Bioethanol production from sweet sorghum bagasse by Mucor hiemalis

The present work deals with production of ethanol from sweet sorghum bagasse by a zygomycetes fungus Mucor hiemalis. The bagasse was treated with phosphoric acid and sodium hydroxide, with or without ultrasonication, prior to enzymatic hydrolysis by commercial cellulase and β-glucosidase enzymes. The phosphoric acid pretreatment was performed at 50 °C for 30 min, while the alkali treatment performed with 12% NaOH at 0 °C for 3 h. The pretreatments resulted in improving the subsequent enzymatic hydrolysis to 79-92% of the theoretical yield. The best hydrolysis performance was obtained after pretreatment by NaOH assisted with ultrasonication. The fungus showed promising results in fermentation of the hydrolyzates. In the best case, the hydrolyzate of NaOH-ultrasound pretreated bagasse followed by 24 h fermentation resulted in about 81% of the corresponding theoretical ethanol yield. Furthermore, the highest volumetric ethanol productivity was observed in the hydrolyzates of NaOH pretreated bagasse, especially after ultrasonication in pretreatment stage.     

Ethanol production from alkali- and ozone-treated cotton stalks using thermotolerant Pichia kudriavzevii HOP-1

In the present study, milled cotton stalks were subjected to alkali pretreatment with NaOH at 1-4% (w/v) concentrations at 121 °C for time ranging from 30 to 90 min. Ozone pretreatment was performed by passing 45 mg/L of ozone gas over 2 mm cotton stalks for 150 min at a flow rate of 0.37 L/min. The residual biomass from 4% alkali pretreatment for 60 min showed 46.6% lignin degradation accompanied by 83.2% increase in glucan content, compared with the untreated biomass. Hydrolysis of 4% alkali-treated and ozone-treated cotton stalks was conducted using enzyme combination of 20 filter paper cellulase units/gram dried substrate (FPU/g-ds), 45 IU/g-ds β-glucosidase and 15 IU/g-ds pectinase. Enzymatic hydrolysis of alkali-treated and ozone-treated biomass after 48 h resulted in 42.29 g/L glucose, 6.82 g/L xylose and 24.13 g/L glucose, 8.3 g/L xylose, respectively. About 99% of glucose was consumed in 24 h by Pichia kudriavzevii HOP-1 cells resulting in 19.82 g/L of ethanol from alkali-treated cotton stalks and 10.96 g/L of ethanol from ozone-treated cotton stalks. Simultaneous saccharification and fermentation of the alkali-treated cotton stalks after 12-h pre-hydrolysis resulted in ethanol concentration, ethanol yield on dry biomass basis and ethanol productivity of 19.48 g/L, 0.21 g/g and 0.41 g/L/h, respectively which holds promise for further scale-up studies. To the best of our knowledge, this is the first study employing SSF for ethanol production from cotton stalks.     

Effects of washing, milling and loading enzymes on the enzymatic hydrolysis of a steam pretreated sugarcane bagasse

Enzymatic hydrolysis of steam pretreated sugarcane bagasse was performed to investigate the production of glucose and xylose. A blend of industrial enzymes (Novozymes) was used. The enzymes were used either without having been washed or having been washed with water or 1% aqueous NaOH solution. The influence of the size of sugarcane bagasse and the proportion of the enzymatic cocktail, which was composed of endoglucanases/cellobiohydrolases (Celluclast 1.5L) and β-glucosidase (Novozym 188) cellulases mixtures, was investigated. The tests were performed at a temperature of 50 °C and at a pH of 4.8 during a period of 72 h. The assays that were conducted with a pretreated sugarcane bagasse that was washed with a 1% aqueous NaOH solution without milling led to the largest amounts of glucose after 72 h, which was independent of the proportions of enzymes used in experiments with the smallest amounts of enzymes. The largest amount of xylose was obtained with a pretreated sugarcane bagasse that was not washed.     

