Resuscitation And Cultivation Of Highly Efficient Degrading Bacteria For Soil PCBs And Enhance Pollution Remediation With Rpf

Polychlorinated biphenyls(PCBs)are complex in composition with a wide range of distribution,which are a type of persistent organic pollutants.Microbial degradation of PCBs is considered as a promising restoration technology,and obtaining the efficient PCBs degrading bacteria is the key to remediate contaminated environments.However,studies showed that more than 99%of the bacteria are viable but non-culturable(VBNC)in the environment,so it is laborious to obtain the VBNC bacteria through traditional culture-depengent methods,which greatly limits the source of PCBs degrading bacteria.Thus,resuscitation of VBNC bacteria by resuscitation-promoting factor(Rpf)from M.luteus opens new eyes on isolation of functional strains.In this study,the high-efficiency PCBs degrading bacteria LS1 was obtained by adding Rpf,and the PCBs degradation performance of this bacteria was studied.The strain LS1 coupled with Rpf was added to the actual PCBs contaminated soil to investigate the changes in PCBs residues and the relative abundance of bphA gene copy number.The major research was to strengthen the soil remediation effect by this combination.The results and conclusions are as follows:(1)At the same biphenyl(BP)concentration,adding Rpf can effectively promote the recovery of the flora in the culture system.Six strains were isolated through resuscitation culture,among which strain LS1 has the best PCBs degradation performance.The metabolic process of strain LS1 on PCBs conforms to the second-order reaction rate equation,and its degradation rate constant is 6.62469×10^-4.Chlorinated2-hydroxy-6-oxo-6-phenylhexyl-2,4-dienoicacid(2-hydroxy-6-oxo-6-phenylhexa-2,2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid,HOPDA),chlorobenzoic acid,muconic acid,it can be inferred that the degradation of PCBs by LS1 conforms to the bphcoding pathway.(2)Strain LS1 is white with irregular edges and shrinkage on the surface of the bacteria.When growing on Luria-Bertani(LB)medium,the subsequent period is short,reaching the maximum growth rate at 6 h.Whole genome analysis showed that the strain LS1 belongs to Bacillus subtilis(Bacillus),and 27 genes are involved in the degradation of exogenous biotin.After the bacterial strain Bacillus sp.LS1 was inoculated into PCBs contaminated soil,the bacterial count showed a trend of first decreasing and then increasing.The bacterial count reached the lowest value at 36 h,which was 4.8×10⁴ CFU/g.After 112 hours of cultivation,it basically recovered to the original bacteria number.After induction,the surface of the cell wall shrinks.(3)Simulate and repair the actual soil contaminated by PCBs.By comparing and studying the degradation of PCBs and the relative abundance of bphA gene in different additive groups(LS1,LS1 and Rpf,Rpf),the best way to enhance the restoration is evaluated.The results show that when LS1 coupled with Rpf is added to the simulated remediation of contaminated soil,the removal rate of PCBs is the highest.Compared with the degradation rate of other treatment groups,the addition of LS1 promoted the degradation of high-chlorinated PCBs,and the addition of Rpf promoted the removal of low-chlorinated PCBs.At 28 days,the copy numbers of bphA gene in LS1,LS1,Rpf and Rpf soils were 9.8×10 ⁴,8.33×10 ⁶ and 4.6×10 ⁷copies/m L,respectively,indicating that the addition of Rpf can effectively enrich PCBs degrading bacteria.The research results of this thesis provide a new idea for strengthening microbial remediation of PCBs contaminated soil,and provide a theoretical basis for re-evaluating the functional bacteria in PCBs contaminated soil.