Submerged Fermentation Research Articles

English

Tannase is an inducible extracellular enzyme produced by filamentous fungi, yeasts, and bacteria by solid-state fermentation (SSF) or submerged fermentation (SmF). Among the filamentous fungi, Aspergillus and Penicillium are recognized as the most efficient producers of tannase. The aims of this study were to evaluate the production of tannase under SSF by isolation from Aspergillus and Penicillium preserved in the Collection of Cultures Micoteca - URM (WDCM604); select the best enzyme producer, and use the crude extract to clarify mangaba and tamarind juices. The optimal conditions were determined by using the Placket-Burman Planning (PB) and response surface methodology (RSM). All tested crops produced activity between 2088.19 and 238.93 U/gds, and Aspergillus carneus URM5577 was the best producer. Through MSR, the best parameters for producing tannase were found to be 70 h of cultivation at pH 6.0, 7% tannic acid at 28°C and, as the response variable, 5449.31 activity U/gds. The optimum purification conditions were the molecular weight of PEG 8000 (g/mol), concentration of PEG 15% (w/w), 25% citrate (w/w), and pH 8.0. Its application in mangaba juice reduced the tannin content by 49.66% after 90 min and in tamarind by 51.82% at 120 min incubation at 37°C. Key words: Tannase, agroforestry waste, Aspergillus, Penicillium.

Extracellular Xylanopectinolytic Enzymes by Bacillus subtilis ADI1 from EFB's Compost.

Microbial xylanase and pectinase are two extremely valuable enzymes, which have captivated much attention. This can be seen from the increased demand for these enzymes by many industrial sectors. This study investigates the isolation and screening of extracellular xylanopectinolytic enzymes-producing bacteria in a submerged fermentation (SmF). Samples are collected from the compost of empty fruit bunch (EFB) at Biocompost Pilot Plant, located at Biorefinery Plant, Universiti Putra Malaysia. From the experiment, out of 20 isolates, 11 isolates show xylanase or/and pectinase activity, and only one isolate (EFB-11) shows the concurrent activities of xylanase and pectinase. These activities are selected for enzyme production under submerged fermentation (quantitative screening). At the 72nd hour of incubation, xylanase and pectinase show the highest production, which ranges about 42.33 U/mL and 62.17 U/mL (with low amount of cellulase present), supplemented with 2% (w/v) of rice bran as carbon source at incubation temperature level, which is 30°C. Meanwhile, the pH of media is shifted to 8.42, which indicates that EFB-11 isolate is alkalotolerant bacteria and identified as Bacillus subtilis ADI1. This strain proves to have potential in agroindustrial bioconversion and has a promising ability to scale up to an industrial scale.

Ganoderma lucidum repress injury of ethanol-induced steatohepatitis via anti-inflammation, anti-oxidation and reducing hepatic lipid in C57BL/6J mice

Ganoderma lucidum has been well recognized as medical herb, which is effective on antitumor, inhibition of lipid peroxidation and downregulation blood-pressure. The aquatic extract of Ganoderma lucidum has many bioactive substances, such as polysaccharides, triterpenes and polysaccharide-peptide complex. However, these curative effects and effective ingredients are fewer known in ethanol-induced fatty liver. In this study, Ganoderma Submerged Fermentation (GSF), the commercial Ganoderma products were investigated treatment in prevention of alcohol-induced liver injury in mice. GLP showed potent antioxidant activities and declined lipid accumulation in ethanol-induced mice, such as liver injury biomarkers (AST, ALT and ALP), triglyceride (TG) and cholesterol (TC) in plasma and hepatic content. Moreover, treatment of GSF calmed the inflammation element expression, including iNOS, COX2, TNF-α NF-κB and IL-6. On the basis of these results, GSF from Ganoderma lucidum possesses potential on anti-oxidant, anti-inflammation, and reduced lipid accumulation in alcoholic liver disease.

Execution of Enriched Rice Bran Medium in Hyper Production of Penicillin V by Penicillium chrysogenum

The antibiotic penicillin is being a major pharmaceutical compound after several years of use. The present research work focuses on the selection of suitable low cost substrate from rice based residues like grains, husk and bran for the seed culturing of Penicillium chrysogenum by solid state fermentation (SSF) and optimization of medium for hyper production of penicillin V (Pen V) by P. chrysogenum under submerged fermentation (SmF). In SSF, significantly highest number of total spores and viable spores were augmented in rice bran media than the rest. The effect of different carbon, nitrogen sources, pH, temperature, incubation time and various concentrations of precursor amino acid (l-valine) were studied for hyper production of Pen V by P. chrysogenum. The results revealed that in SmF, the enriched rice bran medium has had increased the production yield of Pen V at a highest level of 22.1 g/L than the synthetic production medium 19.8 g/L. Hence, the Pen V produced from the enriched low cost rice based substrate is being an alternate for next generation production.

Optimisation of Medium Composition and Culture Conditions of Filter-paperase by Upmc1106 Using Pennisetum Purpureum

Lignocellulosic resources are valuable and widely utilised to produce fermentable sugar. There is potential non-human consumption lignocellulosic material such as Pennisetum sp. available in the tropical and subtropical region. This study was conducted to optimise the medium composition and culture conditions of submerge fermentation of locally isolate UPMC1106, using Pennisetum sp. as the substrate in shake flasks. The Pennisetum sp. or Pennisetum purpureum sp. was collected from Pahang, Malaysia (3.793619° N, 102.523449° E) at the age of three months, processed until milled using Thomas Wiley mill and passed through a 60-mesh sieve. The powder form Pennisetum sp. was used as the sole carbon source in the cultivation medium. Four factors were chosen for the optimisation, as these factors contributing to the cellulosic enzymes production. The factors were Pennisetum sp. concentration, initial medium pH, agitation speed, and inoculum size. For the maximum filter-paperase activity, FPase, (U/mL), the Napier concentration was found to be 4.3 % (w/v), and the initial medium pH was 7.8. The optimum agitation speed and inoculum size were achieved at 170 rpm and 19.7 % (v/v). The optimum values of the respective factors gave 2.895 U/mL of FPase activity. This response was 98.8 % close to the predicted value and 30 % of improvement from the non-optimised factors.