Process optimization for ethanol production from photoperiod-sensitive sorghum: Focus on cellulose conversion

Photoperiod-sensitive sorghum, as a competitive biomass for ethanol production, was investigated to develop an integrated process for improving ethanol yield. Response surface methodology was employed to study the relationship between pretreatment variables (including temperature, sulfuric acid concentration, and reaction time) and cellulose recovery, as well as efficiency of enzymatic hydrolysis (EEH) in the solid part. Recovery yield decreased and EEH increased as the pretreatment temperature, acidic concentration, and reaction time increased. A model was successfully developed to predict total glucose yield with a maximum value of 82.2%. Conditions of co-fermentation were also optimized, and the optimal ethanol yield was obtained with constant-temperature simultaneous saccharification and fermentation at 38 °C. Acetate buffer at a concentration of 50 mM was found helpful for increasing efficiency of enzymatic hydrolysis, as well as ethanol yield. The maximum ethanol yield was 0.21 g ethanol per dry mass at the conditions of 38 °C, 0.05 g yeast/L, and 50 mM acetate buffer. A complete cellulose balance was provided for the whole process.     

Optimization of ethanol production from microwave alkali pretreated rice straw using statistical experimental designs by Saccharomyces cerevisiae

Ethanol production from agro-waste provides an alternative energy-production system. Statistical experimental designs were used for optimization of critical nutrients and process variables for ethanol production. The critical nutrients and process variables were initially selected according to a Placket-Burman (PB) design. Selected factors (inoculum level 1-5%, pH 4.5-7, temperature 25-35 °C and urea concentration 0.25-0.75 g/L) were optimized by response surface methodology (RSM) based on a three-level four-factor Box-Behnken design (BBD). Under optimum conditions of inoculum level 3%, pH 5.75, temperature 30 °C and urea concentration 0.50 g/L maximum ethanol production obtained 13.2 g/L from microwave alkali pretreated rice straw with ethanol productivity 0.33 g/L/h. Under optimum conditions ethanol production studied at fermenter level and obtained ethanol concentration 19.2 g/L, ethanol productivity 0.53 g/L/h and ethanol yield to consumed sugar 0.50 g/g. These results indicated that ethanol production can be enhanced by optimization of nutritional and process variables.     

Utilization of pretreated bagasse for the sustainable bioproduction of cellulase by Aspergillus nidulans MTCC344 using response surface methodology

Response surface methodology (RSM) was used to optimize the conditions for the production of endo β-1,4 glucanase, a component of cellulase by Aspergillus nidulans MTCC344 under solid state fermentation, using bagasse as the chief substrate. A four-factor-five-level central composite design was employed for experimental design and analysis of the results. Maximum cellulase activity (CMCase was 28.96 U g⁻¹) can be attained at the optimum conditions, 16.8 mm bagasse bed height, 60% moisture content, pH 4.25 and temperature 40 °C in the solid state fermenter. These data were rather close to the experimental results obtained (CMCase was 28.84 U g⁻¹). A. nidulans MTCC344 was able to hydrolyze pretreated bagasse completely after 8 days of incubation with significant endo β-1,4 glucanase activities. The results of Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD) and scanning electron microscopy (SEM) of bagasse showed structural changes through pretreatment, in favor of enzymatic hydrolysis. Bagasse with alkali pretreatment using sodium hydroxide is a source of lignocelluloses able to improve the yield of endo β-1,4 glucanase by the strain of A. nidulans. The endo β-1,4 glucanase produced during the bioconversion of cellulose to glucose by A. nidulans MTCC344 is strongly dependent on the pretreatment given before hydrolysis.     

Value-addition of agricultural wastes for augmented cellulase and xylanase production through solid-state tray fermentation employing mixed-culture of fungi

Solid-state fermentation (SSF) was performed to evaluate the potential of agricultural residues for the production of cellulase and hemicellulase using individual and mixed cultures of Aspergillus niger and Trichoderma reseei. The maximum filter paper (FP) cellulase activity of 13.57 IU/gram dry substrate (gds), 22.89 IU/gds and 24.17 IU/gds and β-glucosidase activities of 21.69 IU/gds, 13.58 IU/gds and 24.54 IU/gds were obtained with wheat bran medium at 96 h incubation period with A. niger, T. reseei and mixed-cultures of A. niger and T. reseei, respectively. Mixed-culture SSF using rice straw supplemented with wheat bran in the ratio 3:2 resulted in higher FP cellulase, β-glucosidase, endoglucanase (CMCase) and xylanase activities, compared to the activities obtained using mono-cultures. Similarly, higher FP cellulase, β-glucosidase, CMCase and xylanase activities of 35.8 IU/gds (96 h), 33.71 IU/gds (96 h), 131.34 IU/gds (120 h) and 3106.34 IU/gds (120 h) were achieved in the tray fermentation using rice straw with wheat bran in the ratio of 3:2. Results of present investigation showed that higher cellulase activity and an optimal combination of cellulase and β-glucosidase can be achieved through mixed-culture SSF in trays. The approach of utilizing negative cost agricultural wastes through tray fermentation for cellulase and hemicellulase production is expected to serve the objectives of: (a) management of wastes which would otherwise cause environmental pollution problems; (b) production of hydrolytic enzymes at low cost and; (c) simple technique requiring no sophisticated instruments with practical applications.     

