Thermodynamics of hydrogen metabolism in methanogenic cocultures degrading ethanol or lactate

Pure cultures of Desulfovibrio vulgaris or Pelobacter acetylenicus do not grow with lactate or ethanol, respectively, under obligately proton- reducing conditions. However, a small part of these substrates was oxidized and molecular hydrogen was produced up to 4.2 and 3.2 kPa, respectively. During growth in syntrophic methanogenic cocultures with Methanospirillum hungatei as partner, maximum hydrogen partial pressures were significantly lower (0.7 to 2.5 kPa) than in the corresponding pure cultures. Calculation of Gibbs free energies for the prevailing culture conditions showed that H₂ partial pressures were kept in a range at which both, H₂-producing and H₂-consuming reactions, were thermodynamically permissive in pure as well as in syntrophic mixed cultures.     

Thermodynamics of H2-consuming and H2-producing metabolic reactions in diverse methanogenic environments under in situ conditions

Abstract In situ concentrations of hydrogen and other metabolites involved in H₂-consuming and H₂-producing reactions were measured in anoxic methanogenic lake sediments, sewage sludge and fetid liquid of cottonwood. The data were used to calculate the Gibbs free energies of the metabolic reactions under the conditions prevailing in situ. The thermodynamics of most of the reactions studied were exergonic with Gibbs free energies being more negative for H₂-dependent sulfate reduction methanogenesis acetogenesis and for H₂-producing lactate fermentation ethanol fermentation. Butyrate and propionate fermentation, on the other hand, were endergonic under in situ conditions. This observation is interpreted by suggesting that butyrate and propionate is degraded within microbial clusters which shield the fermentating bacteria from the outside H₂ (and acetate) pool.     

Acetogenesis from H2 and CO2 by methane- and non-methane-producing human colonic bacterial communities

Abstract: The purpose of the study was to define the potential for reductive acetogenesis of colonic microflora from six non-methane- and four methane-excreting human subjects in relation to numbers of the different H₂-utilizing microorganisms. Faecal bacterial suspensions were incubated in the presence of NaH¹³CO₃ and under a gas phase composed of either 100% N₂ (control) or 80% H₂–20% N₂. The effects of a specific methanogenesis inhibitor or of sulfate supplementation were also determined. Quantitative nuclear magnetic resonance showed the presence of both single- and double-labelled acetate in all incubations under hydrogen. H₂/CO₂-acetogenesis appears to be a quantitatively important activity only in the presence of very low numbers of methanogens. Inhibition of methanogenesis induced a large increase in ¹³CO₂ incorporation into acetate in CH₄-producing samples. These results showed that methanogens can efficiently outcompete acetogens in human colonic contents. In contrast, no clear-cut competition for H₂ between acetogenesis and dissimilatory sulfate-reduction could be demonstrated. A slight reduction of the acetogenic activity was only observed at the highest sulfate addition (100 mM).     

Taxonomic identification of Streptomyces exfoliatus K10 and characterization of its poly(3-hydroxybutyrate) depolymerase gene

Abstract Using fungi grown on synthetic agar medium, we evaluated and compared the concentration of various H₂O₂-producing enzymes. Our results showed that oxidase production in solid medium was better than that found in liquid medium and as high as that detected in wood samples. High yields of oxidases made it possible to compare different oxidases in the same culture extracts and under different conditions. Our results also indicated that H₂O₂ production is ubiquitous in the white rot fungi tested and that enzyme levels are influenced by the substrate composition.     

Most probable number enumeration of H2-utilizing acetogenic bacteria from the digestive tract of animals and man

Abstract A method is proposed that allows the enrichment and most probable number estimation of H₂/CO₂-utilizing acetogenic bacteria. It is based on the difference in acetate production for serial dilutions incubated under either a test H₂/CO₂ (4:1), or a control N₂/CO₂ (4:1) headspace atmosphere. A nutritionally non-selective medium was used, containing bromoethane-sulfonic acid as inhibitor of methanogenic archaea and 10% pre-incubated clarified rumen fluid. Acetogenic bacteria were enumerated in rumen and hindgut contents of animals and in human feces. They ranged from below 10² to above 10⁸ per gram wet weight gut content and their population levels were the highest in the absence of methanogenesis. The method described therein should prove useful to better understand the diversity and ecological importance of dominant gut acetogens.     

