Hydrolysis Of Pretreated Sugarcane Bagasse Research Articles

Use of an inexpensive carbon source for the production of a cellulase enzyme complex from Penicillium ucsense S1M29 and enzymatic hydrolysis optimization

AbstractThe high cost of cellulolytic enzyme complexes (CECs) has been a significant impediment to the commercial production of bioproducts from lignocellulose biomass. This study aimed to develop a cost‐effective CEC derived from Penicillium ucsense (former Penicillium echinulatum), utilizing diverse forms of pretreated sugarcane bagasse as the primary carbon/inductor source. Among the different pretreatments used, the hydrothermal pretreatment followed by NaOH delignification (BHD) produced higher FPase and xylanase activities (4.5 FPU mL–1 and 120 IU mL–1) in bioreactor experiments at 20 g BHD L–1 initial concentration. A batch‐mode assay conducted across a range of initial carbon source (5 to 60 g L–1) confirmed the highest FPase activity (4.0 to 5.0 FPU mL–1 at 120 h), in the range of 20–40 g BHD L–1. During these assays the agitation rate, controlled by dissolved O2, tended to stabilize at lower levels, indicating substrate limitation. Conversely, higher initial carbon source concentrations led to an excess of glucose, likely triggering carbon catabolite repression and inhibiting cellulase production. This insight prompted the development of a controlled pulsed fed‐batch strategy, resulting in FPase activity of 11 FPU mL–1 at 220 h using 90 g L–1 BHD controlled fed into the bioreactor. An enzymatic hydrolysis procedure using the generated CEC was also optimized using a central composite rotational design (CCRD). The optimized enzyme hydrolysis conditions achieved a reducing sugar concentration of 80.9 g L–1 in 48 h using 170 g L–1 of BHD as the substrate at a ratio of 15 FPU of enzyme substrate per g of BHD. A preliminary economic assessment demonstrated that, for a first‐ and second‐generation (1G + 2G) ethanol biorefinery, the cost contribution of enzymes would be about US$0.2/L of biofuel. In conclusion, an efficient and highly productive procedure was developed successfully for the production of a CEC. It was particularly effective for the enzymatic hydrolysis of pretreated sugarcane bagasse.

Calcium alginate-immobilized β-glucosidase from Moniliophthora perniciosa: characterization and sugarcane bagasse hydrolysis

The utilization of lignocellulosic materials for second-generation ethanol production via enzymatic catalysts is primarily hindered by enzyme cost. Enzymatic immobilization emerges as a viable solution, enabling enzyme reuse. This study investigated the immobilization of an enzymatic extract obtained from Moniliophthora perniciosa fermentation in calcium alginate spheres using the direct trapping method. Initial tests assessed β-glucosidase activity, showing that a higher concentration of calcium chloride (1 M) alongside larger diameter spheres yielded improved results. The immobilized enzyme was reused for up to 17 cycles without significant loss of activity. The percentage of reducing sugars after 48-h hydrolysis with the supplemented enzymatic extract was 226%, doubling the value achieved with only the free enzymatic extract. The immobilized enzyme retained 50% of its initial activity after 1 h at 80 °C, demonstrating higher activity at pH 6 and 60 °C. These findings suggest that this immobilization technique is simple, economically viable, and effective for the hydrolysis of pretreated sugarcane bagasse.

Stability of the Cellic CTec2 enzymatic preparation immobilized onto magnetic graphene oxide: Assessment of hydrolysis of pretreated sugarcane bagasse

Sugarcane bagasse (SB) was subjected to enzymatic hydrolysis using an enzyme cocktail immobilized on magnetic graphene oxide particles (GO-MNP). The thermal, storage, pH, and operational stabilities of the immobilized enzymes, including exoglucanase, endoglucanase, β-glucosidase, xylanase, and β-xylosidase, were evaluated. The half-life of the biocatalyst (GO-MNP-Enz) was longer than that of the free enzymes at temperatures above 45 °C, with the exception of endoglucanase. SB was subjected to pretreatments generating sulfite-NaOH pretreated SB (SSB) and chlorite pretreated SB (CSB). The enzymatic hydrolysis of SSB and CSB was evaluated using free enzymes and GO-MNP-Enz. The cellulose and xylan conversion of SSB using free enzymes was higher than using GO-MNP-Enz; however, a similar result was obtained after 72 h. This did not occur in the hydrolysis of CSB, in which at the end of 72 h, conversion using GO-MNP-Enz did not reach the levels obtained with free enzymes. GO-MNP-Enz was successfully reused in several SSB hydrolysis cycles, maintaining an efficiency of approximately 80% and presenting the highest turnover frequency when compared with previous results reported in literature. Finally, these results show that the immobilization of cellulases and xylanases improves their operational stability and the obtained GO-MNP-Enz can be used for various SB hydrolysis cycles.

