Study On Stable-isotope Probing Of The Polycyclic Aromatic Hydrocarbon-degrading Bacteria

With the rapid development of the social economy,the pollution of polycyclic aromatic hydrocarbons（PAHs）caused by human activities has become more and more serious.These compounds have the potential to pose serious health risks to all organisms.They are of great environmental concern due to their high toxicity,mutagenicity and carcinogenicity.Bioremediation is considered as a cost-effective and eco-friendly approach for elimination of PAHs from natural environments.The separation of functional microorganisms from complex environmental samples and their detailed genetic and metabolic analysis have been one of the biggest challenges in environmental microbiology.Considerable effort based on traditional cultivation-dependent approaches has focused on isolating and identifying cultivable PAHs degraders to explore the fate of PAHs.These approaches are used to determine the phenotypes and metabolic characteristics of PAH degraders and the functional genes associated with PAH metabolism such as PAH-ring hydroxylating dioxygenase.However,these approaches share a critical drawback in that the majority of microbes are uncultivable,and many autochthonous PAH-degrading microorganisms remain resistant to traditional cultivation approaches.In addition,direct cultivation greatly underestimates the microbial diversity in ecosystems and fails to explain the complex interactions between microorganisms and the natural environment.Therefore,there is an urgent need to develop new biological methods to solve this technical bottleneck.In the present study,DNA-Stable-isotope probing（DNA-SIP）was applied to wastewater samples to link the indigenous bacterial taxa with their PHE biodegradation phenotypes.The PHE-degrading bacteria in wastewater microcosm were successfully characterised using DNA-SIP and the high-throughput sequencing was applied for more complete understanding of the bacterial communities contributing to PHE degradation.Additionally,active PHE degraders were isolated from the indigenous wastewater microbial community with a cultivation-based method in parallel.In addition,wastewater microcosms were established to evaluate the bioremediation potential of autochthonous bioaugmentation（ABA）in PAH-contaminated wastewater with addition of the isolated active PHE degraders.DNA-SIP was used to investigate the changes in the diversity of indigenous PHE degraders by comparing treatments in the presence versus absence of the active PHE degraders,and to link the indigenous bacterial taxa with their PHE biodegradation phenotypes.We hope to provide useful information regarding the mechanisms of ABA in aiding bioremediation of PAH-contaminated wastewater and other potentially polluted environments.Moreover,we also used DNA-SIP to distinguish the role of root exudates in phenanthrene degradation and the associated changes in soil bacterial populations,active phenanthrene degraders,and phenanthrene-degrading genes compared to bulk soil and the rhizosphere.Given the low resolution of SIP in identifying functional microbes,we developed a novel cultivation-independent technique coupling Magnetic-nanoparticle mediated isolation（MMI）and SIP in this study,namely MMI-SIP,to identify and isolate activephenanthrene-degradersfromPAH-contaminatedwastewater.we hypothesized that MMI coupling with SIP can solve this challenge,as a powerful tool to identify and isolate the active functional bacteria from a complex microbial community,with higher resolution and accuracy comparing to either SIP or MMI.The following are the major conclusions:（1）DNA-SIP and the high-throughput sequencing were applied to identify the bacterial taxa responsible for PHE degradation in PAHs-contaminated wastewater.The results provide unequivocal evidence that Acinetobacter,Sphingobium,Kouleothrix and Sandaracinobacter are involved in biodegradation of PHE in wastewater,none of which has been previously reported as indigenous PHE-degrading microorganisms using SIP.Sandaracinobacter and Kouleothrix have not been previously linked to PHE degradation.Moreover,given that few bacteria linked to PHE metabolism have been isolated from real-world habitats,this study identified A.tandoii LJ-5 as a PHE degrader by DNA-SIP and revealed its functions by characterising the functional PHE metabolic genes and pathways.This is the first report of the role of A.tandoii in bioremediation of PAHs-contaminated water.This work provides a successful technology paradigm for in situ exploration and identification of functional microbes.（2）ABA by A.tandoii LJ-5 was first applied as a potential strategy to enhance the remediation of PAH-contaminated wastewater in this study.Besides resulting in a significant increase in the PHE biodegradation efficiency,ABA remarkably modified the functional PHE degrading community of the wastewater.The indigenous microorganisms responsible for PHE degradation were affiliated with Rhodoplanes,Mycobacterium,Xanthomonadaceae and Enterobacteriaceae in the original treatments,whereas five new taxa（Bacillus,Paenibacillus,Ammoniphilus,Sporosarcina and Hyphomicrobium）were acitvated in in situ PHE biodegradation in ABA microcosms.Of all the above PHE degraders,Rhodoplanes,Ammoniphilus,Sporosarcina and Hyphomicrobium were linked to indigenous PHE biodegradation for the first time.Nevertheless,LJ-5 did not participate in indigenous PHE degradation.The change in PAH-RHD_αgene diversity further confirmed our findings by different PAH-RHD_ɑgenes involved in PHE metabolism in the original and ABA treatments.Collectively,our findings raise a new mechanism of ABA,provide new insights into the diversity of PHE-degrading communities,and suggest ABA as a promising in situ strategy for PAH-contaminated wastewater.