulation of the pattern of fermentation in Neocallimastix sp. strain L2 results in changes in enzyme activities but not in the proliferation or disappearance of hydrogenosomes.     

Pentose metabolism byBacteroides ruminicola subsp.brevis strain B14

The fermentation ofd-arabinose byBacteroides ruminicola strain B₁4 occurs in a manner similar to or identical with that shown previously forl-arabinose metabolism by the organism, a combination of hexose resynthesis and the Embden-Meyerhof sequence. The use ofd-arabinose by strain B₁4 was repressed by prior growth in medium containingd-glucose and induced by prior growth in the presence ofl-arabinose ord-xylose. The use ofd-ribose andd-xylose by strain B₁4 is different from that ford-arabinose. During growth in the presence of 1-14C-d-arabinose, labeled acetate, propionate, and succinate were formed, whereas during 1-14C-d-ribose growth only labeled acetate and propionate were obtained. Under the conditions used,d-xylose growth failed to allow formation of acetate, propionate, or succinate. Strain B₁4 incorporates label from 1- or 2-labeled glycine into acetate, propionate, and succinate by a mechanism involving the cleavage of glycine and equilibration of glycine carbons 1 and 2 with different metabolic pools.     

Methanol utilizing <Emphasis Type="Italic">Desulfotomaculum</Emphasis> species utilizes hydrogen in a methanol-fed sulfate-reducing bioreactor

A sulfate-reducing bacterium, strain WW1, was isolated from a thermophilic bioreactor operated at 65°C with methanol as sole energy source in the presence of sulfate. Growth of strain WW1 on methanol or acetate was inhibited at a sulfide concentration of 200 mg l⁻¹, while on H₂/CO₂, no apparent inhibition occurred up to a concentration of 500 mg l⁻¹. When strain WW1 was co-cultured under the same conditions with the methanol-utilizing, non-sulfate-reducing bacteria, Thermotoga lettingae and Moorella mulderi, both originating from the same bioreactor, growth and sulfide formation were observed up to 430 mg l⁻¹. These results indicated that in the co-cultures, a major part of the electron flow was directed from methanol via H₂/CO₂ to the reduction of sulfate to sulfide. Besides methanol, acetate, and hydrogen, strain WW1 was also able to use formate, malate, fumarate, propionate, succinate, butyrate, ethanol, propanol, butanol, isobutanol, with concomitant reduction of sulfate to sulfide. In the absence of sulfate, strain WW1 grew only on pyruvate and lactate. On the basis of 16S rRNA analysis, strain WW1 was most closely related to Desulfotomaculum thermocisternum and Desulfotomaculum australicum. However, physiological properties of strain WW1 differed in some aspects from those of the two related bacteria.     

The new facultatively chemolithoautotrophic, moderately halophilic, sulfate-reducing bacterium Desulfovermiculus halophilus gen. nov., sp. nov., isolated from an oil field

The new mesophilic, chemolithoautotrophic, moderately halophilic, sulfate-reducing bacterium strain 11-6, could grow at a NaCl concentration in the medium of 30–230 g/l, with an optimum at 80–100 g/l. Cells were vibrios motile at the early stages of growth. Lactate, pyruvate, malate, fumarate, succinate, propionate, butyrate, crotonate, ethanol, alanine, formate, and H₂/CO₂ were used in sulfate reduction. Butyrate was degraded completely, without acetate accumulation. In butyrate-grown cells, a high activity of CO dehydrogenase was detected. Additional growth factors were not required. Autotrophic growth occurred, in the presence of sulfate, on H₂/CO₂ or formate without other electron donors. Fermentation of pyruvate and fumarate was possible in the absence of sulfate. Apart from sulfate, sulfite, thiosulfate, and elemental sulfur were able to serve as electron acceptors. The optimal growth temperature was 37°C; the optimum pH was 7.2. Desulfoviridin was not detected. Menaquinone MK-7 was present. The DNA G+C content was 55.2 mol %. Phylogenetically, the bacterium represented a separate branch within the cluster formed by representatives of the family Desulfohalobiaceae in the class Deltaproteobacteria. The bacterium was assigned to a new genus and species, Desulfovermiculus halophilus gen. nov., sp. nov. The type strain is 11-6^T (= VKM B-2364), isolated from the highly mineralized formation water of an oil field.     

