Determination of isotope fractionation factors and quantification of carbon flow by stable carbon isotope signatures in a methanogenic rice root model system

Methanogenic processes can be quantified by stable carbon isotopes, if necessary modeling parameters, especially fractionation factors, are known. Anoxically incubated rice roots are a model system with a dynamic microbial community and thus suitable to investigate principal geochemical processes in anoxic natural systems. Here we applied an inhibitor of acetoclastic methanogenesis (methyl fluoride), calculated the thermodynamics of the involved processes, and analyzed the carbon stable isotope signatures of CO₂, CH₄, propionate, acetate and the methyl carbon of acetate to characterize the carbon flow during anaerobic degradation of rice roots to the final products CO₂ and CH₄. Methyl fluoride inhibited acetoclastic methanogenesis and thus allowed to quantify the fractionation factor of CH₄ production from H₂/CO₂. Since our model system was not affected by H₂ gradients, the fractionation factor could alternatively be determined from the Gibbs free energies of hydrogenotrophic methanogenesis. The fractionation factor of acetoclastic methanogenesis was also experimentally determined. The data were used for successfully modeling the carbon flow. The model results were in agreement with the measured process data, but were sensitive to even small changes in the fractionation factor of hydrogenotrophic methanogenesis. Our study demonstrates that stable carbon isotope signatures are a proper tool to quantify carbon flow, if fractionation factors are determined precisely.     

Biogas production using anaerobic groundwater containing a subterranean microbial community associated with the accretionary prism

In a deep aquifer associated with an accretionary prism, significant methane (CH₄) is produced by a subterranean microbial community. Here, we developed bioreactors for producing CH₄ and hydrogen (H₂) using anaerobic groundwater collected from the deep aquifer. To generate CH₄, the anaerobic groundwater amended with organic substrates was incubated in the bioreactor. At first, H₂ was detected and accumulated in the gas phase of the bioreactor. After the H₂ decreased, rapid CH₄ production was observed. Phylogenetic analysis targeting 16S rRNA genes revealed that the H₂-producing fermentative bacterium and hydrogenotrophic methanogen were predominant in the reactor. The results suggested that syntrophic biodegradation of organic substrates by the H₂-producing fermentative bacterium and the hydrogenotrophic methanogen contributed to the CH₄ production. For H₂ production, the anaerobic groundwater, amended with organic substrates and an inhibitor of methanogens (2-bromoethanesulfonate), was incubated in a bioreactor. After incubation for 24 h, H₂ was detected from the gas phase of the bioreactor and accumulated. Bacterial 16S rRNA gene analysis suggested the dominance of the H₂-producing fermentative bacterium in the reactor. Our study demonstrated a simple and rapid CH₄ and H₂ production utilizing anaerobic groundwater containing an active subterranean microbial community.     

A pilot scale two-stage anaerobic digester treating food waste leachate (FWL): Performance and microbial structure analysis using pyrosequencing

Food waste leachate (FWL) from the food waste recycling facilities in Korea is a serious environmental problem. Much research was done on anaerobic digestion of FWL in a lab-scale; however, there is little information on a large scale anaerobic digestion system (ADS). In this study, a two-phase ADS in a pilot scale was operated using FWL and the ADS performance and microbial structure dynamics using pyrosequencing were investigated. The ADS was operated for 136 days using FWL containing a high concentration of volatile fatty acid (12,435 ± 2203 mg/L), exhibiting volatile acid (VS) removal efficiency of 74–89% and CH₄ yield of 0.39–0.85 Nm³/kg of reduced VS. The microbial structure at 76, 101, and 132 days indicated the methanogen population shift from acetoclastic methanogens (Methanosarcina and Methanosaeta) to hydrogenotrophic methanogens (Methanobacterium and Methanoculleus). The bacterial community also shifted to the taxa syntrophically related with hydrogenotrophic methanogens (Clostridia). The statistical analysis revealed the positive correlation of VS removal efficiency with Methanosarcina, but the negative correlation with Methanobacterium. The results presented here suggest that acetoclastic methanogens and their associated bacteria were more efficient for VS removal in the pilot scale ADS system, providing useful information for FWL treatment in a large scale ADS.     

