Dynamic Transition of a Methanogenic Population in Response to the Concentration of Volatile Fatty Acids in a Thermophilic Anaerobic Digester

In this study, the microbial community succession in a thermophilic methanogenic bioreactor under deteriorative and stable conditions that were induced by acidification and neutralization, respectively, was investigated using PCR-mediated single-strand conformation polymorphism (SSCP) based on the 16S rRNA gene, quantitative PCR, and fluorescence in situ hybridization (FISH). The SSCP analysis indicated that the archaeal community structure was closely correlated with the volatile fatty acid (VFA) concentration, while the bacterial population was impacted by pH. The archaeal community consisted mainly of two species of hydrogenotrophic methanogen (i.e., a Methanoculleus sp. and a Methanothermobacter sp.) and one species of aceticlastic methanogen (i.e., a Methanosarcina sp.). The quantitative PCR of the 16S rRNA gene from each methanogen revealed that the Methanoculleus sp. predominated among the methanogens during operation under stable conditions in the absence of VFAs. Accumulation of VFAs induced a dynamic transition of hydrogenotrophic methanogens, and in particular, a drastic change (i.e., an approximately 10,000-fold increase) in the amount of the 16S rRNA gene from the Methanothermobacter sp. The predominance of the one species of hydrogenotrophic methanogen was replaced by that of the other in response to the VFA concentration, suggesting that the dissolved hydrogen concentration played a decisive role in the predominance. The hydrogenotrophic methanogens existed close to bacteria in aggregates, and a transition of the associated bacteria was also observed by FISH analyses. The degradation of acetate accumulated during operation under deteriorative conditions was concomitant with the selective proliferation of the Methanosarcina sp., indicating effective acetate degradation by the aceticlastic methanogen. The simple methanogenic population in the thermophilic anaerobic digester significantly responded to the environmental conditions, especially to the concentration of VFAs.     

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.     

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.     

Geochemical Influence on Diversity and Microbial Processes in High Temperature Oil Reservoirs

The diversity of thermophilic microbial assemblages detected within two neighboring high temperature petroleum formations was shown to closely parallel the different geochemical regimes existing in each. A high percentage of archaeal 16S rRNA gene sequences, related to thermophilic aceticlastic and hydrogenotrophic methanogens, were detected in the natural gas producing Rincon Formation. In contrast, rRNA gene libraries from the highly sulfidogenic Monterey Formation contained primarily sulfur-utilizing and fermentative archaea and bacteria. In addition to the variations in microbial community structure, microbial activities measured in microcosm experiments using high temperature production fluids from oil-bearing formations also demonstrated fundamental differences in the terminal respiratory and redox processes. Provided with the same suite of basic energy substrates, production fluids from the South Elwood Rincon Formation actively generated methane, while thermophilic microflora within the Monterey production fluids were dominated by hydrogen sulfide producing microorganisms. In both cases, molecular hydrogen appeared to play a central role in the stimulation of carbon and sulfur cycling in these systems. In methanogenic production fluids, the addition of sulfur compounds induced a rapid shift in the terminal electron accepting process, stimulating hydrogen sulfide formation and illustrating the metabolic versatility of the subsurface thermophilic assemblage. The high similarity between microbial community structure and activity corresponding with the prevalent geochemical conditions observed in deep subsurface petroleum reservoirs suggests that the resident microflora have adapted to the subsurface physicochemical conditions and may actively influence the geochemical environment in situ.     