Improvement of cellulase and xylanase production in Penicillium oxalicum under solid-state fermentation by flippase recombination enzyme/ recognition target-mediated genetic engineering of transcription repressors

Penicillium oxalicum has received increasing attention as a potential cellulase-producer. In this study, a copper-controlled flippase recombination enzyme/recognition target (FLP/FRT)-mediated recombination system was constructed in P. oxalicum, to overcome limited availability of antibiotic resistance markers. Using this system, two crucial transcription repressor genes atf1 and cxrC for the production of cellulase and xylanase under solid-state fermentation (SSF) were simultaneously deleted, thereby leading to 2.4- to 29.1-fold higher cellulase and 78.9% to 130.8% higher xylanase production than the parental strain under SSF, respectively. Glucose and xylose released from hydrolysis of pretreated sugarcane bagasse achieved 10.6%–13.5% improvement by using the crude enzymes from the engineered strain Δatf1ΔcxrC::flp under SSF in comparison with that of the parental strain. Consequently, these results provide a feasible strategy for improved cellulase and xylanase production by filamentous fungi.

β-Glucosidase produced by Moniliophthora perniciosa: Characterization and application in the hydrolysis of sugarcane bagasse.

β-Glucosidases (BGLs) belong to the group of enzymes of cellulases and act in the last stage of cellulose degradation, releasing glucose molecules, eliminating the inhibitory effect of cellobiose. This study focused on the production, characterization, and application of BGL from Moniliophthora perniciosa in the hydrolysis of pretreated sugarcane bagasse (3% NaOH + 6% Na2 SO3 ), with varying enzymatic loads and reaction times. The enzyme showed an optimum pH of 4.5 and 60°C. It was stable at all temperatures analyzed (50-90°C) and retained about 100% of its activity at 50°C after 60 min of incubation. Among the ions analyzed, BaCl2 increased BGL activity 9.04 ± 1.41 times. The maximum production of reducing sugars (89.15%) was achieved after 48 h with 10 mg of protein.

Magnetic graphene oxide as a platform for the immobilization of cellulases and xylanases: Ultrastructural characterization and assessment of lignocellulosic biomass hydrolysis

For producing second-generation ethanol (cellulosic ethanol) and other value-added bioproducts, magnetic graphene oxide (GO-MNP) was synthesized in this work and used as the immobilization support for an industrial cellulase-and xylanase-containing preparation. GO-MNP characterization by TEM, SEM and ATR-FTIR spectroscopy showed that the magnetic nanoparticles are homogeneously distributed onto the GO sheets surface. The enzymatic preparation was immobilized by means of carbodiimide cross-linking chemistry using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide (NHS). The supported final biocatalyst (GO-MNP-Enz) showed high activity for the hydrolysis of pretreated sugarcane bagasse (PSB) and presented relative endoglucanase, xylanase, β-glucosidase, and β-xylosidase activities of 70%, 66%, 88%, and 70%, respectively, after 10 cycles of hydrolysis of their respective substrates. The biocatalyst also maintained approximately 50% and 80% of its efficiency for cellulose and xylan hydrolysis, respectively, being the TOF (g.g−1.h−1) the highest observed when compared with previous results reported in literature. These findings suggest that GO-MNP-Enz may be a prospective candidate for industrial applications such as second-generation ethanol production.

Towards a Miniaturized Culture Screening for Cellulolytic Fungi and Their Agricultural Lignocellulosic Degradation.