Acetogenesis and acetotrophy in a hexanoate-catabolizing microbial association isolated from anoxic landfill

Metabolism of the key intermediates, acetate and hydrogen, in anaerobic hexanoate catabolism by an interacting microbial association isolated from a landfill was examined in the presence of sulphate. Hydrogen (the β-oxidation product of hexanoate and butyrate), was competitively utilized by the component sulphate-reducing bacteria whereas in the absence of sulphate an interaction between H₂-utilizing acetogenic and methanogenic bacteria facilitated H₂ removal, with acetate catabolism restricted to methanogenic bacteria. A possible mechanism for energy conservation was suggested in which excess electrons were stored as acetate for subsequent usage as an energy source.     

Formate oxidation by Wolinella recta ATCC 33238 with oxygen as electron acceptor

Abstract The formate oxidizing capacity of Wolinella recta ATCC 33238 was studied in relation to growth under anaerobic and microaerobic conditions. Three distinct activities could be recognized: (a) cyanide-insensitive H₂O₂-producing oxidation of formate; (b) peroxidation of formate (H₂O₂-consuming); (c) oxidation of formate via an electron transport chain with oxygen as the electron acceptor. The contribution of these different formate oxidizing components during the growth of W. recta was dependent on the extent of aeration. It is suggested that due to the relative increase in overall H₂O₂ formation at higher oxygen tensions growth of W. recta appears possible only under anaerobic and microaerobic conditions.     

Vaginal myeloperoxidase and flora in the pig-tailed macaque

Myeloperoxidase (MPO) is an enzyme in neutrophils and monocytes which reacts with H₂O₂ and chloride to kill microbes after phagocytosis. Instillation of MPO into the vagina may augment vaginal defenses against sexually transmitted diseases, since the normal vaginal flora is characterized by the presence of H₂O₂-producing lactobacilli. We assessed the menstrual cycle stage, vaginal flora, pH, macroscopic appearance, and endogenous MPO in the adult female pig-tailed macaque ( Macaca nemestrina ) at baseline (n=26; 60 observations) and at 0, 4, and 24 hours in untreated animals (n=6) or in animals treated with intravaginal MPO gel at time 0 (n=5). Baseline MPO levels were highly variable, and there was no detectable effect of cycle stage. In untreated animals, there was no significant effect of vaginal swab collection on vaginal flora or MPO levels. MPO treatment did not reduce vaginal H₂O₂-producing organisms, and vaginal MPO levels tended to increase at 4 hours in treated animals. Vaginal/cervical colposcopic changes were not detected in either group.     

Selective inhibitors for continuous non-axenic hydrogen production by Rhodobacter capsulatus

To produce H₂ continuously by photosynthetically grown Rhodobacter capsulatus in non-axenic anaerobic reactors, the interaction between the phototroph and possible contaminants was studied and the ecological competitiveness of the Rhodobacter spp. in nitrogen-limited conditions was determined. Experimental test runs showed that blue-green and green algae, sulphate-reducing, acetogenic and methanogenic bacteria significantly interfere with the net amounts of H₂ produced by photobacteria. Therefore, inhibitors to control the growth of those contaminants selectively were screened. By applying a combination of chloroxuron (10mg/l) and cycloheximide (10mg/l) against algae, isohumulones (30 bitterunits/l) and molyb-date (0.5g/l) against sulphate-reducing bacteria and isohumulones and chloroform (10 mg/l) against acetogens and methanogens, photoreactors could be operated in a non-axenic way and continued to produce hydrogen gas at rates depending on the feed quality varying from 333 to 676 ml H₂l reactor/d, for a period of 116d without apparent interference from other microbial contaminants. These findings have a considerable potential for facilitating the isolation of organo-phototrophs and the production of H₂ by these bacteria.     

A strict anaerobic extreme thermophilic hydrogen-producing culture enriched from digested household waste

