Activity and Diversity of Methanogens in a Petroleum Hydrocarbon-Contaminated Aquifer

Methanogenic activity was investigated in a petroleum hydrocarbon-contaminated aquifer by using a series of four push-pull tests with acetate, formate, H₂ plus CO₂, or methanol to target different groups of methanogenic Archaea. Furthermore, the community composition of methanogens in water and aquifer material was explored by molecular analyses, i.e., fluorescence in situ hybridization (FISH), denaturing gradient gel electrophoresis (DGGE) of 16S rRNA genes amplified with the Archaea-specific primer set ARCH915 and UNI-b-rev, and sequencing of DNA from dominant DGGE bands. Molecular analyses were subsequently compared with push-pull test data. Methane was produced in all tests except for a separate test where 2-bromoethanesulfonate, a specific inhibitor of methanogens, was added. Substrate consumption rates were 0.11 mM day⁻¹ for methanol, 0.38 mM day⁻¹ for acetate, 0.90 mM day⁻¹ for H₂, and 1.85 mM day⁻¹ for formate. Substrate consumption and CH₄ production during all tests suggested that at least three different physiologic types of methanogens were present: H₂ plus CO₂ or formate, acetate, and methanol utilizers. The presence of 15 to 20 bands in DGGE profiles indicated a diverse archaeal population. High H₂ and formate consumption rates agreed with a high diversity of methanogenic Archaea consuming these substrates (16S rRNA gene sequences related to several members of the Methanomicrobiaceae) and the detection of Methanomicrobiaceae by using FISH (1.4% of total DAPI [4′,6-diamidino-2-phenylindole]-stained microorganisms in one water sample; probe MG1200). Considerable acetate consumption agreed with the presence of sequences related to the obligate acetate degrader Methanosaeata concilii and the detection of this species by FISH (5 to 22% of total microorganisms; probe Rotcl1). The results suggest that both aceticlastic and CO₂-type substrate-consuming methanogens are likely involved in the terminal step of hydrocarbon degradation, while methanogenesis from methanol plays a minor role. DGGE profiles further indicate similar archaeal community compositions in water and aquifer material. The combination of hydrogeological and molecular methods employed in this study provide improved information on the community and the potential activity of methanogens in a petroleum hydrocarbon-contaminated aquifer.     

Occurrence of Methanogenic Archaea in Highly Polluted Sediments of Tropical Santos–São Vicente Estuary (São Paulo, Brazil)

Little is known about the ability of methanogens to grow and produce methane in estuarine environments. In this study, traditional methods for cultivating strictly anaerobic microorganisms were combined with Fluorescence in situ hybridization (FISH) technique to enrich and identify methanogenic Archaea cultures occurring in highly polluted sediments of tropical Santos–São Vicente Estuary (São Paulo, Brazil). Sediment samples were enriched at 30°C under strict anaerobic and halophilic conditions, using a basal medium containing 2% of sodium chloride and amended with glucose, methanol, and sodium salts of acetate, formate and lactate. High methanogenic activity was detected, as evidenced by the biogas containing 11.5 mmol of methane at 20 days of incubation time and methane yield of 0.138-mmol CH₄/g organic matter/g volatile suspense solids. Cells of methanogenic Archaea were selected by serial dilution in medium amended separately with sodium acetate, sodium formate, or methanol. FISH analysis revealed the presence of Methanobacteriaceae and Methanosarcina sp. cells.     

Fundamentals of methanogenic pathways that are key to the biomethanation of complex biomass

The conversion of biomass to CH4 (biomethanation) involves an anaerobic microbial food chain composed of at least three metabolic groups of which the first two decompose the complex biomass primarily to acetate, formate, and H2. The thermodynamics of these conversions are unfavorable requiring a symbiosis with the CH4-producing group (methanogens) that metabolize the decomposition products to favorable concentrations. The methanogens produce CH4 by two major pathways, conversion of the methyl group of acetate and reduction of CO2 coupled to the oxidation of formate or H2. This review covers recent advances in the fundamental understanding of both methanogenic pathways with the view of stimulating research towards improving the rate and reliability of the overall biomethanation process.     

