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PubMed2004

Effects of several inhibitors on pure cultures of ruminal methanogens.

AIMS: To examine the effects of five inhibitors of methanogenesis, 2-bromoethanesulphonate (BES), 3-bromopropanesulphonate (BPS), lumazine, propynoic acid and ethyl 2-butynoate, on CH4 production of the ruminal methanogens Methanobrevibacter ruminantium, Methanosarcina mazei and Methanomicrobium mobile. METHODS AND RESULTS: Methanogens were grown in MS medium including 25% (v/v) clarified ruminal fluid. Methane production was measured after 4 and 6 days of incubation. Methanobrevibacter ruminantium was the most sensitive species to BES, propynoic acid and ethyl 2-butynoate. Methanosarcina mazei was the least sensitive species to those chemical additives, and Mm. mobile was intermediate. BPS failed to inhibit any of the methanogens. All three species were almost completely inhibited by 50- and 100%-lumazine saturated media, but the inhibition was somewhat lower with a 25%-lumazine saturated media. CONCLUSIONS: There were important differences among species of methanogens regarding their sensitivity to the different inhibitors. In general, Ms. mazei was the most resistant to inhibitors, Mb. ruminantium the least resistant, and Mm. mobile was intermediate. SIGNIFICANCE AND IMPACT OF THE STUDY: Differences among methanogens regarding their resistance to chemical inhibitors should be considered when designing strategies of inhibition of ruminal methanogenesis, as selection of resistant species may result.

Alkanesulfonic Acids↗
PubMed2006

Carriage, quantification, and predominance of methanogens and sulfate-reducing bacteria in faecal samples.

AIMS: To determine carriage rates and densities of methanogens and sulfate-reducing bacteria in adults and children using molecular methods, and to also determine if a reciprocal relationship exists between these organisms. METHODS AND RESULTS: Real-time PCR was used to detect and quantify methanogens and sulfate-reducing bacteria. Real-time PCR was more sensitive than breath methane measurements. Real-time PCR assays were applied to faecal DNA samples collected from 40 children and 12 adults. Methanogens were present in 25% of the children and 42% of the adults studied, and sulfate-reducing bacteria were detected in 15% of the children and 58% of the adults. High levels of sulfate-reducing bacteria were found in two methanogenic adults. CONCLUSIONS: Carriage rates and densities of methanogens and sulfate-reducing bacteria are greater in adults than in children. Competition does not necessarily lead to the predominance of one group in the faecal microflora. SIGNIFICANCE AND IMPACT OF THE STUDY: This study describes sensitive, molecular assays that could be used to monitor these organisms in gastrointestinal disease. Therapeutic exclusion of one group from the bowel would not necessarily lead to the expansion of the other, as there does not appear to be a reciprocal relationship between these groups.

Adult↗
PubMed2000

Molecular phylogeny of methanogens associated with flagellated protists in the gut and with the gut epithelium of termites.

The molecular phylogeny of methanogenic archaea associated with the flagellated protist species Dinenympha and Microjoenia in the gut of termites, Reticulitermes speratus and Hodotermopsis sjoestedti, and those attached to the gut epithelium was examined based on PCR-amplified small-subunit ribosomal RNA genes. The sequences identified were classified into six groups within the genus Methanobrevibacter, including groups of yet uncharacterized novel species. Closely related methanogens were shared between Microjoenia and some Dinenympha cells in each termite. The methanogens harbored by the flagellates were phylogenetically different from the methanogens associated with the gut epithelium, suggesting that distinct methanogen species showed distinct spatial distributions in the termite gut.

Journal Article↗
PubMed2006

Application of pseudomurein endoisopeptidase to fluorescence in situ hybridization of methanogens within the family Methanobacteriaceae.

