Metatranscriptomics-guided genome-scale metabolic reconstruction reveals the carbon flux and trophic interaction in methanogenic communities

Abstract

Background: Despite rapid advances in genomic-resolved metagenomics and remarkable explosion of metagenome-assembled genomes (MAGs), the function of uncultivated anaerobic lineages and their interactions in carbon mineralization remain largely uncertain, which has profound implications in biotechnology and biogeochemistry.

Results: In this study, we combined long-read sequencing and metatranscriptomics-guided metabolic reconstruction to provide a genome-wide perspective of carbon mineralization flow from polymers to methane in an anaerobic bioreactor. Our results showed that incorporating long reads resulted in a substantial improvement in the quality of metagenomic assemblies, enabling the effective recovery of 132 high-quality genomes meeting stringent criteria of minimum information about a metagenome-assembled genome (MIMAG). In addition, hybrid assembly obtained 51% more prokaryotic genes in comparison to the short-read-only assembly. Metatranscriptomics-guided metabolic reconstruction unveiled the remarkable metabolic flexibility of several novel Bacteroidales-affiliated bacteria and populations from Mesotoga sp. in scavenging amino acids and sugars. In addition to recovering two circular genomes of previously known but fragmented syntrophic bacteria, two newly identified bacteria within Syntrophales were found to be highly engaged in fatty acid oxidation through syntrophic relationships with dominant methanogens Methanoregulaceae bin.74 and Methanothrix sp. bin.206. The activity of bin.206 preferring acetate as substrate exceeded that of bin.74 with increasing loading, reinforcing the substrate determinantal role.

Conclusion: Overall, our study uncovered some key active anaerobic lineages and their metabolic functions in this complex anaerobic ecosystem, offering a framework for understanding carbon transformations in anaerobic digestion. These findings advance the understanding of metabolic activities and trophic interactions between anaerobic guilds, providing foundational insights into carbon flux within both engineered and natural ecosystems. Video Abstract.

Keywords: Anaerobic digestion; Long reads; Omics; Reverse electron transfer; Syntrophic bacteria.

Fig. 1

Long-read sequencing improved metagenomic assembly and recovered hidden genetic information in the methanogenic community. A–C The log-scale distribution of contig number and lengths for short-reads-only approach (SR-only) and hybrid assembly (Hybrid). The dashed lines indicate median values. D–F The recovery of repeat regions and predicted genes as well as the proportion of full-length genes in the assembled metagenome-assembled genomes (MAGs) by short-reads-based method (SR-only) and hybrid assembly (Hybrid). G The genomic information of MAG at strain-level by hybrid assembly. H The cumulative relative abundances of high-quality genomes in the anaerobic community. I Unclassified MAGs at different taxonomic levels by GTDB (214 version)

Fig. 2

Phylogenetic genome tree showing the diversity, abundance, and genome quality of the bacterial lineages. The tree is constructed based on the concatenated alignment of 120 single-copy bacterial-specific marker genes by GTDB-Tk. Outer bands with different colors show the phyla of all the bacterial lineages. Inner heatmap indicates the maximum relative abundance of the MAGs in different sampling points. Bar charts and heatmap strips from the inner to the outer sections indicate the genome quality of each MAG. Other detailed information is provided in Supplementary Data 3

Fig. 3

The distribution of metabolic pathways and profiles of functional systems in the top 26 species representatives with a relative transcriptional expression of over 1% in at least one sample of metatranscriptomics. Pathways are considered present only if 100% of steps were identified in a MAG (except 90% for methylmalonyl-CoA pathway). Bar chart demonstrates the number of MAGs encoding the metabolic pathways and energy modules. Heatmap indicates the variation of relative transcriptional expression of each MAG across increased organic loadings

Fig. 4

Carbon flux from (poly) monomers to CH₄ and CO₂ across the organic loading gradient. Headers in the boxes and the line chart represent degradation modules and specific pathways, respectively. Line chart shows the variations in transcriptional activity of anaerobic populations responding to organic loadings. The lines colors represent the species in different phyla. The large circles under line charts colored by organic loadings encompass smaller circles (MAG abundance colored by phylum) represent MAGs encoding the targeted pathways. Circle size indicates MAG relative abundance. Thickness of lines connecting different modules represents the summed transcriptional expression of specific pathways in different MAGs

Fig. 5

Metabolic reconstruction in the key novel syntrophic fatty acid-oxidizing bacteria and their interactions with the most active methanogens. Only the long- and short-chain fatty acid oxidation pathways, the reverse electron transfer mechanism and transporters are presented in the syntrophic bacteria. Light pink and violet cell cartoons present two predominant methanogens of Methanothrix (bin.206) preferring acetate and Methanoregulaceae (bin.74) consuming H₂ and formate generated by the bacteria. The other detailed information of pathway reconstructions is provided in Supplementary Data S12

Fig. 6

Transcriptional expression and reconstructions of the methanogenic pathways in the archaeal communities and MAGs. A Methanogenesis pathway reconstruction of the archaeal MAGs. B The change of transcriptional activities of four methanogenesis pathway with the increased organic loadings. C The change of relative abundance (normalized to the archaeal community) in Methanoregulaceae bin.74 and Methanothrix bin.206. D The change of transcriptional activities of Methanoregulaceae bin.74 and Methanothrix bin.206. E Methanogenesis pathway reconstruction of three representative archaeal MAGs. H15, H7, H4, H2, and H1 means the hydraulic retention times of 15, 7, 4, 2, and 1 day, respectively, and the organic loadings increased with the shortened hydraulic retention times