Production and Optimization of Physicochemical Parameters of Cellulase Using Untreated Orange Waste by Newly Isolated <Emphasis Type="Italic">Emericella variecolor</Emphasis> NS3

Cellulase enzymes have versatile industrial applications. This study was directed towards the isolation, production, and characterization of cellulase enzyme system. Among the five isolated fungal cultures, Emericella variecolor NS3 showed maximum cellulase production using untreated orange peel waste as substrate using solid-state fermentation (SSF). Maximum enzyme production of 31 IU/gds (per gram of dry substrate) was noticed at 6.0 g concentration of orange peel. Further, 50 °C was recorded as the optimum temperature for cellulase activity and the thermal stability for 240 min was observed at this temperature. In addition, the crude enzyme was stable at pH 5.0 and held its complete relative activity in presence of Mn²⁺ and Fe³⁺. This study explored the production of crude enzyme system using biological waste with future potential for research and industrial applications.     

Development of nitrilase-mediated process for phenylacetic acid production from phenylacetonitrile

This study aimed at developing an efficient biotransformation process for phenylacetic acid production from phenylacetonitrile by using recombinant Escherichia coli M15 harboring a double mutant MG nitrilase (I113M/Y199G) from Burkholderia cenocepacia J2315. A yield of 2310 U/mL nitrilase was obtained by fermentation after the optimization of cultivation conditions, with a specific activity of 64 U/mg dcw. The MG nitrilase showed high substrate tolerance and completely hydrolyzed 100 mM phenylacetonitrile in 30 min under optimal conditions. To alleviate substrate inhibition, periodic or continuous batch-feeding of substrate was used during the biotransformation. Up to 164 g/L substrate was completely hydrolyzed in 9 h with continuous batch-feeding using resting cells, corresponding to 400 U/mL of nitrilase activity, and leading to production of 163.4 g/L phenylacetic acid. The hydrolysis process has potential application for phenylacetic acid production on a large scale.     

Prospecting Agro-waste Cocktail: Supplementation for Cellulase Production by a Newly Isolated Thermophilic <Emphasis Type="Italic">B. licheniformis</Emphasis> 2D55

Bacteria isolated from thermophilic environment that can produce cellulase as well as utilise agro-waste biomass have a high potential for developing thermostable cellulase required in the biofuel industry. The cost for cellulase represents a significant challenge in converting lignocellulose to fermentable sugars for biofuel production. Among three potential bacteria examined, Bacillus licheniformis 2D55 (accession no. KT799651) was found to produce the highest cellulolytic activity (CMCase 0.33 U/mL and FPase 0.09 U/mL) at 18–24 h fermentation when grown on microcrystalline cellulose (MCC) as a carbon source in shake flask at 50 °C. Cellulase production process was further conducted on the untreated and NaOH pretreated rice straw (RS), rice husk (RH), sugarcane bagasse (BAG) and empty fruit bunch (EFB). Untreated BAG produced the highest FPase (0.160 U/mL), while the highest CMCase (0.150 U/mL) was supported on the pretreated RH. The mixture of untreated BAG and pretreated RH as agro-waste cocktail has remarkably improved CMCase (3.7- and 1.4-fold) and FPase (2.5- and 11.5-fold) compared to the untreated BAG and pretreated RH, respectively. The mechanism of cellulase production explored through SEM analysis and the location of cellulase enzymes of the isolate was also presented. Agro-waste cocktail supplementation provides an alternative method for an efficient production of cellulase.     

Highly Thermostable and pH-Stable Cellulases from Aspergillus niger NS-2: Properties and Application for Cellulose Hydrolysis

Optimization of cultural conditions for enhanced cellulase production by Aspergillus niger NS-2 were studied under solid-state fermentation. Significant increase in yields (CMCase 463.9?±?20.1 U/g, FPase 101.1?±?3.5 U/g and β-glucosidase 99?±?4.0 U/g) were obtained under optimized conditions. Effect of different nutritional parameters was studied to induce the maximum production of cellulase complex. Scale-up studies for enzyme production process were carried out. Characterization studies showed that enzymes produced by A. niger NS-2 were highly temperature- and pH stable. At 50 °C, the half life for CMCase, FPase, β-glucosidase were approximately 240 h. Cellulases from A. niger NS-2 were stable at 35 °C for 24 h over a broader pH range of 3.0–9.0. We examined the feasibility of using steam pretreatment to increase the saccharification yields from various lignocellulosic residues for sugar release which can potentially be used in bioethanol production. Saccharification of pretreated dry potato peels, carrot peels, composite waste mixture, orange peels, onion peels, banana peels, pineapple peels by crude enzyme extract from A. niger NS-2, resulted in very high cellulose conversion efficiencies of 92–98 %.     

