Groups Of Methanogens Research Articles

Performance and microbial community analysis of a pilot-scale UASB for corn-ethanol wastewater treatment.

The performance and microbial community structure of a pilot-scale upflow anaerobic sludge blanket (UASB) reactor inoculated with flocculent sludge were investigated over 52days. The characteristics of corn-ethanol wastewater were as follows: CODCr, 1,050-4,970mgl(-1); ammonia, 14-298mgl(-1); and alkalinity, 332-2,867mgl(-1). The UASB could start up smoothly with a hydraulic loading rate lower than 180lh(-1) and a ratio of volatile fatty acid versus alkalinity between 0.04 and 0.48. The maximum gas production rate was 432lh(-1) and the highest volumetric loading rate of 7.2kgm(-3)day(-1) was obtained after 48days. The 1mm granules could form a complex network and were composed of many Methanosaeta. Aceticlastic methanogens served as a dominant methanogenic group, which accounted for the relatively high resistance to shock loading.

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.

Methane fluxes and the functional groups of methanotrophs and methanogens in a young Arctic landscape on Disko Island, West Greenland

Arctic soils are known to be important methane (CH4) consumers and sources. This study integrates in situ fluxes of CH4 between upland and wetland soils with potential rates of CH4 oxidation and production as well as abundance and diversity of the methanotrophs and methanogens measured with pyrosequencing of 16S DNA and rRNA fragments in soil and permafrost layers. Here, the spatial patterns of in situ CH4 fluxes for a 2,000 years old Arctic landscape in West Greenland reveal similar CH4 uptake rates (−4 ± 0.3 μmol m−2 h−1) as in other Arctic sites, but lower CH4 emissions (14 ± 1.5 μmol m−2 h−1) at wetland sites compared to other Arctic wetlands. Potential CH4 oxidation was similar for upland and wetland soils, but the wetter soils produced more CH4 in active and permafrost layers. Accordingly, the abundance of methanogenic archaea was highest in wetland soils. The methanotrophic community also differed between upland and wetland soils, with predominant activity of Type II methanotrophs in the active layer for upland soils, but only Type I methanotrophs for the wetland. In the permafrost of upland and wetland soils, activity of the methanotrophs belonging to Type I and Type II as well as methanogens were detected. This study indicates that the magnitude of CH4 oxidation and the direction of the flux, i.e. uptake or emission, are linked to different methanotrophic communities in upland and wetland soils. Also, the observed link between production/consumption rates and the microbial abundance and activity indicates that the age of an Arctic landscape is not important for the CH4 consumption but can be very important for CH4 production. Considering the prevalence of dry landscapes and contrasting ages of high Arctic soils, our results highlight that well-drained soils should not be overlooked as an important component of Arctic net CH4 budget.

Effect of biogas digested slurry-based biochar on methane flux and methanogenic archaeal diversity in paddy soil

A microcosm-based study was conducted to evaluate the effects of varying concentrations of biogas digested slurry-based biochar on CH4 flux, associated methanogenic archaeal community, soil chemical and microbial properties, and plant biomass in paddy soil. Our results showed that biochar application significantly increased the CH4 flux compared to untreated soil, and it increased with the increase in biochar concentration. The NH4+-N concentration also increased significantly in biochar treated soils due to more reducible conditions in the soil which prevented NH4+-N conversion to NO3–-N. The application of biochar significantly increased soluble organic carbon, microbial biomass carbon and soluble total nitrogen content of the soils without affecting microbial biomass nitrogen contents. Paddy plant biomass such as shoot weight, panicle numbers and weight of panicles were also higher in biochar treated soils. Denaturing gradient gel electrophoresis revealed all methanogenic groups with the dominance of Methanosaetaceae and Methanocellales groups, whereas no differences were found in methanogenic archaeal diversity among biochar treated and non-treated soils. Thus, the increase in CH4 flux with the application of varying biochar concentration is possibly due to the variation in the soil variables and plant biomass without affecting methanogenic archaeal diversity.

Evidence of novel phylogenetic lineages of methanogenic archaea from hypersaline microbial mats.