Microbial community of a mesophilic propionate-degrading methanogenic consortium in chemostat cultivation analyzed based on 16S rRNA and acetate kinase genes

Abstract We constructed a mesophilic anaerobic chemostat that was continuously fed with synthetic wastewater containing propionate as the sole source of carbon and energy. Steady-state conditions were achieved below the critical dilution rate of 0.3 d ⁻¹ with almost complete substrate degradation. The propionate-degrading methanogenic communities in the chemostat at dilution rates of 0.01, 0.08, and 0.3 d ⁻¹ were analyzed using molecular biological techniques. Fluorescence in situ hybridization with archaeal and bacterial domain-specific probes showed that archaeal cells predominated throughout the three dilution rates. Archaeal-16S rRNA gene clone library analysis and quantitative real-time polymerase chain reaction studies showed that hydrogenotrophic methanogen rRNA genes closely related to Methanoculleus was detected at a dilution rate of 0.01 d ⁻¹ , whereas rRNA genes closely related to the Methanoculleus and Methanospirillum genera were detected at dilution rates of 0.08 and 0.3 d ⁻¹ . The aceticlastic methanogen, Methanosaeta , was detected throughout the three dilution rates. Bacterial-rRNA gene clone library analysis and denaturing gradient gel electrophoresis demonstrated that rRNA genes affiliated with the genus Syntrophobacter predominated at the low dilution rate, whereas rRNA genes affiliated with the phylum Firmicutes predominated at the higher dilution rates. A significant number of rRNA genes affiliated with the genus Pelotomaculum were detected at dilution rate of 0.3 d ⁻¹ . The diversity of genes encoding acetate kinase agreed closely with the results of the rRNA gene analysis. The dilution rates significantly altered the archaeal and bacterial communities in the propionate-fed chemostat.     

Methanosarcina as the dominant aceticlastic methanogens during mesophilic anaerobic digestion of putrescible waste

Taking into account isotope ¹³C value a mathematical model was developed to describe the dynamics of methanogenic population during mesophilic anaerobic digestion of putrescible solid waste and waste imitating Chinese municipal solid waste. Three groups of methanogens were considered in the model including unified hydrogenotrophic methanogens and two aceticlastic methanogens Methanosaeta sp. and Methanosarcina sp. It was assumed that Methanosaeta sp. and Methanosarcina sp. are inhibited by high volatile fatty acids concentration. The total organic and inorganic carbon concentrations, methane production, methane and carbon dioxide partial pressures as well as the isotope ¹³C incorporation in PSW and CMSW were used for the model calibration and validation. The model showed that in spite of the high initial biomass concentration of Methanosaeta sp. Methanosarcina sp. became the dominant aceticlastic methanogens in the system. This prediction was confirmed by FISH. It is concluded that Methanosarcina sp. forming multicellular aggregates may resist to inhibition by volatile fatty acids (VFAs) because a slow diffusion rate of the acids limits the VFA concentrations inside the Methanosarcina sp. aggregates.     

The molecular diversity of the methanogenic community in a hypereutrophic freshwater lake determined by PCR-RFLP

AIMS: To combine database-held sequence information with a programme of experimental molecular ecology to define the methanogenic community of a hypereutrophic lake by a PCR-restriction fragment length polymorphism (RFLP) analysis. METHODS AND RESULTS: Methanogen diversity in a hypereutrophic freshwater lake was analysed using 16S rDNA PCR-RFLP. Database-held 16S rRNA gene sequences for 76 diverse methanogens were analysed for specific restriction sites that permitted unequivocal differentiation of methanogens. Restriction digestion and agarose gel electrophoresis of the 16S rDNA from selected methanogen pure cultures generated observed restriction profiles that corroborated the expected patterns. This method was then tested by analysing methanogen diversity in samples obtained over 1 year from sediment and water samples taken from the same sampling site. CONCLUSIONS: Restriction analysis of the 16S rRNA gene sequences from 157 methanogen clones generated from lakewater and sediment samples showed that over 50% were similar to Methanoculleus spp. Furthermore, a total of 16 RFLP types (1-16) were identified, eight of which contained no cultured representative archaeal 16S rRNA gene sequences. SIGNIFICANCE AND IMPACT OF THE STUDY: This RFLP strategy provides a robust and reliable means to rapidly identify methanogens in the environment.     

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.     