Carbohydrate degrading enzyme production by plant pathogenic mycelia and microsclerotia isolates of Macrophomina phaseolina through koji fermentation

The ability of hydrolytic enzyme production by two different isolates of Macrophomina phaseolina was studied on apple pomace as a substrate for solid state fermentation (SSF). Initial moisture level, temperature and fermentation period was optimized so as to achieve higher output. Among the two different isolates, microsclerotial (MphP) and mycelial (MphM), MphP was observed as a potential source of different hydrolytic enzymes as compared to MphM. MphP gave higher enzyme activities (IU/gram dry substrate (gds): filter paper cellulase (FPase) activity 196.21 ± 16.3 (120 h), carboxymethyl cellulase (CMCase) 279.34 ± 28.25 (72 h), β-glucosidase (BGL) 129.82 ± 12.41 (96 h), xylanase 2527.88 ± 46.15 (120 h), and amylase 2780.72 ± 38.13 (96 h), respectively at 70% (v/w) IML. The incubation temperature was also found to have impact on the enzyme production ability of Macrophomina strains. The higher enzyme activities were achieved (IU/gds) as follows FPase 276.13 ± 25.02 (40 °C, 120 h), CMCase 278.11 ± 24.47 (35 °C, 144 h), BGL 189.47 ± 15.05 (30 °C, 144 h), xylanase 3845.77 ± 43.38 (35 °C, 144 h) and amylase activity of 3309.45 ± 29.22 (35 °C, 120 h), respectively using MphP at 70% (v/w) IML. This study reports for the first time the potential of carbohydrate degrading enzyme bioproduction by different isolates of M. phaseolina.     

Fractionation of flax shives with pressurized aqueous ethanol

Fractionation of flax shives into major biopolymer constituents, such as cellulose, hemicelluloses, and lignin, was carried out with pressurized aqueous ethanol in a pressurized low-polarity water extractor. The effect of processing parameters such as temperature, ethanol concentration, flow rate, sample size and solvent/feed ratio on the simultaneous extraction of hemicelluloses and lignin was determined. More than 80% of total hemicelluloses and ∼78% of total lignin were removed simultaneously in a single step under the following conditions: 180 °C, 30% (v/v) ethanol concentration, 3 mL/min flow rate, and 45 mL/g solvent/feed ratio. Under these extraction conditions, cellulose degradation was negligible. Further, the separation of lignin from hemicelluloses was carried out using two simple alternative methodologies based on precipitation. Since no acidic or alkali catalysts were used, the degradation of biopolymers was negligible and the oligomer/monomer ratio of sugars was 825:1. Characterization of fractionated biopolymers was carried out with scanning electron microscopy and a Fourier-transform infrared spectrometer (FT-IR). FT-IR spectra of isolated lignin and hemicelluloses showed that both polymers were comparable to commercially available products.     

Release of doxycycline through cellulose acetate symmetric and asymmetric membranes produced from recycled agroindustrial residue: Sugarcane bagasse

Cellulose acetate is one of the components employed in drug controlled-release systems in the form of membranes. The aim of this study was to examine the controlled-release of doxycycline employing cellulose acetate symmetric and asymmetric membranes as matrices. The cellulose triacetate was produced from sugarcane bagasse through a homogeneous acetylation reaction, using acetic acid as the solvent, acetic anhydride as the acetylating agent and sulfuric acid as the catalyst. The viscosity average molecular weight of the cellulose acetate produced was 39,000 g mol⁻¹. The symmetric membranes were produced using a system solvent of dichloromethane/ethanol (9:1, v/v) and the asymmetric membranes were produced from the same solvent system and 10% of water. For the formulation of both, 5% of doxycycline was used. The membranes were characterized by thermal analysis (DSC and TGA) and scanning electron microscopy SEM. The release of doxycycline through cellulose triacetate matrices was examined using spectrophotometric analysis in the ultraviolet-visible region, at 275 nm. The results revealed that asymmetric membranes release 80% of the drug in 100 min, while symmetric membranes release 14% of the drug during the same time interval.     

