Effect of Urea on the Enzymatic Hydrolysis of Lignocellulosic Substrate and Its Mechanism

Effect of hydrogen bond breaker (urea) addition on the enzymatic hydrolysis of Avicel and eucalyptus pretreated by dilute acid (Eu-DA) was investigated. Urea enhanced the enzymatic hydrolysis of Eu-DA at 50 or 30 °C when the concentration of urea was below 60 g/L, while it inhibited the hydrolysis of Avicel. Low concentration urea (<?240 g/L) had little effect on the cellulase spatial structure and its activity. But it decreased cellulase binding to cellulose surface to inhibit the cellulose hydrolysis. Meanwhile, urea obviously prevented the adsorption of cellobiohydrolase I (CBHI) on the lignin in spite of little effect on the adsorption of β-glucosidase (BGL) and two endoglucanases (EGIII and EGV) on lignin. It was proposed that urea enhanced the enzymatic efficiency of Eu-DA by decreasing the cellulase adsorption on lignin surface.     

Enhanced saccharification of alkali-treated rice straw by cellulase from Trametes hirsuta and statistical optimization of hydrolysis conditions by RSM

A white rot fungus, identified as Trametes hirsuta based on morphological and phylogenetic analysis, was found to contain efficient cellulose degrading enzymes. The strain showed maximum endoglucanase (EG), cellobiohydrolase (CBH) and ß-glucosidase (BGL) activities of 55, 0.28 and 5.0 U/mg-protein, respectively. Rice straw was found to be a potentially good substrate for growth of T. hirsuta for cellulase production. Statistical experimental design was used to optimize hydrolysis parameters such as pH, temperature, and concentrations of substrates and enzymes to achieve the highest saccharification yield. Enzyme concentration was identified as the limiting factor for saccharification of rice straw. A maximum saccharification rate of 88% was obtained at an enzyme concentration of 37.5 FPU/g-substrate after optimization of the hydrolysis parameters. The results of a confirmation experiment under the optimum conditions agreed well with model predictions. T. hirsuta may be a good choice for the production of reducing sugars from cellulosic biomass.     

Enhanced cellulase production by <Emphasis Type="Italic">Trichoderma harzianum</Emphasis> by cultivation on glycerol followed by induction on cellulosic substrates

The use of glycerol obtained as an intermediate of the biodiesel manufacturing process as carbon source for microbial growth is a potential alternative strategy for the production of enzymes and other high-value bioproducts. This work evaluates the production of cellulase enzymes using glycerol for high cell density growth of Trichoderma harzianum followed by induction with a cellulosic material. Firstly, the influence of the carbon source used in the pre-culture step was investigated in terms of total protein secretion and fungal morphology. Enzymatic productivity was then determined for cultivation strategies using different types and concentrations of carbon source, as well as different feeding procedures (batch and fed-batch). The best strategy for cellulase production was then further studied on a larger scale using a stirred tank bioreactor. The proposed strategy for cellulase production, using glycerol to achieve high cell density growth followed by induction with pretreated sugarcane bagasse, achieved enzymatic activities up to 2.27 ± 0.37 FPU/mL, 106.40 ± 8.87 IU/mL, and 9.04 ± 0.39 IU/mL of cellulase, xylanase, and β-glucosidase, respectively. These values were 2 times higher when compared to the control experiments using glucose instead of glycerol. This novel strategy proved to be a promising approach for improving cellulolytic enzymes production, and could potentially contribute to adding value to biomass within the biofuels sector.     

Enhanced cellulolytic enzyme production by the synergism between Trichoderma reesei RUT-C30 and Aspergillus niger NL02 and by the addition of surfactants

Two improvement approaches comprising of a mixed culture of Trichoderma reesei and Aspergillus niger and the addition of surfactants were employed in this study in order to enhance cellulolytic enzyme production as well as to improve the composition. Different delay times of A. niger inoculation (0, 24, and 48 h) and inoculum ratios of T. reesei versus A. niger (1:1 and 5:1) derived six mixed culture forms, which were 0 h/1:1, 0 h/5:1, 24 h/1:1, 24 h/5:1, 48 h/1:1, and 48 h/5:1. It was found that the form 48 h/5:1 allowed the highest FPA, 3.30 ± 0.34 IU/mL, and a relatively high BGA, 1.01 ± 0.25 IU/mL, thereafter being selected for the subsequent improvement step addition of surfactants. Among the three surfactants, including Triton X-100, CHAPS and sodium taurocholate, the third one was found to be the best one giving rise to the highest FPA and BGA, 5.02 ± 0.40 and 1.48 ± 0.28 IU/mL, respectively. Differently sourced cellulases were compared in the enzymatic hydrolysis of steam-exploded corn stover (SECS). Moreover, the cellulase produced by the mixed culture form 48 h/5:1 using SECS as a substrate showed the highest yield at 80.93 ± 2.04%, indicating that the composition of this cellulase was improved by the mixed culture of T. reesei and A. niger. The results validate that these two improvement approaches are efficient and applicable in cellulase production.     

