Effect of dilution rate on the microbial structure of a mesophilic butyrate-degrading methanogenic community during continuous cultivation

We constructed two mesophilic anaerobic chemostats that were continuously fed with synthetic wastewater containing butyrate as the sole source of carbon and energy. Steady-state conditions were achieved at dilution rates between 0.025 and 0.7 day⁻¹. Butyrate, fed into the chemostat, was almost completely mineralized to CH₄ and CO₂ at dilution rates below 0.5 day⁻¹. The butyrate-degrading methanogenic communities in the chemostats at dilution rates between 0.025 and 0.7 day⁻¹ were monitored based on the 16S rRNA gene, using molecular biological techniques including clone library analysis, denaturing gradient gel electrophoresis, and quantitative real-time polymerase chain reaction. The aceticlastic methanogen Methanosaeta and the hydrogenotrophic methanogen Methanoculleus dominated in methanogens at low dilution rates, whereas the aceticlastic methanogen Methanosaeta, Methanosarcina, the hydrogenotrophic methanogen Methanoculleus, and Methanospirillum dominated at high dilution rates. Bacteria affiliated with the family Syntrophaceae in the phylum Proteobacteria predominated at the low dilution rate of 0.025 day⁻¹, whereas bacteria affiliated with the phylum Firmicutes and Candidate division OP3 predominated at high dilution rates. A significant quantity of bacteria closely related to the genus Syntrophomonas was detected at high dilution rates. Dilution rate showed an apparent effect on archaeal and bacterial communities in the butyrate-fed chemostats.     

Microbial Community Dynamics and Stability during an Ammonia-Induced Shift to Syntrophic Acetate Oxidation

Anaerobic digesters rely on the diversity and distribution of parallel metabolic pathways mediated by complex syntrophic microbial communities to maintain robust and optimal performance. Using mesophilic swine waste digesters, we experimented with increased ammonia loading to induce a shift from aceticlastic methanogenesis to an alternative acetate-consuming pathway of syntrophic acetate oxidation. In comparison with control digesters, we observed shifts in bacterial 16S rRNA gene content and in functional gene repertoires over the course of the digesters'' 3-year operating period. During the first year, under identical startup conditions, all bioreactors mirrored each other closely in terms of bacterial phylotype content, phylogenetic structure, and evenness. When we perturbed the digesters by increasing the ammonia concentration or temperature, the distribution of bacterial phylotypes became more uneven, followed by a return to more even communities once syntrophic acetate oxidation had allowed the experimental bioreactors to regain stable operation. The emergence of syntrophic acetate oxidation coincided with a partial shift from aceticlastic to hydrogenotrophic methanogens. Our 16S rRNA gene analysis also revealed that acetate-fed enrichment experiments resulted in communities that did not represent the bioreactor community. Analysis of shotgun sequencing of community DNA suggests that syntrophic acetate oxidation was carried out by a heterogeneous community rather than by a specific keystone population with representatives of enriched cultures with this metabolic capacity.     

The genome characteristics and predicted function of methyl-group oxidation pathway in the obligate aceticlastic methanogens, Methanosaeta spp

