Extracellular electron transfer-dependent anaerobic oxidation of ammonium by anammox bacteria

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

• Medium No systematic variation of applied potential between 0.2 and 0.6 V vs. SHE to map the full current density vs. potential curve and identify the minimum potential for sustained activity.

  Paper shows no biofilm at ≤0.2 V and maximum current at 0.6 V, but intermediate potentials (0.3–0.5 V) are only shown as discrete points in Fig. 2a without detailed kinetic analysis. This gap prevents optimization of energy input.

  Try. Chronoamperometry at 0.25, 0.35, 0.45, 0.55 V vs. SHE with mature biofilm; measure current density, NH4+ removal rate, and biofilm thickness over 7 days.

• Medium No investigation of cathode potential or cathodic reaction (H2 evolution) effects on overall system energy balance, despite brief mention that single vs. double-chamber MECs showed no difference.

  Text states 'there was no significant difference in NH4+ oxidation and current production between the different reactor configurations' but provides no data on H2 production, cathode potential, or overall cell voltage. This is critical for energy efficiency assessment.

  Try. Operate paired single- and double-chamber MECs at 0.6 V anode potential; measure cathode potential, H2 production rate, and overall cell voltage; calculate net energy input vs. current output.

• Medium No testing of different NH4+ concentrations to establish the operating window and kinetic parameters (Km, Vmax) for EET-dependent anammox.

  Fig. 2a shows current is 'proportional to NH4+ concentration' but only tests one concentration (4 mM) in detail. No Monod kinetics, substrate inhibition, or minimum viable concentration are reported.

  Try. Feed MEC with NH4+ ranging 0.5–10 mM; measure steady-state current density and removal rate; fit to Monod equation to extract Km and Vmax.

• High No measurement of N2O or NO accumulation during EET-dependent anammox, despite these being critical environmental and safety concerns in nitrogen cycling systems.

  Abstract and text state 'Complete NH4+ oxidation to N2 without accumulation of NO2− and NO3−' and 'NO and N2O were not detected' but detection limits and analytical methods are not specified. This is essential for validating the process as truly clean.

  Try. Use gas chromatography with electron capture detection (GC-ECD) or cavity ring-down spectroscopy (CRDS) to quantify N2O and NO at detection limits <1 ppm; run 7-day continuous operation at 0.6 V vs. SHE.

• Medium No investigation of pH effects on EET-dependent anammox, despite pH being critical for both anammox metabolism and electrode kinetics.

  All experiments appear to be conducted at unspecified pH; text mentions 'OH− ions generated from water' but no pH control or variation is reported. Anammox typically requires pH 6.5–8.5; electrode potential is pH-dependent.

  Try. Operate MECs at pH 6.5, 7.0, 7.5, 8.0, 8.5 with 0.6 V vs. SHE applied; measure current density, NH4+ removal rate, and biofilm composition (FISH) at each pH.

• Medium No long-term stability data (>200 days) for electrode-dependent anammox biofilms, limiting assessment of practical applicability.

  Longest experiment shown is ~144 hours (6 days) in Fig. 3a; Fig. 2a shows ~50 days but with multiple operational changes (NO2− addition/removal). Biofilm fouling, electrode degradation, and microbial drift are not addressed.

  Try. Operate single MEC at constant 0.6 V vs. SHE with continuous NH4+ feed for 180 days; measure current density, NH4+ removal, biofilm thickness (SEM), and community composition (16S rRNA) monthly.