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PubMed1994

Metabolic interactions between anaerobic bacteria in methanogenic environments.

In methanogenic environments organic matter is degraded by associations of fermenting, acetogenic and methanogenic bacteria. Hydrogen and formate consumption, and to some extent also acetate consumption, by methanogens affects the metabolism of the other bacteria. Product formation of fermenting bacteria is shifted to more oxidized products, while acetogenic bacteria are only able to metabolize compounds when methanogens consume hydrogen and formate efficiently. These types of metabolic interaction between anaerobic bacteria is due to the fact that the oxidation of NADH and FADH2 coupled to proton or bicarbonate reduction in thermodynamically only feasible at low hydrogen and formate concentrations. Syntrophic relationships which depend on interspecies hydrogen or formate transfer were described for the degradation of e.g. fatty acids, amino acids and aromatic compounds.

Acetates↗
PubMed1985

Sequence divergence of an archaebacterial gene cloned from a mesophilic and a thermophilic methanogen.

A 1.6-kb fragment of DNA from the thermophilic, methane-producing, anaerobic archaebacterium Methanobacterium thermoautotrophicum delta H has been cloned and sequenced. This DNA complements mutations in both the purE1 and purE2 loci of Escherichia coli. The sequence of the M. thermoautotrophicum DNA predicts that complementation in E. coli results from the synthesis of a polypeptide with a molecular weight of 36,249. A polypeptide apparently of this molecular weight is synthesized in E. coli minicells containing recombinant plasmids that carry the cloned fragment of methanogen DNA. We have previously cloned and sequenced a purE-complementing gene from the mesophilic methanogen Methanobrevibacter smithii. The two methanogen-derived purE-complementing genes are 53% homologous and encode polypeptides that are 45% homologous in their amino acid sequences but would be 74% homologous if conservative amino acid substitutions were considered as maintaining sequence homology. The genome of M. thermoautotrophicum has a molar G + C content of 49.7%, whereas the genome of M. smithii is 30.6% G + C. Conservation of encoded amino acids while accommodating the very different G + C contents is accomplished by use of different codons that encode the same amino acid. The majority of base changes occur at the third codon position. The intergenic regions of the cloned M. thermoautotrophicum DNA contain sequences previously identified as ribosome binding sites and as putative methanogen promoters. Although the two purE-complementing genes are apparently derived from a common ancestor, only the gene from M. smithii maintains a codon usage that conforms to the RNY rule.

Amino Acid Sequence↗
PubMed1999

Biomass Measurement of Methanogens in the Sediments of Tokyo Bay Using Archaeol Lipids.

An archaeal ether-linked lipid, archaeol, was determined to be a biomass indicator for methanogens both in the laboratory enriched culture and in marine sediments. The archaeol measurement method described by Ohtsubo et al. in 1993 was modified and applied to marine sediments. We compared the amount of archaeol with the cell number of methanogens or methane concentration in laboratory enriched culture of methanogens from marine sediment. Good correlations were obtained as follows: (Methane, mmol) = 11.2 x (Archaeol, mg): r =.996 or (Cell number) = 1.13 x 10(11) x (Archaeol, mg): r =.995, respectively. In the sediments of Tokyo Bay, archaeol was measured from approximately 46 to 561 ng/dry g sediment at the entrance to 267 to 4160 ng/dry g sediment at the innermost area. Using the coefficient from the laboratory experiment, these data corresponded to cell numbers of 5.2 x 10(6) to 4.7 x 10(8)/dry g sediment. These values were 1 or 2 orders of magnitude higher than those obtained by culture methods in previous studies. Although dead or decomposed cells might be detected, archaeol measurement is useful for estimating the biomass of methanogens because of the good correlation between methane concentration and archaeol content in marine environments. In this study, we found a correlation of (Methane, mmol) = 0.012 x (Archaeol, mg): r =.932, n = 17 in marine sediments.

Journal Article↗
PubMed2002

Redox-driven proton translocation in methanogenic Archaea.

Methanogenic archaea of the genus Methanosarcina are able to utilize H2 + CO2, methylated C1 compounds or acetate as energy and carbon source, thereby producing methane as the major end product. The methanogenic pathways lead to the formation of a mixed disulfide derived from coenzyme M and coenzyme B. This disulfide is of major importance for methanogens because it is the terminal electron acceptor of a branched respiratory chain. Molecular hydrogen, reduced coenzyme F420 or reduced ferredoxin are used as electron donors. Four enzymes are involved in the membrane-bound electron transport system of Methanosarcina species, all of which are involved in the generation of an electrochemical proton gradient that is used for ATP synthesis. This review focuses on the membrane-bound electron transport chains of Methanosarcina species with respect to the biochemical and genetic characteristics of the unusual energy transducing enzymes. Furthermore, the review addresses questions concerning the relationship between methanogenic proteins and components of respiratory chains found in bacteria and eukarya.

