Study On The Mechanism Of Cometabolic Degradation Of Typical OMPs By ROS-producing Marine Bacteria

In recent years,organic micropollutants(OMPs)represented by pharmaceuticals and personal care products(PPCPs),brominated flame retardants and endocrine disruptors have been widely detected in sewage,surface water,groundwater and even marine environments.Though OMPs was at a low concentration level in the environment,it can easily threat to the ecological environment and human health owing to its teratogenic,carcinogenic,mutagenic,growth toxicity,reproductive toxicity and other multiple toxicities.As an important way to degrade OMPs in the marine environment,marine bacterial biodegradation has received widespread attention.Marine bacteria can produce extracellular reactive oxygen species(ROS)and degrade OMPs through ROS-mediated reaction.However,the current research on the degradation of OMPs by ROS-producing marine bacteria is mostly focused on the degradation ability,and the factors affecting the degradation of OMPs and the degradation mechanism are still unclear.Therefore,in this study,Pseudoalteromonas sp.GCY was used as a representative of ROS-producing bacteria to explore its degradation characteristics of OMPs,and clarify its co-metabolic degradation mechanism by multi-omics techniques.Firstly,the degradation characteristics of OMPs such as Ibuprofen(IBP),Bisphenol A(BPA),and Ampicillin(AMP),by GCY and the effect of these OMPs on ROS production by GCY were studied.The degradation of IBP,BPA,and AMP by GCY are mostly fitted the quasi-first order reaction kinetics,and the degradation rate constants are k _IBP=0.0295 h^-1,k _BPA=0.0352 h^-1,k _AMP=0.0196 h^-1.OMPs can promote superoxide anion radicals(O₂^·-),hydrogen peroxide(H₂O₂),hydroxyl radicals(·OH)and other extracellular ROS production to some degree.Meanwhile,the environmental factors that affect extracellular ROS production by GCY were studied.With the increase of Na⁺concentration,O₂^·-production of GCY increases.Fe²⁺can promote the production of·OH by GCY,but when the Fe²⁺concentration is higher than 100 mg·L^-1,the promotion is not obvious.In a variety of amino acids,L-Leucine has the most significant effect on promoting the production of H₂O₂ by GCY.At the same time,the H₂O₂ production of GCY increases with the increase of amino acid concentration.In the 10 g·L^-1 amino acid system,the H₂O₂ concentration can reach as high as 15.8?mol·L^-1,which is 4.79 times of 1 g·L^-1 system.Secondly,IBP was selected as a typical OMPs to study the active species and their effects in the degradation process.The quenching experiment was used to clarify the active species in the degradation process.The addition of proteinase k to inactivate the enzyme inhibited 29%of IBP degradation;quenching all ROS inhibited 90%of degradation.It shows that the process of GCY degradation of IBP is a process of enzymes and ROS,and ROS dominates.5 intermediate products in the degradation process were identified by LC-MS.It is speculated that GCY degrades IBP mainly through decarboxylation and hydroxylation,subsequently opens the ring of IBP,and finally mineralizes through?-oxidation.Finally,omics technology was used to clarify the mechanism of GCY co-metabolism and degradation of typical OMPs.Whole-genome sequencing revealed that GCY has a large number of genes related to ROS production and oxygenase genes.Transcriptome sequencing results showed that there were 145 differentially expressed genes in the degradation process of IBP,108 genes were up-regulated and 37 genes were down-regulated.Among them,ROS-producing genes(such as nad B,gah A,ldh nqr A-F)and degrading enzyme genes(hmg A,etc.)were significantly up-regulated.GCY also activated the ROS resistance mechanism,the expression of antioxidant protein genes(kat G,kat G1),DNA repair related genes(rad A,rec F,rec N,HSP70,HSP90)and efflux pump related genes(vca M,abc A,bmr A)up.In addition,genes such as liv K and ABC.PA.S in the amino acid uptake system of GCY are up-regulated,which enhances the ability of the strain to utilize amino acids.Based on this,the complete path of IBP degradation by GCY co-metabolism was further constructed.And established a cell model of amino acid-IBP co-metabolism degradation This project revealed the mechanism of GCY co-metabolism and degradation of IBP at the molecular level.