The substantial use of fungal enzymes to degrade lignocellulosic plant biomass has widely been attributed to the extensive requirement of powerful enzyme-producing fungal strains. In this study, a two-step screening procedure for finding cellulolytic fungi, involving a miniaturized culture method with shake-flask fermentation, was proposed and demonstrated. We isolated 297 fungal strains from several cellulose-containing samples found in two different locations in Thailand. By using this screening strategy, we then selected 9 fungal strains based on their potential for cellulase production. Through sequence-based identification of these fungal isolates, 4 species in 4 genera were identified: Aspergillus terreus (3 strains: AG466, AG438 and AG499), Penicillium oxalicum (4 strains: AG452, AG496, AG498 and AG559), Talaromyces siamensis (1 strain: AG548) and Trichoderma afroharzianum (1 strain: AG500). After examining their lignocellulose degradation capacity, our data showed that P. oxalicum AG452 exhibited the highest glucose yield after saccharification of pretreated sugarcane trash, cassava pulp and coffee silverskin. In addition, Ta. siamensis AG548 produced the highest glucose yield after hydrolysis of pretreated sugarcane bagasse. Our study demonstrated that the proposed two-step screening strategy can be further applied for discovering potential cellulolytic fungi isolated from various environmental samples. Meanwhile, the fungal strains isolated in this study will prove useful in the bioconversion of agricultural lignocellulosic residues into valuable biotechnological products.

One-pot synthesis of a chemically functional magnetic carbonaceous acid catalyst for fermentable sugars production from sugarcane bagasse

Magnetic carbonaceous acid catalyst was synthesized from nano-Fe3O4 powder, glucose and hydroxyethyl-sulfonic acid by a novel method of one-pot hydrothermal precipitation-sulfonation. It was found that the prepared C-SO3H@Fe3O4/C catalyst had high acidity (1.23 mmol g−1) and strong magnetism (12.85 Am2/kg). High yields of glucose (62.59%) and total reducing sugar (73.01%) were achieved from NaOH-freeze pretreated sugarcane bagasse by the C-SO3H@Fe3O4/C catalyst at 180 °C in a microwave reactor. In addition, the C-SO3H@Fe3O4/C catalyst could be easily separated and recovered by an external magnetic force at a high recovery rate (93.18%). High yields of glucose (54.47%) and total reducing sugar (60.65%) were also obtained after experiencing five-time cycles. It was demonstrated as an effective hydrolysis of pretreated sugarcane bagasse to fermentable sugars with high yields by the assistance of the C-SO3H@Fe3O4/C catalyst, and the catalyst exhibited an excellent catalytic stability during the conversion process.

Second-generation ethanol production by separate hydrolysis and fermentation from sugarcane bagasse with cellulose hydrolysis using a customized enzyme cocktail

Lignocellulosic materials are a valuable energy source that can be used to reduce dependence on fossil fuels. However, the high cost of enzymes and low efficiency of enzymatic hydrolysis are among the main bottlenecks regarding production of bioethanol. The purpose of the current work is to present the full use of sugarcane bagasse in the production of biofuels: as a carbon source for the production of competitive enzymes and as a platform for conversion of the cellulose and hemicellulose fractions into ethanol. Hydrolysis of pretreated sugarcane bagasse, using an enzymatic cocktail from naturally occurring microorganisms, was carried out, producing cellulose hydrolysate. This hydrolysate was further fermented to ethanol, achieving a concentration of 33 g/L, which corresponded to a fermentation efficiency of 89% and a high volumetric productivity of 11 g/(L·h). From the hemicellulose fraction, 18 g/L of ethanol was produced, which corresponds to a fermentation efficiency of 87%. Hence, from both cellulose and hemicellulose fractions, the overall ethanol concentration was 6.5% (v/v). These results indicate that production of ethanol through separate hydrolysis and fermentation (SHF) is a promising strategy, since it makes it possible to achieve high productivities during fermentation. Also, the possibility of converting xylose to ethanol can lead to an increase in production.

