Comparison of sampling techniques and different media for the enrichment and isolation of cellulolytic organisms from biogas fermenters.

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.