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Identification of syntrophic acetate-oxidizing bacteria in anaerobic digesters by combined protein-based stable isotope probing and metagenomics.

ABSTRACT: Inhibition of anaerobic digestion through accumulation of volatile fatty acids occasionally occurs as the result of unbalanced growth between acidogenic bacteria and methanogens. A fast recovery is a prerequisite for establishing an economical production of biogas. However, very little is known about the microorganisms facilitating this recovery. In this study, we investigated the organisms involved by a novel approach of mapping protein-stable isotope probing (protein-SIP) onto a binned metagenome. Under simulation of acetate accumulation conditions, formations of (13)C-labeled CO2 and CH4 were detected immediately following incubation with [U-(13)C]acetate, indicating high turnover rate of acetate. The identified (13)C-labeled peptides were mapped onto a binned metagenome for improved identification of the organisms involved. The results revealed that Methanosarcina and Methanoculleus were actively involved in acetate turnover, as were five subspecies of Clostridia. The acetate-consuming organisms affiliating with Clostridia all contained the FTFHS gene for formyltetrahydrofolate synthetase, a key enzyme for reductive acetogenesis, indicating that these organisms are possible syntrophic acetate-oxidizing (SAO) bacteria that can facilitate acetate consumption via SAO, coupled with hydrogenotrophic methanogenesis (SAO-HM). This study represents the first study applying protein-SIP for analysis of complex biogas samples, a promising method for identifying key microorganisms utilizing specific pathways.

SUBMITTER: Mosbæk F

PROVIDER: S-EPMC5030692 | biostudies-literature | 2016 Oct

REPOSITORIES: biostudies-literature

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Identification of syntrophic acetate-oxidizing bacteria in anaerobic digesters by combined protein-based stable isotope probing and metagenomics.

Mosbæk Freya F Kjeldal Henrik H Mulat Daniel G DG Albertsen Mads M Ward Alastair J AJ Feilberg Anders A Nielsen Jeppe L JL

The ISME journal 20160429 10

Inhibition of anaerobic digestion through accumulation of volatile fatty acids occasionally occurs as the result of unbalanced growth between acidogenic bacteria and methanogens. A fast recovery is a prerequisite for establishing an economical production of biogas. However, very little is known about the microorganisms facilitating this recovery. In this study, we investigated the organisms involved by a novel approach of mapping protein-stable isotope probing (protein-SIP) onto a binned metagen ...[more]

PMID: 27128991

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Project description: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 >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 minus 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.

Dataset Information

Identification of syntrophic acetate-oxidizing bacteria in anaerobic digesters by combined protein-based stable isotope probing and metagenomics.

Publications

Identification of syntrophic acetate-oxidizing bacteria in anaerobic digesters by combined protein-based stable isotope probing and metagenomics.

Similar Datasets

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