Methanogenesis in hypersaline and high-sulfate environments is typically dominated by methylotrophic methanogens because sulfate reduction is thermodynamically favored over hydrogenotrophic methanogenesis in these environments. We characterized the community composition of methanogenic archaea in both unmanipulated and incubated microbial mats from different hypersaline environments in Baja California Sur, Mexico. Clone libraries of methyl coenzyme-M reductase (mcrA) sequences and DGGE band patterns of 16S rRNA and mcrA sequences showed that the methanogen community in these microbial mats is dominated by methylotrophic methanogens of the genus Methanohalophilus. However, phylogenetic analyses of mcrA sequences from these mats also revealed two new lineages corresponding to putative hydrogenotrophic methanogens related with the strictly hydrogenotrophic order Methanomicrobiales. Stimulated methane production under decreased salinity and sulfate concentrations also suggested the presence of hydrogenotrophic methanogens in these samples. The relative abundance of mcrA gene and transcripts, estimated by SYBR green I qPCR assays, suggested the activity of different phylogenetic groups of methanogens, including the two novel clusters, in unmanipulated samples of hypersaline microbial mats. Using geochemical and molecular approaches, we show that substrate limitation and values of salinity and sulfate higher than 3 % and 25 mM (respectively) are potential environmental constraints for methanogenesis in these environments. Microcosm experiments with modifications of salinity and sulfate concentrations and TMA addition showed that upper salt and sulfate concentrations for occurrence of methylotrophic methanogenesis were 28 % and 263 mM, respectively. This study provides phylogenetic information about uncultivated and undescribed methanogenic archaea from hypersaline environments.

Impact of abrupt temperature increase on the performance of an anaerobic hybrid bioreactor and its intrinsic microbial community

This study aimed to analyse the effect of sudden temperature increases (in the range of 45–65°C) on the performance and the microbial community structure of a hybrid anaerobic reactor. The reactor recovered with time after every temperature shock up to the operating temperature of 55°C. At 55°C, a 10°C shock resulting in an operating temperature of 65°C, deteriorated the reactor’s performance. At this condition, both, the diversity and the relative abundance of methanogenic groups, especially of Methanosaetaceae, were significantly affected as observed by DGGE fingerprinting and quantitative PCR. In contrast, at lower temperatures (i.e., 45 and 55°C), thermal shocks seemed to have less effect due to the presence and maintenance of thermophilic strains, which prevented system deterioration. At 65°C, the absence of any acetoclastic methanogen is assumed to be the cause of system failure.

Source separation increases methane yields for waste-to-energy applications in the personal care product industry

Abstract High-carbon industrial wastewaters are attractive for anaerobic biological waste-to-energy treatment approaches. However, some waste streams, such as those from the personal care product industry (PCP), also can contain inhibitory compounds that impair biogas production (methane; CH4), reducing potential energy recovery. Here we show how source separation of PCP wastes can increase gross CH4 production by shunting inhibitory streams around the methanogenic treatment step. Methane production was compared between shampoo and hair colorant liquid wastes as sources for waste-to-energy applications. Methane yields were five times greater for comparable COD shampoo versus colorant wastes in batch reactors (∼250 versus ∼50 ml CH4/mg COD), suggesting colorant waste constituents impair methanogenesis, although anaerobic treatment reduced the toxicity of both wastes (i.e., Daphnia magna IC50 tolerance values increased from 0.23 ± 0.07 and 0.17 ± 0.04%v/v to 38.0 ± 2.0 and 15.3 ± 8.9%v/v, respectively). To understand methanogenesis inhibition, the colorant waste was separated into sub-streams associated with different production operations and assayed individually, and CH4 production almost tripled when dye and oxidant levels were reduced. To explain CH4 yields, qPCR was used to quantify eubacteria and five methanogen groups among reactors. Colorant-fed units always had lower microbial abundances than shampoo-fed units, but Methanosarcinaceae levels were especially low in the low CH4 reactors. Further, these acetoclasts and hydrogenotrophic Methanobacteriales significantly correlated with CH4 yields across all reactors (r2 = 0.46, p = 0.02 and r2 = 0.68, p = 0.003, respectively). Waste-to-energy opportunities clearly exist for PCP wastes, although source separation is required to maximise gross CH4 yields.

