Temporal and environmental factors drive community structure and function of methanotrophs in volcanic forest soils

Methanotrophs,organisms that obtain oxygen by oxidizing methane,are recognized as the only known biological sink for atmospheric CH_4,and forest soil methanotrophs play crucial roles in mitigating global warming.The succession patterns of methanotrophic communities and functions in Wudalianchi volcano forest soils could provide a basis for the study of evolutionary mechanisms between soil microorganisms,the environment,and carbon cycling of temperate forest ecosystems under climate change.In this study,the characteristics and drivers of methanotrophic community structure and function of two volcanic soils at different stages of development are analyzed,including an old volcano and a new volcano,which most recently erupted 300 years and 17-19×10^(5)years ago,respectively,and a non-volcano hills as control,based on space for time substitution and Miseq sequencing and bioinformation technology.The results showed that CH_(4) fluxes were significantly higher in old-stage volcano forest soils than new-stage forest soils and non-volcano forest soils.There were significant differences in the community composition and diversity of soil methanotrophs from different volcano forest soils.Methylococcus was the dominant genus in all soil samples.Additionally,the relative abundance of Methylococcus,along with Clonothrix,Methyloglobulus,Methylomagum,Methylomonas and Methylosarcina,were the important genera responsible for the differences in methanotrophic community structure in different volcano forest soils.The relative abundance of methanotroph belonging toγ-proteobacteria was significantly higher than that belonging toα-proteobacteria(P<0.05).Chao1,Shannon and Simpson indices of soil methanotrophic community were significantly lower in new-stage volcanos and were significantly affected by bulk density,total porosity,p H,nitrate,dissolved organic carbon and dissolved organic nitrogen.There were significant differences in community structure between new-stage and old-stage volcanoes.Bulk density and p H are important soil properties contributing to the divergence of methanotrophs community structure,and changes in soil properties due to soil development time are important factors driving differences in methanotrophs communities in Wudalianchi volcanic soils.

Biosynthesis of poly-3-hydroxybutyrate with a high molecular weight by methanotroph from methane and methanol

Poly-3-hydroxybutyrate （PHB） can be produced by various species of bacteria. Among the possible carbon sources, both methane and methanol could be a suitable substrate for the production of PHB. Methane is cheap and plentiful not only as natural gas, but also as biogas. Methanol can also maintain methanotrophic activity in some conditions. The methanotrophic strain Methylosinus trichosporium IMV3011 can accumulate PHB with methane and methanol in a brief nonsterile process. Liquid methanol （0.1%） was added to improve the oxidization of methane. The studies were carried out using shake flasks. Cultivation was performed in two stages： a continuous growth phase and a PHB accumulation phase under the conditions short of essential nutrients （ammonium, nitrate, phosphorus, copper, iron （Ⅲ）, magnesium or ethylenediamine tetraacetate （EDTA）） in batch culture. It was found that the most suitable growth time for the cell is 144 h. Then an optimized culture condition for second stage was determined, in which the PHB concentration could be much increased to 0.6 g/L. In order to increase PHB content, citric acid was added as an inhibitor of tricarboxylic acid cycle （TCA）. It was found that citric acid is favorable for the PHB accumulation, and the PHB yield was increased to 40% （w/w） from the initial yield of 12% （w/w） after nutrient deficiency cultivation. The PHB produced is of very high quality with molecular weight up to 1.5 × 10^6Da.

