Department of Microbiology, Technical University of Munich, Emil-Ramann-Str. 4, 85354 Freising, Germany.
Core Facility Microbiome/NGS, ZIEL - Institute for Food & Health, Technical University of Munich, Weihenstephaner Berg 3, 85354 Freising, Germany.
Syst Appl Microbiol. 2019 Jul;42(4):481-487. doi: 10.1016/j.syapm.2019.05.002. Epub 2019 May 23.
Biogas plants achieve its highest yield on plant biomass only with the most efficient hydrolysis of cellulose. This is driven by highly specialized hydrolytic microorganisms, which we have analyzed by investigating enrichment strategies for the isolation of cellulolytic bacteria out of a lab-scale biogas fermenter. We compared three different cultivation media as well as two different inoculation materials: Enrichment on filter paper in nylon bags (in sacco) or raw digestate. Next generation sequencing of the V3/V4 region of the bacterial 16S rRNA of metagenomic DNA from six different enrichment cultures, each in biological triplicates, revealed an average richness of 48 different OTU's with an average evenness of 0.3 in each sample. β-Diversity of the bacterial community revealed significant differences between the two sampling techniques or the different media used. The isolation attempt of single cellulolytic organisms resulted in several clonal pure cultures. Regardless which medium or inoculation material, well-known cellulolytic key players such as Clostridium cellulosi, Herbinix hemicellulosilytica and Hungateiclostridium thermocellum were among the isolates. The inoculation material as well as the cultivation conditions are crucial to cultivate the representative cellulolytic organisms. Taking raw digestate as inoculation material and using the same material, filtered and sterilized, for supplementing media allowed to imitate the natural habitat. Pre-enrichment of cellulolytic organisms directly in their natural habitat led to significant advantages concerning high diversity and high abundance of unknown cellulolytic organisms, which is a key factor for the isolation of hitherto unknown species.
沼气厂只有通过最有效的纤维素水解才能从植物生物质中获得最高产量。这是由高度专业化的水解微生物驱动的，我们通过研究从实验室规模的沼气发酵器中分离纤维素分解菌的富集策略来分析这些微生物。我们比较了三种不同的培养介质以及两种不同的接种材料：在尼龙袋（in sacco）中的滤纸或原始消化物上进行富集。对来自六个不同富集培养物的宏基因组 DNA 的细菌 16S rRNA 的 V3/V4 区进行下一代测序，每个培养物进行了三次生物学重复，结果显示每个样品中平均有 48 个不同的 OTU，均匀度为 0.3。细菌群落的β多样性显示出两种采样技术或使用的不同培养基之间存在显著差异。单一纤维素分解菌的分离尝试导致了几个克隆纯培养物。无论使用哪种培养基或接种材料，众所周知的纤维素分解关键角色，如 Clostridium cellulosi、Herbinix hemicellulosilytica 和 Hungateiclostridium thermocellum 等，都在分离物中。接种材料和培养条件对于培养代表性的纤维素分解菌至关重要。使用原始消化物作为接种材料，并使用相同的材料进行过滤和消毒以补充培养基，可以模拟自然栖息地。在其自然栖息地中直接对纤维素分解菌进行预富集，对于未知纤维素分解菌的高多样性和高丰度具有显著优势，这是分离未知物种的关键因素。