Batch and fed-batch enzymatic hydrolysis of pretreated sugarcane bagasse – Assays and modeling

Choosing the right pretreatment for a specific biomass in order to improve sugar recovery is a key step towards the commercial feasibility of cellulosic ethanol. This article deals with the enzymatic hydrolysis of sugarcane bagasse subjected to either delignification or hydrothermal pretreatment. Two operational strategies were studied: batch experiments were carried out with different solids loads (5–20% w/v) and fed-batch tests reached cumulative solids content of 24% (w/v). The highest glucose concentration (127 g/L) was achieved when working with the delignified material in fed-batch systems. However, the reactions using delignified bagasse presented sharp decrease in conversion when the solids loads exceeded 15% (w/v). During the modeling phase, the hydrolysis behavior was described according to a semi-mechanistic approach. The goodness of fit evaluation showed that the models performed well when compared to the collected data. Finally, fed-batch simulations suggested new feeding polices to increase the final glucose content.

Coconut shell activated carbon as an alternative adsorbent of inhibitors from lignocellulosic biomass pretreatment

The phenolic compounds and furaldehydes released during the pretreatment of lignocellulosic biomass can negatively affect the enzymatic hydrolysis and alcoholic fermentation steps in biorefineries. This work proposes the use of coconut shell activated carbon (CSAC) as an agroindustrial residue-based adsorbent for the removal of the main inhibitors released in the hydrothermal pretreatment of sugarcane bagasse. The prepared CSAC showed a pore distribution favorable for the adsorption of molecules of different sizes, together with a negative surface charge profile in the pH range from 2 to 10. Adsorption isotherms showed that the CSAC presented high affinity for the phenolic compounds vanillin (monomeric) and tannic acid (oligomeric), as well as for the total phenolics present in an inhibitor cocktail. The CSAC also showed efficient adsorption of the phenolic compounds from the pretreatment liquor, without any removal of the sugars, hence displaying a useful high degree of selectivity. The enzymatic hydrolysis of pretreated sugarcane bagasse in the presence of the liquor treated with 2% (m/v) CSAC resulted in a 30% increase in the amount of glucose released, while the alcoholic fermentation efficiency was enhanced up to 14-fold, with complete removal of furaldehydes. These findings indicated that CSAC is an efficient and selective adsorbent of phenolic and furaldehyde inhibitors. It represents an alternative renewable material that could be used to improve the efficiencies of both the enzymatic hydrolysis and the alcoholic fermentation processes in future biorefineries.

Influence of the Chemical Composition on the Enzymatic Hydrolysis of Hot Water and Organosolv Pretreated Sugarcane Bagasse

The aim of this study was to clarify the role of the relative amounts of hemicelluloses and lignin on the enzymatic hydrolysis of pretreated sugarcane bagasse, by using the glucans/lignin ratio and the glucans/xylans ratio as a quick estimation of the enzymes accessibility to cellulose. The samples were selected to contain about 3–20% lignin, 52–90% glucans, and 6–26% xylans (by extraction of hemicelluloses and delignification). The enzyme loads used for saccharification were 10 FPU of cellulases and 20 IU of β-glucosidases per gram of samples. Conversions of glucans to glucose were highly influenced by the chemical composition, varying between 18.1% and 83.4%. The concentration of lignin and xylans in the pretreated bagasse affects the conversion to glucose. High conversions are achieved when the substrates are highly delignified. Less delignified samples can also lead to high conversion when the percentage of xylans are also decreased. Thus, the removal of both lignin and xylans contributes to an increase in the accessibility of cellulases to cellulose.

One-pot strategy for on-site enzyme production, biomass hydrolysis, and ethanol production using the whole solid-state fermentation medium of mixed filamentous fungi.

The efficient use of renewable lignocellulosic feedstocks to obtain biofuels and other bioproducts is a key requirement for a sustainable biobased economy. This requires novel and effective strategies to reduce the cost contribution of the cellulolytic enzymatic cocktails needed to convert the carbohydrates into simple sugars, in order to make large-scale commercial processes economically competitive. Here, we propose the use of the whole solid-state fermentation (SSF) medium of mixed filamentous fungi as an integrated one-pot strategy for on-site enzyme production, biomass hydrolysis, and ethanol production. Ten different individual and mixed cultivations of commonly used industrial filamentous fungi (Aspergillus niger, Aspergillus oryzae, Trichoderma harzianum, and Trichoderma reesei) were performed under SSF and the whole media (without the extraction step) were used in the hydrolysis of pretreated sugarcane bagasse. The cocultivation of T. reesei with A. oryzae increased the amount of glucose released by around 50%, compared with individual cultivations. The release of glucose and reducing sugars achieved using the whole SSF medium was around 3-fold higher than obtained with the enzyme extract. The addition of soybean protein (0.5% w/w) during the hydrolysis reaction further significantly improved the saccharification performance by blocking the lignin and avoiding unproductive adsorption of enzymes. The results of the alcoholic fermentation validated the overall integrated process, with a volumetric ethanol productivity of 4.77 g/L.h, representing 83.5% of the theoretical yield. These findings demonstrate the feasibility of the proposed one-pot integrated strategy using the whole SSF medium of mixed filamentous fungi for on-site enzymes production, biomass hydrolysis, and ethanol production. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:671-680, 2018.