The Variation of Microbial(Methanotroph)Communities in Marine Sediments Due to Aerobic Oxidation of Hydrocarbons

Methanotrophs in marine sediments and overlying water attenuate the emissions of methane into the atmosphere and thus play an important role for the global cycle of this greenhouse gas.However,gas released from natural hydrocarbon seeps are not pure methane but commonly mixed hydrocarbons.Currently,how methanotrophic bacteria behave in the co-presence of methane and heavier hydrocarbons remains unknown.In this paper,the bacteria were cultured aerobically in fresh sediment samples(collected from Bohai Bay in eastern China)at 28℃under the atmospheres of pure methane and methane+ethane+propane mixed gas,respec-tively.The prevailing terrigenous n-alkanes and fatty acids in the original sediment samples varied consistently after incubations,confirming the proceeding of aerobic bacterial activities.The real-time quantitative PCR assay and sequencing of the 16S rRNA and particulate methane monooxygenase(pmoA)genes revealed the changes of microbe communities to a methanotroph-dominating structure after incubations.Particularly,after incubations the family Methylococcaceae(typeⅠmethanotrophs)became dominant with proportions higher than 40%,whereas Methylocystaceae(typeⅡmethanotrophs)nearly disappeared in all incubated samples.More-over,the species of methanotrophs from the samples treated with pure methane were dominated by Methylobacter luteus,whereas Methylobacter whittenburyi took the predominant proportion in the samples treated with mixed gas.The phenomenon suggests that some methanotrophs may also utilize ethane and propane.Collectively,this study may help to gain a better understanding of the ef-fects and contributions of microbial activities in marine hydrocarbon seep ecosystems.

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.

Winter cover crops alter methanotrophs community structure in a double-rice paddy soil

Methanotrophs play a vital role in the mitigation of methane emission from soils. However, the influences of cover crops incorporation on paddy soil methanotrophic community structure have not been fully understood. In this study, the impacts of two winter cover crops（Chinese milk vetch（Astragalus sinicus L.） and ryegrass（Lolium multiflorum Lam.）, representing leguminous and non-leguminous cover crops, respectively） on community structure and abundance of methanotrophs were evaluated by using PCR-DGGE（polymerase chain reaction-denaturing gradient gel electrophoresis） and real-time PCR technology in a double-rice cropping system from South China. Four treatments were established in a completely randomized block design： 1） double-rice cropping without nitrogen fertilizer application, CK; 2） double-rice cropping with chemical nitrogen fertilizer application（200 kg ha^（–1） urea for entire double-rice season）, CF; 3） Chinese milk vetch cropping followed by double-rice cultivation with Chinese milk vetch incorporation, MV; 4） ryegrass cropping followed by double-rice cultivation with ryegrass incorporation, RG. Results showed that cultivating Chinese milk vetch and ryegrass in fallow season decreased soil bulk density and increased rice yield in different extents by comparison with CK. Additionally, methanotrophic bacterial abundance and community structure changed significantly with rice growth. Methanotrophic bacterial pmo A gene copies in four treatments were higher during late-rice season（3.18×10^7 to 10.28×10^7 copies g^–1 dry soil） by comparison with early-rice season（2.1×10^7 to 9.62×10^7 copies g^–1 dry soil）. Type Ⅰ methanotrophs absolutely predominated during early-rice season. However, the advantage of type Ⅰ methanotrophs kept narrowing during entire double-rice season and both types Ⅰ and Ⅱ methanotrophs dominated at later stage of late-rice.

Identification of functionally active aerobic methanotrophs and their methane oxidation potential in sediments from the Shenhu Area,South China Sea

Large amounts of gas hydrate are distributed in the northern slope of the South China Sea,which is a potential threat of methane leakage.Aerobic methane oxidation by methanotrophs,significant methane biotransformation that occurs in sediment surface and water column,can effectively reduce atmospheric emission of hydrate-decomposed methane.To identify active aerobic methanotrophs and their methane oxidation potential in sediments from the Shenhu Area in the South China Sea,multi-day enrichment incubations were conducted in this study.The results show that the methane oxidation rates in the studied sediments were 2.03‒2.36μmol/gdw/d,which were higher than those obtained by sediment incubations from other areas in marine ecosystems.Thus the authors suspect that the methane oxidation potential of methanotrophs was relatively higher in sediments from the Shenhu Area.After the incubations family Methylococcaea(type I methanotrophs)mainly consisted of genus Methylobacter and Methylococcaea_Other were predominant with an increased proportion of 70.3%,whereas Methylocaldum decreased simultaneously in the incubated sediments.Collectively,this study may help to gain a better understanding of the methane biotransformation in the Shenhu Area.

