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.     

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.     

Heavy metal inhibition of methanogenesis by Methanospirillum hungatei GP1

Abstract Washed whole cells of Methanospirillum hungatei incubated in TES buffer retained methanogenic activity in the absence of any reducing agents. Washed cells grown with 80% H₂-20% CO₂ and acetate produced methane from H₂/CO₂ and 50 mM formate at 1.1 to 1.8 and 15 μmol methane · h⁻¹· mg⁻¹ protein, respectively. Cadmium at a concentration of 15 μM and 50 μM mercury, copper or zinc completely inhibited methane production from H₂/CO₂ by M. hungatei . The chelating agent, EDTA, protected the cells from inhibition by cadmium but acetate and citrate did not. The activity of formate dehydrogenase and hydrogenase remaining in cells after incubation with copper, mercury, zinc or cadmium was reduced with formate dehydrogenase being the more sensitive.     

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     

SULPHIDE PRODUCTION FROM SULPHATE-ENRICHED SEWAGE SLUDGES

SUMMARY: Sterilized raw sewage sludge enriched with sulphate and inoculated with pure strains of Desulphovibrio desulphuricans produced negligible sulphide. Unsterilized sludge supplemented with 7% (w/v) CaSO₄.2H₂O and inoculated with crude cultures of sulphate-reducing bacteria obtained from sewage yielded 1·0% S^2- (wt S^2- produced as H₂S/vol. of raw sludge) in 6 months at 30°. By repeated subculture more active cultures developed which produced 1% S^2- in 7 days and 1·2–1·9% in 28 days. Digested sludge yielded only 0·1% S^2-. In semicontinuous fermentations at 30°, raw sludge without added sulphate produced 20 times its own volume of gas containing 70% CH₄ and 30% CO₂. When 5% CaSO₄.2H₂O and an active crude culture of sulphate reducers were added, gas production decreased steadily to zero. There were no differences in pH, temperature and redox potential in sludges producing methane or sulphide. The chief cause of inhibition appeared to be the action of sulphide: 0·02% soluble sulphide (S^2-) totally inhibited methane formation; 0·01% S^2- initially decreased gas production by one-quarter but there was a slow recovery to normal, suggesting acclimatization of the methane-producing organisms to sulphide.
Linked fermentations, in which gas from a methane fermentation swept H₂S from a sulphide fermentation, gave a final gas mixture of about 60% CH₄, 30% CO₂ and 5–10% H₂S. The yield of sulphide depended on the rate of sweeping.     

Competition between reductive acetogenesis and methanogenesis in the pig large-intestinal flora

Washed bacterial suspensions obtained from the pig hindgut were incubated under ¹³CO₂ in a buffer containing NaH¹³CO₃ and carbohydrates. Incorporation of ¹³C into short chain fatty acids was assayed by quantitative nuclear magnetic resonance. The effects of different levels of H₂ added to the gas phase (0, 20 and 80% v/v) and of the specific methanogenesis inhibitor 2-bromoethane-sulphonic acid (BES) were determined. In control incubations increasing the concentration of H₂ markedly increased methane production. Single- and double-labelled acetate and butyrate were formed in all incubations. In the absence of BES, increasing H₂ significantly increased the incorporation of ¹³CO² into butyrate and the proportion of double-labelled acetate in total labelled acetate. The addition of BES proved to be very successful as a methane inhibitor and greatly enhanced the amount of mono- and double-labelled acetate, especially at the highest H₂ partial pressure. The results suggest that methanogenesis inhibited both routes of reductive acetogenesis, i.e. the homoacetate fermentation of hexose (represented for the most part by single labelling) and the synthesis of acetate from external CO₂ and H₂ (represented mostly by double labelling). A highly significant interaction between BES and H₂ concentration was observed. At the highest pH2 BES increased the proportion of labelled acetate in total acetate from 17.1% for the control to 50.9%. It was concluded that although acetogenesis and methanogenesis can occur simultaneously in the pig hindgut, reductive acetogenesis may become a significant pathway of acetate formation in the absence of methanogenesis.     

End products of metabolism in the anaerobic ciliate Trimyema compressum

Abstract The products of anaerobic and micro-aerobic (0.8% O₂) metabolism of the sapropelic ciliate Trimyema compressum strain N were studied. Under anaerobic conditions ethanol was formed in large amounts representing 44% of the total carbon excreted. Acetate, lactate, formate, CO₂ and H₂ were minor products, while succinate was formed in hardly detectable amounts. Under micro-aerobic conditions O₂ was consumed, CO₂ and formate were produced as major end products and no H₂, ethanol and succinate were formed.     

