The Synergism between Methanogens and Methanotrophs and the Nature of their Contributions to the Seasonal Variation of Methane Fluxes in a Wetland:The Case of Dajiuhu Subalpine Peatland

Wetland ecosystems are the most important natural methane(CH_(4))sources,whose fluxes periodically fluctuate.Methanogens(methane producers)and methanotrophs(methane consumers)are considered key factors affecting CH_(4)fluxes in wetlands.However,the symbiotic relationship between methanogens and methanotrophs remains unclear.To help close this research gap,we collected and analyzed samples from four soil depths in the Dajiuhu subalpine peatland in January,April,July,and October 2019 and acquired seasonal methane flux data from an eddy covariance(EC)system,and investigated relationships.A phylogenetic molecular ecological networks(pMENs)analysis was used to identify keystone species and the seasonal variations of the co-occurrence patterns of methanogenic and methanotrophic communities.The results indicate that the seasonal variations of the interactions between methanogenic and methanotrophic communities contributed to CH_(4)emissions in wetlands.The keystone species discerned by the network analysis also showed their importance in mediating CH_(4)fluxes.Methane(CH_(4))emissions in wetlands were lowest in spring;during this period,the most complex interactions between microbes were observed,with intense competition among methanogens while methanotrophs demonstrated better cooperation.Reverse patterns manifested themselves in summer when the highest CH_(4)flux was observed.Methanoregula formicica was negatively correlated with CH_(4)fluxes and occupied the largest ecological niches in the spring network.In contrast,both Methanocella arvoryzae and Methylocystaceae demonstrated positive correlations with CH_(4)fluxes and were better adapted to the microbial community in the summer.In addition,soil temperature and nitrogen were regarded as significant environmental factors to CH_(4)fluxes.This study was successful in explaining the seasonal patterns and microbial driving mechanisms of CH_(4)emissions in wetlands.

The community structure and microbial linkage of rumen protozoa and methanogens in response to the addition of tea seed saponins in the diet of beef cattle

Background: This study investigated changes in rumen protozoal and methanogenic communities, along with the correlations among microbial taxa and methane(CH_4) production of six Belmont Red Composite beef steers fed tea seed saponins(TSS). Animals were fed in three consecutive feeding periods, a high-grain basal diet for 14 d(BD period) then a period of progressive addition of TSS to the basal diet up to 30 g/d for 20 d(TSS period), followed by the basal diet for 13 d without TSS(BDP post-control period).Results: The study found that TSS supplementation decreased the amount of the protozoal genus Entodinium and increased Polyplastron and Eudiplodinium genera. During BDP period, the protozoa community of steers did not return to the protozoal profiles observed in BD period, with higher proportions of Metadinium and Eudiplodinium and lower Isotricha. The addition of TSS was found to change the structure of methanogen community at the subgenus level by decreasing the abundance of methanogens in the SGMT clade and increasing the abundance of methanogens in the RO clade. The correlation analysis indicated that the abundance of SGMT clade methanogens were positively correlated with Isotricha, and Isotricha genus and SGMT clade methanogens were positively correlated with CH_4 production. While RO clade were positively correlated with the proportion of Metadinium genus, which was negatively correlated with CH_4 emission.Conclusions: These results suggest that different genera of rumen protozoa ciliates appear to be selectively inhibited by TSS, and the change in methanogen community at the subgenus level may be due to the mutualistic relationships between methanogens and rumen ciliates.