Microbial community analysis of rectal methanogens and sulfate reducing bacteria in two non-human primate species

Background  Methanogenesis by methanogenic Archaea and sulfate reduction by sulfate reducing bacteria (SRB) are the major hydrogenotrophic pathways in the human colon. Methanogenic status of mammals is suggested to be under evolutionary rather than dietary control. However, information is lacking regarding the dynamics of hydrogenotrophic microbial communities among different primate species.
Methods  Rectal swabs were collected from 10 sooty mangabeys ( Cercocebus atys ) and 10 baboons ( Papio hamadryas ). The diversity and abundance of methanogens and SRB were examined using PCR-denaturing gradient gel electrophoresis (DGGE) and real-time quantitative PCR (qPCR).
Results  The DGGE results revealed that intestinal Archaea and SRB communities differ between mangabeys and baboons. Phylogenetic analyses of Archaea DGGE bands revealed two distinct clusters with one representing a putative novel order of methanogenic Archaea. The qPCR detected a similar abundance of methanogens and SRB.
Conclusions  Intestinal Archaea and SRB coexist in these primates, and the community patterns are host species-specific.     

Thermophilic sulfate reduction and methanogenesis with methanol in a high rate anaerobic reactor

Sulfate reduction outcompeted methanogenesis at 65 degrees C and pH 7.5 in methanol and sulfate-fed expanded granular sludge bed reactors operated at hydraulic retention times (HRT) of 14 and 3.5 h, both under methanol-limiting and methanol-overloading conditions. After 100 and 50 days for the reactors operated at 14 and 3.5 h, respectively, sulfide production accounted for 80% of the methanol-COD consumed by the sludge. The specific methanogenic activity on methanol of the sludge from a reactor operated at HRTs of down to 3.5 h for a period of 4 months gradually decreased from 0. 83 gCOD. gVSS(-1). day(-1) at the start to a value of less than 0.05 gCOD. gVSS(-1). day(-1), showing that the relative number of methanogens decreased and eventually became very low. By contrast, the increase of the specific sulfidogenic activity of sludge from 0. 22 gCOD. gVSS(-1). day(-1) to a final value of 1.05 gCOD. gVSS(-1). day(-1) showed that sulfate reducing bacteria were enriched. Methanol degradation by a methanogenic culture obtained from a reactor by serial dilution of the sludge was inhibited in the presence of vancomycin, indicating that methanogenesis directly from methanol was not important. H(2)/CO(2) and formate, but not acetate, were degraded to methane in the presence of vancomycin. These results indicated that methanol degradation to methane occurs via the intermediates H(2)/CO(2) and formate. The high and low specific methanogenic activity of sludge on H(2)/CO(2) and formate, respectively, indicated that the former substrate probably acts as the main electron donor for the methanogens during methanol degradation. As sulfate reduction in the sludge was also strongly supported by hydrogen, competition between sulfate reducing bacteria and methanogens in the sludge seemed to be mainly for this substrate. Sulfate elimination rates of up to 15 gSO(4)(2-)/L per day were achieved in the reactors. Biomass retention limited the sulfate elimination rate.     

Influence of Metronidazole, CO, CO(2), and Methanogens on the Fermentative Metabolism of the Anaerobic Fungus Neocallimastix sp. Strain L2

The effects of metronidazole, CO, methanogens, and CO(2) on the fermentation of glucose by the anaerobic fungus Neocallimastix sp. strain L2 were investigated. Both metronidazole and CO caused a shift in the fermentation products from predominantly H(2), acetate, and formate to lactate as the major product and caused a lower glucose consumption rate and cell protein yield. An increased lactate dehydrogenase activity and a decreased hydrogenase activity were observed in cells grown under both culture conditions. In metronidazole-grown cells, the amount of hydrogenase protein was decreased compared with the amount in cells grown in the absence of metronidazole. When Neocallimastix sp. strain L2 was cocultured with the methanogenic bacterium Methanobrevibacter smithii, the fermentation pattern changed in the opposite direction: H(2) and acetate production increased at the expense of the electron sink products lactate, succinate, and ethanol. A concomitant decrease in the enzyme activities leading to these electron sink products was observed, as well as an increase in the glucose consumption rate and cell protein yield, compared with those of pure cultures of the fungus. Low levels of CO(2) in the gas phase resulted in increased H(2) and lactate formation and decreased production of formate, acetate, succinate, and ethanol, a decreased glucose consumption rate and cell protein yield, and a decrease in most of the hydrogenosomal enzyme activities. None of the tested culture conditions resulted in changed quantities of hydrogenosomal proteins. The results indicate that manipulation of the pattern of fermentation in Neocallimastix sp. strain L2 results in changes in enzyme activities but not in the proliferation or disappearance of hydrogenosomes.     

