| Dioxins mainly included polychlorinated dibenzo-p-dioxins(PCDDs)and polychlorinated dibenzofurans(PCDFs).It has great harm to human health,due to its persistence,bioaccumulation,toxicity and global dispersion.In spite of that global release of PCDDs/PCDFs has decreased greatly during the last several decades,however,unregulated waste production or improper disposal of historical contaminates still poses a risk to human health.Many sites were regarded as dioxins sink,such as soil,sediment,wastewater treatment system and reservoir et al.Therefore,it is important for human health and environmental protection to remove dioxins from these sites.Compared with soil and sediment,it was lack of research on dioxins treatment in activated sludge system.Not wastewater reuse is closely linked with human life,but the use of excess sludge in agriculture has a risk to bring dioxins in food chain.Therefore,dioxins removal in activated sludge is an important task.Removal of dioxins by microorganisms is a potential method of remediation,especially aerobic bacteria can mineralize the carbon backbones of PCDDs/PCDFs by using a series of enzymes.In our lab,a dioxin-degrading bacterium has been isolated and named Rhodococcus sp.strain p52,which harbors two conjugative dioxin-catabolic plasmids.Strain p52 could not only use non-chlorinated or low chlorinated dioxins,but utilize many other aromatic compounds,such as biphenyl,naphthalene,anthracene and phenanthrene and so on.What is more,the genes for complete dioxins degradation were located on the two conjugative plasmids,which could confer indigenous microorganism the ability to degrade dioxins by conjugative transfer.To excavate the value of strain p52 in persistent organic pollutants degradation,completed genome sequencing analysis was conducted,and our analysis was focused on the chemotactic genes,membrane transport genes and degradation genes,especially those dioxin degrading genes and metabolic pathway.In the genome of strain p52,there were 54 tRNA,12 rRNA,2 sRNA and 453 tandem repeat sequences,which include 399 minisatellite sequences and 17 microsatellite sequences.KEGG annotation of strain p52 genome showed that there were many metabolic pathways of biphenyl,naphthalene,anthracene,fluorene,toluene and xylene et al.Regardless of that strain p52 has no flagellum or pili by transmission electron microscope observation,there were some chemotactic genes in the chromosome and plasmids of strain p52.Drop assays showed that strain p52 showed chemotaxis towards dibenzofuran and its intermediate product such as salicylic acid,and the chemotaxis of strain p52 to dibenzofuran was inducible.The capillary chemotaxis assays showed that strain p52 had chemotaxis towards many substrates in liquidoid.In the genome of strain p52,there were many membrane transport proteins,especially aromatic associated membrane transporters,which suggested the transport ability of strain p52 to many aromatic compounds.On the genome of plasmids pDF01 and pDF02,there were genes for a complete pathway of dibenzofuran degradation,the end-product of which could enter into tricarboxylic acid cycle(TAC).On this basis,we added strain p52 into lab-scale sequence batch reactors(SBRs)to enhance the ability to remove dioxins.The results of gas chromatography showed that bioaugmentation by adding strain p52 could significantly enhance the dioxin degradation.On the 100 days operation,the dioxin removal rate of bioaugmented reactor was 32.6%to 100%higher than non-bioaugmented reactor.By spreading the activated sludge samples on DF plates and PCR confirmation,four kinds transconjugants belonging to Klebsiella(Enterobacteriales),Arthrobacter(Actinomycetales),Corynebacterium(Actinomycetales)and Enterobacter(Enterobacteriales)were isolated.Klebsiella and Enterobacter were affiliated to gammaproteobacterial,and Arthrobacter and Coiynebacterizm were affiliated to actinobacteria.Phylogenetic analysis showed the close relationship between Arthrobacter,Corynebacterium,and strain p52.All these results suggested that strain p52 has a broad transconjugants host range.The results of qPCR indicated that the number of total bacteria was comparable between the bioaugmented and the non-bioaugmented reactors,and fluctuated slightly during the operation.The copy numbers of strain p52 changed obviously,it increased to two-times that of the initial inoculation within the first 10 days,then gradually declined and reduced three order of magnitudes after 80 days,accounted for only 0.05±0.01%of the total bacteria.The copy numbers of transconjugants had a similar trend with strain p52 within 50 days.However,it began to increase,and accounted for 8.2±0.3%of the total bacteria and was 170 times of strain p52 numbers at the 80th day.All these results suggested that the occurrence of conjugative transfer may not rely on selective pressure,but the increase of transconjugants could be enhanced by selective pressure.The Illumina MiSeq sequencing was conducted to investigate the bacterial community structure of sludge after 75 days.The community biodiversity and evenness of bioaugmented reactor were higher than non-bioaugmented reactor.Moreover,heatmap of community composition on genus level indicated that the abundance of transconjugant belonged Arthrobacter,Coiynebacterium and Enterobacter in bioaugmented reactor were higher than non-bioaugmented reactor and seed sludge.A Venn diagram showed that most genera were shared by each sludge sample,and transconjugant belonged Pseudomonas and Enterobacter only exist in bioaugmented reactor.All these results suggested that genetic bioaugmentation could increase biodiversity and evenness of bacterial community,which could enhance the ability to resist environmental disturbance.To further enhance pollutant removal performance of genetic bioaugmentation by strain p52,we concurrently added strain p52 and granular activated carbon(GAC)into reactors.The results of gas chromatography showed that both chemical amendment granular activated carbon(GAC)and biological amendment strain p52 