Amino Acid Sequence↗
PubMed2003

Competition between Fe(III)-reducing and methanogenic bacteria for acetate in iron-rich freshwater sediments.

The kinetics of acetate uptake and the depth distribution of [2-14C]acetate metabolism were examined in iron-rich sediments from a beaver impoundment in northcentral Alabama. The half-saturation constant (Km) determined for acetate uptake in slurries of Fe(III)-reducing sediment (0.8 mM) was more than 10-fold lower than that measured in methanogenic slurries (12 mM) which supported comparable rates of bulk organic carbon metabolism and Vmax values for acetate uptake. The endogenous acetate concentration (Sn) was also substantially lower (1.7 mM) in Fe(III)-reducing vs methanogenic (9.0 mM) slurries. The proportion of [2-14C]acetate converted to 14CH4 increased with depth from ca 0.1 in the upper 0.5 cm to ca 0.8 below 2 cm and was inversely correlated (r2 = 0.99) to a decline in amorphous Fe(III) oxide concentration. The results of the acetate uptake kinetics experiments suggest that differences in the affinity of Fe(III)-reducing bacteria vs methanogens for acetate can account for the preferential conversion of [2-14C]acetate to 14CO2 in Fe(III) oxide-rich surface sediments, and that the downcore increase in conversion of [2-14C]acetate to 14CH4 can be attributed to progressive liberation of methanogens from competition with Fe(III) reducers as Fe(III) oxides are depleted with depth.

Acetates↗
PubMed2003

Analysis of methanogen diversity in a hypereutrophic lake using PCR-RFLP analysis of mcr sequences.

The incidence and diversity of methanogens in Priest Pot, a dynamic and active lake, were monitored by analysing mcrA gene sequences generated from total DNA samples obtained at different times of the year and amplified using the polymerase chain reaction. A number of mcrA clones were analysed by developing an RFLP-based protocol to generate a number of restriction patterns that were assigned to a number of classes. The RFLP patterns for each class were compared with published sequence information for mcrA from cultured methanogens as well as with those from other experimental studies. They could be used to assign tentative identification for some of the Priest Pot clones and also revealed the presence of a number of clones that could not be affiliated to any known methanogens. The limitations of using RFLP profiles of mcrA gene sequences for studying methanogen ecology are discussed.

Bacteria↗
PubMed2004

Methanogenic pathway and archaeal community structure in the sediment of eutrophic Lake Dagow: effect of temperature.

Methanogenic degradation of organic matter is an important microbial process in lake sediments. Temperature may affect not only the rate but also the pathway of CH4 production by changing the activity and the abundance of individual microorganisms. Therefore, we studied the function and structure of a methanogenic community in anoxic sediment of Lake Dagow, a eutrophic lake in north-eastern Germany. Incubation of sediment samples (in situ 7.5 degrees C) at increasing temperatures (4, 10, 15, 25, 30 degrees C) resulted in increasing production rates of CH4 and CO2 and in increasing steady-state concentrations of H2. Thermodynamic conditions for H2/CO2 -dependent methanogenesis were only exergonic at 25 and 30 degrees C. Inhibition of methanogenesis with chloroform resulted in the accumulation of methanogenic precursors, i.e., acetate, propionate, and isobutyrate. Mass balance calculations indicated that less CH4 was formed via H2 at 4 degrees C than at 30 degrees C. Conversion of 14CO2 to 14CH4 also showed that H2/CO2 -dependent methanogenesis contributed less to total CH4 production at 4 degrees C than at 30 degrees C. [2-14 C]Acetate turnover rates at 4 degrees C accounted for a higher percentage of total CH4 production than at 30 degrees C. Collectively, these results showed a higher contribution of H2-dependent methanogenesis and a lower contribution of acetate-dependent methanogenesis at high versus low temperature. The archaeal community was characterized by cloning, sequencing, and phylogenetic analysis of the 16S rRNA genes retrieved from the sediment. Sequences were affiliated with Methanosaetaceae, Methanomicrobiaceae, and three deeply branching euryarchaeotal clusters, i.e., group III, Rice cluster V, and a novel euryarchaeotal cluster, the LDS cluster. Terminal restriction fragment length polymorphism (T-RFLP) analysis showed that 16S rRNA genes affiliated to Methanosaetaceae (20-30%), Methanomicrobiaceae (35-55%), and group III (10-25%) contributed most to the archaeal community. Incubation of the sediment at different temperatures (4-30 degrees C) did not result in a systematic change of the archaeal community composition, indicating that change of temperature primarily affected the activity rather than the structure of the methanogenic community.

