Exercise Behavior And Physiological Response Mechanism Of Aerobic Denitrifying Bacteria Under Environmental Stress

The biofilm formation period of the biofilm reactor is the key stage of the reactor start-up,which has a direct impact on the stability and performance of the reactor.In the initial stage of biofilm formation,bacteria repeatedly contact with the interface to form reversible adhesion through flagella,cilia and other motor organs,after the bacteria attach to the interface,the flagella motor drives the bacteria to rotate and move vertically until the bacteria and the interface form irreversible adhesion by Lectins,polysaccharides and other functions.Therefore,studying the interface movement behavior of wastewater treatment bacteria is helpful to understand the biofilm formation process,which has important guiding significance for engineering application of biofilm method.Bacterial movement behavior is closely related to environmental stimuli.However the antibiotics,high ammonia nitrogen,high salt and other stress environments have unclear effect on the bacterial adhesion process in the early stage of biofilm formation,and its influence mechanism on bacterial movement remains to be explored.Surface plasmon resonance microscopy has the advantages of high sensitivity,high speed and real time imaging.Based on the single bacteria imaging analysis method of surface plasmon resonance microscope(SPRM),this study realize the three-dimensional analysis of single bacteria interface motion(horizontal rotation and vertical jitter).Biological methods and transcriptomics techniques were used to explore the physiological response of interfacial movement behavior to reveal the influence of stress environment on bacterial attachment during biofilm formation.The main research findings are as follows:(1)Taking the model strain E.coli DH5? as the research object,an imaging method of bacterial interface motion behavior based on surface plasmon resonance imaging technology was constructed.By adjusting the angle and intensity of the incident light,and comparing the frame rate,exposure time,sensitivity,signal-to-noise ratio,image number,image resolution and other parameters of the high-speed camera,the operation method of SPRM imaging system is optimized.And the fast,real-time,non-destructive and label-free bacterial interface movement behavior observations are realized through chip modification and selection of bacterial growth stages.(2)Based on the SPRM bacterial interface motion behavior imaging method constructed above,the interface 3D motion behavior of a single aerobic denitrifying bacteria under the stress of polymyxin B(PMB),high ammonia nitrogen and high salt was studied.Couplingwith biology methods,The results showed that: under 0.5 ?g/m L PMB stress,the proportion of dead bacteria increased by only 7.59%,the horizontal movement of the interface was inhibited and stopped rotating,and the variation trend of vertical movement was consistent with that of the proportion of dead bacteria.And the bacteria had both high wastewater treatment capacity and biofilm formation capacity(The biofilm forming capacity was increased by 24.2% compared with the control group).Under 25-200 mg/L ammonia nitrogen stress,flow cytometry results showed that the proportion of dead bacteria increases within 15%,and the horizontal movement of bacteria was inhibited and stopped rotating.The variation trend of vertical movement is also consistent with that of the proportion of dead bacteria.The bacteria have both high wastewater treatment ability and biofilm forming ability(The biofilm forming capacity increased by 17-39% compared with the control group).Under 10 g/L salt stress,flow cytometry results showed that the proportion of dead bacteria increased by only 1.72%,and the horizontal movement of bacteria was inhibited and stopped rotating.The variation trend of vertical movement is also consistent with that of the proportion of dead bacteria.The bacteria have both high wastewater treatment ability and biofilm forming ability(The biofilm forming capacity increased by 7% compared with the control group).(3)Transcriptome analysis showed that under the stress of PMB,high ammonia nitrogen,and high salt,the bacteria's quorum sensing and ABC transporter-related genes were up-regulated,which enhanced the ability of biofilm formation to help the bacteria resist adverse environments.Under high ammonia nitrogen stress,the pathways of bacterial secretion system were down-regulated,and ATPase-related gene Sec A was down-regulated.Bacteria reduced their athletic ability due to lack of energy.Under high-salt stress,genes related to flagellum motor assembly were down-regulated,and pathways of bacterial secretion system were down-regulated.The ATPase-related genes vas G and Sec A in type ? and type ? were down-regulated,and bacteria's energy synthesis was blocked.The overall metabolic activity is weakened,and the bacterial movement ability is therefore inhibited.This research expands the application of SPRM technology in studying interface3 D motion behavior of bacterial.It provides a basis for understanding the interface adhesion of microorganisms,and also provides theoretical support for further research on the start-up and operation of biofilm reactors under common stresses in wastewater.