DOI: 10.25345/c54m91m58
title: MassIVE MSV000091906 - Stable isotope informed metaproteogenomics of an anaerobic syntrophic acetate oxidizing consortium
title: Project description In many anoxic environments, syntrophic acetate oxidation (SAO) is a key pathway mediating the conversion of acetate into methane, and is accomplished through obligate mutualistic cross-feeding between SAO bacteria and methanogenic archaea. This pathway is particularly important in anaerobic digestion (AD) systems operating at thermophilic temperatures and/or with high ammonia. Despite the widespread importance of the SAO functional guild to the stability and efficiency of the AD process, little is known about their in situ ecophysiologies due to their difficulty of isolation as well as low biomass abundances. Here, we performed a long-term (300 day) continuous enrichment of a thermophilic (55C) SAO community from a municipal AD system. Over 80% of the enriched bioreactor metagenome collectively belonged to a three-member consortium, including an acetate-oxidizing DTU068 bacterium encoding for CO2, H2, and formate production, along with two methanogenic Methanothermobacter_A archaeal species. Stable isotope probing was coupled with metaproteogenomics to quantify carbon flux into each community member during acetate conversion and inform metabolic reconstruction and genome-scale modeling. Sample processing After 300 days of chemostat operation, batch microcosms were established in 40 ml glass serum bottles flushed with 80:20 N2:CO2 by anoxically transferring 18 ml of digestate from a single bioreactor (R2) and sealing with butyl rubber septa. Four different incubation conditions were established in triplicate: (1) blank control (e.g. no amendment); (2) 50 mM [12C]-acetate; (3) 50 mM [2 13C]-acetate (e.g. methyl-labeled); (4) 50 mM [1,2 13C]-acetate (universally labeled). Acetate was added to the microcosms (2 ml) as anoxic sterilized basal medium containing [12C], [2 13C], or [1,2 13C] sodium acetate (isotope purity &gt;98%, Cambridge Isotopes). Twelve replicate bottles were established for all universally-labeled acetate-amended microcosms, allowing for three triplicate sets to be sacrificed for protein extraction at 24h, 144 hr, and 408 hr, and a single triplicate set for liquid sampling throughout for VFA analysis. Biomass was pelleted from 10 ml liquid samples via centrifugation (10,000xg) and stored at negatiave 20C until protein extraction. Protein from cell pellet samples were extracted in an 8M urea solution, then reduced and alkylated. Proteins were then digested with trypsin and subsequently desalted using C18 solid phase extraction. MS analysis was performed using 0.1 ug/ul of peptide solution injected into a Q Exactive HF X mass spectrometer (Thermo Scientific). Data processing Mass spectrometry (MS) data for each biological replicate at all time points (n=18) were analyzed using an implementation of OpenMS implemented in KNIME. Briefly, MS/MS spectra were searched using the MS-GF+ tool against a protein database consisting of all ORFs from the de-replicated set of MAGs, concatenated with reversed (decoy) sequences of all protein entries. Peptide spectra matches (PSMs) were filtered at a 5% false discovery rate (FDR) with Percolator.