青藏高原湿地厌氧微生物固体富集及其低温高效产甲烷机制的研究

Biogas fermentation is important for preventing environmental pollution and producing renewable fuel. A common problem that causes biogas reactors to fail is that biogas bacteria are not able to produce sufficient biogas at low temperatures, which hinders practical applications of biogas fermentation. Wetlands in cold-climate areas are the main sources for global biogas emission, and so the fens in the Tibet-Qinghai Plateau are likely to be rich in psychrophilic biogas-producing bacteria. This study aims to isolate these bacteria, and to study the mechanism of biogas production at low temperatures. Adhesion to solid surfaces will be used as a new method for enrichment of bacteria from the Tibet-Qinghai Plateau. Using this method, solid materials will be mixed with peat samples from the Tibet-Qinghai Plateau and more isolates should be recovered compared to traditional isolation methods, such as serial dilution. Biogas fermentation bacteria in a syntrophic relationship interact with each other physiologically and physically, and solid surface adhesion provides physical and chemical supports for their growth and enhances their biogas productivity at the same time. The enriched bacterial communities will be tested for their biogas-producing efficiency at different temperatures between 4 ℃ and 25 ℃, and their species diversity and biogas metabolic pathway will be investigated. Enriched bacteria will also be continuously transferred and the key species for biogas metabolic pathways will be isolated and purified to be used as inocula in practical applications. Based on the results, a theory for biogas production at low temperatures will be proposed, which will help improve efficiency and allow further exploration of the application of biogas fermentation in a cold environment.

沼气发酵技术可有效解决我国环境及能源问题。但在低温条件下，沼气微生物产气量低或不产气制约着该技术的进一步发展。低温湿地是全球重要的甲烷排放源，青藏高原低温湿地可能孕育着独特而丰富的产甲烷微生物，富集分离到这些菌群，阐明其产甲烷机制，将对提高低温沼气发酵效率有重大意义。本项目将利用固体富集技术，即在青藏高原湿地土壤样品中添加固体载体来富集厌氧发酵微生物菌群。与传统技术相比，为微生物提供生长需要的固体附着物富，可以提高从环境中分离到未培养类群的效率，有利于对在生理及空间上相互依赖的互营代谢菌群的富集分离，及其产甲烷效率的提高。通过对固体富集菌群在不同低温（4～25℃）下产甲烷效率、群落结构及产甲烷代谢途径的研究，阐明其高效产甲烷机制，丰富沼气发酵理论。通过固体富集菌群的传代、关键类群的分离纯化，为低温沼气发酵技术提供重要的高效产甲烷菌种资源。

沼气发酵技术可有效解决我国环境及能源问题。但沼气微生物在低温条件下产气量低或不产气制约着该技术的发展与应用。低温湿地是全球重要的甲烷排放源，青藏高原湿地可能孕育着独特而丰富的产甲烷微生物。本项目利用固体富集技术，即在青藏高原湿地土壤样品中添加固体载体（筛选后为片状的几丁质和纤维素）来富集厌氧发酵微生物菌群。通过对固体富集菌群在不同低温（4, 15, 25℃）下产甲烷效率、群落结构及产甲烷代谢途径的研究，阐明其高效产甲烷机制, 同时分离纯化低温沼气发酵菌株。.该项目已筛选出青藏高原6个湿地的微生物群落，它们可低温高效降解纤维素和几丁质并产生产甲烷效率。利用16S rRNA基因高通量测序方法揭示了这6个湿地微生物群落的组成、各类群的定位以及之间的代谢关系：Clostridium III, Clostridium XIVa, Paludibacter, Parcubacteria, Saccharofermentans, Pelotomaculum, Methanosaeta, Methanobrevibacter, Methanoregula, Methanospirillum和 Methanosarcina分别参与了低温条件下纤维素、几丁质的水解、糖酵解、互营代谢和产甲烷作用；乙酸型产甲烷菌主要富集在土浆当中，而氢型产甲烷菌可以富集在土浆和固体表面。通过对若尔盖湿地微生物的富集传代，目前分离纯化出27株菌，其中3株为新的微生物物种 Trichococcus Chengduensis、Trichococcus sichuanensis、Jeotgalibaca zoigenesis和1株为新属新种Chitinipaludosus zoigenesis。通过对Chitinipaludosus zoigenesis基因组和海晏湿地微生物群落的元基因组测序，我们发现有2个几丁质酶基因具有3个催化域，通常一个微生物酶具有一个催化域，这是我们首次发现具有3个催化域的几丁质酶。此外，海晏湿地在元基因组数据中，我们共注释出35个几丁质基因，由于它们的氨基酸序列与NCBI数据库中的一致性较低，因此推测其为新型的几丁质酶。该项工作发现的新微生物物种与新型几丁质酶基因，具有较大的科学研究价值，同时，它们也为低温高效沼气发酵、食品、医学、环保、农业等领域提供了菌株和几丁质酶基因资源。