Elevated and atmospheric-level methane consumption by soil methanotrophs of three grasslands in China

Background:Methane(CH4)oxidation driven by soil aerobic methanotrophs demonstrates the capacity of grassland as a CH4 sink.Methods:In this study,we compared the oxidation characteristics of atmospheric-level and elevated concentration(10%)CH4 in a typical grassland(steppe)on the Loess Plateau,an alpine meadow(meadow)on the Qinghai-Tibet Plateau,and an inland arid-area artificial grassland(pasture)in northwest China and investigated the communities of active methanotrophs and their contribution to CH4 oxidation using DNA-based stable-isotope probing and Illumina Miseq sequencing.Results:The results showed that the oxidation of atmospheric CH4 only occurred in steppe and meadow soils where the USCγgroup of methanotrophs was numerically dominant in the methanotroph community.Pasture soils,with their very low relative abundance of USCγ,did not show atmospheric CH4 oxidation.However,a DNA-stable isotope probing experiment with 10%CH4 indicated that conventional CH4 oxidizers(Methylocaldum and Methylocystis)rather than USCγcommunities assimilated significant amounts of 13CH4 for growth.Conclusions:The CH4 oxidation mechanisms in the three experimental grassland soils varied significantly.The USCγgroup may be obligate oligotrophic microorganisms or their growth requires specific unknown conditions.

Extreme drought with seasonal timing consistently promotes CH_(4) uptake through inconsistent pathways in a temperate grassland, China

Methane(CH_(4))is a potent greenhouse gas that has a substantial impact on global warming due to its substantial influence on the greenhouse effect.Increasing extreme precipitation events,such as drought,attributable to global warming that caused by greenhouse gases,exert a profound impact on the intricate biological processes associated with CH_(4) uptake.Notably,the timing of extreme drought occurrence emerges as a pivotal factor influencing CH_(4) uptake,even when the degree of drought remains constant.However,it is still unclear how the growing season regulates the response of CH_(4) uptake to extreme drought.In an effort to bridge this knowledge gap,we conducted a field manipulative experiment to evaluate the impact of extreme drought on CH_(4) uptake during early,middle,and late growing stages in a temperate steppe of Inner Mongolia Autonomous Region,China.The result showed that all extreme drought consistently exerted positive effects on CH_(4) uptake regardless of seasonal timing.However,the magnitude of this effect varied depending on the timing of season,as evidenced by a stronger effect in early growing stage than in middle and late growing stages.Besides,the pathways of CH_(4) uptake were different from seasonal timing.Extreme drought affected soil physical-chemical properties and aboveground biomass(AGB),consequently leading to changes in CH_(4) uptake.The structural equation model showed that drought both in the early and middle growing stages enhanced CH_(4) uptake due to reduced soil water content(SWC),leading to a decrease in NO_(3)–-N and an increase in pmoA abundance.However,drought in late growing stage primarily enhanced CH_(4) uptake only by decreasing SWC.Our results suggested that seasonal timing significantly contributed to regulate the impacts of extreme drought pathways and magnitudes on CH_(4) uptake.The findings can provide substantial implications for understanding how extreme droughts affect CH_(4) uptake and improve the prediction of potential ecological consequence under future climate change.

