Exploring the microbial diversity and characterization of cellulase and hemicellulase genes in goat rumen: a metagenomic approach.

BACKGROUND: Goat rumen microbial communities are perceived as one of the most potential biochemical reservoirs of multi-functional enzymes, which are applicable to enhance wide array of bioprocesses such as the hydrolysis of cellulose and hemi-cellulose into fermentable sugar for biofuel and other value-added biochemical production. Even though, the limited understanding of rumen microbial genetic diversity and the absence of effective screening culture methods have impeded the full utilization of these potential enzymes. In this study, we applied culture independent metagenomics sequencing approach to isolate, and identify microbial communities in goat rumen, meanwhile, clone and functionally characterize novel cellulase and xylanase genes in goat rumen bacterial communities. RESULTS: Bacterial DNA samples were extracted from goat rumen fluid. Three genomic libraries were sequenced using Illumina HiSeq 2000 for paired-end 100-bp (PE100) and Illumina HiSeq 2500 for paired-end 125-bp (PE125). A total of 435gb raw reads were generated. Taxonomic analysis using Graphlan revealed that Fibrobacter, Prevotella, and Ruminococcus are the most abundant genera of bacteria in goat rumen. SPAdes assembly and prodigal annotation were performed. The contigs were also annotated using the DOE-JGI pipeline. In total, 117,502 CAZymes, comprising endoglucanases, exoglucanases, beta-glucosidases, xylosidases, and xylanases, were detected in all three samples. Two genes with predicted cellulolytic/xylanolytic activities were cloned and expressed in E. coli BL21(DE3). The endoglucanases and xylanase enzymatic activities of the recombinant proteins were confirmed using substrate plate assay and dinitrosalicylic acid (DNS) analysis. The 3D structures of endoglucanase A and endo-1,4-beta xylanase was predicted using the Swiss Model. Based on the 3D structure analysis, the two enzymes isolated from goat's rumen metagenome are unique with only 56-59% similarities to those homologous proteins in protein data bank (PDB) meanwhile, the structures of the enzymes also displayed greater stability, and higher catalytic activity. CONCLUSIONS: In summary, this study provided the database resources of bacterial metagenomes from goat's rumen fluid, including gene sequences with annotated functions and methods for gene isolation and over-expression of cellulolytic enzymes; and a wealth of genes in the metabolic pathways affecting food and nutrition of ruminant animals.

Proteome profile changes during poly-hydroxybutyrate intracellular mobilization in gram positive Bacillus cereus tsu1.

BACKGROUND: Bacillus cereus is a bacterial species which grows efficiently on a wide range of carbon sources and accumulates biopolymer poly-hydroxybutyrate (PHB) up to 80% cell dry weight. PHB is an aliphatic polymer produced and stored intracellularly as a reservoir of carbon and energy, its mobilization is a key biological process for sporulation in Bacillus spp. Previously, B. cereus tsu1 was isolated and cultured on rapeseed cake substrate (RCS), with maximum of PHB accumulation reached within 12 h, and depleted after 48 h. Fore-spore and spore structure were observed after 24 h culture. RESULTS: Quantitative proteomic analysis of B. cereus tsu1 identified 2952 quantifiable proteins, and 244 significantly changed proteins (SCPs) in the 24 h:12 h pair of samples, and 325 SCPs in the 48 h:12 h pair of samples. Based on gene ontology classification analysis, biological processes enriched only in the 24 h:12 h SCPs include purine nucleotide metabolism, protein folding, metal ion homeostasis, response to stress, carboxylic acid catabolism, and cellular amino acid catabolism. The 48 h:12 h SCPs were enriched into processes including carbohydrate metabolism, protein metabolism, oxidative phosphorylation, and formation of translation ternary structure. A key enzyme for PHB metabolism, poly(R)-hydroxyalkanoic acid synthase (PhaC, KGT44865) accumulated significantly higher in 12 h-culture. Sporulation related proteins SigF and SpoEII were significantly higher in 24 h-samples. Enzymes for nitrate respiration and fermentation accumulated to the highest abundance level in 48 h-culture. CONCLUSIONS: Changes in proteome of B. cereus tsu1 during PHB intracellular mobilization were characterized in this study. The key enzyme PhaC for PHB synthesis increased significantly after 12 h-culture which supports the highest PHB accumulation at this time point. The protein abundance level of SpoIIE and SigF also increased, correlating with sporulation in 24 h-culture. Enzymes for nitrate respiration and fermentation were significantly induced in 48 h-culture which indicates the depletion of oxygen at this stage and carbon flow towards fermentative growth. Results from this study provide insights into proteome profile changes during PHB accumulation and reuse, which can be applied to achieve a higher PHB yield and to improve bacterial growth performance and stress resistance.

