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.

Saturated permeability and water retention capacity in biochar-methanotrophs-clay for new landfill cover system

A new landfill cover system,biochar-methanotrophs-clay(BMC)cover is recommended for reducing methane emissions at landfills.It also contributes to decreasing soil permeability and improving soil water retention in a long time,due to highly porous structure of biochar and the growth metabolism of methanotrophs.To determine the effects of biochar content,oxidation aging times and methane-filled days on hydraulic properties,a total of 60 groups of experiments were conducted.The saturated hydraulic conductivity(k_(sat))was obtained by flexible wall permeameter with controllable hydraulic head pressure.The results showed that the k_(sat)of BMC increased with increasing biochar content and oxidation aging times,while decreased with adding methane-filled days.The soil-water characteristic curves(SWCCs)were obtained with soil suction measured by the filter paper method.The results indicated the water retention capacity of MBC reduced with increasing oxidation aging times but increased with adding methane-filled days.Detected by mercury intrusion porosimetry(MIP),fourier transform infrared spectroscopy(FTIR)and scanning electron microscope(SEM),the differences displayed the changes of pore structures and extracellular polymeric substances(EPS).The oxidation aging of biochar increased the volume of pores,resulting in the increased k_(sat)and the decreased water retention capacity.However,the growing of methanotrophs decreased the volume of pores,resulting in the k_(sat)decreased and the water retention capacity increased due to EPS.No matter how many times the oxidation aging process was experienced,the BMC with longer methane-filled days exhibited relatively lower k_(sat)and better water retention capacity.This implied a more stable barrier capacity to reduce water infiltration in the long term.By combing a series of macro and micro experiments,this paper provides theoretical guidance for the application of biochar-methanotroph-clay mixture to landfill covers.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Studies on bio-oxidation of coal mine gas by a biofilter

A new biofilter technology was used to control the methane concentration in the coal mine. The results indicate that the biofilter achieves a steady methane removal capacity of 1 470 mg/（Loh） after 30 days start-up. More than 90% of the methane can be removed with an empty bed retention time （EBRT） of 5.6 min when the inlet concentration of methane （IMC） is lower than 70 mg/L （10%, V/V） and about 80% when IMC is at 105 mg/L （15%, V/V）. The biofilter is still a reliable method to control methane concentration as an auxiliary means to boost coal mine production safety together with aggrandized ventilation and drainage technologies, even though the removal efficiency of methane is not very satisfactory with a high IMC （〉10%） or a short EBRT （〈3.8 min）.

Development of Ice Cover in Water Areas during Methane

In traditional hydrometeorology and ice physics, when analyzing the development of ice, only degree-days of frost are taken into account. Despite the presence of pores in the ice formed during inhomogeneous crystallization and dynamic loads, ice is universally considered monolithic. Situations where ice cannot withstand design loads in frosty conditions are academically inexplicable, although it is common knowledge, for example, porous ice in reeds. Proof of methane accumulations under the ice-fountains of fire over holes in swampy waters. “Culprit” is methane. Methanotrophic microorganisms, structurally and functionally specialize in using it as a source of carbon and energy, and turn monolithic ice into porous, practically without changing its thickness. When rushing, dark products of methanotrophy and detritus raised by gas bubbles appear on the surface of the ice. The albedo of the ice surface decreases, contributing to its melting and the formation of dilutions. The early melting of ice in dilutions and their transformation into vast wormwood is due to methanotrophy products and seismogenic small detritus, which gravitationally slowly settling out of the cold photic layer, thinning it, contributes to an additional insolation warm-up of 2°C ÷ 3°C, freeing the waters from ice months earlier than normal.

