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Using DNA-based stable isotope probing to reveal novel propionate- and acetate-oxidizing bacteria in propionate-fed mesophilic anaerobic chemostats.

ABSTRACT: Propionate is one of the most important intermediates of anaerobic fermentation. Its oxidation performed by syntrophic propionate-oxidizing bacteria coupled with hydrogenotrophic methanogens is considered to be a rate-limiting step for methane production. However, the current understanding of SPOB is limited due to the difficulty of pure culture isolation. In the present study, two anaerobic chemostats fed with propionate as the sole carbon source were operated at different dilution rates (0.05 d-1 and 0.15 d-1). The propionate- and acetate-oxidizing bacteria in the two methanogenic chemostats were investigated combining DNA-stable isotope probing (DNA-SIP) and 16S rRNA gene high-throughput sequencing. The results of DNA-SIP with 13C-propionate/acetate suggested that, Smithella, Syntrophobacter, Cryptanaerobacter, and unclassified Rhodospirillaceae may be putative propionate-oxidizing bacteria; unclassified Spirochaetaceae, unclassified Synergistaceae, unclassified Elusimicrobia, Mesotoga, and Gracilibacter may contribute to acetate oxidation; unclassified Syntrophaceae and Syntrophomonas may be butyrate oxidizers. By DNA-SIP, unclassified OTUs with 16S rRNA gene abundance higher than 62% of total Bacteria in the PL chemostat and 38% in the PH chemostat were revealed to be related to the degradation of propionate. These results suggest that a variety of uncultured bacteria contribute to propionate degradation during anaerobic digestion. The functions and metabolic characteristics of these bacteria require further investigation.

SUBMITTER: Wang HZ

PROVIDER: S-EPMC6874663 | biostudies-literature | 2019 Nov

REPOSITORIES: biostudies-literature

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Using DNA-based stable isotope probing to reveal novel propionate- and acetate-oxidizing bacteria in propionate-fed mesophilic anaerobic chemostats.

Wang Hui-Zhong HZ Lv Xiao-Meng XM Yi Yue Y Zheng Dan D Gou Min M Nie Yong Y Hu Bing B Nobu Masaru K MK Narihiro Takashi T Tang Yue-Qin YQ

Scientific reports 20191122 1

Propionate is one of the most important intermediates of anaerobic fermentation. Its oxidation performed by syntrophic propionate-oxidizing bacteria coupled with hydrogenotrophic methanogens is considered to be a rate-limiting step for methane production. However, the current understanding of SPOB is limited due to the difficulty of pure culture isolation. In the present study, two anaerobic chemostats fed with propionate as the sole carbon source were operated at different dilution rates (0.05 ...[more]

PMID: 31758023

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Project description:Microbial inhibition by high ammonia concentrations is a recurring problem that significantly restricts methane formation from intermediate acids, i.e., propionate and acetate, during anaerobic digestion of protein-rich waste material. Studying the syntrophic communities that perform acid conversion is challenging, due to their relatively low abundance within the microbial communities typically found in biogas processes and disruption of their cooperative behavior in pure cultures. To overcome these limitations, this study examined growth parameters and microbial community dynamics of highly enriched mesophilic and ammonia-tolerant syntrophic propionate and acetate-oxidizing communities and analyzed their metabolic activity and cooperative behavior using metagenomic and metatranscriptomic approaches. Cultivation in batch set-up demonstrated biphasic utilization of propionate, wherein acetate accumulated and underwent oxidation before complete degradation of propionate. Three key species for syntrophic acid degradation were inferred from genomic sequence information and gene expression: a syntrophic propionate-oxidizing bacterium (SPOB) "Candidatus Syntrophopropionicum ammoniitolerans", a syntrophic acetate-oxidizing bacterium (SAOB) Syntrophaceticus schinkii and a novel hydrogenotrophic methanogen, for which we propose the provisional name "Candidatus Methanoculleus ammoniitolerans". The results revealed consistent transcriptional profiles of the SAOB and the methanogen both during propionate and acetate oxidation, regardless of the presence of an active propionate oxidizer. Gene expression indicated versatile capabilities of the two syntrophic bacteria, utilizing both molecular hydrogen and formate as an outlet for reducing equivalents formed during acid oxidation, while conserving energy through build-up of sodium/proton motive force. The methanogen used hydrogen and formate as electron sources. Furthermore, results of the present study provided a framework for future research into ammonia tolerance, mobility, aggregate formation and interspecies cooperation.

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.

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Dataset Information

Using DNA-based stable isotope probing to reveal novel propionate- and acetate-oxidizing bacteria in propionate-fed mesophilic anaerobic chemostats.

Publications

Using DNA-based stable isotope probing to reveal novel propionate- and acetate-oxidizing bacteria in propionate-fed mesophilic anaerobic chemostats.

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