Diversity and distribution of archaea community along a stratigraphic permafrost profile from Qinghai-Tibetan Plateau, China.

Accompanying the thawing permafrost expected to result from the climate change, microbial decomposition of the massive amounts of frozen organic carbon stored in permafrost is a potential emission source of greenhouse gases, possibly leading to positive feedbacks to the greenhouse effect. In this study, the community composition of archaea in stratigraphic soils from an alpine permafrost of Qinghai-Tibetan Plateau was investigated. Phylogenic analysis of 16S rRNA sequences revealed that the community was predominantly constituted by Crenarchaeota and Euryarchaeota. The active layer contained a proportion of Crenarchaeota at 51.2%, with the proportion of Euryarchaeota at 48.8%, whereas the permafrost contained 41.2% Crenarchaeota and 58.8% Euryarchaeota, based on 16S rRNA gene sequence analysis. OTU1 and OTU11, affiliated to Group 1.3b/MCG-A within Crenarchaeota and the unclassified group within Euryarchaeota, respectively, were widely distributed in all sediment layers. However, OTU5 affiliated to Group 1.3b/MCG-A was primarily distributed in the active layers. Sequence analysis of the DGGE bands from the 16S rRNAs of methanogenic archaea showed that the majority of methanogens belonged to Methanosarcinales and Methanomicrobiales affiliated to Euryarchaeota and the uncultured ZC-I cluster affiliated to Methanosarcinales distributed in all the depths along the permafrost profile, which indicated a dominant group of methanogens occurring in the cold ecosystems.

Application of an early warning indicator and CaO to maximize the time–space-yield of an completely mixed waste digester using rape seed oil as co-substrate

In order to increase the organic loading rate (OLR) and hereby the performance of biogas plants an early warning indicator (EWI-VFA/Ca) was applied in a laboratory-scale biogas digester to control process stability and to steer additive dosing. As soon as the EWI-VFA/Ca indicated the change from stable to instable process conditions, calcium oxide was charged as a countermeasure to raise the pH and to bind long-chain fatty acids (LCFAs) by formation of aggregates. An interval of eight days between two increases of the OLR, which corresponded to 38% of the hydraulic residence time (HRT), was sufficient for process adaptation. An OLR increase by a factor of three within six weeks was successfully used for biogas production. The OLR was increased to 9.5kg volatile solids (VS)m−3d−1 with up to 87% of fat. The high loading rates affected neither the microbial community negatively nor the biogas production process. Despite the increase of the organic load to high rates, methane production yielded almost its optimum, amounting to 0.9m3(kgVS)−1. Beneath several uncharacterized members of the phylum Firmicutes mostly belonging to the family Clostridiaceae, a Syntrophomonas-like organism was identified that is known to live in a syntrophic relationship to methanogenic archaea. Within the methanogenic group, microorganisms affiliated to Methanosarcina, Methanoculleus and Methanobacterium dominated the community.

Greenhouse gas emissions from rice microcosms amended with a plant microbial fuel cell

Methane (CH4) release from wetlands is an important source of greenhouse gas emissions. Gas exchange occurs mainly through the aerenchyma of plants, and production of greenhouse gases is heavily dependent on rhizosphere biogeochemical conditions (i.e. substrate availability and redox potential). It is hypothesized that by introducing a biocatalyzed anode electrode in the rhizosphere of wetland plants, a competition for carbon and electrons can be invoked between electrical current-generating bacteria and methanogenic Archaea. The anode electrode is part of a bioelectrochemical system (BES) capable of harvesting electrical current from microbial metabolism. In this work, the anode of a BES was introduced in the rhizosphere of rice plants (Oryza sativa), and the impact on methane emissions was monitored. Microbial current generation was able to outcompete methanogenic processes when the bulk matrix contained low concentrations of organic carbon, provided that the electrical circuit with the effective electroactive microorganisms was in place. When interrupting the electrical circuit or supplying an excess of organic carbon, methanogenic metabolism was able to outcompete current generating metabolism. The qPCR results showed hydrogenotrophic methanogens were the most abundant methanogenic group present, while mixotrophic or acetoclastic methanogens were hardly detected in the bulk rhizosphere or on the electrodes. Competition for electron donor and acceptor were likely the main drivers to lower methane emissions. Overall, electrical current generation with BESs is an interesting option to control CH4 emissions from wetlands but needs to be applied in combination with other mitigation strategies to be successful and feasible in practice.