Effects of amendment with ferrihydrite and gypsum on the structure and activity of methanogenic populations in rice field soil

Methane emission from paddy fields may be reduced by the addition of electron acceptors to stimulate microbial populations competitive to methanogens. We have studied the effects of ferrihydrite and gypsum (CaSO(4). 2H(2)O) amendment on methanogenesis and population dynamics of methanogens after flooding of Italian rice field soil slurries. Changes in methanogen community structure were followed by archaeal small subunit (SSU) ribosomal DNA (rDNA)- and rRNA-based terminal restriction fragment length polymorphism analysis and by quantitative SSU rRNA hybridization probing. Under ferrihydrite amendment, acetate was consumed efficiently (<60 microM) and a rapid but incomplete inhibition of methanogenesis occurred after 3 days. In contrast to unamended controls, the dynamics of Methanosarcina populations were largely suppressed as indicated by rDNA and rRNA analysis. However, the low acetate availability was still sufficient for activation of Methanosaeta spp., as indicated by a strong increase of SSU rRNA but not of relative rDNA frequencies. Unexpectedly, rRNA amounts of the novel rice cluster I (RC-I) methanogens increased significantly, while methanogenesis was low, which may be indicative of transient energy conservation coupled to Fe(III) reduction by these methanogens. Under gypsum addition, hydrogen was rapidly consumed to low levels ( approximately 0.4 Pa), indicating the presence of a competitive population of hydrogenotrophic sulfate-reducing bacteria (SRB). This was paralleled by a suppressed activity of the hydrogenotrophic RC-I methanogens as indicated by the lowest SSU rRNA quantities detected in all experiments. Full inhibition of methanogenesis only became apparent when acetate was depleted to nonpermissive thresholds (<5 microM) after 10 days. Apparently, a competitive, acetotrophic population of SRB was not present initially, and hence, acetotrophic methanosarcinal populations were less suppressed than under ferrihydrite amendment. In conclusion, although methane production was inhibited effectively under both mitigation regimens, different methanogenic populations were either suppressed or stimulated, which demonstrates that functionally similar disturbances of an ecosystem may result in distinct responses of the populations involved.     

Temporal change of composition and potential activity of the thermophilic archaeal community during the composting of organic material

To date, composting has been regarded as an aerobic process but it has been shown that composting piles are often sources of atmospheric methane. In order to gain a more comprehensive view on the diversity of methanogenic Archaea in compost, gas chromatographical methods and molecular cloning were used to study relationships of thermophilic archaeal communities and changes in methane production potential during compost maturation. According to the thermophilic methane production potential, wide differences could be detected between differently aged compost materials. In material derived from 3- and 4-week-old piles, low and no thermophilic methane production potential, respectively, was observed at 50 degrees C. Material from a 6-week-old pile showed the maximum methane production. With compost maturation, the production slowly decreased again with 6 weeks, 8 weeks, and mature compost showing an optimum methane production potential at 60 degrees C. At 70 degrees C, only 6-week-old material showed a comparable high production of methane. The 16S rRNA-based phylogenetic surveys revealed an increase of archaeal diversity with compost maturation. In the 6-week-old material, 86% of the sequences in the archaeal 16S rRNA library had the highest sequence similarities to Methanothermobacter spp. and the remaining 14% of the clones were related to Methanosarcina thermophila. Quantification of methanogens in 6-week-old material, on the basis of the methane production rate, resulted in values of about 2x10(7) cells per gram fresh weight. In 8-week-old and mature compost material, the proportion of sequences similar to Methanothermobacter spp. decreased to 34% and 0%, respectively. The mature compost material showed the highest variation in identified sequences, although 33% could be assigned to as yet uncultured Archaea (e.g. Rice cluster I, III, and IV). Our results indicate that compost harbours a diverse community of thermophilic methanogens, with changing composition during the maturation process, presumably due to altered pile conditions. Likewise, compost may act as a potential carrier for thermophilic methanogens in temperate soils because it is widely used as a soil amendment.     

