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

Abstract

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.