Absolute dominance of hydrogenotrophic methanogens in full-scale anaerobic sewage sludge digesters

Anaerobic digestion (AD) is gaining increasing attention due to the ability to covert organic pollutants into energy-rich biogas and, accordingly, growing interest is paid to the microbial ecology of AD systems. Despite extensive efforts, AD microbial ecology is still limitedly understood, especially due to the lack of quantitative information on the structures and dynamics of AD microbial communities. Such knowledge gap is particularly pronounced in sewage sludge AD processes although treating sewage sludge is among the major practical applications of AD. Therefore, we examined the microbial communities in three full-scale sewage sludge digesters using qualitative and quantitative molecular techniques in combination: denaturing gradient gel electrophoresis (DGGE) and real-time polymerase chain reaction (PCR). Eight out of eleven bacterial sequences retrieved from the DGGE analysis were not affiliated to any known species while all eleven archaeal sequences were assigned to known methanogen species. Quantitative real-time PCR analysis revealed that, based on the 16S rRNA gene abundance, the hydrogenotrophic order Methanomicrobiales is the most dominant methanogen group (> 94% of the total methanogen population) in all digesters. This corresponds well to the prevailing occurrence of the DGGE bands related to Methanolinea and Methanospirillum, both belonging to the order Methanomicrobiales, in all sludge samples. It is therefore suggested that hydrogenotrophic methanogens, especially Methanomicrobiales strains, are likely the major players responsible for biogas production in the digesters studied. Our observation is contrary to the conventional understanding that aceticlastic methanogens generally dominate methanogen communities in stable AD environments, suggesting the need for further studies on the dominance relationship in various AD systems.

Diet Alters Both the Structure and Taxonomy of the Ovine Gut Microbial Ecosystem

We surveyed the ruminal metagenomes of 16 sheep under two different diets using Illumina pair-end DNA sequencing of raw microbial DNA extracted from rumen samples. The resulting sequence data were bioinformatically mapped to known prokaryotic 16S rDNA sequences to identify the taxa present in the samples and then analysed for the presence of potentially new taxa. Strikingly, the majority of the microbial individuals found did not map to known taxa from 16S sequence databases. We used a novel statistical modelling approach to compare the taxonomic distributions between animals fed a forage-based diet and those fed concentrated grains. With this model, we found significant differences between the two groups both in the dominant taxa present in the rumen and in the overall shape of the taxa abundance curves. In general, forage-fed animals have a more diverse microbial ecosystem, whereas the concentrate-fed animals have ruminal systems more heavily dominated by a few taxa. As expected, organisms from methanogenic groups are more prevalent in forage-fed animals. Finally, all of these differences appear to be grounded in an underlying common input of new microbial individuals into the rumen environment, with common organisms from one feed group being present in the other, but at much lower abundance.

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.

Influence of hydrodynamic shear on performance and microbial community structure of a hybrid anaerobic reactor

AbstractBACKGROUNDHydrodynamic shear plays a major role in the formation, maintenance, structure, and metabolism of self‐immobilized microbial communities in reactors. The study attempts to correlate the performance and microbial community structure of granular sludge in a mesophilic hybrid anaerobic reactor under different upflow velocities to fluidize the bed. The reactor was started with an initial upflow velocity of 4 m h‐1, which was increased gradually till system deterioration to observe its effect on granule size, reactor performance, and microbial community structure. Monte Carlo method was used to quantify hydrodynamic shear range at different upflow velocities using a modified Ergun equation for the respective ranges of granule size.RESULTSUp to 6 m h‐1 performance was positively affected, but beyond this upflow velocity, the system started showing reduced performance and completely deteriorated at 10 m h‐1. Significant reduction in the abundance and diversity of both Archaea and Bacteria was observed. Among all methanogenic groups, Methanosaetaceae was most severely affected due to breakage and wash out of granules.CONCLUSIONIt can be concluded that hydrodynamic shear can be employed as a sensitive control parameter to improve reactor performance, and a specified range should be determined experimentally to avoid negative influences associated with this. © 2013 Society of Chemical Industry

Changes in microbial diversity, methanogenesis and fermentation characteristics in the rumen in response to medicinal plant extracts.