Archaea↗
PubMed2003

Methanogen population in a marine biofilm corrosive to mild steel.

This study was conducted to analyze the methanogen population in a corrosive marine biofilm based on 16S rDNA analysis, using a PCR-cloning-sequencing approach. There were 80 methanogen clones developed from the PCR-amplified DNA extracted from the biofilm on the mild steel surface. All clones were categorized into one of five operational taxonomy units (OTUs). Two OTUs (comprising 57 clones) were affiliated with the acetotrophic Methanosaeta genus; the remaining three OTUs (23 clones) were affiliated with the hydrogenotrophic genera of Methanogenium, Methanoplanus and Methanocalculus. The hydrogenotrophic methanogens could directly cause metal corrosion through cathodic depolarization, whereas the acetotrophic methanogens grew syntrophically with corrosion-causing sulfate-reducing bacteria, as observed by fluorescent in situ hybridization, and thus contribute indirectly to metal corrosion.

Base Sequence↗
PubMed2006

Quantification of methanogens by fluorescence in situ hybridization with oligonucleotide probe.

To monitor anaerobic environmental engineering system, new method of quantification for methanogens was tested. It is based on the measurement of specific binding (hybridization) of 16S rRNA-targeted oligonucleotide probe Arc915, performed by fluorescence in situ hybridization (FISH) and quantified by fluorescence spectrometry. Average specific binding of Arc915 probe was 13.4+/-0.5 amol/cell of autofluorescent methanogens. It was 14.3, 13.3, and 12.9 amol/cell at the log phase, at stationary phase and at the period of cell lysis of batch culture, respectively. Specific binding of Arc915 probe per 1 ml of microbial sludge suspension from anaerobic digester linearly correlated with concentration of autofluorescent cells of methanogens. Coefficient of correlation was 0.95. Specific binding of oligonucleotide probe Arc915 can be used for the comparative estimation of methanogens during anaerobic digestion of organic waste. Specific binding of Arc915 probe was linear function of anaerobic sludge concentration when it was between 1.4 and 14.0 mg/ml. Accuracy of the measurements in this region was from 5 to 12%.

Anaerobiosis↗
PubMed2001

Functional patterns and temperature response of cellulose-fermenting microbial cultures containing different methanogenic communities.

The effect of microbial composition on the methanogenic degradation of cellulose was studied using two lines of anaerobic cellulose-fermenting methanogenic microbial cultures at two different temperatures: that at 15 degrees C being dominated by Methanosaeta and that at 30 degrees C by Methanosarcina. In both cultures, CH4 production and acetate consumption were completely inhibited by either 2-bromoethanesulfonate or chloroform, whereas H2 consumption was only inhibited by chloroform, suggesting that homoacetogens utilized H2 concomitantly with methanogens. Hydrogen was the intermediate that was consumed first, while acetate continued to accumulate. At 15 degrees C, acetoclastic methanogenesis smoothly followed H2-dependent CH4 production. Fluorescence in situ hybridization showed that populations of Methanosaeta steadily increased with time from 5 to 25% of total cell counts. At 30 degrees C, two phases of CH4 production were obtained, with acetate consumed after the abrupt increase of Methanosarcina from 0 to 45% of total cell counts. Whereas populations of Methanosaeta were able to adapt after transfer from 15 to 30 degrees C, those of Methanosarcina were not, irrespective of during which phase the cultures were transferred from 30 degrees C to 15 degrees C. Our results thus show that the community structure of methanogens indeed affects the function of a cellulose-fermenting community with respect to temperature response.

Archaea↗
PubMed2003

Growth and activities of sulfate-reducing and methanogenic bacteria in human oral cavity.