Aims:  The aim of this study was to enrich, characterize and identify strict anaerobic extreme thermophilic hydrogen (H₂) producers from digested household solid wastes.
Methods and Results:  A strict anaerobic extreme thermophilic H₂ producing bacterial culture was enriched from a lab-scale digester treating household wastes at 70°C. The enriched mixed culture consisted of two rod-shaped bacterial members growing at an optimal temperature of 80°C and an optimal pH 8·1. The culture was able to utilize glucose, galactose, mannose, xylose, arabinose, maltose, sucrose, pyruvate and glycerol as carbon sources. Growth on glucose produced acetate, H₂ and carbon dioxide. Maximal H₂ production rate on glucose was 1·1 mmol l⁻¹ h⁻¹ with a maximum H₂ yield of 1·9 mole H₂ per mole glucose. 16S ribosomal DNA clone library analyses showed that the culture members were phylogenetically affiliated to the genera Bacillus and Clostridium. Relative abundance of the culture members, assessed by fluorescence in situ hybridization, were 87 ± 5% and 13 ± 5% for Bacillus and Clostridium , respectively.
Conclusions:  An extreme thermophilic, strict anaerobic, mixed microbial culture with H₂-producing potential was enriched from digested household wastes.
Significance and Impact of the Study:  This study provided a culture with a potential to be applied in reactor systems for extreme thermophilic H₂ production from complex organic wastes.     

Characterization of populations of aerobic hydrogen-oxidizing soil bacteria

Abstract Freshly isolated soil bacteria were screened for different characteristics of the H₂ metabolism without prior selection for growth on H₂. The bacteria were isolated from different grain size fractions of a neutral meadow cambisol and an acidic forest cambisol, and then tested (1) for the ability to oxidize H₂, (2) for chemolithoautotrophic growth on H₂ as sole electron donor and energy source, (3) for DNA-DNA-hybridization with two hydrogenase gene fragments from Alcaligenes eutrophus and Rhizobium leguminosarum , and (4) for reduction of 2,3,5-triphenyl-2H-tetrazoliumchloride (TTC) in the presence of H₂. Many (65–90%) of the isolates were able to reduce TTC, but only 30–65% were actually able to oxidize H₂ indicating that the TTC test was not a specific characteristic for H₂ oxidation ability. The TTC test was only reliable in pure cultures of known bacteria with optimized test conditions, here shown for Alcaligenes eutrophus, Bradyrhizobium japonicum and Nocardia opaca , but not in mixed cultures of unknown bacteria. Still less (< 30%) of the isolates were able to grow chemolithoautotrophically indicating that culturable aerobic bacteria with the ability for H₂ oxidation are more abundant than bacteria with the ability for chemolithoautotrophic growth. The DNA-DNA-hybridization test failed to detect many of the bacteria with H₂ oxidation activity, probably since the hydrogenase genes present in the isolates were too diverse to be all detected by the DNA probes applied.     

Effect of dilution on methanogenesis, hydrogen turnover and interspecies hydrogen transfer in anoxic paddy soil

Abstract Dilution of anoxic slurries of paddy soil resulted in a proportional decrease of the rates of total methanogenesis and the rate constants of H₂ turnover per gram soil. Dilution did not affect the fraction of H₂/CO₂-dependent methanogenesis which made up 22% of total CH₄ production. However, dilution resulted in a ten fold decrease of the H₂ steady state partial pressure from approximately 4 to 0.4 Pa indicating that H₂/CO₂-dependent methanogenesis was more or less independent of the H₂ pool. The rates of H₂ production calculated from the H₂ turnover rate constants and the H₂ steady state partial pressures accounted for only < 5% of H₂/CO₂-dependent methanogenesis in undiluted soil slurries and for even less after dilution. Upon dilution, the Gibbs free energy available for H₂/CO₂-dependent methanogenesis decreased from −28.4 to only −5.6 kJ per mol. The results indicate that methane was mainly produced from interspecies H₂ transfer within syntrophic bacterial associations and was not significantly affected by the outside H₂ pool.     

Responses of lactic acid bacteria to oxygen

Abstract A small number of flavoprotein oxidase enzymes are responsible for the direct interaction of lactic acid bacteria (LAB) with oxygen; hydrogen peroxide or water are produced in these reactions. In some cultures exposed to oxygen, hydrogen peroxide accumulates to inhibitory levels.
Through these oxidase enzymes and NADH peroxidase, O₂ and H₂O₂ can accept electrons from sugar metabolism, and thus have a sparing effect on the use of metabolic intermediates, such as pyruvate or acetaldehyde, as electron acceptors. Consequently, sugar metabolism in aerated cultures of LAB can be substantially different from that in unaerated cultures. Energy and biomass yields, end-products of sugar metabolism and the range of substrates which can be metabolised are affected.
Lactic acid bacteria exhibit an inducible oxidative stress response when exposed to sublethal levels of H₂O₂. This response protects them if they are subsequently exposed to lethal concentrations of H₂O₂. The effect appears to be related to other stress responses such as heat-shock and is similar, in some but not all respects, to that previously reported for enteric bacteria.     

