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An anaerobic continuous-flow fermentation system with condensed molasses fermentation soluble (CMS) from monosodium glutamate production as substrate was developed for the biohydrogen production. The production rate of the system was increased, while hydraulic retention time (HRT) was varied from 12 to 3 h and the optimal hydrogen production rate was obtained at HRT 3 h. The major bacterial composition of each HRT was analyzed by 16S rRNA gene-targeted denaturing gradient gel electrophoresis (DGGE). Acidaminococcus sp. that could evolve hydrogen from glutamate was the only hydrogen producer and could be also detected from substrate. Hydrogenase mRNA-targeted reverse transcriptase polymerase chain reaction (RT-PCR) analysis showed that except HRT 3 h, two clusters of clostridial hydrogenase gene sequences were obtained. RT-PCR-DGGE analysis was employed to illustrate the active clostridial strains confirming that clostridial strains were not present at HRT 3 h. Acidaminococcus sp. existing in substrate might be the major strain that contributes to hydrogen production at HRT 3 h.

The Enterprise File Sync and Share (EFSS) is one of the most important services to provide enterprises' employees with cloud file sync, share, and collaboration services. To take enterprises' concerns into account, such as security, privacy, compliance, and regulation, the existing EFSS solutions are either using private (on-premise) or hybrid cloud service model to provide their services. They usually emphasize that files stored in the solutions are encrypted on transfer and at rest and events occurred in the service are logged as the audit trail. However, support of data encryption and audit trail are not capable of protecting enterprise sensitive data from not well addressed security issues of the EFSS service. The security issues, including employee privacy protection, management of share links and synchronized cloud files, and the secure enterprise directory integration, are pointed out in this article. To address these issues, this work proposes and develops a scalable Secure EFSS service which can be deployed on the on-premise Open Stack cloud infrastructure to securely provide employees with EFSS service. Designs of an integrated security approach are introduced in this article, including data and metadata isolations, Distinct Share Link utility, encryption key management for personal and shared files, sandbox-based cloud file synchronization, and out-of-band authentication method.

Background As a strong fermentator, Saccharomyces cerevisiae has the potential to be an excellent host for ethanol production by consolidated bioprocessing. For this purpose, it is necessary to transform cellulose genes into the yeast genome because it contains no cellulose genes. However, heterologous protein expression in S. cerevisiae often suffers from hyper-glycosylation and/or poor secretion. Thus, there is a need to genetically engineer the yeast to reduce its glycosylation strength and to increase its secretion ability. Results Saccharomyces cerevisiae gene-knockout strains were screened for improved extracellular activity of a recombinant exocellulase (PCX) from the cellulose digesting fungus Phanerochaete chrysosporium. Knockout mutants of 47 glycosylation-related genes and 10 protein-trafficking-related genes were transformed with a PCX expression construct and screened for extracellular cellulase activity. Twelve of the screened mutants were found to have a more than 2-fold increase in extracellular PCX activity in comparison with the wild type. The extracellular PCX activities in the glycosylation-related mnn10 and pmt5 null mutants were, respectively, 6 and 4 times higher than that of the wild type; and the extracellular PCX activities in 9 protein-trafficking-related mutants, especially in the chc1, clc1 and vps21 null mutants, were at least 1.5 times higher than the parental strains. Site-directed mutagenesis studies further revealed that the degree of N-glycosylation also plays an important role in heterologous cellulase activity in S. cerevisiae. Conclusions Systematic screening of knockout mutants of glycosylation- and protein trafficking-associated genes in S. cerevisiae revealed that: (1) blocking Golgi-to-endosome transport may force S. cerevisiae to export cellulases; and (2) both over- and under-glycosylation may alter the enzyme activity of cellulases. This systematic gene-knockout screening approach may serve as a convenient means for increasing the extracellular activities of recombinant proteins expressed in S. cerevisiae.

Background Cellulose, which is the most abundant renewable biomass on earth, is a potential bio-resource of alternative energy. The hydrolysis of plant polysaccharides is catalyzed by microbial cellulases, including endo-β-1,4-glucanases, cellobiohydrolases, cellodextrinases, and β-glucosidases. Converting cellobiose by β-glucosidases is the key factor for reducing cellobiose inhibition and enhancing the efficiency of cellulolytic enzymes for cellulosic ethanol production. Results In this study, a cDNA encoding β-glucosidase was isolated from the buffalo rumen fungus Neocallimastix patriciarum W5 and is named NpaBGS. It has a length of 2,331 bp with an open reading frame coding for a protein of 776 amino acid residues, corresponding to a theoretical molecular mass of 85.1 kDa and isoelectric point of 4.4. Two GH3 catalytic domains were found at the N and C terminals of NpaBGS by sequence analysis. The cDNA was expressed in Pichia pastoris and after protein purification, the enzyme displayed a specific activity of 34.5 U/mg against cellobiose as the substrate. Enzymatic assays showed that NpaBGS was active on short cello-oligosaccharides from various substrates. A weak activity in carboxymethyl cellulose (CMC) digestion indicated that the enzyme might also have the function of an endoglucanase. The optimal activity was detected at 40°C and pH 5 ~ 6, showing that the enzyme prefers a weak acid condition. Moreover, its activity could be enhanced at 50°C by adding Mg2+ or Mn2+ ions. Interestingly, in simultaneous saccharification and fermentation (SSF) experiments using Saccharomyces cerevisiae BY4741 or Kluyveromyces marxianus KY3 as the fermentation yeast, NpaBGS showed advantages in cell growth, glucose production, and ethanol production over the commercial enzyme Novo 188. Moreover, we showed that the KY3 strain engineered with the NpaNGS gene can utilize 2 % dry napiergrass as the sole carbon source to produce 3.32 mg/ml ethanol when Celluclast 1.5 L was added to the SSF system. Conclusion Our characterizations of the novel β-glucosidase NpaBGS revealed that it has a preference of weak acidity for optimal yeast fermentation and an optimal temperature of ~40°C. Since NpaBGS performs better than Novo 188 under the living conditions of fermentation yeasts, it has the potential to be a suitable enzyme for SSF.

Background Neocallimastix patriciarum is one of the common anaerobic fungi in the digestive tracts of ruminants that can actively digest cellulosic materials, and its cellulases have great potential for hydrolyzing cellulosic feedstocks. Due to the difficulty in culture and lack of a genome database, it is not easy to gain a global understanding of the glycosyl hydrolases (GHs) produced by this anaerobic fungus. Results We have developed an efficient platform that uses a combination of transcriptomic and proteomic approaches to N. patriciarum to accelerate gene identification, enzyme classification and application in rice straw degradation. By conducting complementary studies of transcriptome (Roche 454 GS and Illumina GA IIx) and secretome (ESI-Trap LC-MS/MS), we identified 219 putative GH contigs and classified them into 25 GH families. The secretome analysis identified four major enzymes involved in rice straw degradation: β-glucosidase, endo-1,4-β-xylanase, xylanase B and Cel48A exoglucanase. From the sequences of assembled contigs, we cloned 19 putative cellulase genes, including the GH1, GH3, GH5, GH6, GH9, GH18, GH43 and GH48 gene families, which were highly expressed in N. patriciarum cultures grown on different feedstocks. Conclusions These GH genes were expressed in Pichia pastoris and/or Saccharomyces cerevisiae for functional characterization. At least five novel cellulases displayed cellulytic activity for glucose production. One β-glucosidase (W5-16143) and one exocellulase (W5-CAT26) showed strong activities and could potentially be developed into commercial enzymes.