Occurrence of sulphate-reducing bacteria in human faeces and the relationship of dissimilatory sulphate reduction to methanogenesis in the large gut

Sulphate-reducing bacteria (SRB) were enumerated in 40 faecal samples obtained from two different human populations in the United Kingdom and rural South Africa. Species able to metabolize acetate, lactate, propionate, butyrate, H2/CO2, succinate, pyruvate, valerate, ethanol and a glutamate/serine/alanine mixture were found in faeces from both populations. Although a variety of nutritionally and morphologically distinct species of SRB belonging to the genera Desulfotomaculum, Desulfobacter, Desulfomonas and Desulfobulbus were identified, Desulfovibrio types always predominated. Significant numbers of SRB were present only in faecal samples from subjects whose breath methane excretion was low or undetectable. Reduced or absent methanogenesis in the presence of SRB was confirmed in fermentation studies with faecal slurries. Fourteen of 20 (70%) British faecal samples contained SRB and the remainder produced methane. The reverse was the case with 20 rural black South Africans, where only three (15%) of the samples had significant levels of SRB; the remaining 85% produced methane. These results suggest that to a large extent, dissimilatory sulphate reduction and methanogenesis are mutually exclusive in the human large gut.     

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.     

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.     

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).     

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.     

Assimilation of methane and inorganic carbon by microbial communities mediating the anaerobic oxidation of methane

The anaerobic oxidation of methane (AOM) is a major sink for methane on Earth and is performed by consortia of methanotrophic archaea (ANME) and sulfate-reducing bacteria (SRB). Here we present a comparative study using in vitro stable isotope probing to examine methane and carbon dioxide assimilation into microbial biomass. Three sediment types comprising different methane-oxidizing communities (ANME-1 and -2 mixture from the Black Sea, ANME-2a from Hydrate Ridge and ANME-2c from the Gullfaks oil field) were incubated in replicate flow-through systems with methane-enriched anaerobic seawater medium for 5–6 months amended with either ¹³CH₄ or H¹³CO₃^-. In all three sediment types methane was anaerobically oxidized in a 1:1 stoichiometric ratio compared with sulfate reduction. Similar amounts of ¹³CH₄ or ¹³CO₂ were assimilated into characteristic archaeal lipids, indicating a direct assimilation of both carbon sources into ANME biomass. Specific bacterial fatty acids assigned to the partner SRB were almost exclusively labelled by ¹³CO₂, but only in the presence of methane as energy source and not during control incubations without methane. This indicates an autotrophic growth of the ANME-associated SRB and supports previous hypotheses of an electron shuttle between the consortium partners. Carbon assimilation efficiencies of the methanotrophic consortia were low, with only 0.25–1.3 mol% of the methane oxidized.     

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.     

Competition for hydrogen between sulphate-reducing bacteria and methanogenic bacteria from the human large intestine

Sulphate-reducing activity in human faecal slurries was followed by measuring sulphide production. Sulphate-reducing bacteria (SRB) were found to outcompete methanogenic bacteria (MB) for the mutual substrate hydrogen in faecal slurries from methane- and non-methane-producing individuals mixed together. When molybdate (20 mmol/l) was added to these slurries, sulphate reduction was inhibited and methanogenesis became the major route of electron disposal. Sulphide production was stimulated by the addition of 20 mmol/l sulphate in non-methanogenic but not in methanogenic slurries. In methanogenic slurries that contained the methanogen inhibitor 2-bromoethanesulphonic acid (BES), hydrogen accumulated whilst sulphide levels were unaffected, confirming the absence of SRB in methanogenic faeces. The addition of nitrate (10 mmol/l) to faecal slurries completely inhibited methanogenesis but only slightly reduced sulphate reduction. The sulphated mucopolysaccharides, chondroitin sulphate and mucin, strongly stimulated sulphide production in non-methanogenic faecal slurries only, suggesting that these substances may be a potential source of sulphate in the large gut.     

Growth and activities of sulphate-reducing bacteria in gut contents of healthy subjects and patients with ulcerative colitis

Abstract During fermentation in the human large intestine, terminal oxidative processes may involve the activities of dissimilatory sulphate-reducing bacteria (SRB). Approximately 50% of healthy individuals harbour significant populations of SRB in faeces. In mixed culture, growth of SRB in vitro was modulated by sulphate availability, with sulphated polysaccharides such as mucin, chondroitin sulphate and carrageenan causing increased growth rates and sulphide production when compared with starch, pectin and arabino-galactan. Rates of H₂S production were higher among SRB isolated from patients with ulccrative colitis in contrast to those present in healthy volunteers. The majority (up to 92%) of SRB in faecal samples belonged to the genus Desulfovibrio . In vitro studies demonstrated that compared to isolates from healthy subjects. Desulfovibrio desulfuricans from colitic individuals were better able to adapt to high dilution rates, which may be associated with the disease. These findings indicate that the metabolic capabilities of SRB isolated from the human large intestine are not uniform and may respond to the type of substrate available in the gut as well as the rate of passage of digesta.     

