Isolation And Characterization Of Nitrobenzene-Degrading Strain And Cloning Of Degrading Genes

Nitrobenzene, a kind of monocylic nitroaromatics compound, is widely used in industrial production as the important chemical material in a past long time. Therefore nitrobenzene was residued in the wastewater of pharmaceutical, aniline, pesticides, dyestuff industry, etc. Nitrobenzene is known to be very stable in its chemical properties and resistant to microbial degradation due to the electron-withdrawing properties of nitro group. Nitrobenzene is easy to volatile in the air and cause a serious environmental pollution for its toxity. Physical and chemical methods have already been used to deal with nitrobenzene pollution, but bioremediation of nitrobenzene pollution is regarded as the most promising approach. Because it is lowcost without secondary pollution and ecological restorative, many researchers paid close attention to bioremediation of environment polluted by nitrobenzene.In these days, a great deal of inorganic or organic pollutant coexists in environment, and that causes combined pollutions. Nitrobenzene often coexists with heavy metals and other organic pollutants such as phenol or aniline in the wastewater. Therefore the aim of this study is to isolate and chose the efficient nitrobenzene-degrading strains which could deal with combined pollutions, and elucidate the degrading pathway, clone the degradation-related genes, at last investigate the feasibility in the use of bioremediation of wastewater by immobilization technique.1. Isolation and identification of nitrobenzene-degrading strainsThe nitrobenzene-degrading strains (D4, B4, C1, F, S7and a3) were isolated from wastewater polluted by nitrobenzene in a chemical factory by the enrichment culture technique. The nitrobenzene-degrading strains were capable to utilize nitrobenzene as sole source of carbon, nitrogen and energy. Otherwise a strain S6-2-9was isolated which could only transform nitrobenzene but could not utilize nitrobenzene as sole carbon source. On the basis of physiological and biochemical characters and16SrRNA gene sequence similarity analysis, strain B4,C1,F, S7and a3were preliminarily identified as Pseudomonas, the strain D4and S6-2-9were preliminarily identified as Cupriavidus.2. Study on the degradation characteristics, degrading pathway of D4and clone of its cnb genesThe nitrobenzene-degrading strain D4was capable to utilize nitrobenzene and4-CNB as sole source of carbon, nitrogen and energy and it also has the resistance to kinds of heavy metals. The strain D4could be a potential degrading strain resource applied in degrading nitrobenzene or4-CNB in presence of heavy metals. The optimal temperature and initial pH of medium for the degradation of nitrobenzene was30℃and pH7.0, respectively. Strain D4could degrade nitrobenzene efficiently in presence of Zn2+,Mn2+,Co2+,Cd2+,Pb2+and AsO43-,but not to Ni2+,Hg2+,Ag+,Cu2+and CrO42-.The metabolites of nitrobenzene and4-CNB degraded by strain D4respectively were investigated, the result implied the two kinds of substrates were all aerobicly degraded by strain D4in patial redcuctive pathway. The cnb genes were cloned by means of PCR with the conserved primers, and it is highly homologic with the reported sequence of related genes from strain CNB-1and ZWL73.The cnb genes of strain D4were located on the two plasmids, that was different from the reported Comamonas sp. strain CNB-1whose cnb genes were located on only one large plasmid pCNB1.The gene of arsenic reductase was also cloned from the strain D4and S6-2-9.And the sequence of arsenic reductase gene from the strain D4and S6-2-9have100%homology with reported strain CNB-1.3. Study on the characteristics of degradation, degrading pathway of nitrobenzene-degrading strain a3and clone of its degrading genesThe nitrobenzene-degrading strains a3was capable to utilize nitrobenzene, aniline and phenol as sole source of carbon and energy. When nitrobenzene was degraded aerobicly as substrate by strain a3, the formation of aniline was found. Which were different from the metabolites in patial reductive pathway. When aniline was degraded as substrate by strain a3, one of the metabolites produced by strain a3were identified as2-aminophenol. And appearance of2-picolinic acid further proved