Molecular Mechanism Of Biofilm Formation In Aquaculture Wastewater By AHLs Mediated Quorum Sensing Engineering Bacteria

Biofilm process is an energy efficient and environmentally friendly biological treatment technology.Biofilm is the core of water treatment and the high concentration of microorganisms on the biofilm and the large specific surface area promote the decomposition of pollutants,including biosorption,degradation,nitrification and denitrification activities,which help to maintain the stability of the biofilm process performance for long-term operation.However,when applying biofilm technology to water treatment in aquaculture,biofilms have the limitations of long start-up periods and structural instability during long-term operation.The quorum sensing(QS)system based on the signalling molecule mediated by acylhomoserine lactones(AHLs)is a central mechanism for regulating biofilm formation in wastewater,and exogenous addition of certain concentrations of AHLs can rapidly promote biofilm formation and improve the water treatment performance of the biofilm process.However,exogenous AHLs are in high demand,costly and not easily stored,making it difficult to apply exogenous AHLs on a large scale to enhance biofilm activity.QS-engineered bacteria modified by genetic engineering techniques can synthesize AHLs efficiently,which can be used as an alternative strategy for exogenous AHLs signalling molecules to regulate biofilm formation,and the application of this aspect in aquaculture is still lacking research.To address the above problems,this paper takes the regulation of biofilm formation by AHLs as the starting point,and realizes the efficient regulation of biofilm AHLs concentration from the endogenous gene and enzymatic levels,and carries out the preliminary molecular mechanism study of bioenhanced QS engineered bacteria to regulate biofilm formation in water treatment systems,with a view to solving the problems of large demand and high cost of AHLs,as well as the long time and poor stability of the initial biofilm formation stage.In addition,this paper designed experiments from the perspective of combining QS and quorum quenching(QQ),forward and reverse,to construct AHLs synthesis engineered bacteria and AHLs quenching engineered bacteria through gene recombination technology,and to verify the ability of engineered bacteria to secrete or degrade AHLs signalling molecules.The effect of the engineered bacteria on biofilm formation was further examined to characterize the response of QS engineered bacteria to the biofilm formation process from the perspectives of AHLs signal molecule concentration,biomass adhesion,biofilm morphological characteristics and microbial community structure,to investigate the regulation of biofilm community succession by QS engineered bacteria and to reveal the molecular mechanism of biofilm formation jointly regulated by QS and QQ.This study explores the real-time regulation of the concentration of biofilm AHLs signalling molecules,which provides an effective method to promote rapid biofilm formation in water treatment systems,improve the water treatment efficiency of biofilm processes and maintain the long-term stability of biofilm performance.The research content and results of this paper are as follows:1.Construction of AHLs signal molecule synthesis engineering bacteria by gene recombination technology.In this experiment,two genes encoding AHLs synthase,Luxl and Lux M,were obtained from the National Center for Biotechnology Information(NCBI),and primers were designed and synthesized.Using the genome DNA of Vibrio harveyi as the template,the target gene fragments were obtained by PCR cloning technology.The recombinant expression vectors p ET-Luxl and p ET-Lux M of AHLs synthase were constructed by using the p ET-28 a plasmid,and the recombinant expression vectors p ET-Luxl and p ET-Lux M of AHLs synthase were selected.The expression strain of E.coli BL21 was selected as the expression strain,and two AHLs synthetic engineering strains(Luxl and Lux M)were successfully constructed.After verification,it was found that the two engineering strains have the ability to efficiently secrete AHLs signal molecules,and can encode and synthesize six AHLs signal molecules,namely C6-HSL 、 C8-HSL 、 C10-HSL 、3-oxo-C10-HSL、3-oxo-C12-HSL、3-oxo-C14-HSL.2.The AHLs signal molecule degrading engineering bacteria were constructed by gene recombination technology.Two AHLs degrading enzyme genes,aii A and Pvd Q,were obtained from the NCBI gene library,respectively encoding and synthesizing aii A lactose and Pvd Q acylase,respectively,using the genomic DNA of Bacillus thuringiensis and Pseudomonas aeruginosa as templates,the synthetic primers were designed,the target gene segments were obtained by PCR cloning technology,the p ET-28 a plasmid was selected,the AHLs degrading enzyme recombinant expression vectors p ET-aii A and p ET-Pvd Q were constructed,and the Escherichia coli BL21 was selected as the expression strain,Two AHLs degrading engineering bacteria(Luxl and Lux M)were successfully constructed.After verification,it was found that the two strains of engineering bacteria have the ability to efficiently degrade AHLs signal molecules,can degrade a variety of AHLs signal molecules,and can inhibit QS.3.Explore the molecular mechanism of AHLs synthesis and degradation engineering bacteria regulating the formation of wastewater biofilm.The engineering bacteria constructed by gene recombination technology have corresponding functional activities,which can control the concentration of AHLs signal molecules in real time,and realize two-way regulation of biofilm formation.In addition,colony sensing and colony quenching engineering bacteria jointly mediate the abundance change of the core community of biofilm,and affect the interaction between communities in the process of biofilm formation.The engineering bacteria can combine quorum sensing and quorum quenching to mediate the formation of biofilm in both directions,providing a new way to grasp the real-time control of the AHLs signal molecular concentration of biofilm,promote the rapid film hanging of aquaculture water treatment system,improve the efficiency of biofilm process water treatment and maintain the long-term stability of biofilm performance.