Priscila da Silva Delabona

Use of a new Trichoderma harzianum strain isolated from the Amazon rainforest with pretreated sugar cane bagasse for on-site cellulase production

The on-site production of cellulases is an important strategy for the development of sustainable second-generation ethanol production processes. This study concerns the use of a specific cellulolytic enzyme complex for hydrolysis of pretreated sugar cane bagasse. Glycosyl hydrolases (FPase, xylanase, and β-glucosidase) were produced using a new strain of Trichoderma harzianum, isolated from the Amazon rainforest and cultivated under different conditions. The influence of the carbon source was first investigated using shake-flask cultures. Selected carbon sources were then further studied under different pH conditions using a stirred tank bioreactor. Enzymatic activities up to 121 FPU/g, 8000 IU/g, and 1730 IU/g of delignified steam-exploded bagasse+sucrose were achieved for cellulase, xylanase and β-glucosidase, respectively. This enzymatic complex was used to hydrolyze pretreated sugar cane bagasse. A comparative evaluation, using an enzymatic extract from Trichoderma reesei RUTC30, indicated similar performance of the T. harzianum enzyme complex, being a potential candidate for on-site production of enzymes.

Understanding the cellulolytic system of Trichoderma harzianum P49P11 and enhancing saccharification of pretreated sugarcane bagasse by supplementation with pectinase and α-L-arabinofuranosidase

Supplementation of cellulase cocktails with accessory enzymes can contribute to a higher hydrolytic capacity in releasing fermentable sugars from plant biomass. This study investigated which enzymes were complementary to the enzyme set of Trichoderma harzianum in the degradation of sugarcane bagasse. Specific activities of T. harzianum extract on different substrates were compared with the extracts of Penicillium echinulatum and Trichoderma reesei, and two commercial cellulase preparations. Complementary analysis of the secretome of T. harzianum was also used to identify which enzymes were produced during growth on pretreated sugarcane bagasse. These analyses enabled the selection of the enzymes pectinase and α-L-arabinofuranosidase (AF) to be further investigated as supplements to the T. harzianum extract. The effect of enzyme supplementation on the efficiency of sugarcane bagasse saccharification was evaluated using response surface methodology. The supplementation of T. harzianum enzymatic extract with pectinase and AF increased the efficiency of hydrolysis by up to 116%.

The capability of endophytic fungi for production of hemicellulases and related enzymes.

BackgroundThere is an imperative necessity for alternative sources of energy able to reduce the world dependence of fossil oil. One of the most successful options is ethanol obtained mainly from sugarcane and corn fermentation. The foremost residue from sugarcane industry is the bagasse, a rich lignocellulosic raw material uses for the production of ethanol second generation (2G). New cellulolytic and hemicellulytic enzymes are needed, in order to optimize the degradation of bagasse and production of ethanol 2G.ResultsThe ability to produce hemicellulases and related enzymes, suitable for lignocellulosic biomass deconstruction, was explored using 110 endophytic fungi and 9 fungi isolated from spoiled books in Brazil. Two initial selections were performed, one employing the esculin gel diffusion assay, and the other by culturing on agar plate media with beechwood xylan and liquor from the hydrothermal pretreatment of sugar cane bagasse. A total of 56 isolates were then grown at 29°C on steam-exploded delignified sugar cane bagasse (DEB) plus soybean bran (SB) (3:1), with measurement of the xylanase, pectinase, β-glucosidase, CMCase, and FPase activities. Twelve strains were selected, and their enzyme extracts were assessed using different substrates. Finally, the best six strains were grown under xylan and pectin, and several glycohydrolases activities were also assessed. These strains were identified morphologically and by sequencing the internal transcribed spacer (ITS) regions and the partial β-tubulin gene (BT2). The best six strains were identified as Aspergillus niger DR02, Trichoderma atroviride DR17 and DR19, Alternaria sp. DR45, Annulohypoxylon stigyum DR47 and Talaromyces wortmannii DR49. These strains produced glycohydrolases with different profiles, and production was highly influenced by the carbon sources in the media.ConclusionsThe selected endophytic fungi Aspergillus niger DR02, Trichoderma atroviride DR17 and DR19, Alternaria sp. DR45, Annulohypoxylon stigyum DR47 and Talaromyces wortmannii DR49 are excellent producers of hydrolytic enzymes to be used as part of blends to decompose sugarcane biomass at industrial level.