The diversity of glycosylation of cellobiohydrolase I from Trichoderma reesei determined with mass spectrometry

Cellulases are glycosylated enzymes that have wide applications in fields like biofuels. It has been widely accepted that glycosylation of cellulases impact their performance. Trichoderma reesei is the most important cellulase-producer and cellobiohydrolase I (CBHI) is the most important cellulase from T. reesei. Therefore, the glycosylation of T. reesei CBHI has been a focus of research. However, investigations have been focused on N-glycosylation of three of the four potential glycosylation sites, as well as O-glycosylation on the linker region, while a full picture of glycosylation of T. reesei CBHI is still needed. In this work, with extensive mass spectrometric investigations on CBHI from two T. reesei strains grown under three conditions, several new discoveries were made: 1) N45 and N64 are N-glycosylated with high mannose type glycans; 2) the catalytic domain of CBHI is extensively O-glycosylated with hexoses and N-acetylhexosamines; 3) experimental evidence on the mannosylation of carbohydrate binding domain (other than the linker adjacent region) was found. With structural analysis, we found several glycosylation sites (such as T383, S8, and S46) are located at the openings of the substrate-binding tunnel, and potentially involve in the binding of cellulose. These investigations provide a full and comprehensive picture on the glycosylation of CBHI from T. reesei, which benefits the engineering of CBHI by raising potential sites for modification.     

Study of a High-Yield Cellulase System Created by Heavy-Ion Irradiation-Induced Mutagenesis of Aspergillus niger and Mixed Fermentation with Trichoderma reesei

The aim of this study was to evaluate and validate the efficiency of ¹²C⁶⁺ irradiation of Aspergillus niger (A. niger) or mutagenesis via mixed Trichoderma viride (T. viride) culturing as well as a liquid cultivation method for cellulase production via mixed Trichoderma reesei (T. reesei) and A. niger culture fermentation. The first mutagenesis approach was employed to optimize yield from a cellulase-producing strain via heavy-ion mutagenesis and high-throughput screening, and the second was to effectively achieve enzymatic hydrolysis of cellulase from a mixed culture of mutant T. viride and A. niger. We found that ¹²C⁶⁺-ion irradiation induced changes in cellulase biosynthesis in A. niger but had no effect on the time course of the synthesis. It is notable that the exoglucanases (CBH) activities of A. niger strains H11-1 and H differed (6.71 U/mL vs. 6.01 U/mL) and were significantly higher than that of A. niger mutant H3-1. Compared with strain H, the filter paper assay (FPA), endoglucanase (EG) and β-glucosidase (BGL) activities of mutant strain H11-1 were increased by 250.26%, 30.26% and 34.91%, respectively. A mixed culture system was successfully optimized, and the best ratio of T. reesei to A. niger was 5:1 for 96 h with simultaneous inoculation. The BGL activity of the mixed culture increased after 72 h. At 96 h, the FPA and BGL activities of the mixed culture were 689.00 and 797.15 U/mL, respectively, significantly higher than those of monocultures, which were 408.70 and 646.98 U/mL for T. reesei and 447.29 and 658.89 U/mL for A. niger, respectively. The EG activity of the mixed culture was 2342.81 U/mL, a value that was significantly higher than that of monocultures at 2206.57 U/mL for T. reesei and 1727.62 U/mL for A. niger. In summary, cellulose production and hydrolysis yields were significantly enhanced by the proposed combination scheme.     