Hydrogenotrophic Methanogenesis by Moderately Acid-Tolerant Methanogens of a Methane-Emitting Acidic Peat

The emission of methane (1.3 mmol of CH₄ m⁻² day⁻¹), precursors of methanogenesis, and the methanogenic microorganisms of acidic bog peat (pH 4.4) from a moderately reduced forest site were investigated by in situ measurements, microcosm incubations, and cultivation methods, respectively. Bog peat produced CH₄ (0.4 to 1.7 μmol g [dry wt] of soil⁻¹ day⁻¹) under anoxic conditions. At in situ pH, supplemental H₂-CO₂, ethanol, and 1-propanol all increased CH₄ production rates while formate, acetate, propionate, and butyrate inhibited the production of CH₄; methanol had no effect. H₂-dependent acetogenesis occurred in H₂-CO₂-supplemented bog peat only after extended incubation periods. Nonsupplemented bog peat initially produced small amounts of H₂ that were subsequently consumed. The accumulation of H₂ was stimulated by ethanol and 1-propanol or by inhibiting methanogenesis with bromoethanesulfonate, and the consumption of ethanol was inhibited by large amounts of H₂; these results collectively indicated that ethanol- or 1-propanol-utilizing bacteria were trophically associated with H₂-utilizing methanogens. A total of 10⁹ anaerobes and 10⁷ hydrogenotrophic methanogens per g (dry weight) of bog peat were enumerated by cultivation techniques. A stable methanogenic enrichment was obtained with an acidic, H₂-CO₂-supplemented, fatty acid-enriched defined medium. CH₄ production rates by the enrichment were similar at pH 4.5 and 6.5, and acetate inhibited methanogenesis at pH 4.5 but not at pH 6.5. A total of 27 different archaeal 16S rRNA gene sequences indicative of Methanobacteriaceae, Methanomicrobiales, and Methanosarcinaceae were retrieved from the highest CH₄-positive serial dilutions of bog peat and methanogenic enrichments. A total of 10 bacterial 16S rRNA gene sequences were also retrieved from the same dilutions and enrichments and were indicative of bacteria that might be responsible for the production of H₂ that could be used by hydrogenotrophic methanogens. These results indicated that in this acidic bog peat, (i) H₂ is an important substrate for acid-tolerant methanogens, (ii) interspecies hydrogen transfer is involved in the degradation of organic carbon, (iii) the accumulation of protonated volatile fatty acids inhibits methanogenesis, and (iv) methanogenesis might be due to the activities of methanogens that are phylogenetic members of the Methanobacteriaceae, Methanomicrobiales, and Methanosarcinaceae.     

Functional and structural response of the methanogenic microbial community in rice field soil to temperature change

The microbial community in anoxic rice field soil produces CH₄ over a wide temperature range up to 55°C. However, at temperatures higher than about 40°C, the methanogenic path changes from CH₄ production by hydrogenotrophic plus acetoclastic methanogenesis to exclusively hydrogenotrophic methanogenesis and simultaneously, the methanogenic community consisting of Methanosarcinaceae, Methanoseataceae, Methanomicrobiales, Methanobacteriales and Rice Cluster I (RC‐1) changes to almost complete dominance of RC‐1. We studied changes in structure and function of the methanogenic community with temperature to see whether microbial members of the community were lost or their function impaired by exposure to high temperature. We characterized the function of the community by the path of CH₄ production measuring δ¹³C in CH₄ and CO₂ and calculating the apparent fractionation factor (α_app) and the structure of the community by analysis of the terminal restriction fragment length polymorphism (T‐RFLP) of the microbial 16S rRNA genes. Shift of the temperature from 45°C to 35°C resulted in a corresponding shift of function and structure, especially when some 35°C soil was added to the 45°C soil. The bacterial community (T‐RFLP patterns), which was much more diverse than the archaeal community, changed in a similar manner upon temperature shift. Incubation of a mixture of 35°C and 50°C pre‐incubated methanogenic rice field soil at different temperatures resulted in functionally and structurally well‐defined communities. Although function changed from a mixture of acetoclastic and hydrogenotrophic methanogenesis to exclusively hydrogenotrophic methanogenesis over a rather narrow temperature range of 42–46°C, each of these temperatures also resulted in only one characteristic function and structure. Our study showed that temperature conditions defined structure and function of the methanogenic microbial community.     