In this work, we report the complete genome sequence of an obligate aceticlastic methanogen, Methanosaeta harundinacea 6Ac. Genome comparison indicated that the three cultured Methanosaeta spp., M. thermophila, M. concilii and M. harundinacea 6Ac, each carry an entire suite of genes encoding the proteins involved in the methyl-group oxidation pathway, a pathway whose function is not well documented in the obligately aceticlastic methanogens. Phylogenetic analysis showed that the methyl-group oxidation-involving proteins, Fwd, Mtd, Mch, and Mer from Methanosaeta strains cluster with the methylotrophic methanogens, and were not closely related to those from the hydrogenotrophic methanogens. Quantitative PCR detected the expression of all genes for this pathway, albeit ten times lower than the genes for aceticlastic methanogenesis in strain 6Ac. Western blots also revealed the expression of fwd and mch, genes involved in methyl-group oxidation. Moreover, (13)C-labeling experiments suggested that the Methanosaeta strains might use the pathway as a methyl oxidation shunt during the aceticlastic metabolism. Because the mch mutants of Methanosarcina barkeri or M. acetivorans failed to grow on acetate, we suggest that Methanosaeta may use methyl-group oxidation pathway to generate reducing equivalents, possibly for biomass synthesis. An fpo operon, which encodes an electron transport complex for the reduction of CoM-CoB heterodisulfide, was found in the three genomes of the Methanosaeta strains. However, an incomplete protein complex lacking the FpoF subunit was predicted, as the gene for this protein was absent. Thus, F(420)H(2) was predicted not to serve as the electron donor. In addition, two gene clusters encoding the two types of heterodisulfide reductase (Hdr), hdrABC, and hdrED, respectively, were found in the three Methanosaeta genomes. Quantitative PCR determined that the expression of hdrED was about ten times higher than hdrABC, suggesting that hdrED plays a major role in aceticlastic methanogenesis.     

基于mcrA基因的沁水盆地煤层气田产甲烷菌群与途径分析

【目的】分析沁水盆地煤层气田不同煤层气井产出水样中产甲烷菌群和生物成因气的生成途径。【方法】以甲基辅酶M还原酶基因(mcr A)作为目标基因,采用454焦磷酸高通量测序方法,同时比对NCBI功能基因文库中的mcr A序列,分析不同煤层气井产出水中的产甲烷菌群。【结果】高通量测序表明,5个出水样产甲烷菌群OTUs(Operational taxonomic units)数为64–157个,共有的为22个,各占样品总数14%-34%;样品共检测到4种已知菌属,即甲烷杆菌属(Methanobacterium)、甲烷微菌属(Methanomicrobium)、甲烷叶菌属(Methanolobus)和甲烷螺菌属(Methanospirillum),优势菌属均为Methanobacterium。系统发育分析表明,未明确地位的菌属主要与Methanobacterium、Methanomicrobium、产甲烷球菌属(Methanococcus)和甲烷囊菌属(Methanoculleus)有较近的亲缘关系。5个样品中菌属所占比例不同,检测到的菌属类别大致相同。所有检测样品生物成因煤层气(Coalbed methane,CBM)的生成途径主要为氢营养型产甲烷途径。【结论】沁水盆地不同煤层气田产甲烷菌群菌种差异比较大,但生物成因气生成途径基本相似,与地理位置和煤藏条件没有相关性。     

Shifts in methanogen community structure and function associated with long-term manipulation of sulfate and salinity in a hypersaline microbial mat

Methanogenesis was characterized in hypersaline microbial mats from Guerrero Negro, Baja California Sur, Mexico both in situ and after long-term manipulation in a greenhouse environment. Substrate addition experiments indicate methanogenesis to occur primarily through the catabolic demethylation of non-competitive substrates, under field conditions. However, evidence for the coexistence of other metabolic guilds of methanogens was obtained during a previous manipulation of sulfate concentrations. To fully characterize methanogenesis in these mats, in the absence of competition for reducing equivalents with sulfate-reducing microorganisms, we maintained microbial mats for longer than 1 year under conditions of lowered sulfate and salinity levels. The goal of this study was to assess whether observed differences in methane production during sulfate and salinity manipulation were accompanied by shifts in the composition of methanogen communities. Culture-independent techniques targeting methyl coenzyme M reductase genes ( mcrA ) were used to assess the dynamics of methanogen assemblages. Clone libraries from mats sampled in situ or maintained at field-like conditions in the greenhouse were exclusively composed of sequences related to methylotrophic members of the Methanosarcinales . Increases in pore water methane concentrations under conditions of low sulfate correlated with an observed increase in the abundance of putatively hydrogenotrophic mcrA , related to Methanomicrobiales . Geochemical and molecular data provide evidence of a significant shift in the metabolic pathway of methanogenesis from a methylotroph-dominated system in high-sulfate environments to a mixed community of methylotrophic and hydrogenotrophic methanogens under low sulfate conditions.     

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.     

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.     