Fusion of cellulose binding domain from Trichoderma reesei CBHI to Cryptococcus sp. S-2 cellulase enhances its binding affinity and its cellulolytic activity to insoluble cellulosic substrates

Cryptococcus sp. S-2 carboxymethyl cellulase (CSCMCase) is active in the acidic pH and lacks a binding domain. The absence of the binding domain makes the enzyme inefficient against insoluble cellulosic substrates. To enhance its binding affinity and its cellulolytic activity to insoluble cellulosic substrates, cellulose binding domain (CBD) of cellobiohydrolase I (CBHI) from Trichoderma reesei belonging to carbohydrate binding module (CBM) family 1 was fused at the C-terminus of CSCMCase. The constructed fusion enzymes (CSCMCase-CBD and CSCMCase-2CBD) were expressed in a newly recombinant expression system of Cryptococcus sp. S-2, purified to homogeneity, and then subject to detailed characterization. The recombinant fusion enzymes displayed optimal pH similar to those of the native enzyme. Compared with rCSCMCase, the recombinant fusion enzymes had acquired an increased binding affinity to insoluble cellulose and the cellulolytic activity toward insoluble cellulosic substrates (SIGMACELL^® and Avicel) was higher than that of native enzyme, confirming the presence of CBDs improve the binding and the cellulolytic activity of CSCMCase on insoluble substrates. This attribute should make CSCMCase an attractive applicant for various application.     

High frequency one-step gene replacement in Trichoderma reesei. I. Endoglucanase I overproduction

The chromosomal cellobiohydrolase 1 locus (cbh1) of the biotechnologically important filamentous fungus Trichoderma reesei was replaced in a single-step procedure by an expression cassette containing an endoglucanase I cDNA (egl1) under control of the cbh1 promoter. CBHI protein was missing from 37–63% of the transformants, showing that targeting of the linear expression cassette to the cbh1 locus was efficient. Studies of expression of the intact cbh1-egl1 cassette at the cbh1 locus revealed that egl1 cDNA is expressed from the cbh1 promoter as efficiently as cbh1 itself. Furthermore, a strain carrying two copies of the cbh1-egl1 expression cassette produced twice as much EG I as the amount of CBHI, the major cellulase protein, produced by the host strain. The level of egl1-specific mRNA in the single-copy transformant was about 10-fold higher than that found in the non transformed host strain, indicating that the cbh1 promoter is about 10 times stronger than the egl1 promoter. The 10-fold increase in the secreted EG I protein, measured with an enzyme-linked immunosorbent assay (ELISA), correlated well with the increase in egl1-specific mRNA.     

Enzyme production by recombinant Trichoderma reesei strains.

The production of both homologous and heterologous proteins with the cellulolytic filamentous fungus Trichoderma reesei is described. Biotechnically important improvements in the production of cellulolytic enzymes have been obtained by genetic engineering methodology to construct strains secreting novel mixtures of cellulases. These improvements have been achieved by gene inactivation and promoter changes. The strong and highly inducible promoter of the gene encoding the major cellulase, cellobiohydrolase I (CBHI) has also been used for the production of eukaryotic heterologous proteins in Trichoderma. The expression and secretion of active calf chymosin is described in detail.     

Cellulolytic enzymes on lignocellulosic substrates in solid state fermentation by <Emphasis Type="Italic">Aspergillus niger</Emphasis>

The production of cellulolytic enzymes by Aspergillus niger on lignocellulosic substrates groundnut fodder, wheat bran, rice bran and sawdust in solid state fermentation in a laboratory scale was compared. Czapek Dox liquid broth amended with cellulose (0.5%) was used to moisten lignocellulosic solid supports for cultivation of Aspergillus niger. The production of filter paperase, carboxymethyl cellulase and -glucosidase were monitored at daily intervals for 5 days. The peak production of the enzymes occurred within 3 days of incubation. Among solid supports used in the study, wheat bran was the best solid matrix followed by groundnut fodder in production of cellulolytic enzymes in solid state fermentation. Groundnut fodder supported significant production of FPase (2.09 FPU/g), CMCase (1.36 U/g) and -glucosidase activity (0.0117 U/g) in solid state fermentation. Considerable secretion of protein (5.10 mg/g) on groundnut fodder at peak time interval 1st day of incubation was recorded.     

