Methanogenic Community Structure Research Articles

Effect of temperature on the structure and activity of a methanogenic archaeal community during rice straw decomposition

In recent years, the rice fields in Sanjiang Plain of Northeast China have drawn more and more attention because of the unique high-latitude location and the important contributions to rice production and methane emission. In the present study, a rice field soil in Sanjiang Plain was anaerobically incubated in presence and absence of rice straw at three temperatures (10 °C, 30 °C and 45 °C). The community structure and activity dynamics of methanogenic archaea were investigated by the terminal restriction fragment length polymorphism analyses in combination with cloning and sequencing of mcrA genes and transcripts and archaeal 16S rRNA genes. We found rice straw addition significantly shortened the lag phase of methanogenesis and enhanced the production of CH4 and the accumulation of methanogenic precursors (CO2, H2 and acetate). The T-RFLP analysis of mcrA gene revealed that structure of methanogenic archaeal community was relatively stable during the anoxic incubation. By contrast, analysis at the transcript level showed a highly dynamic composition of the active methanogens. In the beginning and early stages of incubation, Methanosarcinaceae actively utilized the accumulated acetate and H2/CO2 for CH4 production. Then the different methanogenic community developed along with the incubation time, and each representative methanogen group became predominant in different temperatures and treatments. At 10 °C, Methanobacteriales became more abundant in the soil without straw, while Methanosarcinaceae dominated in the soil with straw. The acetoclastic Methanosaetaceae, in particular, appeared to be active at 30 °C, probably due to the low concentration of acetate. High temperature of 45 °C significantly favored the hydrogenotrophic methanogens, with the increasing abundance of Methanobacteriales early and then the Methanocellales in the later stages.

Rapid fingerprinting of methanogenic communities by high-resolution melting analysis

Characterizing microbial community structure using molecular techniques is becoming a popular approach in studies of waste/wastewater treatment processes. A rapid and robust tool to analyze microbial communities is required for efficient process monitoring and control. In this study, a new community fingerprinting method based on high-resolution melting (HRM) analysis was developed and applied to compare methanogenic community structures of five different anaerobic digesters. The new method produced robust community clustering and ordination results comparable to the results from the commonly used denaturing gradient gel electrophoresis (DGGE) performed in parallel. This method transforms melting peak plots (MPs) of community DNA samples generated by HRM analysis to molecular fingerprints and estimates the relationships between the communities based on the fingerprints. The MP-based fingerprinting would provide a good alternative to monitor variations in microbial community structure especially when handling large sample numbers due to its high-throughput capacity and short analysis time.

Effect of paddy-upland rotation on methanogenic archaeal community structure in paddy field soil.

Methanogenic archaea are strict anaerobes and demand highly reduced conditions to produce methane in paddy field soil. However, methanogenic archaea survive well under upland and aerated conditions in paddy fields and exhibit stable community. In the present study, methanogenic archaeal community was investigated in fields where paddy rice (Oryza sativa L.) under flooded conditions was rotated with soybean (Glycine max [L.] Merr.) under upland conditions at different rotation histories, by polymerase chain reaction (PCR)-denaturing gradient gel electrophoresis (DGGE) and real-time quantitative PCR methods targeting 16S rRNA and mcrA genes, respectively. Soil samples collected from the fields before flooding or seeding, during crop cultivation and after harvest of crops were analyzed. The abundance of the methanogenic archaeal populations decreased to about one-tenth in the rotational plots than in the consecutive paddy (control) plots. The composition of the methanogenic archaeal community also changed. Most members of the methanogenic archaea consisting of the orders Methanosarcinales, Methanocellales, Methanomicrobiales, and Methanobacteriales existed autochthonously in both the control and rotational plots, while some were strongly affected in the rotational plots, with fatal effect to some members belonging to the Methanosarcinales. This study revealed that the upland conversion for one or longer than 1 year in the rotational system affected the methanogenic archaeal community structure and was fatal to some members of methanogenic archaea in paddy field soil.

