Response of methanogenic community and their activity to temperature rise in alpine swamp meadow at different water level of the permafrost wetland on Qinghai-Tibet Plateau.

Wetlands are an important source of atmospheric methane (CH4) and are sensitive to global climate change. Alpine swamp meadows, accounting for ~50% of the natural wetlands on the Qinghai-Tibet Plateau, were considered one of the most important ecosystems. Methanogens are important functional microbes that perform the methane producing process. However, the response of methanogenic community and the main pathways of CH4 production to temperature rise remains unknown in alpine swamp meadow at different water level in permafrost wetlands. In this study, we investigated the response of soil CH4 production and the shift of methanogenic community to temperature rise in the alpine swamp meadow soil samples with different water levels collected from the Qinghai-Tibet Plateau through anaerobic incubation at 5°C, 15°C and 25°C. The results showed that the CH4 contents increased with increasing incubation temperature, and were 5-10 times higher at the high water level sites (GHM1 and GHM2) than that at the low water level site (GHM3). For the high water level sites (GHM1 and GHM2), the change of incubation temperatures had little effect on the methanogenic community structure. Methanotrichaceae (32.44-65.46%), Methanobacteriaceae (19.30-58.86%) and Methanosarcinaceae (3.22-21.24%) were the dominant methanogen groups, with the abundance of Methanotrichaceae and Methanosarcinaceae having a significant positive correlation with CH4 production (p < 0.01). For the low water level site (GHM3), the methanogenic community structure changed greatly at 25°C. The Methanobacteriaceae (59.65-77.33%) was the dominant methanogen group at 5°C and 15°C; In contrast, the Methanosarcinaceae (69.29%) dominated at 25°C, and its abundance showed a significant positive correlation with CH4 production (p < 0.05). Collectively, these findings enhance the understanding of methanogenic community structures and CH4 production in permafrost wetlands with different water levels during the warming process.

Carbon Isotopic Evidence for Gas Hydrate Release and Its Significance on Seasonal Wetland Methane Emission in the Muli Permafrost of the Qinghai-Tibet Plateau.

In order to determine the significant role of gas hydrate in seasonal wetland methane emission at the drilling-affected permafrost, the carbon isotopic monthly field monitoring of methane (CH4), as well as carbon dioxide (CO2), emitted from near-surface soil and a gas hydrate drilling well (DK-8) was conducted in the Muli permafrost of the Qinghai-Tibet Plateau. The methane source effused from the well DK-8 was calculated as -25.9 ± 1.4‱ and -26.5 ± 0.5‱, respectively, by the Keeling and Miller Tans plots, with the carbon isotope fractionation (εC) between CO2 and CH4 from -25.3‱ to -32.1‱. The carbon isotopic signatures are indicative of thermogenic origin associated with gas hydrate dissociation. The near-surface soil-emitted methane has δ13CCH4 values between -52.0 ± 1.2‱ and -43.2 ± 1.8‱ with the heaviest in December and the lightest in July. Further, the εC values of near-surface soil-emitted gases were between 28.6‱ and 47.9‱, significantly correlated with the δ13CCH4 values. The linear correlation between εC and δ13CCH4 values indicated binary end-member of microbial and thermogenic sources control the seasonal variation of wetland methane emission. The thermogenically derived methane was identified as the dominant methane source in autumn and winter, compared with the increasing contribution of microbially derived methane in spring and summer. The finding provides reliable evidence for gas hydrate release on the seasonal wetland methane emission in the Muli permafrost affected by drilling activities. The combined application of εC and δ13CCH4 to distinguish thermogenic from biogenic methane is well established and powerful in complex environments, which can provide an improved constraint on source apportionment for wetland emitted methane in the permafrost of the Qinghai-Tibet Plateau.

Sources of seasonal wetland methane emissions in permafrost regions of the Qinghai-Tibet Plateau.

