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

Johannes Lambrecht,Thomas Hübschmann,Christin Koch,Susann Müller,Hauke Harms,Nicolas Cichocki

doi:10.1186/s12934-017-0793-7

Johannes Lambrecht, Thomas Hübschmann + Show 4 more

Open Access

https://doi.org/10.1186/s12934-017-0793-7

Copy DOI

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.

Highlights

The widely established production of CH4 from renewable biomass in industrial scale anaerobic reactors may play a major role in the future energy supply
autofluorescent cofactor of the methanogenesis (F420) fluorescence in methanogenic archaea Using fluorescence spectroscopy and a test culture of enriched methanogens (MEC) we recorded the characteristic excitation and emission spectra of cofactor F 420 (Fig. 1b). By iterating along these values we tested different flow cytometer configurations to optimize for both, F 420 autofluorescence resolution and mean intensity, in order to discriminate F420 autofluorescent from non-autofluorescent subcommunities of the methanogenic enrichment culture (MEC)
The violet light excites the cofactor F420, which is ubiquitous in methanogenic archaea and unique to this taxon at the identified concentrations [19] (Fig. 1c)

Summary

Introduction

The widely established production of CH4 from renewable biomass in industrial scale anaerobic reactors may play a major role in the future energy supply. DNA sequence-based methods most frequently used today either rely on 16S rDNA specific for the most abundant subgroups of methanogenic archaea [1, 2], metagenomics analysis [3], or the functional mcrA gene coding for the α-subunit of the methyl coenzyme M reductase [4,5,6]. Fingerprinting methods, such as T-RFLP [7] provide qualitative abundance information, while single celllabeling by fluorescent in situ hybridization (FISH) [8] or qPCR methods [9] can be employed for quantification. The steering of running processes based on results of these methods is problematic

Methods

Results

Discussion

Conclusion