Rumen methanogens and mitigation of methane emission by anti-methanogenic compounds and substances

Methanogenic archaea reside primarily in the rumen and the lower segments of the intestines of ruminants, where they utilize the reducing equivalents derived from rumen fermentation to reduce carbon dioxide, formic acid, or methylamines to methane（CH_4）. Research on methanogens in the rumen has attracted great interest in the last decade because CH_4 emission from ruminants contributes to global greenhouse gas emission and represents a loss of feed energy. Some DNA-based phylogenetic studies have depicted a diverse and dynamic community of methanogens in the rumen. In the past decade, researchers have focused on elucidating the underpinning that determines and affects the diversity, composition, structure, and dynamics of methanogen community of the rumen. Concurrently, many researchers have attempted to develop and evaluate interventions to mitigate enteric CH_4 emission. Although much work has been done using plant secondary metabolites, other approaches such as using nitrate and 3-nitrooxy propanol have also yielded promising results. Most of these antimethanogenic compounds or substances often show inconsistent results among studies and also lead to adverse effects on feed intake and digestion and other aspects of rumen fermentation when fed at doses high enough to achieve effective mitigation. This review provides a brief overview of the rumen methanogens and then an appraisal of most of the antimethanogenic compounds and substances that have been evaluated both in vitro and in vivo. Knowledge gaps and future research needs are also discussed with a focus on methanogens and methane mitigation.

Yeast-Derived β-1,3-Glucan Substrate Significantly Increased the Diversity of Methanogens During In vitro Fermentation of Porcine Colonic Digesta

The β-1,3-glucan from yeast has been extensively examined for its immuno-enhancing effects in animals.However,investigation on the relationship among β-glucan,gut microbiota and immune-modulating effects remains limited particularly in pigs.Considering the critical roles of gut methanogens in the microbial fermentation,energy metabolism and disease resistance,we investigated the phylogenetic diversity of methanogens from fermented cultures of porcine colonic digesta with（G） or without（N） yeast β-glucan based on sequences of the archaeal 16S rRNA gene.A total of 145 sequences in the G library were assigned into 8 operational taxonomic units（OTUs） with the majority of sequences（114/145） related to strains Methanobrevibacter millerae or Methanobrevibacter gottschalkii with high identities ranging from 97.9 to 98.6%,followed by 23 sequences to Methanobrevibacter ruminantium,2 sequences to Methanobrevibacter smithii and one sequence to Methanobrevibacter wolinii.The 142 sequences in the N library were assigned to 2 OTUs with most sequences（127/142） related to strains M.millerae or M.gottschalkii with sequence identities ranging from 97.9 to 98.5%,and 15 sequences related to M.gottschalkii with 97.9% identity.Shannon diversity index showed that the G library exhibited significantly higher archaeal diversity（P0.05） and Libshuff analysis indicated the differences in the community structure between the two libraries were significant（P0.0001）.In conclusion,the current study provides evidence that addition of yeast β-glucan significantly increased the diversity of methanogens in in vitro fermented porcine colonic digesta.

Rumen microbial degradation of bromoform from red seaweed(Asparagopsis taxiformis)and the impact on rumen fermentation and methanogenic archaea

Background The red macroalgae Asparagopsis is an effective methanogenesis inhibitor due to the presence of halogenated methane(CH_(4))analogues,primarily bromoform(CHBr_(3)).This study aimed to investigate the degradation process of CHBr3 from A taxiformis in the rumen and whether this process is diet-dependent.An in vitro batch culture system was used according to a 2×2 factorial design,assessing two A taxiformis inclusion rates[0(CTL)and 2%DM diet(AT)]and two diets[high-concentrate(HC)and high-forage diet(HF)].Incubations lasted for 72 h and samples of headspace and fermentation liquid were taken at 0,0.5,1,3,6,8,12,16,24,48 and 72 h to assess the pattern of degradation of CHBr_(3) into dibromomethane(CH_(2)Br_(2))and fermentation parameters.Additionally,an in vitro experiment with pure cultures of seven methanogens strains(Methanobrevibacter smithii,Methanobrevibacter ruminantium,Methanosphaera stadtmanae,Methanosarcina barkeri,Methanobrevibacter millerae,Methanorhermobacter wolfei and Methanobacterium mobile)was conducted to test the effects of increasing concentrations of CHBr3(0.4,2,10and 50μmol/L).Results The addition of AT significantly decreased CH_(4) production(P=0.002)and the acetate:propionate ratio(P=0.003)during a 72-h incubation.The concentrations of CHBr_(3) showed a rapid decrease with nearly 90%degraded within the first 3 h of incubation.On the contrary,CH_(2)Br_(2) concentration quickly increased during the first 6 h and then gradually decreased towards the end of the incubation.Neither CHBr_(3) degradation nor CH_(2)Br_(2) synthesis were affected by the type of diet used as substrate,suggesting that the fermentation rate is not a driving factor involved in CHBr_(3)degradation.The in vitro culture of methanogens showed a dose-response effect of CHBr3 by inhibiting the growth of M.smithii,M.ruminantium,M.stadtmanae,M.barkeri,M.millerae,M.wolfei,and M.mobile.Conclusions The present work demonstrated that CHBr_(3) from A.taxiformis is quickly degraded to CH_(2)Br_(2)in the rumen and that the fermentation rate promoted by different diets is not a driving factor involved in CHBr_(3)degradation.

