Distinguishing responses of acetoclastic and hydrogenotrophic methanogens to ammonia stress in mesophilic mixed cultures

Abstract

A shift from the acetoclastic to the hydrogenotrophic pathway in methanogenesis under ammonia inhibition is a common observation in anaerobic digestion. However, there are still considerable knowledge gaps concerning the differential ammonia tolerance of acetoclastic and hydrogenotrophic methanogens (AMs and HMs), their responses to different ammonia species (NH₄⁺, NH₃), and their recoverability after ammonia inhibition. With the successful enrichment of mesophilic AMs and HMs cultures, this study aimed at addressing the above knowledge gaps through batch inhibition/recovery tests and kinetic modeling under varying total ammonia (TAN, 0.2-10 g N/L) and pH (7.0-8.5) conditions. The results showed that the tolerance level of HMs to free ammonia (FAN, IC₅₀=1345 mg N/L) and NH₄⁺ (IC₅₀=6050 mg N/L) was nearly 11 times and 3 times those of AMs (NH₃, IC₅₀=123 mg N/L; NH₄⁺, IC₅₀=2133 mg N/L), respectively. Consistent with general belief, the AMs were more impacted by FAN. However, the HMs were more adversely affected by NH₄⁺ when the pH was ≤8.0. A low TAN (1.0-4.0 g N/L) could cause irreversible inhibition of the AMs due to significant cell death, whereas the activity of HMs could be fully or even over recovered from severe ammonia stress (FAN≤ 0.9 g N/L or TAN≤10 g N/L; pH ≤8.0). The different tolerance responses of AMs and HMs might be associated with the cell morphology, multiple energy-converting systems, and Gibbs free energy from substrate-level phosphorylation.

Keywords: Acetoclastic methanogens; Ammonia inhibition; Anaerobic digestion; Hydrogenotrophic methanogens; Recovery.