Effect of Inhibition of Acetoclastic Methanogenesis on Growth of Archaeal Populations in an Anoxic Model Environment

Methyl fluoride is frequently used to specifically inhibit acetoclastic methanogenesis, thus allowing determination of the relative contribution of acetate versus H₂/CO₂ to total CH₄ production in natural environments. However, the effect of the inhibitor on growth of the target archaeal population has not yet been studied. Therefore, we incubated rice roots as an environmental model system under anoxic conditions in the presence and absence of CH₃F, measured the activity and Gibbs free energy (ΔG) of CH₄ production, and determined the abundance of individual archaeal populations by using a combination of quantitative (real-time) PCR and analysis of terminal restriction fragment length polymorphism targeting the 16S rRNA gene. It was shown that CH₃F specifically inhibited not only acetoclastic methanogenic activity but also the proliferation of Methanosarcina spp, which were the prevalent acetoclastic methanogens in our environmental model system. Therefore, inhibition experiments with CH₃F seem to be a suitable method for quantifying acetoclastic CH₄ production. It is furthermore shown that the growth and final population size of methanogens were consistent with energetic conditions that at least covered the maintenance requirements of the population.     

Terminal Reactions in the Anaerobic Digestion of Animal Waste

An anaerobic mesophilic digestor was operated using beef cattle waste (diluted to 5.75% volatile solids) as substrate; retention time was 10 days with daily batch feed. Volatile solids destruction was 36%. Daily gas production rate was 1.8 liters of gas (standard temperature and pressure) per liter of digestor contents (0.99 liters of CH₄ per liter of digestor contents). Acetate turnover was measured, and it was calculated that 68% of the CH₄ was derived from the methyl group of acetate. When the methanogenic substrates acetic acid or H₂/CO₂ were added to the digestor on a continuous basis, the microflora were able to adapt and convert them to terminal products while continuing to degrade animal waste to the same extent as without additions. The methanogenic substrates were added at a rate at least 1.5 times the microbial production rate which was measured in the absence of added substrates. Added acetate was converted directly to CH₄ by acetoclastic methanogens; H₂ addition greatly stimulated acetate production in the digestor. A method is described for the measurement of acetate turnover in batch-fed digestors.     

A contribution of hydrogenotrophic methanogenesis to the biogenic coal bed methane reserves of Southern Qinshui Basin,China

The activity of methanogens and related bacteria which inhabit the coal beds is essential for stimulating new biogenic coal bed methane (CBM) production from the coal matrix. In this study, the microbial community structure and methanogenesis were investigated in Southern Qinshui Basin in China, and the composition and stable isotopic ratios of CBM were also determined. Although geochemical analysis suggested a mainly thermogenic origin for CBM, the microbial community structure and activities strongly implied the presence of methanogens in situ. 454 pyrosequencing analysis combined with methyl coenzyme-M reductase (mcrA) gene clone library analysis revealed that the archaeal communities in the water samples from both coal seams were similar, with the dominance of hydrogenotrophic methanogen Methanobacterium. The activity and potential of these populations to produce methane were confirmed by the observation of methane production in enrichments supplemented with H₂ + CO₂ and formate, and the only archaea successfully propagated in the tested water samples was from the genus Methanobacterium. 454 pyrosequencing analysis also recovered the diverse bacterial communities in the water samples, which have the potential to play a role in the coal biodegradation fueling methanogens. These results suggest that the biogenic CBM was generated by coal degradation via the hydrogenotrophic methanogens and related bacteria, which also contribute to the huge CBM reserves in Southern Qinshui Basin, China.     

Co-digestion of manure and whey for in situ biogas upgrading by the addition of H2: process performance and microbial insights

In situ biogas upgrading was conducted by introducing H₂ directly to the anaerobic reactor. As H₂ addition is associated with consumption of the CO₂ in the biogas reactor, pH increased to higher than 8.0 when manure alone was used as substrate. By co-digestion of manure with acidic whey, the pH in the anaerobic reactor with the addition of hydrogen could be maintained below 8.0, which did not have inhibition to the anaerobic process. The H₂ distribution systems (diffusers with different pore sizes) and liquid mixing intensities were demonstrated to affect the gas-liquid mass transfer of H₂ and the biogas composition. The best biogas composition (75:6.6:18.4) was obtained at stirring speed 150 rpm and using ceramic diffuser, while the biogas in the control reactor consisted of CH₄ and CO₂ at a ratio of 55:45. The consumed hydrogen was almost completely converted to CH₄, and there was no significant accumulation of VFA in the effluent. The study showed that addition of hydrogen had positive effect on the methanogenesis, but had no obvious effect on the acetogenesis. Both hydrogenotrophic methanogenic activity and the concentration of coenzyme F₄₂₀ involved in methanogenesis were increased. The archaeal community was also altered with the addition of hydrogen, and a Methanothermobacter thermautotrophicus related band appeared in a denaturing gradient gel electrophoresis gel from the sample of the reactor with hydrogen addition. Though the addition of hydrogen increased the dissolved hydrogen concentration, the degradation of propionate was still thermodynamically feasible at the reactor conditions.     

