Novel syntrophic bacteria in full-scale anaerobic digesters revealed by genome-centric metatranscriptomics

Abstract

Short-chain fatty acid (SCFA) degradation is an important process in methanogenic ecosystems, and is usually catalyzed by SCFA-oxidizing bacteria in syntrophy with methanogens. Current knowledge of this functional guild is mainly based on isolates or enrichment cultures, but these may not reflect the true diversity and in situ activities of the syntrophs predominating in full-scale systems. Here we obtained 182 medium to high quality metagenome-assembled genomes (MAGs) from the microbiome of two full-scale anaerobic digesters. The transcriptomic response of individual MAG was studied after stimulation with low concentrations of acetate, propionate, or butyrate, separately. The most pronounced response to butyrate was observed for two MAGs of the recently described genus Candidatus Phosphitivorax (phylum Desulfobacterota), expressing a butyrate beta-oxidation pathway. For propionate, the largest response was observed for an MAG of a novel genus in the family Pelotomaculaceae, transcribing a methylmalonyl-CoA pathway. All three species were common in anaerobic digesters at Danish wastewater treatment plants as shown by amplicon analysis, and this is the first time their syntrophic features involved in SCFA oxidation were revealed with transcriptomic evidence. Further, they also possessed unique genomic features undescribed in well-characterized syntrophs, including the metabolic pathways for phosphite oxidation, nitrite and sulfate reduction.

Fig. 1. Concentration of acids and net production of methane before and after the SCFA stimuli events.

Sludge slurries originating from digesters at Fredericia and Randers were used. The vertical dotted lines indicate the time points where samples were retrieved for transcriptomic analysis. Distilled water was added instead of SCFA solution for the control. Net methane production from the added SCFAs was calculated by subtracting the average total methane yield of the control reactors from that of the reactors stimulated with SCFAs, and the volume was corrected to standard conditions (20 °C, 1 atm).

Fig. 2. Phylogenetic genome tree of the bacterial MAGs reconstructed from two full-scale anaerobic digesters at Fredericia (black) and Randers (red).

Bootstraps >70% are indicated by the solid black circles. Tree scale indicates evolutionary distance as rate of substitution per site. The MAGs F70, F81, and R76 are indicated by the stars. The average abundance of each MAG in the corresponding digester is shown in the inner circle heatmap. Completeness and contamination of each MAG determined by CheckM are shown in the blue and red barplots, respectively.

Fig. 3. Transcriptional responses of MAGs after the SCFA stimulation.

To the left is a phylogenetic tree of the bacterial MAGs which demonstrated upregulation of any of the genes directly involved in oxidation of acetate, butyrate, and propionate after stimulation by the corresponding SCFA. It is derived from the overall genome tree in Fig. 2. The heatmap to the right shows the transcriptional responses of genes involved in the bioconversion of SCFAs in the bacterial MAGs. 13, 19, and 31 gene families determined by KEGG ontology (KO) numbers were used, representing the key genes directly involved in the aforementioned pathways, which can catalyze bioconversion in 9, 5, and 5 steps (as defined in Fig. S1 and Data S2). The heatmap is colored according to fold change (FC) and the corresponding p value (not corrected for multiple testing to increase sensitivity) of CDSs for the given KO and are categorized as follows: Downregulated: FC ≤ 0.5 and p value < 0.05; Upregulated: FC ≥ 2 and p value < 0.05; Not regulated: 0.5 < FC < 2 or p > 0.05; Not detected: no expression or not encoded. For each MAG, the corresponding SCFA stimuli and control conditions are shown in the upper- and lower-half of the tile, respectively. The three MAGs in bold red were expressing ≥80% of the bioconversion steps in the associated pathways highlighted in black squares, with ≥3 steps upregulated (two steps for R76). The columns represent different genes (KOs), which are clustered into steps based on similar functions in the pathway.

Fig. 4. Metabolic pathways in Ca. Propionivorax syntrophicum F70, Ca. Phosphitivorax anaerolimi F81 and Ca. Phosphitivorax butyraticus R76, and their interactions with methanogens.

The pathways are constructed based on the annotated genome sequences (Data S4). Orange and green cell cartoons symbolize MAGs of Methanothrix (F94, R97, R101, R103) consuming acetate and MAGs of Methanoculleus (F93, F95, R98, R99) and Methanoregulaceae (R100) utilizing H₂ and formate produced by the bacteria. Only the SCFA oxidation pathways with the related electron-transfer systems, transporters, and the potential chemolithotrophic metabolisms are shown for the bacterial MAGs. A more detailed metabolic description is provided in Fig. S3. The reactions and uni-transporters highlighted in green indicate that expressions of the related genes were upregulated after addition of the corresponding SCFA, with fold change > 2 and p value < 0.05. Rnf: Rhodobacter nitrogen fixation complex; Nfn: NADH-dependent Fd_red:NADP⁺ oxidoreductase.

Fig. 5. Relative abundance of known and novel syntrophic bacterial genera in (a) full-scale digesters and (b) the SCFA stimulated reactors.

Data are based on 16S rRNA gene amplicon sequencing of DNA extracted from digester slurry. The full-scale data were based upon 103 samples collected from 30 full-scale digesters at 18 Danish WWTPs in 2016 year with 2 to 12 samples per digester [8]. The numbers in the heatmap are average relative read abundance in percentage for each WWTP. The THP digesters are operated at mesophilic conditions, but the substrates are thermally hydrolyzed prior to anaerobic digestion. The lab-scale data were derived from analyses on the inoculum and biomass sampled 2 h after each SCFA addition, with two samples for each condition. Taxonomic classification was based on the SILVA taxonomy. The novel syntrophic genera described in this study are highlighted in red.