In situ detection of methanogens within the family Methanobacteriaceae is sometimes known to be unsuccessful due to the difficulty in permeability of oligonucleotide probes. Pseudomurein endoisopeptidase (Pei), a lytic enzyme that specifically acts on their cell walls, was applied prior to 16S rRNA-targeting fluorescence in situ hybridization (FISH). For this purpose, pure cultured methanogens within this family, Methanobacterium bryantii, Methanobrevibacter ruminantium, Methanosphaera stadtmanae, and Methanothermobacter thermautotrophicus together with a Methanothermobacter thermautotrophicus-containing syntrophic acetate-oxidizing coculture, endosymbiotic Methanobrevibacter methanogens within an anaerobic ciliate, and an upflow anaerobic sludge blanket (UASB) granule were examined. Even without the Pei treatment, Methanobacterium bryantii and Methanothermobacter thermautotrophicus cells are relatively well hybridized with oligonucleotide probes. However, almost none of the cells of Methanobrevibacter ruminantium, Methanosphaera stadtmanae, cocultured Methanothermobacter thermautotrophicus, and the endosymbiotic methanogens and the cells within UASB granule were hybridized. Pei treatment was able to increase the probe hybridization ratio in every specimen, particularly in the specimen that had shown little hybridization. Interestingly, the hybridizing signal intensity of Methanothermobacter thermautotrophicus cells in coculture with an acetate-oxidizing H(2)-producing syntroph was significantly improved by Pei pretreatment, whereas the probe was well hybridized with the cells of pure culture of the same strain. We found that the difference is attributed to the differences in cell wall thicknesses between the two culture conditions. These results indicate that Pei treatment is effective for FISH analysis of methanogens that show impermeability to the probe.

Animals↗
PubMed1978

Application of the fluorescent-antibody technique to the study of a methanogenic bacterium in lake sediments.

Fluorescent antibody (FA) was prepared for a methanogenic bacterium isolated from Wintergreen Lake pelagic sediment. The isolate resembles Methanobacterium formicicum. The FA did not cross-react with 9 other methanogens, including M. formicicum strains, or 24 heterotrophs, 18 of which had been isolated from Wintergreen Lake sediment. FA-reacting methanogens were detected in heat-fixed smears of several different lake sediments and anaerobic sewage sludge. Pretreatment of all samples with either rhodamine-conjugated geletin or bovine serum albumin adequately controlled nonspecific absorption of the FA. Autofluorescent particles were observed in the sediment samples but, with experience, they could easily be distinguished from FA-reacting bacteria. FA direct counts of the specific methanogen in Wintergreen Lake sediments were made on four different sampling dates and compared with five-tube most-probable-number estimates of the total methanogenic population that was present in the same samples. The FA counts ranged from 3.1 X 10(6) to 1.4 X 10(7)/g of dry sediment. The highest most-probable-number estimates were at least an order ofmagnitude lower.

Anaerobiosis↗
PubMed1984

Methanogenesis in a Thermophilic (58 degrees C) Anaerobic Digestor: Methanothrix sp. as an Important Aceticlastic Methanogen.

Aceticlastic methanogens and other microbial groups were enumerated in a 58 degrees C laboratory-scale (3 liter) anaerobic digestor which was fed air-classified municipal refuse, a lignocellulosic waste (loading rate = 1.8 to 2.7 g of volatile solids per liter per day; retention time = 10 days). Two weeks after start-up, Methanosarcina sp. was present in high numbers (10 to 10 CFU/ml) and autofluorescent Methanosarcina-like clumps were abundant in sludge examined by using epifluorescence microscopy. After about 4 months of digestor operation, numbers of Methanosarcina sp. dropped 2 to 3 orders of magnitude and large numbers (most probable number = 10 to 10/ml) of a thermophilic aceticlastic methanogen morphologically resembing Methanothrix sp. were found. Methanothrix sp. had apparently displaced Methanosarcina sp. as the dominant aceticlastic methanogen in the digestor. During the period when Methanothrix sp. was apparently dominant, acetate concentrations varied between 0.3 and 1.5 mumol/ml during the daily feeding cycle, and acetate was the precursor of 63 to 66% of the methane produced during peak digestor methanogenesis. The apparent K(m) value obtained for methanogenesis from acetate, 0.3 mumol/ml, indicated that the aceticlastic methanogens were nearly saturated for substrate during most of the digestor cycle. CO(2)-reducing methanogens were capable of methanogenesis at rates more than 12 times greater than those usually found in the digestor. Added propionate (4.5 mumol/ml) was metabolized slowly by the digestor populations and slightly inhibited methanogenesis. Added n-butyrate, isobutyrate, or n-valerate (4.5 mumol/ml each) were broken down within 24 h. Isobutyrate was oxidized to acetate, a novel reaction possibly involving isomerization to n-butyrate. The rapid growth rate and versatile metabolism of Methanosarcina sp. make it a likely organism to be involved in start-up, whereas the low K(m) value of Methanothrix sp. for acetate may cause it to be favored in stable digestors operated with long retention times.