Beyond bacteria:Role of non-bacterial gut microbiota species in inflammatory bowel disease and colorectal cancer progression

This letter emphasizes the need to expand discussions on gut microbiome’s role in inflammatory bowel disease(IBD)and colorectal cancer(CRC)by including the often-overlooked non-bacterial components of the human gut flora.It highlights how viral,fungal and archaeal inhabitants of the gut respond towards gut dysbiosis and contribute to disease progression.Viruses such as bacteriophages target certain bacterial species and modulate the immune system.Other viruses found associated include Epstein-Barr virus,human papillomavirus,John Cunningham virus,cytomegalovirus,and human herpes simplex virus type 6.Fungi such as Candida albicans and Malassezia contribute by forming tissue-invasive filaments and producing inflammatory cytokines,respectively.Archaea,mainly methanogens are also found altering the microbial fermentation pathways.This correspondence,thus underscores the significance of considering the pathological and physiological mechanisms of the entire spectrum of the gut microbiota to develop effective therapeutic interventions for both IBD and CRC.

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.

Molecular evolution of methanogens based on their metabolic facets

The information provided by completely sequenced genomes of methanogens can yield insights into a deeper molecular understanding of evolutionary mechanisms.This review describes the advantages of using metabolic pathways to clarify evolutionary correlation of methanogens with archaea and prokaryotes.Metabolic trees can be used to highlight similarities in metabolic networks related to the biology of methanogens.Metabolic genes are among the most modular in the cell and their genes are expected to travel laterally,even in recent evolution.Phylogenetic analysis of protein superfamilies provides a perspective on the evolutionary history of some key metabolic modules of methanogens.Phage-related genes from distantly related organisms typically invade methanogens by horizontal gene transfer.Metabolic modules in methanogenesis are phylogenetically aligned in closely related methanogens.Reverse order reactions of methanogenesis are achieved in methylotrophic methanogens using metabolic and structural modules of key enzymes.A significant evolutionary process is thought to couple the utilization of heavy metal ions with energetic metabolism in methanogens.Over 30 of methanogens genomes have been sequenced to date,and a variety of databases are being developed that will provide for genome annotation and phylogenomic analysis of methanogens.Into the context of the evolutionary hypothesis,the integration of metabolomic and proteomic data into large-scale mathematical models holds promise for fostering rational strategies for strain improvement.

一种快速测定厌氧污泥活性的顶空气相色谱技术

将含有厌氧污泥的样品放入一顶空样品瓶内,在恒定的温度下平衡,并通过多次顶空抽提的方式对样品瓶中所产生的甲烷量随时间的变化进行气相色谱检测,从而实现厌氧污泥比产甲烷活性的检测。实验表明,多次顶空抽提-气相色谱测定厌氧污泥产甲烷活性的技术用约3h就可以得出厌氧污泥产甲烷的线性规律,其结果与对比试验的数据相近。因而该方法适用于厌氧污泥活性的快速预测,可大大提高相关厌氧污泥活性测定及废水处理领域研究的效率。

