Study On Microbiologically Influenced Corrosion Of Stainless Steels By Bacillus Subtilis Via Bidirectional Extracellular Electron Transfer

With the development of social economy and scientific technology,stainless steel with excellent corrosion resistance is widely used.However,due to the complexity of service environments,the corrosion problems of stainless steels are troublesome,and microbial corrosion is one of them.Bacullus subtilis is a kind of Gram-positive bacteria and facultative anaerobic bacteria,which exists widely in various environments.In anaerobic environment,B.subtilis can act as a nitratereducing bacterium and use nitrate as the terminal electron acceptor to complete the respiratory reaction to obtain energy,which will cause serious corrosion of metal materials served in the environment.On the other hand,in the relevant researches of microbiogically influenced corrosion(MIC),extracellular electron transfer(EET)is one of the mechanisms that lead to the failure of metal materials.This mechanism takes into account the biochemical metabolic process of bacteria.It is believed that there is an efficient electron transfer process between biofilm and metal materials,which accelerates the corrosion rate of metals.And in the study of MIC of stainless steels,it is pointed out that the pitting corrosion occurred on the stainless steel is accelerated in the presence of bacteria,but the corrosion mechanism is not clarified.There is still a big gap in the study of MIC mechanism of stainless steels.Therefore,B.subtilis,a nitrate-reducing bacterium containing endogenous electron mediators,is useful to explore its EET mechanism and effect on the MIC of stainless steel,which is of great significance to the microbial control and the corrosion protection of major engineering equipment in the service environments.In this study,304 stainless steel was used as the research object,and the "biocathode" or "bioanode"process between B.subtilis and stainless steel were stimulated by setting up glycrol(as terminal electron donor)or nitrate(as electron acceptor)deficiency conditions.From the aspects of microbial metabolism,biofilm morphology,corrosion characteristics and so on,the corrosion behaviors of stainless steel caused by B.subtilis via bidirectional EET were studied.The steel with native passive surface was set as the passivated state,and the steel with active surface was set as the activated state,which was prepared by artificially removing the native passive surface on the steel surface.The EET processes between B.subtilis biofilm and the steels with two surface states were observed.The differences of corrosion behaviors of the steels with active and passive surfaces caused by B.subtilis via EET pathway under electron donor/acceptor deficiency were compared and analyzed,which further verified the effects of biocathode and bioanode pathways on MIC processes of 304 stainless steel.Meanwhile,in combination with the transcriptomics analysis,the relationship between the endogenous electron mediator(riboflavin)of B.subtilis and its corrosion behavior was revealed from the gene expression level.The main conclusions are as follows:Under electron donor deficiency,B.subtilis tended to aggregate on the surface of 304 stainless steel and formed clusters with small area and large thickness,and the Fe2+ dissolution of the steel was accelerated.It is inferred that under these conditions,the biocathode process of the biofilm was promoted,which mainly obtained electrons by locally dissolving Fe0 of the steel substrate.The results of electrochemistry test showed that when the electron donor was scarce,B.subtilis would preferentially consume organic carbon sources,and then the charge transfer rate between B.subtilis and the steel increased rapidly,and the anode current increased,but the reduction of pitting sensitivity was limited.It indicated that after the biocathode of B.subtilis was promoted,it would mainly accelerate the dissolution of the anode of 304 stainless steel,without causing extensive damage to the passivated film,and only showed relatively serious damage in the local areas.With the decrease of electron acceptor concentrations,the proportion of sessile cells of B.subtilis on the surface of 304 stainless steel increased significantly,and the bacteria were more likely to form biofilms with large biomass and large area.The Fe2+ dissolution was accelerated by B.subtilis,which inferred that the bioanode was promoted,and biofilm transferred electrons to the passive film,further promoting the reduction of passive film.The electrochemical results showed that the pitting potential of 304 stainless steel decreased obviously,while the increase of anode current was limited.It indicated that the reduction of passive film was accelerated by B.subtills via the biocathode process,which further led to the overall deterioration of the passive film and finally increased in its pitting sensitivity.The stainless steel with native passive surface was set as the passivated state,and the steel with active surface was set as the activated state,which was prepared by artificially removing the native passive surface on the steel surface.Furthermore,the role of biocathode and bioanode pathways in the MIC process of 304 stainless steel was studied.The results showed that under electron donor deficiency,the bioanode process was promoted and the biofilm could obtain electrons by accelerating the Fe0 dissolution.Thus,the passive film on the steel surface hindered the direct contact between B.subtilis and Fe0 substrate.When the native surface was removed,the biofilms with large area and large biomass formed on the active surface.The electrochemical results showed that the anode current densities of the steel with passive and active surfaces were significantly increased,while the decrease of the pitting potential was limited.It indicated the acceleration of anode dissolution caused by B.subtilis via the biocathode process,but it wouldn’t cause large area damage to the passive film.Under electron acceptor deficiency,the bioanode process was promoted,and electrons were transferred to the passive film on the steel surface.Thus,the passive film acted as an important role to maintain the bioanode process,and the strain formed biofilms with large biomass and large area on the passive surface.The electrochemical results showed that the pitting potentials of the steels with passive and active surfaces decreased obviously,while the increase of anode current was limited.It indicated that when the EET process of B.subtilis was mainly dominated by bioanode process,B.subtilis biofilm would accelerate the deterioration of passive film,and hindered the regeneration of passive film.Transcriptome analysis showed that under electron donor/acceptor deficiency,B.subtilis had multiple differentially expressed genes,among which the upregulated genes pabB,nfrA1 and nfrA2 related to the riboflavin metabolic pathway were significantly enriched.Meanwhile,the concentration of riboflavin in the metabolites secreted by B.subtilis increased with the enhancement of EET between the biofilm and 304 stainless steel.And the redox peaks of riboflavin appeared in the metabolites under the corresponding conditions.The charge transfer rate between B.subtilis and 304 stainless steel surface was significantly increased and the anodic dissolution was promoted when riboflavin was supplemented to organic carbon source excess deficient medium.Above results indicated that the gene expression and metabolic pathway of riboflavin in B.subtilis were closely related to the EET process of 304 stainless steel.