Start-up strategies for thermophilic anaerobic digestion of pig manure

Sludge physicochemical composition, methane (CH4) yield, and methanogenic community structure and dynamics using quantitative real-time polymerase chain reaction were determined after start-up of anaerobic digestion of pig manure. Eight thermophilic continuous stirred anaerobic digesters were used during 126 days. Four management strategies were investigated: a feedless and a non-feedless period followed by a gradual or an abrupt addition of pig manure (two digesters per strategy). During the first 43 days, VFA (volatile fatty acids) accumulations and low CH4 yield were observed in all digesters. After this period, digesters recovered their initial status being propionic acid the last parameter to be re-established. Non-feedless digesters with an abrupt addition of pig manure showed the best performances (lower VFA accumulation and higher CH4 yield). Differences in microbial orders and dynamics, however, were less evident among treatments. Hydrogenotrophic methanogenesis, Methanomicrobiales first and Methanobacteriales second, was the dominant metabolic pathway in all digesters. Further research is needed to clarify the role and activity of hydrogenotrophic methanogens during the recovery start-up period and to identify the best molecular tools and methodologies to monitor microbial populations and dynamics reliably and accurately in anaerobic digesters.

A half-submerged integrated two-phase anaerobic reactor for agricultural solid waste codigestion

Anaerobic digestion is widely used in bioenergy recovery from waste. In this study, a half-submerged, integrated, two-phase anaerobic reactor consisting of a top roller acting as an acidogenic unit and a recycling bottom reactor acting as a methanogenic unit was developed for the codigestion of wheat straw (WS) and fruit/vegetable waste (FVW). The reactor was operated for 21 batches (nearly 300d). Anaerobic granular sludge was inoculated into the methanogenic unit. The residence time for the mixed waste was maintained as 10d when the operation stabilized, and the temperature was kept at 35°C. The highest organic loading rate was 1.37kg VS/(m3d), and the maximum daily biogas production was 328L/d. Volatile solid removal efficiencies exceeded 85%. WS digestion could be confirmed, and efficiency was affected by both the ratio of WS to FVW and the loading rate. The dominant bacteria were Bacteroides-like species, which are involved in glycan and cellulose decomposition. Methanogenic community structures, pH levels, and volatile fatty acid concentrations in the acidogenic and methanogenic units differed, indicating successful phase separation. This novel reactor can improve the mass transfer and microbial cooperation between acidogenic and methanogenic units and can efficiently and steady codigest solid waste.

Methanogen community composition and rates of methane consumption in Canadian High Arctic permafrost soils

Increasing permafrost thaw, driven by climate change, has the potential to result in organic carbon stores being mineralized into carbon dioxide (CO2) and methane (CH4) through microbial activity. This study examines the effect of increasing temperature on community structure and metabolic activity of methanogens from the Canadian High Arctic, in an attempt to predict how warming will affect microbially controlled CH4 soil flux. In situ CO2 and CH4 flux, measured in 2010 and 2011 from ice-wedge polygons, indicate that these soil formations are a net source of CO2 emissions, but a CH4 sink. Permafrost and active layer soil samples were collected at the same sites and incubated under anaerobic conditions at warmer temperatures, with and without substrate amendment. Gas flux was measured regularly and indicated an increase in CH4 flux after extended incubation. Pyrosequencing was used to examine the effects of an extended thaw cycle on methanogen diversity and the results indicate that in situ methanogen diversity, based on the relative abundance of the 16S ribosomal ribonucleic acid (rRNA) gene associated with known methanogens, is higher in the permafrost than in the active layer. Methanogen diversity was also shown to increase in both the active layer and permafrost soil after an extended thaw. This study provides evidence that although High Arctic ice-wedge polygons are currently a sink for CH4, higher arctic temperatures and anaerobic conditions, a possible result of climate change, could result in this soil becoming a source for CH4 gas flux.