Mesophilic Fermentation of Renewable Biomass: Does Hydraulic Retention Time Regulate Methanogen Diversity?

The present long-term study (about 1,100 days) monitored the diversity of methanogens during the mesophilic, anaerobic digestion of beet silage. Six fermentor samples were analyzed by ribosomal RNA gene restriction analysis, fluorescence in situ hybridization, and fluorescence microscopy. Hydrogenotrophic methanogens dominated within the population in all samples analyzed. Multidimensional scaling revealed that a rapid decrease in hydraulic retention time resulted in increased species richness, which in turn led to slightly higher CH₄ yields.The anaerobic digestion of renewable biomass is a microbial process wherein a community of fermentative and acetogenic bacteria together with acetoclastic and hydrogenotrophic methanogens convert organic matter into CO₂ and CH₄ (26, 36). Wavering microbial populations at one trophic level might cause a metabolic imbalance, leading to an accumulation of intermediates, pH changes, or reduced methane production efficiency (37), which in turn might even cause a process failure. As information on the influence of process parameters upon population dynamics is scarce (21), this study aimed to verify whether either a substrate change or a change in hydraulic retention time (HRT) could impact the diversity of methanogenic Euryarchaeota generating CH₄. Beets were chosen among other potential crops as a model energy crop because they almost completely lack nondigestible lignin and have the highest energy output (45).     

芦岭煤田微生物群落结构和生物成因气的产甲烷类型研究

【目的】揭示芦岭煤田微生物群落组成,并分析其潜在的产甲烷类型及产甲烷途径。【方法】采集芦岭煤田的煤层气样品和产出水样品,分别分析样品的地球化学性质特征;利用Illumina HiSeq高通量测序技术分析产出水中的微生物群落结构;采用添加不同底物的厌氧培养实验进一步证实芦岭煤田生物成因气的产甲烷类型。【结果】该地区煤层气为生物成因和热成因的混合成因气;古菌16S rRNA基因分析表明在产出水中含有乙酸营养型、氢营养型和甲基营养型的产甲烷菌。丰度较高的细菌具有降解煤中芳香族和纤维素衍生化合物的潜力。厌氧富集培养结果表明,添加乙酸盐、甲酸盐、H2+CO2为底物的矿井水样均有明显的甲烷产生。【结论】芦岭煤田具有丰富的生物多样性,该地区同时存在三种产甲烷类型。本研究为利用微生物技术提高煤层气的采收率,实现煤层气的可持续开采提供科学依据。     

Effect of Temperature on Anaerobic Ethanol Oxidation and Methanogenesis in Acidic Peat from a Northern Wetland

The effects of temperature on rates and pathways of CH₄ production and on the abundance and structure of the archaeal community were investigated in acidic peat from a mire in northern Scandinavia (68°N). We monitored the production of CH₄ and CO₂ over time and measured the turnover of Fe(II), ethanol, and organic acids. All experiments were performed with and without specific inhibitors (2-bromoethanesulfonate [BES] for methanogenesis and CH₃F for acetoclastic methanogenesis). The optimum temperature for methanogenesis was 25°C (2.3 μmol CH₄ · g [dry weight]⁻¹ · day⁻¹), but the activity was relatively high even at 4°C (0.25 μmol CH₄ · g [dry weight]⁻¹ · day⁻¹). The theoretical lower limit for methanogenesis was calculated to be at −5°C. The optimum temperature for growth as revealed by real-time PCR was 25°C for both archaea and bacteria. The population structure of archaea was studied by terminal restriction fragment length polymorphism analysis and remained constant over a wide temperature range. Hydrogenotrophic methanogenesis accounted for about 80% of the total methanogenesis. Most 16S rRNA gene sequences that were affiliated with methanogens and all McrA sequences clustered with the exclusively hydrogenotrophic order Methanobacteriales, correlating with the prevalence of hydrogenotrophic methanogenesis. Fe reduction occurred parallel to methanogenesis and was inhibited by BES, suggesting that methanogens were involved in Fe reduction. Based upon the observed balance of substrates and thermodynamic calculations, we concluded that the ethanol pool was oxidized to acetate by the following two processes: syntrophic oxidation with methanogenesis (i) as an H₂ sink and (ii) as a reductant for Fe(III). Acetate accumulated, but a considerable fraction was converted to butyrate, making volatile fatty acids important end products of anaerobic metabolism.     