Archaea and the human gut:New beginning of an old story

Methanogenic archaea are known as human gut inhabitants since more than 30 years ago through the detection of methane in the breath and isolation of two methanogenic species belonging to the order Methanobacteriales, Methanobrevibacter smithii and Methanosphaera stadtmanae. During the last decade, diversity of archaea encountered in the human gastrointestinal tract (GIT) has been extended by sequence identification and culturing of new strains. Here we provide an updated census of the archaeal diversity associated with the human GIT and their possible role in the gut physiology and health. We particularly focus on the still poorly characterized 7th order of methanogens, the Methanomassiliicoccales, associated to aged population. While also largely distributed in non-GIT environments, our actual knowledge on this novel order of methanogens has been mainly revealed through GIT inhabitants. They enlarge the number of final electron acceptors of the gut metabolites to mono- di- and trimethylamine. Trimethylamine is exclusively a microbiota-derived product of nutrients (lecithin, choline, TMAO, L-carnitine) from normal diet, from which seems originate two diseases, trimethylaminuria (or Fish-Odor Syndrome) and cardiovascular disease through the proatherogenic property of its oxidized liver-derived form. This therefore supports interest on these methanogenic species and its use as archaebiotics, a term coined from the notion of archaea-derived probiotics.

Acclimation of CH_(4)emissions from paddy soil to atmospheric CO_(2)enrichment in a growth chamber experiment

Elevated levels of atmospheric CO_(2)(eCO_(2))promote rice growth and increase methane(CH_(4))emissions from rice paddies,because increased input of plant photosynthate to soil stimulates methanogenic archae.However,temporal trends in the effects of eCO_(2)on rice growth and CH_(4)emissions are still unclear.To investigate changes in the effects of eCO_(2)over time,we conducted a two-season pot experiment in a walk-in growth chamber.Positive effects of eCO_(2)on rice leaf photosynthetic rate,biomass,and grain yield were similar between growing seasons.However,the effects of eCO_(2)on CH_(4) emissions decreased over time.Elevated CO_(2)increased CH_(4)emissions by 48%-101%in the first growing season,but only by 28%-30%in the second growing season.We also identified the microbial process underlying the acclimation of CH4 emissions to atmospheric CO_(2)enrichment:eCO_(2)stimulated the abundance of methanotrophs more strongly in soils that had been previously exposed to eCO_(2)than in soils that had not been.These results emphasize the need for long-term eCO_(2)experiments for accurate predictions of terrestrial feedbacks.

Microbial diversity and functionally distinct groups in produced water from the Daqing Oilfield,China

The microbial community structure and functionally distinct groups in three kinds of produced water samples from the shallow,mesothermic and low-salinity Daqing oil reservoir were systematically evaluated using both culture-dependent and culture-independent methods.Sequence analysis of the 16S rRNA genes indicated that the bacterial library was dominated by Acinetobacter and Arcobacter and the archaeal community was dominated by Methanosaeta and Methanolinea.Two isolated methanogens were closely related with Methanothermobacter thermautotrophicus and Methanoculleus receptaculi.The fermentative bacteria were identified as Pseudomonas,Haloanaerobium,Alcalibacter,Arcobacter,and Pannonibacter.The predominant nitrate-reducing bacteria fell within the genus Pseudomonas.The dominant members of the cultured hydrocarbon-oxidizing bacteria were phylogenetically associated with Micrococcus,Pseudomonas,and Bacillus.Enrichments of biosurfactants and biopolymer producing groups mainly yielded Pseudomonas,Bacillus,and Acenitobacter-related members.The functional groups related to polymer degradation were also affiliated with Pseudomonas and Bacillus.Results from this study provide the fresh insight into the diversity of microbial communities in Daqing petroleum reservoirs.The vast pool of functional strains retrieved in this study was presumed to include the promising strains that could be applied in microbial-enhanced oil recovery in future.

