Dataset Information

Characterization of lignocellulolytic activities from a moderate halophile strain of Aspergillus caesiellus isolated from a sugarcane bagasse fermentation.

ABSTRACT: A moderate halophile and thermotolerant fungal strain was isolated from a sugarcane bagasse fermentation in the presence of 2 M NaCl that was set in the laboratory. This strain was identified by polyphasic criteria as Aspergillus caesiellus. The fungus showed an optimal growth rate in media containing 1 M NaCl at 28°C and could grow in media added with up to 2 M NaCl. This strain was able to grow at 37 and 42°C, with or without NaCl. A. caesiellus H1 produced cellulases, xylanases, manganese peroxidase (MnP) and esterases. No laccase activity was detected in the conditions we tested. The cellulase activity was thermostable, halostable, and no differential expression of cellulases was observed in media with different salt concentrations. However, differential band patterns for cellulase and xylanase activities were detected in zymograms when the fungus was grown in different lignocellulosic substrates such as wheat straw, maize stover, agave fibres, sugarcane bagasse and sawdust. Optimal temperature and pH were similar to other cellulases previously described. These results support the potential of this fungus to degrade lignocellulosic materials and its possible use in biotechnological applications.

SUBMITTER: Batista-Garcia RA

PROVIDER: S-EPMC4146556 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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Characterization of lignocellulolytic activities from a moderate halophile strain of Aspergillus caesiellus isolated from a sugarcane bagasse fermentation.

Batista-García Ramón Alberto RA Balcázar-López Edgar E Miranda-Miranda Estefan E Sánchez-Reyes Ayixón A Cuervo-Soto Laura L Aceves-Zamudio Denise D Atriztán-Hernández Karina K Morales-Herrera Catalina C Rodríguez-Hernández Rocío R Folch-Mallol Jorge J

PloS one 20140827 8

A moderate halophile and thermotolerant fungal strain was isolated from a sugarcane bagasse fermentation in the presence of 2 M NaCl that was set in the laboratory. This strain was identified by polyphasic criteria as Aspergillus caesiellus. The fungus showed an optimal growth rate in media containing 1 M NaCl at 28°C and could grow in media added with up to 2 M NaCl. This strain was able to grow at 37 and 42°C, with or without NaCl. A. caesiellus H1 produced cellulases, xylanases, manganese per ...[more]

PMID: 25162614

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Project description:Brazil is the world’s second largest producer of ethanol. Increasing demand for this biofuel has called for investment in new technologies. One example of such technologies is the second-generation (2G) ethanol production. Lignocellulose comprises mainly cellulose and hemicellulose, which consist of high-energy sugars that can be converted into ethanol. Aspergillus sp play an important role in the recycling of lignocellulosic biomass. These species have been investigated as a cell factory to produce industrial enzymes. To improve our understanding of the mechanisms used by Aspergillus sp during degradation of plant biomass and to identify the hydrolytic enzymes secreted by these fungi, we have analyzed the transcriptome and secretome of an Aspergillus species grown on sugarcane bagasse (SEB). A. fumigatus was cultivated in the presence of fructose or SEB. Its cellulolytic and xylanolytic activities depended on time. The maximum activities of endoglucanase and xylanase from the fungus grown on SEB were 0.0029 and 10.82 U mL-1, respectively. Characterization of the transcriptome by RNAseq technology helped to identify genes that participated in the degradation of the biomass demonstrating potential application in the process of enzymatic hydrolysis. The RNAseq data revealed that 2287 genes were differentially expressed in the fungus grown on SEB; 1181 and 1046 of these genes were up- and down-regulated, respectively. There were several CAZymes among the up-regulated genes. Most of these enzymes belonged to the GH family, which includes important hydrolytic and accessory enzymes involved in the degradation of lignocellulose. Similarly, proteomic studies showed that a total of 130 proteins existed in the fungus grown on SEB. These proteins were classified into several groups of secreted extracellular enzymes, and their functional classification demonstrated that 59% of the proteins were CAZymes such as GH45-endoglucanases, GH6 and GH7-cellobiohydrolases, GH3-β-glucosidases, GH10-xylanases, and AA9-LPMO. Data obtained by transcriptome and secretome analyses indicated that A. fumigatus produces a considerable number of CAZymes that participate in the hydrolysis of biomass. These CAZymes are valuable for the lignocellulosic bioenergy industry, provide information for future studies on the degradation of biomass, and improve understanding of the role genes and enzymes play in the degradation process.