Structure and function of methanogens along a short-term restoration chronosequence in the Florida Everglades

The removal of plants and soil to bedrock to eradicate exotic invasive plants within the Hole-in-the-Donut (HID) region, part of the Everglades National Park (Florida), presented a unique opportunity to study the redevelopment of soil and the associated microbial communities in the context of short-term primary succession and ecosystem restoration. The goal of this study was to identify relationships between soil redevelopment and activity and composition of methanogenic assemblages in HID soils. Methane production potentials indicated a general decline in methanogenic activity with restoration age. Microcosm incubations strongly suggested hydrogenotrophic methanogenesis as the most favorable pathway for methane formation in HID soils from all sites. Culture-independent techniques targeting methyl coenzyme M reductase genes (mcrA) were used to assess the dynamics of methanogenic assemblages. Clone libraries were dominated by sequences related to hydrogenotrophic methanogens of the orders Methanobacteriales and Methanococcales and suggested a general decline in the relative abundance of Methanobacteriales mcrA with time since restoration. Terminal restriction fragment length polymorphism analysis indicated methanogenic assemblages remain relatively stable between wet and dry seasons. Interestingly, analysis of soils across the restoration chronosequence indicated a shift in Methanobacteriales populations with restoration age, suggesting genotypic shifts due to site-specific factors.     

Stable Carbon Isotope Fractionation by Methylotrophic Methanogenic Archaea

In natural environments methane is usually produced by aceticlastic and hydrogenotrophic methanogenic archaea. However, some methanogens can use C₁ compounds such as methanol as the substrate. To determine the contributions of individual substrates to methane production, the stable-isotope values of the substrates and the released methane are often used. Additional information can be obtained by using selective inhibitors (e.g., methyl fluoride, a selective inhibitor of acetoclastic methanogenesis). We studied stable carbon isotope fractionation during the conversion of methanol to methane in Methanosarcina acetivorans, Methanosarcina barkeri, and Methanolobus zinderi and generally found large fractionation factors (−83‰ to −72‰). We further tested whether methyl fluoride impairs methylotrophic methanogenesis. Our experiments showed that even though a slight inhibition occurred, the carbon isotope fractionation was not affected. Therefore, the production of isotopically light methane observed in the presence of methyl fluoride may be due to the strong fractionation by methylotrophic methanogens and not only by hydrogenotrophic methanogens as previously assumed.     

The roles of acetotrophic and hydrogenotrophic methanogens during anaerobic conversion of biomass to methane: a review

Among different conversion processes for biomass, biological anaerobic digestion is one of the most economic ways to produce biogas from various biomass substrates. In addition to hydrolysis of polymeric substances, the activity and performance of the methanogenic bacteria is of paramount importance during methanogenesis. The aim of this paper is primarily to review the recent literature about the occurrence of both acetotrophic and hydrogenotrophic methanogens during anaerobic conversion of particulate biomass to methane (not wastewater treatment), while this review does not cover the activity of the acetate oxidizing bacteria. Both acetotrophic and hydrogenotrophic methanogens are essential for the last step of methanogenesis, but the reports about their roles during this phase of the process are very limited. Despite, some conclusions can still be drawn. At low concentrations of acetate, normally filamentous Methanosaeta species dominate, e.g., often observed in sewage sludge. Apparently, high concentrations of toxic ionic agents, like ammonia, hydrogen sulfide (H₂S) and volatile fatty acids (VFA), inhibit preferably Methanosaetaceae and especially allow the growth of Methanosarcina species consisting of irregular cell clumps, e.g., in cattle manure. Thermophilic conditions can favour rod like or coccoid hydrogenotrophic methanogens. Thermophilic Methanosarcina species were also observed, but not thermophilic Methanosaetae. Other environmental factors could favour hydrogentrophic bacteria, e.g., short or low retention times in a biomass reactor. However, no general rules regarding process parameters could be derivated at the moment, which favours hydrogenotrophic methanogens. Presumably, it depends only on the hydrogen concentration, which is generally not mentioned in the literature.     

