Diversity of Bacteria and Glycosyl Hydrolase Family 48 Genes in Cellulolytic Consortia Enriched from Thermophilic Biocompost

Abstract

The enrichment from nature of novel microbial communities with high cellulolytic activity is useful in the identification of novel organisms and novel functions that enhance the fundamental understanding of microbial cellulose degradation. In this work we identify predominant organisms in three cellulolytic enrichment cultures with thermophilic compost as an inoculum. Community structure based on 16S rRNA gene clone libraries featured extensive representation of clostridia from cluster III, with minor representation of clostridial clusters I and XIV and a novel Lutispora species cluster. Our studies reveal different levels of 16S rRNA gene diversity, ranging from 3 to 18 operational taxonomic units (OTUs), as well as variability in community membership across the three enrichment cultures. By comparison, glycosyl hydrolase family 48 (GHF48) diversity analyses revealed a narrower breadth of novel clostridial genes associated with cultured and uncultured cellulose degraders. The novel GHF48 genes identified in this study were related to the novel clostridia Clostridium straminisolvens and Clostridium clariflavum, with one cluster sharing as little as 73% sequence similarity with the closest known relative. In all, 14 new GHF48 gene sequences were added to the known diversity of 35 genes from cultured species.The exploration and understanding of cellulose fermentation capabilities in nature could inform and enable industrial processes converting cellulosic biomass to fuels and other products. Enrichment of microbial communities that can utilize cellulose is useful in this context for the identification of novel organisms, novel metabolisms, and novel functions. Of particular interest are communities that can utilize cellulose at high temperatures and under anaerobic conditions, featuring high rates of solubilization under conditions where the energy and the reducing power of substrates are conserved in potentially useful fermentation products.Some evidence indicates that cocultures may be able to utilize cellulose more fully and produce higher concentrations of ethanol than pure cultures of model cellulolytic organisms such as Clostridium thermocellum and Clostridium straminisolvens (16,20,34). An initial step toward understanding the functional roles of community members in cooperative cellulose degradation is answering the question of what organisms are present in cellulolytic consortia obtained from nature. Currently, diversity estimation methods applied to cellulolytic communities range from traditional methods targeting the 16S rRNA gene (4, 12) to complex metagenomic analyses targeting the breadth of functional genes present in genomes of mixed cultures and the environment (3).From a functional gene standpoint, cellulase systems are complex assemblages of multifunctional glycosyl hydrolases. Even particularly relevant families, such as family 5 and family 9, tend to include hydrolases with multiple substrate specificities, deep evolutionary roots, and extensive sequence diversity within the same organism (19)...

Cellulose- and Xylan-Degrading Thermophilic Anaerobic Bacteria from Biocompost

“…2): (i) isolates No GH48 genes were detected in noncellulolytic isolates. Novel isolates related to C. thermocellum and C. straminisolvens were determined to have both free (celY-like) and cellulosome-bound (celS-like) exoglucanases from glycosyl hydrolase family 48, as seen in reference strains (13). GH48 genes in C. straminisolvens-and C. clariflavum-related isolates displayed very similar grouping, as observed in 16S rRNA gene analyses, suggesting a very strict conservation of this particular family of glycosyl hydrolases within cellulolytic clostridia.…”

“…The lack of such discrepancy in our consortia indicates the narrow metabolic diversity and absence of stressed organisms, which have most probably been lost in the course of consecutive transfers. Low diversity was also detected in the 16S rRNA gene and glycosyl hydrolase family 48 sequences of these consortia (13). Limited diversity can be also explained by high cultivation temperature in combination with cellulose recalcitrance: only limited numbers of species are able to develop under such conditions.…”

“…These mixed cultures are referred to below as "consortia," which are distinct from "primary enrichment," having nonstabilized variable composition. In total, we obtained four anaerobic thermophilic cellulolytic consortia: three (CO-4, CO-5, and CO-6) were derived directly from three respective biocompost samples as described earlier (13), and one, named CT, was obtained from a cellulose trap. Plating of the serially diluted consortium cultures on cellulose agar revealed bacterial colonies with extensive clearing zones, indicating cellulose solubilization by extracellular enzymes (see Fig.…”

“…PCR amplification of the 16S rRNA gene and sequencing were done as described before (33). Amplification of GH48 genes was performed with the GH48F and GH48R degenerate primers as previously described (13). For C. thermocellum and C. straminisolvenstype isolates, coamplification of celS and celY genes was resolved by constructing clone libraries with amplicons by use of the pGEM-T-easy vector (Promega, Madison, WI) as previously described (13).…”

“…In our preceding study (13), we obtained three cellulolytic consortia from self-heated biocompost. They contained cumulatively 30 and 11 operational taxonomic units (OTUs), based on a survey of the 16S rRNA and GH48 (glycosyl hydrolase family 48) gene clone libraries, respectively.…”