Composite materials of thermoplastic starch and fibers from the ethanol-water fractionation of bagasse

The aim of this study was to evaluate the potential of the fibrous material obtained from ethanol-water fractionation of bagasse as reinforcement of thermoplastic starches in order to improve their mechanical properties. The composites were elaborated using matrices of corn and cassava starches plasticized with 30 wt% glycerin. The mixtures (0, 5, 10 and 15 wt% bagasse fiber) were elaborated in a rheometer at 150 °C. The mixtures obtained were pressed on a hot plate press at 155 °C. The test specimens were obtained according to ASTM D638. Tensile tests, moisture absorption tests for 24 days (20-23 °C and 53% RH, ASTM E104), and dynamic-mechanical analyses (DMA) in tensile mode were carried out. Images by scanning electron microscopy (SEM) and X-ray diffraction were obtained. Fibers (10 wt% bagasse fiber) increased tensile strength by 44% and 47% compared to corn and cassava starches, respectively. The reinforcement (15 wt% bagasse fiber) increased more than fourfold the elastic modulus on starch matrices. The storage modulus at 30 °C (E_30 °C′) increased as the bagasse fiber content increased, following the trend of tensile elastic modulus. The results indicate that these fibers have potential applications in the development of biodegradable composite materials.     

Characterization of depolymerized residues from extremely low acid hydrolysis (ELA) of sugarcane bagasse cellulose: Effects of degree of polymerization, crystallinity and crystallite size on thermal decomposition

Sugarcane bagasse cellulose was subjected to the extremely low acid (ELA) hydrolysis in 0.07% H₂SO₄ at 190, 210 and 225 °C for various times. The cellulose residues from this process were characterized by TGA, XRD, GPC, FTIR and SEM. A kinetic study of thermal decomposition of the residues was also carried out, using the ASTM and Kissinger methods. The thermal studies revealed that residues of cellulose hydrolyzed at 190, 210 and 225 °C for 80, 40 and 8 min have initial decomposition temperature and activation energy for the main decomposition step similar to those of Avicel PH-101. XRD studies confirmed this finding by showing that these cellulose residues are similar to Avicel in crystallinity index and crystallite size in relation to the 110 and 200 planes. FTIR spectra revealed no significant changes in the cellulose chemical structure and analysis of SEM micrographs demonstrated that the particle size of the cellulose residues hydrolyzed at 190 and 210 °C were similar to that of Avicel.     

Production of activated carbon from organic by-products from the alcoholic beverage industry: Surface area and hardness optimization by using the response surface methodology

The ability of activated carbon to remove pollutants from water in packed column systems is dependent on granular material with mechanical strength sufficient to avoid attrition caused by stream flow. Therefore, an appropriate balance between surface area and hardness is essential when using activated carbon in real systems. The purpose of this research is to determine the optimal production conditions that generate activated carbon with adequate physical properties to be used in packed systems from agave bagasse, a waste product from the mezcal industries in Mexico. Activated carbons were produced by chemical activation (ZnCl₂ or H₃PO₄). Response surface methodology (RSM) was used to evaluate the effect of the activation temperature (250-550 °C), activation time (0-50 min), and the concentration of activating agent (0.2-1.4; g activating agent/g bagasse) on both surface area and hardness. The production conditions that generated optimal characteristics in the activated carbon were 392 °C, 1.02 g activating agent/g bagasse and 23.8 min for H₃PO₄ activated samples and 456 °C, 1.08 g activating agent/g bagasse and 23.8 min for ZnCl₂ activated samples. The surface area and hardness of the activated carbon produced from bagasse under these conditions were similar to activated commercial carbons (surface area > 800 m²/g and hardness > 85%).     

Conversion of starch from dry common beans (Phaseolus vulgaris L.) to ethanol

Dry common beans (Phaseolus vulgaris L.) were evaluated for potential conversion of starch to ethanol. Eight varieties of beans with average starch content of 46% (db) were assayed in a laboratory-scaled process based upon the commercial corn dry grind fermentation process. Ethanol yield was 0.43-0.51 g ethanol/g glucose (0.19-0.23 g ethanol/g beans). The average ethanol yield for the eight bean types was 92% of maximum theoretical yield, demonstrating that starch from beans could be efficiently converted to ethanol. Ethanol concentration obtained from 20% (w/w) solids loading was 3.5-4.4% (w/v). The residual fermentation solids contained, on a dry basis, 37.1-43.6% crude protein, 10.8-15.1% acid detergent fiber and 19.1-31.3% neutral detergent fiber.     