Viable counts and activities of sulfate-reducing bacteria (SRB) and methanogenic bacteria were determined in the oral cavities of eight volunteers. Of these, seven harbored viable SRB populations, and six harbored viable methanogenic bacterial populations. Two volunteers classified as type III periodontal patients had both SRB and methanogenic bacteria. Six separate sites were sampled: posterior tongue, anterior tongue, mid-buccal mucosa, vestibular mucosa, supragingival plaque, and subgingival plaque. The SRB was found in all areas in one volunteer, and it was mostly present in posterior tongue, anterior tongue, supragingival, and subgingival plaques in many volunteers. The methanogenic bacteria were mostly found in supragingival and subgingival plaques. The activities of sulfate reduction and methane production were determined in randomly selected isolates.

Colony Count, Microbial↗
PubMed1996

Quantitative determination of H2-utilizing acetogenic and sulfate-reducing bacteria and methanogenic archaea from digestive tract of different mammals.

Total number of bacteria, cellulolytic bacteria, and H2-utilizing microbial populations (methanogenic archaea, acetogenic and sulfate-reducing bacteria) were enumerated in fresh rumen samples from sheep, cattle, buffaloes, deer, llamas, and caecal samples from horses. Methanogens and sulfate reducers were found in all samples, whereas acetogenes were not detected in some samples of each animal. Archaea methanogens were the largest H2-utilizing populations in all animals, and a correlation was observed between the numbers of methanogens and those of cellulolytic microorganisms. Higher counts of acetogens were found in horses and llamas (1 x 10(4) and 4 x 10(4) cells ml-1 respectively).

Acetates↗
PubMed2000

The effect of a methanogen, Methanobrevibacter smithii, on the growth rate, organic acid production, and specific ATP activity of three predominant ruminal cellulolytic bacteria.

Three predominant ruminal cellulolytic organisms, Fibrobacter succinogenes S85, Ruminococcus albus 8, and R. flavefaciens FD-1, were cultured with a methanogen, Methanobrevibacter smithii. Growth rates, bacterial protein, organic acids, and methane production were measured. When grown in diculture with the methanogen, a fermentative advantage was observed with F. succinogenes S85 as seen by an increase in specific rate of ATP production and organic acid concentration. The introduction of the methanogen did not improve the growth rate, organic acid yield, or specific rate of ATP production for R. albus 8. The growth rate and amount of organic acid end products increased when R. flavefaciens FD-1 was cultured with the methanogen; however, the specific activity of ATP production did not increase.

Adenosine Triphosphate↗
PubMed2006

Modelling MSW decomposition under landfill conditions considering hydrolytic and methanogenic inhibition.

A landfill typically progresses through a series of microbial degradation phases, in which hydrolysis, production and consumption of fermentation products, such as fatty acids, and methane formation play important roles. For ultimate degradation of the waste, stable methanogenic conditions have to be attained, and maintained for sufficient time. Using experimental data from 100-L landfill simulation reactors containing municipal solid waste from a residential area, a distributed model, which accounts for vertical water flow, was developed. As a first step, the waste was divided into two fractions: readily degradable and recalcitrant waste. Secondly, the general hydrolysis of the recalcitrant waste was accounted for by including a specific, well-defined chemical substance in the model that generally occurs in Municipal Solid Waste (MSW) and is hydrolysed before its further degradation to methane. For this purpose we chose diethyl phthalate and its hydrolysis product monoethyl phthalate, for which leachate data are available from the reactors. The model indicated that inhibition of the hydrolytic and methanogenic processes occurred during the acidogenic phase and that it could be overcome either by improving the chemical environment or by the complete oxidation of the inhibiting, i.e. the easily degraded, fraction of the waste. The generality of the model was confirmed by the patterns of the phthalate di- and monoester transformations obtained. The validity of the model was further confirmed using experimental data from parallel reactors, which were subjected to either leachate exchange with an already methanogenic reactor or to initial aeration to force the reactor into stable methanogenic conditions.

Biodegradation, Environmental↗
PubMed1992

Interrelations between populations of methanogenic archaea and sulfate-reducing bacteria in the human colon.

In humans, CH4 is produced in the colon by methanogenic archaea and is detected in breath samples from approximately 50% of healthy adults, identified as CH4-excretors. Methanogenesis and sulfate reduction have been described as two mutually exclusive processes, potentially regulated by sulfate availability. To determine whether microbial population balances reflected these apparently co-regulated activities, we compared sulfate-reducing bacteria, methanogenic archaea, sulfate and sulfide concentrations in faeces of 10 CH4-excretors (CH4+) and 9 non-CH4-excretors (CH4-). The mean +/- SE of the logarithm of methanogenic archaea per gram wet weight were 9.0 +/- 0.2 and 4.0 +/- 0.7 for CH4+ and CH4-, respectively (P < 0.001). Sulfate-reducing bacterial counts were 6.5 +/- 0.1 and 7.3 +/- 0.2, respectively (P < 0.001). Fecal sulfate and sulfide concentrations did not differ between groups. These results suggest that a competitive interrelation between methanogenic archaea and sulfate-reducing bacteria occurs in the human colon. However, it does not lead to a complete exclusion of the two populations.