Differentiation between sediment and hypolimnion methanogen communities in humic lakes

The traditional view of carbon cycling within the pelagic zone of freshwater lakes has consisted of methane production within the anoxic sediment, followed by diffusive flux and ebullition through the water column. Methanogenic archaea have been shown to be present within the water columns of freshwater lakes; however, little is known about whether these methanogenic communities are distinct from those in the sediment or how these communities change over space and time. We used the methanogen-specific phylogenetic marker mcrA to perform a 3-year study focusing on the community structure of methanogens within the sediment and anoxic hypolimnion water layer of five humic lakes in WI, USA. The hypolimnion and sediment communities were distinct in composition, richness and phylogenetic diversity. Hypolimnion communities displayed a temporally stable biogeographical pattern among lakes, which was driven by both lake-specific environmental variables and barriers to dispersal. We conclude that the hypolimnion comprised communities of methanogens that are distinct from those in the sediment, differentiated among lakes, and likely have unique ecological roles and evolutionary trajectories in these anaerobic environments.

Methane production potential and methanogenic archaeal community structure in tropical irrigated Indian paddy soils

Soil characteristics regulate various belowground microbial processes including methanogenesis and, consequently, affect the structure and function of methanogenic archaeal communities due to change in soil type which in turn influences the CH4 production potential of soils. Thus, five different soil orders (Alfisol, Entisol, Inceptisol, Podzol and Vertisol) were studied to assess their CH4 production potential and also the methanogenic archaeal community structure in dryland irrigated Indian paddy soils. Soil incubation experiments revealed CH4 production to range from 178.4 to 431.2 μg CH4 g-1 dws in all soil orders as: Vertisol<Inceptisol<Entisol<Podzol<Alfisol. The numbers of methanogens as quantified using real-time quantitative polymerase chain reaction (qPCR) targeting mcrA genes varied between 0.06 and 72.97 (×106 copies g-1 dws) and were the highest in Vertisol soil and the least in Alfisol soil. PCR-denaturing gradient gel electrophoresis (DGGE)-based approach targeting 16S rRNA genes revealed diverse methanogenic archaeal communities across all soils. A total of 43 DGGE bands sequenced showed the closely related groups to Methanomicrobiaceae, Methanobacteriaceae, Methanocellales, Methanosarcinaceae, Methanosaetaceae and Crenarchaeota. The composition of methanogenic groups differed among all soils and only the Methanocellales group was common and dominant in all types of soils. The highest diversity of methanogens was found in Inceptisol and Vertisol soils. Methane production potential varied significantly in different soil orders with a positive relationship (p < 0.05) with methanogens population size, permanganate oxidizable C (POXC) and CO2 production. The present study suggested that CH4 production potential of different soils depends on physicochemical properties, methanogenic archaeal community composition and the population size.

Methane production potential and methanogenic archaea community dynamics along the Spartina alterniflora invasion chronosequence in a coastal salt marsh

Invasion by the exotic species Spartina alterniflora, which has high net primary productivity and superior reproductive capacity compared with native plants, has led to rapid organic carbon accumulation and increased methane (CH₄) emission in the coastal salt marsh of China. To elucidate the mechanisms underlying this effect, the methanogen community structure and CH₄ production potential as well as soil organic carbon (SOC), dissolved organic carbon, dissolved organic acids, methylated amines, aboveground biomass, and litter mass were measured during the invasion chronosequence (0-16 years). The CH₄ production potential in the S. alterniflora marsh (range, 2.94-3.95 μg kg(-1) day(-1)) was significantly higher than that in the bare tidal mudflat. CH₄ production potential correlated significantly with SOC, acetate, and trimethylamine concentrations in the 0-20 cm soil layer. The abundance of methanogenic archaea also correlated significantly with SOC, and the dominant species clearly varied with S. alterniflora-driven SOC accumulation. The acetotrophic Methanosaetaceae family members comprised a substantial proportion of the methanogenic archaea in the bare tidal mudflat while Methanosarcinaceae family members utilized methylated amines as substrates in the S. alterniflora marsh. Ordination analysis indicated that trimethylamine concentration was the primary factor inducing the shift in the methanogenic archaea composition, and regressive analysis indicated that the facultative family Methanosarcinaceae increased linearly with trimethylamine concentration in the increasingly sulfate-rich salt marsh. Our results indicate that increased CH₄ production during the S. alterniflora invasion chronosequence was due to increased levels of the non-competitive substrate trimethylamine and a shift in the methanogenic archaea community.

