Harnessing Zero Valent Iron-Derived Hydrogen for Acclimatized Betaproteobacteria-Driven Denitrification in Nitrate-Contaminated Water

Nitrate contamination poses environmental and public-health risks, yet conventional removal technologies (reverse osmosis, ion exchange, nanofiltration) can be costly for small or underserved systems. We evaluated zero-valent iron (ZVI) granules as an inexpensive, safe electron source that generates H₂ in situ to support hydrogenotrophic denitrification by mixed Betaproteobacteria. Duplicate 1-L batch systems (Media-1, Media-2) were run for ~2 months at 36 °C with periodic media replacement. Nitrate was dosed as NaNO₃ (~25–50 mg/L as NO₃–N target) and measured colorimetrically (Hach 353; reported as NO₃–N). Across 32 measurements, median effluent nitrate was 0 mg/L as N (mode = 0; mean = 0.044 mg/L as N), with 78.3% average reduction from influent and final concentrations consistently below the U.S. Environmental Protection Agency (EPA) maximum contaminant level of 10 mg/L as N. Volatile suspended solids (VSS) increased within each growth period, indicating resilient biomass after each media reset. Slopes of nitrate vs. time were negative in both reactors (Media-1: −0.00269; Media-2: −0.00431 mg/L), consistent with acclimation. Improved nitrite reduction supported the conclusion that repeated ZVI exposure enhanced bacterial nitrate-reducing mechanisms. These results support ZVI derived H₂ as a scalable, affordable driver for biological denitrification, particularly where chemical electron donors are impractical. Cost comparisons and long-term stability merit further work.