Use of Aureobasidium pullulans xylanase for simultaneous saccharification and fermentation in second- generation bioethanol production

Use of Aureobasidium pullulans xylanase for simultaneous saccharification and fermentation in second- generation bioethanol production | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Use of Aureobasidium pullulans xylanase for simultaneous saccharification and fermentation in second- generation bioethanol production Carla A. Lara, Evelyn S. Oliveira, Susana Marques, Francisco Gírio, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8022214/v1 This work is licensed under a CC BY 4.0 License Status: Under Revision Version 1 posted 11 You are reading this latest preprint version Abstract The production of second-generation (2G) ethanol from lignocellulosic biomass is a sustainable and economically competitive alternative. Hemicellulolytic enzymatic hydrolysis can be an efficient way to degrade biomass and obtain fermentable sugars. Here, we characterized the extracellular extract of Aureobasidium pullulans UFMG-CM-Y518, and assessed its potential for the enzymatic hydrolysis of pretreated wheat straw and simultaneous saccharification and fermentation (SSF) to produce bioethanol. A. pullulans UFMG-CM-Y518 extract displayed maximum xylanase relative activity, at 40°C and pH ranging from 4.0 to 4.5. Also, presented optimal β-xylosidase activity at 80°C and pH 4.0 to 5.0, with high stability at a moderate temperature of 45°C. A. pullulans UFMG-CM-Y518 extract was evaluated in the hydrolysis of xylan and pretreated wheat straw, in simultaneous saccharification and fermentation processes for the production ethanol. The A. pullulans UFMG-CM-Y518 extract was more efficient in the hydrolysis of beechwood xylan and wheat straw, presenting efficiency 5-times higher than the commercial hemicellulase HTec2. In SSF experiments with the xylose-fermenting yeast Spathaspora passalidarum and beechwood xylan as a substrate, the A. pullulans UFMG-CM-Y518 extract efficiently degraded xylan to xylose with higher yield (66%), when compared to the separate hydrolysis. This resulted in the production of 6.6 g∙L − 1 ethanol with yield of 0.2 g∙g − 1 . A. pullulans UFMG-CM-Y518 extract improved the conversion yield of ethanol from wheat straw by Sp. passalidarum due to the increased xylose available for fermentation. In this way, we can state that this extract has potential for biotechnological applications in the biofuels industry. Aureobasidium pullulans enzymatic hydrolysis Spathaspora passalidarum wheat straw xylan xylanase Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Increases in energy demand, concerns about climate change, and oil prices have stimulated the development of biofuels produced from lignocellulosic biomass, such as second-generation (2G) ethanol, which may contribute to the production of more sustainable (Leal et al. 2013 ; Lynd et al. 2017 ; McCormick and Howard 2013 ; Pereira et al. 2025 ; Song et al. 2016 ) and economically competitive biofuels due to the complete use of sugars. Lignocellulosic biomass consists mainly of cellulose (35–50%), hemicellulose (15–30%), lignin (12–35%), and pectin (2–5%), and is the largest known renewable source of carbon (Kumar et al. 2016 ). The industrial process of 2G bioethanol production requires the efficient degradation of the biomass involving several steps such as pretreatment, enzymatic hydrolysis and alcoholic fermentation (Gírio et al. 2010 ; Fagundes et al. 2024 ; Pereira et al. 2025 ). Pretreatment is essential to overcome biomass recalcitrance and consequently increase the accessibility of the enzymes to the cellulose (Gírio et al. 2010 ; Valdivia et al. 2016 ; Thomas et al. 2016 ). Hydrothermal treatments are considered promising and appropriate for waste biomass (Alvira et al. 2010 ). In this method, the substrate remains a cellulose-rich solid and contains a significant amount of insoluble hemicellulose (Carvalheiro et al. 2008 ). In comparison to chemical hydrolysis, the enzymatic process has important advantages due to its high specificity that prevents the formation of substrate degradation products (from sugars and lignin), maximizing conversion efficiency. During enzymatic hydrolysis, cellulases, hemicellulases, and ancillary enzymes are required to promote the complete conversion of lignocellulose, to increase the yield of simple sugars and reduce the cost of the process (Berlin et al. 2005 ; Maroldi et al. 2024 ; Selig et al. 2008 ). The enzymatic hydrolysis and fermentation stages to obtain ethanol as the final product may be performed sequentially, as separate hydrolysis and fermentation (SHF) processes, or via simultaneous saccharification and fermentation processes (SSF). During enzymatic hydrolysis, the accumulation of products can inhibit enzymes by reducing their hydrolytic activity (Afedzi and Parakulsuksatid 2023 ; Kuma and Wyman 2009 ; Paulova et al. 2015 ). In the SSF, the simple sugars released by hydrolysis will be converted into ethanol, which does not accumulate and minimizes the inhibition of the enzymatic activity. Nonetheless, the SSF process must be performed at intermediate (35°C) to optimal temperatures for enzymatic hydrolysis (approximately 50°C) and fermentation (30°C) as a compromise (Olofsson et al. 2008 ). Saccharomyces cerevisiae is one of the most used microorganisms to produce ethanol. However, this yeast cannot ferment pentose sugars like xylose. Among the species that ferment D-xylose, Spathaspora passalidarum is one of the best producers of ethanol (Cadete and Rosa, 2018 ; Nakamura et al. 2008 ; Racca et al. 2025 ). This yeast can ferment xylose with little or no aeration (Riley et al. 2016 ; Barros et al. 2024 ). Also, it has already been reported in the literature high ethanol yield, around 0.48 g∙g − 1 , which is close to the theoretical maximum value of 0.51 g∙g − 1 (obtained in culture medium) (Cadete and Rosa 2018 ; Pascoli et al. 2021 ; Barros et al. 2024 ).The success in the bioconversion of hemicellulose and cellulose to ethanol is a decisive factor in the economic viability of the process (Chandel et al. 2010 ). The performance of xylanolytic enzymes that remove the insoluble hemicellulose decreases the barrier between the cellulose and lignin, significantly improving the action of cellulase due to greater access of the enzyme to the cellulose (Kuma and Wyman 2009 ; Laureano-Perez et al. 2005 ; Ohgren et al. 2007 ). Enzymatic hydrolysis is usually performed with commercial enzyme cocktails produced from extracellular extracts of selected or genetically modified filamentous fungi. Some hemicellulolytic cocktails that are usually applied in addition to cellulolytic ones are described as xylanase-specific (no cellulase activity) and have optimal activity at 50°C (Duarte and Costa-Ferreira 1994 ; Knob et al. 2010 ). Their performance can be related to xylose levels and is generally limited by the low level of β-xylosidase activity (Coughlan and Hazlewood 1993 ). This behavior may be explained by the inhibition of xylan-degrading enzymes by the hydrolysis products. This limitation leads to incomplete enzymatic hydrolysis of xylan. Also, D-xylose is not naturally fermented by the yeast S. cerevisiae , hindering the application of xylanase in 2G ethanol production processes. The inhibition of the enzymatic hydrolysis of xylan by hydrolysis products discourages their use in SHF. In turn, SSF, which must be performed at a lower temperature (usually at 35°C or below) to allow for fermentation, can lead to inefficient enzymatic hydrolysis of xylan due to the low activity of xylanolytic enzymes at those temperatures. The successful search for xylan-degrading enzymes with high activity at low temperatures could permit the use of these enzymes in xylan simultaneous saccharification and fermentation process. Xylanase and β-xylosidase activities have been identified in yeast and yeast-like fungi with optimal temperatures lower than those of filamentous fungi used to produce commercial cocktails (Lara et al. 2014 ; Morais et al. 2013 ; Romero et al. 2012 ). The yeast-like ascomycetous fungus Aureobasidium pullulans is a xylanase producer with high specific activity and an optimal temperature between 35°C and 50°C (Gautério et al. 2020 ; Leathers 1986 ). The supplementation of the lignocellulosic material, such as pretreated corn stover and pretreated wheat straw, added with cellulolytic enzyme cocktails including xylanases, improve the enzymatic hydrolysis process (Alvira et al. 2011 ; Kuma and Wyman 2009 ; Li et al. 2010 ), thus leading to a higher fermentation yield (Jin et al. 2010 ; Kuma and Wyman 2009 ). The present study aimed to characterize the extracellular extract of Aureobasidium pullulans UFMG-CM-Y518, a strain isolated from water stored in bromeliad tanks, and to evaluate the application of this xylanolytic extract in the enzymatic hydrolysis of xylan and pretreated wheat straw, and to evaluate the performance of this extract in SSF process to produce bioethanol. Materials and Methods Production and characterization of xylanolytic enzymes Aureobasidium pullulans UFMG-CM-Y518 was previously isolated from water stored in bromeliad tanks (phytothelma) (Gomes et al. 2015) and was obtained from the Culture Collection of Microorganisms and Cells at the Federal University of Minas Gerais (UFMG). A. pullulans NRRL Y-2311 was used as a reference strain and was obtained from the National Laboratory of Energy and Geology (Lisbon, Portugal) and from the Culture Collection of the Agriculture Research Service, United States Department of Agriculture. The cells were precultured in a liquid medium (initial pH 5.0) containing yeast nitrogen base (YNB) and D-xylose (YNB 6.7 g∙L − 1 , xylose 30 g∙L − 1 ) at 30°C with agitation at 150 rpm for 24 h. At the end of cultivation, the culture was centrifuged (2,600 × g for 15 min) and the collected cells were inoculated at an initial optical density equal to 4 induced by xylan-YNB medium (yeast nitrogen base 6.7 g∙L − 1 ; xylan, 10 g∙L − 1 ), which were determined at 600 nm. The production of xylan-degrading enzymes was induced in 100 mL Erlenmeyer flasks with 25 mL culture medium at 30°C and agitation at 150 rpm for 72 h. The enzyme extract was collected by centrifugation and used to determine the extracellular enzymatic activities. Enzyme and protein assays Xylanase was assayed according to Bailey et al. ( 1992 ) with a few modifications (Lara et al. 2014 ). The assay was performed with an incubation time of 30 min and a proportion of 1/3 substrate/culture. Cellulase activity was determined using Whatman No. 1 filter paper (Ghose 1987 ). Xylosidase was assayed according to Li et al ( 1993 ). The extracellular protein concentration was measured using the Bicinchoninic Acid Protein Quantitation Assay (Thermo Scientific – Pierce, USA) with bovine serum albumin as the standard. Enzymatic characterization The crude extract of A. pullulans UFMG-CM-Y518 was used to determine the temperature and the optimum pH of the xylanolytic enzymes. The optimum temperature was determined at temperatures ranging from 20 to 90°C in sodium acetate buffer (50 mM, pH 5.5) for 30 min. Sodium acetate and sodium phosphate buffers (both 50 mM) were used to determine the optimal pH over a range of 3.0 to 8.0 at 45 ºC for 30 min. The thermal stability was assessed by incubating the crude extract of the A. pullulans UFMG-CM-Y518 strain in sodium acetate buffer (50 mM, pH 4.8) at 45°C, and was further evaluated by incubation with the substrate (beechwood xylan suspension of 10 g∙L -1 ) at pH 4.8 and 45°C. Aliquots were obtained after 0, 6, 12, 24, 48 and 72 h, which were frozen for later determination of residual activities. Enzymatic hydrolysis Commercial beechwood xylan and pretreated wheat straw were used as substrates. The wheat straw was subjected to a mild hydrothermal pretreatment (190°C for 10 min) and was kindly provided for this study by the Technical University of Denmark. The pretreated wheat straw contained next to 41% of cellulose and 22% of xylan. The extracellular extract of A. pullulans UFMG-CM-Y518 and the commercial preparations Cellic HTec2 and Cellic CTec2 (kindly supplied by Novozymes, Denmark) were used in the enzymatic hydrolysis experiments, simultaneous saccharification and co-fermentation processes (SSCF), and SSF. Cellic HTec2 is a hemicellulase preparation with high endoxylanase activity and residual cellulase activity. Cellic CTec2 is a preparation of cellulases with high β-glucosidase activity and hemicellulolytic activity. Preliminary experiments for enzymatic hydrolysis were conducted with beechwood xylan [2% (w/v)] and with pretreated wheat straw [10% (w/v)] in suspension as substrates. The hydrolysis medium contained substrate (xylan or pretreated wheat straw), 0.08% (w/v) sodium azide, 4 mL of A. pullulans UFMG-CM-Y518 xylanolytic extract or 3 µL of HTec2 [both with activities equivalent to 200 U (endo-xylanase)∙g − 1 xylan], and 50 mM sodium acetate buffer (pH 5.5) at an initial total volume of 20 mL in Erlenmeyer flasks. The flasks were incubated at 50°C with orbital shaking (150 rpm) for 96 h. Aliquots were obtained for analysis after 0, 6, 24, 48, 72, and 96 h of incubation. In the second enzymatic hydrolysis stage (and when in SSF) pretreated wheat straw was used as substrate at a concentration of 15% (w/v) in terms of total solids to verify the effect of supplementation with Cellic CTec2 cellulolytic enzymes with xylanolytic enzymes (extract of A. pullulans UFMG-CM-Y518 strain compared to the Cellic HTec2 