The Exploration Of Microbial Community Structure And Functional Genes In A Lab-Scale Denitrifying Quinoline-Degrading Bioreactor

It is necessary to study the quinoline degradation because Quinoline,a toxic,carcinogenic and recalcitrant nitrogenous heterocyclic compound,is one of the main pollutants in coking wastewater.Recently,using microbial process to remove quinoline has drawn more and more attentions,and denitrification process is often used for degrading aromatic and nitrogenous heterocyclic compounds.So far,researchers from various countries have already isolated several quinoline degrading of bacterial strains such as Pseudomonas,Rhodococcus,Burkholderia and Comamonas,to name but a few.However,these bacteria are aerobic quinoline degrader.When it comes to anoxic or anaerobic degradation of quinoline,especially the denitrification degradation of quinoline,much remains unknown.The Thauera was identified as the functionally important genus in a lab-scale denitrifying degrading bioreactor in our previous work.But several attempts of isolation on the most abundant Thauera bacteria had failed.The only three Thauera isolates from the reactor,strains Q4,Q20-C,and 3-35,nevertheless showed low abundance in the bioreactor community and didn't show any quinoline degrading ability in the medium containing quinoline as the sole carbon source.To understand the potential of other bacteria rather than Thauera in the degrading community,in the first part of this paper,we used streptomycin to disturb the microbial community in order to change the structure after batch culturing.According to the results,streptomycin did not significantly reduce the quinoline removal rate instead of increasing 9.7%.In the meantime,nitrate removing ability was stable.The communities treated by antibiotic showed distinct structure compared to the untreated one in the bray curtis distance PCoA analysis based on the 16 S rRNA gene high-throughput sequencing data.12 OTUs including Pseudomonas?OTU1?,Achromobacter?OTU2?,Brevundimonas?OTU12?,Bosea?OTU24?were significantly enriched while the abundance of 40 OTUs was significantly reduced in the samples treated by streptomycin.Interestingly,the quinoline degradation was not influenced although Thauera almost couldn't be detected.In conclusion,we demonstrate that the function redundancy of a denitrifying quinoline degrading community.Some rare bacteria could become predominant bacteria such as Pseudomonas and might act as a key player for quinoline degradation in the antibiotic reshaped communities.The genes and enzymes involved in anaerobic quinoline catabolism are not known.In the second part of this paper,trying to elucidate this critical step,metatranscriptomic analysis was conducted to compare the genes transcribed during the metabolism of quinoline and 2-hydroxyquinoline via the bacterial community from an anoxic quinoline degrading bioreactor.mRNA was extracted from the quinoline and 2-hydroxyquinoline stimulated samples and sequenced after removing rRNA and built library by Illumina Hiseq.Our main focus was differential gene expression among the treatments.The preliminary results showed that,in quinoline group,a host of genes expression increased significantly,like denitrification genes,degradation genes related with aromatic compounds?such as acyl-CoA dehydrogenase and oxidoreductase?.It is important to note that the gene encode peroxiredoxin?EC: 1.11.1.15?was highly expressed in a variety of denitrifying bacteria,and the expression of this gene was more than 100 times than that in starvation group.Highly expressed genes in quinoline group were identified as the abundant and diverse denitrifying bacteria such as Thauera,Aeromonas,Ralstonia,Burkholderia,Pseudomonas,Sphingobium and Achromobacter.In terms of genus which had the most number of high expression genes,approximately 60 genes from Thauera in quinoline group expressed twice compared with other groups.Tmz1t₁458,Tmz1t₁262,etc.originated from Thauera were defined as function-unknown genes in KEGG and these genes expressed 10-50 times higher when quinoline had been added as carbon source compared with that in starvation group,and these genes may be related to the degradation process of quinoline.In the studies presented herein,we measured quinoline removal rate and assessed bacterial diversity and community structure to compare the communities shifted by using streptomycin.We found that,in the antibiotic reshaped communities,the former rare bacteria such as Pseudomonas become predominant bacteria and replaced the role of Thauera as the key player in the quinoline degradation.On the other hand,RNA-seq was used to explore genes involved in anaerobic quinoline catabolism.And highly expressed genes in quinoline group were related with self-protection,denitrification and degradation.It is noticed that 55.78% sequences in quinoline group could not be comparable to the KEGG,suggesting that these sequences might be new genes and might be associated with quinoline denitrifying degradation.