Responses Of Wetland Plant Rhizobacteria To Long-term Stress Of Anti-inflammatory Drugs And Associated Co-meataolic Degradation Capability

Non-steroidal anti-inflammatory drugs（NSAIDs）are widely detected in surface water environments,posing potential ecological risks and belonging to the emerging pollutants with high priority of water environmental risks in China.How to effectively control the pollution of such pollutants in surface water environments is of great concern.Constructed wetland,which is commonly used in phytoremediation,is widely used to control surface water pollution in China.However,using constructed wetland for the treatment of NSAIDs in surface waters is low efficient and long time consuming.Therefore,it is necessary to conduct studies focusing on the related degradation mechanisms in order to improve the technology for higher removal efficiencies of NSAIDs.In this study,the typical wetland plant Phragmites australis was selected for investigation and five NSAIDs including indometacin（IMC）,ibuprofen（IBP）,naproxen（NPX）,ketoprofen（KTP）and diclofenac（DCF）were used as the target pollutants.This study was based on constructed wetland microcosms and used a number of techniques such as high performance liquid chromatography tandem mass spectrometry,high performance liquid chromatography,gas chromatography,real-time fluorescence quantitative PCR,and high-throughput pyrosequencing.The objectives were to explore the responses of root exudates and rhizosphere microorganisms to the stress of five NSAIDs’mixture at the seedling,vegetative,bolting,heading,and mature stages of Phragmites australis.Besides,the experiment of cultivating rhizosphere NSAIDs-degrading bacteria with artificial root exudates as substrate was studied to clarify the co-metabolic mechanism of NSAIDs by rhizosphere microorganisms mediated by root exudates.The main findings are as follows:（1）Responses of root exudates to NSAIDs stress during the life cycle of Phragmites australis:NSAIDs stress did not significantly affect the growth of Phragmites australis and only brought about adverse effects on chlorophyll at the early growth stages（seedling and vegetative stages）.With the development of Phragmites australis,at the middle and late growth stages（bolting,heading and mature stages）,the photosynthesis was no longer inhibited and the removal efficiencies of NSAIDs were gradually increased.In root exudates,release rates of 10organic acids,12 amino acids,1 monosaccharide,and 1 fatty acid were significantly higher than those free of stress.These root exudates may have the potential to attract beneficial microorganisms to colonize in the rhizosphere to degrade NSAIDs.（2）Responses of rhizosphere bacteria to NSAIDs stress during the life cycle of Phragmites australis:NSAIDs stress significantly increased the number of rhizosphere bacteria at the vegetative and bolting stages of Phragmites australis,but did not have a significant impact on the richness and diversity of rhizosphere bacteria.The rhizosphere bacterial communities at the phylum and genus levels under NSAIDs stress were both significantly different from those in stress-free conditions.There was little difference at the seedling stage,but the difference was gradually increased from the vegetative to heading stages and became smaller again at the mature stage,indicating that the rhizosphere bacteria gradually adapted to NSAIDs stress.At the middle and late growth stages of Phragmites australis,the relative and absolute abundances of the phylum Firmicutes and its genus Hydrogenispora were significantly higher than those without stress,which may have the potential to degrade NSAIDs.（3）Degradation of NSAIDs by rhizosphere microorganisms mediated by root exudates:The artificial root exudates prepared by 10 organic acids had synergistic effects with NSAIDs in promoting the growth of rhizosphere microorganisms,while the artificial root exudates prepared by 12 amino acids had no synergistic effect with NSAIDs.It indicates that the rhizosphere microorganisms had a more obvious substrate preference for organic acids.When the DOC（dissolved organic carbon）concentration added by organic acids or amino acids was10 mg L^-1,the removal efficiencies of NSAIDs were significantly improved.Organic acids were able to significantly improve the degradation efficiencies of NSAIDs in a short time,but amino acids needed a longer time.Under NSAIDs stress,the consumption rates of eight organic acids（oxalic acid,quinic acid,malic acid,citric acid,succinic acid,propionic acid,fumaric acid and anti-aconitine acid）and one amino acid（lysine）were significantly higher than those free of NSAIDs stress.These root exudates might be the effective ones for NSAIDs co-metabolism by rhizosphere microorganisms.The rhizosphere microorganisms cultivated with these effective components(DOC=10 mg·L^-1)could significantly improve the degradation efficiencies of NSAIDs.