hemicellulases) and, simultaneously, the hydrolysis of hemicellulose in the lignocellulosic substrate. Each enzyme preparation was tested separately with pretreated wheat straw. For comparison, beechwood xylan was subjected to hydrolysis by the two hemicellulolytic preparations. A. pullulans UFMG-CM-Y518 or Cellic HTec2 enzyme preparation was used at a dosage equivalent to 1000 U∙g − 1 xylan as a supplement to Cellic CTec2 applied at a dose of 20 filter paper units (FPU)·g − 1 glucan in the hydrolysis of pretreated wheat straw. The 50 mL reaction mixtures in Erlenmeyer flasks in sodium acetate buffer (50 mM, pH 4.8) were incubated at 45°C in orbital shaker (150 rpm) for 96 h. Simultaneous saccharification and fermentation (SSF) and simultaneous saccharification and co-fermentation (SSCF) processes Two strains of microorganisms were used, where Sp. passalidarum UFMG-CM-Y469 ferments xylose and glucose (Cadete et al. 2012 )d cerevisiae Ethanol Red (Fermentis, France) is an industrial strain used in the fuel alcohol industry, once it ferments glucose but not xylose. Preinoculum was prepared by selecting a colony of Sp. passalidarum UFMG-CM-Y469 from Yeast Malt Agar (YM agar (containing yeast extract, peptone, malt extract, glucose)) and inoculating the cells in flasks containing 50 mL of Yeast Extract with Xylose (YPX medium (yeast extract 10 g∙L − 1 , peptone 20 g∙L − 1 , D-xylose 30 g∙L − 1 ) and shaking at 200 rpm and 30°C. S. cerevisiae Ethanol Red was prepared in flasks containing 20 mL of growth medium (glucose 50 g∙L − 1 , yeast extract 2 g∙L − 1 , (NH 4 ) 2 SO 4 2.5 g∙L − 1 , KH 2 PO 4 1 g∙L − 1 , MgSO 4 .7H 2 O 0.3 g∙L − 1 ) and shaking at 200 rpm at 35°C. After 24 h, both precultures were centrifuged and the supernatant was discarded. The cells were weighed and diluted to obtain an inoculum size of 1 g∙L − 1 to begin the fermentation. The xylanolytic extract of the A. pullulans UFMG-CM-Y518, Cellic CTec2, and Cellic HTec2 were used in SSCF and SSF. SSF of xylan and wheat straw with extracellular xylanolytic extract of A. pullulans UFMG-CM-Y518 SSF experiments were initiated by the simultaneous addition of the enzymes for testing and the ethanol production using a lignocellulosic substrate supplemented with nutrients as the culture medium for Sp. passalidarum UFMG-CM-Y469 (10 g L − 1 yeast extract, 20 g∙L − 1 peptone) and S. cerevisiae Ethanol Red (yeast extract 2 g∙L − 1 , (NH 4 ) 2 SO 4 2.5 g∙L − 1 , KH 2 PO 4 1 g∙L − 1 , MgSO 4 .7H 2 O 0.3 g∙L − 1 ). These experiments evaluated ethanol production from beechwood xylan at a concentration of 3% (w/v) and from pretreated wheat straw at a concentration of 15% (w/v) total solids. Three sets of experiments were performed under enzymatic hydrolysis conditions (for the enzymatic dosage) like those previously described, except for temperature (35°C instead of 45°C). The first experiment assessed SSF of beechwood xylan for ethanol using a xylanolytic extract of A. pullulans UFMG-CM-Y518 and the xylose (C5) fermenting yeast Sp. passalidarum UFMG-CM-Y469. The second experiment assessed SSF of pretreated wheat straw for ethanol using Cellic CTec2 supplemented with A. pullulans UFMG-CM-Y518 strain extract or Cellic Htec2 and the S. cerevisiae Ethanol Red industrial yeast that ferments only glucose (C6). The third experiment assessed SSCF of pretreated wheat straw for ethanol using Cellic CTec2 supplemented with A. pullulans UFMG-CM-Y518 strain extract or Cellic Htec2 and Sp. passalidarum UFMG-CM-Y469, a yeast capable of fermenting glucose and xylose (C6/C5). As control, both yeasts were cultured in a medium containing xylose and another containing glucose and xylose. Sp. passalidarum UFMG-CM-Y469 was also cultivated using beechwood xylan alone and the same with A. pullulans UFMG-CM-Y518 extract as substrate at the same concentrations. SSF experiments were performed in duplicate for 96 h using potassium hydrogen phthalate buffer (50 mM, pH 4.8) at 35°C with orbital shaking (200 rpm). Erlenmeyer flasks containing 50 mL of the fermentation medium were used in both fermentation cultures. The flasks were closed with rubber stoppers and coupled to a needle assembly immersed in glycerol to release the gas produced by fermentation without air inlet. Before the start of fermentation, and upon opening the bottle for aliquot removal, nitrogen gas was injected to remove the air (anaerobic conditions). Analytical methods Samples taken from all enzymatic hydrolysis and SSF experiments were heated in boiling water for 10 min (to inactivate enzymes) and centrifuged (2600 × g, 10 min). The collected supernatant was frozen at -20°C, for subsequent analysis of enzymatic hydrolysis (glucose, xylose and xylobiose) and fermentation products (ethanol, glycerol, xylitol, acetate, and ethanol) by high-performance liquid chromatography with a Merck Hitachi chromatograph (Darmstadt, Germany) equipped with an Aminex HPX-87H column (Bio-Rad, USA) at 50°C and a refractive index detector (L-7490; Merck Hitachi, Darmastadt, Germany) using 5 mM H 2 SO 4 as the mobile phase at a flow rate of 0.4 mL∙min − 1 . Fermentation parameter calculation The fermentation parameter Y p/s et (g∙g − 1 ) ethanol yield was experimentally determined. Ethanol Y p/s et (g∙g − 1 ) was calculated following the method suggested by Schmidell et al. ( 2001 ), which correlated ΔP produced (ΔP ethanol) and ΔS consumed (derived by determining the total, initial, and consumed substrate). Statistical analysis Hydrolysis tests of the pretreated wheat straw were analyzed using analysis of variance (ANOVA) (p ≤ 0.05) and the comparison between means by the Duncan test (p ≤ 0.05). Results Production and characterization of xylanolytic enzymes by A. pullulans Extracellular extracts were obtained, and total xylanase activity was determined at pH 5.5 and either 30 ºC or 50 ºC (Fig. 1 ). Extract from A. pullulans UFMG-CM-Y518 was selected for further characterization based on its high xylanase specific activity and purity/specificity in xylan. The temperature and pH profile of xylanase activity were determined between 20 and 90 ºC and a pH range from 3 to 8 (Fig. S1 ). The xylanase extract displayed maximum activity at 40°C (Fig. S1 a), with over 70% of this activity at 30°C and 50°C. Xylanase activity significantly decreased below 30°C and above 50°C, and no activity was detected above 70°C due to heat inactivation of the enzyme. Xylanase activity was highest at pH of 4.0 to 4.5 (Fig. S1 b) and decreased significantly in acidic conditions (pH lower than 3.5) or neutral conditions (pH higher than 5.0). β-xylosidase activity in the A. pullulans UFMG-CM-Y518 extracellular extract was assessed and the optimal activity occurred at 80°C and pH 4.0 (Fig. S2). Figure 1 . Regarding the specificity and extract stability, the extract did not display cellulolytic activity, as determined by Filter Paper Activity (FPase) activity or proteolytic activity. The thermal stability of xylanase was assessed in the absence and presence of substrate (xylan) at 45°C and pH 4.8 for 72 h (Fig. S3). The sample at baseline (t = 0) was not incubated at 45°C. Its enzyme activity at this time was considered as 100%. The stability test indicated that in the absence of substrate, the xylanase enzyme lost the majority (over 60%) of its activity in the first 6 h of incubation (Fig. S3a). However, in the presence of xylan substrate (Fig. S3b) thermal stability was evident, with greater than 80% maximum activity retained for up to 12 h of incubation. The loss of activity was relatively linear over time, and at 72 h was similar to that observed in the absence of xylan at 6 h of incubation (Fig. S3a). The stability of β-xylosidase activity in extracellular extracts of A. pullulans UFMG-CM-Y518 was also determined at 45°C and pH 4.8 for 72 h (Fig.S4). Unlike xylanase activity, β-xylosidase activity was very stable at 45°C, with more than 80% of the maximum activity maintained during the 72 h experiment. Use of xylanolytic extracellular extracts from A. pullulans UFMG-CM-Y518 in the enzymatic hydrolysis of xylan and pretreated wheat straw The cellulolytic (FPase) and xylanolytic activity (xylanase) of enzyme extracts was determined under the conditions applied for the saccharification of substrates, 45°C and pH 4.8. While the Cellic HTec2 and Cellic CTec2 commercial preparations displayed significant xylanase and FPase activities, A. pullulans UFMG-CM-Y518 extract displayed xylanase activity of 104.3 U∙mL − 1 and showed no cellulase activity. The xylanolytic extracellular extract from A. pullulans UFMG-CM-Y518 was evaluated in a preliminary test on beechwood xylan [2% (w/v)] and pretreated wheat straw [10% (w/v) solids; about 2% (w/v) xylan)], in an enzymatic process at 50°C and pH 5.5. The Cellic HTec2 xylanolytic commercial preparation was used for comparison. The hydrolysis efficiency was determined based on the concentration of D-xylose and xylobiose produced from each substrate (Fig. S5). With the use of xylanolytic enzymes of A. pullulans UFMG-CM-Y518, xylan hydrolysis was similar to that obtained with Cellic HTec2 (approximately 25–30%). However, with wheat straw, the A. pullulans UFMG-CM-Y518 extract was more efficient than Cellic HTec2, 60% and 30%, respectively. The concentration of xylose obtained (complete hydrolysis of the xylan) was greater with A. pullulans UFMG-CM-Y518 than with Cellic HTec2, either in xylan or pretreated wheat straw, reaching concentrations of D-xylose that were 3- and 5-fold higher, respectively. Consequently, the yield of xylobiose was higher for substrates treated with HTec2, indicating that the A. pullulans UFMG-CM-Y518 extract possessed more efficient β-xylosidase activity, with a higher yield in the hydrolysis of xylan to xylose, under the conditions chosen for the assay. The activity of A. pullulans UFMG-CM-Y518 extract was evaluated in comparison with Cellic HTec2 hemicellulase using the two substrates [3% beechwood xylan (w/v) and pretreated wheat straw 15% (w/v) total solids] at a higher solids concentration that was equivalent to that used in subsequent enzymatic hydrolysis steps with cellulase and in SSF processes (Fig. 2 ). Figure 2 . The enzyme dosage used in this assay was 1000 U∙g − 1 xylan. In the presence of xylan the xylanolytic extract of A. pullulans UFMG-CM-Y518 and hemicellulase HTec2 exhibited similar conversion efficiency of xylose. HTec2 displayed a higher yield of xylobiose and A. pullulans extract UFMG-CM-Y518 displayed a conversion efficiency of xylose with wheat straw that was approximately 2-fold of that obtained with HTec2. The yield obtained with a higher (50%) substrate concentration led to a lower yield of enzymatic hydrolysis, which did not exceed 25% hydrolysis yield under these conditions, even though the dose of xylanase used was 5-times higher and had more favorable conditions for xylanase activity. Effect of cellulase supplementation with extracellular xylanolytic extract of A. pullulans UFMG-CM-Y518 on the enzymatic hydrolysis of wheat straw Once the potential of A. pullulans xylanolytic extract UFMG-CM-Y518 was demonstrated, new experiments were conducted to evaluate the effect of this extract supplemented with cellulolytic enzymes (Cellic CTec2) to achieve more efficient hydrolysis of wheat straw, using 15% (w/v) of total solids. For comparison, the Cellic CTec2 cellulase preparation was supplemented with the Cellic HTec2 hemicellulolytic enzyme preparation and tested against non-supplemented samples. The conversion yields of xylose and glucose are shown in Fig. 3 . Figure 3 . Supplementation of Cellic CTec2 with xylanolytic enzymes of A. pullulans UFMG-CM-Y518 led to a higher yield in the hydrolysis of both polysaccharide constituents (xylan and cellulose) of pretreated wheat straw, when compared with supplementation with Cellic HTec2 and the isolated use of Cellic CTec2. The xylose conversion yields presented values next to 40% and did not differ significantly (p ≤ 0.05; Duncan's test), for the samples obtained with and without the supplementation of A. pullulans UFMG-CM-Y518 extract with Cellic Ctec2. Ethanol production from xylan and wheat straw by SSF with extracellular xylanolytic extract of A. pullulans UFMG-CM-Y518 strain Ethanol production from xylan was evaluated using the A. pullulans UFMG-CM-Y518 extract to hydrolyze xylan to xylose. Simultaneously, Sp. passalidarum UFMG-CM-Y469 was used to anaerobically ferment xylose to ethanol. In this case, Sp. passalidarum UFMG-CM-Y469 was unable to convert xylan directly to ethanol, indicating that it did not produce xylanolytic enzymes under the conditions used. Importantly, no xylo-oligosaccharides or accumulation of xylose was detected after 96 h. However, when the medium constituted of 30 g∙L − 1 xylan at 35°C (Fig. S6) was supplemented with A. pullulans UFMG-CM-Y518 extract, Sp. passalidarum UFMG-CM-Y469 produced ethanol at a concentration of 6.6 g∙L − 1 , corresponding to a yield of 0.2 g∙g − 1 . At 35°C, Sp. passalidarum UFMG-CM-Y469 produced ethanol from xylose with a conversion yield of 0.3 g∙g − 1 (Fig. S6b). This cultivation was performed in the presence and absence of extracellular extract from A. pullulans UFMG-CM-Y518, indicating that this extract has no direct effect on the metabolism of this yeast. Assuming a yield of 0.3 g·g − 1 for the conversion of xylose to ethanol (from 30 g·L − 1 of xylose under the same conditions), Sp. passalidarum should have fermented 22.5 g·L − 1 of xylose, meaning that the xylanolytic extract A. pullulans UFMG-CM-Y518 should have promoted the hydrolysis of xylan to xylose with an estimated yield