This study evaluated the in vitro effect of medicinal plant extracts on ruminal methanogenesis, four different groups of methanogens and ruminal fermentation characteristics. A fistulated Holstein cow was used as a donor of rumen fluid. Licorice and mugwort extracts (Glycyrrhiza uralensis and Artemisia capillaris, 0.5% and 1% of total substrate DM, respectively), previously used as folk remedies, were added to an in vitro fermentation incubated with buffered-rumen fluid. Total gas production in Glycyrrhiza uralensis extract treatment was not significantly different between treatments (p<0.05) while total gas production in the Artemisia capillaris extract treatment was lower than that of the control. Artemisia capillaris extract and Glycyrrhiza uralensis extract reduced CH4 emission by 14% (p<0.05) and 8% (p<0.05), respectively. Ciliate-associated methanogens population decreased by 18% in the medicinal plant extracts treatments. Medicinal plant extracts also affected the order Methanobacteriales community. Methanobacteriales diversity decreased by 35% in the Glycyrrhiza uralensis extract treatment and 30% in the Artemisia capillaris extract treatment. The order Methanomicrobiales population decreased by 50% in the 0.5% of Glycyrrhiza uralensis extract treatment. These findings demonstrate that medicinal plant extracts have the potential to inhibit in vitro ruminal methanogenesis.

Carbon source — A strong determinant of microbial community structure and performance of an anaerobic reactor

Industrial effluents differ in their organic composition thereby providing different carbon sources to the microbial communities involved in its treatment. This study aimed to investigate the correlation of microbial community structure with wastewater composition and reactor's performance. Self-immobilized granules were developed in simulated wastewater based on different carbon sources (glucose, sugarcane molasses, and milk) in three hybrid anaerobic reactors operated at 37°C. To study archaeal community structure, a polyphasic approach was used with both qualitative and quantitative analysis. While PCR-denaturing gradient gel electrophoresis of 16S rRNA gene did not reveal major shifts in diversity of archaea with change in substrate, quantification of different groups of methanogens and total bacteria by real-time PCR showed variations in relative abundances with the dominance of Methanosaetaceae and Methanobacteriales. These data were supported by differences in the ratio of total counts of archaea and bacteria analyzed by catalyzed reporter deposition – fluorescence in situ hybridization. During hydraulic and organic shocks, the molasses-based reactor showed the best performance followed by the milk- and the glucose-based reactor. The study indicates that carbon source shapes the microbial community structure more in terms of relative abundance with distinct metabolic capacities rather than its diversity itself.

English

Biogas plants continuously convert biological wastes mainly into a mixture of methane, CO2 and H2O a conversion that is carried out by a consortium of bacteria and archaea. The microbial community structure of the fermentation process was evaluated by using oligonucleotide probes complementary to conserved sequences of the 16S rRNAs of phylogenetically defined groups of methanogens. At the beginning of fermentation, Eubacteria are the main group and the count of Archaea then constitutes only an insignificant percentage of the population of microorganisms. During the process of fermentation, we recorded a very significant increase in methanogenic microorganisms after 40 days of the process in progress. It is reflected in the quantity and the composition of released biogas. Key words: Methane fermentation, methanogenic microorganisms, phylogenetic diversity, rRNA.

Structure and function of the methanogenic microbial communities in Uruguayan soils shifted between pasture and irrigated rice fields

