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

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.

was 1 mM at the start of the run for the first 13 min; then it was increased steadily to 3 mM until 26 23 min; subsequently, the concentration was raised to 60 mM at 28 min and kept at this value for 5 min, 24 and then decreased to 1 mM until the end of the program. With this program, nine organic acids 25 commonly produced in anaerobic digestion processes could be analyzed, including: formate, acetate, 26 propionate, butyrate, isobutyrate, valerate, isovalerate, lactate, and hexanoate, and the detection limit 27 was 10 μM.

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Methane production. Methane yield from all the reactors during the 3-day incubation was 29 continuously recorded by the AMPTS II, for which the volume was corrected to standard conditions 30 (20°C, 1 atm). Net methane production from the added SCFAs was calculated by subtracting the 31 average total methane yield of the control reactors from that of the reactors stimulated with SCFAs. 32 33 Two slurry samples from Fredericia (indicated by F-) and five from Randers (indicated by R-) were 34 used for metagenomics studies. F1 and R1 were taken from the SCFA stimulation experiment; F2 35 and R2 were taken from the full-scale digesters 2 months before the SCFA stimulation study; R3, 4, 36 5 derived from the full-scale digester at earlier different time points (Data S1). 37 DNA extraction. DNA used for sequencing was extracted using the FastDNA Spin kit for soil (MP 38 Biomedicals, Santa Ana, CA, USA) as previously described (Kirkegaard et al., 2016). For each of F1, 39 2 and R1, 2, DNA was extracted at two different bead-beating intensities (Protocol 1: 4 m/s for 20 s; sequences were dereplicated using -fastx_uniques with -sizeout -relabel Uniq. Exact amplicon 124 sequence variants (ASVs) were generated using -unoise3 (Edgar, 2016). ASV-tables were created by 125 mapping the raw reads to the ASVs using -otutab with the -zotus and -strand both options. Taxonomy 126 was assigned to ASVs using -sintax with -strand both and -sintax_cutoff 0.8 (Edgar, 2018) with the 127 MiDAS 3.5 reference database (Nierychlo et al., (manuscript in preparation)). Amplicons were 128 mapped to full-length 16S rRNA gene sequences associated with the three MAGs using -129 usearch_global with -id 0.945 -strand both to link ASVs with MAGs (Edgar, 2010). The data was 130 further analyzed in R (R Core Team, 2017) using Ampvis2 (Andersen et al., 2018). The closest relatives of MAGs F70, F81, and R76 were identified by blasting their genomes and 16S 157 rRNA gene sequences in GTDB and NCBI nucleotide database. These genomes, together with those 158 of the known syntrophs, were further compared to search for similarities and differences, as described 159 below.    varied accessory metabolisms for these two species. However, the lack of such "unique" genes could 220 also be due to the incompleteness of the MAGs.

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Methylmalonyl-CoA pathway. The oxaloacetate decarboxylation step can also be performed via a 252 membrane-bound oxaloacetate decarboxylase complex, which extrudes two sodium ions out of the 253 cell while decarboxylating oxaloacetate to pyruvate with proton motive force (PMF) produced, but 254 genes encoding this complex were not highly expressed at any of the studied conditions (Data S4).  with the ones found in Pelotomaculum schinkii, and indicated that they could be other potential PMF 297 generators. However, translocation of protons during menaquinol re-oxidation step could also be 298 linked to succinate oxidation. Succinate oxidation is a thermodynamically unfavorable reaction. It 299 has been generally accepted that a proton gradient across the membrane is needed, which is produced 300 by the hydrolysis of two-thirds of a molecule of ATP for reverse electron transport (RET). In this 301 process, coupling of the electron transfer via menaquinone/menaquinol with the inward movement of 302 protons, catalyzes the production of formate (and/or) H2 outside the cytoplasmic membrane (Stams 303 and Plugge, 2009). Therefore, whether the Hyb-and Fdn-containing enzyme complexes function as 304 PMF generator or consumer need to be further verified.

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The ATP synthase could also function in reverse to hydrolyze ATP and export protons, thereby 306 driving the PMF-requiring reactions like succinate oxidation or rotation of flagellum and pili.        whereby dissimilatory phosphite oxidation drives CO2 reduction to formate, which is then assimilated 378 into biomass via RGP, as described for strain Phox-21 (Figueroa et al., 2018).

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The WLP-related genes in F70 were only expressed and highly upregulated (especially the folD and 380 fhs) with propionate, but were hardly expressed with acetate, indicating that they might not function 381 for SAO, but were probably involved in formate utilization which was produced from SPO. In 382 contrast, the WLP-and RGP-related genes in F81 and R76 were not highly expressed under any 383 condition, except for one acetyl-CoA synthetase (acsE) which was also involved in SBO. Thus, the 384 assimilation of inorganic carbon might be inactive when sufficient butyrate is available.    Table S1. Characteristics of the two anaerobic digesters investigated. The two full-scale 425 anaerobic digesters investigated are located at municipal WWTPs at Randers and Fredericia and have 426 been operated for more than 5 years under stable conditions. The digester at Randers WWTP treats  Fred-9 56 FredS12 Fred-12 69 FredCo1 Fred-3, 6, 9, 12 135 FredCo2 Fred-3, 6, 9, 12 128 Randers RandS1 Rand-1 73 1007 103