of 66% throughout the SSF process. Therefore, the extent of xylan hydrolysis was significantly improved when coupled with fermentation, as when the same extract was applied to xylan at the same substrate and A. pullulans UFMG-CM-Y518 doses, a hydrolytic yield, or rather, xylan to xylose conversion, of less than 20% was obtained. Production of cellulosic ethanol from pretreated wheat straw by SSF with cellulase supplemented with xylanolytic extract of A. pullulans UFMG-CM-Y518strain The effect of supplementation of Cellic CTec2 cellulase with hemicellulolytic enzymes ( A. pullulans UFMG-CM-Y518 extract compared with the Cellic HTec2 commercial enzyme preparation) on ethanol production from the cellulose fraction of wheat straw pretreated with S. cerevisiae Ethanol Red was investigated (Fig. S7). Under the test conditions, the addition of A. pullulans UFMG-CM-Y518 extract did not lead to a better performance of Cellic CTec2 cellulase concerning the extent of cellulose hydrolysis achieved from wheat straw. There was no significant effect on ethanol production, unlike supplementation with Cellic HTec2 (Fig. S7 and Table 1 ). Nonetheless, the addition of extract A. pullulans UFMG-CM-Y518 led to a higher concentration of accumulated xylose (Table 1 ), indicating that the efficiency of xylanolytic enzymes was superior when supplemented with A. pullulans UFMG-CM-Y518 extract. Table 1 Maximum ethanol production after 96 h by SSF with S. cerevisiae Ethanol Red at 35 ºC and 150 rpmunder anaerobic conditions from the cellulose fraction of wheat strawusing Cellic CTec2without supplementation, supplementation with Cellic HTec2, and supplementation with Aureobasidium pullulans UFMG-CM-Y518 extract (XBro) Enzymes [Ethanol] max (g L − 1 ) Yield* (% max theoretical) [Xylose] accumulated (g L − 1 ) Cellic CTec2 12.6 ± 2.18 23.1 (35.7) 11.5 ± 0.13 Cellic CTec2 + Cellic HTec2 15.0 ± 1.30 27.4 (42.4) 12.1 ± 1.09 Cellic CTec2 + UFMG-CM-Y518 13.1 ± 0.45 23.9 (36.9) 13.2 ± 0.72 *Calculated relative to the maximum possible concentration of ethanol obtained by fermentation from glucose and xylose resulting from the complete hydrolysis of cellulose and xylan existing in pretreated wheat straw (in brackets the yield value considering only the fraction of cellulose). Production of lignocellulosic ethanol from pretreated wheat straw by SSCF cellulase supplemented with A. pullulans UFMG -CM-Y518 and the yeast Sp. passalidarum UFMG-CM-Y469. Table 1 . Finally, we studied the effect of Cellic CTec2 cellulase that was not supplemented with xylanolytic enzymes ( A. pullulans UFMG-CM-Y518 extract compared to Cellic HTec2 hemicellulase) in the production of ethanol from the cellulose and hemicellulose fractions of wheat straw pretreated with Sp. passalidarum UFMG-CM-Y469 (Fig. 4 , Table 2 ). Under the tested conditions, the usage of A. pullulans UFMG -CM-Y518 promoted a better performance of Cellic CTec2, where the maximum ethanol production is approximately 1.13-fold higher, as well as the yield, which is 1.13-fold higher. On regard of the accumulated xylose, values 1.24-fold higher were obtained. Table 2 Maximum ethanol production after 96 hby SSCF with Spathaspora passalidarum UFMG-CM-Y469, at 35°C and 150 rpm under anaerobic conditions from the cellulose and hemicellulose fractions of wheat straw using Cellic CTec2without supplementation, supplementation with Cellic HTec2, and supplementation with Aureobasidium pullulans UFMG-CM-Y518 extract Enzymes [Ethanol] max (g L − 1 ) Yield* (% max theoretical) [Xylose] accumulated (g L − 1 ) Cellic CTec2 14.3 ± 0.33 26.1 12.1 ± 0.57 Cellic CTec2 + Cellic HTec2 14.8 ± 1.22 27.1 11.4 ± 0.38 Cellic CTec2 + UFMG-CM-Y518 16.7 ± 0.45 30.5 14.1 ± 0.95 *Calculated relative to the maximum possible concentration of ethanol obtained by fermentation from glucose and xylose, resulting from the complete hydrolysis of cellulose and xylan in the pretreated wheat straw. Table 2 . Figure 4 . Discussion Some important aspects of the production of xylanases by the A. pullulans UFMG-CM-Y518 and NRRL Y-2311 strains were evaluated after induction by xylan. One of these aspects is the activity profiles at different pH and temperature conditions. The results presented above were very similar to those observed for the A. pullulans NRRL Y-2311 strain (Leathers 1986 ). The optimal β-xylosidase activity in the A. pullulans UFMG-CM-Y518 extracellular extract was consistent with values reported in the extracellular extract of A. pullulans CBS 58475 (80°C and pH 4.5 with maximum activity values in xylan concentrations of 0.04 and 0.20 U∙mL-1, respectively) (Dobberstein and Emeis 1991). This result is in accordance with the literature once the β-xylosidase activity in fungi generally presents higher optimum temperatures than endo-xylanase activity, usually 60°C or above (Knob et al. 2010 ). Also, the enzymatic extract of A. pullulans UFMG-CM-Y518 strain displayed xylanolytic activities. This factor is extremely important once extracts of A. pullulans are cellulase-free (Leathers 1986 ), making them applicable in the selective hydrolysis of hemicelluloses with important applications in the pulp and paper industry, bleaching of pulp (Gangwar et al. 2014 ), and in the baking industry, when this extract does not possess proteolytic activity (Collins et al. 2005 ). Due to its wide range of commercial applications, one important factor that must be highlighted is the fact that higher substrate concentration (50%) led to a lower yield of enzymatic hydrolysis (lower than 25%), even though these conditions are more favorable for xylanase activity. There are some explanations for this effect. First, the higher solid concentrations could inhibit an efficient stirring and homogeneous access of enzymes to the substrate. This scenario is supported by the observation of markedly reduced enzymatic hydrolysis efficiency when using wheat straw with 15% solids instead of 10%. Besides that, we can also hypothesize that the enzymes interacted with the structure of the solid, undergoing a sorption process, and consequently being less available for reaction. Other explanation is end-product inhibition of the enzymatic hydrolysis, which can be explained by the lower hydrolysis yield of 3% xylan with the A. pullulans UFMG-CM-Y518 extract, compared to that observed by hydrolysis yield of 2% xylan (approximately 22% versus 26%, respectively).The most dramatic effect observed was the increase of wheat straw solids with A. pullulans UFMG-CM-Y518 extract, which could be explained by the combination of rheological (agitation and the enzyme access to substrate) and biochemical (product inhibition) effects. One of the applications of this enzymatic extract is for the bioethanol production through the process of saccharification and fermentation process. It was already reported that Sp. passalidarum UFMG-CM-Y469 can efficiently ferment xylose to ethanol, with reported yields of approximately 0.4 g∙g − 1 at 30°C (Cadete et al. 2012 ) and with lower yield at 35°C (Melo 2013 ). In fact, 35 ºC was described as the maximum fermentation temperature by the Sp. passalidarum UFMG-CM-Y469 (Melo 2013 ). As described above, Sp. passalidarum UFMG-CM-Y469 was unable to convert xylan to ethanol, so we can state that the ethanol produced probably should be obtained from xylan, because when Sp. passalidarum UFMG-CM-Y469 was cultured only with A. pullulans UFMG-CM-Y518 extract as substrate (containing only 0.12 g∙L − 1 of xylose) no fermentation product was obtained. These results are good indicators of the efficiency of xylanolytic enzymes when supplemented with A. pullulans UFMG-CM-Y518 extract. The use of a yeast capable of fermenting both C6 and C5 fractions (glucose and xylose), such as Sp. passalidarum UFMG-CM-Y469, led to an improvement in the ethanol conversion yield from wheat straw with supplementation of the A. pullulans UFMG-CM-Y518 extract. Sp. passalidarum UFMG-CM-Y469, being able to ferment xylose, increases the ethanol yields obtained from lignocellulosic material by allowing the fermentation of the hemicellulosic fraction in addition to the cellulose fraction. On the other hand, the positive effect on hemicellulose hydrolysis allowed for greater availability of xylose for fermentation, with a more pronounced effect with the A. pullulans UFMG-CM-Y518 extract. This effect is also reflected in the increased accumulation of xylose throughout the process. However, the conversion of D-xylose to ethanol could have been more efficient since under the tested conditions there was a latency period, during which the Sp. passalidarum UFMG-CM-Y469 yeast took at least 24 h to initiate ethanol production. Thus, there was an accumulation of sugar (glucose and xylose), with preferential consumption of glucose thereafter. This latency may be related to the fact that Sp. passalidarum UFMG-CM-Y469 operates at a temperature (35 ºC) that may affect its optimal performance, as this is the maximum fermentation temperature for this strain (Melo 2013 ). Conducting the process at a lower temperature could avoid latency, although it would decrease the cellulase activity of Cellic CTec2. On the other hand, prolonging the fermentation could have resulted in higher ethanol yields than those determined, as there was still xylose available for fermentation. This study shows that the extracellular extract of A. pullulans UFMG-CM-Y518 has substrate specificity (xylan) with no cellulolytic or proteolytic activity. Higher efficiency in the hydrolysis of beechwood xylan (2% w/v) was found compared to pretreated wheat straw (10% w/v solids), with a xylose yield 5-fold greater compared with commercial Cellic HTec2 hemicellulase. Concerning enzymatic hydrolysis, supplementation of commercial cellulase (Cellic CTec2) with hemicellulases (Cellic HTec2) and A. pullulans UFMG-CM-Y518 extract increased the yields of xylan hydrolysis and cellulose from pretreated wheat straw. These results highlight the importance of xylanase in the degradation and valuation of lignocellulosic biomass. The results indicate the potential of the extracellular extract of A. pullulans UFMG-CM-Y518 in SSF, due to its high activity at temperatures of 30–35°C (Fig. 1 ). In SSF experiments using beechwood xylan as a substrate, A. pullulans UFMG-CM-Y518 extract was essential in the production of ethanol by Sp. passalidarum UFMG-CM-Y469 (yield 0.2 g∙g − 1 ), with an efficient degradation of xylan to xylose and a hydrolysis yield estimated at 66% in SSF, compared with the yield of 17% in enzymatic hydrolysis. These findings demonstrate that SSF processes increase the extent of enzymatic hydrolysis by the removal of inhibitory hydrolysis products. The association of Sp. passalidarum UFMG-CM-Y469 with a commercial cellulase (Cellic CTec2) supplemented with the A. pullulans UFMG-CM-Y518 extract also led to an improvement in the conversion yield of wheat straw to ethanol compared to S. cerevisiae Ethanol Red and Cellic Ctec2, as the extract may have increased the availability of xylose that could be fermented by Sp. passalidarum . The extracellular extract of A. pullulans UFMG-CM-Y518 showed maximum activity at 40°C and retains over 70% of this activity at 30°C. The optimal pH range (4–5) favors its application in simultaneous saccharification and fermentation (SSF) processes. Enzyme-substrate adsorption resulted in greater stability of the xylanolytic enzymes from the extracellular extract of A. pullulans UFMG-CM-Y518, showing substrate specificity (xylan) without cellulolytic or proteolytic activity. Additionally, it exhibited higher efficiency in hydrolyzing beechwood xylan (2% w/v) and pre-treated wheat straw (10% w/v solids) compared to the commercial hemicellulase Cellic HTec2 (xylanase), achieving a 5-fold higher xylose yield when applied to pre-treated wheat straw. Supplementation of commercial cellulases (e.g., Cellic CTec2) with hemicellulases like Cellic HTec2 and the extract A. pullulans UFMG-CM-Y518 increases not only xylan hydrolysis yield but also cellulose hydrolysis yield in pre-treated wheat straw. This underscores the importance of xylanases in the degradation and valorization of lignocellulosic biomass. The extracellular extract of A. pullulans UFMG-CM-Y518 has high potential for use in SSF experiments due to its high activity at temperatures of 30–35°C. In SSF experiments, efficient degradation of xylan to xylose was observed, 66% compared to 17% in separate enzymatic hydrolysis. This demonstrates that simultaneous saccharification and fermentation increase the extent of enzymatic hydrolysis by reducing inhibition by the hydrolysis products. The combination of the yeast Sp. passalidarum UFMG-CM-Y469 with a commercial cellulase (Cellic CTec2) supplemented with the extract A. pullulans UFMG-CM-Y518 also improved the conversion yield of wheat straw to ethanol (compared to Sc. cerevisiae Ethanol Red and Cellic Ctec2) since the extract increased the availability of xylose, which could be fermented by Sp. passalidarum. Declarations Conflict of Interest The authors declare that they have no conflict of interest. The authors have no relevant financial or non-financial interests to disclose. Funding This study was funded by the European Commission in the framework of EUBrazil Project ProEthanol2G “Integration of Biology and Engineering into an Economical and Energy-Efficient 2G Bioethanol Biorefinery” (FP7-251151), by Conselho Nacional de Desenvolvimento Cientifico e Tecnológico (CNPq, Process Nos. 0457499/2014-1, 141586/2017-6, 313088/2020-9 and 408733/2021). This work is part of the project “INCT Yeasts: Biodiversity, preservation and biotechnological innovation”, funded by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Brazil, grant #406564/2022-1. This work was also funded by Fundação do Amparo a Pesquisa do Estado de Minas Gerais (FAPEMIG, process numbers APQ-01525-14, and APQ-02552-15, APQ-03071–17), and Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP/BIOEN/FAPEMIG). Author Contribution Conceived of study: Carla Lara, Susana Marques, Francisco Gírio, César Fonseca and Carlos Rosa; Performed research, Carla Lara, Susana Marques, César Fonseca; Analyzed data: Carla Lara, Evelyn S. Oliveira, Susana Marques, Francisco Gírio, Giordana Arend, César Fonseca and Carlos Rosa; Wrote the paper: Carla Lara, Giordana Arend, César Fonseca and Carlos Rosa. Data Availability The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request. 