Prediction of overall glucose yield in hydrolysis of pretreated sugarcane bagasse using a single artificial neural network: good insight for process development

AbstractBACKGROUNDIn this work a single artificial neural network (ANN) was used to model the overall yield of glucose (YGLC) as a function of a wide range of operating conditions of both pretreatment and enzymatic hydrolysis.RESULTSThe model was validated experimentally and presented good predictions of YGLC. Sensitivity analysis using the ANN model indicated that most of the operating parameters, except for pretreatment time, were statistically significant (P‐value <0.05). Experiments showed that the processing of sugarcane bagasse (in natura) results in a satisfactory glucose yield of 69.34% when pretreated for 60 min with low initial biomass concentration and acid concentration (10% and 1.0% w/v), and followed by enzymatic hydrolysis for 72 h with 3.0% w/v substrate loading and 60 FPU per gWIS enzyme concentration.CONCLUSIONThis study demonstrated how pretreatment and enzymatic hydrolysis data can be used to parameterize a single ANN model. Acceptable predictions of YGLC are achieved in terms of RSD, MSE and R2. Supported by the model, this study provided a good insight for process development. © 2017 Society of Chemical Industry

Using in-situ viscosimetry and morphogranulometry to explore hydrolysis mechanisms of filter paper and pretreated sugarcane bagasse under semi-dilute suspensions

The relationship between in-situ viscosimetry, in- and ex-situ morphogranulometry, and biochemistry has been investigated during enzymatic hydrolysis of pretreated sugarcane bagasse (SCB, 3% w/v) and filter paper (FP, 1.5% w/v) at various enzyme dosages (0.3–25FPU/g cellulose). Semi-dilute conditions (1.5–2 times higher than critical concentrations) were considered in order to generate non-Newtonian rheological behaviors and to avoid neglecting particle–particle interactions in opposition to dilute conditions. Observed phenomena as well as hydrolysis mechanisms including solvation, separation, fragmentation, and solubilization appeared to depend strongly on initial substrate properties. For both FP and SCB, suspension viscosities were correlated with particle size distributions and volume fractions during hydrolysis, but they exhibited strongly different trends. With SCB, a viscosity overtaking was clearly observed at enzyme loading ≤10FPU/g cellulose. This phenomenon is explained by the separation of agglomerates into individual fragments, leading to an increase in the total number of particles and a morphological shift of particles from sphere-like into fiber-like. With FP, the viscosity collapsed at the initial stage in relation to the volume reduction of coarse particles and the number increase of fine particles. Suspension viscosity was strongly dependent on the fraction of coarse population and nearly independent from the glucose conversion yield.

Soybean protein as a cost-effective lignin-blocking additive for the saccharification of sugarcane bagasse

Addition of surfactants, polymers, and non-catalytic proteins can improve the enzymatic hydrolysis of lignocellulosic materials by blocking the exposed lignin surfaces, but involves extra expense. Here, soybean protein, one of the cheapest proteins available, was evaluated as an alternative additive for the enzymatic hydrolysis of pretreated sugarcane bagasse. The effect of the enzyme source was investigated using enzymatic cocktails from A. niger and T. reesei cultivated under solid-state, submerged, and sequential fermentation. The use of soybean protein led to approximately 2-fold increases in hydrolysis, relative to the control, for both A. niger and T. reesei enzymatic cocktails from solid-state fermentation. The effect was comparable to that of BSA. Moreover, the use of soybean protein and a 1:1 combination of A. niger and T. reesei enzymatic cocktails resulted in 54% higher glucose release, compared to the control. Soybean protein is a potential cost-effective additive for use in the biomass conversion process.