Conversion of Food Waste into 2,3-Butanediol via Thermophilic Fermentation: Effects of Carbohydrate Content and Nutrient Supplementation.

Fermentation of food waste into 2,3-butanediol (2,3-BDO), a high-value chemical, is environmentally sustainable and an inexpensive method to recycle waste. Compared to traditional mesophilic fermentation, thermophilic fermentation can inhibit the growth of contaminant bacteria, thereby improving the success of food waste fermentation. However, the effects of sugar and nutrient concentrations in thermophilic food waste fermentations are currently unclear. Here, we investigated the effects of sugar and nutrients (yeast extract (YE) and peptone) concentrations on 2,3-BDO production from fermenting glucose and food waste media using the newly isolated thermophilic Bacillus licheniformis YNP5-TSU. When glucose media was used, fermentation was greatly affected by sugar and nutrient concentrations: excessive glucose (>70 g/L) slowed down the fermentation and low nutrients (2 g/L YE and 1 g/L peptone) caused fermentation failure. However, when food waste media were used with low nutrient addition, the bacteria consumed all 57.8 g/L sugars within 24 h and produced 24.2 g/L 2,3-BDO, equivalent to a fermentation yield of 0.42 g/g. An increase in initial sugar content (72.9 g/L) led to a higher 2,3-BDO titer of 36.7 g/L with a nearly theoretical yield of 0.47 g/g. These findings may provide fundamental knowledge for designing cost-effective food waste fermentation to produce 2,3-BDO.

Compositional Characterization of Different Industrial White and Red Grape Pomaces in Virginia and the Potential Valorization of the Major Components.

To better evaluate potential uses for grape pomace (GP) waste, a comprehensive chemical composition analysis of GP in Virginia was conducted. Eight commercial white and red pomace samples (cv. Viognier, Vidal Blanc, Niagara, Petit Manseng, Petit Verdot, Merlot, Cabernet Franc, and Chambourcin) obtained from different wineries in Virginia, USA were used. For extractives, GPs contained 2.89%-4.66% titratable acids, 4.32%-6.60% ash, 4.62%-12.5% lipids with linoleic acid being the predominant (59.0%-70.9%) fatty acid, 10.4-64.8 g total phenolic content (gallic acid equivalents)/kg GP, 2.09-53.3 g glucose/kg GP, 3.79-52.9 g fructose/kg GP, and trace sucrose. As for non-extractives, GPs contained 25.2%-44.5% lignin, 8.04%-12.7% glucan, 4.42%-7.05% xylan, and trace amounts of galactan, arabinan, and mannan (less than 3% in total). Potential usages of these components were further examined to provide information on better valorization of GP. Considering the valuable extractives (e.g., polyphenols and oil) and non-extractives (e.g., lignin), designing a biorefinery process aiming at fully recover and/or utilize these components is of future significance.

Draft Genome Sequence of Bacillus licheniformis Strain YNP1-TSU Isolated from Whiterock Springs in Yellowstone National Park.

Novel cellulolytic microorganisms can potentially influence second-generation biofuel production. This paper reports the draft genome sequence of Bacillus licheniformis strain YNP1-TSU, isolated from hydrothermal-vegetative microbiomes inside Yellowstone National Park. The assembled sequence contigs predicted 4,230 coding genes, 66 tRNAs, and 10 rRNAs through automated annotation.

Draft Genome Sequences of Three Cellulolytic Bacillus licheniformis Strains Isolated from Imperial Geyser, Amphitheater Springs, and Whiterock Springs inside Yellowstone National Park.

Novel cellulolytic microorganisms are becoming more important for rapidly growing biofuel industries. This paper reports the draft genome sequences of Bacillus licheniformis strains YNP2-TSU, YNP3-TSU, and YNP5-TSU. These cellulolytic isolates were collected from several hydrothermal features inside Yellowstone National Park.