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Oliveira","email":"","orcid":"","institution":"Universidade Federal de Minas Gerais","correspondingAuthor":false,"prefix":"","firstName":"Evelyn","middleName":"S.","lastName":"Oliveira","suffix":""},{"id":544466674,"identity":"396e4dd1-d85f-44a5-bb12-706f2900793a","order_by":2,"name":"Susana Marques","email":"","orcid":"","institution":"National Laboratory of Energy and Geology","correspondingAuthor":false,"prefix":"","firstName":"Susana","middleName":"","lastName":"Marques","suffix":""},{"id":544466681,"identity":"1d6863a1-ba26-4e3f-bf38-83f6da65a98b","order_by":3,"name":"Francisco Gírio","email":"","orcid":"","institution":"National Laboratory of Energy and Geology","correspondingAuthor":false,"prefix":"","firstName":"Francisco","middleName":"","lastName":"Gírio","suffix":""},{"id":544466682,"identity":"3d5ba83a-fc34-4595-9683-a14a6454cf50","order_by":4,"name":"Giordana D. 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Rosa","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA2klEQVRIiWNgGAWjYHACxgMgkp+ZgQ1IyQExD2E9YC2SzWAtxiRoMThArBb+9rMPDldU1MkbH2d+9oCxzSBa3oH32Ad8WiTOpBscPHPmsOG2w2zmBkAtuRsP8CXPwKfFgCGN4WBj2wHGbYd52CQY2/7kbmzgMcbrMAP+ZyAtdfabm8FaDIjQIgG2hTlxAzNUy3wGAlokbgBtaThzOHkGyC8J5wxyNzDzJePVwt+fxviwoaLOtr//8LMHH8qAtrT3HsarBRUkgJxKigYIkG8gWcsoGAWjYBQMcwAAvehGVDVv1dEAAAAASUVORK5CYII=","orcid":"","institution":"Universidade Federal de Minas Gerais","correspondingAuthor":true,"prefix":"","firstName":"Carlos","middleName":"A.","lastName":"Rosa","suffix":""}],"badges":[],"createdAt":"2025-11-03 19:38:16","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8022214/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8022214/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":96266922,"identity":"961a6094-9d30-408d-b45a-84ccd83935bc","added_by":"auto","created_at":"2025-11-19 08:43:49","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":281780,"visible":true,"origin":"","legend":"","description":"","filename":"Aureobasidiumpullulansmanuscript.docx","url":"https://assets-eu.researchsquare.com/files/rs-8022214/v1/aba8c926e21debe737e35c45.docx"},{"id":96364250,"identity":"d23198ad-4e3e-432b-8e46-5505237536ad","added_by":"auto","created_at":"2025-11-20 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08:43:49","extension":"jpeg","order_by":7,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":30298,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8022214/v1/29ffc961814faeb0c8faee63.jpeg"},{"id":96363308,"identity":"5299a85b-4d99-4068-a550-96d01146c9fe","added_by":"auto","created_at":"2025-11-20 10:06:10","extension":"xml","order_by":12,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":142472,"visible":true,"origin":"","legend":"","description":"","filename":"f33b6a57379d47ffa1e296267283ce961structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-8022214/v1/acb820d2bb59dea2fa44205a.xml"},{"id":96266943,"identity":"d32d5659-b130-46c4-94f9-eb32c5b21782","added_by":"auto","created_at":"2025-11-19 08:43:49","extension":"html","order_by":13,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":150482,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8022214/v1/89408c964f2ec25c31efd882.html"},{"id":96266921,"identity":"0f105e0e-64fd-4ab7-94a3-64bafee5079d","added_by":"auto","created_at":"2025-11-19 08:43:49","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":12209,"visible":true,"origin":"","legend":"\u003cp\u003eProduction and characterization of extracellular xylanolytic enzymes by \u003cem\u003eAureobasidium pullulans \u003c/em\u003eUFMG-CM-Y518 and NRRL Y-2311.YY: Volumetric activity (U mL\u003csup\u003e-1\u003c/sup\u003e) YY: Specific activity (U mg\u003csup\u003e-1\u003c/sup\u003e).\u003cstrong\u003e\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Onlinefloatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-8022214/v1/fbc67a9971f2082622a26d74.png"},{"id":96363562,"identity":"d9bfc23d-f57e-430a-bb0c-4c83ee250f8b","added_by":"auto","created_at":"2025-11-20 10:07:19","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":5275,"visible":true,"origin":"","legend":"\u003cp\u003eEfficiency of extracellular extract of \u003cem\u003eAureobasidium pullulans \u003c/em\u003eUFMG-CM-Y518 (XBro) and commercial hemicellulase Cellic HTec2 at 1000 U g\u003csup\u003e-1\u003c/sup\u003e (xylan) in the hydrolysis of beechwood xylan [3% (w/v)] and pretreated wheat straw [15% (w/v) total solids] at 45 °C and pH 4.8. The results are the conversion efficiency to xylose and xylobiose.\u003c/p\u003e","description":"","filename":"Onlinefloatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-8022214/v1/1d179b2139f8a22ab31d7832.png"},{"id":96266923,"identity":"b5127a96-b003-4012-b4de-3b2e71140326","added_by":"auto","created_at":"2025-11-19 08:43:49","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":24437,"visible":true,"origin":"","legend":"\u003cp\u003eYield in xylose (\u003cstrong\u003eA\u003c/strong\u003e) and glucose (\u003cstrong\u003eB\u003c/strong\u003e) from the enzymatic hydrolysis of xylan and the pretreated wheat straw cellulose by supplementation of Cellic CTec2 (CT) cellulase with the extracellular extract of \u003cem\u003eAureobasidium pullulans \u003c/em\u003eUFMG-CM-Y518 (XBro) or commercial Cellic HTec2 (HT) hemicellulase at 45 °C and pH 4.8.\u003c/p\u003e","description":"","filename":"Onlinefloatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-8022214/v1/1e236f02db4604b94f546c02.png"},{"id":96266929,"identity":"9a088dae-f338-4c97-b4bb-8dc2e4ed2638","added_by":"auto","created_at":"2025-11-19 08:43:49","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":16479,"visible":true,"origin":"","legend":"\u003cp\u003eEthanol production by SSCF with \u003cem\u003eSpathaspora passalidarum \u003c/em\u003eUFMG-UFMG-CM-Y469 at 35 °C and 150 rpm under anaerobic conditions from the cellulose and hemicellulose fractions of wheat straw using the cellulase Cellic CTec2 [(-▲-) without supplementation; (-♦-) supplemented with Cellic HTec2; (-■-) supplemented with \u003cem\u003eAureobasidium pullulans\u003c/em\u003eUFMG-CM-Y518 (XBro) extract].\u003c/p\u003e","description":"","filename":"Onlinefloatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-8022214/v1/6df3dd761f257fd12fd1812a.png"},{"id":96369170,"identity":"6c448db5-4970-4284-acb6-96e78558b9cf","added_by":"auto","created_at":"2025-11-20 10:19:52","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":931758,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8022214/v1/6fc40ecb-1e4d-4df8-acc8-1d1893c20366.pdf"},{"id":96363672,"identity":"fe57ab02-9851-49a9-9779-72c0edd9c50a","added_by":"auto","created_at":"2025-11-20 10:07:41","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":147869,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarymaterial.docx","url":"https://assets-eu.researchsquare.com/files/rs-8022214/v1/63e4162d3fbbec8a3da85be8.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Use of Aureobasidium pullulans xylanase for simultaneous saccharification and fermentation in second- generation bioethanol production","fulltext":[{"header":"Introduction","content":"\u003cp\u003eIncreases in energy demand, concerns about climate change, and oil prices have stimulated the development of biofuels produced from lignocellulosic biomass, such as second-generation (2G) ethanol, which may contribute to the production of more sustainable (Leal et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Lynd et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; McCormick and Howard \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Pereira et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2025\u003c/span\u003e; Song et al. \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2016\u003c/span\u003e) and economically competitive biofuels due to the complete use of sugars. Lignocellulosic biomass consists mainly of cellulose (35\u0026ndash;50%), hemicellulose (15\u0026ndash;30%), lignin (12\u0026ndash;35%), and pectin (2\u0026ndash;5%), and is the largest known renewable source of carbon (Kumar et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). The industrial process of 2G bioethanol production requires the efficient degradation of the biomass involving several steps such as pretreatment, enzymatic hydrolysis and alcoholic fermentation (G\u0026iacute;rio et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Fagundes et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Pereira et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Pretreatment is essential to overcome biomass recalcitrance and consequently increase the accessibility of the enzymes to the cellulose (G\u0026iacute;rio et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Valdivia et al. \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Thomas et al. \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Hydrothermal treatments are considered promising and appropriate for waste biomass (Alvira et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). In this method, the substrate remains a cellulose-rich solid and contains a significant amount of insoluble hemicellulose (Carvalheiro et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). In comparison to chemical hydrolysis, the enzymatic process has important advantages due to its high specificity that prevents the formation of substrate degradation products (from sugars and lignin), maximizing conversion efficiency. During enzymatic hydrolysis, cellulases, hemicellulases, and ancillary enzymes are required to promote the complete conversion of lignocellulose, to increase the yield of simple sugars and reduce the cost of the process (Berlin et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Maroldi et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Selig et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2008\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe enzymatic hydrolysis and fermentation stages to obtain ethanol as the final product may be performed sequentially, as separate hydrolysis and fermentation (SHF) processes, or via simultaneous saccharification and fermentation processes (SSF). During enzymatic hydrolysis, the accumulation of products can inhibit enzymes by reducing their hydrolytic activity (Afedzi and Parakulsuksatid \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Kuma and Wyman \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Paulova et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). In the SSF, the simple sugars released by hydrolysis will be converted into ethanol, which does not accumulate and minimizes the inhibition of the enzymatic activity. Nonetheless, the SSF process must be performed at intermediate (35\u0026deg;C) to optimal temperatures for enzymatic hydrolysis (approximately 50\u0026deg;C) and fermentation (30\u0026deg;C) as a compromise (Olofsson et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2008\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e is one of the most used microorganisms to produce ethanol. However, this yeast cannot ferment pentose sugars like xylose. Among the species that ferment D-xylose, \u003cem\u003eSpathaspora passalidarum\u003c/em\u003e is one of the best producers of ethanol (Cadete and Rosa, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Nakamura et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Racca et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). This yeast can ferment xylose with little or no aeration (Riley et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Barros et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Also, it has already been reported in the literature high ethanol yield, around 0.48 g∙g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, which is close to the theoretical maximum value of 0.51 g∙g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (obtained in culture medium) (Cadete and Rosa \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Pascoli et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Barros et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).The success in the bioconversion of hemicellulose and cellulose to ethanol is a decisive factor in the economic viability of the process (Chandel et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). The performance of xylanolytic enzymes that remove the insoluble hemicellulose decreases the barrier between the cellulose and lignin, significantly improving the action of cellulase due to greater access of the enzyme to the cellulose (Kuma and Wyman \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Laureano-Perez et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Ohgren et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2007\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eEnzymatic hydrolysis is usually performed with commercial enzyme cocktails produced from extracellular extracts of selected or genetically modified filamentous fungi. Some hemicellulolytic cocktails that are usually applied in addition to cellulolytic ones are described as xylanase-specific (no cellulase activity) and have optimal activity at 50\u0026deg;C (Duarte and Costa-Ferreira \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e1994\u003c/span\u003e; Knob et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Their performance can be related to xylose levels and is generally limited by the low level of β-xylosidase activity (Coughlan and Hazlewood \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e1993\u003c/span\u003e). This behavior may be explained by the inhibition of xylan-degrading enzymes by the hydrolysis products. This limitation leads to incomplete enzymatic hydrolysis of xylan. Also, D-xylose is not naturally fermented by the yeast \u003cem\u003eS. cerevisiae\u003c/em\u003e, hindering the application of xylanase in 2G ethanol production processes. The inhibition of the enzymatic hydrolysis of xylan by hydrolysis products discourages their use in SHF. In turn, SSF, which must be performed at a lower temperature (usually at 35\u0026deg;C or below) to allow for fermentation, can lead to inefficient enzymatic hydrolysis of xylan due to the low activity of xylanolytic enzymes at those temperatures.