Hailin Tian

Acclimation to extremely high ammonia levels in continuous biomethanation process and the associated microbial community dynamics

Acclimatized anaerobic communities to high ammonia levels can offer a solution to the ammonia toxicity problem in biogas reactors. In the current study, a stepwise acclimation strategy up to 10g NH4+-N L-1, was performed in mesophilic (37±1°C) continuously stirred tank reactors. The reactors were co-digesting (20/80 based on volatile solid) cattle slurry and microalgae, a protein-rich, 3rd generation biomass. Throughout the acclimation period, methane production was stable with more than 95% of the uninhibited yield. Next generation 16S rRNA gene sequencing revealed a dramatic microbiome change throughout the ammonia acclimation process. Clostridium ultunense, a syntrophic acetate oxidizing bacteria, increased significantly alongside with hydrogenotrophic methanogen Methanoculleus spp., indicating strong hydrogenotrophic methanogenic activity at extreme ammonia levels (>7g NH4+-N L-1). Overall, this study demonstrated for the first time that acclimation of methanogenic communities to extreme ammonia levels in continuous AD process is possible, by developing a specialised acclimation AD microbiome.

Different cultivation methods to acclimatise ammonia-tolerant methanogenic consortia

Bioaugmentation with ammonia tolerant-methanogenic consortia was proposed as a solution to overcome ammonia inhibition during anaerobic digestion process recently. However, appropriate technology to generate ammonia tolerant methanogenic consortia is still lacking. In this study, three basic reactors (i.e. batch, fed-batch and continuous stirred-tank reactors (CSTR)) operated at mesophilic (37°C) and thermophilic (55°C) conditions were assessed, based on methane production efficiency, incubation time, TAN/FAN (total ammonium nitrogen/free ammonia nitrogen) levels and maximum methanogenic activity. Overall, fed-batch cultivation was clearly the most efficient method compared to batch and CSTR. Specifically, by saving incubation time up to 150%, fed-batch reactors were acclimatised to nearly 2-fold higher FAN levels with a 37%-153% methanogenic activity improvement, compared to batch method. Meanwhile, CSTR reactors were inhibited at lower ammonia levels. Finally, specific methanogenic activity test showed that hydrogenotrophic methanogens were more active than aceticlastic methanogens in all FAN levels above 540mgNH3-NL-1.

16s rRNA gene sequencing and radioisotopic analysis reveal the composition of ammonia acclimatized methanogenic consortia

Different mesophilic and thermophilic methanogenic consortia were acclimatised and enriched to extreme total ammonia (9.0 and 5.0 g NH4+-N L-1, respectively) and free ammonia (1.0 and 1.4 g NH3-N L-1, respectively) levels in this study. [2-14C] acetate radioisotopic analyses showed the dominance of aceticlastic methanogenesis in all enriched consortia. According to 16S rRNA gene sequencing result, in mesophilic consortia, methylotrophic Methanomassiliicoccus luminyensis was predominant, followed by aceticlastic Methanosarcina soligelidi. A possible scenario explaining the dominance of M. luminyensis includes the use of methylamine produced by Tissierella spp. and biomass build-up by metabolizing acetate. Nevertheless, further studies are needed to pinpoint the exact metabolic pathway of M. luminyensis. In thermophilic consortia, aceticlastic Methanosarcina thermophila was the sole dominant methanogen. Overall, results derived from this study demonstrated the efficient biomethanation ability of these ammonia-tolerant methanogenic consortia, indicating a potential application of these consortia to solve ammonia toxicity problems in future full-scale reactors.