APECS junior research group, Leibniz Institute for Agricultural Engineering, Max-Eyth-Allee 100, 14469 Potsdam, Germany.
BMC Microbiol. 2013 Dec 4;13:278. doi: 10.1186/1471-2180-13-278.
The production of bio-methane from renewable raw material is of high interest because of the increasing scarcity of fossil fuels. The process of biomethanation is based on the inter- and intraspecific metabolic activity of a highly diverse and dynamic microbial community. The community structure of the microbial biocenosis varies between different biogas reactors and the knowledge about these microbial communities is still fragmentary. However, up to now no approaches are available allowing a fast and reliable access to the microbial community structure. Hence, the aim of this study was to originate a Flow-FISH protocol, namely a combination of flow cytometry and fluorescence in situ hybridization, for the analysis of the metabolically active microorganisms in biogas reactor samples. With respect to the heterogenic texture of biogas reactor samples and to collect all cells including those of cell aggregates and biofilms the development of a preceding purification procedure was indispensable.
Six different purification procedures with in total 29 modifications were tested. The optimized purification procedure combines the use of the detergent sodium hexametaphosphate with ultrasonic treatment and a final filtration step. By this treatment, the detachment of microbial cells from particles as well as the disbandment of cell aggregates was obtained at minimized cell loss. A Flow-FISH protocol was developed avoiding dehydration and minimizing centrifugation steps. In the exemplary application of this protocol on pure cultures as well as biogas reactor samples high hybridization rates were achieved for commonly established domain specific oligonucleotide probes enabling the specific detection of metabolically active bacteria and archaea. Cross hybridization and autofluorescence effects could be excluded by the use of a nonsense probe and negative controls, respectively.
The approach described in this study enables for the first time the analysis of the metabolically active fraction of the microbial communities within biogas reactors by Flow-FISH.
由于化石燃料的日益稀缺，从可再生原料中生产生物甲烷引起了人们的极大兴趣。生物甲烷化过程基于高度多样和动态的微生物群落的种间和种内代谢活性。微生物生物群落的群落结构在不同的沼气反应器之间有所不同，并且对这些微生物群落的了解仍然是零散的。然而，到目前为止，还没有方法可以快速可靠地了解微生物群落结构。因此，本研究的目的是开发一种流式荧光原位杂交（Flow-FISH）方案，即流式细胞术和荧光原位杂交的组合，用于分析沼气反应器样品中代谢活跃的微生物。鉴于沼气反应器样品的异质质地，并收集所有细胞，包括细胞聚集体和生物膜的细胞，开发了一个预先的纯化程序是必不可少的。
总共测试了 6 种不同的纯化程序，共 29 种改进方法。优化的纯化程序结合使用了去垢剂六偏磷酸钠、超声处理和最终过滤步骤。通过这种处理，可以在最小化细胞损失的情况下，从颗粒上分离微生物细胞并解散细胞聚集体。开发了一种 Flow-FISH 方案，避免了脱水并尽量减少离心步骤。在对纯培养物和沼气反应器样品的示例应用中，该方案实现了高杂交率，对于常用的建立特定领域的寡核苷酸探针，能够特异性检测代谢活跃的细菌和古菌。通过使用无意义探针和阴性对照，可以排除交叉杂交和自发荧光效应。
本研究中描述的方法首次能够通过 Flow-FISH 分析沼气反应器中微生物群落的代谢活跃部分。
