基于中度嗜盐菌的活性污泥耐盐降解分子机制

Recently, huge quantity of hypersaline organic wastewater discharged every year in China, which was great harmful to the ecological environment and became the big problem in environmental engineering field. Moderate halophiles possessing strong metabolism ability widely existed in the habitats with extended range of salinity, which had great importance to the activitaed sludge in the biotreatment of hypersaline effluents. This project focused on the mechnisn of moderate halophiles optimizing the activited sludge's ability for salt tolorance and oganic biodegradation. This study plans to analyze the osmoprotection and degradation microbial ecosystem in biotreatment process, illustrate the moderately halophilic bacterial community succession, and reveal the community composition, ecological distribution and mechanism of environmental adaption in different salinity levels. The aim was to illustrate the correlations between the structure and function of moderate halophiles community, to clarify the molecular mechanism of osmoprotection and resistance of salinity fluctuation, to explain the osmoprotection and degradation functional gene diversity and their horizontal gene transfer mechanism, to explore the bioaugmentation degradatin process by the recombinant clone of exogenous functional gene transposon, and construct the regulation stratege for moderate halophiles community function by adjusting compatible solutes and environmental factors. The results will deep into the understand of the coupled effect of "Moderate halophile bacteria-activited sludge ecosystem-regulation mechanism of osmoprotection and biodegradation", which contributes to provide scientific basis for breaking through the bottlenecks of the hypersaline organic wastewater biotreatment theory and technology.

我国目前高盐有机废水年排放量巨大、处理效率低，严重影响生态环境安全，已经成为环境工程领域亟待解决的难题。中度嗜盐菌抗盐分波动范围宽、代谢能力强，对处理高盐有机废水的活性污泥具有重要作用。本项目针对中度嗜盐菌强化活性污泥耐盐降解性能的机理问题，分析高盐有机废水生物处理过程的耐盐降解微生物生态系统，查明中度嗜盐菌群的演替规律，揭示盐分波动条件下中度嗜盐菌群的组成、生态分布及环境响应机制；阐明中度嗜盐菌群结构和功能的相互关系，揭示耐受高盐胁迫和抵抗盐分波动冲击的分子机制，研究耐盐降解功能基因多样性和水平基因转移机制，探索外源功能基因转座子重组克隆强化耐盐降解性能的可行性，调整相容性溶质和环境因子建立中度嗜盐菌群优化活性污泥的功能调控机制。结果将为深入理解"中度嗜盐菌群－活性污泥生态系统－耐盐降解功能调控机制"耦合作用的科学问题，突破高盐有机废水生物处理理论的瓶颈问题提供科学依据。

我国高盐有机废水具有排放量大、影响面广、污染严重等特点，已成为环境工程领域亟待解决的难题之一。针对高盐有机废水生物处理效率较低的现状，引入耐盐范围宽、代谢能力强的中度嗜盐菌，开展基于中度嗜盐菌的活性污泥耐盐降解分子机制研究。通过四年研究，取得主要进展如下：.1）成功培育并构建了基于中度嗜盐菌的高效耐盐活性污泥系统。该系统在10-120 g/L NaCl盐度范围内可有效降解苯酚、氯酚等有机污染物。当盐度为80 g/L，氯酚浓度在1000 mg/L以上时，有机物去除率仍可高达98%。.2）揭示了高效耐盐活性污泥系统优势功能菌演替规律及代谢特征。PCR-克隆测序分析表明，Halomonas和Alcaligenes分别是耐盐降解苯酚和氯酚的优势功能菌；两种菌群同时含有编码（氯代）邻苯二酚 1,2-双加氧酶和（氯代）2,3-双加氧酶的相应基因，具有对难降解有机物多样化降解潜能。.3）阐明了中度嗜盐菌耐盐降解及调控生理机制。构建高通量筛菌体系，分离获得两株中度嗜盐苯酚降解菌，16S rDNA测序鉴定为Virgibacillus sp. PDB-F2和Brachybacterium sp. PDB-G1。前者可通过超量积累四氢嘧啶达到耐盐目的，NaCl浓度为12%时，其胞内四氢嘧啶浓度可达73.12 mg/g细胞干重。.4）揭示了中度嗜盐菌群耐盐功能基因（簇）多样性特征。宏基因组分析表明，合成甜菜碱、四氢嘧啶和羟基四氢嘧啶等相容性溶质的功能基因丰度随盐度增加有明显增长。胞外表达试验显示，ABC及BCCT转运蛋白家族可能存在协同主转运机制。.5）成功构建了高效耐盐抗逆基因工程菌。以耐辐射球菌全基因组为模板，通过密码子优化，合成扩增耐盐抗逆基因PPRI，电击导入底盘微生物，成功构建一株耐盐抗逆基因菌。高盐条件下，苯甲酸钠降解效能高达90%以上。.本项目对基于中度嗜盐菌活性污泥系统的微生物群落和耐盐分子机制进行了深入研究，重点突破了中度嗜盐菌占优势的活性污泥构建、群落演替、菌群环境适应、相容性溶质合成与运输及耐盐降解性能调控等高盐有机废水生物处理基础性难题。上述成果为高盐有机废水生物处理技术的开发提供了理论支撑。