In Situ Real-time Monitoring Of Metabolic Activity In Biofilm Of Sulfate-reducing Bacteria And Its Preliminary Application

As the typical,studied,and most corrosive microorganism,sulfate-reducing bacteria(SRB)influenced corrosion was closely related with the formation of biofilms on the surface of metal materials.Traditional methods for measuring SRB activity in biofilms cannot distinguish the metabolic activity difference between sessile SRB in the biofilm and planktonic SRB in bulk solution,and it is difficult to apply in the field application.In order to solve these problems,two types of all-solid ion selective electrochemical microprobes were prepared in this thesis,and used for construction the metabolic activity detection platforms for a continuous,in situ detection of the SRB metabolic activity in biofilms by selectively recognition the characteristic metabolic substances.In addition,the feasibility of the SRB metabolic activity detection performances were verified by two organic fluorescent probes.The metabolic activity of sessile SRB in biofilm on the surface of inert material and planktonic SRB in bulk solution were continuous measured,and the test results preliminarily revealed the changes and differences of metabolic activity between SRB cells in the biofilm and free SRB cells.Subsequently,the metabolic activity of sessile SRB in biofilm on the surface of metal material and planktonic SRB in bulk solution were continuous measured,and the results were discussed in this thesis.This study was of great significance for revealing the variation regulation and difference characteristics of SRB metabolic activity inside and outside the biofilm,and held significant meaning for the research of SRB biofilm corrosive mechanism.The main finding were as follows:(1)An all-solid sulfide selective microprobe for specific recognition of sulfide produced by SRB metabolism was constructed.The microprobe was fabricated on a flexible silver wire as the conductive substrate,and subsequently layered reduced graphene sheets as the ion to electron transfer layer and nanostructured Ag₂S as the sulfide selective membrane were successively constructed by electrochemical method.The electro-chemical deposition process was optimized and the corresponding surface morphology was characterized.The linear range,stability,selectivity,and other detection properties of the all-solid sulfide selective microprobe were systematically studied.Finally,the all-solid sulfide selective microprobe was verified in seawater and tap water samples testing.The results showed that the linearly detection range of the all-solid sulfide selective microprobe was 5×10^-7-1×10^-2M,with the detection limit of1.78×10^-7 M.The highly flexible all-solid sulfide selective microprobe has good selectivity and stability,and held great application potential in material interface research and deep-sea environment detection.(2)An all-solid hydrogen ion selective microprobe was constructed.The microprobe was fabricated on flexible silver wire as the conductive substrate,and then a layered reduced graphene sheet as the ion to electron conversion layer was constructed by an electrochemical deposition method.Next,on the surface of graphene layer,tri-n-dodecyl amine(TDDA)as a neutral carrier,was mixed with potassium tetraphenylborate(KTPB),dioctyl sebacate(DOS)and polyvinyl chloride(PVC)to fabricate a neutral carrier film for selective hydrogen ion recognition.The linear range,stability,selectivity,and other detection properties of the all-solid hydrogen ion selective microprobe were systematically studied.Finally,the all-solid hydrogen ion selective microprobe was verified in seawater,tap water and culture medium samples.The results showed that the linearly response of the all-solid hydrogen ion selective microprobe for p H was ranged from 2.5 to 12,with excellent selectivity and stability,and exhibited good application performance in practical samples.The highly flexible all-solid hydrogen ion selective microprobe has great application potential in the study of material interfaces and deep-sea environment detection.(3)Based on the developed all-solid sulfide selective microprobe,a real-time,in situ,continuous detection platform for SRB metabolic activity in the biofilm on the surface of inert material was constructed.Consequently,successive detection of metabolic activity of sessile SRB in the biofilm and planktonic SRB in bulk solution was achieved.In addition,two types of organic fluorescent probes were used to analyze SRB metabolic activity and cell distribution in biofilms,and therefore to evaluate the practical application performance of the all-solid sulfide selective microprobe for in situ metabolic activity in SRB biofilms.The effect of addition of D-methyl sulfide amino acid(D-Met)on the metabolic activity of SRB on the surface of inert material was measured.According to the research results,the sulfide concentration produced by SRB metabolism in the biofilm on the surface of the inert material was 0.4-0.6 m M higher than that of the planktonic SRB in bulk,and the same change trend of the metabolic activity of planktonic SRB was 1-2 days delayed compared with that of in biofilm.The fluorescence measurement results showed that the metabolic activity of SRB in the biofilm and the spatial distribution of cells were very similar,and the changes trend of fluorescence intensity was consistent with that of in the all-solid sulfide selective microprobe,which verified the feasibility and accuracy of the all-solid sulfide selective microprobe in the detection of SRB metabolic activity on the surface of inert material.In addition,the results of electrochemical tests showed that the addition of D-Met caused the decrease in the SRB metabolic activity in the biofilm,which was also confirmed by the results of fluorescence probes.This detection platform provides a new strategy for in situ,real-time detection of SRB metabolic activity in biofilms in the marine environment.(4)Based on the developed all-solid sulfide selective microprobe,a real-time,in situ,continuous detection platform for SRB metabolic activity in the biofilm on the surface of metal material was constructed.The continuous measurements of metabolic activity of sessile SRB in the biofilm on the surface of metal material and planktonic SRB in bulk solution were conducted.Next,two types of organic fluorescent probes were used to analyze SRB metabolic activity and cell distribution in biofilms.The results showed that the SRB metabolic activity in the biofilm on the metal surface was much higher than that of free SRB in bulk solution.In addition,compared with the SRB metabolic activity on surface of inert material,the variation trend of SRB metabolic activity in the biofilm on the metal surface was more complicated,and the metal material significantly improved the metabolic activity of SRB in the biofilm.The feasibility and accuracy of the electrochemical monitoring method were verified by the results of fluorescence microscopy measurements.