典型溴化阻燃剂和重金属联合作用对微生物反硝化的影响及机理研究

It is well-known that the global nitrogen cycle, greenhouse gas (such as N2O) emissions and water eutrophication are closely related to the metabolism of denitrifying bacteria, which are widely survived in nature. Recently, with the inefficient electronic waste recycling process, a large number of brominated flame retardants and heavy metals, which were two important components of electronic equipment and electronic waste, flowed into the environment, and brought significant influence on organisms and the ecosystem. However, to date, there were few studies concerned with the effects of brominated flame retardants and the combined effects of brominated flame retardants and heavy metals on denitrifying bacteria. Therefore, this project firstly investigated the combined effects of brominated flame retardants and heavy metals on the growth and surface properties of model denitrifying bacteria, and the detailed mechanisms was investigated from cellular morphology, extracellular polymeric substances, lipid peroxidation, etc. Then, the combined effects on the carbon and nitrogen metabolism of denitrifying bacteria were investigated, and the mechanisms were explored from transmembrane transport, gene transcription regulation, metabolic pathway analysis, etc. Finally, the combined effects of brominated flame retardants and heavy metals on real denitrifying system were testified using sediments as the inoculation. Simutanously, the variation of denitrifying enzymes activity, the copies of key denitrifying function genes, and the diversity of microbial population on the sediments were also investigated. The results of the project can provide the theoretical basis for comprehensive evaluation about the responses of microbial denitrification on the combined effects of brominated flame retardants and heavy metals and its mechanisms.

微生物反硝化广泛存在于自然界，与氮素循环、温室气体（N2O）排放、水体富营养化等密切相关。近年来，溴化阻燃剂和重金属随电子废物拆解回收大量进入环境，对生物和生态环境产生了重要影响。但有关溴化阻燃剂以及溴化阻燃剂和重金属联合作用对反硝化微生物的影响尚未见报道。故本项目拟首先选用模式反硝化菌为研究对象，考察典型溴化阻燃剂和重金属联合作用对反硝化菌的生长和表面性质的影响，并从细胞膜结构、胞外聚合物、脂质过氧化等角度进行机理分析；然后重点考察两者联合作用对反硝化菌碳源和硝酸盐代谢的影响，并从物质跨膜转运、基因转录调控、代谢通路分析、关键酶活性等方面探索其机理；在以上研究基础上，最后探讨两者联合作用对实际反硝化体系（以沉积物为代表）的短期和长期影响，并从反硝化酶活性、微生物种群结构、关键基因表达等方面进行机理分析。研究结果可为评价典型溴化阻燃剂和重金属联合作用对微生物反硝化的影响及机理提供科学依据。

近年来，由电子垃圾引起的溴系阻燃剂和重金属大量流入环境，由此引发的环境问题得到广泛关注。反硝化过程是一个与氮循环、富营养化等密切相关的重要生物地球化学过程。然而，目前有关微生物，尤其对反硝化微生物对溴化阻燃剂以及溴化阻燃剂和重金属联合作用的响应，均未见报道。本项目分别以模式反硝化微生物和沉积物为研究对象，分别考察了典型溴系阻燃剂以及与重金属联合作用对反硝化菌及反硝化过程的影响，得出主要研究结论如下：.（1）高浓度（≥ 0.5 mg/L）TBBPA显著抑制反硝化菌生长及反硝化性能。研究发现TBBPA通过抑制反硝化菌反硝化能力以及生物量影响反硝化过程。深入分析表明TBBPA通过调控胞内碳源代谢过程关键酶的基因表达（如6-磷酸葡萄糖异构酶，丙酮酸脱氢酶、异柠檬酸裂解酶等），抑制糖酵解和磷酸戊糖通路、激活乙醛酸循环途径，抑制葡萄糖代谢，减少胞内NADH产生，从而抑制反硝化能力。作为电子供体的NADH不足，引起各反硝化酶电子竞争、反硝化中间产物（NO2––N和N2O）积累，从而引起胞内活性氮（RNS）大量积累，抑制反硝化菌生长和增加凋亡，导致反硝化菌生物量受到抑制。.（2）进一步研究了TBBPA和Cu联合作用对反硝化过程的影响，发现0.05 mg/L 铜显著增强了TBBPA对反硝化菌的毒性，反硝化功能受到更强抑制。TBBPA和铜可降低反硝化菌胞内毒素抗毒素系统稳定性，造成细菌程序性死亡；与TBBPA单独作用相比，联合作用可引起细菌胞外聚合物显著增加，造成碳源——葡萄糖转运能力下降，使细菌摄取葡萄糖的能力减弱，最终影响细菌的生长和反硝化功能。.（3）利用沉积物的短期和长期实验进一步验证了前续实验结论，同样发现典型溴系阻燃剂、溴系阻燃剂和重金属联合作用均会显著抑制反硝化过程，并会随着长期培养而毒性增强。.本次研究可为评价典型溴化阻燃剂和重金属联合作用对微生物反硝化的影响及机理提供科学依据。