Journal Article↗
PubMed1984

Rapidly growing rumen methanogenic organism that synthesizes coenzyme M and has a high affinity for formate.

Methanogenic bacteria with a coccobacillus morphology similar to Methanobrevibacter ruminantium were isolated from the bovine rumen. One isolate, 10-16B, represented a previously undescribed rumen population that, unlike M. ruminantium, synthesized coenzyme M, grew rapidly (mu = 0.24 h-1) on H2-CO2 in a complex medium, had simple nutritional requirements, and metabolized formate at reported rumen concentrations. H2 was metabolized to partial pressures 10-fold lower than those reported for the rumen. After H2 starvation for 26 h, strain 10-16B rapidly resumed growth when H2 was made available. The minimum concentrations of acetate (6 mM) and ammonia (less than 7 mM) that were required for optimal growth were lower than the reported acetate and ammonia concentrations in the rumen. Isoleucine and leucine stimulated growth, but only at concentrations (greater than 50 microM) higher than those reported for the rumen. Another coccobacillary methanogenic organism that synthesized coenzyme M was isolated from a different animal as were organisms that required an exogenous supply of coenzyme M. In general, methanogenic bacteria that required an exogenous supply of coenzyme M had lower maximum growth rates and more complex nutritional requirements than organisms that synthesized the cofactor. The ability of all isolates to metabolize formate below the detection limit of 10 microM indicated that, in contrast to previous reports, methanogenic bacteria have the potential to directly metabolize formate in the rumen. This study demonstrated that there are physiologically diverse populations of coccobacillary methanogenic bacteria in the rumen that can interact competitively and cooperatively.

Animals↗
PubMed1988

Quantitative immunologic analysis of the methanogenic flora of digestors reveals a considerable diversity.

To determine which methanogens occur in digestors, we performed a quantitative immunologic analysis of a variety of samples. A comprehensive panel of calibrated polyclonal antibody probes of predefined specificity spectra was used. This allowed precise identification of bacteria by antigenic fingerprinting. A considerable diversity of methanogens was uncovered, much larger than previously reported, encompassing at least 14 strains of 11 species. Strategies were developed to measure the load of any given methanogen in a sample and to compare samples quantitatively. Two methanogens were found to predominate which were antigenically closely related with either Methanobacterium formicicum MF or Methanobrevibacter arboriphilus AZ. Fundamental data, probes, and methods are now available to monitor methanogenic subpopulations during digestor operation and thus learn about their respective roles and predictive significance.

Antibodies, Archaeal↗
PubMed1991

Biodegradation of dichloromethane and its utilization as a growth substrate under methanogenic conditions.

Biodegradation of dichloromethane (DCM) to environmentally acceptable products was demonstrated under methanogenic conditions (35 degrees C). When DCM was supplied to enrichment cultures as the sole organic compound at a low enough concentration to avoid inhibition of methanogenesis, the molar ratio of CH4 formed to DCM consumed (0.473) was very close to the amount predicted by stoichiometric conservation of electrons. DCM degradation was also demonstrated when methanogenesis was partially inhibited (with 0.5 to 1.5 mM 2-bromoethanesulfonate or approximately 2 mM DCM) or completely stopped (with 50 to 55.5 mM 2-bromoethanesulfonate). Addition of a eubacterial inhibitor (vancomycin, 100 mg/liter) greatly reduced the rate of DCM degradation. 14CO2 was the principal product of [14C]DCM degradation, followed by 14CH4 (when methanogenesis was uninhibited) or 14CH3COOH (when methanogenesis was partially or completely inhibited). Hydrogen accumulated during DCM degradation and then returned to background levels when DCM was consumed. These results suggested that nonmethanogenic organisms mediated DCM degradation, oxidizing a portion to CO2 and fermenting the remainder to acetate; acetate formation suggested involvement of an acetogen. Methanogens in the enrichment culture then converted the products of DCM degradation to CH4. Aceticlastic methanogens were more easily inhibited by 2-bromoethanesulfonate and DCM than were CO2-reducing methanogens. When DCM was the sole organic-carbon and electron donor source supplied, its use as a growth substrate was demonstrated. The highest observed yield was 0.085 g of suspended organic carbon formed per g of DCM carbon consumed. Approximately 85% of the biomass formed was attributable to the growth of nonmethanogens, and 15% was attributable to methanogens.