\u003c/p\u003e\u003cp\u003eThe successful search for xylan-degrading enzymes with high activity at low temperatures could permit the use of these enzymes in xylan simultaneous saccharification and fermentation process. Xylanase and β-xylosidase activities have been identified in yeast and yeast-like fungi with optimal temperatures lower than those of filamentous fungi used to produce commercial cocktails (Lara et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Morais et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Romero et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). The yeast-like ascomycetous fungus \u003cem\u003eAureobasidium pullulans\u003c/em\u003e is a xylanase producer with high specific activity and an optimal temperature between 35\u0026deg;C and 50\u0026deg;C (Gaut\u0026eacute;rio et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Leathers \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e1986\u003c/span\u003e). The supplementation of the lignocellulosic material, such as pretreated corn stover and pretreated wheat straw, added with cellulolytic enzyme cocktails including xylanases, improve the enzymatic hydrolysis process (Alvira et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Kuma and Wyman \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Li et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2010\u003c/span\u003e), thus leading to a higher fermentation yield (Jin et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Kuma and Wyman \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). The present study aimed to characterize the extracellular extract of \u003cem\u003eAureobasidium pullulans\u003c/em\u003e UFMG-CM-Y518, a strain isolated from water stored in bromeliad tanks, and to evaluate the application of this xylanolytic extract in the enzymatic hydrolysis of xylan and pretreated wheat straw, and to evaluate the performance of this extract in SSF process to produce bioethanol.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eProduction and characterization of xylanolytic enzymes\u003c/h2\u003e\u003cp\u003e\u003cem\u003eAureobasidium pullulans\u003c/em\u003e UFMG-CM-Y518 was previously isolated from water stored in bromeliad tanks (phytothelma) (Gomes et al. 2015) and was obtained from the Culture Collection of Microorganisms and Cells at the Federal University of Minas Gerais (UFMG). \u003cem\u003eA. pullulans\u003c/em\u003e NRRL Y-2311 was used as a reference strain and was obtained from the National Laboratory of Energy and Geology (Lisbon, Portugal) and from the Culture Collection of the Agriculture Research Service, United States Department of Agriculture.\u003c/p\u003e\u003cp\u003eThe cells were precultured in a liquid medium (initial pH 5.0) containing yeast nitrogen base (YNB) and D-xylose (YNB 6.7 g∙L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, xylose 30 g∙L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) at 30\u0026deg;C with agitation at 150 rpm for 24 h. At the end of cultivation, the culture was centrifuged (2,600 \u0026times; \u003cem\u003eg\u003c/em\u003e for 15 min) and the collected cells were inoculated at an initial optical density equal to 4 induced by xylan-YNB medium (yeast nitrogen base 6.7 g∙L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e; xylan, 10 g∙L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), which were determined at 600 nm. The production of xylan-degrading enzymes was induced in 100 mL Erlenmeyer flasks with 25 mL culture medium at 30\u0026deg;C and agitation at 150 rpm for 72 h. The enzyme extract was collected by centrifugation and used to determine the extracellular enzymatic activities.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eEnzyme and protein assays\u003c/h3\u003e\n\u003cp\u003eXylanase was assayed according to Bailey et al. (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e1992\u003c/span\u003e) with a few modifications (Lara et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). The assay was performed with an incubation time of 30 min and a proportion of 1/3 substrate/culture. Cellulase activity was determined using Whatman No. 1 filter paper (Ghose \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e1987\u003c/span\u003e). Xylosidase was assayed according to Li et al (\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e1993\u003c/span\u003e). The extracellular protein concentration was measured using the Bicinchoninic Acid Protein Quantitation Assay (Thermo Scientific \u0026ndash; Pierce, USA) with bovine serum albumin as the standard.\u003c/p\u003e\n\u003ch3\u003eEnzymatic characterization\u003c/h3\u003e\n\u003cp\u003eThe crude extract of \u003cem\u003eA. pullulans\u003c/em\u003e UFMG-CM-Y518 was used to determine the temperature and the optimum pH of the xylanolytic enzymes. The optimum temperature was determined at temperatures ranging from 20 to 90\u0026deg;C in sodium acetate buffer (50 mM, pH 5.5) for 30 min. Sodium acetate and sodium phosphate buffers (both 50 mM) were used to determine the optimal pH over a range of 3.0 to 8.0 at 45 \u0026ordm;C for 30 min. The thermal stability was assessed by incubating the crude extract of the \u003cem\u003eA. pullulans\u003c/em\u003e UFMG-CM-Y518 strain in sodium acetate buffer (50 mM, pH 4.8) at 45\u0026deg;C, and was further evaluated by incubation with the substrate (beechwood xylan suspension of 10 g∙L\u003csup\u003e-1\u003c/sup\u003e) at pH 4.8 and 45\u0026deg;C. Aliquots were obtained after 0, 6, 12, 24, 48 and 72 h, which were frozen for later determination of residual activities.\u003c/p\u003e\n\u003ch3\u003eEnzymatic hydrolysis\u003c/h3\u003e\n\u003cp\u003eCommercial beechwood xylan and pretreated wheat straw were used as substrates. The wheat straw was subjected to a mild hydrothermal pretreatment (190\u0026deg;C for 10 min) and was kindly provided for this study by the Technical University of Denmark. The pretreated wheat straw contained next to 41% of cellulose and 22% of xylan. The extracellular extract of \u003cem\u003eA. pullulans\u003c/em\u003e UFMG-CM-Y518 and the commercial preparations Cellic HTec2 and Cellic CTec2 (kindly supplied by Novozymes, Denmark) were used in the enzymatic hydrolysis experiments, simultaneous saccharification and co-fermentation processes (SSCF), and SSF. Cellic HTec2 is a hemicellulase preparation with high endoxylanase activity and residual cellulase activity. Cellic CTec2 is a preparation of cellulases with high β-glucosidase activity and hemicellulolytic activity.\u003c/p\u003e\u003cp\u003ePreliminary experiments for enzymatic hydrolysis were conducted with beechwood xylan [2% (w/v)] and with pretreated wheat straw [10% (w/v)] in suspension as substrates. The hydrolysis medium contained substrate (xylan or pretreated wheat straw), 0.08% (w/v) sodium azide, 4 mL of \u003cem\u003eA. pullulans\u003c/em\u003e UFMG-CM-Y518 xylanolytic extract or 3 \u0026micro;L of HTec2 [both with activities equivalent to 200 U (endo-xylanase)∙g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e xylan], and 50 mM sodium acetate buffer (pH 5.5) at an initial total volume of 20 mL in Erlenmeyer flasks. The flasks were incubated at 50\u0026deg;C with orbital shaking (150 rpm) for 96 h. Aliquots were obtained for analysis after 0, 6, 24, 48, 72, and 96 h of incubation.\u003c/p\u003e\u003cp\u003eIn the second enzymatic hydrolysis stage (and when in SSF) pretreated wheat straw was used as substrate at a concentration of 15% (w/v) in terms of total solids to verify the effect of supplementation with Cellic CTec2 cellulolytic enzymes with xylanolytic enzymes (extract of \u003cem\u003eA. pullulans\u003c/em\u003e UFMG-CM-Y518 strain compared to the Cellic HTec2 hemicellulases) and, simultaneously, the hydrolysis of hemicellulose in the lignocellulosic substrate. Each enzyme preparation was tested separately with pretreated wheat straw. For comparison, beechwood xylan was subjected to hydrolysis by the two hemicellulolytic preparations. \u003cem\u003eA. pullulans\u003c/em\u003e UFMG-CM-Y518 or Cellic HTec2 enzyme preparation was used at a dosage equivalent to 1000 U∙g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e xylan as a supplement to Cellic CTec2 applied at a dose of 20 filter paper units (FPU)\u0026middot;g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e glucan in the hydrolysis of pretreated wheat straw. The 50 mL reaction mixtures in Erlenmeyer flasks in sodium acetate buffer (50 mM, pH 4.8) were incubated at 45\u0026deg;C in orbital shaker (150 rpm) for 96 h.\u003c/p\u003e\n\u003ch3\u003eSimultaneous saccharification and fermentation (SSF) and simultaneous saccharification and co-fermentation (SSCF) processes\u003c/h3\u003e\n\u003cp\u003eTwo strains of microorganisms were used, where \u003cem\u003eSp. passalidarum\u003c/em\u003e UFMG-CM-Y469 ferments xylose and glucose (Cadete et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2012\u003c/span\u003e)d \u003cem\u003ecerevisiae\u003c/em\u003e Ethanol Red (Fermentis, France) is an industrial strain used in the fuel alcohol industry, once it ferments glucose but not xylose. Preinoculum was prepared by selecting a colony of \u003cem\u003eSp. passalidarum\u003c/em\u003e UFMG-CM-Y469 from Yeast Malt Agar (YM agar (containing yeast extract, peptone, malt extract, glucose)) and inoculating the cells in flasks containing 50 mL of Yeast Extract with Xylose (YPX medium (yeast extract 10 g∙L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, peptone 20 g∙L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, D-xylose 30 g∙L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) and shaking at 200 rpm and 30\u0026deg;C. \u003cem\u003eS. cerevisiae\u003c/em\u003e Ethanol Red was prepared in flasks containing 20 mL of growth medium (glucose 50 g∙L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, yeast extract 2 g∙L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, (NH\u003csub\u003e4\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e 2.5 g∙L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, KH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e 1 g∙L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, MgSO\u003csub\u003e4\u003c/sub\u003e.7H\u003csub\u003e2\u003c/sub\u003eO 0.3 g∙L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) and shaking at 200 rpm at 35\u0026deg;C. After 24 h, both precultures were centrifuged and the supernatant was discarded. The cells were weighed and diluted to obtain an inoculum size of 1 g∙L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e to begin the fermentation. The xylanolytic extract of the \u003cem\u003eA. pullulans\u003c/em\u003e UFMG-CM-Y518, Cellic CTec2, and Cellic HTec2 were used in SSCF and SSF.\u003c/p\u003e\u003cp\u003e\u003cem\u003eSSF of xylan and wheat straw with extracellular xylanolytic extract of A. pullulans\u003c/em\u003e UFMG-CM-Y518\u003c/p\u003e\u003cp\u003eSSF experiments were initiated by the simultaneous addition of the enzymes for testing and the ethanol production using a lignocellulosic substrate supplemented with nutrients as the culture medium for \u003cem\u003eSp. passalidarum\u003c/em\u003e UFMG-CM-Y469 (10 g L \u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e yeast extract, 20 g∙L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e peptone) and \u003cem\u003eS. cerevisiae\u003c/em\u003e Ethanol Red (yeast extract 2 g∙L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, (NH\u003csub\u003e4\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e 2.5 g∙L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, KH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e 1 g∙L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, MgSO\u003csub\u003e4\u003c/sub\u003e.7H\u003csub\u003e2\u003c/sub\u003eO 0.3 g∙L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e). These experiments evaluated ethanol production from beechwood xylan at a concentration of 3% (w/v) and from pretreated wheat straw at a concentration of 15% (w/v) total solids.