水稻品种间作对甲烷排放的影响

作物品种多样化种植是提高农业生态系统功能的有效措施,但不同品种混合种植能否影响温室气体排放仍然缺乏研究。本研究以水稻为例,通过原位盆栽试验,研究品种间隔种植(间作)对甲烷排放的影响。以甲烷高排放品种常农粳8号、皖稻153以及甲烷低排放品种苏香粳100、Ⅱ优084为供试材料,设置8个处理,包括4个水稻品种单一种植(单作),即常农粳8号单作(记作CN)、皖稻153单作(WD)、苏香粳100单作(SX)、Ⅱ优084单作(ⅡY),以及2个甲烷高排放品种与2个甲烷低排放品种间作,即常农粳8号+Ⅱ优084间作(CN+ⅡY)、常农粳8号+苏香粳100间作(CN+SX)、皖稻153+Ⅱ优084间作(WD+ⅡY)、皖稻153+苏香粳100间作(WD+SX)。间作品种按1∶1的株数比例间隔种植。结果表明,4个间作处理均能显著增加或维持水稻产量。不同间作处理的甲烷排放通量存在显著差异,与期望值相比,CN+SX处理显著降低甲烷的排放,而CN+ⅡY、WD+ⅡY处理则显著增加甲烷排放。与甲烷高排放品种的单作处理相比,苏香粳100与2个甲烷高排放品种间作时均能显著降低生长季土壤产甲烷古菌mcrA基因平均丰度,但Ⅱ优084仅在与皖稻153间作时有显著作用。除CN+SX外,其余3个间作处理下生长季土壤甲烷氧化菌pmoA基因平均丰度均显著低于所对应的单作处理。本研究认为可以通过水稻品种间作在获得增产的同时降低甲烷排放,但品种间作组合需要仔细筛选。

Diversity of methanotrophs in Zoige wetland soils under both anaerobic and aerobic conditions

Zoige wetland is one of the most important methane emission centers in China. The oxidation of methane in the wetland affects global warming, soil ecology and atmospheric chemistry. Despite their global significance, microorganisms that consume methane in Zoige wetland remain poorly characterized. In this study, we investigated methanotrophs diversity in soil samples from both anaerobic site and aerobic site in Zoige wetland using pmoA gene as a molecular marker. The cloning library was constructed according to the pmoA sequences detected. Four clusters of methanotrophs were detected. The phylogenetic tree showed that all four clusters detected were affiliated to type I methanotrophs. Two novel clusters （cluster 1, cluster 2） were found to relate to none of the recognized genera of methanotrophs. These clusters have no cultured representatives and reveal an ecological adaptation of particular uncultured methanotrophs in Zoige wetland. Two clusters were belonging to Methylobacter and Methylococcus separately. Denaturing gradient gel electrophoresis gel bands pattern retrieved from these two samples revealed that the community compositions of anaerobic soil and aerobic soil were different from each other while anaerobic soil showed a higher metanotrophs diversity. Real-time PCR assays of the two samples demonstrated that aerobic soil sample in Zoige wetland was 1.5 times as much copy numbers as anaerobic soil. These data illustrated that methanotrophs are a group of microorganisms influence the methane consumption in Zoige wetland.

Do land utilization patterns affect methanotrophic communities in a Chinese upland red soil?

Soil samples were collected from three plots under different land utilization patterns including degradation, farming, and restoration. The abundances of methanotrophs were quantified using real-time polymerase chain reaction （PCR） based on the pmoA and 16S rRNA genes, and the community fingerprint was analyzed using denaturing gradient gel electrophoresis （DGGE） aiming at pmoA gene. Significantly lower 16S rRNA and pmoA genes copies were found in the degradation treatment than in farming and restoration. Higher abundances of Type I than those of Type II methanotrophs were detected in all treatments, The treatment of farming was clearly separated from degradation and restoration according to the DGGE profile by cluster analysis. The lowest diversity indices were observed in the F （farming plot）, suggesting that the community structure was strongly affected by farming activities. There were significantly positive correlations between the copy numbers of pmoA also Type II-related 16S rRNA genes and soil available K content. Strong negative and positive correlations were found between Type I and soil pH, and available P content, respectively. We concluded that the vegetation cover or not, soil characteristics including pH and nutrients of P and K as a result of anthropogenic disturbance may be key factors affecting methanotrophic communities in upland soil.