End Products of Anaerobic Chitin Degradation by Salt Marsh Bacteria as Substrates for Dissimilatory Sulfate Reduction and Methanogenesis

The anaerobic pathway of chitin decomposition by chitinoclastic bacteria was examined with an emphasis on end product coupling to other salt marsh bacteria. Actively growing chitinoclastic bacterial isolates produced primarily acetate, H₂, and CO₂ in broth culture. No sulfate-reducing or methanogenic isolates grew on chitin as sole carbon source or produced any measurable degradation products. Mixed cultures of chitin degraders with sulfate reducers resulted in positive sulfide production. Mixed cultures of chitin-degrading isolates with methanogens resulted in the production of CH₄ with reductions in headspace CO₂ and H₂. The combination of all three metabolic types resulted in the simultaneous production of methane and sulfide, with more methane being produced in mixed cultures containing CO₂-reducing methanogens and acetoclastic sulfate reducers because of less interspecific H₂ competition.     

Archaeal Community Structure and Pathway of Methane Formation on Rice Roots

The community structure of methanogenic Archaea on anoxically incubated rice roots was investigated by amplification, sequencing, and phylogenetic analysis of 16S rRNA and methyl-coenzyme M reductase (mcrA) genes. Both genes demonstrated the presence of Methanomicrobiaceae, Methanobacteriaceae, Methanosarcinaceae, Methanosaetaceae, and Rice cluster I, an uncultured methanogenic lineage. The pathway of CH₄ formation was determined from the ¹³C-isotopic signatures of the produced CH₄, CO₂ and acetate. Conditions and duration of incubation clearly affected the methanogenic community structure and the pathway of CH₄ formation. Methane was initially produced from reduction of CO₂ exclusively, resulting in accumulation of millimolar concentrations of acetate. Simultaneously, the relative abundance of the acetoclastic methanogens (Methanosarcinaceae, Methanosaetaceae), as determined by T-RFLP analysis of 16S rRNA genes, was low during the initial phase of CH₄ production. Later on, however, acetate was converted to CH₄ so that about 40% of the produced CH₄ originated from acetate. Most striking was the observed relative increase of a population of Methanosarcina spp. (but not of Methanosaeta spp.) briefly before acetate concentrations started to decrease. Both acetoclastic methanogenesis and Methanosarcina populations were suppressed by high phosphate concentrations, as observed under application of different buffer systems. Our results demonstrate the parallel change of microbial community structure and function in a complex environment, i.e., the increase of acetoclastic Methanosarcina spp. when high acetate concentrations become available.     

Coal methanogenesis: a review of the need of complex microbial consortia and culture conditions for the effective bioconversion of coal into methane

Microbial biodegradation of coal into low-molecular-weight compounds such as methane has been extensively researched in the last two decades because of the underlying environmental and industrial applications of this technique as compared to the chemical and physical methods of coal conversions. However, the irregular structure of coal and the need for complex microbial consortia under specific culture conditions do not make this biotransformation an ideal process for the development of anaerobic bioreactors. The most abundant species in a methanogenic culture are acetoclastic and hydrogenotrophic methanogens which utilize acetate and H₂+CO₂, respectively. Medium- to low-rank coals such as high-volatile bituminous, sub-bituminous and lignite are more promising in this bioconversion as compared to semi- and meta-anthracite coals. While covering the details of the ideal culture conditions, this review enlightens the need of research setups to explore the complex microbial consortia and culture conditions for maximum methane production through coal methanogenesis.     

Trophic links between fermenters and methanogens in a moderately acidic fen soil

Trophic links between fermentation and methanogenesis of soil derived from a methane‐emitting, moderately acidic temperate fen (pH 4.5) were investigated. Initial CO₂:CH₄ production ratios in anoxic microcosms indicated that methanogenesis was concomitant to other terminal anaerobic processes. Methane production in anoxic microcosms at in situ pH was stimulated by supplemental H₂–CO₂, formate or methanol; supplemental acetate did not stimulate methanogenesis. Supplemental H₂–CO₂, formate or methanol also stimulated the formation of acetate, indicating that the fen harbours moderately acid‐tolerant acetogens. Supplemental monosaccharides (glucose, N‐acetylglucosamine and xylose) stimulated the production of CO₂, H₂, acetate and other fermentation products when methanogenesis was inhibited with 2‐bromoethane sulfonate 20 mM. Glucose stimulated methanogenesis in the absence of BES. Upper soil depths yielded higher anaerobic activities and also higher numbers of cells. Detected archaeal 16S rRNA genes were indicative of H₂–CO₂‐ and formate‐consuming methanogens (Methanomicrobiaceae), obligate acetoclastic methanogens (Methanosaetaceae) and crenarchaeotes (groups I.1a, I.1c and I.3). Molecular analyses of partial sequences of 16S rRNA genes revealed the presence of Acidobacteria, Nitrospirales, Clamydiales, Clostridiales, Alpha‐, Gamma‐, Deltaproteobacteria and Cyanobacteria. These collective results suggest that this moderately acidic fen harbours phylogenetically diverse, moderately acid tolerant fermenters (both facultative aerobes and obligate anaerobes) that are trophically linked to methanogenesis.     