Anaerobiosis↗
PubMed1999

Estimation of methanogen biomass by quantitation of coenzyme M.

Determination of the role of methanogenic bacteria in an anaerobic ecosystem often requires quantitation of the organisms. Because of the extreme oxygen sensitivity of these organisms and the inherent limitations of cultural techniques, an accurate biomass value is very difficult to obtain. We standardized a simple method for estimating methanogen biomass in a variety of environmental matrices. In this procedure we used the thiol biomarker coenzyme M (CoM) (2-mercaptoethanesulfonic acid), which is known to be present in all methanogenic bacteria. A high-performance liquid chromatography-based method for detecting thiols in pore water (A. Vairavamurthy and M. Mopper, Anal. Chim. Acta 78:363-370, 1990) was modified in order to quantify CoM in pure cultures, sediments, and sewage water samples. The identity of the CoM derivative was verified by using liquid chromatography-mass spectroscopy. The assay was linear for CoM amounts ranging from 2 to 2,000 pmol, and the detection limit was 2 pmol of CoM/ml of sample. CoM was not adsorbed to sediments. The methanogens tested contained an average of 19.5 nmol of CoM/mg of protein and 0.39 +/- 0.07 fmol of CoM/cell. Environmental samples contained an average of 0.41 +/- 0.17 fmol/cell based on most-probable-number estimates. CoM was extracted by using 1% tri-(N)-butylphosphine in isopropanol. More than 90% of the CoM was recovered from pure cultures and environmental samples. We observed no interference from sediments in the CoM recovery process, and the method could be completed aerobically within 3 h. Freezing sediment samples resulted in 46 to 83% decreases in the amounts of detectable CoM, whereas freezing had no effect on the amounts of CoM determined in pure cultures. The method described here provides a quick and relatively simple way to estimate methanogenic biomass.

Biomass↗
PubMed1999

Anaerobic degradation of phthalate isomers by methanogenic consortia.

Three methanogenic enrichment cultures, grown on ortho-phthalate, iso-phthalate, or terephthalate were obtained from digested sewage sludge or methanogenic granular sludge. Cultures grown on one of the phthalate isomers were not capable of degrading the other phthalate isomers. All three cultures had the ability to degrade benzoate. Maximum specific growth rates (microseconds max) and biomass yields (YXtotS) of the mixed cultures were determined by using both the phthalate isomers and benzoate as substrates. Comparable values for these parameters were found for all three cultures. Values for microseconds max and YXtotS were higher for growth on benzoate compared to the phthalate isomers. Based on measured and estimated values for the microbial yield of the methanogens in the mixed culture, specific yields for the phthalate and benzoate fermenting organisms were calculated. A kinetic model, involving three microbial species, was developed to predict intermediate acetate and hydrogen accumulation and the final production of methane. Values for the ratio of the concentrations of methanogenic organisms, versus the phthalate isomer and benzoate fermenting organisms, and apparent half-saturation constants (KS) for the methanogens were calculated. By using this combination of measured and estimated parameter values, a reasonable description of intermediate accumulation and methane formation was obtained, with the initial concentration of phthalate fermenting organisms being the only variable. The energetic efficiency for growth of the fermenting organisms on the phthalate isomers was calculated to be significantly smaller than for growth on benzoate.

Acetates↗
PubMed1999

Role of methanogens and other bacteria in degradation of dimethyl sulfide and methanethiol in anoxic freshwater sediments.