（3）Although both ryegrass rhizoshpere and root exedutes influenced the abundance and diversity of PAH-degrading bacteria in PAH-contaminated soils,no significant difference was observed between phenanthrene degradation efficiencies with and without root exudates.Furthermore,PAH-RHD_αgene abundance exhibited similar trends and was significantly lower in the root exudate microcosms than the rhizosphere,consistent with our results regarding the abundance of active phenanthrene degraders.These results suggest that root exudates offer a minor contribution to accelerated in situ phenanthrene remediation within the rhizosphere.Moreover,the differences in the phenanthrene degradation efficiencies were attributed to the distinct communities of active phenanthrene degraders.The indigenous microorganisms responsible for phenanthrene degradation belonged to eight bacterial classes,and only two key classes（Alphaproteobacteria and Nitrososphaeria）wereobservedinallthreetreatments.Instead,phenanthrene-degrading Sphingobacteriia and Actinobacteria were active in phenanthrene mineralisation in two treatments（AD+RG and RG+RE,respectively）,whereas the other four phenanthrene-degrading microbes were only observed in the AD or RE treatments.These findings suggest that active phenanthrene degraders within the same microbial community might be altered by environmental changes.Of the phenanthrene degraders,Blastomonas,Sphingoaurantiacus,Ramlibacter,Mucilaginibacter,Pedobacter,Nitrososphaera,and unclassified Pyrinomonadaceae,Chitinophagaceae,and Chthoniobacteraceae were found,for the first time,to be directly responsible for indigenous phenanthrene biodegradation.The correlation analysis further indicated that the PAH-RHD_αgenes and the abundance of phenanthrene degraders had strong correlations with the phenanthrene degradation efficiency.This study provides theoretical insights into the in situ phytoremediation of PAH-contaminated sites and helps to clarify the roles of root exudates in the phenanthrene biodegradation process within the rhizosphere,while simultaneously providing a more complete understanding of the diversity of phenanthrene-degrading communities in the rhizosphere during PAH degradation.（4）A novel MMI-SIP method was developed and employed to identify and isolate active phenanthrene-degraders from PAH-contaminated wastewater.Microbes affiliated to Pseudomonas and Sphingobium were responsible for phenanthrene mineralization from SIP results.MMI-SIP significantly increased the enrichment of Pseudomonas and Sphingobium in the heavy DNA fractions and found a new active phenanthrene degrader Pigmentiphaga.Further evidence was also found from the higher enrichment of PAH-RHD_αgenes in the heavy DNA fraction of ¹³C_MMI-SIP than that in the ¹³C_SIP microcosms.Our results suggested that MMI-SIP is a reliable cultivation-independent approach to identify functional-yet-uncultivable microbes from complex microbiota,with higher resolution and accuracy comparing to MMI or SIP alone.Additionally,MMI-SIP successfully isolated the active phenanthrene-degrading consortium and significantly increased the phenanthrene removal efficiency,attributing to the isolation and enrichment of living phenanthrene-degraders by MMI.Our study indicates that MMI-SIP approach is both cultivation-independent tool and practical bioaugmentation strategy,distinguishing the in situ active phenanthrene degrading microorganisms and accelerating phenanthrene degradation simultaneously.It might provide a more precise map and new mechanisms of active microbial degraders for other organic pollutants and help in understanding their ecological roles and influential factors during the microbial degradation processes.In summary,combine stable isotope probes and high-throughput sequencing technologies,it is possible to effectively conduct in-situ exploration of functional microbial populations involved in the phenanthrene degradation process in polluted environments,and to successfully confirm and isolate a representative active PHE degrader（Acinetobacter tandoii LJ-5）from the indigenous wastewater microbial community with a cultivation-based method in parallel.Although the ABA strategy with LJ-5 inoculum encouraged PHE biodegradation,the participation of A.tandoii LJ-5 in PHE degradation in situ was questioned due to their limited enrichment in the heavy DNA fraction according to the DNA-SIP results.Actually,the roles of the autochthonous strain LJ-5 in ABA strategy was to modify the diversity of indigenous PHE degraders instead of participating in in situ PAH degradation.In addition,we also used SIP to clarify the roles of root exudates in the phenanthrene biodegradation process within the rhizosphere.In order to improve the resolution of SIP in identification and isolation of the active functional degraders from a complex microbial community,we boldly developed a novel cultivation-independent technique coupling MMI and SIP in this study,namely MMI-SIP,to identify and isolate active phenanthrene-degraders from PAH-contaminated wastewater.It greatly improved the accuracy and resolution of SIP technology in exploration of functional bacteria,and is a typical innovation in related research fields.