Utilisation of wheat bran as a substrate for bioethanol production using recombinant cellulases and amylolytic yeast

Wheat bran, generated from the milling of wheat, represents a promising feedstock for the production of bioethanol. This substrate consists of three main components: starch, hemicellulose and cellulose. The optimal conditions for wheat bran hydrolysis have been determined using a recombinant cellulase cocktail (RCC), which contains two cellobiohydrolases, an endoglucanase and a beta-glucosidase. The 10% (w/v, expressed in terms of dry matter) substrate loading yielded the most glucose, while the 2% loading gave the best hydrolysis efficiency (degree of saccharification) using unmilled wheat bran. The ethanol production of two industrial amylolytic Saccharomyces cerevisiae strains, MEL2[TLG1-SFA1] and M2n [TLG1-SFA1], were compared in a simultaneous saccharification and fermentation (SSF) for 10% wheat bran loading with or without the supplementation of optimised RCC. The recombinant yeasts. cerevisiae MEL2[TLG1-SFA1] and M2n[TLG1-SFA1] completely hydrolysed wheat bran's starch producing similar amounts of ethanol (5.3 +/- 0.14 g/L and 5.0 +/- 0.09 g/L, respectively). Supplementing SSF with RCC resulted in additional ethanol production of about 2.0 g/L. Scanning electron microscopy confirmed the effectiveness of both RCC and engineered amylolytic strains in terms of cellulose and starch depolymerisatio

Enzymatic Hydrolysis of Softwood Derived Paper Sludge by an In Vitro Recombinant Cellulase Cocktail for the Production of Fermentable Sugars

CITATION:Malgas S, Rose SH, van Zyl WH, Pletschke BI. Enzymatic Hydrolysis of Softwood Derived Paper Sludge by an In Vitro Recombinant Cellulase Cocktail for the Production of Fermentable Sugars. Catalysts. 2020; 10(7):775. https://doi.org/10.3390/catal10070775Abstract: Paper sludge is an attractive biomass feedstock for bioconversion to ethanol due to its low cost and the lack of pretreatment required for its bioprocessing. This study assessed the use of a recombinant cellulase cocktail (mono-components: S. cerevisiae-derived PcBGL1B (BGL), TeCel7A (CBHI), ClCel6A (CBHII) and TrCel5A (EGII) mono-component cellulase enzymes) for the efficient saccharification of softwood-derived paper sludge to produce fermentable sugars. The paper sludge mainly contained 74.3% moisture and 89.7% (per dry mass (DM)) glucan with a crystallinity index of 91.5%. The optimal protein ratio for paper sludge hydrolysis was observed at 9.4: 30.2: 30.2: 30.2% for BGL: CBHI: CBHII: EGII. At a protein loading of 7.5 mg/g DW paper sludge, the yield from hydrolysis was approximately 80%, based on glucan, with scanning electron microscopy micrographs indicating a significant alteration in the microfibril size (length reduced from ≥ 2 mm to 93 µm) of the paper sludge. The paper sludge hydrolysis potential of the Opt CelMix (formulated cellulase cocktail) was similar to the commercial Cellic CTec2® and Celluclast® 1.5 L cellulase preparations and better than Viscozyme® L. Low enzyme loadings (15 mg/g paper sludge) of the Opt CelMix and solid loadings ranging between 1 to 10% (w/v) rendered over 80% glucan conversion. The high glucose yields attained on the paper sludge by the low enzyme loading of the Opt CelMix demonstrated the value of enzyme cocktail optimisation on specific substrates for efficient cellulose conversion to fermentable sugars

Enrichment of maize and triticale bran with recombinant Aspergillus tubingensis ferulic acid esterase