Effect of Land Use History on Methane Emission and Methanogenic Flora in Flooded Soils

The characteristics of methane emission were compared among six types of upland and paddy soils developed from different parent materials with distinct physics and chemical properties after planting rice. The fluxes of methane emission in submerged soils from the upland were obviously lower than those from the paddy rice field. The flux of methane emission in the paddy soil developed from fluvo-aquic soil was the largest among all the types of soils. Planting of rice was helpful to emission of methane in soils. The amounts of various groups of methanogenic flora were conformed with the deferences among the fluxes of methane emission in various types of soils. Methane formation was observed in each type Of air-dried soils stored for a long time after addition of water and incubation at 35℃.

Long-term open circuit microbial electrosynthesis system promotes methanogenesis

Microbial electrosynthesis(MES) can potentially provide a mean for storing renewable energy surpluses as chemical energy. However, the fluctuating nature of these energy sources may represent a threat to MES, as the microbial communities that develop on the biocathode rely on the continuous existence of a polarized electrode. This work assesses how MES performance, product generation and microbial community evolution are affected by a long-period(6 weeks) power off(open circuit). Acetogenic and H2-producing bacteria activity recovered after reconnection. However, few days later syntrophic acetate oxidation bacteria and H2-consuming methanogens became dominant, producing CH4 as the main product, via electromethanogenesis and the syntrophic interaction between eubacterial and archaeal communities which consume both the acetic acid and the hydrogen present in the cathode environment. Thus,the system proved to be resilient to a long-term power interruption in terms of electroactivity. At the same time, these results demonstrated that the system could be extensively affected in both end product generation and microbial communities.

Relationship between Microbial Community Characteristics and Flooding Efficiency in Microbial Enhanced Oil Recovery

Microbial enhanced oil recovery (MEOR) is the research focus in the field of energy development as an environmentally friendly and low cost technology. MEOR can bes divided into indigenous microbial oil recovery and exogenous microbial oil recovery. The ultimate goal of indigenous microbial flooding is to enhance oil recovery via stimulation of specific indigenous microorganisms by injecting optimal nutrients. For studying the specific rule to activate the indigenous community during the long-term injection period, a series of indigenous displacement flooding experiments were carried out by using the long-core physical simulation test. The experimental results have shown that the movement of nutrients components (i.e., carbon/nitrogen/phosphorus) differed from the consumption of them. Moreover, there was a positive relationship between the nutrients concentration and bacteria concentration once observed in the produced fluid. And the trend of concentration of acetic acid was consistent with that of methanogens. When adding same activators, the impacts of selective activators to stimulate the indigenous microorganisms became worse along with the injection period, which led to less oil recovery efficiency.

Effect of inorganic carbon on anaerobic ammonium oxidation enriched in sequencing batch reactor

The present lab-scale research reveals the enrichment of anaerobic ammonium oxidation microorganism from methanogenic anaerobic granular sludge and the effect of inorganic carbon(sodium bicarbonate)on anaerobic ammonium oxidation.The enrichment of anammox bacteria was carried out in a 7.0-L sequencing batch reactor(SBR)and the effect of bicarbonate on anammox was conducted in a 3.0-L SBR.Research results,especially the biomass,showed first signs of anammox activity after 54 d cultivation with synthetic wast...

Batch methanogenic fermentation experiments of wastewater from a brown coal low-temperature coke plant

Coke plant effluents with high contents of organic compounds are mainly treated by biological aerobic fermentation after physical pre-treatment. In this study, a brown coal condensate wastewater from a low temperature coking process was fermented under methanogenic conditions in discontinuous experiments. By this fermentation, acetate, propionate, and the main polyphenolic compounds （catechol, resorcinol and hydroquinone） were degraded to a level below the detection limit. The COD was reduced by 72% with a residual concentration of 2.1 g/L. This anaerobic fermented wastewater had a residual BOD5 of 0.66 g/L and 2.2 L CH4 were formed per litre of wastewater. An abiotic pre-treatment for this wastewater with air had a negative effect on the COD reduction and decrease of colour on the methanogenic fermentation due to the autoxidation of polyphenolic compounds to humic-like compounds. This study showed that methanogenic fermentations in the treatment sequence of brown coal coking wastewaters could reduce energy consumption for aeration in further treatment processes and had the potential for a better effluent quality due to a less formation of recalcitrant humic-like compounds.

