2.1. SAMFC construction and microorganism acclimation
Cube-shaped plexiglass with a volume of 84 mL was used as the setup of SAMFCs. The anodes were graphite brushes with 2 cm in diameter and 2.5 cm in length. The air-cathodes were fabricated by rolling method as previously described (Liu et al., 2014) using activated carbon as oxygen reduction reaction catalyst. Nine SAMFCs were built and divided into three groups with three in each group. The three groups of SAMFCs run at conditions of open-circuit (OC), 1000Ω load and 20Ω load (denoted as SAMFC-OC, SAMFC-1000Ω and SAMFC-20Ω), respectively. These SAMFCs were inoculated from SAMFC that inoculated with domestic wastewater from treatment plant (Qingshan, Nanchang) and run for more than one year. The SAMFCs were firstly operated by feeding 50 mM phosphate buffer solution (PBS) containing 1.64 g·L-1 sodium acetate (equal to 1280 mg·L-1 COD) for 30 days to acclimate exoelectrogens onto the anodes, then run by feeding the 50 mM PBS containing 1.64 g·L-1 and extra 1.21 g·L-1 sodium acetate (200 mg·L-1NO3--N) to acclimate denitrifers. The detailed acclimation and characterization process of the SAMFCs is shown in Figure S1.
All the SAMFCs were operated in fed-batch mode at a constant temperature of 35±1°C. During the acclimation process, the anode potentials and cell voltages were automatically recorded every 5 min using a data acquisition system (digital multimeter, Keithley 2700, USA) connected to a personal computer. Medium was refreshed when the anode potential rose to over 0 V. All the electrode potentials were measured versus the Ag/AgCl reference electrode (+0.198 V versus SHE, saturated KCl). The concentrations of NO3--N in the SAMFCs were measured at the 20th (t20), 40th (t40), 60th(t60), 80th (t80), and 100th (t100) day, respectively. The microbial electrocatalytic activity and community analyses of the microorganisms at the anodes were conducted at the initial (t0), 20th (t20) and 100th (t100) day, respectively. Microbial community analysis can damage the anode biofilm, therefore one of the three SAMFCs in each group was used for NO3--N concentration, CV characterization and final microbial community analysis, while the other two SAMFCs were only used for microbial community analysis.