Thermophilic Bacillus coagulans Requires Less Cellulases for Simultaneous Saccharification and Fermentation of Cellulose to Products than Mesophilic Microbial Biocatalysts

Ethanol production from lignocellulosic biomass depends on simultaneous saccharification of cellulose to glucose by fungal cellulases and fermentation of glucose to ethanol by microbial biocatalysts (SSF). The cost of cellulase enzymes represents a significant challenge for the commercial conversion of lignocellulosic biomass into renewable chemicals such as ethanol and monomers for plastics. The cellulase concentration for optimum SSF of crystalline cellulose with fungal enzymes and a moderate thermophile, Bacillus coagulans, was determined to be about 7.5 FPU g^?1 cellulose. This is about three times lower than the amount of cellulase required for SSF with Saccharomyces cerevisiae, Zymomonas mobilis, or Lactococcus lactis subsp. lactis whose growth and fermentation temperature optimum is significantly lower than that of the fungal cellulase activity. In addition, B. coagulans also converted about 80% of the theoretical yield of products from 40 g/L of crystalline cellulose in about 48 h of SSF with 10 FPU g^?1 cellulose while yeast, during the same period, only produced about 50% of the highest yield produced at end of 7 days of SSF. These results show that a match in the temperature optima for cellulase activity and fermentation is essential for decreasing the cost of cellulase in cellulosic ethanol production.     

Production of l-asparaginase, an anticancer agent, from Aspergillus niger using agricultural waste in solid state fermentation

This article reports the production of high levels of l-asparaginase from a new isolate of Aspergillus niger in solid state fermentation (SSF) using agrowastes from three leguminous crops (bran of Cajanus cajan, Phaseolus mungo, and Glycine max). When used as the sole source for growth in SSF, bran of G. max showed maximum enzyme production followed by that of P. mungo and C. cajan. A 96-h fermentation time under aerobic condition with moisture content of 70%, 30 min of cooking time and 1205–1405 μ range of particle size in SSF appeared optimal for enzyme production. Enzyme yield was maximum (40.9±3.35 U/g of dry substrate) at pH 6.5 and temperature 30±2°C. The optimum temperature and pH for enzyme activity were 40°C and 6.5, respectively. The study suggests that choosing an appropriate substrate when coupled with process level optimization improves enzyme production markedly. Developing an asparaginase production process based on bran of G. max as a substrate in SSF is economically attractive as it is a cheap and readily available raw material in agriculture-based countries.     

High Cell Density Process for Constitutive Production of a Recombinant Phytase in Thermotolerant Methylotrophic Yeast <Emphasis Type="Italic">Ogataea thermomethanolica</Emphasis> Using Table Sugar as Carbon Source

The yeast Ogataea thermomethanolica has recently emerged as a potential host for heterologous protein expression at elevated temperature. To evaluate the feasibility of O. thermomethanolica as heterologous host in large-scale fermentation, constitutive production of fungal phytase was investigated in fed-batch fermentation. The effect of different temperatures, substrate feeding strategies, and carbon sources on phytase production was investigated. It was found that O. thermomethanolica can grow in the temperature up to 40 °C and optimal at 34 °C. However, the maximum phytase production was observed at 30 °C and slightly decreased at 34 °C. The DOT stat control was the most efficient feeding strategy to obtain high cell density and avoid by-product formation. The table sugar can be used as an alternative substrate for phytase production in O. thermomethanolica. The highest phytase activity (134 U/mL) was obtained from table sugar at 34 °C which was 20-fold higher than batch culture (5.7 U/mL). At a higher cultivation temperature of 38 °C, table sugar can be used as a low-cost substrate for the production of phytase which was expressed with an acceptable yield (85 U/mL). Lastly, the results from this study reveal the industrial favorable benefits of employing O. thermomethanolica as a host for heterologous protein production.     

