Isolation and characterization of an anaerobic benzoate-degrading spore-forming sulfate-reducing bacterium, Desulfotomaculum sapomandens sp. nov.

Abstract Spore-forming sulfate-reducing bacteria (SRB) were enriched selectively from various kinds of aerobic soils with fatty acids as the sole carbon and energy source. A Gram-negative motile rod-shaped bacterium, which produced gas vacuoles during sporulation was isolated. It degraded alcohols, aromatic and n-fatty acids (up to C₁₈) except for propionate, completely to CO₂. Sulfate, sulfite, thiosulfate or elemental sulfur served as electron acceptors. Because of its sensitivity to H₂S, the isolate never produced more than 8 mM dissolved sulfide at pH 7.0. G + C-content of the DNA was 48.0 mol %. The isolated strain Pato is described as a new species Desulfotomaculum sapomandens .     

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.     

Methanogenesis in the hypersaline Solar Lake (Sinai)

Abstract Enrichment studies on microbial mat sediments (potential stromatolites) from the hypersaline Solar Lake (Sinai) indicated high numbers of methanogenic bacteria (up to 10⁵ ml⁻¹ sediment) in spite of the high sulfate reduction rate, sulfate concentration and salinity. Among H₂/CO₂, acetate and monomethylamine, the methylated amine was the preferred substrate. The predominant species enriched was a Methanosarcina sp. The findings indicate that methanogenic bacteria play an important role in hypersaline sulfate-enriched anoxic sediments and stromatolithic microbial mats.     

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.     

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.     

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.     

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.     

Monoxenic cultivation of the anaerobic amoeboflagellate Psalteriomonas lanterna and isolation of the methanogenic endosymbiont

Abstract The endosymbiont of the anaerobic amoeboflagellate Psalteriomonas lanterna was isolated and identified as Methanobacterium formicicum , a H₂/CO₂- and formate-utilizing methanogen. A monoxenic culture of P. lanterna was established in a complex liquid medium with Pseudomonas fluorescenss as food bacterium.     

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.     

Growth, CO2 consumption and H2 production of Anabaena variabilis ATCC 29413-U under different irradiances and CO2 concentrations

Aims:  The objective of this study is to develop kinetic models based on batch experiments describing the growth, CO₂ consumption, and H₂ production of Anabaena variabilis ATCC 29413-U^TM as functions of irradiance and CO₂ concentration.
Methods and Results:  A parametric experimental study is performed for irradiances from 1120 to 16100 lux and for initial CO₂ mole fractions from 0·03 to 0·20 in argon at pH 7·0 ± 0·4 with nitrate in the medium. Kinetic models are successfully developed based on the Monod model and on a novel scaling analysis employing the CO₂ consumption half-time as the time scale.
Conclusions:  Monod models predict the growth, CO₂ consumption and O₂ production within 30%. Moreover, the CO₂ consumption half-time is an appropriate time scale for analysing all experimental data. In addition, the optimum initial CO₂ mole fraction is 0·05 for maximum growth and CO₂ consumption rates. Finally, the saturation irradiance is determined to be 5170 lux for CO₂ consumption and growth whereas, the maximum H₂ production rate occurs around 10 000 lux.
Significance and Impact of the Study:  The study presents kinetic models predicting the growth, CO₂ consumption and H₂ production of A. variabilis . The experimental and scaling analysis methods can be generalized to other micro-organisms.     

The inhibition of photosynthesis and photorespiration in isolated mesophyll cells of Phaseolus and Lycopersicon by reduced osmotic potentials

Mesophyll cells isolated from Phaseolus vulgaris and Lycopersicon esculentum show decreasing photosynthetic rates when suspended in media containing increasing concentrations of osmoticum. The photosynthetic activity was sensitive to small changes in osmotic potential over a range of sorbitol concentrations from 0.44 M (−1.08 MPa) to 0.77 M (−1.88 MPa). Photorespiration assayed by ¹⁴CO₂ release in CO₂-free air and by ¹⁴CO₂ release from the oxidation of [1–¹⁴C] glycolate also decreased as the osmotic potential of the incubation medium was reduced. The CO₂ compensation points of the cells increased with increasing concentration of osmoticum from approximately 60 μ I⁻¹1 at −1.08 MPa to 130 μl 1⁻¹ for cells stressed at −1.88 MPa. Changes in photosynthetic and photorespiratory activities occurred at moderate osmotic potentials in these cells suggesting that in whole leaves during a reduction in water potential, non- stomatal inhibition of CO₂ assimilation and glycolate pathway metabolism occurs simultaneously with stomatal closure.     

Sodium bioenergetics in methanogens and acetogens

Abstract Recent investigations with Methanosarcina barkeri elucidated the role of sodium ions in the energy metabolism of methanogenic bacteria and provided evidence for a novel mechanism of energy transduction with Na⁺ as the coupling ion. During methanogenesis from methanol, an eletrochemical sodium gradient generated by a Na⁺/H⁺ antiporter is used as the driving force for the thermodynamically unfavourable oxidation of methanol to the formal redox level of formaldehyde. During methanogenesis from H₂+ CO₂, the reverse reaction, the reduction of formaldehyde to the level of methanol, is accompanied by a primary, electron transport-driven sodium extrusion. Acetogenesis from H₂+ CO₂ as carried out by Acetobacterium woodii is a sodium-dependent process and is accompanied by the generation of a transmembrane sodium gradient with the reduction of formaldehyde to the level of methanol as the sodium-dependent step.     

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.     

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.     