\u003c/p\u003e\u003cp\u003eThree sets of experiments were performed under enzymatic hydrolysis conditions (for the enzymatic dosage) like those previously described, except for temperature (35\u0026deg;C instead of 45\u0026deg;C). The first experiment assessed SSF of beechwood xylan for ethanol using a xylanolytic extract of \u003cem\u003eA. pullulans\u003c/em\u003e UFMG-CM-Y518 and the xylose (C5) fermenting yeast \u003cem\u003eSp. passalidarum\u003c/em\u003e UFMG-CM-Y469. The second experiment assessed SSF of pretreated wheat straw for ethanol using Cellic CTec2 supplemented with \u003cem\u003eA. pullulans\u003c/em\u003e UFMG-CM-Y518 strain extract or Cellic Htec2 and the \u003cem\u003eS. cerevisiae\u003c/em\u003e Ethanol Red industrial yeast that ferments only glucose (C6). The third experiment assessed SSCF of pretreated wheat straw for ethanol using Cellic CTec2 supplemented with \u003cem\u003eA. pullulans\u003c/em\u003e UFMG-CM-Y518 strain extract or Cellic Htec2 and \u003cem\u003eSp. passalidarum\u003c/em\u003e UFMG-CM-Y469, a yeast capable of fermenting glucose and xylose (C6/C5). As control, both yeasts were cultured in a medium containing xylose and another containing glucose and xylose. \u003cem\u003eSp. passalidarum\u003c/em\u003e UFMG-CM-Y469 was also cultivated using beechwood xylan alone and the same with \u003cem\u003eA. pullulans\u003c/em\u003e UFMG-CM-Y518 extract as substrate at the same concentrations.\u003c/p\u003e\u003cp\u003eSSF experiments were performed in duplicate for 96 h using potassium hydrogen phthalate buffer (50 mM, pH 4.8) at 35\u0026deg;C with orbital shaking (200 rpm). Erlenmeyer flasks containing 50 mL of the fermentation medium were used in both fermentation cultures. The flasks were closed with rubber stoppers and coupled to a needle assembly immersed in glycerol to release the gas produced by fermentation without air inlet. Before the start of fermentation, and upon opening the bottle for aliquot removal, nitrogen gas was injected to remove the air (anaerobic conditions).\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eAnalytical methods\u003c/h2\u003e\u003cp\u003eSamples taken from all enzymatic hydrolysis and SSF experiments were heated in boiling water for 10 min (to inactivate enzymes) and centrifuged (2600 \u0026times; g, 10 min). The collected supernatant was frozen at -20\u0026deg;C, for subsequent analysis of enzymatic hydrolysis (glucose, xylose and xylobiose) and fermentation products (ethanol, glycerol, xylitol, acetate, and ethanol) by high-performance liquid chromatography with a Merck Hitachi chromatograph (Darmstadt, Germany) equipped with an Aminex HPX-87H column (Bio-Rad, USA) at 50\u0026deg;C and a refractive index detector (L-7490; Merck Hitachi, Darmastadt, Germany) using 5 mM H\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e as the mobile phase at a flow rate of 0.4 mL∙min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eFermentation parameter calculation\u003c/h3\u003e\n\u003cp\u003eThe fermentation parameter Y\u003csub\u003ep/s\u003c/sub\u003e\u003csup\u003eet\u003c/sup\u003e (g∙g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) ethanol yield was experimentally determined. Ethanol Y\u003csub\u003ep/s\u003c/sub\u003e\u003csup\u003eet\u003c/sup\u003e (g∙g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) was calculated following the method suggested by Schmidell et al. (\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2001\u003c/span\u003e), which correlated ΔP produced (ΔP ethanol) and ΔS consumed (derived by determining the total, initial, and consumed substrate).\u003c/p\u003e\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003eStatistical analysis\u003c/h2\u003e\u003cp\u003eHydrolysis tests of the pretreated wheat straw were analyzed using analysis of variance (ANOVA) (p\u0026thinsp;\u0026le;\u0026thinsp;0.05) and the comparison between means by the Duncan test (p\u0026thinsp;\u0026le;\u0026thinsp;0.05).\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eProduction and characterization of xylanolytic enzymes by A. pullulans\u003c/h2\u003e\u003cp\u003eExtracellular extracts were obtained, and total xylanase activity was determined at pH 5.5 and either 30 \u0026ordm;C or 50 \u0026ordm;C (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Extract from \u003cem\u003eA. pullulans\u003c/em\u003e UFMG-CM-Y518 was selected for further characterization based on its high xylanase specific activity and purity/specificity in xylan. The temperature and pH profile of xylanase activity were determined between 20 and 90 \u0026ordm;C and a pH range from 3 to 8 (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). The xylanase extract displayed maximum activity at 40\u0026deg;C (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003ea), with over 70% of this activity at 30\u0026deg;C and 50\u0026deg;C. Xylanase activity significantly decreased below 30\u0026deg;C and above 50\u0026deg;C, and no activity was detected above 70\u0026deg;C due to heat inactivation of the enzyme. Xylanase activity was highest at pH of 4.0 to 4.5 (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eb) and decreased significantly in acidic conditions (pH lower than 3.5) or neutral conditions (pH higher than 5.0). β-xylosidase activity in the \u003cem\u003eA. pullulans\u003c/em\u003e UFMG-CM-Y518 extracellular extract was assessed and the optimal activity occurred at 80\u0026deg;C and pH 4.0 (Fig. S2).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e\u003cp\u003eRegarding the specificity and extract stability, the extract did not display cellulolytic activity, as determined by Filter Paper Activity (FPase) activity or proteolytic activity. The thermal stability of xylanase was assessed in the absence and presence of substrate (xylan) at 45\u0026deg;C and pH 4.8 for 72 h (Fig. S3). The sample at baseline (t\u0026thinsp;=\u0026thinsp;0) was not incubated at 45\u0026deg;C. Its enzyme activity at this time was considered as 100%. The stability test indicated that in the absence of substrate, the xylanase enzyme lost the majority (over 60%) of its activity in the first 6 h of incubation (Fig. S3a). However, in the presence of xylan substrate (Fig. S3b) thermal stability was evident, with greater than 80% maximum activity retained for up to 12 h of incubation. The loss of activity was relatively linear over time, and at 72 h was similar to that observed in the absence of xylan at 6 h of incubation (Fig. S3a). The stability of β-xylosidase activity in extracellular extracts of \u003cem\u003eA. pullulans\u003c/em\u003e UFMG-CM-Y518 was also determined at 45\u0026deg;C and pH 4.8 for 72 h (Fig.S4). Unlike xylanase activity, β-xylosidase activity was very stable at 45\u0026deg;C, with more than 80% of the maximum activity maintained during the 72 h experiment.\u003c/p\u003e\u003cp\u003e\u003cem\u003eUse of xylanolytic extracellular extracts from A. pullulans UFMG-CM-Y518 in the enzymatic hydrolysis of xylan and pretreated wheat straw\u003c/em\u003e\u003c/p\u003e\u003cp\u003eThe cellulolytic (FPase) and xylanolytic activity (xylanase) of enzyme extracts was determined under the conditions applied for the saccharification of substrates, 45\u0026deg;C and pH 4.8. While the Cellic HTec2 and Cellic CTec2 commercial preparations displayed significant xylanase and FPase activities, \u003cem\u003eA. pullulans\u003c/em\u003e UFMG-CM-Y518 extract displayed xylanase activity of 104.3 U∙mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and showed no cellulase activity.\u003c/p\u003e\u003cp\u003eThe xylanolytic extracellular extract from \u003cem\u003eA. pullulans\u003c/em\u003e UFMG-CM-Y518 was evaluated in a preliminary test on beechwood xylan [2% (w/v)] and pretreated wheat straw [10% (w/v) solids; about 2% (w/v) xylan)], in an enzymatic process at 50\u0026deg;C and pH 5.5. The Cellic HTec2 xylanolytic commercial preparation was used for comparison. The hydrolysis efficiency was determined based on the concentration of D-xylose and xylobiose produced from each substrate (Fig. S5).\u003c/p\u003e\u003cp\u003eWith the use of xylanolytic enzymes of \u003cem\u003eA. pullulans\u003c/em\u003e UFMG-CM-Y518, xylan hydrolysis was similar to that obtained with Cellic HTec2 (approximately 25\u0026ndash;30%). However, with wheat straw, the \u003cem\u003eA. pullulans\u003c/em\u003e UFMG-CM-Y518 extract was more efficient than Cellic HTec2, 60% and 30%, respectively. The concentration of xylose obtained (complete hydrolysis of the xylan) was greater with \u003cem\u003eA. pullulans\u003c/em\u003e UFMG-CM-Y518 than with Cellic HTec2, either in xylan or pretreated wheat straw, reaching concentrations of D-xylose that were 3- and 5-fold higher, respectively. Consequently, the yield of xylobiose was higher for substrates treated with HTec2, indicating that the \u003cem\u003eA. pullulans\u003c/em\u003e UFMG-CM-Y518 extract possessed more efficient β-xylosidase activity, with a higher yield in the hydrolysis of xylan to xylose, under the conditions chosen for the assay.\u003c/p\u003e\u003cp\u003eThe activity of \u003cem\u003eA. pullulans\u003c/em\u003e UFMG-CM-Y518 extract was evaluated in comparison with Cellic HTec2 hemicellulase using the two substrates [3% beechwood xylan (w/v) and pretreated wheat straw 15% (w/v) total solids] at a higher solids concentration that was equivalent to that used in subsequent enzymatic hydrolysis steps with cellulase and in SSF processes (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e.\u003c/p\u003e\u003cp\u003eThe enzyme dosage used in this assay was 1000 U∙g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e xylan. In the presence of xylan the xylanolytic extract of \u003cem\u003eA. pullulans\u003c/em\u003e UFMG-CM-Y518 and hemicellulase HTec2 exhibited similar conversion efficiency of xylose. HTec2 displayed a higher yield of xylobiose and \u003cem\u003eA. pullulans\u003c/em\u003e extract UFMG-CM-Y518 displayed a conversion efficiency of xylose with wheat straw that was approximately 2-fold of that obtained with HTec2. The yield obtained with a higher (50%) substrate concentration led to a lower yield of enzymatic hydrolysis, which did not exceed 25% hydrolysis yield under these conditions, even though the dose of xylanase used was 5-times higher and had more favorable conditions for xylanase activity.\u003c/p\u003e\u003cp\u003e\u003cem\u003eEffect of cellulase supplementation with extracellular xylanolytic extract of A. pullulans\u003c/em\u003e UFMG-CM-Y518 \u003cem\u003eon the enzymatic hydrolysis of wheat straw\u003c/em\u003e\u003c/p\u003e\u003cp\u003eOnce the potential of \u003cem\u003eA. pullulans\u003c/em\u003e xylanolytic extract UFMG-CM-Y518 was demonstrated, new experiments were conducted to evaluate the effect of this extract supplemented with cellulolytic enzymes (Cellic CTec2) to achieve more efficient hydrolysis of wheat straw, using 15% (w/v) of total solids. For comparison, the Cellic CTec2 cellulase preparation was supplemented with the Cellic HTec2 hemicellulolytic enzyme preparation and tested against non-supplemented samples. The conversion yields of xylose and glucose are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e.\u003c/p\u003e\u003cp\u003eSupplementation of Cellic CTec2 with xylanolytic enzymes of \u003cem\u003eA. pullulans\u003c/em\u003e UFMG-CM-Y518 led to a higher yield in the hydrolysis of both polysaccharide constituents (xylan and cellulose) of pretreated wheat straw, when compared with supplementation with Cellic HTec2 and the isolated use of Cellic CTec2. The xylose conversion yields presented values next to 40% and did not differ significantly (p\u0026thinsp;\u0026le;\u0026thinsp;0.05; Duncan's test), for the samples obtained with and without the supplementation of \u003cem\u003eA. pullulans\u003c/em\u003e UFMG-CM-Y518 extract with Cellic Ctec2.\u003c/p\u003e\u003cp\u003e\u003cem\u003eEthanol production from xylan and wheat straw by SSF with extracellular xylanolytic extract of A. pullulans UFMG-CM-Y518 strain\u003c/em\u003e\u003c/p\u003e\u003cp\u003eEthanol production from xylan was evaluated using the \u003cem\u003eA. pullulans\u003c/em\u003e UFMG-CM-Y518 extract to hydrolyze xylan to xylose. Simultaneously, \u003cem\u003eSp. passalidarum\u003c/em\u003e UFMG-CM-Y469 was used to anaerobically ferment xylose to ethanol. In this case, \u003cem\u003eSp. passalidarum\u003c/em\u003e UFMG-CM-Y469 was unable to convert xylan directly to ethanol, indicating that it did not produce xylanolytic enzymes under the conditions used. Importantly, no xylo-oligosaccharides or accumulation of xylose was detected after 96 h. However, when the medium constituted of 30 g∙L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e xylan at 35\u0026deg;C (Fig. S6) was supplemented with \u003cem\u003eA. pullulans\u003c/em\u003e UFMG-CM-Y518 extract, \u003cem\u003eSp. passalidarum\u003c/em\u003e UFMG-CM-Y469 produced ethanol at a concentration of 6.6 g∙L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, corresponding to a yield of 0.2 g∙g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. At 35\u0026deg;C, \u003cem\u003eSp. passalidarum\u003c/em\u003e UFMG-CM-Y469 produced ethanol from xylose with a conversion yield of 0.3 g∙g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (Fig. S6b). This cultivation was performed in the presence and absence of extracellular extract from \u003cem\u003eA. pullulans\u003c/em\u003e UFMG-CM-Y518, indicating that this extract has no direct effect on the metabolism of this yeast.