近海微生物驱动的木质素降解及其碳循环机制

Marine sediments are the largest organic carbon and microbial resource storage on the earth. Structural polymers such as lignin, cellulose and hemicellulose are important components of terrestrial organic matters, accounting for about one third of the organic matters buried in the ocean. The offshore area is the most diverse hydrosphere containing the active material transformation and energy flow, and the most diverse forms of interaction between microbes and organics. At present, there is no research report on the carbon material circulation mechanism of marine microbe-mediated lignin degradation and its carbon cycle mechanism. This project will take two aerobic degrading bacteria consortia selected from the sediments of the East China Sea and the South China Sea as the research objects. The lignin model compound alkali lignin will be used as the substrate to study material changes and the carbon cycle during the process of the lignin degradation by marine bacteria. The diversity of strains from the consortia will be analyzed basing on 16s rRNA genes, and the synergistic interaction between the strains within the degradation consortia will also be explored. In addition, the single cell and the pure culture with degradation capability will be obtained by Raman Activated Single Cell Ejection System and the conventional isolation and culture technique, respectively. The single cell and pure culture genomes will be sequenced and the potential lignin catabolic genes will be mined by bioinformatics analysis. And then the function of catabolic genes and enzymatic properties of key enzymes will be studied at the molecular and biochemical levels to analyze lignin degradation pathway and its carbon cycle mechanism. Eventually the abundance and distribution of degrading bacteria and degrading enzyme-encoding genes in the offshore area will be analyzed to explore the relationship between microbes in the offshore and the microbe-mediated lignin degradation.

海洋沉积物是地球上最大的有机碳库和微生物资源库，以木质素、纤维素、半纤维素为主的结构性聚合物是陆源有机质的重要组分，约占海洋埋藏有机质的三分之一。近海区是物质转化与能量流动最活跃、微生物与有机物交互作用形式最为多样的水圈环境。目前海洋微生物驱动的木质素的碳循环机制尚无研究报道。本项目以东海和南海沉积物中筛选的两种降解菌群为研究对象，以木质素模式化合物碱木质素为底物，研究木质素降解过程中物质变化和碳循环机制，分析降解菌群的菌株多样性及其协同互作关系；通过拉曼光谱单细胞分选和传统分离培养技术分别获得具有降解功能的单细胞和降解菌纯培养；利用单细胞和纯培养基因组测序和分析，挖掘潜在的木质素降解基因，在分子和生化水平研究降解基因的功能和关键酶的特性，解析木质素降解途径及其碳循环机制；分析降解菌和降解酶编码基因在近海环境中的丰度与分布，探究近海中微生物与其驱动的木质素降解及物质循环的关系。

木质素等陆地有机碳是全球海洋碳的重要组成部分。据推测，细菌在海洋系统中对木质素降解起着重要作用。然而，海洋微生物参与木质素降解的程度及其对海洋碳循环的贡献仍不清楚。在本研究中，从东海和南海的近岸沉积物中富集了两个能够降解碱木质素（木质素的模式化合物）的细菌群落，命名为菌群LIG-B和LIG-S。菌群LIG-B和LIG-S主要由变形杆菌门组成，分别为硝化还原菌属（71.6%）和盐单胞菌属（91.6%）。发现菌群LIG-B（最大降解率为57%）比LIG-S（最大18%）更利于木质素降解。能够将木质素分解成较小片段的木质素分解酶漆酶（Lac）、锰过氧化物酶（MnP）和木质素过氧化物酶（LiP）在这两个菌群中都具有活性。经过两个菌群处理的碱木质素（AL）中新出现的低分子量芳香族化合物、有机酸和其他木质素衍生化合物证明木质素可以被两个菌群解聚。发现菌群LIG-S中的木质素途径在构建的降解网络中表现出更高的完整性。进一步的研究揭示，通过原儿茶酸盐和邻苯二酚的多种途径参与木质素及其衍生物的降解基因，不仅存在于菌群LIG-B和LIG-S中，还存在于来自9个近岸地区的783个公开可用的宏基因组组装基因组中。同时，从菌群LIG-B和LIG-S中分离出11株细菌，并对其利用AL的生长及降解进行了研究。7株菌株表现出AL的能力，其中盐单胞菌NyZ327和盐单胞杆菌NyZ357在24小时的降解率分别为2.61%和4.95%。扫描电子显微镜显示，这两种菌株可以破坏AL的微观形态并形成颗粒，并且某些化学基团的吸收峰变弱。GC-MS结果表明，AL降解为芳烃。对菌株NyZ357基因组的分析表明，该菌株可能具有代谢木质素衍生化合物香草醛、4-羟基苯甲酸酯和苯甲酸酯的途径。相关降解基因在大肠杆菌中表达并进行了功能验证。本研究为解析海洋细菌降解木质素的机理并探讨海洋环境中的碳循环提供了理论依据。