Production, oxidation and emission of methane in rice paddies

Abstract Production and emission of methane from submerged paddy soil was studied in laboratory rice cultures and in Italian paddy fields. Up to 80% of the CH₄ produced in the paddy soil did not reach the atmosphere but was apparently oxidized in the rhizosphere. CH₄ emission through the rice plants was inhibited by an atmosphere of pure O₂ but was stimulated by an atmosphere of pure N₂ or an atmosphere containing 5% acetylene. Gas bubbles taken from the submerged soil contained up to 60% CH₄, but only < 1% CH₄ after the bubbles had passed the soil-water interface or had entered the intercellular gas space system of the rice plants. CH₄ oxidation activities were detected in the oxic surface layer of the submerged paddy soil. Flooding the paddy soil with water containing > 0.15% sea salt (0.01% sulfate) resulted in a strong inhibition of the rates of methanogenesis and a decrease in the rates of CH₄ emission. This result explains the observation of relatively low CH₄ emission rates in rice paddy areas flooded with brackish water.     

Methanogenesis in a saltern in the Bretagne (France)

Abstract Salterns in the Bretagne (France), exhibiting different stages of salinity of 5% to 33% evolved up to 0.7 mmol methane per m² per day. Methane concentrations of up to 0.4 mM were found. High methane evolution rates and increased methane concetration were restricted to basins of up to 7% and more than 15% salinity, and to the upper 10 cm of the sediments, where high sulfate concentrations (50 to 100 mM) occurred as well. Basins of 10–15% salinity exhibited only low methane evolution rates (less than 0.05 mmol methane per m² per day) and low methane concentrations (less than 0.02 mM). Gas bubbles arising during times of increased photosynthetic activity from the microbial mats covering the sediments of the saltern basins contained up to 2% methane. Addition of methylated amines and methanol, but not of H₂/CO₂, formate or acetate, to sediments slurries from basins of up to 12% salinity resulted in a rapid enrichment of methanogenic populations. Enriched methanogenic bacteria did not grow at salinities exceeding 15% or temperatures exceeding 45°C, and showed characteristics similar to those documented for Methanococcus halophilus and strain SF1 (DSM 3243). No enrichments were obtained from basins of more than 20% salinity in spite of methane being produced and evolved from those basins.     

Competition for hydrogen between sulphate-reducing bacteria and methanogenic bacteria from the human large intestine

Sulphate-reducing activity in human faecal slurries was followed by measuring sulphide production. Sulphate-reducing bacteria (SRB) were found to outcompete methanogenic bacteria (MB) for the mutual substrate hydrogen in faecal slurries from methane- and non-methane-producing individuals mixed together. When molybdate (20mmol/l) was added to these slurries, sulphate reduction was inhibited and methanogenesis became the major route of electron disposal. Sulphide production was stimulated by the addition of 20 mmol/1 sulphate in non-methanogenic but not in methanogenic slurries. In methanogenic slurries that contained the methanogen inhibitor 2-bromoethanesulphonic acid (BES), hydrogen accumulated whilst sulphide levels were unaffected, confirming the absence of SRB in methanogenic faeces. The addition of nitrate (10 mmol/l) to faecal slurries completely inhibited methanogenesis but only slightly reduced sulphate reduction. The sulphated mucopolysaccharides, chondroitin sulphate and mucin, strongly stimulated sulphide production in non-methanogenic faecal slurries only, suggesting that these substances may be a potential source of sulphate in the large gut.     

Methanogenesis from methanol in Methanosarcina barkeri studied using membrane inlet mass spectrometry

Abstract A mass spectrometer with membrane inlet was used to study methanol metabolism by Methanosarcina barkeri strain MS. The addition of methanol to methanol grown culture samples in the mass spectrometer vessel stimulated methanogenesis and hydrogen production. The apparent K _s for methanol was determined as 0.5 mM and the V _max as 8.14 mmol g (dry weight) h⁻¹. The V _max for methane production was fairly constant during growth of the culture on methanol implying that growth is tightly coupled to methanogenesis. The addition of methanol to culture samples in the mass spectrometer vessel stimulated methanogenesis with no lag which indicated that methanogenesis can be uncoupled from growth. Exposure of the culture sample in the mass spectrometer vessel to an atmosphere of 2 kPa oxygen for 80 min resulted in a decrease in the rate of methanogenesis from methanol but on returning the atmosphere to nitrogen the addition of further methanol stimulated methanogenesis. The effect of other inhibitors of methanogenesis (2-bromoethane sulphonate and monensin); K _j values 21.5 μM and 0.3 mM, respectively) were also studied.     

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.     