Irrigated rice fields in Uruguay are temporarily established on soils used as cattle pastures. Typically, 4 years of cattle pasture are alternated with 2 years of irrigated rice cultivation. Thus, oxic upland conditions are rotated with seasonally anoxic wetland conditions. Only the latter conditions are suitable for the production of CH4 from anaerobic degradation of organic matter. We studied soil from a permanent pasture as well as soils from different years of the pasture-rice rotation hypothesizing that activity and structure of the bacterial and archaeal communities involved in production of CH4 change systematically with the duration of either oxic or anoxic conditions. Soil samples were taken from drained fields, air-dried and used for the experiments. Indeed, methanogenic archaeal gene copy numbers (16S rRNA, mcrA) were lower in soil from the permanent pasture than from the pasture-rice alternation fields, but within the latter, there was no significant difference. Methane production started to accumulate after 16 days and 7 days of anoxic incubation in soil from the permanent pasture and the pasture-rice alternation fields respectively. Then, CH4 production rates were slightly higher in the soils used for pasture than for rice production. Analysis of δ(13) C in CH4, CO2 and acetate in the presence and absence of methyl fluoride, an inhibitor of aceticlastic methanogenesis, indicated that CH4 was mainly (58-75%) produced from acetate, except in the permanent pasture soil (42%). Terminal restriction fragment length polymorphism (T-RFLP) of archaeal 16S rRNA genes showed no difference among the soils from the pasture-rice alternation fields with Methanocellaceae and Methanosarcinaceae as the main groups of methanogens, but in the permanent pasture soil, Methanocellaceae were relatively less abundant. T-RFLP analysis of bacterial 16S rRNA genes allowed the distinction of permanent pasture and fields from the pasture-rice rotation, but nevertheless with a high similarity. Pyrosequencing of bacterial 16S rRNA genes generally revealed Firmicutes as the dominant bacterial phylum, followed by Proteobacteria, Acidobacteria and Actinobacteria. We conclude that a stable methanogenic microbial community established once pastures have been turned into management by pasture-rice alternation despite the fact that 2 years of wetland conditions were followed by 4 years of upland conditions that were not suitable for CH4 production.

The Effect of Plant Extracts on In-vitro Ruminal Fermentation, Methanogenesis and Methane-related Microbes in the Rumen.

The effect on methanogens attached to the surface of rumen ciliate protozoa by the addition of plant extracts (pine needles and ginkgo leaves) was studied with particular reference to their effectiveness for decreasing methane emission. The plant extracts (pine needles and ginkgo leaves) were added to an in vitro fermentation incubated with rumen fluid. The microbial population including bacteria, ciliated-associated methanogen, four different groups of methanogens and Fibrobacter succinogenes were quantified by using the real-time PCR. Gas profiles including methane, carbon dioxide and hydrogen, and runinal fermentation characteristics were observed in vitro. The methane emission from samples with an addition of individual juices from pine needles, ginkgo leaves and 70% ethanol extract from ginko leaves was significantly lower (p<0.05, 27.1, 28.1 and 28.1 vs 34.0 ml/g DM) than that of the control, respectively. Total VFAs in samples with an addition of any of the plant extracts were significantly lower than that of the control (p<0.05) as well. The order Methanococcales and the order Methanosarcinales were not detected by using PCR in any incubated mixtures. The ciliate-associated methanogens population decreased from 25% to 49% in the plant extacts as compared to control. We speculate that the supplementation of juice from pine needles and ginkgo leaves extract (70% ethanol extract) decreased the protozoa population resulting in a reduction of methane emission in the rumen and thus inhibiting methanogenesis. The order Methanobacteriales community was affected by addition of all plant extracts and decreased to less than the control, while the order Methanomicrobiales population showed an increase to more than that of the control. The F. succinogenes, the major fibrolytic microorganism, population in all added plant extracts was increased to greater than that of the control. In conclusion, pine needles and ginkgo leaves extracts appear to have properties that decrease methanogenesis by inhibiting protozoa species and may have a potential for use as additives for ruminants.

Use of artificial DNA with multiple probe sites as reference DNA templates for quantitative real-time PCR to examine methanogen communities

Isolation of reference DNA templates for quantitative real-time PCR assays is an expensive, labor-intensive and time-consuming process if they are not readily available. Two artificial DNA templates with multiple probe sites were designed for quantifying methanogens and their 10 subgroups, based on the methyl coenzyme M reductase gene (mcrA). Their standards were comparable to each other. PCR amplification efficiencies (cycle vs. cumulative fluorescence) of the artificial DNAs were also comparable to those of the observed methanogen groups from anaerobic digesters. The artificial templates can be alternatives to the actual references.