Studies On Isolation And Identification Of Pentachloronitrob Enzenedegrading Bacteria, And Degradation Characteristics, Metabolic Pathway,SufB Gene Function Of Strain DH19

Pentachloronitrobenzene(PCNB), a typical organochlorine fungicide, has been applied widely in the production because of its broad-spectrum control effect and low price. However,PCNB has long residual duration and hardly degradation, and can produce a variety of toxic substances in environment, which brings risks to crop production quality and environmental safety, and restricts the international trade of crop production. Therefore, it is necessary to develop bioremediation process to degrade and eliminate PCNB contaminantion in the environment. In this study, the soil samples were collected from perennial PCNB-contaminanted ginseng field. The bacteria strains used PCNB as sole source of carbon were isolated from the soil samples. The strains were purified and identified. The degradation characteristics, optimum fermentation conditions, and degradation mechanism were studied. In addition, degradation metabolites of PCNB were identified, and a possible degradation pathway was deduced. The function of PCNB-degrading gene was studied by gene recombination method. Therefore, the results of this study could provide theoretical support and reasonable basis for the bioremediation of contaminated environments, lay foundation for further development and utilization of industrialized bacteria formulation, improve the product quality of kinds of crops including ginseng, and relieve the embarrassing situation of severe restriction of export trade,which has important significance to safeguard effectively product quality safety, expand exportation trade, and preserve the ecological environment. Research results were as follows:1. The soil samples from perennial PCNB-contaminanted ginseng field were isolated by enrichment acclimation and re-isolation method. The 403 bacteria strains were obtained in this study. 64 degrading strains used PCNB as sole source of carbon were screened primarily by transparent zones and isolated from the soil. The degradation rates of the strains to PCNB were tested by ultraviolet spectrophotometry method. Ten strains with high degradation rates were obtained. At 7 days after application, strain QTH3,CB8,DH19,JA30,BS34,HL37,SJH42,CB44,FS49 and CB56 could degrade PCNB with the degradation rates of 88.89%, 57.13%,90.86%, 83.30%, 86.68%, 83.46%, 84.36%, 86.97%, 78.06% and 70.99%, respectively.2. PCNB-degrading strains named QTH3,CB8,DH19,JA30,BS34,HL37,SJH42,CB44,FS49 and CB56 were identified based on morphological, physiological and biochemical tests,molecular identification and BIOLOG Automated Microbiology System as Pseudomonas putida,Brevibacterium sanguines, Arthrobacter nicotianae, Rhodococcus erythropolis, Arthrobacternicotianae, Brevibacterium antarcticum, Rhodococcus ruber, Pseudomonas stutzeri,Rhodococcus erythropolis and Gordonia amicalis, respectively.3. The degradation dynamics of DH19 to PCNB were determined by using gas chromatography coupled with triple quadrupole mass spectrometry(GC-MS/MS). The results showed that the degradation effects of strain DH19 to penatchioorniortbenzene(PCNB), pentachloroaniline(PCA) and pentachlorothioanisole(PCTA) were obvious. At 14 days, degradation rate of DH19 to PCNB was 98.64%, the PCA residue was not detected and PCTA residue was 0.30 mg/kg.4. To clarify the degradation characteristics of strain DH19 in different conditions, the optimum conditions(pH 6.85, 30 ℃ and inoculum biomass amount of 1.45 g/L) were obtained by response surface methodology with three-level, three-variable design based on single factor experiment. Under optimum conditions, the growth amount and degradation curves were obtained. At 7 days after application, the degradation rate of strain DH19 to PCNB was above90%, and the bacteria amount was increasing gradually with the time prolongation. The results showed that strain DH19 could use PCNB as the sole source of carbon and energy.5. The degradations of strain DH19 to hexachlorocyclohexane, dichlorodiphenyl trichloroethane(DDT), cypermethrin and cyhalothrin were determined by using GC-MS/MS. Within 5 days and7 days, the degradation rates of strain DH19 to hexachlorocyclohexane, dichlorodiphenyl trichloroethane, cypermethrin and cyhalothrin were more than 80% and 95%. The results showed that strain DH19 could degrade efficiently hexachlorocyclohexane, DDT, cypermethrin and cyhalothrin.6. The PCNB-degrading enzymatic reaction system was established in this study. The results showed that the crude intracellular enzyme played main role in degradation effect with degradation rate 73.47%. However, the degradation rate of the crude extracellular protein enzyme was 4.7%. The results showed that the optimum conditions of fermentation medium were as follows: carbon source of glucose 8 g/L, nitrogen source of beef extract 1.6 g/L, the time for culturing seed liquid of 16 hours, the time for culturing fermentation liquid of 18 hours,initial pH value of 8, inoculum biomass amount of 2%. Under the optimum conditions, strain DH19 had best degradation effect with the highest relative activity of intracellular degradation enzyme.7. The fragment ions produced in metabolic process of strain DH19 to PCNB were determined by using GC-MS/MS, and contrasted with NIST database. The possible degradation process was deduced based on five possible metabolic products including 2, 3, 4, 5, 6-pentachloroaniline, 3,5-dichloroaniline, 3, 4-dichloroaniline, aniline and catechol. In addition, the metabolic pathway from PCNB to H2 O and CO2 was deduced.8. SufB, SufC and SufD gene belonging to iron-sulfur cluster were obtained through PCR amplification of partial gene in strain DH19 genome. SufB gene was cloned and analyzed, and its promoter and terminator were predicted in this study. In addition, the gene mutant strain △Suf B-DH19(KmRand GmR) and complementary strain ▲SufB-DH19(SpRand SmR) were constructed. The degradation effects of wild strain DH19, the SufB gene mutant strain△Suf B-DH19(KmR and GmR), and complementary strain ▲Suf B-DH19(SpR and SmR) were contrasted. At 7 days after culture, through degradation of wild strain to PCNB, the residue of PCNB was 9.88 mg/kg, the degradation rate was 89.71%. Through degradation of complementary strain ▲SufB-DH19 to PCNB, the residue of PCNB was 25.77 mg/kg, the degradation rate was 73.16%. Through degradation of the gene mutant strain △SufB-DH19 to PCNB, the residue of PCNB was 34.75 mg/kg, the degradation rate was 63.80%. Therefore,Suf B gene belonging to iron-sulfur cluster could positively regulate the degradation of strain DH19 to PCNB.9. According to the colonization trends of strain DH19 marked with rifampicin, strain DH19 could colonize stably into the soil containing PCNB. According to field degradation trial, the degradation effects of strain DH19 to PCNB, PCA and PCTA were obvious. Therefore, the strain has great potential and applicative prospect for degrading PCNB, PCA and PCTA in production.