Methanotrophic community structure of aged refuse and its capability for methane bio-oxidation

Aged refuse from waste landfills closed for eight years was examined and found to contain rich methanotrophs capable of biooxidation for methane. Specially, community structure and methane oxidation capability of methanotrophs in the aged refuse were studied. The amount of methanotrophs ranged 61.97×10^3-632.91×10^3 cells/g （in dry basis） in aged refuse from Shanghai Laogang Landfill. Type I and II methanotrophs were found in the aged refuse in the presence of sterilized sewage sludge and only Type I methanotrophs were detected in the presence of nitrate minimal salt medium （NMS）. The clone sequences of the pmoA gene obtained from the aged refuse were similar to the pmoA gene of Methylobacter, Methylocaldum, and Methylocystis, and two clones were distinct with known genera of Type I methanotrophs according to phylogenetic analysis. Aged refuse enriched with NMS was used for methane biological oxidation and over 93% conversions were obtained.

Enrichments of methanotrophic–heterotrophic cultures with high poly-β-hydroxybutyrate(PHB) accumulation capacities

Methanotrophic–heterotrophic communities were selectively enriched from sewage sludge to obtain a mixed culture with high levels of poly-β-hydroxybutyrate（PHB）accumulation capacity from methane.Methane was used as the carbon source,N2as sole nitrogen source,and oxygen and Cu content were varied.Copper proved essential for PHB synthesis.All cultures enriched with Cu could accumulate high content of PHB（43.2%–45.9%）,while only small amounts of PHB were accumulated by cultures enriched without Cu（11.9%–17.5%）.Batch assays revealed that communities grown with Cu and a higher O2content synthesized more PHB,which had a wider optimal CH4：O2range and produced a high PHB content（48.7%）even though in the presence of N2.In all methanotrophic–heterotrophic communities,both methanotrophic and heterotrophic populations showed the ability to accumulate PHB.Although methane was added as the sole carbon source,heterotrophs dominated with abundances between 77.2%and 85.6%.All methanotrophs detected belonged to type II genera,which formed stable communities with heterotrophs of different PHB production capacities.

From nature to nurture:Essence and methods to isolate robust methanotrophic bacteria

Methanotrophic bacteria are entities with innate biocatalytic potential to biofilter and oxidize methane into simpler compounds concomitantly conserving energy,which can contribute to copious industrial applications.The future and efficacy of such industrial applications relies upon acquiring and/or securing robust methanotrophs with taxonomic and phenotypic diversity.Despite several dramatic advances,isolation of robust methanotrophs is still a long-way challenging task with several lacunae to be filled in sequentially.Methanotrophs with high tolerance to methane can be isolated and cultivated by mimicking natural environs,and adopting strategies like adaptive metabolic evolution.This review summarizes existent and innovative methods for methanotrophic isolation and purification,and their respective applications.A comprehensive description of new insights shedding light upon how to isolate and concomitantly augment robust methanotrophic metabolism in an orchestrated fashion follows.

Influence of biomass density and food to microorganisms ratio on the mixed culture type I methanotrophs enriched from activated sludge

Methanotrophic based process can be the remedy to offset the wastewater treatment facilities increasing energy requirements due to methanotroph＇s unique ability to integrate methane assimilation with multiple biotechnological applications like biological nitrogen removal and methanol production. Regardless of the methanotrophic process end product, the challenge to maintain stable microbial growth in the methanotrophs cultivation bioreactor at higher cell densities is one of the major obstacles facing the process upscaling. Therefore, a series of consecutive batch tests were performed to attentively investigate the biomass density influence on type I methanotrophs bacterial growth. In addition, food to microorganisms（F/M）, carbon to nitrogen（C/N） and nitrogen to microorganisms（N/M） ratio effect on the microbial activity was studied for the first time. It was clarified that the F/M ratio is the most influencing factor on the microbial growth at higher biomass densities rather than the biomass density increase, whereas C/N and N/M ratio change, while using nitrate as the nitrogen source,does not influence methanotrophs microbial growth. These study results would facilitate the scaling up of methanotrophic based biotechnology by identifying that F/M ratio as the key parameter that influences methanotrophs cultivation at high biomass densities.

