pH control and microbial community analysis with HCl or CO₂ addition in H₂-based autotrophic denitrification

H₂-based autotrophic denitrification is promising to remove nitrate from water or wastewater lacking organic carbon sources, and pH is one of its most important process parameters. HCl and CO₂ addition are known as adequate pH control methods for practical purposes. However, because of H₂, added CO₂ may participate in microbial metabolisms and affect denitrification mechanisms. Here, a combined micro-electrolysis and autotrophic denitrification (CEAD) reactor, in which H₂ is generated based on galvanic-cell reactions between zero-valent iron and carbon, was optimized and continuously operated for 233 days by adding HCl or CO₂ to control pH in the range of 7.2-8.2. Microbial communities were compared between the two pH-control methods through high-throughput sequencing of 16S rRNA, nirS, and nirK genes. Under a low COD/N ratio of 0.5 in the influent (with ∼36 mgNO₃^--N/L), when adding HCl, the total nitrogen (TN) removal efficiency reached 91.4% ± 0.9% with a 28-h hydraulic retention time (HRT). When adding CO₂, the TN removal efficiency was improved to 96.5% ± 1.7% with 24-h HRT. Significant differences of 16S rRNA and nirS genes between the two pH-control stages indicated the variation of microbial communities and nirS-type denitrifiers. With HCl addition, Thiobacillus, unclassified Comamonadaceae, Arenimonas, Limnobacter, and Thermomonas, which were reported previously as likely autotrophic or heterotrophic denitrifiers, were most dominant in the biofilms. With CO₂ addition, the biofilms became dominated by Anaerolineaceae and Methylocystaceae (related to organic carbon metabolism), Denitratisoma (likely heterotrophic denitrifier), and uncultured bacteria TK10 and AKYG587. The results suggest that the added CO₂ not only contributed to pH control but also participated in microbial metabolisms. This study provides useful insights into microbial mechanisms and further optimization of H₂-based autotrophic denitrification in water and wastewater treatment.