Adolescent↗
PubMed2004

Methanogenic symbionts of anaerobic ciliates and their contribution to methanogenesis in an anoxic rice field soil.

Methanogenesis in rice field soils starts soon after flooding while potentially competing processes like reduction of sulphate and iron take place. Early methanogenesis is mainly driven by hydrogen, while later in the season acetate tends to become more important. Anaerobic ciliates are abundant during this period, and their endosymbionts use hydrogen produced by the ciliates to reduce carbon dioxide to methane. These endosymbiotic methanogens are protected from the competition for substrates with other bacteria that may control methanogenesis outside the protozoan cells. Thus, we focussed on early methanogenesis and on the potential contribution from ciliates and their endosymbionts. Only ciliates of the genus Metopus were found to harbour methanogens, as identified by the F(420)-fluorescence of the endosymbionts. We followed the population dynamics of the ciliates with time, and calculated the ratio of symbiotic methane production to overall methanogenesis. Symbiotic methane production was calculated from the species-specific numbers of methanogenic endosymbionts times the cell-specific methane production of the symbionts. According to this calculation, the symbionts' contribution to overall methane production was only 6.4% at the beginning and decreased with time. In a second experiment, colchicine and cycloheximide were used to inhibit all eukaryotes, comparing the remaining methane production rate to a control without inhibitors. In the inhibition experiment, the contribution from symbionts decreased from 40% to 6% during the first days after flooding, and dropped to near zero within 2 weeks. However, nearly all methane produced from H(2)/CO(2) could be attributed to the ciliates' symbionts between days 5 and 10 after flooding. Both experiments showed that the contribution of methanogenic symbionts to overall methane production is a transient phenomenon, restricted to the first 2 weeks.

Animals↗
PubMed2005

Trophic interactions in the methanogenic microbial community of low-temperature terrestrial ecosystems.

The formation of methane in various ecosystems is due to the functioning of an anaerobic community, which combines trophically different groups of microorganisms. The methanogenic microbial community is a complex biological system, which responds to low temperatures by changes in its trophic structure resulting in redistributing matter flows. The enhanced activity of homoacetogenic bacteria at low temperature plays a significant role in this redistribution. Due to their relatively high growth rates and metabolic versatility, homoacetogens can successfully compete with fermenting bacteria and hydrogenotrophic methanogenic archaea for common substrates. The concentration of hydrogen is an important regulatory factor in the psychroactive methanogenic community. At low temperature methanogenic archaea possessing a higher affinity for hydrogen than homoacetogens provide for interspecies H2 transport in syntrophic reactions of fatty acid decomposition. The formation of a balanced community at low temperature is a longtime process. Cold terrestrial ecosystems are dominated by psychroactive (psychrotolerant) microorganisms, which can grow over a wide range of ambient temperatures.

Archaea↗
PubMed2005

PCR-based DGGE fingerprinting and identification of methanogens detected in three different types of UASB granules.

Methane is produced by various methanogenic bacteria present in upflow anaerobic sludge blanket (UASB) bioreactors. Methane can be used to predict and improve UASB bioreactor efficiency. The methanogen population in the granules can be influenced by the composition of the substrate. The aim of this study was to fingerprint and identify the methanogens present in three different types of UASB granules that had been used to treat winery, brewery and peach-lye canning effluents. This was done using polymerase chain reaction (PCR)-based denaturing gradient gel electrophoresis (DGGE) and DNA sequence analysis. The DGGE fingerprints obtained from the methanogen reference cultures of Methanosaeta concilii, Methanosaeta thermophila, Methanosarcina barkeri, Methanosarcina mazeii and Methanobacterium formicicum were compared to the DGGE profiles of the Archaea in the different granules. The positions of the DGGE bands that did not correspond well to the bands of the known species were sequenced and compared to sequences available on GenBank using the Blastn search option. The aligned DNA sequences were used to construct a phylogenetic tree. Based on the data obtained, a DGGE marker was constructed which was used to provide a quick method to identify the Archaeal members of the microbial consortium in UASB granules.

Base Sequence↗