Determination of cellular redox state and ATP concentration

Two-chamber microbial electrosynthesis cells with a volume of 100 ml of each chamber were constructed as previously described (45). Graphite plates (1 cm × 3 cm × 4 cm) served as the anode and cathode, and Hg|Hg₂Cl₂ (sat. KCl) electrode acting as the reference electrode was applied in the cathode chamber, and the electrolyte was NB medium with 30 mM nitrate supplied in the catholyte. To start electroautotrophic growth, 8 ml of R. palustris cells grown in 0259 medium was collected and washed using 0.9% NaCl solution and subsequently inoculated into the cathode chamber with the cathode poised at 0.1 V (versus SHE) using a multichannel potentiostat (1000C, CH Instruments Inc., USA). Cells were run at 30°C under darkness, and the current was monitored accordingly.

For measurements of NAD⁺/NADH, NADP⁺/NADPH, and ATP, cells were harvested from the cathode and treated with appropriate buffers as indicated by the providers. In particular, NAD⁺/NADH and NADP⁺/NADPH ratios were determined using an NAD⁺/NADH Quantification kit (Millipore Sigma, Missouri, USA) and a NADP⁺/NADPH Assay kit (Solarbio, Beijing, China) and were detected using a colorimetric assay under a microplate reader (Eon, BioTek Instruments Inc., Vermont, USA) with detection wavelengths of 450 and 570 nm, respectively. The ATP concentration was calculated using an enhanced ATP assay kit (Beyotime Biotechnology, Shanghai, China) by measuring chemiluminescence with a luminometer plate reader (Promega Biotech Co. Ltd., Beijing, China).