Anode potential-dependent protection of electroactive biofilms against metal ion shock via regulating extracellular polymeric substances

Extracellular polymeric substances (EPS) have been considered as a barrier for toxic species penetration into the cells, but their function in protecting electroactive biofilms (EABs) had been rarely revealed. In this study, the anode potential was used to regulate the EPS quantity and components in mixed-culture EABs, where their resistance to Ag+ shock was assessed. The results showed that the EAB grown at 0 V showed the highest anti-shock capability by the Ag+ exposure compared to those grown at -0.2, 0.2, and 0.4 V. The EAB produced at 0 V had both of the highest amounts of loosely bound EPS (LB-EPS; 61.9 mg-EPS/g-VSS) and tightly bound EPS (TB-EPS; 74.8 mg-EPS/g-VSS) than those grown under other potentials, where proteins and humic acid were the predominated components. The abundance of genes associated with EPS biosynthesis were also confirmed to be related with the applied anode potentials, based on the metagenomic analysis. Considering proteins and humic acid in LB-EPS showed positive linearity with the current recovery and viability of the EABs, these two main components might play important roles in reducing the Ag+ toxicity. Synchronous fourier transform infrared (FTIR) spectroscopy integrated two-dimensional correlation spectroscopy (2D-COS) analyses further confirmed that the oxygen and nitrogen moieties (i.e. amide, carbonyl CO, phenolic, and C-O-C) in proteins and humic acid of the LB-EPS were response for the binding with the Ag+ to prevent the penetration into the cells. The underlying molecular mechanisms of EPS in protecting EABs from the Ag+ shock explored in this study can provide implications for developing new methods to construct highly stable EABs.