Green Synthesized Iron Oxide Nanoparticles Effect on Fermentative Hydrogen Production by Clostridium acetobutylicum

A green synthesis of iron oxide nanoparticles (FeNPs) was developed using Murraya koenigii leaf extract as reducing and stabilizing agent. UV–vis spectra show that the absorption band centred at a wavelength of 277 nm which corresponds to the surface plasmon resonances of synthesized FeNPs. Fourier transform infrared spectroscopy spectrum exhibits that the characteristic band at 580 cm^?1 is assigned to Fe–O of γ-Fe₂O₃. Transmission electron microscopy image confirms that the spherical with irregular shaped aggregates and average size of nanoparticles was found to be ～59 nm. The effect of synthesized FeNPs on fermentative hydrogen production was evaluated from glucose by Clostridium acetobutylicum NCIM 2337. The hydrogen yield in control experiment was obtained as 1.74?±?0.08 mol H₂/mol glucose whereas the highest hydrogen yield in FeNPs supplemented experiment was achieved as 2.33?±?0.09 mol H₂/mol glucose at 175 mg/L of FeNPs. In addition, the hydrogen content and hydrogen production rate were also increased from 34?±?0.8 to 52?±?0.8 % and 23 to 25.3 mL/h, respectively. The effect of FeNPs was compared with supplementation of FeSO₄ on fermentative process. The supplementation of FeNPs enhanced the hydrogen production in comparison with control and FeSO₄. The supplementation of FeNPs led to the change of the metabolic pathway towards high hydrogen production due to the enhancement of ferredoxin activity. The fermentation type was shifted from butyrate to acetate/butyrate fermentation type at the addition of FeNPs.     

Optimization of Endoglucanase and Xylanase Activities from Fusarium verticillioides for Simultaneous Saccharification and Fermentation of Sugarcane Bagasse

Enzymatic hydrolysis is an important but expensive step in the production of ethanol from biomass. Thus, the production of efficient enzymatic cocktails is of great interest for this biotechnological application. The production of endoglucanase and xylanase activites from F. verticillioides were optimized in a factorial design (2⁵) followed by a CCDR design. Endoglucanase and xylanase activities increased from 2.8 to 8.0 U/mL and from 13.4 to 114 U/mL, respectively. The optimal pH and temperature were determined for endoglucanase (5.6, 80 °C), cellobiase (5.6, 60 °C), FPase (6.0, 55 °C) and xylanase (7.0, 50 °C). The optimized crude extract was applied in saccharification and fermentation of sugarcane bagasse from which 9.7 g/L of ethanol was produced at an ethanol/biomass yield of 0.19.     

Production, Purification, and Characterization of Exoglucanase by Aspergillus fumigatus

Fungi are considered good producers of industrially valuable enzymes with higher enzymatic activities. Among these cellulases are group of extracellular enzymes commonly employed in many industries for the hydrolysis of cellulolytic material. Aspergillus fumigatus produced exoglucanase having high enzymatic activity (83 U/gds) during the solid-state fermentation of wheat straw under optimum physical and nutritional conditions. Maximum production was obtained after 72 h of fermentation, at 55 °C temperature, pH 5.5, 80 % moisture level, and 2 mL fungal inoculum. Production was further increased by the addition of fructose (0.3 %) as additional carbon source, peptone (0.4 %) as nitrogen source, Tween-80 (0.3 %) as surfactant, and ammonium sulfate (0.2 %) in media. Exoglucanase was 2.30-folds purified by adding 40 % ammonium sulfate with volumetric activity 95.4 U/gds and specific activity 14.74 U/mg. Further, it was 5.18-folds purified by gel filtration chromatography with volumetric activity 115.2 U/gds and specific activity 33.10 U/mg. Purified exoglucanase has maximum activity at 55 °C and pH 4.8 using 1 % Avicel aqueous solution as substrate. The K _m and V _max were 4.34 mM and 7.29 μM/min, respectively. Calcium, magnesium, and zinc ions have positive effect on exoglucanase activity.     

