基于复苏VBNC菌的强化微生物修复PCBs污染土壤及其作用机制

The environmental friendly microbial bioremediation technology has attracted much attention due to its great potential of development and application in the treatment of polychlorinated biphenyls (PCBs) contaminated soils. However, the indigenous microorganisms exhibiting a lower metabolic activity as a result of entry into the viable but non-culturable (VBNC) state is the bottleneck limiting microbial bioremediation. How to resuscitate VBNC bacteria for stimulating the degradation efficiency of pollutants from indigenous microorganisms is a key issue needed to be solved. Now it has been widely reported in the medical field that the resuscitation-promoting factor (Rpf) from Micrococcus luteus has a significant role in promoting the resuscitation of VBNC bacteria, but so far, very few studies have been performed on the resuscitation of VBNC bacteria using Rpf in contaminated soils for enhanced microbial remediation. The objective of this work is to investigate the effect of Rpf on the PCBs degradation, and obtain microorganisms with a high efficiency of degrading PCBs from VBNC bacteria. Furthermore, the key factors of inducing the PCB-degrading bacteria into the VBNC state will be uncovered, and the mechanisms involved in the VBNC formation and resuscitation will be preliminary revealed. On the basis of these results, simulation in resuscitation of potential PCB-degrading bacteria in the VBNC state in PCB-contaminated soil will be further performed, and hopefully the best method of resuscitating indigenous PCB-degrading bacteria will be established. We anticipate this work can provide a new way for enhancing microbial bioremediation of PCB-contaminated soil and screening high-efficiency PCB-degrading bacteria.

环境友好的微生物修复技术在多氯联苯(PCBs)污染土壤治理中因极具发展应用潜力而备受关注，然而土著微生物因处于活的但非可培养(VBNC)状态而具有较低代谢活性是制约微生物修复效果的瓶颈。如何复苏VBNC菌以激发土著微生物的降解效能是微生物修复亟需解决的关键问题。藤黄球菌复苏促进因子(Rpf)对VBNC菌具有显著的复苏促进作用已在医学领域中广泛报道，但迄今为止，利用Rpf复苏VBNC菌以强化微生物修复污染土壤的研究较为罕见。本项目拟考察Rpf对PCBs降解菌群的复苏促进效能，获得高效降解PCBs的VBNC菌种资源，明确诱导PCBs降解菌进入VBNC状态的关键因素，初步阐明PCBs降解菌VBNC状态形成与复苏的机制，在此基础上模拟VBNC状态的PCBs降解菌在土壤中的复苏，建立最优复苏PCBs污染土壤中VBNC菌的方法，从而为强化微生物修复PCBs污染土壤及筛选高效PCBs降解菌提供新的途径。

在多氯联苯(PCBs)污染土壤微生物修复中，绝大多数土著或外源接种微生物因进入低代谢活性的活的非可培养（VBNC）状态，难以发挥其PCBs降解效能。如何激发功能菌群活性，挖掘潜在的VBNC菌种资源是污染环境生态修复领域的重要科学问题。本项目利用藤黄球菌复苏促进因子（Rpf）可复苏VBNC状态菌使其恢复功能及可培养性能等作用，开展复苏VBNC菌以强化微生物修复PCBs污染土壤的研究。通过响应面分析法优化Rpf制备条件，Rpf产量达0.42mg/mL，且具有溶菌酶结构域。以PCBs污染土壤中微生物菌群为研究对象，添加Rpf的富集培养体，其Aroclor1242的降解效率最高约提高35%，且Rpf的添加对具有最低PCBs浓度土壤中的微生物菌群的复苏促进效果最显著。高通量测序分析表明，对Rpf响应的最主要菌群为Alpha-变形菌纲，其次是Beta-变形菌纲。从样品中分离获得23株PCBs降解菌，其中11株为Rpf添加组所特有菌株。从具有bphA基因的菌株中，筛选出高效降解PCBs的复苏菌株卡斯特兰尼氏菌SPC4，当PCB77浓度为50mg/L时，84h的降解率达74.5%，且对PCB77的降解符合Edward模型，其降解途径为先通过bph编码的联苯途径，后通过3,4-二氯苯酸盐矿化途径转化其代谢物。同时，对复苏菌株链球菌SPC0进行PCBs降解性能及固定化研究：SPC0具有降解异型生物质的潜力，对PCB18的降解效果最佳，PCB52次之，PCB77较差。当包埋菌量、小球添加量和PCBs初始浓度分别为15mg/L、15%和15mg/L时，固定化小球对PCBs的降解效果最佳。另外，对复苏菌株芽孢杆菌LS1进行PCBs降解性能及VBNC状态诱导研究，结果表明LS1降解性能与SPC0相似。将LS1接种至PCBs污染土壤，70d后其可培养菌数下降约一个数量级，表明复苏的VBNC菌株重新接种至污染土壤，仅有少部分细胞进入VBNC状态。基于此，将复苏VBNC菌的方法应用于模拟强化修复实际PCBs污染土壤，结果发现，Rpf可激发土壤体系中土著微生物的代谢活性，强化其PCBs降解性能，且利用Rpf可获得处于VBNC状态的菌种资源，为PCBs污染土壤微生物修复技术提供优良的菌种资源储备。本研究为强化微生物修复PCBs污染土壤及挖掘潜在的功能菌种资源提供了重要科学依据。