Department of Environmental Microbiology, Helmholtz Centre for Environmental Research-UFZ, Permoserstr. 15, 04318, Leipzig, Germany.
Microb Cell Fact. 2017 Oct 30;16(1):180. doi: 10.1186/s12934-017-0793-7.
The widely established production of CH 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.
In 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 F in a range from 3.7 × 10 (± 3.3 × 10) cells mL and 1.8 x 10 (± 1.1 × 10) cells mL. 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.
The 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.
从可再生生物量在工业规模厌氧反应器中广泛生产 CH 可能在未来的能源供应中发挥主要作用。它依赖于产甲烷古菌作为关键生物，这些生物是该过程的瓶颈。这些生物的定量分析可以帮助最大限度地提高工艺性能，在失效前发现干扰，并最终导致基于群落的过程控制方案。现有的 qPCR 和荧光显微镜方法非常有吸引力，但成本高且费力。
在这项研究中，我们提出了一种基于自发荧光的流式细胞术方法，用于快速、低成本地定量复杂微生物群落和粗基质中的产甲烷古菌。该方法应用于产甲烷富集培养物（MEC）和消化器样品（DS）。使用其辅助因子 F 的独特荧光对产甲烷古菌进行定量，范围从 3.7 ± 3.3 × 10 个细胞 mL 和 1.8 x 10 (± 1.1 x 10) 个细胞 mL。我们评估了不同的固定方法并测试了样品稳定性。在 6°C 的 PBS 中进行有氧储存时，可稳定记录长达 26 天的丰度和荧光强度。通过 SYBR Green I 染色可以区分整个微生物群落与普遍存在的颗粒噪声，从而能够计算 MEC 中产甲烷古菌的相对丰度高达 9.64 ± 0.23%，DS 中高达 4.43 ± 0.74%。mcrA 基因的元分析增强了结果。
该方法可在真实消化器条件下快速可靠地定量微生物群落中的产甲烷古菌，因此可用于沼气消化器的过程监测和控制。