The roles of several trophic groups of organisms (methanogens and sulfate- and nitrate-reducing bacteria) in the microbial degradation of methanethiol (MT) and dimethyl sulfide (DMS) were studied in freshwater sediments. The incubation of DMS- and MT-amended slurries revealed that methanogens are the dominant DMS and MT utilizers in sulfate-poor freshwater systems. In sediment slurries, which were depleted of sulfate, 75 micromol of DMS was stoichiometrically converted into 112 micromol of methane. The addition of methanol or MT to DMS-degrading slurries at concentrations similar to that of DMS reduced DMS degradation rates. This indicates that the methanogens in freshwater sediments, which degrade DMS, are also consumers of methanol and MT. To verify whether a competition between sulfate-reducing and methanogenic bacteria for DMS or MT takes place in sulfate-rich freshwater systems, the effects of sulfate and inhibitors, like bromoethanesulfonic acid, molybdate, and tungstate, on the degradation of MT and DMS were studied. The results for these sulfate-rich and sulfate-amended slurry incubations clearly demonstrated that besides methanogens, sulfate-reducing bacteria take part in MT and DMS degradation in freshwater sediments, provided that sulfate is available. The possible involvement of an interspecies hydrogen transfer in these processes is discussed. In general, our study provides evidence for methanogenesis as a major sink for MT and DMS in freshwater sediments.

Anaerobiosis↗
PubMed1991

Distribution of compatible solutes in the halophilic methanogenic archaebacteria.

Accumulation of compatible solutes, by uptake or de novo synthesis, enables bacteria to reduce the difference between osmotic potentials of the cell cytoplasm and the extracellular environment. To examine this process in the halophilic and halotolerant methanogenic archaebacteria, 14 strains were tested for the accumulation of compatible solutes in response to growth in various extracellular concentrations of NaCl. In external NaCl concentrations of 0.7 to 3.4 M, the halophilic methanogens accumulated K+ ion and low-molecular-weight organic compounds. beta-Glutamate was detected in two halotolerant strains that grew below 1.5 M NaCl. Two unusual beta-amino acids, N epsilon-acetyl-beta-lysine and beta-glutamine (3-aminoglutaramic acid), as well as L-alpha-glutamate were compatible solutes among all of these strains. De novo synthesis of glycine betaine was also detected in several strains of moderately and extremely halophilic methanogens. The zwitterionic compounds (beta-glutamine, N epsilon-acetyl-beta-lysine, and glycine betaine) and potassium were the predominant compatible solutes among the moderately and extremely halophilic methanogens. This is the first report of beta-glutamine as a compatible solute and de novo biosynthesis of glycine betaine in the methanogenic archaebacteria.

Amino Acids↗
PubMedDate not supplied

Reductive decolorization of a textile reactive dyebath under methanogenic conditions.

The objective of the present study was to assess the biological decolorization of an industrial, spent reactive dyebath and its three dye components (Reactive Blue 19 [RB 19], Reactive Blue 21 [RB 21], and Reactive Red 198 [RR 198]) under methanogenic conditions. Using a mixed, methanogenic culture, batch assays were performed to evaluate the rate and extent of color removal as well as any potential toxic effects. Overall, a high rate and extent of color removal (>10 mg/[L.h] and 88%, respectively) were observed in cultures amended with either RB 19 (an anthraquinone dye) or spent dyebath at an initial dye concentration of 300 mg/L (expressed as RB 19 equivalent) and 30 g/L of NaCl. Inhibition of acidogenesis and, to a larger degree, of methanogenesis resulting in accumulation of volatile fatty acids was observed in both RB 19- and spent dyebath-amended cultures. RB 21 (a phthalocyanine dye) and RR 198 (an azo dye) tested at an initial concentration of 300 mg/L did not result in any significant inhibition of the mixed methanogenic culture. Based on results obtained with cultures amended with RB 19 with and without NaCl, as well as a control culture amended with 30 g/L of NaCl, salt was less inhibitory than either RB 19 or the dyebath. Therefore, the toxic effect of the spent dyebath is at least partially attributed to its major dye component RB 19 and NaCl. Further testing of the effect of RB 19 decolorization products on the methanogenic activity in the absence of NaCl demonstrated that these products are much less inhibitory than the parent dye. Although color removal occurred despite the severe culture inhibition, biological decolorization of full-strength reactive spent dyebaths using methanogenic cultures in a repetitive, closed-loop system is not deemed feasible. For this reason, a fermentative and halotolerant culture was developed and successfully used in our laboratory for the decolorization of industrial reactive dyebaths with 100 g/L of NaCl.