Molecular diversity and tools for deciphering the methanogen community structure and diversity in freshwater sediments

Methanogenic archaeal communities existing in freshwater sediments are responsible for approximately 50% of the total global emission of methane. This process contributes significantly to global warming and, hence, necessitates interventional control measures to limit its emission. Unfortunately, the diversity and functional interactions of methanogenic populations occurring in these habitats are yet to be fully characterized. Considering several disadvantages of conventional culture-based methodologies, in recent years, impetus is given to molecular biology approaches to determine the community structure of freshwater sedimentary methanogenic archaea. 16S rRNA and methyl coenzyme M reductase (mcrA) gene-based cloning techniques are the first choice for this purpose. In addition, electrophoresis-based (denaturing gradient gel electrophoresis, temperature gradient gel electrophoresis, and terminal restriction fragment length polymorphism) and quantitative real-time polymerase chain reaction techniques have also found extensive applications. These techniques are highly sensitive, rapid, and reliable as compared to traditional culture-dependent approaches. Molecular diversity studies revealed the dominance of the orders Methanomicrobiales and Methanosarcinales of methanogens in freshwater sediments. The present review discusses in detail the status of the diversity of methanogens and the molecular approaches applied in this area of research.

Thermo-alkaline pretreatment of waste activated sludge at low-temperatures: Effects on sludge disintegration, methane production, and methanogen community structure

Low-temperature thermo-alkaline pretreatment of waste activated sludge (WAS) was studied, within the region of 0-0.2 M NaOH and 60-90°C, for the effects of NaOH concentration and temperature on sludge degradability in anaerobic digestion (AD). Significant disintegration of sludge solids (up to 75.6%) and an increase in methane production (up to 70.6%) were observed in the pretreatment trials. Two quadratic models were successfully generated by response surface analysis (R(2)>0.9, p<0.05) to approximate how the degree of sludge disintegration (SD) and methane production (MP) respond to changes in the pretreatment conditions. The maximum responses of SD (77.8%) and MP (73.9% increase over the control) were shown at [0.16 M NaOH, 90°C] and [0.10 M NaOH, 73.7°C], respectively. NaOH addition showed a significant influence on the evolution of methanogen community structure during AD, whereas temperature did not. Aceticlastic Methanosaeta and Methanosarcina speceies were likely the major methanogens.

Characterization of the methanogen community in a household anaerobic digester fed with swine manure in China

Household anaerobic digesters have been installed across rural China for biogas production, but information on methanogen community structure in these small biogas units is sparsely available. By creating clone libraries for 16S rRNA and methyl coenzyme M reductase alpha subunit (mcrA) genes, we investigated the methanogenic consortia in a household biogas digester treating swine manure. Operational taxonomic units (OTUs) were defined by comparative sequence analysis, seven OTUs were identified in the 16S rRNA gene library, and ten OTUs were identified in the mcrA gene library. Both libraries were dominated by clones highly related to the type strain Methanocorpusculum labreanum Z, 64.0% for 16S rRNA gene clones and 64.3% for mcrA gene clones. Additionally, gas chromatography assays showed that formic acid was 84.54% of the total volatile fatty acids and methane was 57.20% of the biogas composition. Our results may help further isolation and characterization of methanogenic starter strains for industrial biogas production.

Comparative study of fermentation and methanogen community structure in the digestive tract of goats and rabbits

Methane is the most important anthropogenic contribution to climate change after carbon dioxide and represents a loss of feed energy for the animal, mainly for herbivorous species. However, our knowledge about the ecology of Archaea, the microbial group responsible for methane synthesis in the gut, is very poor. Moreover, it is well known that hindgut fermentation differs from rumen fermentation. The composition of archaeal communities in fermentation compartments of goats and rabbits were investigated using DGGE to generate fingerprints of archaeal 16S rRNA gene. Ruminal contents and faeces from five Murciano-Granadina goats and caecal contents of five commercial White New Zealand rabbits were compared. Diversity profile of methanogenic archaea was carried out by PCR-DGGE. Quantification of methanogenic archaea and the abundance relative to bacteria was determined by real-time PCR. Methanogenic archaeal species were relatively constant across species. Dendrogram from DGGE of the methanogen community showed one cluster for goat samples with two sub-clusters by type of sample (ruminal and faeces). In a second cluster, samples from rabbit were grouped. No differences were found either in richness or Shannon index as diversity indexes. Although the primer sets used was developed to investigate rumen methanogenic archaeal community, primers specificity did not affect the assessment of rabbit methanogen community structure. Rumen content showed the highest number or methanogenic archaea (log₁₀ 9.36), followed by faeces (log₁₀ 8.52) and showing rabbit caecum the lower values (log₁₀ 5.52). DGGE profile showed that pre-gastric and hindgut fermenters hold a very different methanogen community. Rabbits hold a microbial community of similar complexity than that in ruminants but less abundant, which agrees with the type of fermentation profile.

