Study On The Formation Of Microbial VBNC State Under Pollutants Stess And Resuscitation Of Functional Bacteria By Rpf

Viable but non-culturable(VBNC)status,as a survival mechanism for microorganisms to resist adverse environments,is widespread in polluted environments.Numerous bacteria can not be isolated and cultured because of entering VBNC state,and it is difficult to play pollutant degradation function.Thereby,it is of great significance to study the recovery and cultivation of VBNC state and to explore its potential environmental functions.However,current researches on VBNC status are mainly focused on the medical field,and there is hardly any relevant literature on functional VBNC status bacteria in the natural environment.In view of this,for the first time,the present study explored the changes of culturability,morphology,physiology,biochemistry and function of phenol degrading fungi induced by high concentration of pollutants(phenol),and determined its phenol degradation and bioavailability after removing phenol stress.Secondly,the purified resuscitation promoting factor(Rpf)was obtained by metal affinity chromatography.According to the amino acid sequence of Rpf protein,the spatial protein structure was constructed to determine the lysozyme like domain,and the recovery promoting effect of Rpf protein on VBNC state bacteria was explored by enzyme spectrum analysis and peptidoglycan hydrolase activity analysis.Finally,the high ammonia nitrogen wastewater was used as the enrichment source,and the unique strains of the Rpf protein-added group were isolated,cultured and identified.High-efficiency aerobic denitrifying bacteria were screened and their nitrogen removal performance was determined to explore the potential of the regenerated bacteria to degrade pollutants.The results are as follows:(1)A highly efficient phenol-degrading strain LN1 was obtained by separation and screening,which could completely degrade 1000 mg/L phenol in 48 h.The whole genome sequencing showed that LN1 belonged to Candida and had the catA gene related to phenol degradation.The results showed that strain Candida sp.LN1 entered VBNC state after being induced by 6000 mg/L phenol for 14 hours,and showed the changes of cell membrane surface shrinkage,cell length shortening(from 3.44 ?m to 1.62 ?m),DNA damage,protein structure change and key oxidation.After the high concentration of phenol stress was removed,the LN1 strain was inoculated into the LB culture medium,and its culture performance was restored after 12 h.The recovery of cultured cells has almost the same cell growth and phenol degradation performance as normal cells,but has a longer lag period than normal cells.(2)The recombinant Rpf protein expressed in Escherichia coli BL21(DE3),which showed the apparent molecular weight of 25-27 kDa in sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE).Secondly,based on the amino acid sequence of Rpf,the spatial structure of protein constructed in the website of phyre 2 had a credibility of 99.8%.This predicted structure model showed 48% identity with the "d1xsfa1".Meanwhile,analysis of zymogram and peptidoglycanase activity showed that Rpf protein exhibited maximum hydrolase activity at p H 5.0 and temperature of about 50 °C.(3)The high ammonia nitrogen wastewater was used as the enrichment source,the most probable numbe(MPN)method was used to count live bacteria.By comparing the Rpf-added group and the inactivated group,it was found that the addition of Rpf could increase the number of living cells by 58.1%.Four strains of VBNC were isolated by MPN and dilution coating.ZYR51 belongs to Gordonia,CHZYN52,CHZYR61 and CHZYR63 belong to Pseudomonas.Compared with other resuscitation strains,ZYR51 has the strongest nitrogen removal performance.ZYR51 has an ammonia nitrogen removal rate of 86.7% and a total nitrogen removal rate of 55% in 120 h.The results of this thesis not only provide a theoretical basis for exploring the formation and recovery of microbial VBNC status in polluted environments,but also open up a new method for expanding microbial resources.At the same time,it lays a certain foundation for the application of Rpf in the field of bioremediation.