Project description:BACKGROUND:As one of the most abundant agricultural wastes, sugarcane bagasse is largely under-exploited, but it possesses a great potential for the biofuel, fermentation, and cellulosic biorefinery industries. It also provides a unique ecological niche, as the microbes in this lignocellulose-rich environment thrive in relatively high temperatures (50°C) with varying microenvironments of aerobic surface to anoxic interior. The microbial community in bagasse thus presents a good resource for the discovery and characterization of new biomass-degrading enzymes; however, it remains largely unexplored. RESULTS:We have constructed a fosmid library of sugarcane bagasse and obtained the largest bagasse metagenome to date. A taxonomic classification of the bagasse metagenome reviews the predominance of Proteobacteria, which are also found in high abundance in other aerobic environments. Based on the functional characterization of biomass-degrading enzymes, we have demonstrated that the bagasse microbial community benefits from a large repertoire of lignocellulolytic enzymes, which allows them to digest different components of lignocelluoses into single molecule sugars. Comparative genomic analyses with other lignocellulolytic and non-lignocellulolytic metagenomes show that microbial communities are taxonomically separable by their aerobic "open" or anoxic "closed" environments. Importantly, a functional analysis of lignocellulose-active genes (based on the CAZy classifications) reveals core enzymes highly conserved within the lignocellulolytic group, regardless of their taxonomic compositions. Cellulases, in particular, are markedly more pronounced compared to the non-lignocellulolytic group. In addition to the core enzymes, the bagasse fosmid library also contains some uniquely enriched glycoside hydrolases, as well as a large repertoire of the newly defined auxiliary activity proteins. CONCLUSIONS:Our study demonstrates a conservation and diversification of carbohydrate-active genes among diverse microbial species in different biomass-degrading niches, and signifies the importance of taking a global approach to functionally investigate a microbial community as a whole, as compared to focusing on individual organisms.

Project description:The production of bioethanol from non-food agricultural residues represents an alternative energy source to fossil fuels for incorporation into the world's economy. Within the context of bioconversion of plant biomass into renewable energy using improved enzymatic cocktails, Illumina RNA-seq transcriptome profiling was conducted on a strain of Aspergillus tamarii, efficient in biomass polysaccharide degradation, in order to identify genes encoding proteins involved in plant biomass saccharification. Enzyme production and gene expression was compared following growth in liquid and semi-solid culture with steam-exploded sugarcane bagasse (SB) (1% w/v) and glucose (1% w/v) employed as contrasting sole carbon sources. Enzyme production following growth in liquid minimum medium supplemented with SB resulted in 0.626 and 0.711 UI.mL-1 xylanases after 24 and 48 h incubation, respectively. Transcriptome profiling revealed expression of over 7120 genes, with groups of genes modulated according to solid or semi-solid culture, as well as according to carbon source. Gene ontology analysis of genes expressed following SB hydrolysis revealed enrichment in xyloglucan metabolic process and xylan, pectin and glucan catabolic process, indicating up-regulation of genes involved in xylanase secretion. According to carbohydrate-active enzyme (CAZy) classification, 209 CAZyme-encoding genes were identified with significant differential expression on liquid or semi-solid SB, in comparison to equivalent growth on glucose as carbon source. Up-regulated CAZyme-encoding genes related to cellulases (CelA, CelB, CelC, CelD) and hemicellulases (XynG1, XynG2, XynF1, XylA, AxeA, arabinofuranosidase) showed up to a 10-fold log2FoldChange in expression levels. Five genes from the AA9 (GH61) family, related to lytic polysaccharide monooxygenase (LPMO), were also identified with significant expression up-regulation. The transcription factor gene XlnR, involved in induction of hemicellulases, showed up-regulation on liquid and semi-solid SB culture. Similarly, the gene ClrA, responsible for regulation of cellulases, showed increased expression on liquid SB culture. Over 150 potential transporter genes were also identified with increased expression on liquid and semi-solid SB culture. This first comprehensive analysis of the transcriptome of A. tamarii contributes to our understanding of genes and regulatory systems involved in cellulose and hemicellulose degradation in this fungus, offering potential for application in improved enzymatic cocktail development for plant biomass degradation in biorefinery applications.

Project description:BACKGROUND:Sugarcane bagasse has been proposed as a lignocellulosic residue for second-generation ethanol (2G) produced by breaking down biomass into fermentable sugars. The enzymatic cocktails for biomass degradation are mostly produced by fungi, but low cost and high efficiency can consolidate 2G technologies. A. fumigatus plays an important role in plant biomass degradation capabilities and recycling. To gain more insight into the divergence in gene expression during steam-exploded bagasse (SEB) breakdown, this study profiled the transcriptome of A. fumigatus by RNA sequencing to compare transcriptional profiles of A. fumigatus grown on media containing SEB or fructose as the sole carbon source. Secretome analysis was also performed using SDS-PAGE and LC-MS/MS. RESULTS:The maximum activities of cellulases (0.032 U mL-1), endo-1,4-β--xylanase (10.82 U mL-1) and endo-1,3-β glucanases (0.77 U mL-1) showed that functional CAZymes (carbohydrate-active enzymes) were secreted in the SEB culture conditions. Correlations between transcriptome and secretome data identified several CAZymes in A. fumigatus. Particular attention was given to CAZymes related to lignocellulose degradation and sugar transporters. Genes encoding glycoside hydrolase classes commonly expressed during the breakdown of cellulose, such as GH-5, 6, 7, 43, 45, and hemicellulose, such as GH-2, 10, 11, 30, 43, were found to be highly expressed in SEB conditions. Lytic polysaccharide monooxygenases (LPMO) classified as auxiliary activity families AA9 (GH61), CE (1, 4, 8, 15, 16), PL (1, 3, 4, 20) and GT (1, 2, 4, 8, 20, 35, 48) were also differentially expressed in this condition. Similarly, the most important enzymes related to biomass degradation, including endoxylanases, xyloglucanases, β-xylosidases, LPMOs, α-arabinofuranosidases, cellobiohydrolases, endoglucanases and β-glucosidases, were also identified in the secretome. CONCLUSIONS:This is the first report of a transcriptome and secretome experiment of Aspergillus fumigatus in the degradation of pretreated sugarcane bagasse. The results suggest that this strain employs important strategies for this complex degradation process. It was possible to identify a set of genes and proteins that might be applied in several biotechnology fields. This knowledge can be exploited for the improvement of 2G ethanol production by the rational design of enzymatic cocktails.