Aceticlastic and NaCl-requiring methanogen "Methanosaeta pelagica" sp. nov., isolated from marine tidal flat sediment

Among methanogens, only 2 genera, Methanosaeta and Methanosarcina, are known to contribute to methanogenesis from acetate, and Methanosaeta is a specialist that uses acetate specifically. However, Methanosaeta strains so far have mainly been isolated from anaerobic digesters, despite the fact that it is widespread, not only in anaerobic methanogenic reactors and freshwater environments, but also in marine environments, based upon extensive 16S rRNA gene-cloning analyses. In this study, we isolated an aceticlastic methanogen, designated strain 03d30q(T), from a tidal flat sediment. Phylogenetic analyses based on 16S rRNA and mcrA genes revealed that the isolate belongs to the genus Methanosaeta. Unlike the other known Methanosaeta species, this isolate grows at Na(+) concentrations of 0.20 to 0.80 M, with an optimum concentration of 0.28 M. Quantitative estimation using real-time PCR detected the 16S rRNA gene of the genus Methanosaeta in the marine sediment, and relative abundance ranged from 3.9% to 11.8% of the total archaeal 16S rRNA genes. In addition, the number of Methanosaeta organisms increased with increasing depth and was much higher than that of Methanosarcina organisms, suggesting that aceticlastic methanogens contribute to acetate metabolism to a greater extent than previously thought in marine environments, where sulfate-reducing acetate oxidation prevails. This is the first report on marine Methanosaeta species, and based on phylogenetic and characteristic studies, the name "Methanosaeta pelagica" sp. nov. is proposed for this novel species, with type strain 03d30q.     

The structure of the bacterial and archaeal community in a biogas digester as revealed by denaturing gradient gel electrophoresis and 16S rDNA sequencing analysis

Aims:  To identify the bacterial and archaeal composition in a mesophilic biogas digester treating pig manure and to compare the consistency of two 16S rDNA-based methods to investigate the microbial structure.
Methods and results:  Sixty-nine bacterial operational taxonomic units (OTU) and 25 archaeal OTU were identified by sequencing two 16S rDNA clone libraries. Most bacterial OTU were identified as phyla of Firmicutes (47·2% of total clones), Bacteroides (35·4%) and Spirochaetes (13·2%). Methanoculleus bourgensis (29·0%), Methanosarcina barkeri (27·4%) and Methanospirillum hungatei (10·8%) were the dominant methanogens. Only 9% of bacterial and 20% of archaeal OTU matched cultured isolates at a similarity index of ≥97%. About 78% of the dominant bacterial (with abundance >3%) and 83% of archaeal OTU were recovered from the denaturing gradient gel electrophoresis (DGGE) bands of V3 regions in 16S rDNAs.
Conclusions:  In the digester, most bacterial and archaeal species were uncultured; bacteria belonging to Firmicutes , Bacteroides and Spirochaetes seem to take charge of cellulolysis, proteolysis, acidogenesis, sulfur-reducing and homoacetogenesis; the most methanogens were typical hydrogenotrophic or hydrogenotrophic/aceticlastic; DGGE profiles reflected the dominant microbiota.
Significance and Impact of the Study:  This study gave a first insight of the overall microbial structure in a rural biogas digester and also indicated DGGE was useful in displaying its dominant microbiota.     

Composition of Archaeal Community in a Paddy Field as Affected by Rice Cultivar and N Fertilizer

Methanogenesis in paddy fields is significantly influenced by environmental and field management factors such as rice cultivar and nitrogenous fertilizer. However, it has been unclear whether such effects are reflected in the structure of methanogenic archaeal populations. In the present study, molecular analyses including cloning and sequencing and terminal restriction fragment length polymorphism (T-RFLP) fingerprinting of archaeal 16S rRNA genes were used to characterize the methanogenic archaeal assemblages and to identify the effect of environmental variables including rice cultivar and N fertilizer on archaeal community compositions in a Chinese paddy field soil. The correlation between methanogenic archaeal composition and environmental variables was explored by correspondence analysis. The results showed that the spatial or niche factor (rice roots versus rhizosphere, surface, and the deeper layer soils) had the greatest influence on the archaeal community composition. There was an obvious enrichment or selection of hydrogenotrophic as opposed to acetoclastic methanogens by rice roots. The archaeal community also changed, though slightly, between the rhizosphere and bulk soils and between the surface soil and the deeper layer soil. However, rice cultivar and N fertilizer appear to have an effect only on methanogens tightly associated with rice roots.     