d-Pentose metabolism byBacteroides vulgatus strain 8482

Bacteroides vulgatus strain 8482 metabolizedd-arabinose by a mechanism involving a 32 (top to bottom) cleavage of the arabinose carbon skeleton. During growth in the presence of 1-¹⁴C-d-arabinose, acetate, propionate, and succinate were labeled, but during growth in the presence of 5-labeledd-arabinose, only labeled acetate and succinate were formed. The metabolism ofd-ribose by strain 8482 differed from that ford-arabinose. Strain 8482 converted glycolic acid and glycine to acetate and succinate, but not propionate, by a mechanism involving cleavage of the glycine and glycolic acid carbon skeletons and equilibration of carbons 1 and 2 of glycolic acid and glycine with nonequivalent metabolic pools. The metabolism ofd-arabinose,d-ribose,d-glycine, andd-glycolic acid by strain 8482 was similar, in some respects, to that ofBacteroides fragilis strain 2044, but differed substantially from the metabolism of the same substances byBacteroides ruminicola strain B₁4.     

Succinic acid production by <Emphasis Type="Italic">Actinobacillus succinogenes</Emphasis> from batch fermentation of mixed sugars

Succinic acid production from the monosaccharides xylose, arabinose, glucose, mannose and galactose was studied using the bacterium Actinobacillus succinogenes. In Duran bottle cultures, containing 10 g/L of each of sugar, succinic acid was produced from all sugars except for galactose. The highest succinate yield, 0.56 g/g, was obtained with glucose, whereas the succinate yield was 0.42, 0.38 and 0.44 g/g for xylose, mannose and arabinose, respectively. The specific succinate productivity was 0.7 g/g h for glucose, but below 0.2 g/g h for the other sugars. Batch bioreactor fermentations were carried out using a sugar mixture of the five sugars giving a total concentration of 50 g/L, mimicking the distribution of sugars in spent sulfite liquor (SSL) from Eucalyptus which is rich in xylose. In this mixture, an almost complete conversion of all sugars (except galactose) was achieved resulting in a final succinate concentration of 21.8–26.8 g/L and a total yield of 0.59–0.68 g/g. There was evidence of co-consumption of glucose and xylose, whereas mannose was consumed after glucose. The main by-products were acetate 0.14–0.20 g/g and formate 0.08–0.13 g/g. NADH balance calculations suggested that NADH required for succinate production was not met solely from formate and acetate production, but other means of NADH production was necessary. Results from mixed sugar fermentations were verified using SSL as substrate resulting in a succinate yield of 0.60 g/g. In addition, it was found that CO₂ sparging could replace carbonate supply in the form of MgCO₃ without affecting the succinate yield.     

Propionate formation from alcohols or aldehydes by Desulfobulbus propionicus in the absence of sulfate

Fermentative degradation of alcohols and aldehydes in the absence of sulfate was investigated using a propionate-oxidizing, sulfate-reducing bacterium, Desulfobulbus propionicus strain MUD (DSM 6523). The organism converted ethanol plus CO₂ to acetate and propionate. The conversion was not affected by the presence of hydrogen. Strain MUD converted propanol plus acetate to propionate. Acetaldehyde and propionaldehyde were also converted with a dismutation reaction in the absence of sulfate. The products were propionate and acetate from acetaldehyde, and propionate from propionaldehyde plus acetate.     

