Syntrophic entanglements for propionate and acetate oxidation under thermophilic and high-ammonia conditions

Abstract

Propionate is a key intermediate in anaerobic digestion processes and often accumulates in association with perturbations, such as elevated levels of ammonia. Under such conditions, syntrophic ammonia-tolerant microorganisms play a key role in propionate degradation. Despite their importance, little is known about these syntrophic microorganisms and their cross-species interactions. Here, we present metagenomes and metatranscriptomic data for novel thermophilic and ammonia-tolerant syntrophic bacteria and the partner methanogens enriched in propionate-fed reactors. A metagenome for a novel bacterium for which we propose the provisional name 'Candidatus Thermosyntrophopropionicum ammoniitolerans' was recovered, together with mapping of its highly expressed methylmalonyl-CoA pathway for syntrophic propionate degradation. Acetate was degraded by a novel thermophilic syntrophic acetate-oxidising candidate bacterium. Electron removal associated with syntrophic propionate and acetate oxidation was mediated by the hydrogen/formate-utilising methanogens Methanoculleus sp. and Methanothermobacter sp., with the latter observed to be critical for efficient propionate degradation. Similar dependence on Methanothermobacter was not seen for acetate degradation. Expression-based analyses indicated use of both H₂ and formate for electron transfer, including cross-species reciprocation with sulphuric compounds and microbial nanotube-mediated interspecies interactions. Batch cultivation demonstrated degradation rates of up to 0.16 g propionate L^-1 day^-1 at hydrogen partial pressure 4-30 Pa and available energy was around -20 mol^-1 propionate. These observations outline the multiple syntrophic interactions required for propionate oxidation and represent a first step in increasing knowledge of acid accumulation in high-ammonia biogas production systems.

Fig. 1. Microbial community structure resolving the exclusive presence of Pelotomaculaceae in the propionate-fed continuous reactors.

Bubble plot showing percentage relative abundance (>2%) of microbial communities at family level in the acetate-fed (RA1, RA2) and propionate-fed (RP1, RP2) enrichment reactors.

Fig. 2. Metabolic reconstruction of syntrophic propionate and acetate oxidation and the interspecies hydrogen/formate transfer with hydrogenotrophic methanogens employed under thermophilic and high ammonia conditions.

Visualisation of the molecular exchange anchored interplay and metabolic pathways employed by the multiple syntrophic bacteria and their methanogenic partner during syntrophic propionate degradation under thermophilic and high-ammonia conditions. The figure highlighted the cooperation of syntrophic propionate oxidising bacteria (SPOB, MAG4) via acetate assimilation by syntrophic acetate oxidising bacteria (SAOB, MAG9). These SPOB and SAOB further obligately establish formate or hydrogen pivoted syntrophic network to circumvent the reducing potential which is used by hydrogenotrophic methanogen (MAG1) to reduce carbon dioxide and generate methane.

Fig. 3. Gene expression profile of the methylmalonyl CoA pathway for propionate oxidation by the SPOB candidate.

Metatranscriptomics expression profile of the methylmalonyl CoA (MMC) pathway of propionate metabolism (based on transcripts per million (TPM) counts) in propionate versus acetate batch assay (B01, B03 and B09) for the novel syntrophic propionate-oxidising bacteria (SPOB) `Candidatus Thermosyntrophopropionicum ammoniitolerans´ (MAG4). The numerical values with the enzyme name denote the step in the MMC pathway. The values on heatmap represented are the aggregated TPM counts of all copies and subunits for respective gene present and expressed in the metagenome assembled genome.

Fig. 4. Gene expression profile of the Wood-Ljungdahl pathway by the SAOB candidate.

Metatranscriptomics expression profile of the Wood-Ljungdahl pathway and other genes of relevance for acetate metabolism (based on transcripts per million (TPM) counts) in propionate versus acetate batch assay (B01, B03 and B09) for the novel syntrophic acetate-oxidising bacteria (SAOB) MAG9 and for MAG5 belonging to the genus Acetomicrobium. The numerical values with the enzyme name denote the step in the pathway. The values on heatmap represented are the aggregated TPM counts of all copies and subunits for respective gene present and expressed in the metagenome assembled genome.