Response of anaerobes to methyl fluoride, 2-bromoethanesulfonate and hydrogen during acetate degradation

To use the selective inhibition method for quantitative analysis of acetate metabolism in methanogenic systems, the responses of microbial communities and metabolic activities, which were involved in anaerobic degradation of acetate, to the addition of methyl fluoride (CH3F), 2-bromoethanesulfonate (BES) and hydrogen were investigated in a thermophilic batch experiment. Both the methanogenic inhibitors, i.e., CH3F and BES, showed their effectiveness on inhibiting CH4 production, whereas acetate metabolism other than acetoclastic methanogenesis was stimulated by BES, as reflected by the fluctuated acetate concentration. Syntrophic acetate oxidation was thermodynamically blocked by hydrogen (H2), while H2-utilizing reactions as hydrogenotrophic methanogenesis and homoacetogenesis were correspondingly promoted. Results of PCR-DGGE fingerprinting showed that, CH3F did not influence the microbial populations significantly. However, the BES and hydrogen notably altered the bacterial community structures and increased the diversity. BES gradually changed the methanogenic community structure by affecting the existence of different populations to different levels, whilst H2 greatly changed the abundance of different methanogenic populations, and induced growth of new species.

Behavior of methanogens during start-up of farm-scale anaerobic digester treating swine wastewater

The aim of this study was to monitor the changes in methanogenic community structures in an anaerobic digester (250m3 working volume) during start-up including prolonged starvation periods. Redundancy analysis was performed to investigate the correlations between environmental variables and microbial community structures. The anaerobic digester was operated for 591 days at alternating operating temperatures. In initial start-up period at stage I (35°C), growth of various species of mesophilic aceticlastic methanogens (AMs) and hydrogenotrophic methanogens (HMs) was observed. Methanobacteriales species survived better than other methanogens under long-term starvation conditions. In stage II (50°C), HMs became dominant over AMs as the operating temperature changed from mesophilic to thermophilic due to increase of ammonia inhibition. In stage III (35°C), only the Methanomicrobiales population significantly increased during 50 days of HRT while Methanobacteriales dominated over 15 days of HRT. The influent pH negatively correlated with all methanogenic populations especially in stage II.

Methanosarcina domination in anaerobic sequencing batch reactor at short hydraulic retention time

The Archaea population of anaerobic sequential batch reactor (ASBR) featuring cycle operations under varying hydraulic retention time (HRT) was evaluated for treating a dilute waste stream. Terminal-Restriction Length Polymorphism and clone libraries for both 16S rRNA gene and mcrA gene were employed to characterize the methanogenic community structure. Results revealed that a Methanosarcina dominated methanogenic community was successfully established when using an ASBR digester at short HRT. It was revealed that both 16S rRNA and mcrA clone library could not provide complete community structure, while combination of two different clone libraries could capture more archaea diversity. Thermodynamic calculations confirmed a preference for the observed population structure. The results both experimentally and theoretically confirmed that Methanosarcina dominance emphasizing ASBR's important role in treating low strength wastewater as Methanosarcina will be more adept at overcoming temperature and shock loadings experienced with treating this type of wastewater.