Cellulase On-Site Production from Sugar Cane Bagasse Using Penicillium echinulatum

Penicillium echinulatum was evaluated as a cellulolytic enzyme producer in shaking flasks and bioreactor submerged culture using sugarcane bagasse as carbon source. Sodium hydroxide delignified steam-exploded pretreated bagasse (SDB) and hydrothermal pretreated bagasse had a maximum filter paper activity (FPase) of 2.4 and 2.6 FPU/mL, respectively. Delignified acid pretreated bagasse and Celufloc 200TM (CE) carbon sources displayed maximum FPase of 1.3 and 1.6 FPU/mL while in natura bagasse (INB) provided the lowest enzyme activity, ca. 0.4 FPU/mL. Measurement of surface specific area of lignocellulosic material and scanning electron microscopic images showed a possible correlation between fungal mycelia accessibility to lignocellulosic particles and obtained cellulolytic enzyme activity of fermentation broth. Fed-batch experiments performed in a controlled bioreactor attained the highest value of FPase of 3.7 FPU/mL, enzyme productivity of 25.7 FPU/L h, and enzyme yield from cellulose equal to 134 FPU/g with SDB. Enzyme hydrolysis of steam-pretreated bagasse accomplished with the obtained supernatant of fermentation broth (10 FPU/g of biomass and 5 % w/v) performed better than commercial cellulose complex. The results showed that P. echinulatum has potential to be used as an on-site enzyme platform aiming second bioethanol production from sugarcane lignocellulosic residue.     

Heterologous expression of Neurospora crassa cbh1 gene in Pichia pastoris resulted in production of a neutral cellobiohydrolase I

The high production cost of cellulase is one of the limitations that hinder the commercialization of lignocellulose-based biorefineries. As one of the important cellulases, Neurospora crassa cellulase is not so intensively investigated as T. reesei cellulase. In this study, the cbh1gene (NCU07340) cloned from N. crassa was expressed in Pichia pastoris under the control of alcohol oxidase 1 (AOX1) promotor. Six transformants with the highest resistance to G418 were selected by two rounds of transformant screening, among which the most robust producer of the recombinant cellobiohydrolase I (CBHI) has an Avicelase activity of 0.22 U/mL. After fermentation optimization, it was improved to 0.30 U/mL. Interestingly, the optimal temperature and pH of the recombinant CBHI were 60°C and 7.2, respectively, and it was quite stable within the wide ranges of temperature and pH. This work is a good example for the future improvement and optimization of N. crassa cellulase.     

Direct production of 4-hydroxybenzoic acid from cellulose using cellulase-displaying Pichia pastoris

4-hydroxybenzoic acid (4-HBA) is an industrially important aromatic compound, and there is an urgent need to establish a bioprocess to produce this compound in a sustainable and environmentally friendly manner from renewable feedstocks such as cellulosic biomass. Here, we developed a bioprocess to directly produce 4-HBA from cellulose using a recombinant Pichia pastoris strain that displays heterologous cellulolytic enzymes on its cell surface via the glycosylphosphatidylinositol (GPI)-anchoring system. β-glucosidase (BGL) from Aspergillus aculeatus, endoglucanase (EG) from Trichoderma reesei, and cellobiohydrolase (CBH) from Talaromyces emersonii were co-displayed on the cell surface of P. pastoris using an appropriate GPI-anchoring domain for each enzyme. The cell-surface cellulase activity was further enhanced using P. pastoris SPI1 promoter- and secretion signal sequences. The resulting strains efficiently hydrolyzed phosphoric acid swollen cellulose (PASC) to glucose. Then, we expressed a highly 4-HBA-resistant chorismate pyruvate-lyase (UbiC) from Providencia rustigianii in the cellulase-displaying strain. This strain produced 975 mg/L of 4-HBA from PASC, which corresponding to 36.8% of the theoretical maximum yield, after 96 h of batch fermentation without the addition of commercial cellulase. This 4-HBA yield was over two times higher than that obtained from glucose (12.3% of the theoretical maximum yield). To our knowledge, this is the first report on the direct production of an aromatic compound from cellulose using cellulase-displaying yeast.     

Ethanol production from sunflower meal biomass by simultaneous saccharification and fermentation (SSF) with Kluyveromyces marxianus ATCC 36907

The lignocellulosic materials are considered promising renewable resources for ethanol production, but improvements in the processes should be studied to reduce operating costs. Thus, the appropriate enzyme loading for cellulose saccharification is critical for process economics. This study aimed at evaluating the concentration of cellulase and β-glucosidase in the production of bioethanol by simultaneous saccharification and fermentation (SSF) of sunflower meal biomass. The sunflower biomass was pretreated with 6 % H₂SO₄ (w/v), at 121 °C, for 20 min, for hemicellulose removal and delignificated with 1 % NaOH. SSF was performed with Kluyveromyces marxianus ATCC 36907, at 38 °C, 150 rpm, for 72 h, with different enzyme concentrations (Cellulase Complex NS22086-10, 15 and 20 FPU/g_substrate and β-Glucosidase NS22118, with a cellulase to β-glucosidase ratio of 1.5:1; 2:1 and 3:1). The best condition for ethanol production was cellulase 20 FPU/g_substrate and β-glucosidase 13.3 CBU/g_substrate, resulting in 27.88 g/L ethanol, yield of 0.47 g/g and productivity of 0.38 g/L h. Under this condition the highest enzymatic conversion of cellulose to glucose was attained (87.06 %).     

