Methanogen community in Zoige wetland of Tibetan plateau and phenotypic characterization of a dominant uncultured methanogen cluster ZC‐I

Abstract

Zoige wetland of Tibetan plateau is characterized by being located at a low latitude (33 degrees 56'N, 102 degrees 52'E) region and under the annual temperature around 1 degrees C. Previous studies indicated that Zoige wetland was one of the CH(4) emission centres in Qinghai-Tibetan plateau; in this study, the methanogen community in this low-latitude wetland was analysed based on the homology of 16S rRNA and mcrA genes retrieved from the soil. The results indicated that members of Methanosarcinales and Methanomicrobiales constituted the majority of methanogens, and a novel uncultured methanogen cluster, Zoige cluster I (ZC-I) affiliated to Methanosarcinales, could be dominant. Using quantitative polymerase chain reaction (qPCR) assay, ZC-I methanogens were estimated to be 10(7) cells per gram of soil, accounting for about 30% of the total Archeae. By combining culturable enrichment with qPCR assay, the quantity of ZC-I methanogens in the methanogenic enrichment with acetate, H2/CO(2), methanol or trimethylamine was determined to increase to 10(8) cells ml(-1), but not with formate, which indicated that ZC-I methanogens could use the four methanogenic substrates. The growth rates at 30 degrees C and 15 degrees C were not pronounced different, implying ZC-I to be the cold-adaptive methanogens. The broad substrate spectrum identified the ZC-I methanogens to be a member of Methanosarcinaceae, and could represent a novel sub-branch specifically inhabited in cold ecosystems. Fluorescence in situ hybridization (FISH) images also visualized ZC-I methanogens the sarcina-like aggregate of the spherical cells. The prevalence and flexibility in substrate utilization and growth temperature suggested ZC-I methanogens to be an important player in the methanogenesis of Zoige wetland.

Methanogenic System of a Small Lowland Stream Sitka, Czech Republic

“…Our results from the DGGE analysis are supported by cloning and sequencing of methyl coenzyme M reductase (mcrA) gene which also retrieved relatively rich diversity (25 different mcrA gene clones) of the methanogenic community in the Sitka stream hyporheic sediments. Similar richness in number of clones was also mentioned in a methanogenic community in Zoige wetland, where 21 different clones were found (Zhang et al 2008a), while 20 clones were described in the methane cycle of a meromictic lake in France (Biderre-Petit et al 2011). In addition, soils from Ljubljana marsh (Slovenia) showed 17 clones (Jerman et al 2009), for example.…”

Intense methane ebullition from open water area of a shallow peatland lake on the eastern Tibetan Plateau

“…Temperature is thought to be an important factor controlling methane production (Zeikus and Winfrey, 1976;Whiting and Chanton, 1993;Wik et al, 2014). However, it was reported that the majority of methanogens in Zoige wetlands was cold adapted and not sensitive to normal temperature changes (Zhang et al, 2008;Liu et al, 2011). Therefore, production rate of methane affected by temperature could have made a limited contribution to the variation of methane ebullition.…”

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

“…The temperature profile was identical to the study of Zhang et al (2008): initial denaturation at 94°C for 7 min followed by 30 cycles of denaturation at 94°C for 60 sec, annealing at 50°C for 45 sec and extension at 72°C for 90 sec, and a final elongation at 72°C for 7 min. PCR reaction mixture contains 5 μL Taq buffer, 3 μL dNTP (1.5 mmol/L, final concentration), 1 μL of each primer (0.4 mmol/L, final concentration), 0.2 μL BSA (10 mg/mL), 1 μL template DNA, 0.4 μL Taq DNA polymerase (5 U/ μL, Takara, Japan) and add RNase free dH 2 O (Takara, Japan) to a final volume of 50 μL.…”