Resuscitation Of Nonculturable Bacteria In PCB-contaminated Environmental Samples And The Mechanism Of Formation Of Nonculturable State

The state of viable but non-culturable(VBNC)is a survival strategy adopted by microorganisms when exposed to stressing environment.VBNC bacteria could be resuscitated by resuscitation-promoting factor(Rpf)which was secreted by Micococcus luteus as well as other homologous microorganisms.At present,research on formation and resuscitation of VBNC bacteria is mainly focused on pathogenic bacteria and epidemiology.Numerous bacteria entered the VBNC state due to stresses in xenobiotic-contaminated soils and sediments,it is therefore of great significance to resuscitate VBNC bacteria in xenobiotic-contaminated environments in order to explore high efficient pollutant-degrading bacteria and new microbial resources,and uncover the mechanisms of induction and resuscitation of VBNC bacteria.In this study,extracellular organic matter(EOM)from Micococcus luteus containing Rpf was used to investigate the non-culturable bacteria in PCB-contaminated environments.Firstly,response surface methodology(RSM)was adopted to optimize medium compositions and culture conditions to achieve maximum protein yield in EOM.Secondly,the effects of EOM on the cell growth,biphenyl(BP)-degading capability,bacterial community composition and structure were investigated in each enrichment culture of four different polychlorinated biphenyl(PCB)-contaminated soils and sediments.Thirdly,isolating and identifying colonies that were unique in the group with EOM addition were performed.Fourthly,the strain with the highest BP/PCB-degrading capability was chosen for polyphasic taxonomic investigation in order to propose a novel species.At last,the VBNC state of the new species bacterium was induced under ligotrophic and low temperature conditions.Meanwhile,for uncovering the mechanism of VBNC formation,the changes of morphology,enzymatic activity and gene expressions that might underline such state were investigated.The primary results of this study were summaried as follows:(1)The most important four variables from medium compositions and culture conditions,which influenced the protein production were determined using the Plackett-Burman design(PBD).The optimal levels for the four significant variables and the effects of their interactions on protein production were further investigated by the central composite design(CCD)and response surface methodology(RSM).The maximum protein production could be achieved when the variables of mineral solution,lithium L-lactate,initial pH and incubation time were set at 1.5 ml/L,8.75 g/L,7.5 and 48 h,respectively.The EOM contains both polysaccharides and proteins,and their contents were 405.74 and 25.13 mg/L,respectively.Gel permeation chromatography(GPC)analysis demonstrated that the molecular weights of proteins were 11489 Da and 9562 Da,and polysaccharides were mainly containing oligosaccharides with low molecular weight of 4422 Da.Molecular colonig and expression of the rpf gene were performed.The molecular weight of the recombinant Rpf protein was 45 KDa,which was 1.25 times greater than the theoretical value.(2)For different enrichment cultures of PCB-contaminated soil and sediment samples,addition of EOM significantly enhanced BP-degrading capability and bacterial community diversity,and changed the bacterial community structure and composition.Meanwhile,compared with the group with autoclaved EOM addition,the better performance of the group with EOM addition could be attributed to the proteins in EOM.The results of PCR-DGGE(polymerase chain reaction and denaturing gradient gel electrophoresis)and 16S rRNA gene sequencing indicated that EOM significantly stimulated the growth of the genera of Rhodococcus and Pseudomonas which belonged to the phyla Actinobacteria and Proteobacteria,respectively.Furthermore,EOM had the greatest effect on the sediment sample with the highest PCB concentration.The genus of Rhodococcus and other non-culturable bacteria belonged to the phylum Actinobacteria could be the high-efficient BP/PCB-degrading microorganisms which were resuscitated in high PCB-concentration sites.The unique strains in EOM addition groups belonging to nine genera,and most of them belonged to the phylum Actinobacteria.Among them,Rhodococcus strain TG9 was the most efficient BP/PCB-degrading strain,and it could tolerate BP at concentration up to 3000 mg/L,followed by Rhodococcus strain TG13.Both of them could degrade benzoate that was dead-end intermediate during the BP biodegradation.(3)The most efficient BP/PCB-degrading strain TG9T was selected forpolyphasic taxonomic analysis.The strain TG9T only shared more than 98%16S rRNA gene sequence similarity with four type strains including Rhodococcus gordoniae DSM 44689T,Rhodococcus pyridinivorans DSM 44555T,Rhodococcus rhodochrous DSM 43241T and Rhodococcus artemisiae DSM 45380T.There were differences between the strain TG9T and the four type strains in phenotypic characteristics,including the range of temperature,pH and NaCl concentration,the use of carbon and nitrogen sources,acid production,hydrolysis,antibiotic sensitivity,ect..The results of chemotaxonomic analysis were as follows:The diagnostic amino acid and sugars in the cell wall were meso-diaminiopimelic acid,arabinose and galactose(cell-wall chemotype?);The main composition of fatty acid was C16:0;The polar lipids contained phospholipids,glycolipids and unknown lipids;MK-8(H2)was the only menaquinone detected;Mycolic acids were presented.The genomic DNA G+C content of the strain TG9T was 62.8 mol%.The DNA-DNA homology of the strain TG9T with the four type strains were below 50%.Evidently,on the basis of the distinctive morphological,physiological,chemotaxonomic and molecular analyses,the type strain TG9T represented a novel species of the genus Rhodococcus,for which the name Rhodococcus biphenylivorans sp.nov.is proposed.(4)The VBNC state of R.biphenylivorans TG9T was induced by oligotrophic and low temperature conditions.The number of culturable cells counted by visible colonies decreased to undetectable levels(<0.1 CFU/mL)after 145 days,which verified the presence of VBNC cells.Resuscitation from the VBNC state could be achieved by plating VBNC cells on the Luria-Bertani(LB)agar plate at 30?for 3 days.The VBNC cells exhibited significant dwarfing and weaker fluorescence intensities in comparison with exponential-phase cells.The size and fluorescence intensity of resuscitated cells were between the VBNC and exponential-phase cells.The enzyme activities of the three different cells were assessed by using API ZYM kit.The activities of eight enzymes were all identified in the three different cells,and these enzymes in the VBNC cells exhibited lower activities in comparison with those in exponential-phase cells.Based on the transcriptome data,a total of 2097 differentially expressed genes(DEGs)were detected during the VBNC state formation,of which 1452 were up-regulated and 645 were down-regulated.Those genes with expression fold change of at least 20-fold up or down-regulation were 17 of and 42,repectively.Combining the enrichment results of GO(Gene Ontology)functions and KEGG(Kyoto Encyclopedia of Genes and Genomes)pathways,the up-regulated genes were mainly involved in ATP accumulation,protein modification,peptidoglycan biosynthesis and RNA polymerase,and the down-regulated genes were related to oxidation-reduction process and amino acid metabolism.In summary,the results of the study not only open up a new avenue for exploring novel high-efficiency xenobiotic-degrading bacteria,but also provide a scientific basis for enhanced bioremediation of xenobiotic-contaminated soils by resuscitation of VBNC bacteria.Meanwhile,the obtained results provide basic information on revealing the mechanisms of induction and resuscitation of the VBNC state of xenobiotic-degrading bacteria.