塑料降解昆虫中肠道微生物的固氮作用和机制

The “White pollution” brought by plastic wastes has become a global environmental concern. Our long-term research has demonstrated that the plastic-eating insects are able to effectively degrade and subsist on petroleum-based plastic. However, plastic diet is rich in carbon but deficient in nitrogen. Nitrogen deficiency occurs in the plastic degradation process. Nitrogen is necessary for the biosynthesis of proteins and nucleotides, as well as other essential metabolites. Hence, it is interesting to investigate how the plastic-degrading insects overcome the nitrogen deficiency. As showed in the previous research, nitrogen fixation by the gut microbiota was the important way to obtain nitrogen for the insects on diets with high carbon to nitrogen (C : N) ratios. Several putative diazotrophic bacteria have been observed in the guts of plastic-degrading insects, indicating that the plastic-degrading insects could overcome the nitrogen deficiency via nitrogen fixation by the symbiotic gut microbiota. In the present proposal, we aim to assess the nitrogen-fixing ability of the gut microbiota in the polystyrene-degrading mealworms by the acetylene reduction assay and stable isotopic tracer, and characterize the diversity of diazotrophic microbial community and the in situ expression of nitrogenase by the molecular biology techniques. We expect to uncover the significant contributions of gut microbiota to the nitrogen-fixation, and provide theoretical support for the adequately utilization of the diverse metabolic abilities of the plastic-degrading insects.

塑料废物造成的"白色污染"是全球关注的环境问题。我们长期的研究证实啮食塑料昆虫能高效降解石油基塑料并维持正常生存。但塑料仅能提供碳源却不能提供氮源，塑料降解过程中存在缺乏氮源的问题。氮是合成蛋白质、核苷酸等生长代谢产物不可或缺的元素。因此，急需探究啮食塑料昆虫在降解塑料过程中获取氮源的途径和机制。已有研究结果显示，肠道微生物作用下的生物固氮功能是寡氮营养型昆虫获取氮源的重要途径，啮食塑料昆虫肠道内存有潜在的固氮细菌，可能具有进行生物固氮以获取氮素维持生长代谢的能力。本项目拟以啮食降解聚苯乙烯塑料的黄粉虫为研究对象，拟用乙炔还原法结合同位素示踪技术，确定黄粉虫具有肠道微生物作用下的生物固氮功能；拟用分子生物学技术结合传统分离纯培养方法，解析肠道区系中固氮细菌的多样性，原位验证固氮酶的表达。本项目将揭示塑料降解昆虫肠道微生物的生物固氮机制，为充分利用塑料降解昆虫的代谢能力提供重要的理论支持。

塑料废物造成的“白色污染”已经成为全球关注的环境问题。生物降解作为一种富有潜力的塑料废物处理方式，已经成为科学研究的热点。我们前期发现黄粉虫能够啮食聚苯乙烯塑料，并能以塑料作为唯一食物维持正常生存。但是，塑料只能提供碳源，存在氮源缺乏的问题。已有研究证明，肠道微生物的生物固氮功能是寡氮食物型昆虫获取氮源的重要途径，因此，肠道微生物的生物固氮作用可能是啮食塑料昆虫获取氮源的重要途径。在本项目中，首先，我们采用乙炔还原和同位素法，获得了十分确凿的证据，证实了黄粉虫的固氮活性。进一步，通过抗生素抑制法，发现肠道微生物被抑制后，固氮活性明显下降，证实了肠道微生物对黄粉虫固氮活性的决定性贡献。从肠道微生物宏基因组中扩增出了nifH基因，并利用无氮培养基在有氧条件下从黄粉虫肠道中分离纯化到101株细菌，其中鉴定出一株细菌新种Mixta tenebrionis BIT-26(T)，并发现Klebsiella属肠道菌具有较高的固氮活性，并从该肠道固氮菌基因组中也扩增出了nifH基因，确认了黄粉虫肠道存在固氮微生物；此外，从肠道微生物中提取RNA，利用RT-PCR扩增出nifH基因，证明了肠道微生物固氮酶的表达。本项目首次全面揭示啮食降解聚苯乙烯的黄粉虫肠道菌群生物固氮能力和微生物学机制，为阐明啮食塑料昆虫降解塑料过程中的供氮机制提供了实验证据，使昆虫降解塑料的研究迈上新的高度，为全面利用塑料降解昆虫资源，构建碳氮协同代谢的塑料生物降解技术提供重要的理论支持。