Effect of 2-bromo-ethane sulfonate, molybdate and chloroform on acetate consumption by methanogenic and sulfate-reducing populations in freshwater sediment

The relative importance of methanogenesis and sulfate reduction in freshwater sediment supplemented with acetate was investigated. Addition of acetate stimulated both methane formation and sulfate reduction, indicating that an active aceticlastic population of methanogens and sulfate reducers was present in the sediment. Sulfate reducers were most important in the consumption of acetate. However, when sulfate reducers were inhibited, acetate was metabolised at a similar rate by methanogens. Acetate, propionate and valerate accumulated only when both processes were inhibited by the combined addition of 2-bromo-ethane sulfonate and molybdate. The relative amounts of acetate, propionate and valerate were 93, 6 and 1 mol%, respectively. These results demonstrate the role of acetate as a key intermediate in the terminal step of organic matter mineralisation in the sediment. Addition of chloroform inhibited both methanogenesis and sulfate reduction. We studied the inhibitory effect of CHCl(3) on homoacetogenic bacteria, sulfate-reducing bacteria and methanogens. The results showed that inhibition by CHCl(3) correlates with microorganisms, which operate the acetyl-CoA cleavage pathway. We propose that chloroform can be used to elucidate the role of different metabolic types of sulfate reducers to sulfate reduction in natural environments.     

Thermophilic anaerobic digestion of livestock waste: the effect of ammonia

Ammonia concentrations of 4 g N/l or more inhibited thermophilic digestion of cattle manure. A stable digestion of cattle manure could be maintained with ammonia concentrations up to 6 g N/l after 6 months of operation. However, the methane yield was reduced and the concentration of volatile fatty acids increased from 1 to 3 g/l as acetate, compared to controls with an ammonia concentration of 2.5 g N/l. The temporary strong inhibition following an one-step increase in ammonia concentration was reduced by applying a gradual increase. The specific methanogenic activity of ammonia-inhibited reactors (6 g N/l) with acetate or hydrogen as substrate was reduced by 73 and 52%, respectively. Tests of ammonia toxicity on the acetate- and hydrogen-utilizing populations showed a higher sensitivity of the aceticlastic compared to the hydrogenotrophic methanogens; the specific growth rate for the aceticlastic methanogens was halved at ammonia concentrations of 3.5 g N/l, compared to 7 g N/l for the hydrogenotrophic methanogens. Correspondence to: B. K. Ahring     

Archaea diversity within a commercial biogas plant utilizing herbal biomass determined by 16S rDNA and mcrA analysis

Aims: The Archaea diversity was evaluated in an agricultural biogas plant supplied with cattle liquid manure and maize silage under mesophilic conditions. Methods and Results: Two different genes (16S rRNA; methyl‐coenzyme‐M‐reductase, MCR) targeted by three different PCR primer sets were selected and used for the construction of three clone libraries comprising between 104 and 118 clones. The clone libraries were analysed by restriction fragment polymorphism (RFLP). Between 11 and 31 operational taxonomic units (OTUs) were detected and assigned to orders Methanomicrobiales, Methanosarcinales and Methanobacteriales. Over 70% of all Archaea OTUs belong to the order Methanomicrobiales which mostly include hydrogenotrophic methanogens. Acetotrophic methanogens were detected in minor rates. Similar relative values were obtained by a quantitative real‐time PCR analysis. Conclusions: The results implied that in this biogas plant the most of the methane formation resulted from the conversion of H₂ and CO₂. Significance and Impact of the Study: This study reports, for the first time, a molecular analysis of the archaeal community in this type of agricultural biogas plants. Therein the hydrogenotrophic methanogenesis seems to be the major pathway of methane formation. These results are in contrast with the common thesis that in biogas fermentations the primary substrate for methanogenesis is acetate.     