Sugarcane bagasse whiskers: Extraction and characterizations

This work evaluates the use of sugarcane bagasse (SCB) as a source of cellulose to obtain whiskers. These fibers were extracted after SCB underwent alkaline peroxide pre-treatment followed by acid hydrolysis at 45 °C. The influence of extraction time (30 and 75 min) on the properties of the nanofibers was investigated. Sugarcane bagasse whiskers (SCBW) were analyzed by transmission electron microscopy (TEM), X-ray diffraction (XRD) and thermogravimetric analysis (TGA) in air atmosphere. The results showed that SCB could be used as source to obtain cellulose whiskers and they had needle-like structures with an average length (L) of 255 ± 55 nm and diameter (D) of 4 ± 2 nm, giving an aspect ratio (L/D) around 64. More drastic hydrolysis conditions (75 min) resulted in less thermally stable whiskers and caused some damage on the crystal structure of the cellulose as observed by XRD analysis.     

Composition and oxidative stability of crude oil extracts of corn germ and distillers grains

The fatty acid composition, Acid Value, and the content and composition of tocopherols, tocotrienols, carotenoids, phytosterols, and steryl ferulates were determined in corn germ oil and four post-fermentation corn oils from the ethanol dry grind process. The oxidative stability index at 110 °C was determined for the five oils, and four oils were compared for their stability during storage at 40 °C as determined by peroxide value and hexanal content. The fatty acid composition of all five oils was typical for corn oil. The Acid Value (and percentage of free fatty acids) was highest (28.3 mg KOH/g oil) in corn oil extracted centrifugally from a conventional dry grind ethanol processing facility and for oil extracted, using hexane, from distillers dried grains with solubles (DDGS) from a raw starch ethanol processing facility (20.8 mg KOH/g oil). Acid Value was lowest in two oils extracted centrifugally from thin stillage in a raw starch ethanol facility (5.7 and 6.9 mg KOH/g oil). Tocopherols were highest in corn germ oil (∼1400 μg/g), but tocotrienols, phytosterols, steryl ferulates, and carotenoids were higher in all of the post-fermentation corn oils. Hexane extracted oil from DDGS was the most oxidatively stable as evaluated by OSI and storage test at 40 °C, followed by centrifugally extracted thin stillage oil from the raw starch ethanol process, and centrifugally extracted thin stillage oil from the conventional dry grind ethanol process. Corn germ oil was the least oxidatively stable. When stored at room temperature, the peroxide value of centrifugally extracted thin stillage oil from the raw starch ethanol process did not significantly increase until after six weeks of storage, and was less than 2.0 mequiv. peroxide/kg oil after three months of storage. These results indicate that post-fermentation corn oils have higher content of valuable functional lipids than corn germ oil. Some of these functional lipids have antioxidant activity which increases the oxidative stability of the post-fermentation oils.     

Extraction of flax shive using sodium ethoxide catalyst in anhydrous ethanol

This work investigated the yield and nature of solvent-soluble organic compounds extracted from flax shive using a room temperature reaction (20 °C) with sodium ethoxide catalyst at four different concentrations (0.2, 0.5, 0.7, and 1.0 M) in anhydrous ethanol. Results were compared with the use of aqueous sodium hydroxide (1.0 M) at two different reaction temperatures (20 °C and 100 °C). Quantitative yield from flax shive varied linearly with sodium ethoxide concentration and averaged 54.5 mg/g on a dry-mass basis (db) at 1.0 M. In contrast, the quantitative yield using 1.0 M sodium hydroxide was much lower, averaging 2.2 mg/g (db). Yield did not differ significantly due to changes of particle size in either case, or due to changes of temperature over the range considered in the case of sodium hydroxide.Analyses using proton nuclear magnetic resonance (¹H NMR) confirmed all extracts to contain aromatic compounds, thus likely lignin derived, but found differences in chemical characteristics between the two extraction methods. One key difference was the presence of compounds with methyl ether groups in sodium hydroxide extracts that were absent in the case of sodium ethoxide extracts. Given that flax contains a mixed guaiacyl-syringyl lignin, methyl ether groups would be expected to be present. Control reactions on three model compounds were carried out to confirm that transesterification occurred with sodium ethoxide. These control reactions also demonstrated that methyl ether groups would be expected to remain intact under the extraction conditions reported here. In light of the higher yield of solvent soluble compounds recovered by extraction with basic ethanol, flax shive may represent a source of value-added phenolic constituents. This processing method may also represent a useful pre-treatment prior to the production of biofuels by cellulose degrading organisms.     

