Isolation And Characterization Of A Novel High Effectively Quinoline Degrading Bacterial Strain Alcaligenes Sp. WUST-qu

Quinoline and its derivatives are widely-used industirial raw materials,meanwhile,are also a variety of carcinogenic,teratogenic,mutagenic and recalcitrant organic pollutants.Since the direct discharge of large amount of quinoline containing wastewaters produced by industries will definitely pose serious risks to soils and waters,as well as the organisms living in them;the effective treatment of quinoline containing industrial wastewaters arouses more and more concerns.At present,many technologies aiming at removing quinoilne from contaminated environment had been developed,mainly including physiochemical and biological methods.Compared with the former,the latter wins more attention for its lower costs and less secondary pollution in operation.In this study,a bacterial strain which high effectively degrades quinoline was isolated from the aeration tank of the waste water treatment system of Wugang Coke plant,and was named as WUST-qu.The morphological,physiological and biochemical properties of the strain were revealed,and its taxonomic identification was accomplished mainly by comparing the nucleotide homology of its 16S rRNA gene with other bacterial strains.In addition,the quinoline degrading properties at differert initial pH were explored.The substrate broad-spectrum and antibiotic resistant characteristics of strain WUST-qu were also examined.The kinetic models of the strain was preliminarily studied during the process of degrading quinoline.The main research results of this study are as follows:?1?The strain WUST-qu is Gram negative and sporulating with a length of1.15±0.08?m?n=20?,and a width of 0.26±0.01?m?n=20?.Partial 16S rDNA sequences?1435 bp?alignment indicated that WUST-qu belongs to the genus of Alcaligenes with a 99%identity to 16S rDNA sequences of Alcaligenes.sp M14 and Alcaligenes.sp CD243.?2?Since pH is one of the most influential factors to determine the bioremediation of the strain at source,the characteristics of quinoline degradation by strain WUST-qu at different initial pH were explored.The results domenstrate that when the strain is incubated at 35?and 150 rpm in a orbital shaker with a inocula of 5%?v/v??the optical density of the suspension at 600 nm is 1.0?,the strain could effectively degrade quinoline at initial pH of MSM between pH 5.0 and 9.0,but the optimal pH for the strain to degrade quinoline is neutral or slightly basic?7.0⁸.0?.When the initial pH of MSM is 5.0,the strain could completely degrade quinoline at 400 mg·L^-1 within 60 h upon inoculation;when the initial pH of MSM is 6.0,the strain could thoroughly degrade quinoline at 600 mg·L^-1 within 54 h after inoculation;when the initial pH of MSM is 7.0 and 8.0,respectively,the strain could degrade quinoline at 700 mg L^-1 to an undetectable level within 48 h after inoculation;when the initial pH of MSM is 9.0,the strain could degrade quinoline at 700 mg·L^-1 within 72 h completely upon inoculation.700 mg·L^-1 is the highest concentration of quinoline the strain could completely degrade among the tested concentrations in this study.?3?The heavy metal ions pose unfavorable roles on the growth and metabolism of microorganisms.The effects of heavy metal ions,Fe³⁺,Cr⁶⁺,Cd²⁺,Cu²⁺,Hg²⁺,Ag⁺,Ni²⁺,Cd²⁺,Mn²⁺,Pb²⁺,Ba²⁺,Zn²⁺,on the growth of the strain WUST-qu and quinoline degradation were tested under cultural conditions of 35?,150 rpm,a neutral initial pH 7.0 of MSM,and an initial concentration of quinoline 300 mg L^-1.The results show that Fe³⁺,Cr⁶⁺,Cd²⁺,Cu²⁺,Hg²⁺,Ag⁺,Ni²⁺have obvious inhibiting effects on growth of the strain,and thus seriously inhibit the degradation of quinoline.Cd²⁺shows the most toxic effect on the growth of the strain.Mn²⁺,Pb²⁺,Ba²⁺have no obvious effects on the growh and degradation properties of the strain,and Zn²⁺promotes both the grwoth and degradation of quinoline of the strain.?4?Due to the tremendous application of antibiotics,the residues of them are found in both soils and waters?including industrial wastewaters?.The resistance of the strain WUST-qu against five kinds of widely used antibiotics in medical treatment and animal husbandry on agar-MSM plates containing quinoline(300 mg·L^-1)and the effects of the antibiotics on the degradation of quinoline in MSM were examined.The results indicated that the strain WUST-qu is resistant to ampicillin,but is highly sensitive to streptomycin,tetramycin,kanamycin,and chloromycetin at their working concentrations.Quinoline at a concentration of 500 mg·L^-1 can be degraded to an undetectable lelvel by the strain WUST-qu within 20 h in MSM containing 50?g·mL^-1 penicillin when incubated at 35?,150 rpm,an initial pH 7.0 of MSM.?5?Growth substrate experiments demonstrated that among 21 of the most frequently seen pollutants in industrial wastewaters tested in this study,including methylbenzene,paraxylene,pyridine,indole,acetic acid,catechol,n-propyl alcohol,isopropyl alcohol,formic acid,nitrobenzene,diethyl ether,ethylbenzene,n-hexane,phenol,di-butyl phthalate,diphenylamine,o-cresol,ethyl acetate,acetone,aniline,and alcohol,taking an increase of biomass at least 1 order of magnitude at OD₆₀₀00 after incubation for 72 h was considered as true positive growth,the strain WUST-py can also utilize phenol,acetic acid,and n-propyl alcohol as the sole carbon source to grow.The properties of the strain WUST-qu to degrade phenol with phenol as the sole carbon and energy source were tested,and the results indicated that when it was incubated under 35?,150 rpm,an initial pH of MSM 8.0,an inocula of 5%?v/v?(OD ₆₀₀=1.0),the strain could completely degrade 700 mg·L^-1 phenol within 32 h upon inoculation.?6?Haldane kinetic equation was used to describe the growth kinetics of the strain WUST-qu.The specific growth rates were estimated by performing a linear least-squares regression of the semilogarithmic plot of biomass concentration against cultivation time during the exponential growth phase,and nonlinear least-squares regression analysis was used to estimate the Haldane kinetic parameters as follows:?_m=1.6576 h^-1,K_s=36.42 mg·L^-1,K_i=81.418 mg·L^-1,R²=0.968.When the concentration of quinoline was less than 500 mg·L^-1,the degradation of quinoline fits zero-order reaction kinetics.?7?GC-MS was adopted to monitor the metabolites during the degradation process,and the presence of 2-hydroxyquinoline was confirmed.The quinoline nirtrgen was mainly released through annonium-nitrogen,in addition to meet the requirement of bacterial growth.When the exogenous electron donor such as oxalic acid was added to the reaction system,NH⁴⁺-N could be further converted.