GH53 Endo-Beta-1,4-Galactanase from a Newly Isolated Bacillus licheniformis CBMAI 1609 as an Enzymatic Cocktail Supplement for Biomass Saccharification.

Galactanases (endo-β-1,4-galactanases-EC 3.2.1.89) catalyze the hydrolysis of β-1,4 galactosidic bonds in arabinogalactan and galactan side chains found in type I rhamnogalacturan. The aim of this work was to understand the catalytic function, biophysical properties, and use of a recombinant GH53 endo-beta-1,4-galactanase for commercial cocktail supplementation. The nucleotide sequence of the endo-β-1,4-galactanase from Bacillus licheniformis CBMAI 1609 (Bl1609Gal) was cloned and expressed in Escherichia coli, and the biochemical and biophysical properties of the enzyme were characterized. The optimum pH range and temperature of Bl1609Gal activity were 6.5-8 and 40°C, respectively. Furthermore, Bl1609Gal showed remarkable pH stability, retaining more than 75% activity even after 24h of incubation at pH 4-10. The enzyme was thermostable, retaining nearly 100% activity after 1-h incubation at pH 7.0 at 25-45°C. The enzymatic efficiency (K cat /K m ) against potato galactan under optimum conditions was 241.2s(-1)mg(-1)mL. Capillary zone electrophoresis demonstrated that the pattern of galactan hydrolysis by Bl1609Gal was consistent with that of endogalactanases. Supplementation of the commercial cocktail ACCELLERASE(®)1500 with recombinant Bl1609Gal increased hydrolysis of pretreated sugarcane bagasse by 25%.

An approach to cellulase recovery from enzymatic hydrolysis of pretreated sugarcane bagasse with high lignin content

The possibility of recovering the cellulases used for enzymatic hydrolysis of sugarcane bagasse was evaluated. A strategy was adopted to maximize the enzyme recovery: desorption of the enzymes adsorbed in the solid residue after hydrolysis, and re-adsorption of the enzymes from the liquid medium onto a fresh substrate. The use of surfactant during the enzymatic hydrolysis was important to improve the glucose release from the material structure and also to facilitate the enzyme desorption from the solid residue after hydrolysis. The temperature and pH used during desorption influenced the enzymes recovery, with the best results (90% adsorbed cellulase) being achieved at 45 °C and pH 5.5. The enzymes present in the liquid medium after enzymatic hydrolysis were partially recovered (77%) by adsorption onto the fresh substrate and used in new enzymatic hydrolysis batches. It was concluded that it is possible to recycle cellulases from an enzymatic medium for use in subsequent hydrolysis processes.

Increased enzymatic hydrolysis of sugarcane bagasse from enzyme recycling.

BackgroundDevelopment of efficient methods for production of renewable fuels from lignocellulosic biomass is necessary to maximize yields and reduce operating costs. One of the main challenges to industrial application of the lignocellulosic conversion process is the high costs of cellulolytic enzymes. Recycling of enzymes may present a potential solution to alleviate this problem. In the present study enzymes associated with the insoluble fraction were recycled after enzymatic hydrolysis of pretreated sugarcane bagasse, utilizing different processing conditions, enzyme loadings, and solid loadings.ResultsIt was found that the enzyme blend from Chrysoporthe cubensis and Penicillium pinophilum was efficient for enzymatic hydrolysis and that a significant portion of enzyme activity could be recovered upon recycling of the insoluble fraction. Enzyme productivity values (g glucose/mg enzyme protein) over all recycle periods were 2.4 and 3.7 for application of 15 and 30 FPU/g of glucan, representing an increase in excess of ten times that obtained in a batch process with the same enzyme blend and an even greater increase compared to commercial cellulase enzymes.ConclusionsContrary to what may be expected, increasing lignin concentrations throughout the recycle period did not negatively influence hydrolysis efficiency, but conversion efficiencies continuously improved. Recycling of the entire insoluble solids fraction was sufficient for recycling of adhered enzymes together with biomass, indicative of an effective method to increase enzyme productivity.

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

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