Betaine Fermentation and Oxidation by Marine Desulfuromonas Strains

Two bacterial strains were dominant in anaerobic enrichment cultures with betaine (N,N,N-trimethylglycine) as a substrate and intertidal mud as an inoculum. One was a coccoid bacterium which was a trimethylamine (TMA)-fermenting methanogen similar to Methanococcoides methylutens. The other strain, a rod-shaped, gram-negative, motile bacterium, fermented betaine. On the basis of its ability to oxidize acetate and ethanol to CO₂ with sulfur as an electron acceptor, its inability to reduce sulfate and sulfite, its morphology, the presence of c-type cytochromes, and other characteristics, the isolated strain PM1 was identified as Desulfuromonas acetoxidans. Although only malate and fumarate were known as substrates for fermentative growth of this species, the type strain (DSM 684) also fermented betaine. Strain PM1 grew with a doubling time of 9.5 h at 30°C on betaine and produced approximately 1 mol of TMA per mol of betaine, 0.75 mol of acetate, and presumably CO₂ as fermentation products but only in the presence of selenite (100 nM). In this fermentation, betaine is probably reductively cleaved to TMA and acetate, and part of the acetate is then oxidized to CO₂ to provide the reducing equivalents for the initial cleavage reaction. In the presence of sulfur, betaine was converted to TMA and presumably CO₂ with the formation of sulfide; then, only traces of acetate were produced.     

d-arabinose metabolism byBacteroides fragilis strain 2044

During growth in the presence of 1-14C-d-arabinose,Bacteroides fragilis strain 2044 formed labeled succinic, acetic, and propionic acis. Degradation of the acids by the Schmidt reaction revealed that at least 89% of the succinate radioactivity was found in the methylene carbons and that 75% and 84% of the label in propionate and acetate were found in the noncarboxyl carbons of these molecules. No label was found in acetate, propionate, or succinate during growth of strain 2044 in the presence of 5-3H-d-arabinose. Strain 2044 converted radioactivity from 1- or 2-labeled glycolic acid and glycine to succinate by a mechanism involving cleavage of the glycine and glycolic acid carbon skeletons. Label from 1- or 2-labeled glycine and 2 but not 1-labeled glycolic acid was found in acetate. Uniformly labeled 14C-glyoxalate gave rise to labeled acetate, but not succinate.Bacteroides fragilis strain 2044 metabolizesd-arabinose by a mechanism involving a 32 cleavage of the molecule.     

Engineering of Acetate Recycling and Citrate Synthase to Improve Aerobic Succinate Production in Corynebacterium glutamicum

Corynebacterium glutamicum lacking the succinate dehydrogenase complex can produce succinate aerobically with acetate representing the major byproduct. Efforts to increase succinate production involved deletion of acetate formation pathways and overexpression of anaplerotic pathways, but acetate formation could not be completely eliminated. To address this issue, we constructed a pathway for recycling wasted carbon in succinate-producing C. glutamicum. The acetyl-CoA synthetase from Bacillus subtilis was heterologously introduced into C. glutamicum for the first time. The engineered strain ZX1 (pEacsA) did not secrete acetate and produced succinate with a yield of 0.50 mol (mol glucose)⁻¹. Moreover, in order to drive more carbon towards succinate biosynthesis, the native citrate synthase encoded by gltA was overexpressed, leading to strain ZX1 (pEacsAgltA), which showed a 22% increase in succinate yield and a 62% decrease in pyruvate yield compared to strain ZX1 (pEacsA). In fed-batch cultivations, strain ZX1 (pEacsAgltA) produced 241 mM succinate with an average volumetric productivity of 3.55 mM h⁻¹ and an average yield of 0.63 mol (mol glucose) ⁻¹, making it a promising platform for the aerobic production of succinate at large scale.     

Anaerobic degradation of methoxylated aromatic compounds by Clostridium methoxybenzovorans and a nitrate-reducing bacterium Thauera sp. strain Cin3,4