The acetyl-CoA pathway of autotrophic growth

Abstract The most direct conceivable route for synthesis of multicarbon compounds from CO₂ is to join two molecules of CO₂ together to make a 2-carbon compound and then polymerize the 2-carbon compound or add CO₂ successively to the 2-carbon compound to make multicarbon compounds. Recently, it has been demonstrated that the bacterium, Clostridium thermoaceticum , grows autotrophically by such a process. The mechanism involves the reduction of one molecule of CO₂ to a methyl group and then its combination with a second molecule of CO₂ and CoA to form acetyl-CoA. We have designated this autotrophic pathway the acetyl-CoA pathway [1]. Evidence is accumulating that this pathway is utilized by other bacteria that grow with CO₂ and H₂ as the source of carbon and energy. This group includes bacteria which, like C. thermoaceticum , produce acetate as a major end product and are called acetogens or acetogenic bacteria. It also includes the methane-producing bacteria and sulfate-reducing bacteria.
The purpose of this review is to examine critically the evidence that the acetyl-CoA pathway occurs in other bacteria by a mechanism that is the same or similar to that found in C. thermoaceticum . For this purpose, the mechanism of the acetyl-CoA pathway, as found in C. thermoaceticum , is described and hypothetical mechanisms for other organisms are presented based on the acetyl-CoA pathway of C. thermoaceticum . The available data have been reviewed to determine if the hypothetical schemes are in accord with presently known facts. We conclude that the formation of acetyl-CoA by other acetogens, the methanogens and sulphate-reducing bacteria occurs by a mechanism very similar to that of C. thermoaceticum .     

Hydrogen turnover by psychrotrophic homoacetogenic and mesophilic methanogenic bacteria in anoxic paddy soil and lake sediment

Abstract The effect of temperature on CH₄ production, turnover of dissolved H₂, and enrichment of H₂-utilizing anaerobic bacteria was studied in anoxic paddy soil and sediment of Lake Constance. When anoxic paddy soil was incubated under an atmosphere of H₂/CO₂, rates of CH₄ production increased 25°C, but decreased at temperatures lower than 20°C. Chloroform completely inhibited methano-genesis in anoxic paddy soil and lake sediment, but did not or only partially inhibit the turnover of dissolved H₂, especially at low incubation temperatures. Cultures with H₂ as energy source resulted in the enrichment of chemolithotrophic homoacetogenic bacteria whenever incubation temperatures were lower than 20°C. Hydrogenotrophic methanogens could only be enriched at 30°C from anoxic paddy soil. A homoacetogen     

Soils contain two different activities for oxidation of hydrogen

Abstract Hydrogen oxidation rates were measured in a neutral compost soil and an acidic sandy loam at H₂ mixing ratios of 0.01 to 5000 ppmv. The kinetics were biphasic showing two different K _m values for H₂, one at about 10–40 nM dissolved H₂, the other at about 1.2–1.4 μM H₂. The low- K _m activity was less sensitive to chloroform fumigation than the high- K _m activity. If sterile soil was amended with Paracoccus denitrificans or a H₂-oxidizing strain isolated from compost soil, it exhibited only a high- K _m (0.7–0.9 μM) activity. It also failed to utilize H₂ mixing ratios below a threshold of 1.6–3.0 ppmv H₂ (160–300 mPa). A similar result was obtained when fresh soil samples were suspended in water, and H₂ oxidation was determined from the decrease of dissolved H₂. However, H₂ was again utilized to mixing ratios lower than 0.05 ppmv, if the supernatant of the soil suspension or the settled soil particles were dried onto sterile soil or purified quarz sand. Obviously, soils contain two different activities for oxidation of H₂: (1) a high- K _m, high-threshold activity which apparently is due to aerobic H₂-oxidizing bacteria, and (2) a low- K _m, low-threshold activity whose origin is unknown but presumably is due to soil enzymes.     