Archaeal communities in High Arctic wetlands at Spitsbergen, Norway (78 degrees N) as characterized by 16S rRNA gene fingerprinting

Emissions of the greenhouse gas methane from Arctic wetlands have been studied extensively, though little is known about the ecology and community structure of methanogenic archaea that catalyze the methane production. As part of a project addressing microbial transformations of methane in Arctic wetlands, we studied archaeal communities in two wetlands (Solvatnet and Stuphallet) at Spitsbergen, Norway (78 degrees N) during two summer seasons. Directly extracted peat community DNA and enrichment cultures of methanogenic archaea were analyzed by nested PCR combined with denaturing gradient gel electrophoresis and subsequent sequencing of 16S rRNA gene fragments. Sequences affiliated with Methanomicrobiales, Methanobacteriaceae, Methanosaeta and Group I.3b of the uncultured crenarchaeota were detected at both sites. Sequences affiliated with Methanosarcina were recovered only from the site Solvatnet, while sequences affiliated with the euryarchaeotal clusters Rice Cluster II and Sediment 1 were detected only at the site Stuphallet. The phylogenetic affiliation of the recovered sequences suggested a potential of both hydrogenotrophic and acetoclastic methanogenesis at both sites. At Solvatnet, there were clear temporal trends in the archaeal community structure over the Arctic summer season. The archaeal community composition was significantly affected by factors influencing the activity of the overall bacterial community, as measured by in situ emissions of CO2. Methane emissions at both sites were influenced more by peat temperatures and thaw depth than by the archaeal community structure. Enrichment cultures for methanogenic archaea determined that most of the methanogens detected directly in peat could grow in culture at 10 degrees C. Culture based biases were indicated in later enrichment steps by the abundant growth of a Methanosarcina strain that was not detected directly in peat samples.     

Post-translational modifications in the active site region of methyl-coenzyme M reductase from methanogenic and methanotrophic archaea

Methyl-coenzyme M reductase (MCR) catalyzes the methane-forming step in methanogenic archaea. Isoenzyme I from Methanothermobacter marburgensiswas shown to contain a thioxo peptide bond and four methylated amino acids in the active site region. We report here that MCRs from all methanogens investigated contain the thioxo peptide bond, but that the enzymes differ in their post-translational methylations. The MS analysis included MCR I and MCR II from Methanothermobacter marburgensis, MCR I from Methanocaldococcus jannaschii and Methanoculleus thermophilus, and MCR from Methanococcus voltae, Methanopyrus kandleri and Methanosarcina barkeri. Two MCRs isolated from Black Sea mats containing mainly methanotrophic archaea of the ANME-1 cluster were also analyzed.     

Traditional cattle manure application determines abundance, diversity and activity of methanogenic Archaea in arable European soil

