扎龙盐碱湿地芦苇根际土的优势产甲烷途径分析

Predominant methanogenic pathway in the rhizosphere soil of reed in saline-alkaline Zhalong wetland

摘要:

【背景】芦苇湿地是甲烷主要的排放源之一，产甲烷古菌是唯一产生大量甲烷的生物，而盐碱湿地芦苇根际土优势甲烷途径鲜有研究。【目的】调查扎龙低温盐碱湿地芦苇根际土中的优势产甲烷途径。【方法】通过16S rRNA基因扩增子测序，分析扎龙湿地芦苇生长季根际土壤深度0–20 cm的产甲烷古菌和细菌组成。用已知的产甲烷底物三甲胺、甲醇、乙酸和H₂/CO₂，以及高盐环境植物和细菌的相似相容物质——甜菜碱(被细菌还原成三甲胺)在pH 8.0培养获得芦苇根际土的产甲烷富集物。测定各种富集物的产甲烷速率鉴定芦苇根际土的优势产甲烷途径；测定甜菜碱富集物中的16S rRNA基因多样性，并用RT-qPCR定量优势细菌和古菌的物种组成，从而推测协同代谢甜菜碱产甲烷的细菌和古菌类群。【结果】扎龙盐碱湿地芦苇根际土含有氢营养型的甲烷杆菌属(Methanobacterium, 36.42%)、偏好低氢的Rice Cluster II (11.55%)、乙酸营养型的甲烷鬃菌属(Methanosaeta, 11.29%)、甲基营养型的甲烷八叠球菌属(Methanosarcina, 6.53%)、H₂还原甲基物质的甲烷马赛球菌属(Methanomassiliicoccus, 4.05%)和高比例的未培养厌氧甲烷氧化古菌-噬甲烷菌属(Candidatus Methanoperedens, 35.06%)。优势细菌包括绿弯菌门(Chloroflexi, 21.55%)、变形杆菌门(Proteobacteria, 16.88%)、放线菌门(Actinobacteria, 13.37%)和酸杆菌门(Acidobacteria, 10.0%)。扎龙湿地的三甲胺和甜菜碱富集物产甲烷速率最高，富集物中代谢甜菜碱的优势细菌包括鼠胞菌科(Sporomusaceae)、沉积杆菌科(Sedimentibacteraceae)、亨氏梭菌科(Hungateiclostridiaceae)和梭菌科(Clostridiaceae)。从三甲胺产甲烷的优势古菌包括甲基营养型的甲烷八叠球菌属和H₂还原甲基物质产甲烷的甲烷马赛球菌属。【结论】以相似相容物质甜菜碱的还原产物三甲胺为前体的甲烷代谢途径是扎龙低温盐碱湿地芦苇根际土的优势产甲烷途径。

Abstract:

[Background] Reed wetland is one of the main methane emission sources, and methanogenic archaea are the only known organisms producing ample methane. Whereas, the dominant methane-production pathways in the rhizosphere soil of reed in saline-alkali wetland are unknown. [Objective] To reveal the dominant methanogenic pathway in the saline-alkaline Zhalong wetland. [Methods] High-throughput sequencing of 16S rRNA gene was employed to study the diversity of methanogenic archaea and bacteria in the rhizosphere soil (0–20 cm depth) of reed in Zhalong wetland. The known methanogenic substrates including trimethylamine (TMA), methanol, betaine, acetate, and H₂/CO₂ were used to enrich the methanogenic microorganisms in the wetland soil. The methanogenic rate of each microorganism was measured to determine the predominant methanogenic pathway in the rhizosphere soil, and qPCR was employed to quantify the bacterial and archaeal groups and further predict the bacteria and archaea that jointly convert betaine to CH₄. [Results] The dominant methanoarchaea were determined to be the CO₂-reducing Methanobacterium (36.42%) and Rice Cluster II (11.55%), the methane anaerobic oxidizer Candidatus Methanoperedens (35.06%), the aceticlastic methanogen Methanosaeta (11.29%), methylotrophic Methanosarcina (6.53%), and the H₂-dependent methylotrophic methanogen Methanomassiliicoccus(4.05%). The predominant bacteria were Chloroflexi (21.55%), Proteobacteria (16.88%), Actinobacteria (13.37%), and Acidobacteria (10.00%). The highest methane-producing rate was observed in the media with the addition of TMA and betaine. The dominant bacteria reducing betaine to TMA included Sporomusaceae, Sedimentibacteraceae, Hungateiclostridiaceae, and Clostridiaceae and the dominant archaea producing CH₄ from TMA included Methanosarcina and Methanomassiliicoccus. [Conclusion] Methylotrophic methanogenesis based on the TMA from betaine reduction is the dominant methanogenic pathway in the rhizosphere soil of reed in the low-temperature Zhalong wetland.