that2-aminophenol underwent meta ring-cleavage to form2-aminomuconic-6-semialdehyde. Therefore it is inferred that the aniline degrading pathway of strain a3is novel and different from others. The hydroxylase gene was inserted by a suicide shuttle plasmid pJQ200L500to form mutant of strain a3whose hydroxylase was inactive. The mutant of strain a3could no more degrade nitrobenzene, aniline, phenol, etc. So the hydroxylase was identified as the related enzyme for degrading nitrobenzene, aniline, etc..Then the sequence of multiple components hydroxylase was amplified by SEFA-PCR, and the gene sequences of upstream and downstream the multiple component hydroxylase were the regulator gene of hydroxylase and ring-cleavage enzyme gene respectively, the whole gene cluster was named pll gene.To find the reason why the strain a3could hydroxylate aniline, the analysis of pll gene cluster was presented. The gene pllKMNOPL of the multiple components hydroxylase was conserved contrasted from other related genes. In general the substrates of the multiple components hydroxylase were phenol or its derivates. In this study aniline was hydroxylated by the hydroxylase of strain a3. That was different from other strains who have the same hydroxylase.Then the analysis of pllR (belong to Ntrc regulator family and N-terminal of regulator is an inducer binding domain) implied that the regulator gene was different from other related genes and it is ascribed to the different substrates as the specific inducer for hydroxylase.4. The study on the degradation of nitrobenzene by strain a3immobilized in PVA-SAIn recent years, the researches of wastewater treatment techniques were focused on the immobilization of cells.The immobilized cells have advantages in high density and reaction speed, and could protect the cells embedded away from impact of toxic wastewater.For the reason that nitrobenzene wastewater mixed with other toxic compounds usually, the immobilization technique is a good choice for improvment of the bioremediation of nitrobenzene mixed wastrwater.Active carbon and sodium-algin(SA) were taken as immobilization supports for immobilization of strain a3, but the two kinds of supports were so weak in mechanical strength that they were abandoned to be used as supports. So the mixture of PVA and SA was chosen to immobilize cells of strain a3for its good mechanical strength. The optimal scheme was determined by cross experiment for immobilization:SA3%, PVA9%, CaCl21%and embedding ratio4:25。 The optimal temperature and initial pH of medium for the degradation of nitrobenzene was30℃and pH7.0, respectively. The immobilization of strain a3could efficiently degrade nitrobenzene, aniline and phenol respectively. The immobilized cells of strain a3could degrade300mg/L nitrobenzene within8h and was not influenced by adding phenol or aniline, at the same time the aniline and phenol were degraded completely. The free cells of strain a3were greatly influenced in biodegradation by adding other substrates. The immobilized strain a3had higher degradation rate than free cells in degrading nitrobenzene or nitrobenzene mixed with aniline and phenol. In experiments of reusing immobilizing cells and simulated aeration, the results revealed that the immobilized cells maintain active and good mechanical strength in periods of operation. All the results revealed that, the immobilized strain a3had the potential in treating nitrobenzene and other aromatics co-contaminated wastewater.In the present study, nitrobenzene-degrading strains were isolated by the enrichment culture technique. Attentions were paid to two strains:One is Cupriavidus sp.D4, which could degrade nitrobenzene and4-chloronitrobenzene as sole carbon, nitrogen, and energy with resistance to heavy metals; The other is Pseudomonas sp.a3, which could degrade nitrobenzene, aniline and phenol. Novel nitrobenzene and aniline-degrading pathway were found in the strain a3.The multiple components hydroxylase gene cluster was amplified by SEFA-PCR. Analysis of the sequence implied there was a novel regulator gene at the upstream of hydroxylase. The strain a3was immobilized in PVA-SA mixed immobilization support, and the immobilized cells were more effective in degrading nitrobenzene or mixture with kinds of aromatics than the free cells.