Impact of protein blocking on enzymatic saccharification of bagasse from sugarcane clones

Lignin plays an important functional and structural role in plants, but also contributes to the recalcitrance of lignocellulosic biomass to hydrolysis. This study addresses the influence of lignin in hydrolysis of sugarcane bagasse from conventional bred lines (UFV260 and UFV204) that were selected from 432 field-grown clones. In addition to higher sugar production, bagasse clone UFV204 had a small, but statistically significant, lower insoluble lignin content compared with clone UFV260 (15.5% vs, 16.6%) and also exhibited a significantly higher cellulose conversion to glucose (81.3% vs. 63.3%) at a cellulase loading of 5 (filter paper unit) FPU/g of glucan or 3 FPU/g total solids for liquid hot water pretreated bagasse (200°C, 10 min). The enzyme loading was further decreased by 50% to 2.5 FPU/g glucan and resulted in a similar glucan conversion (88.5%) for clone UFV204 when the bagasse was preincubated with bovine serum albumin at pH 4.8 and nonproductive binding of cellulase components was blocked. Comparison of Langmuir adsorption isotherms and differential adsorption of the three major cellulolytic enzyme components endoglucanase, cellobiohydrolase, and β-glucosidase help to explain differences due to lignin content.     

Development of a cellulolytic Saccharomyces cerevisiae strain with enhanced cellobiohydrolase activity

Consolidated bioprocessing (CBP) is a promising technology for lignocellulosic ethanol production, and the key is the engineering of a microorganism that can efficiently utilize cellulose. Development of Saccharomyces cerevisiae for CBP requires high level expression of cellulases, particularly cellobiohydrolases (CBH). In this study, to construct a CBP-enabling yeast with enhanced CBH activity, three cassettes containing constitutively expressed CBH-encoding genes (cbh1 from Aspergillus aculeatus, cbh1 and cbh2 from Trichoderma reesei) were constructed. T. reesei eg2, A. aculeatus bgl1, and the three CBH-encoding genes were then sequentially integrated into the S. cerevisiae W303-1A chromosome via δ-sequence-mediated integration. The resultant strains W1, W2, and W3, expressing uni-, bi-, and trifunctional cellulases, respectively, exhibited corresponding cellulase activities. Furthermore, both the activities and glucose producing activity ascended. The growth test on cellulose containing plates indicated that CBH was a necessary component for successful utilization of crystalline cellulose. The three recombinant strains and the control strains W303-1A and AADY were evaluated in acid- and alkali-pretreated corncob containing media with 5 FPU exogenous cellulase/g biomass loading. The highest ethanol titer (g/l) within 7 days was 5.92 ± 0.51, 18.60 ± 0.81, 28.20 ± 0.84, 1.40 ± 0.12, and 2.12 ± 0.35, respectively. Compared with the control strains, W3 efficiently fermented pretreated corncob to ethanol. To our knowledge, this is the first study aimed at creating cellulolytic yeast with enhanced CBH activity by integrating three types of CBH-encoding gene with a strong constitutive promoter Ptpi.     

Enhancement of <Emphasis Type="Italic">Penicillium echinulatum</Emphasis> glycoside hydrolase enzyme complex

