Mechanism Of Duckweed-functional Bacteria Coupled Degradation Of P-tert-butylphenol In Water

p-tert-Butylphenol（PTBP）is an alkylphenol compounds commonly used in daily life,industrial production and pesticide production.It is widely spread in water environment,and difficult to be removed.Because of its persistency,PTBP is easily accumulate in the organism through the food chain.It is toxic to aquatic organisms and interferes the endocrine system fuction,which seriously threatens the organisms health and ecological safety.Therefore,how to remove PTBP effectively in water has aroused widespread attention.Plant-microbial remediation is a technology that remove environmental pollutants using the cooperation of plants and microorganisms.The organic components in plant root exudates can be utilized by rhizosphere microorganisms for cell growth and maintance of cell metabolic activity.In return,rhizosphere microorganisms degrade pollutants to reduce the environmental toxicity of pollutants,or enhance plant resistance to external environmental stress to promote plant growth.It has been proved that plant-microbe cooperation can remove pollutants efficiently,continuously,and without pollution.To be mentioned is the performance of plant-microbe remediation is be restricted by different factors.At first,the selection of suitable plants and microorganisms is the decisive factor of the remediation efficiency.The degrading activity of the degrading bacteria is of the most important.Secondly,the physical and chemical properties of the surrounding water environment and its indigenous microbial flora also directly or indirectly affect the growth of plants and the survival and metabolic activity of microorganisms.Duckweed,is an wide-spread aquatic plant with the characteristics of fast growth speed,easily handly and high starch and protein content.It is a model plant that frequently used in the remediation of eutrophication and heavy metal polluted water.In this study,duckweed was used as the test plant,and high-efficiency PTBP degrading bacterial strain isolated from activated sludge was used as the test strain.By constructing a duckweed-functional bacteria association,the ability of plant-microbe combination to remove PTBP,its mechanism and influencing factors were further investigated.The outcome of this study will provide useful information for optimization the functional flora and improving the efficiency of plant-microbe remediation of polluted water bodies.The main results of the research are as follows:（1）A PTBP degradating bacteria Tas13 was successfully isolated from activated sludge by spreading plate plate method.It was gram negative,and belonged to Sphingobium phenoxybenzoativorans.Strain Tas13 can degrade 75 mg/L PTBP within 16 h,and use PTBP as the only carbon and energy source,and.The metabolites were analyzed by high performance liquid chromatography（HPLC）and gas chromatography mass spectrometry（GC-MS）.The results showed that the degradation pathway of PTBP by Tas13 was through hydroxylation of benzene ring to p-tert-butylcatechol,and then through interposition decomposition of benzene ring to 3,3-dimethyl-2-butanone and 2,2-dimethyl-propionic acid.The genome length of strain Tas13 was 4828758 bp,consisting of one chromosome and three plasmids.In total,there has 4731 genes,and among which 4298,6329 and 4246 genes were annotated in COG,GO and KEGG databases,respectively.There are 28 genes involved in metabolic pathways related to the degradation of aromatic compounds,including the interposition of PTBP benzene ring to degrade catechol 2,3 dioxygenase.（2）Duckweed-strain Tas13 association can accelerate the removal of PTBP in water.The removal of PTBP is mainly due to the degradation of the strain Tas13,not the uptake of plants.In the sequencing batch degradation experiment,the association can repeatedly,efficiently and continuously degrade 5 mg/L PTBP within 10 days,and the degradation rate increases with the extension of the cultivating time,and the colonization ability of strain Tas13 remains stable during the whole experimental period.（3）The application of duckweed-Tas13 association showed great potential in remediation of polluted water in different environmental water.the duckweed-degrading bacteria association showed higher PTBP degradation activity than the control treatments without inoculation and only with duckweed inoculation,and colud effectively reduce the concentrations of COD,TN and NH₄⁺-N in water and increased the biomass of duckweed.The degradation activity of duckweed-Tas13 coupling system was inhibited by the inoculation growth-promoting bacteria and inhibitory bacteria,but the degradation rate of PTBP increased with the extension of cultivating time.In this study,by integrating whole genome sequencing and GC-MS techniques together,we discovered 28 genes related to the metabolic pathway of aromatic compounds in strain Tas13,and proposed the interposition degradation pathway of benzene ring in the degrading strain.At the same time,constructed duckweed-degrading bacteria association displayed great potential PTBP removal in different environmental water bodies.The further investigation of mechanism of duckweed-functional bacteria interaction will give guidance on theoretical foundation and pratical application of plant-microbe association in removal of organic pollutants in water bodies.