Methanotrophy-driven accumulation of organic carbon in four paddy soils of Bangladesh

Biological methane oxidation is a crucial process in the global carbon cycle that reduces methane emissions from paddy fields and natural wetlands into the atmosphere.However,soil organic carbon accumulation associated with microbial methane oxidation is poorly understood.Therefore,to investigate methane-derived carbon incorporation into soil organic matter,paddy soils originated from different parent materials(Inceptisol,Entisol,and Alfisol) were collected after rice harvesting from four major rice-producing regions in Bangladesh.Following microcosm incubation with 5%(volume/volume)^(13) CH_(4),soil^(13) C-atom abundances significantly increased from background level of 1.08% to 1.88%–2.78%,leading to a net methane-derived accumulation of soil organic carbon ranging from 120 to 307 mg kg^(-1).Approximately 23.6%–60.0% of the methane consumed was converted to soil organic carbon during microbial methane oxidation.The phylogeny of^(13) C-labeled pmoA(enconding the alpha subunit of the particulate methane monooxygenase) and 16 S rRNA genes further revealed that canonical α(type II) and γ(type I) Proteobacteria were active methane oxidizers.Members within the Methylobacter-and Methylosarcina-affiliated type Ia lineages dominated active methane-oxidizing communities that were responsible for the majority of methane-derived carbon accumulation in all three paddy soils,while Methylocystis-affiliated type IIa lineage was the key contributor in one paddy soil of Inceptisol origin.These results suggest that methanotroph-mediated synthesis of biomass plays an important role in soil organic matter accumulation.This study thus supports the concept that methanotrophs not only consume the greenhouse gas methane but also serve as a key biotic factor in maintaining soil fertility.

Diversity of methanotrophs in a simulated modified biocover reactor

A simulated landfill biocover microcosm consisting of a modifying ceramsite material and compost were investigated.Results show that the mixture can improve the material porosity and achieve a stable and highly efficient （100%） methane oxidation over an extended operating period.The diversity of the methanotrophic community in the microcosm was assessed.Type I methanotrophs were enhanced in the microcosm due to the increased air diffusion and distribution,whereas the microbial diversity and population density of type II methanotrophs were not significantly affected.Moreover,the type I methanotrophic community structure significantly varied with the reactor height,whereas that of type II methanotrophic communities did not exhibit a spatial variation.Phylogenetic analysis showed that type I methanotroph-based nested polymerase chain reaction-denaturing gradient gel electrophoresis （PCR-DGGE） resulted in the detection of eight different populations,most of which are related to Methylobacter sp.,whereas that of type II resulted in the detection of nine different populations,most of which are related to Methylocystaceae.Methanotrophic community analysis also indicated that a number of new methanotrophic genera not closely related to any known methanotrophic populations were present.

Methanogenesis and Methanotrophy in Soil: A Review

Global warming,as a result of an increase in the mean temperature of the planet,might lead to catastrophic events for humanity.This temperature increase is mainly the result of an increase in the atmospheric greenhouse gases(GHG)concentration.Water vapor,carbon dioxide(CO2),methane(CH4)and nitrous oxide(N2O)are the most important GHG,and human activities,such as industry,livestock and agriculture,contribute to the production of these gases.Methane,at an atmospheric concentration of 1.7μmol mol-1currently,is responsible for 16%of the global warming due to its relatively high global warming potential.Soils play an important role in the CH4cycle as methanotrophy(oxidation of CH4)and methanogenesis(production of CH4)take place in them.Understanding methanogenesis and methanotrophy is essential to establish new agriculture techniques and industrial processes that contribute to a better balance of GHG.The current knowledge of methanogenesis and methanotrophy in soils,anaerobic CH4 oxidation and methanotrophy in extreme environments is also discussed.