Floating plants reduced methane fluxes from wetlands by creating a habitat conducive to methane oxidation

Wetlands are one of the important natural sources of atmospheric methane (CH_4),as an important part of wetlands,floating plants can be expected to affect methane release.However,the effects of floating plants on methane release are limited.In this study,methane fluxes,physiochemical properties of the overlying water,methane oxidation potential and rhizospheric bacterial community were investigated in simulated wetlands with floating plants Eichhornia crassipes,Hydrocharis dubia,and Trapa natans.We found that E.crassipes,H.dubia,and T.natans plants could inhibit 84.31%-97.31%,4.98%-88.91%and 43.62%-92.51%of methane fluxes at interface of water-atmosphere compared to Control,respectively.Methane fluxes were negatively related to nutrients concentration in water column but positively related to the aerenchyma proportions of roots,stems,and leaves.At the same biomass,root of E.crassipes (36.44%) had the highest methane oxidation potential,followed by H.dubia (12.99%) and T.natans (11.23%).Forty-five bacterial phyla in total were identified on roots of three plants and 7 bacterial genera (2.10%-3.33%) were known methanotrophs.Type I methanotrophs accounted for 95.07%of total methanotrophs.The pmoA gene abundances ranged from 1.90×10^(16)to 2.30×10^(18)copies/g fresh weight of root biofilms.Abundances of pmoA gene was significantly positively correlated with environmental parameters.Methylotrophy (5.40%) and methanotrophy (3.75%) function were closely related to methane oxidation.This study highlights that floating plant restoration can purify water and promote carbon neutrality partially by reducing methane fluxes through methane oxidation in wetlands.

Construction of a broad-host-range Anderson promoter series and particulate methane monooxygenase promoter variants expand the methanotroph genetic toolbox

Methanotrophic bacteria are currently used industrially for the bioconversion of methane-rich natural gas and anaerobic digestion-derived biogas to valuable products.These bacteria may also serve to mitigate the negative effects of climate change by capturing atmospheric greenhouse gases.Several genetic tools have previously been developed for genetic and metabolic engineering of methanotrophs.However,the available tools for use in methanotrophs are significantly underdeveloped compared to many other industrially relevant bacteria,which hinders genetic and metabolic engineering of these biocatalysts.As such,expansion of the methanotroph genetic toolbox is needed to further our understanding of methanotrophy and develop biotechnologies that leverage these unique microbes for mitigation and conversion of methane to valuable products.Here,we determined the copy number of three broad-host-range plasmids in Methylococcus capsulatus Bath and Methylosinus trichosporium OB3b,representing phylogenetically diverse Gammaproteobacterial and Alphaproteobacterial methanotrophs,respectively.Further,we show that the commonly used synthetic Anderson series promoters are functional and exhibit similar relative activity in M.capsulatus and M.trichosporium OB3b,but the synthetic series had limited range.Thus,we mutagenized the native M.capsulatus particulate methane monooxygenase promoter and identified variants with activity that expand the activity range of synthetic,constitutive promoters functional not only in M.capsulatus,but also in Escherichia coli.Collectively,the tools developed here advance the methanotroph genetic engineering toolbox and represent additional synthetic genetic parts that may have broad applicability in Pseudomonadota bacteria.

Characterization of Methylocystis strain JTA1 isolated from aged refuse and its tolerance to chloroform

To accelerate the efficiency of methane biodegradation in landfills, a Gram-negative, rod-shaped, non-motile, non-spore-forming bacterium, JTA1, which can utilize methane as well as acetate, was isolated from the Laogang MSW landfills, Shanghai, China. Strain JTA1 was a member of genus Methylocystis on the basis of 16S rRNA and pmoA gene sequence similarity. The maximum specific cell growth rates （μmax= 0.042 hr-1, R2= 0.995） was derived through Boltzmann simulation, and the apparent half-saturation constants （Km（app） = 7.08 mmol/L, R2 = 0.982） was calculated according to Michaelis-Menton hyperbolic model, indicating that Methylocystis strain JTA1 had higher-affinity potential for methane oxidation than other reported methanotrophs. By way of adding the strain JTA1 culture, the methane consumption of aged refuse reached 115 mL, almost two times of control experiment. In addition, high tolerance of Methylocystis strain JTA1 to chloroform could facilitate the methane oxidation of aged refuse bio-covers. At the chloroform concentration of 50 mg/L, the methane-oxidation rate of bio-cover reached 0.114 mL/（day.g）, much higher than the highest rate, 0.0135 mL/（day.g）, of reported bio-covers. In conclusion, strain JTA1 opens up a new possibility for environmental biotechnology, such as soil or landfills bioremediation and wastewater decontamination.

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.