In this study, systematic soil methane cycle geochemical monitoring was carried out in a typical gas hydrate region in the Qinghai-Tibet Plateau. Soil gas samples were collected for hydrocarbon components and carbon isotope analysis. Meanwhile, soil-methane fluxes from the upper active layer (20-30 cm) were monitored during six months of one year. The results of this research provide evidence of a new source of methane emission from wetland soils in permafrost regions: gas hydrate release. Sites with large methane emissions were found using flux monitoring, the characteristics of thermogenic methane were identified using carbon isotope tracing, and the relationship between emission by soils and effusion from gas hydrates was determined through correlation analyses of soil-adsorbed hydrocarbons. Seasonal variation of methane emissions are also discussed by considering the emission of bacterial methane, thermogenic methane, and the absorption of methane from the soil active layer. These comprehensive findings provide valuable information for carbon cycle research of wetlands in permafrost regions.

Diversity and Distribution of Methanogenic Community Between Two Typical Alpine Ecosystems on the Qinghai-Tibetan Plateau.

Alpine permafrost regions are important sources of biogenic CH4 and methanogens play an important role in the methane-producing process. The alpine permafrost on the Qinghai-Tibetan plateau comprises about one-sixth of China's land area, and there are various types of alpine ecosystems. However, the methanogenic communities in the typical alpine ecosystems are poorly understood. In this study, the active layers and permafrost layers of the natural ecosystem of alpine grassland (DZ2-1) and alpine swamp meadow (DZ2-5) were selected to investigate the diversity and abundance of methanogenic communities. Methanobacterium (63.65%) are overwhelmingly dominant in the active layer of the alpine grassland (DZ2-1A). ZC-I cluster (26.13%), RC-I cluster (19.56%), and Methanobacterium (15.02%) are the dominant groups in the permafrost layer of the alpine grassland (DZ2-1P). Methanosaeta (32.92%), Fen cluster (29.59%), Methanosarcina (16.33%), and Methanobacterium (13.95%) are the dominant groups in the active layer of the alpine swamp meadow (DZ2-5A), whereas the Fen cluster (50.85%), ZC-I cluster (27.63%), and RC-I cluster (14.15%) are relatively abundant in the permafrost layer of the alpine swamp meadow (DZ2-5P). qPCR data showed that the abundance of methanogens was higher in the natural ecosystem of alpine swamp meadow than in alpine grassland. We found that the community characteristics of methanogens were related to environmental factors. Pearson correlation analyses indicated that the relative abundance of Methanobacterium had a significantly positive correlation with hydrogen concentration (P < 0.01), while the relative abundances of Methanosaeta and Methanosarcina were positively correlated with acetate concentration (P < 0.05). This study will help us to understand the methanogenic communities and their surrounding environments in alpine ecosystems.

Comparative evaluation of three archaeal primer pairs for exploring archaeal communities in deep-sea sediments and permafrost soils.

16S rRNA gene profiling is a powerful method for characterizing microbial communities; however, no universal primer pair can target all bacteria and archaea, resulting in different primer pairs which may impact the diversity profile obtained. Here, we evaluated three pairs of high-throughput sequencing primers for characterizing archaeal communities from deep-sea sediments and permafrost soils. The results show that primer pair Arch519/Arch915 (V4-V5 regions) produced the highest alpha diversity estimates, followed by Arch349f/Arch806r (V3-V4 regions) and A751f/AU1204r (V5-V7 regions) in both sample types. The archaeal taxonomic compositions and the relative abundance estimates of archaeal communities are influenced by the primer pairs. Beta diversity of the archaeal community detected by the three primer pairs reveals that primer pairs Arch349f/Arch806r and Arch519f/Arch915r are biased toward detection of Halobacteriales, Methanobacteriales and MBG-E/Hydrothermarchaeota, whereas the primer pairs Arch519f/Arch915r and A751f/UA1204r are biased to detect MBG-B/Lokiarchaeota, and the primers pairs Arch349f/Arch806r and A751f/UA1204r are biased to detect Methanomicrobiales and Methanosarcinales. The data suggest that the alpha and beta diversities of archaeal communities as well as the community compositions are influenced by the primer pair choice. This finding provides researchers with valuable experimental insight for selection of appropriate archaeal primer pairs to characterize archaeal communities.

Shifts of methanogenic communities in response to permafrost thaw results in rising methane emissions and soil property changes.

