High Pressure Hydrogenotrophic Denitrification (HPHD) provided a promising alternative for efficient and clean nitrate removal. In particular, the denitrification rates at low temperature could be compensated by elevated H₂ partial pressure. However, nitrite reduction was strongly inhibited while nitrate reduction was barely affected at low temperature. In this study, the nitrate reduction gradually recovered under long-term low temperature stress, while nitrite accumulation increased from 0.1 to 41.0 mg N/L. The activities of the electron transport system (ETS), nitrate reductase (NAR), and nitrite reductase (NIR) decreased by 45.8 %, 27.3 %, and 39.3 %, respectively, as the temperature dropped from 30 °C to 15 °C. Real time quantitative PCR analysis revealed that the denitrifying gene expression rather than gene abundance regulated nitrogen biotransformation. The substantial nitrite accumulation was attributed to the significant up-regulation by 54.7 % of narG gene expression and down-regulation by 73.7 % of nirS gene expression in hydrogenotrophic denitrifiers. In addition, the nirS-gene-bearing denitrifiers were more sensitive to low temperature compared to those bearing nirK gene. The dominant populations shifted from the genera Paracoccus to Hydrogenophaga under long-term low temperature stress. Overall, this study revealed the microbial mechanism of high nitrite accumulation in hydrogenotrophic denitrification at low temperature.

In the envisaged hydrogen economy, H₂ could be an interesting alternative electron donor for the denitrification of drinking water or wastewater. The main obstacle to engineering the hydrogenotrophic denitrification process is the low solubility of H₂ in water under atmospheric pressure, which limits denitrification rate and nitrogen removal efficiency. In this paper, we demonstrated a novel configuration of hydrogenotrophic denitrification, namely High Pressure Hydrogenotrophic Denitrification (HPHD). Elevated H₂ partial pressure (pH₂) was employed to increase dissolved H₂ concentration and concomitantly enhance denitrification rate. Our results showed that the specific denitrification rate increased from 9.6 mg N/(gVSS·h) at 0.5 bars to 51.0 mg N/(gVSS·h) at 9 bars in HPHD. The denitrification effect could be retained with elevated pH₂ at a low temperature. The specific denitrification rate at 3 bars and 15 °C was 20.5 mg N/(gVSS·h), approximately 1.5 times that at 1 bar and 30 °C, which was quite beneficial for hydrogenotrophic denitrification under cold conditions. Different from nitrite reduction, less impact was observed on nitrate reduction by low temperature, which explained high nitrite accumulation in HPHD at 15 °C. Overall, our investigations shed light on the role of pH₂ in the promising solution for nitrogen removal in HPHD.