Effect of fumarate reducing bacteria on in vitro rumen fermentation,methane mitigation and microbial diversity

The metabolic pathways involved in hydrogen (H₂) production, utilization and the activity of methanogens are the important factors that should be considered in controlling methane (CH₄) emissions by ruminants. H₂ as one of the major substrate for CH₄ production is therefore should be controlled. One of the strategies on reducing CH₄ is through the use of hydrogenotrophic microorganisms such as fumarate reducing bacteria. This study determined the effect of fumarate reducing bacteria, Mitsuokella jalaludinii, supplementation on in vitro rumen fermentation, CH4 production, diversity and quantity. M. jalaludinii significantly reduced CH₄ at 48 and 72 h of incubation and significantly increased succinate at 24 h. Although not significantly different, propionate was found to be highest in treatment containing M. jalaludinii at 12 and 48 h of incubation. These results suggest that supplementation of fumarate reducing bacteria to ruminal fermentation reduces CH₄ production and quantity, increases succinate and changes the rumen microbial diversity.     

Functional-Structural Analysis of Nitrogen-Cycle Bacteria in a Hypersaline Mat from the Omani Desert

Anaerobic microbial activity in northern peat soils most often results in more carbon dioxide (CO ₂ ) production than methane (CH₄) production. This study examined why methanogenic conditions (i.e., equal molar amounts of CH₄ production and CO₂ production) prevail so infrequently. We used peat soils from two ombrotrophic bogs and from two rheotrophic fens. The former two represented a relatively dry bog hummock and a wet bog hollow, and the latter two represented a forested fen and a sedge-dominated fen. We quantified gas production rates in soil samples incubated in vitro with and without added metabolic substrates (glucose, ethanol, H₂/CO₂). None of the peat soils exhibited methanogenic conditions when incubated in vitro for a short time (< 5 days) and without added substrates. Incubating some samples > 50 days without added substrates led to methanogenic conditions in only one of four experiments. The anaerobic CO₂:CH₄ production ratio ranged from 5:1 to 40:1 in peat soil without additions and was larger in samples from the dry bog hummock and forested fen than the wet bog hollow and sedge fen. Adding ethanol or glucose separately to peat soils led to methanogenic conditions within 5 days after the addition by stimulating rates of CH₄ production, suggesting CH₄ production from both hydrogenotrophic and acetoclastic methanogenesis. Our results suggest that methanogenic conditions in peat soils rely on a constant supply of easily decomposable metabolic substrates. Sample handling and incubation procedures might obscure methanogenic conditions in peat soil incubated in vitro.     

Methanogen genotypes involved in methane formation during anaerobic decomposition of Microcystis blooms at different temperatures

The main goal of this work was to determine which methanogens were present during the anaerobic degradation of Microcystis biomass in the water columns of freshwater lakes. Simulation experiments were performed in which 30 ml Microcystis slurries were anaerobically incubated in 60 ml airtight bottles at three temperatures (15, 25, and 35 °C) for over 90 days. The production of CH₄ was monitored, and the methanogenic community was analyzed by cloning and sequencing the mcrA genes in samples incubated at the three different temperatures. In total, four clusters were detected at different temperatures by phylogenetic analysis of mcrA genes; these included members of Methanomicrobiales, Methanobacteriaceae, and Methanosarcina. An apparent linkage between temperature and phylogeny of the methanogenic community was observed: Methanomicrobiales and Methanobacteriaceae dominated the incubation system at the lower temperatures of 15 and 25 °C, whereas Methanosarcina prevailed at 35 °C. The dominance of these hydrogenotrophic methanogens suggested that, at least at lower temperatures, H₂ and CO₂ might be the primary substrates for CH₄ production during Microcystis anaerobic decomposition.