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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.

SUBMITTER: Izquierdo JA

PROVIDER: S-EPMC2876464 | biostudies-literature | 2010 Jun

REPOSITORIES: biostudies-literature

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Diversity of bacteria and glycosyl hydrolase family 48 genes in cellulolytic consortia enriched from thermophilic biocompost.

Izquierdo Javier A JA Sizova Maria V MV Lynd Lee R LR

Applied and environmental microbiology 20100409 11

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 ...[more]

PMID: 20382819

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Project description:BackgroundHemicellulose is one of the copious polymer in lignocellulosic biomass (LCB). It is primarily composed of xylan linked by β-1,4 glycosidic bonds. Xylanase preferentially cleaves the β-1,4-glycosidic bonds in the xylan backbone resulting in complete hydrolysis of the biomass. Thermostable variants of glycoside hydrolases act as robust catalysts, not only in degradation but also during processing, to obtain specific carbohydrate-containing chemicals and materials (Ramasamy et al. in Madras Agric J 107(special):1. https://doi.org/10.29321/MAJ.2020.000382 , 2020).ResultsThe xylanase production by two thermophilic bacteria isolated from thermal springs was evaluated. In addition, the gene encoding this industrially vital enzyme was isolated and characterized, and its protein structure was analyzed. The thermophilic bacteria producing xylanases were isolated from augmented sawdust and banana fiber biomass from hot springs of Himachal Pradesh and identified as Bacillus subtilis VSDB5 and Bacillus licheniformis KBFB4 using 16S rRNA gene sequencing. The persistent xylanase activity revealed that the enzyme is secreted extracellularly with the maximum activity of 0.76 IU mL-1 and 1.0 IU mL-1 at 6 h and 12 h of growth by KBFB4 and VSDB5, respectively, under submerged fermentation. Both the strains exhibited the maximum activity at pH 6 and a temperature of 50 °C. The xylanases of KBFB4 and VSDB5 were thermostable and retained 40% of their activity at 60 °C after incubation for 30 min. Xylanase of VSDB5 had wide thermotolerance and retained 20% of its activity from 60 to 80 °C, whereas xylanase of KBFB4 showed wide alkali tolerance and retained 80% of its activity until pH 10. The xylanase (xynA)-encoding gene (650 bp) cloned from both the strains using specific primers showed 98 to 99% homology to β-1,4-endoxylanase gene. Further in silico analysis predicted that the xylanase protein, with a molecular weight of 23 kDa, had a high pI (9.44-9.65), which explained the alkaline nature of the enzyme and greater aliphatic index (56.29). This finding suggested that the protein is thermostable. Multiple sequence alignment and homology modeling of the protein sequence revealed that the gene product belonged to the GH11 family, indicating its possible application in bioconversion.ConclusionThe strains B. subtilis VSDB5 and B. licheniformis KBFB4 obtained from hot springs of Himachal Pradesh produced potent and alkali-tolerant thermostable xylanases, which belong to the GH11 family. The enzyme can be supplemented in industrial applications for biomass conversion at high temperatures and pH (or in processes involving alkali treatment).

Project description:BackgroundThe rumen harbors a complex microbial ecosystem for efficient hydrolysis of plant polysaccharides which are the main constituent of the diet. Xylanase is crucial for hemicellulose hydrolysis and plays an important role in the plant cell wall degradation. Xylanases of ruminal strains were widely studied, but few studies have focused on their diversity in rumen microenvironment.Methodology/principal findingsWe explored the genetic diversity of xylanases belonging to two major glycosyl hydrolase families (GH 10 and 11) in goat rumen contents by analyzing the amplicons generated with two degenerate primer sets. Fifty-two distinct GH 10 and 35 GH 11 xylanase gene fragments (similarity <95%) were retrieved, and most had low identities with known sequences. Based on phylogenetic analysis, all GH 10 xylanase sequences fell into seven clusters, and 88.5% of them were related to xylanases from Bacteroidetes. Five clusters of GH 11 xylanase sequences were identified. Of these, 85.7% were related to xylanases from Firmicutes, and 14.3% were related to those of rumen fungi. Two full-length xylanase genes (one for each family) were directly cloned and expressed in Escherichia coli. Both the recombinant enzymes showed substantial xylanase activity, and were purified and characterized. Combined with the results of sheep rumen, Bacteroidetes and Firmicutes are the two major phyla of xylan-degrading microorganisms in rumen, which is distinct from the representatives of other environments such as soil and termite hindgut, suggesting that xylan-degrading microorganisms are environment specific.Conclusion/significanceThe numerous new xylanase genes suggested the functional diversity of xylanase in the rumen microenvironment which may have great potential applications in industry and agriculture. The phylogenetic diversity and different distributions of xylanase genes will help us understand their roles in plant cell wall degradation in the rumen microenvironment.

Dataset Information

Diversity of bacteria and glycosyl hydrolase family 48 genes in cellulolytic consortia enriched from thermophilic biocompost.

Publications

Diversity of bacteria and glycosyl hydrolase family 48 genes in cellulolytic consortia enriched from thermophilic biocompost.

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