Detection of hydrogen peroxide produced by meat lactic starter cultures

Twelve strains of meat lactic starter cultures (Pediococcus spp. and Lactobacillus plantarum) were found to produce hydrogen peroxide in vitro. The (cumulative) amounts of H₂O₂ produced were measured through the peroxidative action of catalase on H₂O₂ and oxidation of added formate to CO₂ by the H₂O₂-catalase complex formed. There was a problem in building a calibration curve for converting values of formate oxidation into amounts of H₂O₂, either by adding H₂O₂ directly to the assay mixture or having it produced via a glucose-glucose oxidase system.     

H2 and CO2 production from methanol or formaldehyde by the methanogenic bacterium strain Gö1 treated with 2-bromoethanesulfonic acid

Abstract In cell suspensions of the methanogenic bacterium strain Gö1 or Methanosarcina barkeri H₂ formation from methanol in the presence of 2-bromoethanesulfonic acid (BES) was strictly dependent on sodium ions; apparent K _S for Na⁺, 1.3±0.3 mM.H₂ formation was inhibited by the uncoupler tetrachlorosalicylanilide (TCS), but this inhibition could be temporarily overcome, when a sodium pulse (100 mM) was given to the cell suspension. On the other hand, H₂ formation from formaldehyde in the presence of BES (rate: 300 nmol H₂/h·mg protein as compared to 25 nmol H₂/h·mg protein from methanol) was not sodium-dependent, not TCS-sensitive and not inhibited by addition of monensin. H₂ formation was accompanied by CO₂ formation in stoichiometric amounts, 3 H₂:1 CO₂ for methanol and 2 H₂:1 CO₂ for formaldehyde oxidation.     

Influence of freshwater sediment on the survival of Escherichia coli and Salmonella sp. as measured by three methods of enumeration

The thermophilic hydrogen-oxidizing eubacterium, Hydrogenobacter thermophilus was grown with a semi-continuous gas feed in a 150 1 fermentor, on different ratios of H₂, O₂ (air) and CO₂ and on media of variable mineral salts composition. The best gas ratio during the exponential phase was approximately 0.5:1.0:0.03 atm of H₂: air: CO₂. Cell densities were increased by increasing the concentration of ammonium and phosphate in the medium. The maximum cell density obtained under the most favourable conditions was 2.62 g wet wt 1^-1, corresponding to an absorption of 1.6 at 600 nm. The maximum specific growth rate ( u ) was 0.44 h^-1.     

Methyl sulfides as intermediates in the anaerobic oxidation of methane

While it is clear that microbial consortia containing Archaea and sulfate-reducing bacteria (SRB) can mediate the anaerobic oxidation of methane (AOM), the interplay between these microorganisms remains unknown. The leading explanation of the AOM metabolism is 'reverse methanogenesis' by which a methanogenesis substrate is produced and transferred between species. Conceptually, the reversal of methanogenesis requires low H₂ concentrations for energetic favourability. We used ¹³C-labelled CH₄ as a tracer to test the effects of elevated H₂ pressures on incubations of active AOM sediments from both the Eel River basin and Hydrate Ridge. In the presence of H₂, we observed a minimal reduction in the rate of CH₄ oxidation, and conclude H₂ does not play an interspecies role in AOM. Based on these results, as well as previous work, we propose a new model for substrate transfer in AOM. In this model, methyl sulfides produced by the Archaea from both CH₄ oxidation and CO₂ reduction are transferred to the SRB. Metabolically, CH₄ oxidation provides electrons for the energy-yielding reduction of CO₂ to a methyl group ('methylogenesis'). Methylogenesis is a dominantly reductive pathway utilizing most methanogenesis enzymes in their forward direction. Incubations of seep sediments demonstrate, as would be expected from this model, that methanethiol inhibits AOM and that CO can be substituted for CH₄ as the electron donor for methylogenesis.     

Microbial H2/CO2 acetogenesis in animal guts: nature and nutritional significance

Abstract The intestinal tract of invertebrate and vertebrate animals, including man, is an anoxic habitat wherein microbial formation of acetate from H₂+ CO₂ is often a major H₂-consuming reaction. This paper will discuss the magnitude and microbiology of H₂/CO₂ acetogenesis in animal guts, its impact on host animal nutrition, competition for H₂ between anaerobic microbes, and the global significance of intestinal H₂/CO₂ acetogenesis.     

