Excavation And Phylogenetic Studies Of Un-cultivated Microbial Resources In Environment

Pharmaceutical wastewater contained a large number of organic pollutants pollution,and governancing was also very difficult.It can be only obtained which the cultivated bacteria from environment less than 0.01-10%by used traditional pure cultivating method,most of them were in un-culturable or viable but non-culturable(VBNC),state.This thesis focused on resuscitation promoting factor(Rpf)to resuscitation and isolation of VBNC state bacteria.First extracted the rpf(resuscitation promoting factor)gene of Micrococcus luteus IAM 14879(= NCIMB 13267),672 bp in length,and construct the recombinant strains by used pET-15b,then transformation and expressionwere carried out by using Escherichia coli BL21(DE3).Added 1 mmol/L IPTG induced culture,in Ni-NTA affinity column,carried out the concentration gradient elution,under 100mM concentration of Rpf protein was eluted,SDS-P AGE protein induced expression verification,finally obtained the Rpf recombinant protein.To add pure Rpf recombinant protein in medium,by using MPN(most probable number)culture system method to evaluate the effect of recovery growth.Results showed that VBNC state bacteria can be recoveried by the Rpf recombinant protein,its capacity get up about to 10 times.The molecular weight of the Rpf recombinant protein was 26.3 KDa,the concentration was 2 mg/mL.It showed the Rpf protein in connection with the VBNC state bacteria have the effect of promoting the recovery growth.A total of 28 VBNC state bacteria were obtained by Rpf recovery,and one of them was multidimensional taxonomy as a new species of bacteria.A Gram-staining negative,aerobic,motile,rod-shaped bacterium(0.3-0.5x1.0-1.2 ?m),with one polar-flagellum,designated as ZYSR67-ZT,was isolated from pharmaceutical wastewater in Jinhua city,Zhejiang Province,Eastern China.Supernatant Rpf(1%,v/v)was added to the MPN(most probable number)culture system to isolate the VBNC state bacterium,strain ZYSR67-ZT.The strain grew at 4-43?(optimum,35 ?),pH5.0-9.0(optimum,6.0)and 0-5.0%(w/v)NaCl(optimum,2.0%).Phylogenetic analysis based on 16S rRNA gene sequence revealed that ZYSR67--ZT belonged to the genus Pseudomonas,and pairwise sequence similarity to Pseudomonas guguanensis CC-G9AT(98.4%),Pseudomonas alcaligenes NBRC 14159T(98.1%),Pseudomonas indoloxydans IPL-1T(98.2%),Pseudomonas chengduensis MBRT(98.0%),Pseudomonas oleovorans subsp.lubricantis RS1T(98.0%),Pseudomonas alcaliphila AL15-21T(97.9%),Pseudomonas mendocina NBRC 141 62T(97.33%),Pseudomonas tuomuerensis JCM 14085T(97.2%)and Pseudomonas flexibilis ATCC 29606T(97.1%).Based on the results of three housekeeping genes(rpoB,gyrB and rpoD),it was found that it had higher resolution at the species level.Besides,strain ZYSR67-ZT possessed C12:0,C16:0,C18:1 ?7c and summed features 3 as predominant fatty acids.The major polar lipids of strain ZYSR67-ZT were phosphatidylethanolamine(PE),phosphatidylglycerol(PG),diphosphatidylglycerol(DPG),phosphatidylcholine(PC),one unidentified aminophospholipid(APL),two unknown glycolipids(GL1,GL2),one unidentified aminolipid(AL)and one unidentified phospholipid(PL).Additionally,the predominant quinone system was ubiquinone-9,and presence of trace amounts of ubiquinone-8.The genomic DNA G+C content was 59.7 mol%.DNA-DNA hybridization values of strain ZYSR67-ZT with Pseudomonas guguanensis CC-G9AT and Pseudomonas alcaligenes NBRC 14159T were 46.4%and 33.6%,respectively.On the basis of the morphological,phylogenetic,physiological and chemotaxonomic characteristics,strain ZYSR67-ZT was proposed to represent a novel species of the genus Pseudomonas,named Pseudomonas pharmacopolae sp.nov.The type strain is ZYSR67-ZT(? CGMCC 1.15498T= JCM 31306T).