Field-scale isotopic labeling of phospholipid fatty acids from acetate-degrading sulfate-reducing bacteria

Isotopic labeling of biomarker molecules is a technique applied to link microbial community structure with activity. Previously, we successfully labeled phospholipid fatty acids (PLFA) of suspended nitrate-reducing bacteria in an aquifer. However, the application of the method to low energy-yielding processes such as sulfate reduction, and extension of the analysis to attached communities remained to be studied. To test the feasibility of the latter application, an anoxic test solution of 500 l of groundwater with addition of 0.5 mM Br- as a conservative tracer, 1.1 mM SO4(2-), and 2.0 mM [2-13C]acetate was injected in the transition zone of a petroleum hydrocarbon-contaminated aquifer where sulfate-reducing and methanogenic conditions prevailed. Thousand liters of test solution/groundwater mixture were extracted in a stepwise fashion after 2-46 h incubation. Computed apparent first-order rate coefficients were 0.31+/-0.04 day(-1) for acetate and 0.34+/-0.05 day(-1) for SO4(2-) consumption. The delta13C increased from -71.03 per thousand to +3352.50 per thousand in CH4 and from -16.15 per thousand to +32.13 per thousand in dissolved inorganic carbon (DIC). A mass balance suggested that 43% of the acetate-derived (13)C appeared in DIC and 57% appeared in CH4. Thus, acetate oxidation coupled to sulfate reduction and acetoclastic methanogenesis occurred simultaneously. The delta13C of PLFA increased on average by 27 per thousand in groundwater samples and 4 per thousand in sediment samples. Hence, both suspended and attached communities actively degraded acetate. The PLFA labeling patterns and fluorescent in situ hybridization (FISH) analyses of sediment and groundwater samples suggested that the main sulfate-reducing bacteria degrading the acetate were Desulfotomaculum acetoxidans and Desulfobacter sp. in groundwater, and D. acetoxidans in sediment.     

Activity and diversity of sulfate-reducing bacteria in a petroleum hydrocarbon-contaminated aquifer

Microbial sulfate reduction is an important metabolic activity in petroleum hydrocarbon (PHC)-contaminated aquifers. We quantified carbon source-enhanced microbial SO(4)(2-) reduction in a PHC-contaminated aquifer by using single-well push-pull tests and related the consumption of sulfate and added carbon sources to the presence of certain genera of sulfate-reducing bacteria (SRB). We also used molecular methods to assess suspended SRB diversity. In four consecutive tests, we injected anoxic test solutions (1,000 liters) containing bromide as a conservative tracer, sulfate, and either propionate, butyrate, lactate, or acetate as reactants into an existing monitoring well. After an initial incubation period, 1,000 liters of test solution-groundwater mixture was extracted from the same well. Average total test duration was 71 h. We measured concentrations of bromide, sulfate, and carbon sources in native groundwater as well as in injection and extraction phase samples and characterized the SRB population by using fluorescence in situ hybridization (FISH) and denaturing gradient gel electrophoresis (DGGE). Enhanced sulfate reduction concomitant with carbon source degradation was observed in all tests. Computed first-order rate coefficients ranged from 0.19 to 0.32 day(-1) for sulfate reduction and from 0.13 to 0.60 day(-1) for carbon source degradation. Sulfur isotope fractionation in unconsumed sulfate indicated that sulfate reduction was microbially mediated. Enhancement of sulfate reduction due to carbon source additions in all tests and variability of rate coefficients suggested the presence of specific SRB genera and a high diversity of SRB. We confirmed this by using FISH and DGGE. A large fraction of suspended bacteria hybridized with SRB-targeting probes SRB385 plus SRB385-Db (11 to 24% of total cells). FISH results showed that the activity of these bacteria was enhanced by addition of sulfate and carbon sources during push-pull tests. However, DGGE profiles indicated that the bacterial community structure of the dominant species did not change during the tests. Thus, the combination of push-pull tests with molecular methods provided valuable insights into microbial processes, activities, and diversity in the sulfate-reducing zone of a PHC-contaminated aquifer.     