Inhibition of Methanosarcina barkeri strain 227 by cadmium

Abstract The effect of cadmium (Cd) on methane formation from methanol and/or H₂–CO₂ by Methanosarcina barkeri was examined in a defined growth medium and in a simplified buffer system containing 50 mM Tes with or without 2 mM dithiothreitol (DTT). No inhibition of methanogenesis by high concentrations of cadmium was observed in growth medium. Similarly, little inhibition of methanogenesis by whole cells in the Tes buffer system was observed in the presence of 430 μM Cd or 370 μM mercury (Hg) with 2 mM DTT. When the concentration of DTT was reduced to 0.4 mM, almost complete inhibition of methanogenesis from H₂–CO₂ and methanol by 600 μM Cd was observed. In the absence of DTT, 150 μM Cd inhibited methanogenesis from H₂–CO₂ completely and from methanol by 97%. Methanogenesis from H₂–CO₂ was more sensitive to Cd than that from methanol.     

Theory and practice of a portable photosynthesis instrument

Abstract. Theory and practice of non-steady-state portable photosynthesis instruments (LI-6000 and 6200, LI-COR Inc., Nebraska, U.S.A.) are presented. Mass balance equations for the time dependence of H₂O and CO₂ mol fractions within the leaf chamber were used to describe instrument function. Measurements for each run were fitted to an exponential function to estimate average rates of CO₂ assimilation and transpiration during the measurement period. Stomatal conductances and intercellular CO₂ mol fractions were also computed. Linear data analysis used in the LI-6200 produced similar results for assimilation rates, stomatal conductances and intercellular CO₂ concentrations compared to a more rigorous nonlinear analysis, provided humidity within the chamber was kept constant during the measurement period. Instrument performance for CO₂ fluxes was confirmed by injecting pure CO₂ at steady rates from a microsyringe into the chamber. Miniature evaporimeters were designed to check H₂O flux measurements. Significant discrepancies were observed between LI-6200 estimates of H₂O fluxes and direct measurement and errors were attributed to adsorption desorption of water vapour on chamber walls or to leaks. The leaf chamber should be stored at humidities and temperatures similar to those during measurement conditions for maximum reliability of results.     

Bicarbonate-dependent production and methanogenic consumption of acetate in anoxic paddy soil

Abstract Acetate turnover was measured in slurries of anoxic methanogenic paddy soil after addition of carrier-free [2-¹⁴C]-acetate. Acetate concentrations stayed fairly constant for about 1–2 days indicating steady state between production and consumption reactions. Depending on the experiment, acetate concentrations were between 100 and 3000 μM. Turnover rates were determined from the logarithmic decrease of [2-¹⁴C]-acetate or from the accumulation of acetate in the presence of chloroform resulting in similar values, i.e. 12–13 nmol h⁻¹g⁻¹d.w. soil at 17°C and 36–88 nmol h⁻¹g⁻¹d.w. at 30°C. Acetate consumption was completely inhibited by chloroform. The respiratory index (RI) was < 0.27. Hence, acetate was apparently consumed by methanogenic bacteria. About 80–90% of the CH₄ produced originated from the methyl group of acetate. The role of homoacetogenesis for acetate production was studied by measuring the incorporation of radioactive bicarbonate into acetate. In different experiments, CO₂ incorporation accounted for fractions of 1–60% of the acetate produced, about 10% being the most likely value for steady-state conditions. The fraction increased at high H₂ concentrations and decreased at high acetate concentrations. The rate of H₂ production that was required for chemolithotrophic acetate production from CO₂ was two orders of magnitude higher than the actually measured rate. Hence, most of the CO₂ incorporation into acetate was caused by electron donors other than H₂ (e.g., carbohydrates) and/or by exchange reactions.     