The coupling of growth of the o-demethylating bacterium, Clostridium methoxybenzovorans SR3, with a nitrate-reducing bacterium able to degrade aromatic compounds, Thauera sp. Cin3,4, allowed complete mineralization of poorly oxidizable methoxylated aromatic compounds such as vanillate, isovanillate, vanilline, anisate, ferulate and veratrate. C. methoxybenzovorans o-demethylated these aromatic compounds to their corresponding hydroxylated derivatives and fermented the side chains to acetate and butyrate. The hydroxylated compounds and the fermentation end-products in the C. methoxybenzovorans spent growth medium were then completely metabolized to CO₂ on inoculation with the Thauera strain. Kinetic studies with veratrate indicated that C. methoxybenzovorans initially o-demethylated the substrate to vanillate and then further to protocatechuate together with the production of acetate and butyrate from the demethylated side chains. Protocatechuate, acetate and butyrate were then utilized as a carbon source by the Thauera strain aerobically or anaerobically in the presence of nitrate. The results therefore suggest that mono- or dimethoxylated aromatic compounds can be completely mineralized by coupling the growth of a fermentative bacterium with a nitrate-reducing bacterium, and a metabolic pathway for this is proposed.     

Ferribacterium limneticum, gen. nov., sp. nov., an Fe(III)-reducing microorganism isolated from mining-impacted freshwater lake sediments

A dissimilatory Fe(III)-reducing bacterium was isolated from mining-impacted lake sediments and designated strain CdA-1. The strain was isolated from a 4-month enrichment culture with acetate and Fe(III)-oxyhydroxide. Strain CdA-1 is a motile, obligately anaerobic rod, capable of coupling the oxidation of acetate and other organic acids to the reduction of ferric iron. Fe(III) reduction was not observed using methanol, ethanol, isopropanol, propionate, succinate, fumarate, H₂, citrate, glucose, or phenol as potential electron donors. With acetate as an electron donor, strain CdA-1 also grew by reducing nitrate or fumarate. Growth was not observed with acetate as electron donor and O₂, sulfoxyanions, nitrite, trimethylamine N-oxide, Mn(IV), As(V), or Se(VI) as potential terminal electron acceptors. Comparative 16 S rRNA gene sequence analyses show strain CdA-1 to be most closely related (93.6% sequence similarity) to Rhodocyclus tenuis. However, R. tenuis did not grow heterotrophically by Fe(III) reduction, nor did strain CdA-1 grow photrophically. We propose that strain CdA-1 represents a new genus and species, Ferribacterium limneticum. Strain CdA-1 represents the first dissimilatory Fe(III) reducer in the β subclass of Proteobacteria, as well as the first Fe(III) reducer isolated from mine wastes. Received: 14 July 1998 / Accepted: 14 December 1998     

Corynebacterium glutamicum,a natural overproducer of succinic acid?

Corynebacterium glutamicum is well known as an important industrial amino acid producer. For a few years, its ability to produce organic acids, under micro‐aerobic or anaerobic conditions was demonstrated. This study is focused on the identification of the culture parameters influencing the organic acids production and, in particular, the succinate production, by this bacterium. Corynebacterium glutamicum 2262, used throughout this study, was a wild‐type strain, which was not genetically designed for the production of succinate. The oxygenation level and the residual glucose concentration appeared as two critical parameters for the organic acids production. The maximal succinate concentration (4.9 g L^?1) corresponded to the lower k_La value of 5 h^?1. Above 5 h^?1, a transient accumulation of the succinate was observed. Interestingly, the stop in the succinate production was concomitant with a lower threshold glucose concentration of 9 g L^?1. Taking into account this threshold, a fed‐batch culture was performed to optimize the succinate production with C. glutamicum 2262. The results showed that this wild‐type strain was able to produce 93.6 g L^?1 of succinate, which is one of the highest concentration reported in the literature.     

Beneckea gazogenes sp. nov., a red,facultatively anaerobic,marine bacterium

A red, facultatively anaerobic marine bacterium was isolated from samples of saltwater marsh mud. The Gram-negative microorganism was rod-shaped and possessed a single sheathed polar flagellum. The red pigment that the cells produced was identified as prodigiosin. THe bacteria grew readily on synthetic seawater media containing carbohydrates, amino acids, organic acids, or tricarboxylic acid cycle intermediates as sole sources of carbon and energy. Na⁺ was required for growth. Cells fermented glucose to CO₂, H₂, ethanol, acetate, succinate, and pyruvate. The guanine-plus-cytosine content of the strain’s DNA was 47.1 mole%. The morphological and physiological characteristics of this bacterium, together with the mole % G+C of its DNA, place it in the genusBeneckea. The main features of this strain are sufficiently different from previously described members of the genus to place it in a new species for which the nameBeneckea gazogenes is proposed.     