Anthraquinones↗
PubMed2006

Population dynamics of anaerobic microbial consortia in thermophilic methanogenic sludge treating paper-containing solid waste.

To maintain a stable thermophilic (55 degrees C) anaerobic digestion treating toilet paper-containing garbage, it is necessary to operate the digester at long hydraulic retention time (HRT) and low organic loading rate (OLR). Critical conditions of the digestion were investigated by operating the digester at HRT 23 days and OLR 3.4 gCOD(Cr)/L/d (R1) or HRT 14 days and OLR 5.6 gCOD/Cr)/L/d (R2) separately. Characteristics of methanogenesis of the two digesters were examined by measuring gas generation volume and volatile fatty acids (VFA) concentration, and the populations of four anaerobic acidogens and three methanogens were analyzed using quantitative PCR method. In digester R1, methanogenic activity was unstable but it could be recovered by stopping feeding as though VFA accumulation occurred. The population of acidogens and two methanogens were maintained at 10(11) - 10(13) copies/L, however, the population of Methanoculleus could not be recovered after methanogenesis recovering. In digester R2, the period of methanogenesis was significantly shorter than that in digester R1. Both the acidogens and the methanogens could not be maintained at a stable concentration. It is suggested that the critical HRT to sustain the population of acidogens in this process should be longer than 14 days and for all kinds of methanogens, HRT should be longer than 23 days.

Acids↗
PubMed2000

Nitrogen fixation in methanogens: the archaeal perspective.

The methanogenic Archaea bring a broadened perspective to the field of nitrogen fixation. Biochemical and genetic studies show that nitrogen fixation in Archaea is evolutionarily related to nitrogen fixation in Bacteria and operates by the same fundamental mechanism. At least six nif genes present in Bacteria (nif H, D, K, E, N and X) are also found in the diazotrophic methanogens. Most nitrogenases in methanogens are probably of the molybdenum type. However, differences exist in gene organization and regulation. All six known nif genes of methanogens, plus two homologues of the bacterial nitrogen sensor-regulator glnB, occur in a single operon in Methanococcus maripaludis. nif gene transcription in methanogens is regulated by what appears to be a classical prokaryotic repression mechanism. At least one aspect of regulation, post-transcriptional ammonia switch-off, involves novel members of the glnB family. Phylogenetic analysis suggests that nitrogen fixation may have originated in a common ancestor of the Bacteria and the Archaea.

Amino Acid Sequence↗
PubMed2003

Influence of feedstock and bleaching technologies on methanogenic toxicity of kraft mill wastewater.

Chlorine-bleached effluent is problematic for anaerobic wastewater treatment due to its high toxicity against methanogenic bacteria. To date, alternative bleaching processes are being introduced, such as elemental chlorine-free (ECF) and total chlorine-free (TCF). The purpose of this study was to investigate the influence of bleaching technologies and different types of feedstock on methanogenic activity. In order to compare the toxicity of the different bleaching sequences per unit of COD, the 50%-inhibitory concentration (50% IC) for methanogenic bacteria of the different bleaching effluents were compared. The results show that there is no direct relationship between the effluent's methanogenic toxicity and the bleaching sequences. Methanogenic toxicity ranged from 0.6 to 2.4 g COD/L.

Bacteria, Anaerobic↗
PubMed1998

Methanogenic bacteria: presence in foodstuffs.

Methanogenic bacteria are anaerobic, oxygen-intolerant microorganisms, and it is only by studying the different habitats of such bacteria that fundamental information about their ecology becomes available. This research has evaluated methanogenic bacteria in apparently aerobic ecosystems, in foodstuffs not subjected to chemical-physical reclamation processes, where the presence of methanogenic bacteria has never been investigated. Methanogenic bacteria, ascribable to the Methanogenium, Methanobacterium and Methanosarcina genera, were found in vegetables, meat, fish and cheese but were generally absent in confectionery products and fruit. The microorganisms appear to be chance contaminants, usually being present in only very low numbers. It should be noted that none of the tested foods showed the presence of Methanobrevibacter smithii, M. oralis or Methanosphaera stadtmaneae, methanogenic bacteria sometimes present in the human digestive tract.

Animals↗