Study On The Structure And Function Of A Denitrifying Quinoline Degrading Microbial Community

Quinoline is a N-heterocyclic compound often found in chemical,pharmaceutical,pesticides and dyeing industries as raw materials and solvents,which have the intense carcinogenesis and tetratogenesis.Aerobic degradation of quinoline is well documented.However heavily polluted environments are ofen oxygen defcient,thus restricting the aerobic degradation of quinoline unless oxygen is injected.But the knowledge of the anaerobic transformation of quinoline is limited.Our understanding of the bacterial community and its key players is scarce.Our laboratory has maintained an efficient quinoline-degrading microbial community for several years,but the quinoline-degrading microorganism has never been isolated.First of all,we found that COD/N ratio affected the quinoline removal efficiency.In the condition(COD/N=5.1),the highest removal efficiency was obtained.If the COD/N ratio>5.1,the quinoline removal efficiency would decrease,otherwise,if the COD/N ratio<5.1,the nitrite accumulation would happened.By the robotized incubation system(ROBOT),we obtained a detailed picture of quinoline degradation.In the optimum condition(COD/N=5.1),the quinoline degrdation experienced two different stages(denitrification and methanogenesis).In the denitrification stage,nitrate reduction occurred concomitantly the nitrite accumulation,When the added electron acceptor was completely used up,all of the NO3--N were reduced to N2-N;then the process of methane production began.The results obtained from time-resolved community analysis by the 16 S ribosomal DNA and reverse transcribed ribosomal RNA pyrosequencing demonstrated that a group of bacteria involved in quinoline and subsequent degradation reactions in the batch culture.It was clear that Thauera species were enriched into the dominant populations with relative abundances of 12%-30% in DNA-based libraries and 10%-50%in RNA-based libraries.Phylotypes related to the genus Rhodococus and Desulfobacterium showed remarkably active.All together,76 different phylotypes were found to be responsible for quinoline degradation,and clustered into 13 different co-abundance groups(CAGs).Each CAG contained two to nine bacterial phylotypes and showed similar dynamic pattern in quinoline degrading-process.Based on the dynamic study on the microbial community,gas products and chemical parameters,we suspected Thauera as a core bacterium,responsed to the initial degrade of quinoline;then the members belonged to Rhodococcus,Desulfobacterium,Rhodocyclaceae and Syntrophobacteraceae assisted Thauera to finish the subsequent steps untill Methanosaeta involved in the methane production.In order to gain a deeper understanding about how the COD/N ratio affected the quinoline degradation process,a higher COD/N ratio 12.2and a lower COD/N ratio 2.4 were studied.Through a robotized incubation and sampling system,we found N2 O and nitrite cumulated in the lower COD/N ratio condition.While in the higher COD/N ratio condition(12.2),the quinoline degrdation also experienced two different stages,denitrification and methanogenesis.Time-resolved community analysis showed the different COD/N ratio did not change the structure of quinoline-degrading microbial community.Comparing to the 76 key OTUs which identified in optimum condition,55 OTUs were same,the number was 58 in lower COD/N treatment.Some low abundance OTUs were identified linking to quinoline degradation.We also studied how the nitrite influenced the quinoline degradation process.When nitrite as electronic acceptor with COD/N ratio 5.1,the quinoline degrdation also experienced two stages,denitrification and methanogenesis degradation.Because the accept electrons capacity of the nitrite is less than nitrate,the quinoline removing efficiency was 44.7%,but all the nitrite reduced to nitrogen.However,nitrite as electron acceptors has much impacted on the microbial community structure.Comparing to the 76 key OTUs which identified in optimum condition,49 OTUs were same,other members belonged to Chloroflexi and Deltaproteobacteria were identified linking to quinoline degradation.The denitrifiers community structure was also investigated by four PCR-clone libraries(nos Z?nar G?nir S and nir K).Phylogenetic analysis from nitrous oxide reductase gene(nos Z)and the dissimilatory nitrite reductase gene(nar G)clone libraries showed the dominant denitrifiers belonged to Betaproteobacteria,distributed into the genus Rhizobium?Thiobacillus?Acidovorax and Alicycliphilus,which were identified as key players in quinoline-degrading process.The clones which showed high similarity to Thauera species,existed in nar G,nir S and nos Z gene clone library,nevertheless,their abundance was unconspicuous.The present study supplies more detailed knowledge on the linkage between quinoline biodegradation and denitrification in this enrichment.In conclusion,by using a robotized incubation and sampling system,a quinoline degrading consortium with fine division and cooperation was dissected.The quinoline degradation depended on the cooperation of different function organisms.We also found different COD/N ratio significantly affected function of consortium,such as the N2 O accumulation.Nitrite as a harmful intermediate metabolite,affected the the quinoline removing efficiency and the rate.Our research lay the foundation for intense research in the mechanism of quinoline biodegradation.