Resuscitation Culture And Degradation Characteristics Of High Efficiency PCBs Degrading Bacteria Based On Rpf From Micrococcus Luteus

Vialb but non-culturanle?VBNC?state has attracted more and more attention in the field of microbial resources because of its wide distribution and easy resuscitation.Polychlorinated biphenyls?PCBs?are a widely distributed persistent organic pollutant with complex composition and stable properties.Biodegradable PCBs technology is a research hotspot.Obtaining efficient PCBs degrading bacteria is the key of research.However,the ability of screening PCBs-degrading bacteria is limited by culture-dependent.The study of functional bacteria in VBNC will open up a new prospect.In to study,the optimal preparation conditions of resuscitation promotion factor?Rpf?for resuscitation of VBNC bacteria were explored by response surface methodology?RSA?of central composite design?CCD?.Using Aroclor 1242 as carbon source,the effects of Rpf on the activity and structure of bacteria in contaminated soil were studied by enrichment and domestication culture combined with 16S rDNA high-throughput sequence technology.The most probable number?MPN?was used to isolate VBNC bacteria.In addition,3,3',4,4'-tetrachlorobiphenyl as substrate,real-time quantitative PCR?qPCR?was used to study the expression of bphA gene.The growth and degradation kinetics were studied with PCB 77,and its intermediate metabolites were analyzed by gas chromatography-mass spectrometry?GC-MS?.The following results were obtained:?1?Design-Expert software was used to obtain the optimal conditions for the preparation of Rpf by central combination design response surface analysis.The final concentration of Isopropyl beta-D-thiogalactopyranoside?IPTG?was 33.9 mg/L,the concentration of induced bacteria(OD₆₀₀)was 0.4,the induction temperature was20.3?,and the induction time was 7.0 h.Repetitive experiments verify the reliability of the model.?2?At the same concentration of Aroclor 1242,the addition of Rpf could effectively improve the activity of bacteria in culture medium.When the concentration of Aroclor 1242 increased to 100 mg/L,the effect of Rpf was the most significant,and then the effect of Rpf decreased.When the concentration of Aroclor1242 increased to 200 mg/L,the recovery effect of Rpf was limited,and there was no significant difference between the treatment group and the control group.The results of 16S rDNA high throughput sequence showed that Rpf could effectively improve the diversity of functional bacteria.Bacteria of genus Sphingomonas,Pseudomonas and Burkholderia commonly used as PCBs transformants,and their relative abundance increase from the addition of Rpf.Proteobacteria is the most sensitive phylum to Rpf addition from different taxonomic levels.Alphaproteobacteria and Betaproteobacteria,as well as most of the Psedomonadaceae belonging to Gammaproteobacteria,have PCBs degradation effects.?3?Twenty-three functional strains were successfully isolated and revived,in which Castellaniella sp.SPC4 showed excellent degradation effect of PCB 77?at 50mg/L PCB 77 concentration,the degradation rate was over 40%within two days?and the presence of bphA gene was detected.The expression of bphA gene in the process of Castellaniella sp.SPC4 metabolism of PCB 77 showed a trend of increasing at first and then decreasing,which confirmed the involvement in bphA gene in this process.?4?At different PCB 77 concentrations,the growth of Castellaniella sp.SPC4bacteria and the degradation rate of PCB 77 indicated that there was only substrate inhibition and no product inhibition in the system.The metabolic process of PCB 77by Castellaniella sp.SPC4 conforms to the first-order reaction rate equation,and a kinetic model of PCB 77 microbial degradation was constructed.The model matched the Edward model.The fitting goodness of R² was 0.8055,the maximum specific degradation rate q_max was 0.9315 1/h,the substrate affinity constant K_S was 11.33mg/L,and the substrate inhibition constant K_I was 11.41 mg/L.The presence of chlorobenzoic acid and chlorinated 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid?HOPDA?in the degradation of PCB 77 by Castellaniella sp.SPC4 was detected by gas chromatography-mass spectrometry?GC-MS?,which provided evidence for the pathway of PCB 77 degradation.In this study,the optimum conditions for preparation of Rpf from Micrococcus luteus were explored to provide material basis of its potential application as a bioremediation assistant.The effective effects of Rpf on the activity of PCBs degrading bacteria and the structure of functional bacteria in PCBs contaminated soil samples were investigated,which provided a theoretical basis for its use as a bioremediation assistant.Successful resuscitation and isolation of high-efficient functional bacteria in VBNC state will explore the range of various high-efficient functional bacteria from conventional culturable bacteria to non-culturable areas.The degradation kinetics of PCB 77 in degradation process is discussed,which will provide theoretical support for the promotion and application of bioremediation of persistent organic pollutants,especially polychlorinated biphenyls.