Weaning methods affect ruminal methanogenic archaea composition and diversity in Holstein calves

The objective of the present study was to examine the effect of different weaning methods on the ruminal methanogenic archaea composition and diversity in Holstein calves.Thirty-six newborn Holstein bull calves were assigned to 1 of 3 treatments:(1)conventional weaning(d 56)and fed a high proportion of solid feed(CWS);(2)conventional weaning(d 56)and fed a high proportion of liquid feed(CWL);(3)early weaning(d 42)and fed with a high proportion of solid feed(EWS).High-throughput sequencing of the methyl coenzyme M reductase(mcr A)gene,which encodes theα-subunit of methyl coenzyme M reductase-the enzyme that catalyzes the final step in methanogenesis was used to determine the composition and diversity of rumen methanogens.No significant difference(P>0.05)was observed for operational taxonomic units(OTUs)or richness indices,but diversity indices increased(P<0.05)for calves fed high dietary solids.Predominant families across the three treatments were Methanobacteriaceae,Thermoplasmataceae and Methanomassiliicoccaceae.Calves in the EWS treatment had a higher(P<0.05)relative abundance of Methanobrevibacter sp.strain AbM4 and Methanosphaera stadtmanae,while calves in the CWL treatment had a higher(P<0.05)abundance of Methanosphaera sp.strain SM9.A positive(P<0.05)relationship was identified between butyrate and Methanobrevibacter sp.strain AbM4.In conclusion,the composition and diversity of methanogens in the rumen of Holstein calves varied under the different weaning methods.This study identified a positive relationship between butyrate and Methanobrevibacter sp.strain AbM4,potentially reflecting correlations between ruminal fermentation variables and methanogenesis function.These in-depth analyses provide further understanding of weaning methods for intensified production systems.

Inhibitory effect of ammonia nitrogen on specific methanogenic activity of anaerobic granular sludge

A series of batch experiments were conducted in 125 mL serum bottles to assess the toxicity of different concentrations of ammonia nitrogen to the specific methanogenic activity of anaerobic granular sludge from upflow anaerobic sludge bed（UASB） and expanded granular sludge bed（EGSB） reactors. The effects of pH value and temperature on toxicity of ammonia nitrogen to anaerobes were investigated. The results show that the specific methanogenic activity of anaerobic granular sludge suffers inhibition from ammonia nitrogen, the concentrations of ammonia nitrogen that produce 50 % inhibition of specific methanogenic activity for sludge from UASB and EGSB reactor are 2.35 and 2.75 g/L, respectively. Hydrogen utilizing methanogens suffers less inhibition from ammonia mtrogen than that of acetate utilizing methanogens. Hydrogen-producing acetogens that utilize propionate and butyrate as substrates suffer serious inhibition from ammonia nitrogen. The toxicity of ammonia nitrogen to anaerobic granular sludge enhances when pH value and temperature increase. Anaerobic granular sludge can bear higher concentrations of ammonia nitrogen after being acclimated by ammonia nitrogen for 7 d.

Effect of sulfate on the methanogenic activity of a bacterial culture from a brewery wastewater during glucose degradation

The maximum specific methanogenic activity （SMA） of a sludge originating from a brewery wastewater treatment plant on the degradation of glucose was investigated at various levels of sulfate on a specific loading basis. Batch experiments were conducted in serum bottles at pH 7 and 35℃. A comparison of the values indicates that the SMA of this mixed culture was increased and reached its highest level of 0.128 g CH4 gas COD/（g VSS.d） when biomass was in contact with sulfate at a ratio of 1：0.114 by weight.