\u003c/p\u003e\u003cp\u003eAssuming a yield of 0.3 g\u0026middot;g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e for the conversion of xylose to ethanol (from 30 g\u0026middot;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e of xylose under the same conditions), \u003cem\u003eSp. passalidarum\u003c/em\u003e should have fermented 22.5 g\u0026middot;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e of xylose, meaning that the xylanolytic extract \u003cem\u003eA. pullulans\u003c/em\u003e UFMG-CM-Y518 should have promoted the hydrolysis of xylan to xylose with an estimated yield of 66% throughout the SSF process. Therefore, the extent of xylan hydrolysis was significantly improved when coupled with fermentation, as when the same extract was applied to xylan at the same substrate and \u003cem\u003eA. pullulans\u003c/em\u003e UFMG-CM-Y518 doses, a hydrolytic yield, or rather, xylan to xylose conversion, of less than 20% was obtained.\u003c/p\u003e\u003cp\u003e\u003cem\u003eProduction of cellulosic ethanol from pretreated wheat straw by SSF with cellulase supplemented with xylanolytic extract of A. pullulans UFMG-CM-Y518strain\u003c/em\u003e\u003c/p\u003e\u003cp\u003eThe effect of supplementation of Cellic CTec2 cellulase with hemicellulolytic enzymes (\u003cem\u003eA. pullulans\u003c/em\u003e UFMG-CM-Y518 extract compared with the Cellic HTec2 commercial enzyme preparation) on ethanol production from the cellulose fraction of wheat straw pretreated with \u003cem\u003eS. cerevisiae\u003c/em\u003e Ethanol Red was investigated (Fig. S7). Under the test conditions, the addition of \u003cem\u003eA. pullulans\u003c/em\u003e UFMG-CM-Y518 extract did not lead to a better performance of Cellic CTec2 cellulase concerning the extent of cellulose hydrolysis achieved from wheat straw. There was no significant effect on ethanol production, unlike supplementation with Cellic HTec2 (Fig. S7 and Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Nonetheless, the addition of extract \u003cem\u003eA. pullulans\u003c/em\u003e UFMG-CM-Y518 led to a higher concentration of accumulated xylose (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), indicating that the efficiency of xylanolytic enzymes was superior when supplemented with \u003cem\u003eA. pullulans\u003c/em\u003e UFMG-CM-Y518 extract.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eMaximum ethanol production after 96 h by SSF with \u003cem\u003eS. cerevisiae\u003c/em\u003e Ethanol Red at 35 \u0026ordm;C and 150 rpmunder anaerobic conditions from the cellulose fraction of wheat strawusing Cellic CTec2without supplementation, supplementation with Cellic HTec2, and supplementation with \u003cem\u003eAureobasidium pullulans\u003c/em\u003eUFMG-CM-Y518 extract (XBro)\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"4\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eEnzymes\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003e[Ethanol]\u003csub\u003emax\u003c/sub\u003e\u003c/p\u003e\u003cp\u003e(g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eYield*\u003c/p\u003e\u003cp\u003e(% max theoretical)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003e[Xylose]\u003csub\u003eaccumulated\u003c/sub\u003e\u003c/p\u003e\u003cp\u003e(g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCellic CTec2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e12.6\u0026thinsp;\u0026plusmn;\u0026thinsp;2.18\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e23.1 (35.7)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e11.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.13\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCellic CTec2\u0026thinsp;+\u0026thinsp;Cellic HTec2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e15.0\u0026thinsp;\u0026plusmn;\u0026thinsp;1.30\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e27.4 (42.4)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e12.1\u0026thinsp;\u0026plusmn;\u0026thinsp;1.09\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCellic CTec2\u0026thinsp;+\u0026thinsp;UFMG-CM-Y518\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e13.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.45\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e23.9 (36.9)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e13.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.72\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"4\"\u003e*Calculated relative to the maximum possible concentration of ethanol obtained by fermentation from glucose and xylose resulting from the complete hydrolysis of cellulose and xylan existing in pretreated wheat straw (in brackets the yield value considering only the fraction of cellulose).\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cem\u003eProduction of lignocellulosic ethanol from pretreated wheat straw by SSCF cellulase supplemented with A. pullulans UFMG\u003c/em\u003e-CM-Y518 \u003cem\u003eand the yeast Sp. passalidarum\u003c/em\u003e UFMG-CM-Y469.\u003c/p\u003e\u003cp\u003eTable\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e\u003cp\u003eFinally, we studied the effect of Cellic CTec2 cellulase that was not supplemented with xylanolytic enzymes (\u003cem\u003eA. pullulans\u003c/em\u003e UFMG-CM-Y518 extract compared to Cellic HTec2 hemicellulase) in the production of ethanol from the cellulose and hemicellulose fractions of wheat straw pretreated with \u003cem\u003eSp. passalidarum\u003c/em\u003e UFMG-CM-Y469 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Under the tested conditions, the usage of \u003cem\u003eA. pullulans UFMG\u003c/em\u003e-CM-Y518 promoted a better performance of Cellic CTec2, where the maximum ethanol production is approximately 1.13-fold higher, as well as the yield, which is 1.13-fold higher. On regard of the accumulated xylose, values 1.24-fold higher were obtained.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eMaximum ethanol production after 96 hby SSCF with \u003cem\u003eSpathaspora passalidarum\u003c/em\u003eUFMG-CM-Y469, at 35\u0026deg;C and 150 rpm under anaerobic conditions from the cellulose and hemicellulose fractions of wheat straw using Cellic CTec2without supplementation, supplementation with Cellic HTec2, and supplementation with \u003cem\u003eAureobasidium pullulans\u003c/em\u003eUFMG-CM-Y518 extract\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"4\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eEnzymes\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003e[Ethanol]\u003csub\u003emax\u003c/sub\u003e\u003c/p\u003e\u003cp\u003e(g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eYield*\u003c/p\u003e\u003cp\u003e(% max theoretical)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003e[Xylose]\u003csub\u003eaccumulated\u003c/sub\u003e\u003c/p\u003e\u003cp\u003e(g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCellic CTec2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e14.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.33\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e26.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e12.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.57\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCellic CTec2\u0026thinsp;+\u0026thinsp;Cellic HTec2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e14.8\u0026thinsp;\u0026plusmn;\u0026thinsp;1.22\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e27.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e11.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.38\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCellic CTec2\u0026thinsp;+\u0026thinsp;UFMG-CM-Y518\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e16.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.45\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e30.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e14.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.95\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"4\"\u003e*Calculated relative to the maximum possible concentration of ethanol obtained by fermentation from glucose and xylose, resulting from the complete hydrolysis of cellulose and xylan in the pretreated wheat straw.\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eTable\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e.\u003c/p\u003e\u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e.\u003c/p\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eSome important aspects of the production of xylanases by the \u003cem\u003eA. pullulans\u003c/em\u003e UFMG-CM-Y518 and NRRL Y-2311 strains were evaluated after induction by xylan. One of these aspects is the activity profiles at different pH and temperature conditions. The results presented above were very similar to those observed for the \u003cem\u003eA. pullulans\u003c/em\u003e NRRL Y-2311 strain (Leathers \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e1986\u003c/span\u003e). The optimal β-xylosidase activity in the \u003cem\u003eA. pullulans\u003c/em\u003e UFMG-CM-Y518 extracellular extract was consistent with values reported in the extracellular extract of A. pullulans CBS 58475 (80\u0026deg;C and pH 4.5 with maximum activity values in xylan concentrations of 0.04 and 0.20 U∙mL-1, respectively) (Dobberstein and Emeis 1991). This result is in accordance with the literature once the β-xylosidase activity in fungi generally presents higher optimum temperatures than endo-xylanase activity, usually 60\u0026deg;C or above (Knob et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2010\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eAlso, the enzymatic extract of \u003cem\u003eA. pullulans\u003c/em\u003e UFMG-CM-Y518 strain displayed xylanolytic activities. This factor is extremely important once extracts of A. pullulans are cellulase-free (Leathers \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e1986\u003c/span\u003e), making them applicable in the selective hydrolysis of hemicelluloses with important applications in the pulp and paper industry, bleaching of pulp (Gangwar et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2014\u003c/span\u003e), and in the baking industry, when this extract does not possess proteolytic activity (Collins et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2005\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eDue to its wide range of commercial applications, one important factor that must be highlighted is the fact that higher substrate concentration (50%) led to a lower yield of enzymatic hydrolysis (lower than 25%), even though these conditions are more favorable for xylanase activity. There are some explanations for this effect. First, the higher solid concentrations could inhibit an efficient stirring and homogeneous access of enzymes to the substrate. This scenario is supported by the observation of markedly reduced enzymatic hydrolysis efficiency when using wheat straw with 15% solids instead of 10%. Besides that, we can also hypothesize that the enzymes interacted with the structure of the solid, undergoing a sorption process, and consequently being less available for reaction. Other explanation is end-product inhibition of the enzymatic hydrolysis, which can be explained by the lower hydrolysis yield of 3% xylan with the \u003cem\u003eA. pullulans\u003c/em\u003e UFMG-CM-Y518 extract, compared to that observed by hydrolysis yield of 2% xylan (approximately 22% versus 26%, respectively).The most dramatic effect observed was the increase of wheat straw solids with \u003cem\u003eA. pullulans\u003c/em\u003e UFMG-CM-Y518 extract, which could be explained by the combination of rheological (agitation and the enzyme access to substrate) and biochemical (product inhibition) effects.\u003c/p\u003e\u003cp\u003eOne of the applications of this enzymatic extract is for the bioethanol production through the process of saccharification and fermentation process. It was already reported that \u003cem\u003eSp. passalidarum\u003c/em\u003e UFMG-CM-Y469 can efficiently ferment xylose to ethanol, with reported yields of approximately 0.4 g∙g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e at 30\u0026deg;C (Cadete et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2012\u003c/span\u003e) and with lower yield at 35\u0026deg;C (Melo \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). In fact, 35 \u0026ordm;C was described as the maximum fermentation temperature by the \u003cem\u003eSp. passalidarum\u003c/em\u003e UFMG-CM-Y469 (Melo \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). As described above, \u003cem\u003eSp. passalidarum\u003c/em\u003e UFMG-CM-Y469 was unable to convert xylan to ethanol, so we can state that the ethanol produced probably should be obtained from xylan, because when \u003cem\u003eSp. passalidarum\u003c/em\u003e UFMG-CM-Y469 was cultured only with \u003cem\u003eA. pullulans\u003c/em\u003e UFMG-CM-Y518 extract as substrate (containing only 0.12 g∙L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e of xylose) no fermentation product was obtained. These results are good indicators of the efficiency of xylanolytic enzymes when supplemented with \u003cem\u003eA. pullulans\u003c/em\u003e UFMG-CM-Y518 extract.