Addition of feruloyl esterase and xylanase produced on-site improves sugarcane bagasse hydrolysis

Accessory enzymes that assist biomass degradation could be used to improve the recovery of fermentable sugar for use in biorefineries. In this study, different fungal strains isolated from the Amazon rainforest were evaluated in terms of their ability to produce feruloyl esterase (FAE) and xylanase enzymes, and an assessment was made of the contributions of the enzymes in the hydrolysis of pretreated sugarcane bagasse. In the selection step, screening using plate assays was followed by shake flask submerged cultivations. After carbon source selection and cultivation in a stirred-tank bioreactor, Aspergillusoryzae P21C3 proved to be a promising strain for production of the enzymes. Supplementation of a commercial enzyme preparation with 30% (v/v) crude enzymatic complex from A. oryzae P21C3 increased the conversion of cellulose derived from pretreated sugarcane bagasse by 36%. Supplementation with FAE and xylanase enzymes produced on-site can therefore be used to improve the hydrolysis of sugarcane bagasse.

Increased production of cellulases and xylanases by Penicillium echinulatum S1M29 in batch and fed-batch culture.

The development of more productive strains of microorganisms and processes that increase enzyme levels can contribute to the economically efficient production of second generation ethanol. To this end, cellulases and xylanases were produced with the S1M29 mutant strain of Penicillium echinulatum, using different concentrations of cellulose (20, 40, and 60 g L(-1)) in batch and fed-batch processes. The highest activities of FPase (8.3 U mL(-1)), endoglucanases (37.3 U mL(-1)), and xylanases (177 U mL(-1)) were obtained in fed-batch cultivation with 40 g L(-1) of cellulose. The P. echinulatum enzymatic broth and the commercial enzyme Cellic CTec2 were tested for hydrolysis of pretreated sugar cane bagasse. Maximum concentrations of glucose and xylose were achieved after 72 h of hydrolysis. Glucose yields of 28.0% and 27.0% were obtained using the P. echinulatum enzymatic extract and Cellic CTec2, respectively.

Experimental mixture design as a tool to enhance glycosyl hydrolases production by a new Trichoderma harzianum P49P11 strain cultivated under controlled bioreactor submerged fermentation.

This work investigates the glycosyl hydrolase (GH) profile of a new Trichoderma harzianum strain cultivated under controlled bioreactor submerged fermentation. The influence of different medium components (delignified steam-exploded sugarcane bagasse, sucrose, and soybean flour) on GH biosynthesis was assessed using experimental mixture design (EMD). Additionally, the effect of increased component concentrations in culture media selected from the EMD was studied. It was found that that a mixed culture medium could significantly maximize GH biosynthesis rate, especially for xylanase enzymes which achieved a 2-fold increment. Overall, it was demonstrated that T. harzianumP49P11 enzymes have a great potential to be used in the deconstruction of biomass.

Transcriptome profile of Trichoderma harzianum IOC-3844 induced by sugarcane bagasse.

Profiling the transcriptome that underlies biomass degradation by the fungus Trichoderma harzianum allows the identification of gene sequences with potential application in enzymatic hydrolysis processing. In the present study, the transcriptome of T. harzianum IOC-3844 was analyzed using RNA-seq technology. The sequencing generated 14.7 Gbp for downstream analyses. De novo assembly resulted in 32,396 contigs, which were submitted for identification and classified according to their identities. This analysis allowed us to define a principal set of T. harzianum genes that are involved in the degradation of cellulose and hemicellulose and the accessory genes that are involved in the depolymerization of biomass. An additional analysis of expression levels identified a set of carbohydrate-active enzymes that are upregulated under different conditions. The present study provides valuable information for future studies on biomass degradation and contributes to a better understanding of the role of the genes that are involved in this process.

