Effects of free ammonia nitrogen on in-situ biomethanation and microbial communities

Abstract

This study aims to evaluate the impact of free ammonia on in-situ biomethanation process using submerged biofilm reactors and accesses the resistance and changes in the microbial communities. Specifically, the study focuses on susceptibility of acetoclastic methanogenesis.

In this study, ammonia inhibition on in-situ biomethanation process is investigated using cattle manure-based inoculum. A batch assay was conducted using 500 ml of inoculum at thermophilic temperature (51oC), while nine bottles filled with 140-gram carrier materials to establish submerged biofilm. In-situ biomethanation was performed using a mixture of CO2 and H2 in a ratio 4:1 (volume based). After a 4-weeks of adaption period, ammonia was added to maintain free ammonia concentration at 0, 2.5 and 5 g/L in the submerged biofilm reactor, respectively. Additionally, reactors (with added ammonia but no carrier materials) and blank reactors (inoculum only) were prepared. Gas consumption and composition (H2, CH4, CO2) were monitored throughout the experiment.

Literature suggests that acetoclastic methanogens are particularly susceptible to ammonia, prompting carbon isotope analysis to evaluate potential pathway shifts. Results indicated that under ammonia inhibition (5 g/L), the process shifted towards hydrogenotrophic methanogenesis (α_C > 1.065), although this pathway was not dominant due to the relatively short experimental period. Similarly, reactors without ammonia injection showed an average ratio of 1.032, slightly above the range for acetoclastic pathways (1.025), indicating significant impact from ammonia inhibition on methanogenic pathways.

The addition of ammonia up to 5 g/L demonstrated strong inhibition of in-situ biomethanation. Batch assays without ammonia achieved up to 63% CH4 production, while those with ammonia of 5 g/L reached only around 33%. The submerged biofilm reactor helped alleviate inhibition, with normalized CH4 production reaching 33% in the reactor with 5 g/L ammonia, which is 8% higher than the one without biocarriers. Similarly, in reactors with carriers and ammonia up to 2.5 g/L, CH4 production reached around 36%.

Future studies should focus on the long-term impact of free ammonia and strategies for recovering from ammonia inhibition in biomethanation processes