Enhanced production of xylanase from locally isolated fungal strain using agro-industrial residues under solid-state fermentation

This study is related to the isolation of fungal strain for xylanase production using agro-industrial residues. Forty fungal strains with xylanolytic potential were isolated by using xylan agar plates and quantitatively screened in solid-state fermentation. Of all the tested isolates, the strain showing highest ability to produce xylanase was assigned the code Aspergillus niger LCBT-14. For the enhanced production of the enzyme, five different fermentation media were evaluated. Out of all media, M4 containing wheat bran gave maximum enzyme production. Effect of different variables including incubation time, temperature, pH, carbon and nitrogen sources has been investigated. The optimum enzyme production was obtained after 72 h at 30°C and pH 4. Glucose as a carbon source while ammonium sulphate and yeast extract as nitrogen sources gave maximum xylanase production (946 U/mL/min). This study was successful in producing xylanase by A. niger LCBT-14 economically by utilising cheap indigenous substrate.     

Application of Endo-β-1,4,d-mannanase and Cellulase for the Release of Mannooligosaccharides from Steam-Pretreated Spent Coffee Ground

Spent coffee ground (SCG), a present waste stream from instant coffee production, represents a potential feedstock for mannooligosaccharides (MOS) production. MOS can be used in nutraceutical products for humans/animals or added to instant coffee, increasing process yield and improving product health properties. The SCG was evaluated for MOS production by steam pretreatment and enzymatic hydrolysis with a recombinant mannanase and a commercial cellulase cocktail (Acremonium, Bioshigen Co. Ltd, Japan). The mannanase was produced using a recombinant strain of Yarrowia lipolytica, used to produce and secrete endo-1,4-β,d-mannanase from Aspergillus aculeatus in bioreactor cultures. Endo-1,4-β,d-mannanase was produced with an activity of 183.5 U/mL and 0.23 mg protein/mL. The enzyme had an optimum temperature of 80 °C, and the activity in the supernatant was improved by 150 % by supplementation with 0.2 % sodium benzoate and 35 % sorbitol as a preservative and stabiliser, respectively. The steam pretreatment of SCG improved the enzymatic digestibility of SCG, thus reducing the required enzyme dosage for MOS release. Combined enzymatic hydrolysis of untreated or steam-pretreated (150, 190 and 200 °C for 10 min) SCG with mannanase and cellulase cocktail resulted in 36–57 % (based on mannan content) of MOS production with a degree of polymerization of up to 6. The untreated material required at least 1 % of both mannanase and cellulase loading. The optimum mannanase and cellulase loadings for pretreated SCG hydrolysis were between 0.3 and 1 and 0.4 and 0.8 %, respectively. Statistical analysis suggested additive effect between cellulase cocktail and mannanase on MOS release, with no indication of synergism observed.     

Kinetic characterization of extracellular α-amylase from a derepressed mutant ofBacillus licheniformis

Three strains ofBacillus licheniformis were isolated and screened for α-amylase production by solid-state fermentation. Of these, IS-2 gave relatively higher enzyme production (32±2.3 U/[g·min]) and was selected for improvement after treatment withN-methylN-nitroN-nitroso guanidine (NG) or nitrous acid (NA) to enhance its hydrolytic potential. Among the mutant variants, NA-14 gave higher enzyme production (98±1.6 U/[g·min]), and hence, was selected for kinetic and thermal characterization. M1 as a moistening agent (pH 7.0, optimized) supported 2.65-fold improved amylolytic activity by the derepressed mutant 72 h after inoculation. The values of product yield coefficient (Y _p/x=1833.3 U/g) and specific rate constant (q _p=25.46 U/[g·h]) with starch were severalfold improved over those from other carbon sources and the other cultures. The purified enzyme from NA-14 was most active at 40°C; however, the activity remained almost constant up to 44°C. The NA-induced random mutagenesis substantially improved the enthalpy (ΔH _D=94.5±11 kJ/mol) and entropy of activation (ΔS=−284±22 J/[mol·K]) for α-amylase activity and substrate binding for starch hydrolysis. The results of this study (117.8±5.5 U/[g·min]) revealed a concomitant improvement in the endogenous metabolism of the mutant culture for α-amylase production.     

