Development of Electroactive and Anaerobic Ammonium-Oxidizing (Anammox) Biofilms from Digestate in Microbial Fuel Cells

Specific gaps in this paper, with an experiment that would close each.

• High No measurement of anode or cathode potential (vs. Ag/AgCl or SHE); operating window for anammox enrichment and nitrogen removal is unknown.

  Section 2.2 states 'fixed external resistance, Rext, of 180 Ω' but does not report actual anode or cathode potentials. Polarization curves (Figure 3) show voltage and current but not electrode potentials. Anammox bacteria have known redox preferences; without potential data, the electrochemical conditions driving their enrichment cannot be replicated or optimized.

  Try. Measure anode and cathode potential (vs. Ag/AgCl) throughout batch cycles; vary anode potential (−0.2 to +0.2 V) and quantify anammox 16S rRNA gene abundance and nitrogen removal rate.

• High No dissolved oxygen (DO) measurement in the anode chamber during operation; anaerobic conditions assumed but not verified.

  Section 2.4 lists DO measurement capability ('dissolved oxygen was measured with a 913 OXY oximeter') but Section 3.3 only states 'dissolved oxygen in the anodic chambers was always lower than 0.05 mg/L' without time-series data or sampling frequency. Anammox requires strict anaerobiosis; DO leakage would shift nitrogen removal toward nitrification-denitrification.

  Try. Log DO continuously in anode chamber; correlate with anammox gene abundance and nitrite/nitrate accumulation; test membrane integrity and N2 flushing frequency.

• High No measurement of nitrite (NO2−) or nitrate (NO3−) accumulation in the anode chamber; only stated as 'negligible' at cycle end.

  Section 3.3 states 'nitrates and nitrites at the end of each cycle were negligible in the anodic and cathodic chambers' but provides no time-series data, detection limits, or quantitative values. Anammox consumes NO2− and produces N2; transient NO2− accumulation would confirm anammox activity but is not tracked.

  Try. Measure NO2− and NO3− daily by ion chromatography (method already available, Section 2.4); plot against TKN removal and anammox gene expression; test whether NO2− limitation constrains nitrogen removal.

• Medium No variation in C/N ratio or HRT; single digestate dilution (1:10) and weekly feeding tested.

  Section 2.1 and 2.2 describe a fixed feed composition (1:10 digestate dilution, weekly replacement of 150 mL of 200 mL anode volume, ~7-day HRT). No exploration of how C/N ratio or residence time affects anammox enrichment or nitrogen removal efficiency. Anammox typically requires low C/N and long HRT.

  Try. Test digestate dilutions (1:5, 1:10, 1:20) and feeding intervals (3, 7, 14 days); measure TKN removal, anammox gene copy number, and power output.

• Medium No measurement of N2O or N2 gas production; nitrogen balance incomplete.

  Section 3.3 states 'we did not detect CH4 production' but does not report N2O or N2 measurement. Anammox produces N2; denitrification can produce N2O. Without gas chromatography or isotope tracing, the fate of 32–40% removed TKN is unknown—it could be biomass incorporation, N2 loss, or N2O accumulation.

  Try. Measure headspace N2O by GC-ECD and N2 by GC-TCD or isotope-labeled 15N tracing; quantify nitrogen balance and confirm anammox stoichiometry (NH4+ + NO2− → N2 + 2H2O).

• Medium No catholyte pH control or monitoring; neutral pH assumed but not reported.

  Section 2.2 states cathode filled with 'potassium phosphate buffer (100 mM pH 7.5)' but no pH time-series or post-operation pH reported. Cathode pH can drift due to proton consumption during reduction; anammox in cathode chamber (if any) would be pH-sensitive.

  Try. Monitor cathode pH continuously; test pH range (6.5–8.5) and measure anammox activity and nitrogen removal in cathode chamber if applicable.