Effect Of Applied Voltage On Stress Tolerance Of Electroactive Biofilm And Its Mechanism

Extracellular polymeric substances(EPS)have been considered as the barrier for toxic species penetration into the cells,but their function in protecting electroactive biofilms(EABs)had been rarely revealed.EPS containing groups(such as amino,carboxyl,and phenol)can complex various toxicants to increase biofilm resistance.In this study,the anode potentials were used to regulate the EPS quantity and composition in mixed-culture EABs,where their resistance to Ag~+shock was assessed.Through scanning electron microscopy,three-dimensional fluorescence,infrared spectroscopy,two-dimensional fluorescence,and two-dimensional infrared technology,we systematically explored the stress tolerance performance and mechanism of electroactive biofilms at different voltages.By comparing the physical and chemical properties of electroactive biofilms at different anode potentials,it is concluded that the current density of EAB at 0.2 V is 2.7±0.3 m A/cm~2,which is higher than the current density at other potentials,and the thickness of the biofilm reached 60μm.As far as the total amount of EPS is concerned,the total amount of EPS of 0V biofilm is the largest.And in tightly bound EPS(TB-EPS)and loosely bound EPS(LB-EPS),the content of protein and humic acid was the highest.The abundance of genes associated with EPS biosynthesis was also confirmed to be related with the applied anode potentials,based on the metagenomic analysis.In the study of stress tolerance of EAB to silver ions cultivated at different anode potentials,it was found that after 3 h of Ag~+solution stimulation,the current recovery rates of EAB at four potentials were 3.2%,40.0%,18.5%and 15.8%,respectively.Combined with the electricity generation capacity,the EAB cultured at 0 V achieved a maximum current density of 1.0±0.1 m A/cm~2.The proportion of viable in biofilms at different voltages has the same trend as the current recovery rate.These results indicate that EAB at 0 V may have stronger stress tolerance than that at 0.2 VFrom the correlation between EPS components and current recovery rate of EAB at different anode potentials,it was found that the current recovery rate of EAB after exposure to silver ion solution was weakly correlated with the polysaccharides and DNA of LB-EPS and TB-EPS(R~2<0.21;P<0.05),LB-EPS polysaccharides and DNA may have a limited effect on the anti-shock properties of EAB,but there is a significant correlation between the protein or humic acid of TB-EPS in EAB and the current recovery rate(R~2=0.93 and 0.95;P<0.05).At the same time,the protein and humic acid of LB-EPS also have a strong positive correlation(R~2=0.51 and 0.46;P<0.05),which indicates that these two main components may play an important role in reducing Ag~+toxicity.Structural analysis showed that LB-EPS was the main component affecting the anti-shock ability of EABs.Chemical analysis showed that the protein and humic acid of LB-EPS were the main active components.Synchronous fluorescence and FTIR associated with two-dimensional correlation spectroscopy(2D-COS)further confirmed that the oxygen and nitrogen moieties(i.e.amide,carbonyl C=O,phenolic,and C-O-C)of the LB-EPS were response for the binding with the Ag~+to prevent the penetration into the cells.Spectral analysis and 2D-COS analysis showed that during the process of binding with silver ions,the sequence of EPS main chain changes follows the protein’s amine group>carbonyl group>phenol group>polysaccharide C-O-C.The potential molecular mechanism of EPS protecting EABs from Ag~+shocks explored in this study provides some inspiration for the development of new methods for constructing highly stable EABs.