Improving the extraction conditions of endoglucanase produced by Aspergillus niger under solid-state fermentation

Production of cellulases under solid-state fermentation (SSF) is a promising technique that can help to reduce costs. Besides optimizing the production process, it is also important to consider enzyme recovery during the extraction step. Here, an experimental design methodology was used to investigate the effects of the operational parameters solid to liquid ratio (1:3, 1:6 and 1:9), stirring rate (80, 120 and 160 rpm), and temperature (10, 22 and 35 °C) on the recovery of endoglucanases produced by Aspergillus niger cultivated under SSF. Statistical analysis revealed that only the solid to liquid ratio had a significant influence on endoglucanase extraction. The highest endoglucanase recovery (35.7 U/g) was achieved using 0.2 mol/L acetate buffer at pH 4.8, together with a solid to liquid ratio of 1:9 and an agitation time of 10 minutes. In sequential extraction experiments, it was shown that most of the enzyme was recovered during the first extraction. The procedure adopted increased the efficiency of endoglucanase extraction by 70%, emphasizing the importance of selection of suitable operational conditions during SSF processes.

Myceliophthora thermophila M77 utilizes hydrolytic and oxidative mechanisms to deconstruct biomass

Biomass is abundant, renewable and useful for biofuel production as well as chemical priming for plastics and composites. Deconstruction of biomass by enzymes is perceived as recalcitrant while an inclusive breakdown mechanism remains to be discovered. Fungi such as Myceliophthora thermophila M77 appear to decompose natural biomass sources quite well. This work reports on this fungus fermentation property while producing cellulolytic enzymes using natural biomass substrates. Little hydrolytic activity was detected, insufficient to explain the large amount of biomass depleted in the process. Furthermore, this work makes a comprehensive account of extracellular proteins and describes how secretomes redirect their qualitative protein content based on the nature and chemistry of the nutritional source. Fungus grown on purified cellulose or on natural biomass produced secretomes constituted by: cellobiohydrolases, cellobiose dehydrogenase, β-1,3 glucanase, β-glucosidases, aldose epimerase, glyoxal oxidase, GH74 xyloglucanase, galactosidase, aldolactonase and polysaccharide monooxygenases. Fungus grown on a mixture of purified hemicellulose fractions (xylans, arabinans and arabinoxylans) produced many enzymes, some of which are listed here: xylosidase, mixed β-1,3(4) glucanase, β-1,3 glucanases, β-glucosidases, β-mannosidase, β-glucosidases, galactosidase, chitinases, polysaccharide lyase, endo β-1,6 galactanase and aldose epimerase. Secretomes produced on natural biomass displayed a comprehensive set of enzymes involved in hydrolysis and oxidation of cellulose, hemicellulose-pectin and lignin. The participation of oxidation reactions coupled to lignin decomposition in the breakdown of natural biomass may explain the discrepancy observed for cellulose decomposition in relation to natural biomass fermentation experiments.

Enhancing of sugar cane bagasse hydrolysis by Annulohypoxylon stygium glycohydrolases.

The aim of this study was to develop a bioprocess for the production of β-glucosidase and pectinase from the fungus Annulohypoxylon stygium DR47. Media optimization and bioreactor cultivation using citrus bagasse and soybean bran were explored and revealed a maximum production of 6.26 U/mL of pectinase at pH 4.0 and 10.13 U/mL of β-glucosidase at pH 5.0. In addition, the enzymes extracts were able to replace partially Celluclast 1.5L in sugar cane bagasse hydrolysis. Proteomic analysis from A. stygium cultures revealed accessory enzymes, mainly belong to the families GH3 and GH54, that would support enhancement of commercial cocktail saccharification yields. This is the first report describing bioreactor optimization for enzyme production from A. stygium with a view for more efficient degradation of sugar cane bagasse.