Temporal change of gas metabolism by hydrogen-syntrophic methanogenic bacterial associations in anoxic paddy soil

Abstract Interspecies H₂ transfer within methanogenic bacterial associations (MBA) accounted for 95–97% of the conversion of ¹⁴CO₂ to ¹⁴CH₄ in anoxic paddy soil. Only 3–5% of the ¹⁴CH₄ were produced from the turnover of dissolved H₂. The H₂-syntrophic MBA developed within 5 days after the paddy soil had been submerged and placed under anoxic atmosphere. Afterwards, both the contribution of MBA to H₂-dependent methanogenesis and the turnover of dissolved H₂ did not change significantly for up to 7 months of incubation. However, while the rates of H₂-dependent methanogenesis stayed relatively constant, the rates of total methanogenesis decreased. The contribution of MBA to H₂-dependent methanogenesis was further enhanced to 99% when the temperature was shifted from 30°C to 17°C, or when the soil had been planted with rice. This enhancement was partially due to an increased utilization of dissolved H₂ by chloroform-insensitive non-methanogenic bacteria, most probably homoacetogens, so that CH₄ production was almost completely restricted to H₂-syntrophic MBA. The activity of MBA, as measured by the conversion of ¹⁴CO₂ to ¹⁴CH₄, was stimulated by glucose, lactate, and ethanol to a similar or greater extent than by exogenous H₂. Propionate and acetate had no effect.     

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.     

Kinetics of two complementary hydrogen sink reactions in a defined 3-chlorobenzoate degrading methanogenic co-culture

Abstract The interrelationships between an obligate hydrogen-producing and two different hydrogen-scavenging populations grown as synthrophic members of a 3-chlorobenzoate degrading methanogenic consortium were studied. The hydrogen producer was a benzoate degrader (strain BZ-2), and the hydrogen consumers were a 3-chlorobenzoate dechlorinating bacterium ( Desulfomonile tiedjei ) and a hydrogenotropic methanogen ( Methanospirillum strain PM-1). When a mixture of 3-chlorobenzoate plus benzoate was added to this consortium, the rate of benzoate degradation was 50% higher, at slightly lower H₂ concentrations, than when benzoate alone was added. The enhanced benzoate degradation rate was apparantly triggered by the lower H₂ concentration, as the rate of benzoate degradation was shown to be a function of the H₂ concentration. By offering a hydrogen sink, in addition to methanogenesis, the dechlorinating hydrogen-scavenging population stimulated the rate of benzoate degradation. The lowering of the H₂ concentration was very small, which was in agreement with the observation that the rate of methanogenesis was hardly affected by this lower hydrogen concentration. Thus there was no significant competition for H₂ between the two hydrogen-scavenging populations in the consortium, as they practically complemented each other's hydrogen-scavenging potential at in situ hydrogen concentrations during the degradation of 3-chlorobenzoate. The H₂ concentrations at which hydrogen driven methanogenesis by Methanospirillum occurred in the consortium were well below the threshold concentration extrapolated for this methanogen after growth at high H₂ concentrations.     

Comparison of fermentation reactions in different regions of the human colon

Colonic contents were obtained from two human sudden-death victims within 3 h of death. One of the subjects (1) was methanogenic, the other (2) was a non-CH, producer. Measurements of bacterial fermentation products showed that in both individuals short-chain fatty acids, lactate and ethanol concentrations were highest in the caecum and ascending colon. In contrast, products of protein fermentation, such as ammonia, branched chain fatty acids and phenolic compounds, progressively increased from the right to the left colon, as did the pH of gut contents. In Subject 1, cell population densities of methanogenic bacteria (MB) increased distally through the gut and methanogenic activity was lower in the right (0.78–1–18 μmol CH₄ produced/h/g dry wt contents) than in the left colon (1.34 μmol CH₄ produced/h/g dry wt contents). Methane production rates did not correlate with MB numbers.
Sulphate-reducing bacteria (SRB) were not found and dissimilatory sulphate reduction was not detected in any region of the colon. Methanogenic bacteria did not occur in subject 2, but high numbers of SRB were present throughout the gut ( ca 10⁹/g dry wt contents). Sulphate reduction rates were maximal in the ascending and transverse colons (0.24 and 0.22 μmol ³⁵SO^2–₄ reduced/h/g dry wt contents, respectively). Short-chain fatty acid production by caecal contents was up to eight-fold higher than contents from the sigmoid/rectum. These findings demonstrate significant differences in fermentation reactions in different regions of the large gut.     

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.     

Chemostat enrichment of sulphate-reducing bacteria from the large gut

Electron donor-limited chemostat enrichments were used to isolate sulphate-reducing bacteria (SRB) from human faeces. When acetate, lactate, propionate, butyrate or amino acids were used as electron donors, SRB identical to those found using traditional isolational methods (agar shake dilution series) were obtained. However, chemostat enrichments facilitated the isolation of SRB able to metabolize mixtures of alcohols (C₁-C₅) and mixtures of fatty acids (C₂-C₆) which had not been detected by the direct isolation techniques.