Flow cytometric quantification, sorting and sequencing of methanogenic archaea based on F420 autofluorescence

Abstract

BackgroundThe widely established production of CH4 from renewable biomass in industrial scale anaerobic reactors may play a major role in the future energy supply. It relies on methanogenic archaea as key organisms which represent the bottleneck in the process. The quantitative analysis of these organisms can help to maximize process performance, uncover disturbances before failure, and may ultimately lead to community-based process control schemes. Existing qPCR and fluorescence microscopy-based methods are very attractive but can be cost-intensive and laborious.ResultsIn this study we present an autofluorescence-based, flow cytometric method for the fast low-cost quantification of methanogenic archaea in complex microbial communities and crude substrates. The method was applied to a methanogenic enrichment culture (MEC) and digester samples (DS). The methanogenic archaea were quantified using the distinct fluorescence of their cofactor F420 in a range from 3.7 × 108 (± 3.3 × 106) cells mL−1 and 1.8 x 109 (± 1.1 × 108) cells mL−1. We evaluated different fixation methods and tested the sample stability. Stable abundance and fluorescence intensity were recorded up to 26 days during aerobic storage in PBS at 6 °C. The discrimination of the whole microbial community from the ubiquitous particle noise was facilitated by SYBR Green I staining and enabled calculation of relative abundances of methanogenic archaea of up to 9.64 ± 0.23% in the MEC and up to 4.43 ± 0.74% in the DS. The metaprofiling of the mcrA gene reinforced the results.ConclusionsThe presented method allows for fast and reliable quantification of methanogenic archaea in microbial communities under authentic digester conditions and can thus be useful for process monitoring and control in biogas digesters.Electronic supplementary materialThe online version of this article (10.1186/s12934-017-0793-7) contains supplementary material, which is available to authorized users.

Neutral mechanisms and niche differentiation in steady‐state insular microbial communities revealed by single cell analysis

“…Thus, a unique subcommunity (gate) usually contains several distinct phylotypes but can also be mono‐dominant (Zimmermann et al ., ). This has been verified by cell sorting combined with 16S rRNA gene amplicon sequencing in earlier studies (Lambrecht et al ., , van Gelder et al ., 2018, Liu et al ., ) and also in this study to reveal community and subcommunity compositions (Supporting Information S12). The used state variables were the numbers of gates (summed up to 68 gates, Supporting Information S5, Fig.…”

Control of biogas production process by enzymatic and fluorescent image analysis

“…Each of the four microbial functional groups has different optimal ambient parameters. The key micro-organisms of biogas production are the methanogens [5]. They belong to the domain Archaea and are particularly sensitive to changes in the environment such as temperature and pH fluctuations.…”

Syntrophy via interspecies H2transfer betweenChristensenellaandMethanobrevibacterunderlies their global co-occurrence in the human gut

“…For confocal microscopy, SYBR ®□ Green I staining was performed as previously described (61) with the modifications described in Appendix 1. Imaging by confocal microscopy (LSM 780 NLO, Zeiss) was used to detect the autofluorescence emission of coenzyme F 420 of M. smithii and the emission of SYBR Green I (Appendix 1).…”

“…For confocal microscopy, SYBR ®️ Green I staining was performed as previously described (61) with the following modifications: 0.5 mL of culture were sampled and pelleted by centrifugation for 6 min at 6,000 xg (Benchtop centrifuge, Eppendorf, Hamburg, Germany) and pellets were resuspended in a solution containing 744 μL 1x PBS, 16 μL 25x SYBR ®️ Green I (Sigma-Aldrich, Merck, Germany) and 40 μL 70% v/v ethanol. Samples were pelleted and resuspended before imagining in 100 μL 1x PBS, of which 5 μL were immobilized on 50 μL solid agar (1.5% noble agar in distilled water) (67).…”