Enumeration of H2-utilizing methanogenic archaea, acetogenic and sulfate-reducing bacteria from human feces

Abstract: Fecal specimens from 19 healthy humans were used to enumerate H₂-utilizing microbial populations of methanogenic archaea (MA), acetogenic bacteria (AB) and sulfate-reducing bacteria (SRB). Eight subjects were methane (CH₄) excretors (CH₄+) and 11 non CH₄-excretors (CH₄−), based on breath methane concentrations. The mean ± S.E. of the logarithm of MA per gram wet weight feces were 8.8 ± 0.21 and 2.6 ± 0.39 for CH₄+ and CH₄−, respectively ( P < 0.001). SRB counts were 7.1 ± 0.43 and 7.3 ± 0.39, respectively (NS), while counts of AB were 4.6 ± 0.75 and 6.6 ± 0.38, respectively ( P < 0.02). Counts of AB were negatively correlated with counts of MA (r = −0.53; P < 0.05). These results confirm the potential importance of AB in the human colon, especially for CH₄— subjects, and suggest that a much greater competitive interrelation occurs in the human colon between MA and AB than between the former and SRB. We further report on the isolation of representatives of the dominant     acetogenic population. Three strains from two CH₄— subjects were characterized from 10⁻⁵-10⁻⁷ dilutions. They all consumed     and several carbohydrates to produce acetate as the sole metabolite. Phenotypically related to the species Peptostreptococcus productus , the strains used     via the acetyl-CoA pathway.     

Energetics of syntrophic fatty acid oxidation

Abstract: Fatty acids are key intermediates in methanogenic degradation of organic matter in sediments as well as in anaerobic reactors. Conversion of butyrate or propionate to acetate, (CO₂), and hydrogen is endergonic under standard conditions, and becomes possible only at low hydrogen concentrations (10^-4-10^-5 bar). A model of energy sharing between fermenting and methanogenic bacteria attributes a maximum amount of about 20 kJ per mol reaction to each partner in this syntrophic cooperation system. This amount corresponds to synthesis of only a fraction (one-third) of an ATP to be synthesized per reaction. Recent studies on the biochemistry of syntrophic fatty acid-oxidizing bacteria have revealed that hydrogen release from butyrate by these bacteria is inhibited by a protonophore or the ATPase inhibitor DCCD ( N , N '-dicyclohexyl carbodiimide), indicating that a reversed electron transport step is involved in butyrate or propionate oxidation. Hydrogenase, butyryl-CoA dehydrogenase, and succinate dehydrogenase acitivities were found to be partially associated with the cytoplasmic membrane fraction. Also glycolic acid is degraded to methane and CO₂ by a defined syntrophic coculture. Here the most difficult step for hydrogen release is the glycolate dehydrogenase reaction ( E '₀=−92 mV). Glycolate dehydrogenase, hydrogenase, and ATPase were found to be membrane-bound enzymes. Membrane vesicles produced hydrogen from glycolate only in the presence of ATP; protonophores and DCCD inhibited this hydrogen release. This system provides a suitable model to study reversed electron transport in interspecies hydrogen transfer between fermenting and methanogenic bacteria in methanogenic biomass degradation.     

H2 oxidation in the presence of thiosulfate, by a Thermoanaerobacter strain isolated from an oil-producing well

Abstract A thermophilic rod (strain SEBR 5268), isolated from an oil-producing well, was identified as a Thermoanaerobacter strain that was phenotypically related to T. finnii . Both SEBR 5268 and T. finnii oxidized H₂ by reducing thiosulfate to sulfide using yeast extract as growth substrate. H₂ oxidation in the presence of thiosulfate was significant at the end of the exponential growth of SEBR 5268 and was maintained during the lysis phase. In the absence of thiosulfate, H₂ was inhibitory for both strains. The role of H₂ consumption by these bacteria is discussed with regard to their metabolism on organic compounds.     

OPTIMIZATION OF CULTURE CONDITIONS FOR ENUMERATION OF H2S BACTERIA IN THE FLESH OF SEAFISH

H₂S bacteria of seafish flesh are weakly halophilic and require on average 1.68% NaCl according to statistical studies. Enumeration is optimal on PCA-H₂S(a PCA medium supplemented with sulfur sources and increased NaCl concentrations) incubated at 25C. Total aerobic bacteria can be counted simultaneously on this medium. The proportion of H₂S bacteria relative to total aerobic bacteria increased slightly during prolonged storage of the fish, but was highly variable. Models relating H₂S bacterial counts to spoilage of fish are sigmoidal and showed that when the count exceeds 10,000 CFU/g, whole or filleted fish stored in ice at 0C are unfit for consumption. Shewanella putrefaciens accounted for 69% of the H₂S bacteria at the fifth day of storage and 100% at the fifteenth.