Production of Cellulase from Kraft Paper Mill Sludge by Trichoderma Reesei Rut C-30

Paper mill sludge is a solid waste material generated from pulping and papermaking operations. Because of high glucan content and its well-dispersed structure, paper mill sludges are well suited for bioconversion into value-added products. It also has high ash content originated from inorganic additives used in papermaking, which causes hindrance to bioconversion. In this study, paper mill sludges from Kraft process were de-ashed by a centrifugal cleaner and successive treatment by sulfuric acid and sodium hydroxide, and used as a substrate for cellulase production. The treated sludge was the only carbon source for cellulase production, and predominantly inorganic nutrients were used as the nitrogen source for this bioprocess. The cellulase enzyme produced from the de-ashed sludge exhibited cellulase activity of 8 filter paper unit (FPU)/mL, close to that obtainable from pure cellulosic substrates. The yield of cellulase enzyme was 307 FPU/g glucan of de-ashed sludge. Specific activity was 8.0 FPU/mg protein. In activity tests conducted against the corn stover and α-cellulose, the xylanse activity was found to be higher than that of a commercial cellulase. Relatively high xylan content in the sludge appears to have induced high xylanase production. Simultaneous saccharification and fermentation (SSF) was performed using partially de-ashed sludge as the feedstock for ethanol production using Sacharomyces cerevisiae and the cellulase produced in-house from the sludge. With 6% (w/v) glucan feed, ethanol yield of 72% of theoretical maximum and 24.4 g/L ethanol concentration were achieved. These results were identical to those of the SSF using commercial cellulases.     

Waste Biogas Residue from Cassava Dregs as Carbon Source to Produce Galactomyces sp. Cczu11-1 Cellulase and its Enzymatic Saccharification

In the conversion of cassava starch dregs to biogas by anaerobic fermentation, the biogas residue (BR) containing lignocellulosic materials still remained in the environment. In order to effectively utilize BR, the complexed 1-methyl-3-methylimidazolium dimethyl phosphate ([Mmim]DMP) media were used for pretreating cellulosic materials. After the optimization of pretreatment, the IL [Mmim]DMP-HCl-water (78.5:1.5:20, w/w/w) pretreament media were used for pretreating BR at 130 °C for 30 min. Furthermore, BR pretreated could be effectively saccharified by cellulase of Galactomyces sp. CCZU11-1. Moreover, BR could be used as a cheap carbon source for the production of Galactomyces sp. CCZU11-1 cellulase. After the culture optimization, the optimal culture conditions were obtained as follows: BR 5 g/L, (NH₄)₂SO₄ 5 g/L, K₂HPO₄ 2 g/L, MgSO₄ 0.2 g/L, NaCl 1 g/L, PEG6000 4 g/L, pH 5.5, and culture temperature 30 °C. After the fermentation for 6 days, the FPA and CMCase were 26.2 and 52.8 U/mL, respectively. In conclusion, waste BR could be chosen as a promising feedstock for biofuels.     

Biorefining of Waste Paper Biomass: Increasing the Concentration of Glucose by Optimising Enzymatic Hydrolysis

Waste copier paper is a potential substrate for the production of glucose relevant for manufacture of platform chemicals and intermediates, being composed of 51 % glucan. The yield and concentration of glucose arising from the enzymatic saccharification of solid ink-free copier paper as cellulosic substrate was studied using a range of commercial cellulase preparations. The results show that in all cellulase preparations examined, maximum hydrolysis was only achieved with the addition of beta-glucosidase, despite its presence in the enzyme mixtures. With the use of Accellerase® (cellulase), high substrate loading decreased conversion yield. However, this was overcome if the enzyme was added between 12.5 and 20 FPU g substrate^?1. Furthermore, this reaction condition facilitated continual stirring and enabled sequential additions (up to 50 % w/v) of paper to be made to the hydrolysis reaction, degrading nearly all (99 %) of the cellulose fibres and increasing the final concentration of glucose whilst simultaneously making high substrate concentrations achievable. Under optimal conditions (50 °C, pH 5.0, 72 h), digestions facilitate the production of glucose to much improved concentrations of up to 1.33 mol l^?1.     

Crude Cellulase from Oil Palm Empty Fruit Bunch by Trichoderma asperellum UPM1 and Aspergillus fumigatus UPM2 for Fermentable Sugars Production