Ferulic acid is a natural antioxidant found in various plants and serves as a precursor for various fine chemicals, including the flavouring agent vanillin. However, expensive extraction methods have limited the commercial application of ferulic acid, in particular for the enrichment of food substrates. A recombinant Aspergillus tubingensis ferulic acid esterase Type A (FAEA) was expressed in Aspergillus niger D15#26 and purified with anion-exchange chromatography (3487 U/mg, Km = 0.43 mM, Kcat = 0.48/min on methyl ferulate). The 36-kDa AtFAEA protein showed maximum ferulic acid esterase activity at 50 C and pH 6, suggesting potential application in industrial processes. A crude AtFAEA preparation extracted 26.56 and 8.86 mg/g ferulic acid from maize bran and triticale bran, respectively, and also significantly increased the levels of p-coumaric and caffeic acid from triticale bran. The cost-effective production of AtFAEA could therefore allow for the enrichment of brans generally used as food and fodder, or for the production of fine chemicals (such as ferulic and p-coumaric acid) from plant substrates. The potential for larger-scale production of AtFAEA was demonstrated with the A. niger D15[AtfaeA] strain yielding a higher enzyme activity (185.14 vs.83.48 U/ml) and volumetric productivity (3.86 vs. 1.74 U/ml/h) in fed-batch than batch fermentation.In part by the National Research Foundation of South Africa (Grant 76597 to MVB and Grant 86423 to WHvZ).http://link.springer.com/journal/131972018-03-31hb2017Food Scienc

Construction of industrial Saccharomyces cerevisiae strains for the efficient consolidated bioprocessing of raw starch

CITATION: Cripwell, R. A., et al. 2019. Construction of industrial Saccharomyces cerevisiae strains for the efficient consolidated bioprocessing of raw starch. Biotechnology for Biofuels, 12:201, doi:10.1186/s13068-019-1541-5.Background: Consolidated bioprocessing (CBP) combines enzyme production, saccharification and fermentation into a one-step process. This strategy represents a promising alternative for economic ethanol production from starchy biomass with the use of amylolytic industrial yeast strains. Results: Recombinant Saccharomyces cerevisiae Y294 laboratory strains simultaneously expressing an α-amylase and glucoamylase gene were screened to identify the best enzyme combination for raw starch hydrolysis. The codon optimised Talaromyces emersonii glucoamylase encoding gene (temG_Opt) and the native T. emersonii α-amylase encoding gene (temA) were selected for expression in two industrial S. cerevisiae yeast strains, namely Ethanol Red™ (hereafter referred to as the ER) and M2n. Two δ-integration gene cassettes were constructed to allow for the simultaneous multiple integrations of the temG_Opt and temA genes into the yeasts’ genomes. During the fermentation of 200 g l−1 raw corn starch, the amylolytic industrial strains were able to ferment raw corn starch to ethanol in a single step with high ethanol yields. After 192 h at 30 °C, the S. cerevisiae ER T12 and M2n T1 strains (containing integrated temA and temG_Opt gene cassettes) produced 89.35 and 98.13 g l−1 ethanol, respectively, corresponding to estimated carbon conversions of 87 and 94%, respectively. The addition of a commercial granular starch enzyme cocktail in combination with the amylolytic yeast allowed for a 90% reduction in exogenous enzyme dosage, compared to the conventional simultaneous saccharification and fermentation (SSF) control experiment with the parental industrial host strains. Conclusions: A novel amylolytic enzyme combination has been produced by two industrial S. cerevisiae strains. These recombinant strains represent potential drop-in CBP yeast substitutes for the existing conventional and raw starch fermentation processes.https://biotechnologyforbiofuels.biomedcentral.com/articles/10.1186/s13068-019-1541-5Publisher's versio

Raw starch conversion by Saccharomyces cerevisiae expressing Aspergillus tubingensis amylases

Publication of this article was funded by the Stellenbosch University Open Access Fund.The original publication is available at :Viktor, M. J., Rose, S. H., Van Zyl, W. H. & Viljoen-Bloom, M. 2013. Raw starch conversion by Saccharomyces cerevisiae expressing Aspergillus tubingensis amylases. Biotechnology for Biofuels, 6:167, doi:10.1186/1754-6834-6-167.The original publication is available at http://www.biotechnologyforbiofuels.com/Background: Starch is one of the most abundant organic polysaccharides available for the production of bio-ethanol as an alternative transport fuel. Cost-effective utilisation of starch requires consolidated bioprocessing (CBP) where a single microorganism can produce the enzymes required for hydrolysis of starch, and also convert the glucose monomers to ethanol. Results: The Aspergillus tubingensis T8.4 α-amylase (amyA) and glucoamylase (glaA) genes were cloned and expressed in the laboratory strain Saccharomyces cerevisiae Y294 and the semi-industrial strain, S. cerevisiae Mnuα1. The recombinant AmyA and GlaA displayed protein sizes of 110–150 kDa and 90 kDa, respectively, suggesting significant glycosylation in S. cerevisiae. The Mnuα1[AmyA-GlaA] and Y294[AmyA-GlaA] strains were able to utilise 20 g l⁻¹ raw corn starch as sole carbohydrate source, with ethanol titers of 9.03 and 6.67 g l⁻¹ (0.038 and 0.028 g l⁻¹ h⁻¹), respectively, after 10 days. With a substrate load of 200 g l⁻¹ raw corn starch, Mnuα1[AmyA-GlaA] yielded 70.07 g l⁻¹ ethanol (0.58 g l⁻¹ h⁻¹) after 120 h of fermentation, whereas Y294[AmyA-GlaA] was less efficient at 43.33 g l-1 ethanol (0.36 g l⁻¹ h⁻¹). Conclusions: In a semi-industrial amylolytic S. cerevisiae strain expressing the A. tubingensis α-amylase and glucoamylase genes, 200 g l⁻¹ raw starch was completely hydrolysed (saccharified) in 120 hours with 74% converted to released sugars plus fermentation products and the remainder presumably to biomass. The single-step conversion of raw starch represents significant progress towards the realisation of CBP without the need for any heat pretreatment. Furthermore, the amylases were produced and secreted by the host strain, thus circumventing the need for exogenous amylases.Stellenbosch UniversityPublishers' Versio