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 is 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.     

Cultivation of methanogens from shallow marine sediments at Hydrate Ridge, Oregon

Little is known about the methanogenic degradation of acetate, the fate of molecular hydrogen and formate or the ability of methanogens to grow and produce methane in cold, anoxic marine sediments. The microbes that produce methane were examined in permanently cold, anoxic marine sediments at Hydrate Ridge (44 degrees 35' N, 125 degrees 10' W, depth 800 m). Sediment samples (15 to 35 cm deep) were collected from areas of active methane ebullition or areas where methane hydrates occurred. The samples were diluted into enrichment medium with formate, acetate or trimethylamine as catabolic substrate. After 2 years of incubation at 4 degrees C to 15 degrees C, enrichment cultures produced methane. PCR amplification and sequencing of the rRNA genes from the highest dilutions with growth suggested that each enrichment culture contained a single strain of methanogen. The level of sequence similarity (91 to 98%) to previously characterized prokaryotes suggested that these methanogens belonged to novel genera or species within the orders Methanomicrobiales and Methanosarcinales. Analysis of the 16S rRNA gene libraries from DNA extracted directly from the sediment samples revealed phylotypes that were either distantly related to cultivated methanogens or possible anaerobic methane oxidizers related to the ANME-1 and ANME-2 groups of the Archaea. However, no methanogenic sequences were detected, suggesting that methanogens represented only a small proportion of the archaeal community.     

Methanogenic activity and diversity in the centre of the Amsterdam Mud Volcano, Eastern Mediterranean Sea

Marine mud volcanoes are geological structures emitting large amounts of methane from their active centres. The Amsterdam mud volcano (AMV), located in the Anaximander Mountains south of Turkey, is characterized by intense active methane seepage produced in part by methanogens. To date, information about the diversity or the metabolic pathways used by the methanogens in active centres of marine mud volcanoes is limited. (14)C-radiotracer measurements showed that methylamines/methanol, H(2)/CO(2) and acetate were used for methanogenesis in the AMV. Methylotrophic methanogenesis was measured all along the sediment core, Methanosarcinales affiliated sequences were detected using archaeal 16S PCR-DGGE and mcrA gene libraries, and enrichments of methanogens showed the presence of Methanococcoides in the shallow sediment layers. Overall acetoclastic methanogenesis was higher than hydrogenotrophic methanogenesis, which is unusual for cold seep sediments. Interestingly, acetate porewater concentrations were extremely high in the AMV sediments. This might be the result of organic matter cracking in deeper hotter sediment layers. Methane was also produced from hexadecanes. For the most part, the methanogenic community diversity was in accordance with the depth distribution of the H(2)/CO(2) and acetate methanogenesis. These results demonstrate the importance of methanogenic communities in the centres of marine mud volcanoes.     

Additional characteristics of one-carbon-compound utilization by Eubacterium limosum and Acetobacterium woodii

Growth characteristics of Eubacterium limosum and Acetobacterium woodii during one-carbon-compound utilization were investigated. E. limosum RF grew with formate as the sole energy source. Formate also replaced a requirement for CO2 during growth with methanol. Growth with methanol required either rumen fluid, yeast extract, or acetate, but their effects were not additive. Cultures were adapted to grow in concentrations of methanol of up to 494 mM. Growth occurred with methanol in the presence of elevated levels of Na+ (576 mM). The pH optima for growth with methanol, H2-CO2, and carbon monoxide were similar (7.0 to 7.2). Growth occurred with glucose at a pH of 4.7, but not at 4.0. The apparent Km values for methanol and hydrogen were 2.7 and 0.34 mM, respectively. The apparent Vmax values for methanol and hydrogen were 1.7 and 0.11 mumol/mg of protein X min-1, respectively. The Ks value for CO was estimated to be less than 75 microM. Cellular growth yields were 70.5, 7.1, 3.38, and 0.84 g (dry weight) per mol utilized for glucose, methanol, CO, and hydrogen (in H2-CO2), respectively. E. limosum was also able to grow with methoxylated aromatic compounds as energy sources. Glucose apparently repressed the ability of E. limosum to use methanol, hydrogen, or isoleucine but not CO. Growth with mixtures of methanol, H2, CO, or isoleucine was not diauxic. The results, especially the relatively high apparent Km values for H2 and methanol, may indicate why E. limosum does not usually compete with rumen methanogens for these energy sources.(ABSTRACT TRUNCATED AT 250 WORDS)     