Permafrost thaw can bring negative consequences in terms of ecosystems, resulting in permafrost collapse, waterlogging, thermokarst lake development, and species composition changes. Little is known about how permafrost thaw influences microbial community shifts and their activities. Here, we show that the dominant archaeal community shifts from Methanomicrobiales to Methanosarcinales in response to the permafrost thaw, and the increase in methane emission is found to be associated with the methanogenic archaea, which rapidly bloom with nearly tenfold increase in total number. The mcrA gene clone libraries analyses indicate that Methanocellales/Rice Cluster I was predominant both in the original permafrost and in the thawed permafrost. However, only species belonging to Methanosarcinales showed higher transcriptional activities in the thawed permafrost, indicating a shift of methanogens from hydrogenotrophic to partly acetoclastic methane-generating metabolic processes. In addition, data also show the soil texture and features change as a result of microbial reproduction and activity induced by this permafrost thaw. Those data indicate that microbial ecology under warming permafrost has potential impacts on ecosystem and methane emissions.

Comparative Analyses of Methanogenic and Methanotrophic Communities Between Two Different Water Regimes in Controlled Wetlands on the Qinghai-Tibetan Plateau, China.

Wetlands are an important methane (CH4) emission source. CH4 is mainly produced during the biogeochemical process, in which methanogens and methanotrophs both play important roles. However, little is known how these two microbial communities change under different water regimes. In this study, the diversity and abundance of methanogens and methanotrophs in wetlands on Qinghai-Tibetan Plateau with different water contents (a high water content site DZ2-14-3 and a low water content site DZ2-14-4) were studied by using phylogenetic analysis and quantitative PCR based on mcrA gene and pmoA gene. A total of 16 methanogenic operational taxonomic units (OTUs) and 9 methanotrophic OTUs are obtained. For methanogens, Fen cluster (58.0%) and Methanosaetaceae (20.3%) are the dominant groups in high moisture samples, whereas Methanosaetaceae (32.4%), Methanosarcinaceae (29.4%), and Methanobacteriaceae (22.1%) are prevalent in low moisture samples. Methylobacter (90.0%) of type I methanotrophs are overwhelmingly dominant in high moisture samples, while Methylocystis (53.3%) and Methylomonas (42.2%) belonging to types II and I methanotrophs are the predominant groups in low moisture samples. Furthermore, qPCR analysis revealed that the abundance of methanogens and methanotrophs were higher in high moisture samples than that in low moisture samples. Overall, this comparative study between wetlands controlled by two different water regimes on the Qinghai-Tibetan Plateau provides fundamental data for further research on microbial functions within extreme ecosystems.

Qipengyuania sediminis gen. nov., sp. nov., a member of the family Erythrobacteraceae isolated from subterrestrial sediment.

A Gram-reaction-negative, non-motile, facultatively aerobic bacterium, designated strain M1T, was isolated from a subterrestrial sediment sample of Qiangtang Basin in Qinghai-Tibetan plateau, China. The strain formed rough yellow colonies on R2A plates. Cells were oval or short rod-shaped, catalase-positive and oxidase-negative. Phylogenetic analyses based on 16S rRNA gene sequences indicated that the isolate belonged to the family Erythrobacteraceae and showed 96.2­96.4 % 16S rRNA gene sequence similarities to its closest relatives. Chemotaxonomic analysis revealed ubiquinone-10 (Q10) as the dominant respiratory quinone of strain M1T and C17 : 1ω6c (44.2 %) and C18 : 1ω7c (13.7 %) as the major fatty acids. The major polar lipids were phosphatidylethanolamine, phosphatidylcholine, phosphatidylglycerol, diphosphatidylglycerol, sphingoglycolipid, three unidentified glycolipids, one unidentified phosphoglycolipid and one unidentified lipid. The DNA G+C content of strain M1T was 73.7 mol%. On the basis of phenotypic, phylogenetic and genotypic data presented in this study, strain M1T represents a novel species of a new genus in the family Erythrobacteraceae, for which the name Qipengyuania sediminis gen. nov., sp. nov. is proposed. The type strain of the type species is M1T ( = CGMCC 1.12928T = JCM 30182T).

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.