大于80年自然风干保存土壤中甲烷氧化菌的复苏与富集

本研究通过对长期保存的土壤标本中甲烷氧化菌的复苏和富集培养,明确了活性甲烷氧化菌类群及其氧化甲烷潜力,同时分析了对长期干旱胁迫有较强抗逆性的甲烷氧化菌种类。研究主要针对自然风干保存83~87年的采集自福建龙岩荒地、江西南昌水田、四川华阳旱地、甘肃榆中林地、青海湟源和青海都兰牧场的6个土壤标本进行了高浓度甲烷条件下的微宇宙培养,利用气相色谱法测定了甲烷气体浓度动态变化情况以研究其甲烷氧化能力;在获得富集培养物后提取基因组DNA,利用16S rRNA和pmo A基因高通量测序分析了整体微生物和甲烷氧化菌的群落结构;同时,利用两种基因的长片段克隆测序对优势甲烷氧化菌序列进行了系统发育分析。结果显示:经过短暂的复苏,6例土壤档案样品均表现出强烈的甲烷氧化活性,前两代富集液甲烷氧化速率较慢,甲烷氧化速率仅为1.4~3.8μg/(mL·h),而第三代富集液的甲烷氧化速率达到了4.9~7.7μg/(mL·h)。其中,福建龙岩、江西南昌、青海都兰3个样品的富集物还可以将高浓度甲烷持续氧化到大气甲烷浓度(1.8μL/L)以下。高通量测序结果表明,6种富集物中甲烷氧化菌在细菌总量中的占比为39%~85%;pmoA功能基因分析表明,富集物中的优势甲烷氧化菌均为Methylocystis(TypeⅡa)。克隆序列的系统发育分析表明,富集到的Methylocystis种类和已知的Methylocystis rosea strain BRCS1亲缘关系最近,16S rRNA基因相似性为99.86%,pmoA基因相似性为99.05%。可见,TypeⅡa型Methylocystis属甲烷氧化菌具有强大的抗干旱胁迫的能力,经80年的风干保存后其活性仍可以迅速恢复,TypeⅡ甲烷氧化菌在未来将拥有更大的应用空间和价值。此外,长期保存的土壤档案样品仍具有较高的潜在生物活性,因此风干保存的土壤样品同样具有重要的微生物学研究价值。

中亚热带人工幼林土壤甲烷通量月动态及影响因素

亚热带森林土壤是重要的大气甲烷(CH_(4))汇。本研究以中亚热带人工幼林为研究对象,采用静态箱-气相色谱法对土壤CH_(4)通量进行为期1年的野外原位观测,并探究其与环境因子、土壤理化性质及土壤微生物的关系。结果表明:研究区内人工幼林土壤为甲烷汇,土壤CH_(4)月均吸收通量变化范围为10~75.6μg·m^(-2)·h^(-1)。土壤CH_(4)吸收通量呈显著的月动态,最高值出现在3月,最低值是5月。土壤含水率、林内气温和空气湿度是控制甲烷吸收的重要环境因子,能解释土壤CH_(4)月吸收通量变化的36.4%,土壤CH_(4)月吸收通量与土壤含水率呈负相关关系,与林内气温和空气湿度呈正相关关系。土壤pH值、土壤可溶性有机氮、铵态氮与土壤CH_(4)吸收通量为显著的正相关,能解释其变异的23%;土壤CH_(4)吸收通量与甲烷氧化菌(18∶1ω7c)浓度无显著的相关性。此外,土壤微生物生物量氮是影响甲烷氧化菌(18∶1ω7c)的主要因子。研究结果证实了亚热带人工幼林具有重要的土壤甲烷吸收作用,土壤CH_(4)吸收通量主要受环境因子和土壤理化的影响(约占60%)。本研究对中亚热带森林土壤甲烷汇的估算具有重要意义,也为森林碳循环和固碳潜力的发展提供理论支持。