\u003c/p\u003e\u003cp\u003eThe use of a yeast capable of fermenting both C6 and C5 fractions (glucose and xylose), such as \u003cem\u003eSp. passalidarum\u003c/em\u003e UFMG-CM-Y469, led to an improvement in the ethanol conversion yield from wheat straw with supplementation of the A. pullulans UFMG-CM-Y518 extract. \u003cem\u003eSp. passalidarum\u003c/em\u003e UFMG-CM-Y469, being able to ferment xylose, increases the ethanol yields obtained from lignocellulosic material by allowing the fermentation of the hemicellulosic fraction in addition to the cellulose fraction. On the other hand, the positive effect on hemicellulose hydrolysis allowed for greater availability of xylose for fermentation, with a more pronounced effect with the \u003cem\u003eA. pullulans\u003c/em\u003e UFMG-CM-Y518 extract. This effect is also reflected in the increased accumulation of xylose throughout the process. However, the conversion of D-xylose to ethanol could have been more efficient since under the tested conditions there was a latency period, during which the \u003cem\u003eSp. passalidarum\u003c/em\u003e UFMG-CM-Y469 yeast took at least 24 h to initiate ethanol production. Thus, there was an accumulation of sugar (glucose and xylose), with preferential consumption of glucose thereafter. This latency may be related to the fact that \u003cem\u003eSp. passalidarum\u003c/em\u003e UFMG-CM-Y469 operates at a temperature (35 \u0026ordm;C) that may affect its optimal performance, as this is the maximum fermentation temperature for this strain (Melo \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Conducting the process at a lower temperature could avoid latency, although it would decrease the cellulase activity of Cellic CTec2. On the other hand, prolonging the fermentation could have resulted in higher ethanol yields than those determined, as there was still xylose available for fermentation.\u003c/p\u003e\u003cp\u003eThis study shows that the extracellular extract of \u003cem\u003eA. pullulans\u003c/em\u003e UFMG-CM-Y518 has substrate specificity (xylan) with no cellulolytic or proteolytic activity. Higher efficiency in the hydrolysis of beechwood xylan (2% w/v) was found compared to pretreated wheat straw (10% w/v solids), with a xylose yield 5-fold greater compared with commercial Cellic HTec2 hemicellulase. Concerning enzymatic hydrolysis, supplementation of commercial cellulase (Cellic CTec2) with hemicellulases (Cellic HTec2) and \u003cem\u003eA. pullulans\u003c/em\u003e UFMG-CM-Y518 extract increased the yields of xylan hydrolysis and cellulose from pretreated wheat straw. These results highlight the importance of xylanase in the degradation and valuation of lignocellulosic biomass. The results indicate the potential of the extracellular extract of \u003cem\u003eA. pullulans\u003c/em\u003e UFMG-CM-Y518 in SSF, due to its high activity at temperatures of 30\u0026ndash;35\u0026deg;C (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). In SSF experiments using beechwood xylan as a substrate, \u003cem\u003eA. pullulans\u003c/em\u003e UFMG-CM-Y518 extract was essential in the production of ethanol by \u003cem\u003eSp. passalidarum\u003c/em\u003e UFMG-CM-Y469 (yield 0.2 g∙g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), with an efficient degradation of xylan to xylose and a hydrolysis yield estimated at 66% in SSF, compared with the yield of 17% in enzymatic hydrolysis. These findings demonstrate that SSF processes increase the extent of enzymatic hydrolysis by the removal of inhibitory hydrolysis products. The association of \u003cem\u003eSp. passalidarum\u003c/em\u003e UFMG-CM-Y469 with a commercial cellulase (Cellic CTec2) supplemented with the \u003cem\u003eA. pullulans\u003c/em\u003e UFMG-CM-Y518 extract also led to an improvement in the conversion yield of wheat straw to ethanol compared to \u003cem\u003eS. cerevisiae\u003c/em\u003e Ethanol Red and Cellic Ctec2, as the extract may have increased the availability of xylose that could be fermented by \u003cem\u003eSp. passalidarum\u003c/em\u003e.\u003c/p\u003e\u003cp\u003eThe extracellular extract of \u003cem\u003eA. pullulans\u003c/em\u003e UFMG-CM-Y518 showed maximum activity at 40\u0026deg;C and retains over 70% of this activity at 30\u0026deg;C. The optimal pH range (4\u0026ndash;5) favors its application in simultaneous saccharification and fermentation (SSF) processes. Enzyme-substrate adsorption resulted in greater stability of the xylanolytic enzymes from the extracellular extract of \u003cem\u003eA. pullulans\u003c/em\u003e UFMG-CM-Y518, showing substrate specificity (xylan) without cellulolytic or proteolytic activity. Additionally, it exhibited higher efficiency in hydrolyzing beechwood xylan (2% w/v) and pre-treated wheat straw (10% w/v solids) compared to the commercial hemicellulase Cellic HTec2 (xylanase), achieving a 5-fold higher xylose yield when applied to pre-treated wheat straw. Supplementation of commercial cellulases (e.g., Cellic CTec2) with hemicellulases like Cellic HTec2 and the extract \u003cem\u003eA. pullulans\u003c/em\u003e UFMG-CM-Y518 increases not only xylan hydrolysis yield but also cellulose hydrolysis yield in pre-treated wheat straw. This underscores the importance of xylanases in the degradation and valorization of lignocellulosic biomass.\u003c/p\u003e\u003cp\u003eThe extracellular extract of \u003cem\u003eA. pullulans\u003c/em\u003e UFMG-CM-Y518 has high potential for use in SSF experiments due to its high activity at temperatures of 30\u0026ndash;35\u0026deg;C. In SSF experiments, efficient degradation of xylan to xylose was observed, 66% compared to 17% in separate enzymatic hydrolysis. This demonstrates that simultaneous saccharification and fermentation increase the extent of enzymatic hydrolysis by reducing inhibition by the hydrolysis products. The combination of the yeast \u003cem\u003eSp. passalidarum\u003c/em\u003e UFMG-CM-Y469 with a commercial cellulase (Cellic CTec2) supplemented with the extract \u003cem\u003eA. pullulans\u003c/em\u003e UFMG-CM-Y518 also improved the conversion yield of wheat straw to ethanol (compared to \u003cem\u003eSc. cerevisiae\u003c/em\u003e Ethanol Red and Cellic Ctec2) since the extract increased the availability of xylose, which could be fermented by \u003cem\u003eSp. passalidarum.\u003c/em\u003e\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003ch2\u003eConflict of Interest\u003c/h2\u003e\u003cp\u003eThe authors declare that they have no conflict of interest. The authors have no relevant financial or non-financial interests to disclose.\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e\u003cp\u003eThis study was funded by the European Commission in the framework of EUBrazil Project ProEthanol2G \u0026ldquo;Integration of Biology and Engineering into an Economical and Energy-Efficient 2G Bioethanol Biorefinery\u0026rdquo; (FP7-251151), by Conselho Nacional de Desenvolvimento Cientifico e Tecnol\u0026oacute;gico (CNPq, Process Nos. 0457499/2014-1, 141586/2017-6, 313088/2020-9 and 408733/2021). This work is part of the project \u0026ldquo;INCT Yeasts: Biodiversity, preservation and biotechnological innovation\u0026rdquo;, funded by Conselho Nacional de Desenvolvimento Cient\u0026iacute;fico e Tecnol\u0026oacute;gico (CNPq), Brazil, grant #406564/2022-1. This work was also funded by Funda\u0026ccedil;\u0026atilde;o do Amparo a Pesquisa do Estado de Minas Gerais (FAPEMIG, process numbers APQ-01525-14, and APQ-02552-15, APQ-03071\u0026ndash;17), and Funda\u0026ccedil;\u0026atilde;o de Amparo \u0026agrave; Pesquisa do Estado de S\u0026atilde;o Paulo (FAPESP/BIOEN/FAPEMIG).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eConceived of study: Carla Lara, Susana Marques, Francisco G\u0026iacute;rio, C\u0026eacute;sar Fonseca and Carlos Rosa; Performed research, Carla Lara, Susana Marques, C\u0026eacute;sar Fonseca; Analyzed data: Carla Lara, Evelyn S. Oliveira, Susana Marques, Francisco G\u0026iacute;rio, Giordana Arend, C\u0026eacute;sar Fonseca and Carlos Rosa; Wrote the paper: Carla Lara, Giordana Arend, C\u0026eacute;sar Fonseca and Carlos Rosa.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAfedzi AEK and Parakulsuksatid P (2023) Recent advances in process modifications of simultaneous saccharification and fermentation (SSF) of lignocellulosic biomass for bioethanol production. Biocatal. Agric. Biotechnol., 54: 102961. https://doi.org/10.1016/j.bcab.2023.102961\u003c/li\u003e\n\u003cli\u003eAlvira P, Tom\u0026aacute;s-Pej\u0026oacute; E, Ballesteros M, Negro MJ (2010) Pretreatment technologies for an efficient bioethanol production process based on enzymatic hydrolysis: A review. 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(2016) Hydrolysis of pretreated rice straw by an enzyme cocktail comprising acidic xylanase from Aspergillus sp. for bioethanol production. Renew. Energy, 98:9-15. https://doi.org/10.1016/j.renene.2016.05.011\u003c/li\u003e\n\u003cli\u003eValdivia M, Galan JL, Laffarga J, Ramos JL (2016) Biofuels 2020: Biorefineries based on lignocellulosic materials. Microb. Biotechnol., 9:585-594. doi: 10.1111/1751-7915.12387\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"world-journal-of-microbiology-and-biotechnology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"wibi","sideBox":"Learn more about [World Journal of Microbiology and Biotechnology](https://www.springer.com/journal/11274)","snPcode":"11274","submissionUrl":"https://submission.nature.com/new-submission/11274/3","title":"World Journal of Microbiology and Biotechnology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Aureobasidium pullulans, enzymatic hydrolysis, Spathaspora passalidarum, wheat straw, xylan, xylanase","lastPublishedDoi":"10.21203/rs.3.rs-8022214/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8022214/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe production of second-generation (2G) ethanol from lignocellulosic biomass is a sustainable and economically competitive alternative. Hemicellulolytic enzymatic hydrolysis can be an efficient way to degrade biomass and obtain fermentable sugars. Here, we characterized the extracellular extract of \u003cem\u003eAureobasidium pullulans\u003c/em\u003e UFMG-CM-Y518, and assessed its potential for the enzymatic hydrolysis of pretreated wheat straw and simultaneous saccharification and fermentation (SSF) to produce bioethanol. \u003cem\u003eA. pullulans\u003c/em\u003e UFMG-CM-Y518 extract displayed maximum xylanase relative activity, at 40\u0026deg;C and pH ranging from 4.0 to 4.5. Also, presented optimal β-xylosidase activity at 80\u0026deg;C and pH 4.0 to 5.0, with high stability at a moderate temperature of 45\u0026deg;C. \u003cem\u003eA. pullulans\u003c/em\u003e UFMG-CM-Y518 extract was evaluated in the hydrolysis of xylan and pretreated wheat straw, in simultaneous saccharification and fermentation processes for the production ethanol. The \u003cem\u003eA. pullulans\u003c/em\u003e UFMG-CM-Y518 extract was more efficient in the hydrolysis of beechwood xylan and wheat straw, presenting efficiency 5-times higher than the commercial hemicellulase HTec2. In SSF experiments with the xylose-fermenting yeast \u003cem\u003eSpathaspora passalidarum\u003c/em\u003e and beechwood xylan as a substrate, the \u003cem\u003eA. pullulans\u003c/em\u003e UFMG-CM-Y518 extract efficiently degraded xylan to xylose with higher yield (66%), when compared to the separate hydrolysis. This resulted in the production of 6.6 g∙L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e ethanol with yield of 0.2 g∙g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. \u003cem\u003eA. pullulans\u003c/em\u003e UFMG-CM-Y518 extract improved the conversion yield of ethanol from wheat straw by \u003cem\u003eSp. passalidarum\u003c/em\u003e due to the increased xylose available for fermentation. In this way, we can state that this extract has potential for biotechnological applications in the biofuels industry.\u003c/p\u003e","manuscriptTitle":"Use of Aureobasidium pullulans xylanase for simultaneous saccharification and fermentation in second- generation bioethanol production","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-19 08:43:44","doi":"10.21203/rs.3.rs-8022214/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-12-19T06:20:02+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-11-25T00:17:25+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-11-24T21:18:32+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-11-13T10:41:15+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"293316897277742449762122748406780901233","date":"2025-11-12T12:32:09+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"331567155576758683202349191830731206869","date":"2025-11-10T17:07:07+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"323404280094625409970485485709835969600","date":"2025-11-10T06:48:18+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-11-10T04:46:21+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-11-05T04:21:29+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-11-04T13:37:06+00:00","index":"","fulltext":""},{"type":"submitted","content":"World Journal of Microbiology and Biotechnology","date":"2025-11-03T19:29:34+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"world-journal-of-microbiology-and-biotechnology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"wibi","sideBox":"Learn more about [World Journal of Microbiology and Biotechnology](https://www.springer.com/journal/11274)","snPcode":"11274","submissionUrl":"https://submission.nature.com/new-submission/11274/3","title":"World Journal of Microbiology and Biotechnology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"bb7fa1ad-4ecb-4836-beda-06a8e450203f","owner":[],"postedDate":"November 19th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"in-revision","subjectAreas":[],"tags":[],"updatedAt":"2025-12-19T06:23:55+00:00","versionOfRecord":[],"versionCreatedAt":"2025-11-19 08:43:44","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8022214","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8022214","identity":"rs-8022214","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}