Metagenomic and cultivation-based description of a syntrophic butyrate-oxidizing bacterium from a thermophilic and high-ammonia biogas process.

Abstract

Background: Ammonia inhibition in anaerobic digestion can lead to butyrate accumulation and reduced methane yield. Despite the importance of syntrophic butyrate oxidation in mitigating this effect, the microorganisms and interactions involved under high-ammonia conditions remain poorly understood. Here, we combine metagenomics and cultivation studies to describe a novel ammonia-tolerant syntrophic butyrate-oxidizing bacterium and its interactions with acetate-oxidizing bacteria and hydrogenotrophic methanogens enriched from a high-ammonia, thermophilic biogas process.

Results: The enrichment culture degraded butyrate at rates of 0.12-0.47 mmol/day. Amplicon sequencing and phylogenetic analyses of a retrieved metagenome-assembled genome (MAG) assigned the putative syntrophic butyrate-oxidizing bacterium (SBOB) to the genus Syntrophothermus, for which we propose the provisional species name 'Candidatus Syntrophothermus ammoniitolerans'. Metagenomic analyses revealed the genomic potential for β-oxidation and essential electron transfer pathways associated with syntrophic energy conservation. Furthermore, one additional MAG (MAG9) possessed a complete β-oxidation pathway but lacked key genes associated with reverse electron transfer, making its role as a SBOB uncertain. Acetate produced during butyrate oxidation was further oxidized by syntrophic acetate-oxidizing bacteria and ultimately converted to methane by hydrogenotrophic methanogens, illustrating a tightly coupled metabolic network that supports butyrate degradation under high-ammonia conditions. Three methanogenic MAGs, affiliated with the genera Methanoculleus and Methanothermobacter, were identified as potential hydrogen- or formate-consuming partners.

Conclusions: Together, these results identify a novel syntrophic butyrate-oxidizing candidate that enables butyrate degradation under high-ammonia conditions via tightly coupled interactions with acetate-oxidizing bacteria and hydrogenotrophic methanogens, sustaining methane production under ammonia stress.