Effect of permafrost properties on gas hydrate petroleum system in the Qilian Mountains, Qinghai, Northwest China.

The gas hydrate petroleum system in the permafrost of the Qilian Mountains, which exists as an epigenetic hydrocarbon reservoir above a deep-seated hydrocarbon reservoir, has been dynamic since the end of the Late Pleistocene because of climate change. The permafrost limits the occurrence of gas hydrate reservoirs by changing the pressure-temperature (P-T) conditions, and it affects the migration of the underlying hydrocarbon gas because of its strong sealing ability. In this study, we reconstructed the permafrost structure of the Qilian Mountains using a combination of methods and measured methane permeability in ice-bearing sediment permafrost. A relationship between the ice saturation of permafrost and methane permeability was established, which permitted the quantitative evaluation of the sealing ability of permafrost with regard to methane migration. The test results showed that when ice saturation is >80%, methane gas can be completely sealed within the permafrost. Based on the permafrost properties and genesis of shallow gas, we suggest that a shallow "gas pool" occurred in the gas hydrate petroleum system in the Qilian Mountains. Its formation was related to a metastable gas hydrate reservoir controlled by the P-T conditions, sealing ability of the permafrost, fault system, and climatic warming. From an energy perspective, the increasing volume of the gas pool means that it will likely become a shallow gas resource available for exploitation; however, for the environment, the gas pool is an underground "time bomb" that is a potential source of greenhouse gas.

Flavobacterium qiangtangensis sp. nov., isolated from Qiangtang basin in Qinghai-Tibetan Plateau, China.

A flexirubin-type yellow-pigmented, non-gliding, non-flagellated, gram-negative bacterium strain, designated F3(T), was isolated from a drilling core sample of the Qiangtang basin, Qinghai-Tibetan plateau, China. Phylogenetic analysis based on 16S rRNA gene sequences showed that the strain F3(T) belongs to the genus Flavobacterium, with the highest 16S rRNA gene sequence similarity to the Flavobacterium noncentrifugens CGMCC 1.10076(T) (94.92 %). Strain F3(T) grew optimally at temperature about 20 °C, at pH about 7.0-8.0, at NaCl concentration 0 % (w/v). The DNA G+C content of the isolate was 35.5 mol%. The major polar lipid was phosphatidylethanolamine, predominant cellular fatty acids of the strain was iso-C15:0 (22.02 %), while the major menaquinone was menaquinone 6. Due to the phenotypic and genetic distinctiveness and several other characteristic studied in this article, we consider F3(T) as a novel species of the genus Flavobacterium, and propose to name it Flavobacterium qiangtangensis sp. nov. The type strain is F3(T) (=CGMCC 1.12706(T) = JCM 19739(T)).

Hymenobacter qilianensis sp. nov., isolated from a subsurface sandstone sediment in the permafrost region of Qilian Mountains, China and emended description of the genus Hymenobacter.

A red-pink, Gram-negative, rod-shaped, non-motile, non-spore-forming bacterium, designated strain DK6-37 was isolated from the permafrost region of Qilian Mountains in northwest of China. Phylogenetic analysis based on 16S rRNA gene sequencing indicated that this isolate represents a novel member of the genus Hymenobacter, with low sequence similarities (<97 %) to recognized Hymenobacter species. Optimum growth was observed at 28 °C, pH 7.0 and 0 % NaCl. The strain was found to contain MK-7 as the predominant menaquinone. The polar lipids were identified as phosphatidylethanolanmine, two unknown aminophospholipids, one unknown aminolipid and three unknown polar lipids. The major fatty acids were identified as summed feature 3 (C16:1 ω7c/C16:1 ω6c as defined by MIDI), summed feature 4 (anteiso-C17:1 B/iso-C17:1 I), C16:1 ω5c, iso-C17:0 3-OH, iso-C15:0 and C18:0. The DNA G + C content was determined to be 67.4 mol %. On the basis of the polyphasic evidence presented, it is proposed that strain DK6-37 represents a novel species of the genus Hymenobacter, for which the name Hymenobacter qilianensis sp. nov. is proposed. The type strain is DK6-37(T) (= CGMCC 1.12720(T) = JCM 19763(T)).