Recombinant glutamine synthetase originating from high ammonium-assimilating Bacillus subtilis effectively reduces NH3 emissions and enhances the conversion into amino acids in liquid manure

Abstract

Excessive ammonia (NH₃) emissions from livestock manure contribute to environmental pollution and nitrogen (N) loss, yet effective mitigation strategies remain limited. In this study, Bacillus strains capable of utilizing ammonia nitrogen (NH₄⁺) were screened from 166 probiotic strains. Nine strains exhibited utilization rates above 65.0 %, with B. subtilis M246 exceeding 80.0 %. Genome sequencing revealed the absence of key nitrification genes (e.g., amoCAB), suggesting NH₄⁺ is metabolized primarily through assimilation via the GS/GOGAT pathway. The glnA gene, encoding glutamine synthetase (GS), was identified as central to this process but was transcriptionally repressed under high NH₄⁺ concentrations (100 mM). To overcome this, glnA was heterologously expressed in Pichia pastoris, and the recombinant GS applied to liquid manure. GS treatments reduced NH₃ emissions by 19.57 %–45.91 %, outperforming M246 (6.45 %), and significantly decreasing total nitrogen loss (18.53 %–24.30 %) compared to CK (32.71 %, p < 0.001) and M246 (29.31 %, p < 0.001 for 1 % and 5 % GS; p = 0.003 for 2 % GS). Moreover, GS reduced nitrous oxide emissions by 18.29 %–33.59 % relative to CK, and increased readily degradable proteins, promoting their conversion into humic substances and significantly elevating glutamine (p < 0.001) and glutamate (p = 0.001). GS significantly enriched microbial genera associated with N retention and mitigated emissions (p = 0.049); Acholeplasma and Sedimentibacter were enriched in both GS and M246 treatments, while Sphaerochaeta was specific to GS. Therefore, exogenous GS effectively reduces gaseous emissions, enhances N retention, promotes NH₄⁺ conversion into amino acids, and improves the biofertilizer quality of liquid manure.