Abstract
The formyltetrahydrofolate synthetase (FTHFS) gene is a molecular marker of choice to study the diversity of acetogenic communities. However, current analyses are limited due to lack of a high-throughput sequencing approach for FTHFS gene amplicons and a dedicated bioinformatics pipeline for data analysis, including taxonomic annotation and visualization of the sequence data. In the present study, we combined the barcode approach for multiplexed sequencing with unsupervised data analysis to visualize acetogenic community structure. We used samples from a biogas digester to develop proof-of-principle for our combined approach. We successfully generated high-throughput sequence data for the partial FTHFS gene and performed unsupervised data analysis using the novel bioinformatics pipeline “AcetoScan” presented in this study, which resulted in taxonomically annotated OTUs, phylogenetic tree, abundance plots and diversity indices. The results demonstrated that high-throughput sequencing can be used to sequence the FTHFS amplicons from a pool of samples, while the analysis pipeline AcetoScan can be reliably used to process the raw sequence data and visualize acetogenic community structure. The method and analysis pipeline described in this paper can assist in the identification and quantification of known or potentially new acetogens. The AcetoScan pipeline is freely available at https://github.com/abhijeetsingh1704/AcetoScan.
Highlights
Acetogens are a group of bacteria that (1) use the WoodLjungdahl pathway (WLP) for energy conservation, (2) generate acetyl-Coenzyme A (CoA) by reduction of two molecules of carbon dioxide (CO2), (3) may or may not produce acetate as the main end product of carbon fixation by WLP and (4) are obligate anaerobes, with some members having tolerance to periods of aerobiosis (Wagner et al, 1996; Drake and Küsel, 2003; Küsel and Drake, 2011; Schuchmann and Müller, 2016)
For the 80% clustering threshold, only one phylum showed misidentification (3.4% of operational taxonomic units (OTU) belonging to phylum Actinobacteria were misidentified as phylum Firmicutes)
The formyltetrahydrofolate synthetase (FTHFS) OTUs generated in the AcetoScan pipeline do not claim that the detected OTUs is an ‘acetogen,’ and we propose a threestep procedure which must be followed before an OTU can be defined as an acetogen: (1) Identification of the bacterial candidate with the FTHFS gene; (2) identification of the WLP genes in the genome of the candidate; and (3) experimental validation of acetogenic metabolism in an environment which sustains acetogenesis
Summary
Acetogens are a group of bacteria that (1) use the WoodLjungdahl pathway (WLP) for energy conservation, (2) generate acetyl-Coenzyme A (CoA) by reduction of two molecules of carbon dioxide (CO2), (3) may or may not produce acetate as the main end product of carbon fixation by WLP and (4) are obligate anaerobes, with some members having tolerance to periods of aerobiosis (Wagner et al, 1996; Drake and Küsel, 2003; Küsel and Drake, 2011; Schuchmann and Müller, 2016). WLP is one of the most ancient biological pathways known (Pereló et al, 1999; Russell and Martin, 2004) It is used mainly by acetogens, and by some archaea, syntrophic acetateoxidizing bacteria and sulfate-reducing bacteria (Drake, 1994; Drake et al, 1997; Ragsdale and Pierce, 2008; Poehlein et al, 2012; Sakimoto et al, 2016). Acetogens are phylogenetically very diverse and consist of more than 100 species in more than 23 genera (Drake et al, 2013; Müller and Frerichs, 2013; Shin et al, 2016) They are metabolically very dexterous and, in addition to acetate, may produce ethanol, butyrate, lactate etc. Acetogens are of importance in constructed anaerobic digestion systems, where they are important key players for efficient and stable methane production (e.g., Xu et al, 2009; Weiland, 2010; Hori et al, 2011; Wang et al, 2013; Moestedt et al, 2016; Müller et al, 2016; Westerholm et al, 2018; Fischer et al, 2019)
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