Detection of novel syntrophic acetate‐oxidizing bacteria from biogas processes by continuous acetate enrichment approaches

To enrich syntrophic acetate‐oxidizing bacteria (SAOB), duplicate chemostats were inoculated with sludge from syntrophic acetate oxidation (SAO)‐dominated systems and continuously supplied with acetate (0.4 or 7.5 g l^?1) at high‐ammonia levels. The chemostats were operated under mesophilic (37°C) or thermophilic (52°C) temperature for about six hydraulic retention times (HRT 28 days) and were sampled over time. Irrespective of temperature, a methane content of 64–69% and effluent acetate level of 0.4–1.0 g l^?1 were recorded in chemostats fed high acetate. Low methane production in the low‐acetate chemostats indicated that the substrate supply was below the threshold for methanization of acetate via SAO. Novel representatives within the family Clostridiales and genus Syntrophaceticus (class Clostridia) were identified to represent putative SAOB candidates in mesophilic and thermophilic conditions respectively. Known SAOB persisted at low relative abundance in all chemostats. The hydrogenotrophic methanogens Methanoculleus bourgensis (mesophilic) and Methanothermobacter thermautotrophicus (thermophilic) dominated archaeal communities in the high‐acetate chemostats. In line with the restricted methane production in the low‐acetate chemostats, methanogens persisted at considerably lower abundance in these chemostats. These findings strongly indicate involvement in SAO and tolerance to high ammonia levels of the species identified here, and have implications for understanding community function in stressed anaerobic processes.     

Estimates of biogenic methane production rates in deep marine sediments at Hydrate Ridge, Cascadia margin

Methane hydrate found in marine sediments is thought to contain gigaton quantities of methane and is considered an important potential fuel source and climate-forcing agent. Much of the methane in hydrates is biogenic, so models that predict the presence and distribution of hydrates require accurate rates of in situ methanogenesis. We estimated the in situ methanogenesis rates in Hydrate Ridge (HR) sediments by coupling experimentally derived minimal rates of methanogenesis to methanogen biomass determinations for discrete locations in the sediment column. When starved in a biomass recycle reactor, Methanoculleus submarinus produced ca. 0.017 fmol methane/cell/day. Quantitative PCR (QPCR) directed at the methyl coenzyme M reductase subunit A gene (mcrA) indicated that 75% of the HR sediments analyzed contained <1,000 methanogens/g. The highest numbers of methanogens were found mostly from sediments <10 m below seafloor. By considering methanogenesis rates for starved methanogens (adjusted to account for in situ temperatures) and the numbers of methanogens at selected depths, we derived an upper estimate of <4.25 fmol methane produced/g sediment/day for the samples with fewer methanogens than the QPCR method could detect. The actual rates could vary depending on the real number of methanogens and various seafloor parameters that influence microbial activity. However, our calculated rate is lower than rates previously reported for such sediments and close to the rate derived using geochemical modeling of the sediments. These data will help to improve models that predict microbial gas generation in marine sediments and determine the potential influence of this source of methane on the global carbon cycle.     

Characterization of microbial biofilms in a thermophilic biogas system by high-throughput metagenome sequencing

DNAs of two biofilms of a thermophilic two-phase leach-bed biogas reactor fed with rye silage and winter barley straw were sequenced by 454-pyrosequencing technology to assess the biofilm-based microbial community and their genetic potential for anaerobic digestion. The studied biofilms matured on the surface of the substrates in the hydrolysis reactor (HR) and on the packing in the anaerobic filter reactor (AF). The classification of metagenome reads showed Clostridium as most prevalent bacteria in the HR, indicating a predominant role for plant material digestion. Notably, insights into the genetic potential of plant-degrading bacteria were determined as well as further bacterial groups, which may assist Clostridium in carbohydrate degradation. Methanosarcina and Methanothermobacter were determined as most prevalent methanogenic archaea. In consequence, the biofilm-based methanogenesis in this system might be driven by the hydrogenotrophic pathway but also by the aceticlastic methanogenesis depending on metabolite concentrations such as the acetic acid concentration. Moreover, bacteria, which are capable of acetate oxidation in syntrophic interaction with methanogens, were also predicted. Finally, the metagenome analysis unveiled a large number of reads with unidentified microbial origin, indicating that the anaerobic degradation process may also be conducted by up to now unknown species.     