Carbon and water fluxes in a calcareous grassland under elevated CO2

1. As part of a long-term study of the effects of elevated CO₂ on biodiversity and ecosystem function in a calcareous grassland, we measured ecosystem carbon dioxide and water-vapour fluxes over 24-h periods during the 1994 and 1995 growing seasons. Data were used to derive CO₂ and H₂O gas-exchange response functions to quantum flux density (QFD).
2. The relative increase in net ecosystem CO₂ flux (NEC) owing to CO₂ enrichment increased as QFD rose. Daytime NEC at high QFD under elevated CO₂ increased by 25% to 60%, with the greatest increases in the spring and after mowing in June when above-ground biomass was lowest. There was much less stimulation of NEC in early June and again in October when the canopy was fully developed. Night-time NEC was not significantly altered under elevated CO₂.
3. Short-term reversal of CO₂ concentrations between treatments after two seasons of CO₂ exposure provided evidence for a 50% downward adjustment of NEC expressed per unit above-ground plant dry weight. However, when expressed on a land area basis, this difference disappeared because of a c. 20% increase in above-ground biomass under elevated CO₂.
4. Ecosystem evapotranspiration (ET) was not significantly altered by elevated CO₂ when averaged over all measurement dates and positions. However, ET was reduced 3–18% at high QFD in plots at the top of the slope at our study site. In summary, CO₂ enrichment resulted in a large stimulation of ecosystem CO₂ capture, especially during periods of a large demand of carbon in relationship to its supply, and resulted in a relatively small and variable effect on ecosystem water consumption.     

Biological oxidation of hydrogen in soils flushed with a mixture of H2, CO2, O2 and N2

Abstract A stainless steel cylinder filled with soil was flushed upstream with a H₂/CO₂/air mixture. The consequence was a strong enrichment of the aerobic, autotrophic hydrogen-oxidising microflora, which reached densities enabling them to oxidize 84.5 ml H₂· dm⁻²· h⁻¹ in the first 25-cm layer. H₂ concentration profiles, hydrogen uptake activity and cell numbers correlated well with each other. Most of the organisms isolated were dinitrogen fixers. Thus, soils containing hydrogen-oxidising bacteria may act as a biological shield between H₂-rich environments and air, and may be utilized as biofilters, e.g., in the waste-processing industry.     

A Simple Test for Differentiating Streptococcus bovis from other Streptococci

S ummary . Streptococcus bovis grew in acid media, which are normally inhibitory for this organism, when incubated in an atmosphere containing 5% (v/v) CO₂ or if the CO₂ produced by the organism itself was prevented from escaping from the media. Since no other streptococcus grew in acid media under these conditions, this finding enables Strep. bovis to be differentiated from other streptococci by a simple test.     

Methanofuran-b is required for CO2 formation from formaldehyde by Methanosarcina barkeri

Abstract Extracts of Methanosarcina barkeri strain Fasaro oxidized formaldehyde to CO₂ with methyl-coenzyme M as the natural terminal electron acceptor resulting in methanogenesis. A combination of the artificial electron acceptors methylviologen and metronidazole could substitute for methyl-coenzyme M. The rate of formaldehyde oxidation was thereby increased. Taking advantage of this artificial electron acceptor system the role of cofactors in formaldehyde oxidation was investigated. Cofactor-free extract of M. barkeri did not catalyze the oxidation of formaldehyde. CO₂ formation could be restored by the addition of tetrahydromethanopterin-b (H₄MPT-b) and methanofuran-b (MFR-b) from M. barkeri . Other low molecular weight or heat-resistant compounds stimulating formaldehyde oxidation were not found. Formaldehyde oxidation seems, therefore, to proceed via H₄ MPT-b and MFR-b-derivatives already shown to be involved in methanogenesis from H₂+ CO₂.     

Combined monitoring of changes in δ13CH4 and archaeal community structure during mesophilic methanization of municipal solid waste

Reconstituted municipal solid waste (MSW) with varying contents of putrescible and cellulosic waste was incubated anaerobically under mesophilic conditions. Standard physicochemical parameters were monitored, together with stable isotopic signatures of produced CH₄ and CO₂. δ¹³C values for CH₄ indicated a change of methanogenic metabolism with time. CH₄ was predominantly produced from H₂/CO₂ at the beginning of the incubations. This period was associated with important shifts in archaeal communities monitored by automated ribosomal intergenic spacer analysis (ARISA) and FISH of oligonucleotidic probes targeting specifically 16S rRNA gene of various methanogenic groups. The onset of the active methane generation phase was characterized by an increase of CH₄δ¹³C, indicating a progressive shift toward an aceticlastic metabolism. When the methane production levelled off, a decrease in the isotopic signature was observed toward values characteristics of hydrogenotrophic metabolism. ARISA profiles were, however, found to be stable from the beginning of the active methane generation phase until the end of the experiment. FISH observation indicated that members of the family Methanosarcinaceae were predominant in the archaeal community during this period, suggesting that these methanogens might exhibit a high metabolic versatility during methanization of waste.