整合宏组学方法研究番茄秸秆堆肥生境中的关键微生物及其功能

The enormous quantity of biomass from vegetable waste is not only a rich resource but also a huge pollutant that need wide research to be recycled. Compost offered a good choice and it was proved to play important roles in enhancing soil fertility as well as in promoting restoration of the damaged ecological environment. However, little study has been carried out on the effective biodegradation of vegetable waste, leading no common rules on developing functioning microbial inoculums by now. In the proposed project, the compost habitat of tomato straw, which owns the largest quantity of biomass in the circumstance of intensive vegetable production, was selected as the study object, and the new integrated meta-omics was applied in probing the succession of microbial community structure and functional enzymes. The effective-degraded layer in thermophilic period was continuously monitored and the dynamic responds of microbial community structure in time were fully displayed by the culture-dependent method as well as technologies of metagenomics; while, populations of the active enzymes and their catalytic properties were demonstrated by the combination of functional Native-PAGE of glycoside hydrolase and metaproteomics. Then, dominant microbes and their corresponding metabolism that truly determined to the fermentation process can be further uncovered comprehensively, authenticly and effectively by the integrated analysis above. Results of the research will surely provide more scientific guidance for the vegetable waste recycling.

针对蔬菜废弃物环境压力大、资源价值广、研究相对贫乏的生产实际，结合秸秆堆肥在培肥地力、促进受损生态环境修复方面的显著作用但相应功能菌剂开发落后的现实，本项目拟以集约化蔬菜栽培中产生量最大的番茄秸秆为研究对象进行堆肥，采用一种新的整合宏组学方法系统研究影响其堆肥发酵进程的关键功能微生物及其生命活动。以山东寿光集约化蔬菜生产基地为平台，通过整合番茄秸秆高温堆腐期高效降解层微生物的传统分离培养技术及宏基因组学技术，系统全面地揭示微生物区系随发酵进程的动态演替；通过糖苷水解酶活性电泳及宏蛋白质组学技术，对相关活性酶组分及其功能进行高通量表征；通过系统整合以上宏组学结果，全面、真实、有效地探明关键微生物及其功能。研究结果对蔬菜废弃物资源的有效开发利用具有重要的科学指导意义。

我国是蔬菜生产大国，与蔬菜产量和种植面积同比增长的是蔬菜秸秆产生量和由此造成的环境压力。针对蔬菜废弃物环境压力大、资源价值广、研究相对贫乏的生产实际，结合秸秆堆肥在培肥地力、促进受损生态环境修复方面的显著作用但相应功能菌剂开发落后的现实，本项目以集约化蔬菜栽培中产生量最大的番茄秸秆为研究对象进行堆肥。通过堆肥理化性质的监测并结合整合宏组学方法，系统研究影响堆肥发酵进程的关键功能微生物及其生命活动。结果表明，以玉米秸秆为调理剂，与番茄秸秆按质量比1：3混合共堆肥，物料第一天即进入高温期，前十天为升温期，之后进入降温期，50℃以上的高温期持续20天，之后进入腐熟期。各时期微生物群落结构差异较大，升温期受高温等环境压力影响，微生物多样性较低，主要以Actinobacteria（如Thermobifida和Saccharomonospora）、Firmicutes(如Bacillus)和Ascomycota（Aspergillus）为主，降温期微生物多样性显著增加，主要以Proteobacteria、Bacteroidetes、Ascomycota（Thermomyces）和Basidiomycota (如Coprinopsis)为主。质谱鉴定结果表明，Thermobifida功能多样，既能降解纤维素（Thermobifida是该时期唯一的微生物降解者），又能降解半纤维素；除Thermobifida外，其他丰度较高的微生物功能都较单一，如Saccharomonospora、Bacillus、Streptomyces、Planifilum和Aspergillus主要参与半纤维素的降解，Planifilum和Sphingobacterium则主要降解蛋白质。降温期不仅增加了微生物的多样性，同时丰富了糖苷水解酶的家族数量，Thermobifida的重要性开始减弱，如除Thermobifida外，Glycomyces、Nocardiopsis和Streptomyces同样开始降解纤维素，半纤维素降解者主要为Thermobifida、Saccharomonospora、Streptomyces和Chaetomium；蛋白质主要由Thermobifida、Saccharomonospora、Norcadiopsis和Bacillus降解，同时该时期还鉴定到Acremonium和