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     

Fermentation characteristics of Clostridium pasteurianum LMG 3285 grown on glucose and mannitol

Clostridium pasteurianum fermented glucose to acetate, butyrate, CO₂ and H₂. In batch cultures the fermentation pattern was only slightly affected by culture pH over the range 8·0 to 5·5. The acetate/butyrate ratio was always higher than or equal to one. Between 2·14 and 2·33 mol H₂ was produced per mol glucose fermented. At unregulated pH, more butanol and less butyrate was formed. In a carbon-limited chemostat, the steady-state acetate/butyrate ratio was always lower than one. H₂ production was approximately 1·70 mol per mol glucose consumed. Substantial amounts of extracellular protein were formed. With decreasing pH, acetate and formate production decreased, while H₂ production was highest at pH 6.0. With increasing dilution rate ( D ), the product spectrum hardly changed, but more biomass was formed. Y _glucose^max and Y _ATP^max were 55·97 and 31·48 g dry weight per mol glucose or ATP respectively. With increasing glucose input the formation of fatty acids and H₂ slightly decreased.
Continuous cultures fermented mannitol to acetate, butyrate, butanol, CO₂ and H₂. With acetate as co-substrate, butanol production and molar growth yields, Y _mannitol and Y _ATP, markedly decreased, while the butyrate and H₂ production increased. The latter reached a value of 2·21 mol H₂ per mol mannitol consumed.     

Spirochaeta smaragdinae sp. nov., a new mesophilic strictly anaerobic spirochete from an oil field

An obligately anaerobic spirochete designated strain SEBR 4228^T (T = type strain) was isolated from an oil field of Congo, Central Africa. The strain grew optimally with a sodium chloride concentration of 5% (sodium chloride concentration growth range 1.0–10%) at 37°C (growth temperature range 20–40°C) and pH of 7.0–7.2 (pH growth range pH 5.5–8.0). Strain SEBR 4228^T grew on carbohydrates (glucose, fructose, ribose, d -xylose, galactose, mannitol and mannose), glycerol, fumarate, peptides and yeast extract. Yeast extract was required for growth and could not be replaced by vitamins. It reduced thiosulfate and sulfur, to H₂S. Glucose was oxidised to lactate, acetate, CO₂ and H₂S in the presence of thiosulfate but in its absence lactate, ethanol, CO₂ and H₂ were produced. Fumarate was fermented to acetate and succinate. The G+C content of strain SEBR 4228^T was 50%. Strain SEBR 4228^T was spiral shaped measuring 5–30 by 0.3–0.5 μm and was motile with a corkscrew-like motion. Electron microscopy revealed the presence of periplasmic flagella in a 1-2-1 arrangement. Strain SEBR 4228^T possessed features typical of the members of the genus Spirochaeta . 16S rRNA sequence analysis revealed that it was closely related to Spirochaeta bajacaliforniensis (similarity 98.6%). The lack of DNA homology with S. bajacaliforniensis (38%), together with other phenotypic differences, indicated that strain SEBR 4228^T is a new species, which we have designated Spirochaeta smaragdinae . The type strain is SEBR 4228^T (= DSM 11293).     

Biomass Production and Carbohydrate Content of Arabidopsis thaliana at Atmospheric CO2 Concentrations from 390 to 1680 μl l-1

Abstract: The concentration dependency of the impact of elevated atmospheric CO₂ concentrations on Arabidopsis thaliana L. was studied. Plants were exposed to nearly ambient (390), 560, 810, 1240 and 1680 μl I^-1 CO₂ during the vegetative growth phase for 8 days. Shoot biomass production and dry matter content were increased upon exposure to elevated CO₂. Maximal increase in shoot fresh and dry weight was obtained at 560 μl I^-1 CU₂, which was due to a transient stimulation of the relative growth rate for up to 3 days. The shoot starch content increased with increasing CO₂ concentrations up to two-fold at 1680 μl I^-1 CO₂, whereas the contents of soluble sugars and phenolic compounds were hardly affected by elevated CO₂. The chlorophyll and carotenoid contents were not substantially affected at elevated CO₂ and the chlorophyll a/b ratio remained unaltered. There was no acclimation of photosynthesis at elevated CO₂; the photosynthetic capacity of leaves, which had completely developed at elevated CO₂ was similar to that of leaves developed in ambient air. The possible consequences of an elevated atmospheric CO₂ concentration to Arabidopsis thaliana in its natural habitat is discussed.     

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.     

Carbon Dioxide Exchange Between the Atmosphere and an Alpine Shrubland Meadow During the Growing Season on the Qinghai-Tibetan Plateau

Abstract: In the present study, we used the eddy covariance method to measure CO₂ exchange between the atmosphere and an alpine shrubland meadow ecosystem (37°36'N, 101°18'E; 3 250 m a.s.l.) on the Qinghai-Tibetan Plateau, China, during the growing season in 2003, from 20 April to 30 September. This meadow is dominated by formations of Potentilla fruticosa L. The soil is Mol-Cryic Cambisols. During the study period, the meadow was not grazed. The maximum rates of CO₂ uptake and release derived from the diurnal course of CO₂ flux were -9.38 and 5.02 μmol·m^-2·s^-1, respectively. The largest daily CO₂ uptake was 1.7 g C·m^-2·d^-1 on 14 July, which is less than half that of an alpine Kobresia meadow ecosystem at similar latitudes. Daily CO₂ uptake during the measurement period indicated that the alpine shrubland meadow ecosystem may behave as a sink of atmospheric CO₂ during the growing season. The daytime CO₂ uptake was correlated exponentially or linearly with the daily photo synthetic photon flux density each month. The daytime average water use efficiency of the ecosystem was 6.47 mg CO₂/g H₂O. The efficiency of the ecosystem increased with a decrease in vapor pressure deficit.
(Managing editor: Ya-Qin HAN)