Project description:BackgroundBiofuels obtained from first-generation (1G) sugars-starch streams have been proven unsustainable as their constant consumption is not only significantly costly for commercial scale production systems, but it could potentially lead to problems associated with extortionate food items for human usage. In this regard, biofuels' production in alkali-thermophilic environs from second-generation (2G) bio-waste would not only be markedly feasible, but these extreme conditions might be able to sustain aseptic fermentations without spending much for sterilization.ResultsPresent investigation deals with the valuation of ethanologenic potential of locally isolated moderate alkali-thermophilic fermentative bacterium, Bacillus licheniformis KU886221 employing sugarcane cane bagasse (SCB) as substrate. A standard 2-factor central composite response surface design was used to estimate the optimized cellulolytic and hemicellulolytic enzymatic hydrolysis of SCB into maximum fermentable sugars. After elucidation of optimized levels of fermentation factors affecting ethanol fermentation using Taguchi OA L27 (3^13) experimental design, free cell batch culture was carried out in bench-scale stirred-tank bioreactor for ethanol fermentation. Succeeding fermentation modifications included subsequent substrate addition, immobilized cells fibrous-bed bioreactor (FBB) incorporation to the basic setup, and performance of in situ gas stripping for attaining improved ethanol yield. Highest ethanol yield of 1.1406 mol ethanol/mol of equivalent sugars consumed was obtained when gas stripping was performed during fed-batch fermentation involving FBB under aseptic conditions. Despite the fact that under non-aseptic conditions, 30.5% lesser ethanol was formed, still, reduced yield might be considered influential as it saved the cost of sterilization for ethanol production.ConclusionEffectual utilization of low-priced abundantly available lignocellulosic waste sugarcane bagasse under non-aseptic moderate alkali-thermophilic fermentation conditions as directed in this study has appeared very promising for large-scale cost-effective bioethanol generation processes.

Project description:In this study, bagasse was pretreated with ionic liquid (IL) 1-butyl-3-methylimidazolium chloride ([Bmim]Cl) and 1% NaOH solution for initial activation of bagasse. A mixed fermentation of treated bagasse by Aspergillus niger and Candida shehatae showed the optimal conditions with the addition of C. shehatae 12 h later at a 1:1 proportion to A. niger. To further improve the ethanol production and obtain optimal fermentation conditions, a Plackett-Burman design was applied to screen the significant formulation and process variables. The optimal ethanol fermentation conditions with IL pretreated bagasse were determined using response surface methodology by Box-Behnken design. Three variables "initial pH, (NH4)2SO4, fermentation time" were regarded as significant factors in the optimization study. The resulting optimum fermentation conditions for bioethanol was identified as: initial pH of 5.89, (NH4)2SO4 concentration of 0.40 g/50 mL, and fermentation time of 3.60 days. The verification experimental ethanol concentration was 8.14 g/L, which agreed with the predicted value. An enhancement of approximately 153.58% compared with initial fermentation conditions in ethanol production was found using optimized conditions. It demonstrated that optimization methodology had a positive effect on the improvement of ethanol production. Under the optimal fermentation medium and conditions, the ethanol production with IL-pretreated bagasse and untreated bagasse was 8.14 g/L and 5.03 g/L, respectively, which exhibited 62% increase, compared to initial conditions with production of 3.21 g/L and 2.67 g/L, respectively, which displayed 20% increase. Both under optimal and original fermentation conditions, compared to the fermentation medium with untreated bagasse, all the results indicated that IL-pretreated bagasse resulted in higher ethanol production than untreated bagasse, demonstrating that IL-pretreated bagasse successfully increased the ethanol production in the mixed fermentation by A. niger and C. shehatae.

Dataset Information

Characterization of lignocellulolytic activities from a moderate halophile strain of Aspergillus caesiellus isolated from a sugarcane bagasse fermentation.

Publications

Characterization of lignocellulolytic activities from a moderate halophile strain of Aspergillus caesiellus isolated from a sugarcane bagasse fermentation.

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