The bioenergetics of methanogenesis

The reduction of CO2 or any other methanogenic substrate to methane serves the same function as the reduction of oxygen, nitrate or sulfate to more reduced products. These exergonic reactions are coupled to the production of usable energy generated through a charge separation and a protonmotive-force-driven ATPase. For the understanding of how methanogens derive energy from C-1 unit reduction one must study the biochemistry of the chemical reactions involved and how these are coupled to the production of a charge separation and subsequent electron transport phosphorylation. Data on methanogenesis by a variety of organisms indicates ubiquitous use of CH3-S-CoM as the final electron acceptor in the production of methane through the methyl CoM reductase and of 5-deazaflavin as a primary source of reducing equivalents. Three known enzymes serve as catalysts in the production of reduced 5-deazaflavin: hydrogenase, formate dehydrogenase and CO dehydrogenase. All three are potential candidates for proton pumps. In the organisms that must oxidize some of their substrate to obtain electrons for the reduction of another portion of the substrate to methane (e.g., those using formate, methanol or acetate), the latter two enzymes may operate in the oxidizing direction. CO2 is the most frequent substrate for methanogenesis but is the only substrate that obligately requires the presence of H2 and hydrogenase. Growth on methanol requires a B12-containing methanol-CoM methyl transferase and does not necessarily need any other methanogenic enzymes besides the methyl-CoM reductase system when hydrogenase is present. When bacteria grow on methanol alone it is not yet clear if they get their reducing equivalents from a reversal of methanogenic enzymes, thus oxidizing methyl groups to CO2. An alternative (since these and acetate-catabolizing methanogens possess cytochrome b) is electron transport and possible proton pumping via a cytochrome-containing electron transport chain. Several of the actual components of the methanogenic pathway from CO2 have been characterized. Methanofuran is apparently the first carbon-carrying cofactor in the pathway, forming carboxy-methanofuran. Formyl-FAF or formyl-methanopterin (YFC, a very rapidly labelled compound during 14C pulse labeling) has been implicated as an obligate intermediate in methanogenesis, since methanopterin or FAF is an essential component of the carbon dioxide reducing factor in dialyzed extract methanogenesis. FAF also carries the carbon at the methylene and methyl oxidation levels.(ABSTRACT TRUNCATED AT 400 WORDS)     

Additional characteristics of one-carbon-compound utilization by Eubacterium limosum and Acetobacterium woodii.

Growth characteristics of Eubacterium limosum and Acetobacterium woodii during one-carbon-compound utilization were investigated. E. limosum RF grew with formate as the sole energy source. Formate also replaced a requirement for CO2 during growth with methanol. Growth with methanol required either rumen fluid, yeast extract, or acetate, but their effects were not additive. Cultures were adapted to grow in concentrations of methanol of up to 494 mM. Growth occurred with methanol in the presence of elevated levels of Na+ (576 mM). The pH optima for growth with methanol, H2-CO2, and carbon monoxide were similar (7.0 to 7.2). Growth occurred with glucose at a pH of 4.7, but not at 4.0. The apparent Km values for methanol and hydrogen were 2.7 and 0.34 mM, respectively. The apparent Vmax values for methanol and hydrogen were 1.7 and 0.11 mumol/mg of protein X min-1, respectively. The Ks value for CO was estimated to be less than 75 microM. Cellular growth yields were 70.5, 7.1, 3.38, and 0.84 g (dry weight) per mol utilized for glucose, methanol, CO, and hydrogen (in H2-CO2), respectively. E. limosum was also able to grow with methoxylated aromatic compounds as energy sources. Glucose apparently repressed the ability of E. limosum to use methanol, hydrogen, or isoleucine but not CO. Growth with mixtures of methanol, H2, CO, or isoleucine was not diauxic. The results, especially the relatively high apparent Km values for H2 and methanol, may indicate why E. limosum does not usually compete with rumen methanogens for these energy sources.(ABSTRACT TRUNCATED AT 250 WORDS)     

Adaptation of mesophilic anaerobic sewage fermentor populations to thermophilic temperatures.