Cellulase Production from Spent Lignocellulose Hydrolysates by Recombinant Aspergillus niger▿

A recombinant Aspergillus niger strain expressing the Hypocrea jecorina endoglucanase Cel7B was grown on spent hydrolysates (stillage) from sugarcane bagasse and spruce wood. The spent hydrolysates served as excellent growth media for the Cel7B-producing strain, A. niger D15[egI], which displayed higher endoglucanase activities in the spent hydrolysates than in standard medium with a comparable monosaccharide content (e.g., 2,100 nkat/ml in spent bagasse hydrolysate compared to 480 nkat/ml in standard glucose-based medium). In addition, A. niger D15[egI] was also able to consume or convert other lignocellulose-derived compounds, such as acetic acid, furan aldehydes, and phenolic compounds, which are recognized as inhibitors of yeast during ethanolic fermentation. The results indicate that enzymes can be produced from the stillage stream as a high-value coproduct in second-generation bioethanol plants in a way that also facilitates recirculation of process water

Engineering industrial yeast strains for Consolidated Bioprocessing of starchy substrates and by-products to ethanol.

Introduction. Commercial bioethanol is currently obtained from starchy substrates, with a relatively mature technology for corn in the USA. However, starch-to-ethanol processes are still expensive and the development of a Consolidated Bioprocessing (CBP) through amylolytic yeast could considerably reduce commercial costs (van Zyl et al. 2012). This research project aimed to construct efficient amylolytic CBP Saccharomyces cerevisiae strains for the industrial ethanol production from starchy feedstock. Materials and methods. In the last eight years, ten fungal glucoamylase and alpha-amylase genes, native and codon-optimized, were screened in different combinations for their high activity into the laboratory strain S. cerevisiae Y294. The most proficient sequences were \u3b4-integrated into industrial yeast strains (van Zyl et al. 2011; Favaro et al., 2012; Favaro et al. 2015). Results. This report gives an overview of the research outcomes we obtained towards the CBP of starchy materials into ethanol. So far, the most effective raw starch-hydrolyzing combination was found to be the codon-optimized glucoamylase of Thermomyces lanuginosus glucoamylase (TLG1) and \u3b1-amylase of Saccharomycopsis fibuligera (SFA1) and their gene were \u3b4-integrated into the industrial S. cerevisiae strains M2n and MEL2. The resulting recombinant yeast displayed high activities on raw starch (up to 4461 nkat/g dry cell weight) and produced in a bioreactor about 64 g/L ethanol from 200 g/L raw corn starch, corresponding to 55% of the theoretical yield (g of ethanol/g of glucose equivalent). Their starch conversion efficiencies were even higher on sorghum and triticale (62 and 73% of the theoretical yield, respectively). Moreover, both recombinant strains were efficiently used also for the CBP of starchy by-products, such as wheat bran and rice husk, where starch content is about 10-30% of the biomass. Supplementing the CBP with recombinant cellulases was beneficial to hydrolyze also the cellulose content of the agricultural residues, thus increasing the overall ethanol yield. Discussion. This is the first report of CBP from natural starchy substrates and by-products using industrial yeast strains co-secreting glucoamylase and \u3b1-amylase. The high ethanol yields achieved at bioreactor scale pave the way for their large scale CBP applications