Comparison of methanogenic community structure and anaerobic process performance treating swine wastewater between pilot and optimized lab scale bioreactors

To investigate methanogenic community structure and process performance of anaerobic digestion treating swine wastewater at different scale, a pilot plant with 20 m(3) of effective working volume and lab scale methanogenic digester with 6L working volume were operated for 71 days and 6 turnover periods, respectively. During the steady state of anaerobic digestion, COD and VS removal efficiency in pilot plant were 65.3±3.2, 51.6±4.3%, respectively, which was similar to those in lab scale. However, calculated VFAs removal efficiency and methane yield were lower in pilot plant than in lab scale digester. Also, organics removal efficiencies, which consist of total carbohydrates, proteins, and lipids, were different between pilot and lab scale. These results were thought to be due to the ratio of carbohydrates to proteins in the raw swine wastewater. As a result of qualitative microbial analysis, Methanoculleus receptaculii, and Methanoculleus bourgensis, were commonly concerned with methane production.

Methane emissions from wetlands: biogeochemical, microbial, and modeling perspectives from local to global scales

Understanding the dynamics of methane (CH4 ) emissions is of paramount importance because CH4 has 25 times the global warming potential of carbon dioxide (CO2 ) and is currently the second most important anthropogenic greenhouse gas. Wetlands are the single largest natural CH4 source with median emissions from published studies of 164 Tgyr(-1) , which is about a third of total global emissions. We provide a perspective on important new frontiers in obtaining a better understanding of CH4 dynamics in natural systems, with a focus on wetlands. One of the most exciting recent developments in this field is the attempt to integrate the different methodologies and spatial scales of biogeochemistry, molecular microbiology, and modeling, and thus this is a major focus of this review. Our specific objectives are to provide an up-to-date synthesis of estimates of global CH4 emissions from wetlands and other freshwater aquatic ecosystems, briefly summarize major biogeophysical controls over CH4 emissions from wetlands, suggest new frontiers in CH4 biogeochemistry, examine relationships between methanogen community structure and CH4 dynamics in situ, and to review the current generation of CH4 models. We highlight throughout some of the most pressing issues concerning global change and feedbacks on CH4 emissions from natural ecosystems. Major uncertainties in estimating current and future CH4 emissions from natural ecosystems include the following: (i) A number of important controls over CH4 production, consumption, and transport have not been, or are inadequately, incorporated into existing CH4 biogeochemistry models. (ii) Significant errors in regional and global emission estimates are derived from large spatial-scale extrapolations from highly heterogeneous and often poorly mapped wetland complexes. (iii) The limited number of observations of CH4 fluxes and their associated environmental variables loosely constrains the parameterization of process-based biogeochemistry models.

Changes in Microbial Community Structure During Anaerobic Repeated-Batch Treatment of Cheese-Processing Wastewater

This study investigated changes in microbial community structure, associated with changes in process performance, in an anaerobic bioreactor treating cheese-processing wastewater in repeated-batch mode. During the operation of three batch cycles, the overall reaction rate increased with the increase in starting microbial population size over cycles, indicating a positive effect of biomass accumulation on process performance. Bacterial community structure varied significantly over cycles in the reactor. In contrast, methanogen community structure was maintained almost unchanged (>90% similarity) during the experiment. This indicates that changes in bacterial community structure interestingly had little influence on the formation of methanogen community structure in the reactor. Another notable point is that the reactor methanogen community was greatly dominated by hydrogenotrophic methanogens (up to 90% of the total population), likely suggesting H2-utilizing pathway as the major methanogenesis route in the reactor. This is contrary to the conventional wisdom that aceticlastic methanogens are the main drivers of methanogenesis in anaerobic digesters under normal conditions.

Comparative study of changes in reaction profile and microbial community structure in two anaerobic repeated-batch reactors started up with different seed sludges

Microbial community structure and dynamics were examined in two anaerobic reactors run in repeated-batch mode to treat whey permeate. Despite being started up using different seeding sources, the reactors showed generally similar reaction patterns and performances. During the repeated-batch operation for three cycles, the overall reaction rate increased with the increase in the initial population size of both bacteria and methanogens over cycles. Clostridium- and Methanospirillum-related microorganisms were likely the main acidogenic and methanogenic populations, respectively, in both reactors. Bacterial community structure shifted dynamically over cycles, while little change was observed in methanogen community structure throughout the operation. This means that the changes in bacterial community structure changes had little influence on the formation and evolution of methanogen community structure in the reactors. The increased methanogenesis rate with cycles seemed therefore more likely due to the effect of the increase in methanogen abundance rather than the alteration of community structure.