Thermophilic (50 degrees C) and obligately thermophilic (60 degrees C) anaerobic carbohydrate- and protein-digesting and methanogenic bacterial populations were enumerated in a mesophilic (35 degrees C) fermentor anaerobically digesting municipal primary sludge. Of the total bacterial population in the mesophilic fementor, 9% were thermophiles (36 x 10(6)/ml) and 1% were obligate thermophiles (4.5 x 10(6)/ml). Of these 10%, the percentages of bacteria (thermophiles and obligate thermophiles, respectively) able to use specific substrates were further enumerated as follows: bacteria able to digest albumin, casein, starch, and mono- and disaccharides, 30 and 10%; pectin degraders, 10 and 0.2%; cellulose degraders, 2 and 0.06%; methanogens that grow with H2 and CO2, methanol, and dimethylamine, 9 and 1%; methanogens that grow with formate, 8 and 5%; and methanogens that grow with acetate, 25 and less than 0.8%. Shortly after the temperature was elevated from 35 to 50 or 60 degrees C, the digestion of albumin, casein, starch, and mono- and disaccharides was detected, and methane was produced from H2 and CO2. Methane produced from acetate was not delayed at 50 degrees C, but was delayed by 29 days at 60 degrees C. Methane produced from formate was delayed by 3 days, from methanol by 7 days, and from dimethylamine by 5 days at 50 and 60 degrees C. A 10- and 20-day acclimation period was required for hydrolysis of pectin and cellulose, respectively, at 50 degrees C. Digestion of pectin required 20 days and cellulose longer than 85 days when the temperature was elevated abruptly from 35 to 60 degrees C. The acclimation period for the digestion of pectin and cellulose at 60 degrees C was shortened to 3 and 15 days, respectively, by seeding with a small amount of a culture acclimated to 50 degrees C. The data suggest that enrichment of cellulolytic, pectinolytic, and acetate-utilizing bacteria is crucial for the digestion of sewage sludge at 60 degrees C.     

Methanogenic communities in permafrost-affected soils of the Laptev Sea coast, Siberian Arctic, characterized by 16S rRNA gene fingerprints

Permafrost environments in the Arctic are characterized by extreme environmental conditions that demand a specific resistance from microorganisms to enable them to survive. In order to understand the carbon dynamics in the climate-sensitive Arctic permafrost environments, the activity and diversity of methanogenic communities were studied in three different permafrost soils of the Siberian Laptev Sea coast. The effect of temperature and the availability of methanogenic substrates on CH4 production was analysed. In addition, the diversity of methanogens was analysed by PCR with specific methanogenic primers and by denaturing gradient gel electrophoresis (DGGE) followed by sequencing of DGGE bands reamplified from the gel. Our results demonstrated methanogenesis with a distinct vertical profile in each investigated permafrost soil. The soils on Samoylov Island showed at least two optima of CH4 production activity, which indicated a shift in the methanogenic community from mesophilic to psychrotolerant methanogens with increasing soil depth. Furthermore, it was shown that CH4 production in permafrost soils is substrate-limited, although these soils are characterized by the accumulation of organic matter. Sequence analyses revealed a distinct diversity of methanogenic archaea affiliated to Methanomicrobiaceae, Methanosarcinaceae and Methanosaetaceae. However, a relationship between the activity and diversity of methanogens in permafrost soils could not be shown.     

Distribution of cytochromes in methanogenic bacteria

Abstract Various methanogenic bacteria belonging to the orders Methanobacteriales, Methanococcales , and Methanomicrobiales were examined for the presence of cytochromes. Those methanogens which are capable of growing only on H ₂+ CO ₂ or formate were found to lack cytochromes. However, membrane-bound cytochromes were detected in species able to utilize methanol, methylamines or acetate.     