Modeling the effects of solid state fermentation operating conditions on endoglucanase production using an instrumented bioreactor

Cellulase production is one of the most critical steps in the economics of second generation ethanol. Although solid-state fermentation (SSF) is an attractive process for the production of enzymes, SSF is highly limited by the difficulty in controlling the operating variables which affect microbial growth and metabolites production. In this context, this work evaluates the effects of operational conditions on endoglucanase production by a selected strain of Aspergillus niger cultivated under SSF using an instrumented lab-scale bioreactor equipped with an on-line automated monitoring and control system. The effects of air flow rate, inlet air relative humidity and substrate initial moisture on endoglucanase production were evaluated using a statistical design methodology. A correlation coefficient of 0.9106 and a calculated value of F, 5.46 folds higher than the listed value (P-value < 0.05) allowed the modeling of endoglucanase production under different process conditions. Higher endoglucanase production (56.1 U/g) was achieved for a selected condition of substrate initial moisture of 72%, air inlet humidity of 70%, and flow rate of 20 mL/min. A significant increase in endoglucanase production was also found to be achieved under forced aeration conditions (50.2 IU/g) compared to static conditions (29.8 IU/g) after 72 h of cultivation. Besides, respirometric analysis revealed that the total amount of CO₂ produced was linearly correlated with enzyme production (R² of 0.988). The bioreactor system used, as well as the methodology employed herein, was very effective in evaluating the influence of operational variables on enzymes production under SSF.     

Biological pretreatment of sugar cane bagasse for the production of cellulases and xylanases by Penicillium echinulatum

In this study, sugar cane bagasse was pretreated with the white rot fungus Pleurotus sajor-caju PS 2001, and this biomass was subsequently used in the production of cellulases and xylanases by the fungus Penicillium echinulatum. Despite the environmental advantages offered by this type of pretreatment, the enzymatic activity obtained with biologically pretreated sugar cane bagasse (PSCB) was lower than that of the control treatments, which were carried out with untreated sugar cane bagasse (SCB) and cellulose. For medium supplemented with PSCB, the average peak activities obtained were 0.13, 1.0, 0.18, and 0.33 U ml^?1 for FPA, endoglucanase, β-glucosidases, and xylanases, respectively. For the cellulose, control values of 0.52, 1.20, 0.20, and 1.46 U ml^?1, and SCB values of 0.95, 1.60, 0.21, and 1.49 U ml^?1 were obtained, respectively. Although the enzymatic activities of the culture with biologically pretreated sugar cane bagasse were lower than the cultures carried out with untreated sugar cane bagasse, it should be noted that production of enzymes of the cellulase and hemicellulase complex after production of the mushrooms is another way to add value to this agricultural residue.     

Value added waste of Jatropha curcas residue: Optimization of protease production in solid state fermentation by Taguchi DOE methodology

The oil extraction of Jatropha curcas created the large amount of the by-product from its seeds. An application of solid-state fermentation (SSF) was considered to be of value to these raw materials. This study investigated the potential of a utilization of deoiled J. curcas seed cake as substrate for protease productions by Aspergillus oryzae. While various parameters for SSF was conventionally individually optimized, five parameters were simultaneously examined based on Taguchi method. The effect of three different levels of five factors, including moisture content of substrate, inoculums size, incubation temperature, type of porous substrate and incubation time were examined. The optimum conditions for the protease production by A. oryzae obtained from this experiment were 45% moisture content of substrate, 10% inoculums size, 30 °C incubation temperature, deoiled J. curcas seed cake mixed with cassava bagasse ratio 4:1 as porous substrate at 84 h of incubation time. By adjusting the conditions to these optimum levels, the protease production increased up to 4.6 times as many as the protease yield from the non-optimizing experiment. The use of statistical approach, Taguchi method, provided a satisfactory outcome in defining the optimum conditions for protease production by A. oryzae. Further, the utilization of deoiled J. curcas seed cake as substrate for SSF was proven as the suitable practice for this agricultural waste, in order to develop for an industrial use.