Cellulase is an enzyme that converts the polymer structure of polysaccharides into fermentable sugars. The high market demand for this enzyme together with the variety of applications in the industry has brought the research on cellulase into focus. In this study, crude cellulase was produced from oil palm empty fruit bunch (OPEFB) pretreated with 2 % NaOH with autoclave, which was composed of 59.7 % cellulose, 21.6 % hemicellulose, and 12.3 % lignin using Trichoderma asperellum UPM1 and Aspergillus fumigatus UPM2. Approximately 0.8 U/ml of FPase, 24.7 U/ml of CMCase and 5.0 U/ml of β-glucosidase were produced by T. asperellum UPM1 at a temperature of 35 °C and at an initial pH of 7.0. A 1.7 U/ml of FPase, 24.2 U/ml of CMCase, and 1.1 U/ml of β-glucosidase were produced by A. fumigatus UPM2 at a temperature of 45 °C and at initial pH of 6.0. The crude cellulase was best produced at 1 % of substrate concentration for both T. asperellum UPM1 and A. fumigatus UPM2. The hydrolysis percentage of pretreated OPEFB using 5 % of crude cellulase concentration from T. asperellum UPM1 and A. fumigatus UPM2 were 3.33 % and 19.11 %, with the reducing sugars concentration of 1.47 and 8.63 g/l, respectively.     

Random mutagenesis and media optimisation for hyperproduction of cellulase from Bacillus species using proximally analysed Saccharum spontaneum in submerged fermentation

This study deals with the isolation of novel mutant of Bacillus and optimisation of media for the hyperproduction of cellulase. Cellulase-producing Bacillus PC-BC6 was subjected to physical and chemical mutagenesis to enhance the cellulolytic potential. Later, mutagenesis isolates were screened both qualitatively and quantitatively. Among all the tested isolates, Bacillus N3 yielded maximum (CMCase 1250 IU/mL/min and FPase 629 IU/mL/min) activity. The Bacillus N3 strain exhibited 1.7-fold more enzyme production as compared with the parental strain. Proximate analysis of untreated and pretreated Saccharum spontaneum was carried out to improve cellulase production. Three different media were tested for the production of cellulase, among which M2 medium containing MgSO₄, pretreated S. spontaneum, K₂HPO₄, (NH₄)₂SO₄ and peptone was found to be the best for maximum enzyme production by mutant Bacillus N3.     

Direct Fermentation of Gelatinized Cassava Starch to Acetone,Butanol, and Ethanol Using Clostridium acetobutylicum Mutant Obtained by Atmospheric and Room Temperature Plasma

The mutant strain designated as ART18, obtained from the wild-type strain Clostridium acetobutylicum PW12 treated by atmospheric and room temperature plasma, showed higher solvent tolerance and butanol production than that of the wild-type strain. The production of butanol was 11.3?±?0.5 g/L, 31 % higher than that of the wild-type strain when it was used for acetone, butanol, and ethanol fermentation in P2 medium. Furthermore, the effects of cassava flour concentration, pH regulators, and vitamins on the ABE production were also investigated. The highest butanol production of 15.8?±?0.8 g/L and butanol yield (0.31 g/g) were achieved after the above factors were optimized. When acetone, butanol, and ethanol fermentation by ART18 was carried out in a 15-L bioreactor, the butanol production, the productivity of butanol, and the total solvent were 16.3?±?0.9, 0.19, and 0.28 g/L^/h, respectively. These results indicate that ART18 is a promising industrial producer in ABE fermentation.     

Effect of Physical and Chemical Properties of Oil Palm Empty Fruit Bunch,Decanter Cake and Sago Pith Residue on Cellulases Production by Trichoderma asperellum UPM1 and Aspergillus fumigatus UPM2

The effect of cultivation condition of two locally isolated ascomycetes strains namely Trichoderma asperellum UPM1 and Aspergillus fumigatus UPM2 were compared in submerged and solid state fermentation. Physical evaluation on water absorption index, solubility index and chemical properties of lignin, hemicellulose and cellulose content as well as the cellulose structure on crystallinity and amorphous region of treated oil palm empty fruit bunch (OPEFB) (resulted in partial removal of lignin), sago pith residues (SPR) and oil palm decanter cake towards cellulases production were determined. Submerged fermentation shows significant cellulases production for both strains in all types of substrates. Crystallinity of cellulose and its chemical composition mainly holocellulose components was found to significantly affect the total cellulase synthesis in submerged fermentation as the higher crystallinity index, and holocellulose composition will increase cellulase production. Treated OPEFB apparently induced the total cellulases from T. asperellum UPM1 and A. fumigatus UPM2 with 0.66 U/mg FPase, 53.79 U/mg CMCase, 0.92 U/mg β-glucosidase and 0.67 U/mg FPase, 47.56 U/mg and 0.14 U/mg β-glucosidase, respectively. Physical properties of water absorption and solubility for OPEFB and SPR also had shown significant correlation on the cellulases production.