Investigation of the methanogen population structure and activity in a brackish lake sediment

The methanogen community in sediment from the edge of a small brackish lake connected to the Beaulieu Estuary (Hampshire, UK) was investigated by analysis of 16S rRNA gene diversity using new methanogen-specific primers plus Archaea-specific primers. 16S rRNA gene primers previously used for polymerase chain reaction (PCR) detection of methanogenic Archaea from a variety of environments were evaluated by in silico testing. The primers displayed variable coverage of the four main orders of methanogens, highlighting the importance of this type of primer evaluation. Three PCR primer sets were designed using novel reverse primers to facilitate specific amplification of the orders Methanomicrobiales/Methanosarcinales, Methanobacteriales and Methanococcales. Diversity of the methanogen functional gene, methyl coenzyme M reductase (mcrA), was also studied. All gene libraries constructed from this sediment indicated that Methanomicrobiales and Methanosarcinales were the only methanogens detected. There was good agreement between the relative sequence abundances in the methanogen-specific 16S rRNA gene library and terminal restriction fragment length polymorphism (T-RFLP) profiling, suggesting that the population was dominated by putative H2 CO2 utilizing Methanomicrobiales, although acetate-utilizing methanogens were also present. The methanogen population analyses were in agreement with methanogenic activity measurements, which indicated that bicarbonate methanogenesis was higher than acetate methanogenesis at all depths measured and overall there was a significant difference (P = 0.001) between the rates of the two pathways. This study demonstrates the utility of new 16S rRNA gene PCR primers targeting specific methanogenic orders, and the combined results suggest that the CO2 reduction pathway dominates methanogenesis in the brackish sediment investigated.     

Azo dye reduction by mesophilic and thermophilic anaerobic consortia

The reduction of the azo dye model compounds Reactive Red 2 (RR2) and Reactive Orange 14 (RO14) by mesophilic (30 degrees C) and thermophilic (55 degrees C) anaerobic consortia was studied in batch assays. The contribution of fermentative and methanogenic microorganisms in both temperatures was evaluated in the presence of the fermentative substrate glucose and the methanogenic substrates acetate, H2/CO2, methanol, and formate. Additionally, the effect of the redox mediator riboflavin on electron shuttling was assessed. We concluded that the application of thermophilic anaerobic treatment is an interesting option for the reductive decolorization of azo dyes compared to mesophilic conditions. The use of high temperature may decrease or even take the place of the need for continuous redox mediator dosage in bioreactors, contrarily to the evident effect of those compounds on dye reduction under mesophilic conditions. Both fermenters and methanogens may play an important role during reductive decolorization of dyes, in which mediators are important not only for allowing the different microbes to participate more effectively in this complex reductive biochemistry but also for assisting in the competition for electrons between dyes and other organic and inorganic electron acceptors.     

Relationship of formate to growth and methanogenesis by Methanococcus thermolithotrophicus

Methanococcus thermolithotrophicus is a methanogenic archaebacterium that can use either H2 or formate as its source of electrons for reduction of CO2 to methane. Growth and suspended-whole-cell experiments show that H2 plus CO2 methanogenesis was constitutive, while formate methanogenesis required adaptation time; selenium was necessary for formate utilization. Cells grown on formate had 20 to 100 times higher methanogenesis rates on formate than cells grown on H2-CO2 and transferred into formate medium. Enzyme assays with crude extracts and with F420 or methyl viologen as the electron acceptor revealed that hydrogenase was constitutive, while formate dehydrogenase was regulated. Cells grown on formate had 10 to 70 times higher formate dehydrogenase activity than cells grown on H2-CO2 with Se present in the medium; when no Se was added to H2-CO2 cultures, even lower activities were observed. Adaptation to and growth on formate were pH dependent, with an optimal pH for both about one pH unit above that optimal for H2-CO2 (pH 5.8 to 6.5). When cells were grown on H2-CO2 in the presence of formate, formate (greater than or equal to 50 mM) inhibited both growth and methanogenesis at pH 5.8 to 6.2, but not at pH greater than 6.6. Both acetate and propionate produced similar inhibition. Formate inhibition was also observed in Methanospirillum hungatei.