Microbial Community and Metabolic Activity in Thiocyanate Degrading Low Temperature Microbial Fuel Cells

Tensions the paper itself points to. Each side quotes the passage it comes from.

• Within the paper Thiobacillus species are reported to fix CO₂ via the Calvin-Benson-Bassham (CBB) cycle, but RuBisCO (the key CBB enzyme) mRNA was absent.

  One side

  All described species in the Thiobacillus genus fix carbon dioxide for cellular growth and contain CBB cycle genes

  “All described species in the Thiobacillus genus fix carbon dioxide for cellular growth (Boden et al., 2017) and reconstruction of the thiocyanate degrading Thiobacillus-like species genome contained genes coding for the Calvin-Benson-Bassha”

  Other side

  mRNA reads encoding the key CBB cycle enzyme ribulose biphosphate carboxylase (RuBisCO) were missing

  “mRNA reads encoding the key CBB cycle enzyme ribulose biphosphate carboxylase (RuBisCO) were missing although seven other genes in the cycle...were identified”

  Authors’ reading. Authors suggest alternative carbon fixation pathways (reductive TCA cycle, Wood-Ljungdahl pathway) were active instead, despite genomic presence of CBB genes; indicates post-transcriptional regulation or pathway switching under MFC conditions.

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

• High No measurement of dissolved oxygen concentration in anolyte or cathode; authors note 'trace levels of oxygen...can diffuse into the MFC' as a competing electron acceptor but do not quantify impact on CE.

  Authors identify oxygen diffusion as a major CE loss (2.5% vs potential higher efficiency) but provide no O₂ measurements or sensitivity analysis; sealing strategy effectiveness is untested.

  Try. Measure dissolved O₂ in anolyte over time with and without N₂ sparging; correlate O₂ concentration to current density and CE to quantify oxygen's contribution to electron loss.

• High No investigation of N₂O or NO₂⁻ accumulation despite Thiobacillus denitrificans dominance and presence of denitrification genes in the inoculum (from prior work by Broman et al., 2017).

  The inoculum was from 'a previously described low temperature, autotrophic denitrifying culture' but nitrate was removed from the anolyte; no analysis of whether denitrification genes were expressed or whether N₂O was produced as a side product.

  Try. Measure N₂O, NO₂⁻, and NO₃⁻ in anolyte and gas headspace; sequence denitrification genes (narG, nirS, norB, nosZ) in metatranscriptome to confirm they are silent or active under MFC conditions.

• High No systematic variation of anode potential or cathode potential to map the operating window for SCN⁻ degradation rate, current density, and CE.

  Anode potential remained suboptimal (more positive than theoretical) throughout; no potentiostatic or galvanostatic sweep was performed to identify optimal electrode potentials for this psychrophilic consortium.

  Try. Operate duplicate MFCs at fixed anode potentials (e.g., −400, −300, −200 mV vs Ag/AgCl) and measure SCN⁻ degradation rate, current density, and CE; identify trade-offs between kinetics and efficiency.

• Medium No measurement of soluble electron shuttle (menaquinone, riboflavin) concentrations in anolyte; hypothesis of shuttle-mediated EET is inferred from gene expression and current recovery after medium replacement, not direct evidence.

  Authors state 'presence of mRNA reads for...menaquinone plus riboflavin biosynthesis suggested...EET using soluble electron shuttles' and note current recovery after medium replacement, but do not quantify shuttle levels or test inhibitors.

  Try. Measure menaquinone and riboflavin concentrations in anolyte by HPLC or LC-MS; add shuttle inhibitors (e.g., HOQO for quinones) and measure impact on current density and anode potential.

• Medium No analysis of biofilm formation on graphite felt despite authors noting 'no biofilm formation was observed' as a factor limiting electron transfer kinetics.

  Lack of biofilm is presented as a limitation but not quantified (e.g., biomass per unit electrode area, EPS composition); no attempt to promote biofilm formation via electrode material or operational changes.

  Try. Examine anode felt by SEM/confocal microscopy at multiple time points; quantify attached biomass and EPS; test alternative electrode materials (carbon cloth, 3D-printed carbon) to promote biofilm.

• Medium No variation of SCN⁻ concentration or C/N ratio; initial SCN⁻ was ~6 mM (based on industrial wastewater) but no dose-response or inhibition study was performed.

  Single initial SCN⁻ concentration (6 mM) was chosen based on industrial data; no exploration of whether higher or lower concentrations affect lag phase, degradation rate, or product selectivity (e.g., sulfate vs elemental sulfur).

  Try. Operate MFCs with SCN⁻ ranging from 1–20 mM; measure degradation kinetics, current density, and sulfur product distribution (SO₄²⁻, S⁰, S₂O₃²⁻) to identify optimal concentration window.