Response Characteristics of Nitrifying Bacteria and Archaea Community Involved in Nitrogen Removal and Bioelectricity Generation in Integrated Tidal Flow Constructed Wetland-Microbial Fuel Cell

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

• High No measurement of N₂O or N₂ production; paper reports NO₃⁻-N, NO₂⁻-N, and NH₄⁺-N removal but does not close the nitrogen mass balance or quantify gaseous losses.

  Text states 'TN removal rate' but does not specify whether TN is removed as N₂ (complete denitrification), N₂O (incomplete), or accumulated as NO₃⁻. Figure 2B shows TN removal but Figure 2D shows NO₃⁻ accumulation, implying a gap in the balance.

  Try. Install gas chromatography or membrane inlet mass spectrometry to measure N₂ and N₂O flux at anode and cathode under both C/N ratios; quantify the fraction of TN removed as gas versus accumulated as NO₃⁻.

• High No cathode potential or anode potential measurement; paper reports voltage output but not the individual electrode potentials or current density distribution.

  Text reports 'maximum power densities' and 'current densities' but does not specify anode or cathode potential. This prevents understanding whether the cathode is operating in a denitrification window (−0.3 to −0.6 V vs SHE) or nitrification window (>+0.4 V).

  Try. Measure anode and cathode potential versus Ag/AgCl reference electrode during operation at both C/N ratios; correlate potential with NOB and denitrifier abundance; test if cathode potential can be controlled to favor denitrification over nitrification.

• Medium No intermediate C/N ratios tested between 5:1 and 10:1; paper compares only two extremes, missing the optimal C/N for balancing TN removal, NO₃⁻ reduction, and power output.

  Text states 'the recommended optimal influent C/N ratio for the removal of nitrogen (from approximately 80% TN) by CW-MFC is 5.37' but this study tests only 5:1 and 10:1. Figure 2 shows NO₃⁻ removal increases with C/N, suggesting an optimum exists between 5 and 10.

  Try. Operate TFCW-MFC at C/N ratios of 6:1, 7:1, 8:1, and 9:1 for 90 days; measure TN, NO₃⁻-N, NO₂⁻-N removal, voltage, and microbial community (AOA, AOB, NOB) to identify the C/N that maximizes both TN removal and denitrification.

• Medium No variation in tidal cycle parameters (flood duration, drain duration, rest duration); paper uses fixed 5 h flood, 12 min drain, 4 h rest, 2 h recirculation but does not test whether shorter or longer cycles affect nitrification/denitrification balance.

  Text states 'tidal flow operation' increases DO and TN removal, but does not isolate the effect of cycle frequency. Shorter cycles may increase oxygen transfer but reduce denitrification time; longer cycles may favor denitrification but reduce aeration.

  Try. Test tidal cycles of 3 h flood / 2 h rest, 5 h flood / 4 h rest (current), and 8 h flood / 8 h rest at C/N 7:1; measure DO profile, NO₃⁻-N removal, and denitrifier abundance to find the cycle that maximizes NO₃⁻ reduction.

• Medium No measurement of pH in anode and cathode layers; paper reports effluent pH but not the spatial pH gradient, which drives nitrification and denitrification kinetics.

  Text states 'differences of pH, dissolved oxygen (DO), and nitrogen concentration' control NOB distribution, but only effluent pH is reported. Anode pH can drop to <6 (inhibiting AOB) and cathode pH can rise (favoring NOB).

  Try. Install pH probes in anode, cathode, and middle layers; measure pH profile during flood and drain phases at both C/N ratios; correlate local pH with AOA, AOB, NOB abundance.

• Low No test of external carbon addition to boost NO₃⁻-N removal at low C/N; paper shows C/N 5:1 limits denitrification but does not test whether supplementing acetate or methanol can recover NO₃⁻ removal without changing the influent C/N ratio.

  Text states 'the loss of carbon source may lead to incomplete denitrification, thus causing the accumulation of NO₃⁻-N' but does not propose or test a remedy. This is a practical design option for operators.

  Try. At C/N 5:1, inject acetate or methanol into the anode layer at rates of 50, 100, and 200 mg/L; measure NO₃⁻-N removal, voltage, and denitrifier abundance to determine if external carbon can restore denitrification without inhibiting power output.