Based on lipid analyses, 16S rRNA/rRNA gene single-strand conformation polymorphism fingerprints and methane flux measurements, influences of the fertilization regime on abundance and diversity of archaeal communities were investigated in soil samples from the long-term (103 years) field trial in Bad Lauchst?dt, Germany. The investigated plots followed a gradient of increasing fertilization beginning at no fertilization and ending at the 'cattle manure' itself. The archaeal phospholipid etherlipid (PLEL) concentration was used as an indicator for archaeal biomass and increased with the gradient of increasing fertilization, whereby the concentrations determined for organically fertilized soils were well above previously reported values. Methane emission, although at a low level, were occasionally only observed in organically fertilized soils, whereas the other treatments showed significant methane uptake. Euryarchaeotal organisms were abundant in all investigated samples but 16S rRNA analysis also demonstrated the presence of Crenarchaeota in fertilized soils. Lowest molecular archaeal diversity was found in highest fertilized treatments. Archaea phylogenetically most closely related to cultured methanogens were abundant in these fertilized soils, whereas Archaea with low relatedness to cultured microorganisms dominated in non-fertilized soils. Relatives of Methanoculleus spp. were found almost exclusively in organically fertilized soils or cattle manure. Methanosarcina-related microorganisms were detected in all soils as well as in the cattle manure, but soils with highest organic application rate were specifically dominated by a close phylogenetic relative of Methanosarcina thermophila. Our findings suggest that regular application of cattle manure increased archaeal biomass, but reduced archaeal diversity and selected for methanogenic Methanoculleus and Methanosarcina strains, leading to the circumstance that high organic fertilized soils did not function as a methane sink at the investigated site anymore.     

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.     

Isolation and characterization of Methanothermobacter crinale sp. nov., a novel hydrogenotrophic methanogen from the Shengli oil field

Syntrophic acetate oxidation coupled with hydrogenotrophic methanogenesis is an alternative methanogenic pathway in certain thermophilic anaerobic environments such as high-temperature oil reservoirs and thermophilic biogas reactors. In these environments, the dominant thermophilic methanogens were generally related to uncultured organisms of the genus Methanothermobacter. Here we isolated two representative strains, Tm2(T) and HMD, from the oil sands and oil production water in the Shengli oil field in the People's Republic of China. The type strain, Tm2(T), was nonmotile and stained Gram positive. The cells were straight to slightly curved rods (0.3 μm in width and 2.2 to 5.9 μm in length), but some of them possessed a coccal shape connecting with the rods at the ends. Strain Tm2(T) grew with H(2)-CO(2), but acetate is required. Optimum growth of strain Tm2(T) occurred in the presence of 0.025 g/liter NaCl at pH 6.9 and a temperature of 65°C. The G+C content of the genomic DNA was 40.1 mol% ± 1.3 mol% (by the thermal denaturation method) or 41.1 mol% (by high-performance liquid chromatography). Analysis of the 16S rRNA gene sequence indicated that Tm2(T) was most closely related to Methanothermobacter thermautotrophicus ΔH(T) and Methanothermobacter wolfeii VKM B-1829(T) (both with a sequence similarity of 96.4%). Based on these phenotypic and phylogenic characteristics, a novel species was proposed and named Methanothermobacter crinale sp. nov. The type strain is Tm2(T) (ACCC 00699(T) = JCM 17393(T)).     

Microbial population dynamics during start-up and overload conditions of anaerobic digesters treating municipal solid waste and sewage sludge

Microbial population dynamics were investigated during start-up and during periods of overload conditions in anaerobic co-digesters treating municipal solid waste and sewage sludge. Changes in community structure were monitored using ribosomal RNA-based oligonucleotide probe hybridization to measure the abundance of syntrophic propionate-oxidizing bacteria (SPOB), saturated fatty acid-beta-oxidizing syntrophs (SFAS), and methanogens. These changes were linked to traditional performance parameters such as biogas production and volatile fatty acid (VFA) concentrations. Digesters with high levels of Archaea started up successfully. Methanosaeta concilii was the dominant aceticlastic methanogen in these systems. In contrast, digesters that experienced a difficult start-up period had lower levels of Archaea with proportionally more abundant Methanosarcina spp. Syntrophic propionate-oxidizing bacteria and saturated fatty acid-beta-oxidizing syntrophs were present at low levels in all digesters, and SPOB appeared to play a role in stabilizing propionate levels during start-up of one digester. Digesters with a history of poor performance tolerated a severe organic overload event better than digesters that had previously performed well. It is hypothesized that higher levels of SPOB and SFAS and their methanogenic partners in previously unstable digesters are responsible for this behavior.