Cultivation-independent and cultivation-dependent metagenomes reveal genetic and enzymatic potential of microbial community involved in the degradation of a complex microbial polymer
Authors: Costa Ohana Y. A. , de Hollander Mattias , Pijl Agata , Liu Binbin , Kuramae Eiko E. 
Published: June 2020

Abstract
Background
Cultivation-independent methods, including metagenomics, are tools for the exploration and discovery of biotechnological compounds produced by microbes in natural environments. Glycoside hydrolases (GHs) enzymes are extremely desired and important in the industry of production for goods and biofuel and removal of problematic biofilms and exopolysaccharide (EPS). Biofilms and EPS are complex, requiring a wide range of enzymes for a complete degradation. The aim of this study was to identify potential GH microbial producers and GH genes with biotechnological potential, using EPS-complex structure (WH15EPS) of Acidobacteria Granulicella sp. strain WH15 as an enrichment factor, in cultivation-independent and cultivation-dependent methods. We performed stable isotope probing (SIP) combined with metagenomics on topsoil litter amended with WH15EPS and coupled solid culture-EPS amended medium with metagenomics.

Results
SIP metagenome analysis of the soil litter demonstrated that phyla Proteobacteria, Actinobacteria, Acidobacteria, and Planctomycetes were the most abundant in WH15EPS amended and unamended treatments. The enrichment cultures in solid culture medium coupled to metagenomics demonstrated an enrichment in Proteobacteria, and the metagenome assembly of this enrichment cultures resulted in 4 metagenome-assembled genomes (MAGs) of microbes with low identity (42–86%) to known microorganisms. Among all carbohydrate-active enzymes (CAZymes) retrieved genes, glycoside transferase (GT) was the most abundant family, either in culture-independent or culture-based metagenome datasets. Within the glycoside hydrolases (GHs), GH13 was the most abundant family in both metagenome datasets. In the “heavy” fraction of the culture-independent metagenome SIP dataset, GH109 (α-N-acetylgalactosaminidases), GH117 (agarases), GH50 (agarases), GH32 (invertases and inulinases), GH17 (endoglucanases), and GH71 (mutanases) families were more abundant in comparison with the controls. Those GH families are affiliated to microorganism that are probably capable to degrade WH15EPS and potentially applicable for biofilm deconstruction. Subsequent in culture-based metagenome, the assembled 4 MAGs (unclassified Proteobacteria) also contained GH families of interest, involving mannosidases, lysozymes, galactosidases, and chitinases.

Conclusions
We demonstrated that functional diversity induced by the presence of WH15EPS in both culture-independent and culture-dependent approaches was enriched in GHs, such as amylases and endoglucanases that could be applied in chemical, pharmaceutical, and food industrial sectors. Furthermore, WH15EPS may be used for the investigation and isolation of yet unknown taxa, such as unclassified Proteobacteria and Planctomycetes, increasing the number of current cultured bacterial representatives with potential biotechnological traits.