showed an improvement on dioxins removal,especially the combination of biological and chemical amendment,which could enhance the removal rate 92.12%more for dibenzofuran and 84.90%more for dibenzo-p-dioxin than control.The results of qPCR revealed that there was a similar pattern of total bacteria and strain p52 to the previous study.However,the conjugative frequency in biofilm on the surface of GAC was significantly higher than suspended sludge.The proportion of transconjugants harbored plasmid pDF01 to total bacteria in biofilm samples was 14.27 times(R2)and 25.66(R3)times of suspended sludge.Similarly,the proportion of transconjugants harbored plasmid pDF02 to total bacteria in biofilm samples was 32.00 times(R2)and 45.73(R3)times of suspended sludge.By spreading the activated sludge samples on DF plates and PCR confirming,10 kinds transconjugants were isolated,belonging to Pseudomonas,Corynebacterium,Rhodococcus,Glutamicibacter,Citrobacter,Klebsiella,Enterobacter,Arthrobacter,Agrobacterium,and Acinetobacter,respectively.Among these transconjugants,Agrobacterium and Acinetobacter were only detected in biofilm samples,and Pseudomonas,Corynebacterium,Citrobacter and Enterobacter were only detected in suspended sludge.Phylogenetic analysis showed the close relationship between transconjugants and gammaproteobacterial and actinobacteria of core microbiome.However,transconjugants were not from the most abundant alphaproteobacterial,which indicated that abundance of indigenous microbe may not the necessary factor.Bray-Curtis matrix-based Principal Coordinate Analysis(PCoA)and analysis of similarity(ANOSIM)results revealed that strain p52 was the main factor to influence the bacterial community,and GAC almost had no effect on the microbial community structure compared with the corresponding reactor without GAC amendment.Alpha diversity showed that bioaugmentation by addition of strain p52 could enhance the biodiversity of bacterial community over the time.The biodiversity of bacterial community of bioaugmented reactors was significantly higher than non-bioaugmented reactors from the 40th day,and it began to be stable after the 60th day Pairwise correlation analysis revealed that genetic bioaugmentation by catabolic plasmids facilitated the recovery of microbial community disturbed by environmental element(i.e.,toxic compounds).Beta diversity showed that the abundance of actinobacteria,gammaproteobacterial and flavobacteria in non-bioaugmented reactors was higher than bioaugmented reactors.The abundance of sphingobacteriia,deltaproteobacteria and betaproteobacteria in bioaugmented reactors was higher than non-bioaugmented reactors.On the genus level,we analyzed the 'sensitive responders','sensitive responders' to strain p52 were much higher than that to GAC.and the'sensitive responders' to strain p52 were almost negative responders.Molecular ecological networks(MENs)revealed that the MENs complexity of all reactors gradually decreased over time,and the position of transconjugants affiliated taxonomic groups in MENs also decreased.Topological properties of each species showed that module hubs and connectors in the network of bioaugmented reactors were much more than that in the networks of non-bioaugmented reactors.Furthermore,there was one module hub in the network of bioaugmented reactors affiliating to genus Coiynebacterium,which indicated that transconjugants might play an important role in the microbial community of bioaugmented reactors.Soil is an important sink of dioxins,and strain p52 was isolated from oil polluted soil.Therefore,the application of bioaugmentation by strain p52 in the field of soil bioremediation is potential.We simulated soil in-situ bioremediation by adding strain p52 to remove dioxins,and investigated the influence of abiotic factors on dioxin degradation and conjugative transfer.When temperature was the sole factor,we found that higher temperature(25?-35?)facilitated dioxin degradation.However,lower temperature contributed to high frequency of plasmids conjugative transfer.When moisture was the sole factor,the results showed that dioxin removal rate had positive correlation with water content under 30%,and dioxin removal rate became lower with 45%water content.Moreover,15%and 30%water content were optimal condition for strain p52 and transconjugants survival,respectively.The concentration of pollutants played an important role on strain p52 survival,but there was no direct correlation between pollutant concentration and strain p52 number.Number of strain p52 was higher when the concentration was 100 mg/kg than 1000 mg/kg and 0 mg/kg.However,the number of transconjugants had positive correlation with pollutant concentration,which confirmed the results that high select pressure contributed to the proliferation of transconjugants.There was a good performance of genetic bioaugmentation to remove dioxins in all kinds of soils we collected.However,the soil type may have an influence on strain p52 survival and transconjugants numbers.By spreading the soil samples on DF plates and PCR confirming,transconjugants affiliated 5 genera were isolated,belonging to Shewanella,Rhodobacteraceae,Pseudomonas,Sediminibacter and Polaribacter,respectively.Pseudomonas was the same genus with transconjugants isolated from the activated sludge systems of our previous studies,which indicated that plasmids host may be overlapped among different environments.The foundation of new kinds of transconjugants confirmed that strain p52 had a broad host range.Eighteen non-transconjugants colonies on DF plates were identified,and they belonged to Aqarivorans,ncultureed Bacteroidetes/Alqibacter and Pseudarthrobacter,which were different from transconjugants.In conclusion,strain p52 has shown great application potential based on not only genome analysis but also activated sludge treatment and soil bioremediation tests Therefore,genetic bioaugmentation with catabolic plasmids harbored by strain p52 to environment dioxins cleanup is a promising remediation technique. |
PDF Full Text Request