Hydrogen Gas Production by an Ectothiorhodospira vacuolata Strain

A hydrogen gas (H₂)-producing strain of Ectothiorhodospira vacuolata isolated from Soap Lake, Washington, possessed nitrogenase activity. Increasing evolution of H₂ with decreasing ammonium chloride concentrations provided evidence that nitrogenase was the catalyst in gas production. Cells were grown in a mineral medium plus 0.2% acetate with sodium sulfide as an electron donor. Factors increasing H₂ production included addition of reduced carbon compounds such as propionate and succinate, increased reducing power by increasing sodium sulfide concentrations, and increased energy charge (ATP) by increasing light intensity.     

Acetic acid from H2 and CO2

Cell extracts of a nonsporeforming strictly anaerobic bacterium, Acetobacterium woodii produced acetate in N-tris(Hydroxymethyl)methyl-2-aminoethane sulfonic acid or phosphate buffers from hydrogen and carbon dioxide. The formation of acetate was not dependent on the presence of ATP in the reaction mixture; ADP also did not influence the acetate production. Since acetic acid is the main fermentation product during growth of A. woodii with H₂ and CO₂, ATP must be synthesized in the course of acetate formation. The possible sites of ATP synthesis are discussed.     

Anaerobic Degradation of Propionate by a Mesophilic Acetogenic Bacterium in Coculture and Triculture with Different Methanogens

A mesophilic acetogenic bacterium (MPOB) oxidized propionate to acetate and CO₂ in cocultures with the formate- and hydrogen-utilizing methanogens Methanospirillum hungatei and Methanobacterium formicicum. Propionate oxidation did not occur in cocultures with two Methanobrevibacter strains, which grew only with hydrogen. Tricultures consisting of MPOB, one of the Methanobrevibacter strains, and organisms which are able to convert formate into H₂ plus CO₂ (Desulfovibrio strain G11 or the homoacetogenic bacterium EE121) also degraded propionate. The MPOB, in the absence of methanogens, was able to couple propionate conversion to fumarate reduction. This propionate conversion was inhibited by hydrogen and by formate. Formate and hydrogen blocked the energetically unfavorable succinate oxidation to fumarate involved in propionate catabolism. Low formate and hydrogen concentrations are required for the syntrophic degradation of propionate by MPOB. In triculture with Methanospirillum hungatei and the aceticlastic Methanothrix soehngenii, propionate was degraded faster than in biculture with Methanospirillum hungatei, indicating that low acetate concentrations are favorable for propionate oxidation as well.     

Aerobiosis–anaerobiosis transition has a significant impact on organic acid production by Corynebacterium glutamicum

Corynebacterium glutamicum is known to produce organic acids under anaerobic culture conditions, in particular, lactic, succinic, and acetic acids. Our study is focused on acetic and succinic acid production using a lactate dehydrogenase-deficient strain of C. glutamicum. Usually, with this bacterium, the organic acid production process is based on an initial aerobic growth phase, followed by a rapid deoxygenation and an anaerobic production phase. In our study, we demonstrated that this strategy was unfavorable for the production of organic acids. Conversely, we showed that applying the best transition strategy based on progressive deoxygenation significantly increased the concentration of organic acids up to 640%. This was observed either by applying controlled dissolved oxygen concentrations or by decreasing the steps of gas flow rates. Our results also showed that applying constant oxygen transfer flux throughout the culture, and thus in the absence of the anaerobic phase, promoted constant production yields (approximately 0.5 mol of succinate or acetate per mole of glucose). In this case, acetate production (120 mM) was favored over succinate production (132 mM), resulting in a decrease in the molar ratio of products (succinate/acetate) from 4.8 to 1.1 between progressive deoxygenation and constant OTR cultures.