氢营养型产甲烷代谢途径研究进展

产甲烷古菌是一类极端厌氧的古菌域微生物,可以利用CO_2、甲醇、乙酸等简单化合物产甲烷并获得能量。目前能够培养的氢营养型(CO_2/H_2)产甲烷古菌的种类较多,而且在三类产甲烷代谢类型中,氢营养型产甲烷途径的产能效率最高,并具有多种模式的特殊能量利用系统。近年来,随着质谱、光谱和晶体技术的发展与运用,人们对产甲烷代谢途径的研究进一步深入,尤其是对氢营养型产甲烷途径的生化机制有了新的认识,揭示了产甲烷古菌在能量极限条件下独特、高效的能量利用模式。本文从能量储存、代谢途径、蛋白功能与催化机制等方面概述产甲烷古菌利用CO_2/H_2产甲烷的详细过程,并对产甲烷古菌代谢途径的研究方向与技术发展进行展望。     

Methanol as the Primary Methanogenic and Acetogenic Precursor in the Cold Zoige Wetland at Tibetan Plateau

Previous studies suggested that methanol and acetate were the likely methanogenic precursors in the cold Zoige wetland. In this study, the contribution of the two substances to methanogenesis and the conversion in Zoige wetland were analyzed. It was determined that methanol supported the highest CH₄ formation rate in the enrichments of the soil grown with Eleocharis valleculosa, and even higher at 15°C than at 30°C; while hydrogenotrophic methanogenesis was higher at 30°C. Both methanol- and acetate-using methanogens were counted at the highest (10⁷ g⁻¹) in the soil, whereas methanol-using acetogens (10⁸ g⁻¹) were ten times more abundant than either methanol- or acetate-using methanogens. Both methanol and acetate were detected in the methanogenesis-inhibited soil samples, so that both could be the primary methanogenic precursors in E. valleculosa soil. However, the levels of methanol and acetate accumulated in 2-bromoethane-sulfonate (BES)- and CHCl₃-treated soils were in reverse, i.e., higher methanol in CHCl₃- and higher acetate in BES-treated soil, so that methanol-derived methanogenesis could be underestimated due to the consumption by acetogens. Analysis of the soil 16S rRNA genes revealed Acetobacterum bakii and Trichococcus pasteurii to be the dominant methanol-using acetogens in the soil, and a strain of T. pasteurii was isolated, which showed the high conversion of methanol to acetate at 15°C.     

Immobilization patterns and dynamics of acetate-utilizing methanogens immobilized in sterile granular sludge in upflow anaerobic sludge blanket reactors

Sterile granular sludge was inoculated with either Methanosarcina mazeii S-6, Methanosaeta concilii GP-6, or both species in acetate-fed upflow anaerobic sludge blanket (UASB) reactors to investigate the immobilization patterns and dynamics of aceticlastic methanogens in granular sludge. After several months of reactor operation, the methanogens were immobilized, either separately or together. The fastest immobilization was observed in the reactor containing M. mazeii S-6. The highest effluent concentration of acetate was observed in the reactor with only M. mazeii S-6 immobilized, while the lowest effluent concentration of acetate was observed in the reactor where both types of methanogens were immobilized together. No changes were observed in the kinetic parameters (Ks and mumax) of immobilized M. concilii GP-6 or M. mazeii S-6 compared with suspended cultures, indicating that immobilization does not affect the growth kinetics of these methanogens. An enzyme-linked immunosorbent assay using polyclonal antibodies against either M. concilii GP-6 or M. mazeii S-6 showed significant variations in the two methanogenic populations in the different reactors. Polyclonal antibodies were further used to study the spatial distribution of the two methanogens. M. concilii GP-6 was immobilized only on existing support material without any specific pattern. M. mazeii S-6, however, showed a different immobilization pattern: large clumps were formed when the concentration of acetate was high, but where the acetate concentration was low this strain was immobilized on support material as single cells or small clumps. The data clearly show that the two aceticlastic methanogens immobilize differently in UASB systems, depending on the conditions found throughout the UASB reactor.