Isolation,Identification And Characterization Of Functional Bacteria From A Denitrifying Quinoline-degrading Bioreactor

Quinoline and its derivatives are N-heterocyclic compounds with characteristics of strong toxicity,carcinogenicity,solubility and recalcitrance.They are widely distributed in coking wastewater environment as the typical pollutant,which has aroused especial attention.A lab-scale denitrifying bioreactor for quinoline degradation has been maintained efficiently for several years.Previous studies have found that Thauera was significantly enriched among the consortium with the proceeding of quinoline degradation.Therefore,isolation and identification of key organisms for quinoline degradation as well as the analysis of their physiological function,can contribute to reveal the mechanism of the bioreactor operation at strain level.The quinoline-degrading bacterial consortium was analysed through Illumina Mi Seq sequencing of the 16S r RNA gene V3-V4 region.The results showed that the most predominant OTU(OTU1)whose abundance was 22.5%,belonged to Thauera.While the second abundant OTU(OTU2),which accounted for 12.5%,was affiliated to Rhodococcus.It might suggest that the organisms of OTU1 and OTU2 could be actively involved in quinoline degradation.In previous work,all attempts to isolate organisms with the highest abundance in the reactor,which were supposed as the main degraders,have proved unsuccessful.Therefore,the most dominant OTU specific PCR primer was designed to perform the sequence-guided isolation for the target organisms.Four target strains whose 16S r RNA gene sequences were identical with OTU1 from the reactor and belonging to Thauera aminoaromatica,were finally obtained.Further tests on utilization of carbon source demonstrated that none of the strains could strive on quinoline.However,all of them could efficiently degrade 2-hydroxyquinoline,which was the primary intermediate of quinoline catabolism,under nitrate-reducing condition.Meanwhile,no intermediate was detected by HPLC.In addition,Rhodococcus pyridinivorans YF3,isolated from the same reactor,was shown to convert quinoline into 2-hydroxyquinoline under specific oxygen-containing condition.Interestingly,the final products and removal rate of quinoline transformation by Rhodococcus were varied with different content of oxygen.Firstly,YF3 could degrade quinoine without the accumlation of2-hydroxyquinoline under aerobic;whereas,under anaerobic condition,quinoline was persistent;notably,0.23 m M quinoline was transformed into equivalent 2-hydroxyquinoline without further transformation when 0.5%volume of oxygen supplied,which demonstrated that Rhodococcus sp.YF3 was responsible for the initial attack of quinoline under specific condition.To investigate the cooperative degradation of quinoline,a synthetic consortium of Thauera aminoaromatica R2 and Rhodococcus pyridinivorans YF3 was constructed.Results for this co-cultivating trial showed that 0.8%O₂ could be the optimum condition for complete degradation of quinoline.Specifically among this consortium,Thauera sp.R2 could cross-feed on the 2-hydroxyquinoline released by Rhodococcus sp.YF3.In addition,we found that the concentration of2-hydroxyquinoline increased gradually in parallel to quinoline consumption by Rhodococcus sp.YF3 under strictly anaerobic condition,if YF3 was pre-incubated with quinoline,2-hydroxyquinoline or phenol under 0.5%oxygen condition for 24 h and then transferred to fresh minimum mineral quinoline medium.While in the mixture of Thauera and Rhodococcus,2-hydroxyquinoline accumulated firstly and then declined completely under denitrifying condition.It was speculated that quinoline and oxygen could induce the expression of hydroxylase in Rhodococcus sp.YF3,which could still exert catalytic activity later even without molecular oxygen involvement.In order to further understand the high performance of quinoline degradation in the influent pipeline of the bioreactor,the potential quinoline-degrading bacteria in the biofilm of the pipeline were also isolated and characterized.The medium with quinoline as sole carbon and energy source was adopted to enrich,screen and purify degrading strains.Phylogenetic analysis was performed through 16S r RNA gene sequencing.The characteristics of quinoline degradation by different strains were investigated under different p H and temperature conditions.Based on the phylogenetic analysis,four isolates Q1,Q3,Q7 and Q8 were identified as the genera of Sphingobium,Massilia.Rhodococcus and Dyadobacter,respectively.The results showed that 50 mg/L quinoline could be completely removed within 48 h by all of the isolates.And they exhibited different characteristics on degradation of quinoline,such as the accumulation of 2-hydroxyquinoline was only detected during the incubation of strain Q1,Q3 and Q8.Notably,the genera of Sphingobium,Massilia and Dyadobacter have not been reported yet on quinoline degradation.In conclusion,we constructed a model on the relay reaction of denitrifying quinoline digestion by two cross-feeding bacterial strains.Thus the mechanism of microbial interaction inside the bioreactor was revealed at the population level.In addition,the four quinoline-degrading bacteria isolated from the influent pipeline of bioreactor provided novel strain resources for decontaminating industrial wastewater containing quinoline,which may have potential applications in bioaugmentation.