The enhancement of enzyme complex produced by Penicillium echinulatum grown in several culture media components (bagasse sugarcane pretreated by various methods, soybean meal, wheat bran, sucrose, and yeast extract) was studied to increment FPase, xylanase, pectinase, and β-glucosidase enzyme activities. The present results indicated that culture media composed with 10 g/L of the various bagasse pretreatment methods did not have any substantial influence with respect to the FPase, xylanase, and β-glucosidase attained maximum values of, respectively, 2.68 FPU/mL, 2.04, and 115.4 IU/mL. On the other hand, proposed culture media to enhance β-glucosidase production composed of 10 g/L steam-exploded bagasse supplemented with soybean flour 5.0 g/L, yeast extract 1.0 g/L, and sucrose 10.0 g/L attained, respectively, 3.19 FPU/mL and 3.06 IU/mL while xylanase was maintained at the same level. The proteomes obtained from the optimized culture media for enhanced FPase, xylanase, pectinase, and β-glucosidase production were analyzed using mass spectrometry and a panel of GH enzyme activities against 16 different substrates. Culture medium designed to enhance β-glucosidase activity achieved higher enzymatic activities values (13 measured activities), compared to the culture media for FPase/pectinase (9 measured activities) and xylanase (7 measured activities), when tested against the 16 substrates. Mass spectrometry analyses of secretome showed a consistent result and the greatest number of spectral counts of Cazy family enzymes was found in designed β-glucosidase culture medium, followed by FPase/pectinase and xylanase. Most of the Cazy identified protein was cellobiohydrolase (GH6 and GH7), endoglucanase (GH5), and endo-1,4-β-xylanase (GH10). Enzymatic hydrolysis of hydrothermally pretreated sugarcane bagasse performed with β-glucosidase enhanced cocktail achieved 51.4 % glucose yield with 10 % w/v insoluble solids at enzyme load of 15 FPU/g material. Collectively the results demonstrated that it was possible to rationally modulate the GH activity of the enzymatic complex secreted by P. echinulatum using adjustment of the culture medium composition. The proposed strategy may contribute to increase enzymatic hydrolysis of lignocellulosic materials.     

Characterization of commercial cellulases and their use in the saccharification of a sugarcane bagasse sample pretreated with dilute sulfuric acid

This study aimed to correlate the efficiency of enzymatic hydrolysis of the cellulose contained in a sugarcane bagasse sample pretreated with dilute H₂SO₄ with the levels of independent variables such as initial content of solids and loadings of enzymes and surfactant (Tween 20), for two cellulolytic commercial preparations. The preparations, designated cellulase I and cellulase II, were characterized regarding the activities of total cellulases, endoglucanase, cellobiohydrolase, cellobiase, β-glucosidase, xylanase, and phenoloxidases (laccase, manganese and lignin peroxidases), as well as protein contents. Both extracts showed complete cellulolytic complexes and considerable activities of xylanases, without activities of phenoloxidases. For the enzymatic hydrolyses, two 2³ central composite full factorial designs were employed to evaluate the effects caused by the initial content of solids (1.19–4.81%, w/w) and loadings of enzymes (1.9–38.1 FPU/g bagasse) and Tween 20 (0.0–0.1 g/g bagasse) on the cellulose digestibility. Within 24 h of enzymatic hydrolysis, all three independent variables influenced the conversion of cellulose by cellulase I. Using cellulase II, only enzyme and surfactant loadings showed significant effects on cellulose conversion. An additional experiment demonstrated the possibility of increasing the initial content of solids to values much higher than 4.81% (w/w) without compromising the efficiency of cellulose conversion, consequently improving the glucose concentration in the hydrolysate.     

Production and partial purification of cellulase from a novel fungus,Aspergillus flavus BS1

This study describes the isolation and characterization of a novel fungus, Aspergillus flavus BS1 and its cellulolytic activities with special emphasis on endoglucanase production. Preliminary screening studies showed that A. flavus BS1 was a potent strain for the production of cellulase. To study the cellulolytic activities in detail by submerged fermentation (SmF), productions of endoglucanase, exoglucanase, and β-glucosidase were estimated from the basal salt medium (BSM) supplemented with 1 % carboxy methyl cellulose (CMC). CMC medium supported the maximum yield of endoglucanase (2,793 U/ml) on day 5 of incubation at 28 °C and 150 rpm, which was higher than that obtained with naturally available supplements (flour) from banana, tapioca, potato, or banana peel. During cellulase production by solid-state fermentation, 10 % (w/w) tapioca flour in sawdust (teak wood) moisturized with BSM (1:2, w/v) supported maximum cellulase yield (5,408 U/g dry substrate) on day 3 at 28 °C, which was 2-fold higher than that obtained during SmF. The active cellulase was qualitatively estimated by polyacrylamide gel electrophoresis (PAGE). Native-PAGE (0.25 % CMC impregnated on the 10 % gel) activity staining with congo-red showed a clear zone for CMCase activity, whereas SDS-PAGE showed a distinct band. In conclusion, this study showed that A. flavus strain BS1 is a potent strain for the production of cellulase on lignocellulosic media, the hot enzyme for bioethanol production from the lignocellulosic biomass by SSF.