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. 2022 Oct 26;7(5):e0033922.
doi: 10.1128/msystems.00339-22. Epub 2022 Sep 8.

Syntrophic Acetate-Oxidizing Microbial Consortia Enriched from Full-Scale Mesophilic Food Waste Anaerobic Digesters Showing High Biodiversity and Functional Redundancy

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Syntrophic Acetate-Oxidizing Microbial Consortia Enriched from Full-Scale Mesophilic Food Waste Anaerobic Digesters Showing High Biodiversity and Functional Redundancy

Chao Li et al. mSystems. .

Abstract

Syntrophic acetate oxidation (SAO) coupled with hydrogenotrophic methanogenesis (HM) plays a vital role in the anaerobic digestion of protein-rich feedstocks such as food wastes. However, current knowledge of the biodiversity and genetic potential of the involved microbial participants, especially syntrophic acetate-oxidizing bacteria (SAOB), is limited due to the low abundance of these microorganisms and challenges in their isolation. The intent of this study was to enrich and identify potential SAOB. Therefore, we conducted continuous acetate feeding under high ammonia concentrations using two separate inoculum consortia of microorganisms that originated from full-scale mesophilic food waste digesters, which lasted for more than 200 days. Using 16S rRNA gene amplicon and metagenomic analyses, we observed a convergence of the experimental microbial communities during the enrichment regarding taxonomic composition and metabolic functional composition. Stable carbon isotope analyses of biogas indicated that SAO-HM was the dominant methanogenic pathway during the enrichment process. The hydrogenotrophic methanogen Methanoculleus dominated the archaeal community. The enriched SAO community featured high biodiversity and metabolic functional redundancy. By analyzing the metagenome-assembled genomes, the known SAOB Syntrophaceticus schinkii and six uncultured populations were identified to have the genetic potential to perform SAO through the conventional reversed Wood-Ljungdahl pathway, while another six bacteria were found to encode the reversed Wood-Ljungdahl pathway combined with a glycine cleavage system as novel SAOB candidates. These results showed that the food waste anaerobic digesters harbor diverse SAOB and highlighted the importance of the glycine cleavage system for acetate oxidation. IMPORTANCE Syntrophic acetate oxidation to CO2 and H2, together with hydrogenotrophic methanogenesis, contributes to much of the carbon flux in the anaerobic digestion of organic wastes, especially at high ammonia concentrations. A deep understanding of the biodiversity, metabolic genetic potential, and ecology of the SAO community can help to improve biomethane production from wastes for clean energy production. Here, we enriched the SAO-HM functional guild obtained from full-scale food waste anaerobic digesters and recorded dynamic changes in community taxonomic composition and functional profiles. By reconstructing the metabolic pathways, diverse known and novel bacterial members were found to have SAO potential via the reversed Wood-Ljungdahl (WL) pathway alone, or via the reversed WL pathway with a glycine cleavage system (WLP-GCS), and those catalyzing WLP-GCS showed higher microbial abundance. This study revealed the biodiversity and metabolic functional redundancy of SAOB in full-scale anaerobic digester systems and provided inspiration for further genome-centric studies.

Keywords: anaerobic digestion; biodiversity; functional redundancy; metagenomic analysis; methanogenic pathway; syntrophic acetate oxidation.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

FIG 1
FIG 1
Reactor performance during the enrichment process for the CZ reactor (a) and LM reactor (b), represented by concentrations of dissolved ammonium nitrogen (DAN), free ammonia nitrogen (FAN), and pH; gas production and methane content; and concentrations of dissolved organic carbon (DOC) and acetate. The dashed red line indicates the added acetate concentration by the daily feedstock (240 mg of C/L).
FIG 2
FIG 2
Dynamic changes of microbial community diversity and composition in CZ and LM reactors during the enrichment process, determined by 16S rRNA gene amplicon sequencing. Genus-level taxa with any occurrence of >1% are shown. Relative abundance was calculated as log2(reads percentage + 1) for visualization, which was also applied to Fig. 5 and Fig. 6.
FIG 3
FIG 3
PCoA of microbial community structure derived from 16S rRNA gene analysis using weighted UniFrac distance (a) and functional composition derived from metagenomic analysis using Bray-Curtis distance (b). Numbers of KEGG orthogroups enriched (after/before ratio > 2.0) or degenerated (after/before ratio < 0.5) during the enrichment process (c) and KEGG orthogroups specifically participated in energy metabolism (d). The six metagenomes were obtained by sequencing samples taken 2 months before (CZ1 and LM1, from the full-scale digesters), just at the start (CZ2 and LM2, from the full-scale digesters), and after (CZ3 and LM3, from the lab-scale reactors) the enrichment process. Functional category information was obtained by referring to the KEGG BRITE database.
FIG 4
FIG 4
Simultaneous monitoring of stable carbon isotope signature of biogas in lab-scale acetate-fed reactors and full-scale food waste digesters providing the inoculating microbiota at the same period. A green dotted line was added to highlight the specific value of αC (1.065) to distinguish the predominance of hydrogenotrophic methanogenesis. Day 0 represented initiation of the lab-scale reactors, and the biogas samples taken together with the inoculating microbiota from the full-scale digesters on that date were analyzed.
FIG 5
FIG 5
(a) Methanogenic pathways detected in the metagenomes and the abundance change of enzyme-encoding genes before and after the enrichment (represented by the after/before ratio). For enzyme encoded by more than one gene, it was denoted as colored triangles if the after/before ratio of all corresponding essential genes were >2; but as long as the after/before ratio of one essential gene was <0.5, it was labeled with an inverted color triangle. Colored boxes are used in cases that are different from the aforementioned two situations (details can be found in Data Set S3). (b) Phylogenetic tree of 10 archaeal MAGs (completeness > 80% and contamination < 5%). The relative abundance of each MAG in the two digester groups, before and after the enrichment, is shown in the heatmap. Existence data for genes associated with different methanogenic pathways in these MAGs are shown as colored rectangles.
FIG 6
FIG 6
Phylogenetic tree of 146 bacterial MAGs (completeness > 80% and contamination < 5%). SAOB candidates are labeled with a star. The relative abundance of each MAG in the two digester groups, before and after the enrichment, is shown in the heatmap.
FIG 7
FIG 7
Acetate oxidation metabolism through (a) the reversed Wood-Ljungdahl (WL) pathway, and (b) the reversed Wood-Ljungdahl pathway combined with the glycine cleavage system (WLP-GCS) reconstructed from the MAGs of SAO community. The existence of related genes in these MAGs is indicated by colored rectangles. The numbers next to the genes represent the reaction steps in the pathway; details are provided in Data Set S3. The relative abundance of SAOB candidates based on metagenomic analysis (see the bottom axis) is expressed by a rectangle (open rectangles for before the enrichment and solid rectangles for after the enrichment), while the after/before ratio of the relative abundance (see the top axis) is indicated by a triangle (red triangles for >1 and green triangles for <1).

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