Optimization of bioprocess variables for production of a thermostable and wide range pH stable carboxymethyl cellulase from Bacillus subtilis MS 54 under solid state fermentation

Vast application spectrum of cellulases especially for bioconversion of lignocellulosics into simple sugars for production of biofuel, chemicals, and other industrial products in an environmentally benign manner, faces bottleneck due to unavailability of process‐apt cellulases, and high production cost of enzymes. Solid state fermentation (SSF) offers several advantages over submerged fermentation (SmF) including the cost‐effective production of enzymes particularly using agricultural residues as substrates. Bacillus subtilis MS 54, a recently isolated bacterium was previously shown to produce thermostable and wide range pH stable cellulase under SmF. This study reports the Design of experiments (DoE) based optimization of process variables for cellulase production from B. subtilis MS 54 under SSF. Plackett–Burman (PB) design and response surface methodology (RSM) were used, respectively, for selection of significant process variables, and for optimization of the selected variables. Three process variables, that is, moisture content, maize bran and almond shell, earmarked based on PB‐design, were optimized by RSM to achieve cellulase yield enhancement by 2.02‐fold as compared with that under unoptimized conditions. Application of maize bran and almond shells as substrates for bacterial cellulase production under SSF is being reported for the first time. Solid‐state fermentation coupled with DoE tools represents a cost‐effective strategy for production of industrial enzymes. © 2017 American Institute of Chemical Engineers Environ Prog, 36: 1123–1130, 2017

Gibberellic Acid Production by Different Fermentation Systems Using Citric Pulp as Substrate/Support.

Gibberellic acid (GA3) is an important phytohormone, a member of gibberellins family, which acts as a promoter and regulator of plant growth. This study aimed to evaluate GA3 production by Fusarium moniliforme LPB03 and Gibberella fujikuroi LPB06 using different techniques of fermentation, solid state fermentation (SSF), submerged fermentation (SmF), and semisolid state fermentation (SSSF), and different types of bioreactors. In all techniques, citric pulp (CP), a subproduct obtained from the extraction of orange juice, was employed as the substrate/support. GA3 production by SSF reached 7.60 g kg−1 and 7.34 g kg−1 in Erlenmeyer flasks and column bioreactors, respectively. For SmF, the highest concentration of GA3 obtained was 236.00 mg L−1 in Erlenmeyer flasks, 273.00 mg L−1 in a 10 L stirred tank reactor (STR), and 203.00 mg L−1 in a 1.5 L bubble column reactor (BCR). SSSF was conducted with a CP suspension. In this case, GA3 concentration reached 331.00 mg L−1 in Erlenmeyer flasks and 208 mg L−1 in a BCR. The choice of the fermentation technique is undoubtedly linked to the characteristics and productivity of each process. The methods studied are inexpensive and were found to produce good proportions of GA3, making them suitable for several applications.

Synergistic Effect of Bacterial Consortium for Enhanced Laccase Productionby Submerged Fermentation

Industrialisation is rapidly changing the pace of economy by leaps and bounds. At the same time, the effects of pollution are evident in terms of infiltration and accumulation of hazardous substances in environment at large. Rajasthan has witnessed tremendous growth in small scale industries, one of them being the handmade paper industry. Globally, the finished product famously known as “Sanganeri handmade paper” is being appreciated for its ethnic hues and multi-usage. The current practices of paper manufacturing rely on intensive mechanical pulping process utilising an array of raw materials finally leading to enormous volumes of effluent. A combination of mechanical and chemical pulping process has certain identifiable gaps in the form of high production cost, high energy consumption and generation of large volumes of solid waste and effluents rich in high BOD,COD, synthetic dyes, heavy metals, bleaching agents, lignins and diversified range of xenobiotic compounds; thereby posing an environmental threat. Considering this fact, we proposed a pilot study aimed at cleaner and greener production of handmade paper by bioprospecting of indigenous micro flora. For this study, soil samples were collected in accordance with standard procedures from local handmade paper industry located at Sanganer, Jaipur. Preliminarily, the samples were screened for bacterial isolates capable of producing laccase, an important enzyme responsible for delignification). Laccases (EC 1.10.3.2) are copper-containing oxidase enzymes found in many plants, fungi, and microorganisms. Furthermore, synergistic effect of bacterial consortium was explored for enhanced laccase production through submerged fermentation. Laccase activity as monitored in Cell Free Extract (CFE), was found to be maximum of 60.9 U/ml for bacterial consortium and was highly significant (p<0.05) with respect to abiotic control. This pilot study suggested the role of autochthonous micro flora in delignification of raw materials thereby obliterating the energy and cost intensive chemico-mechanical pulping process.

Research on the Solid State Fermentation of Jerusalem Artichoke Pomace for Producing R,R-2,3-Butanediol by Paenibacillus polymyxa ZJ-9.

R,R-2,3-butanediol (R,R-2,3-BD) was produced by Paenibacillus polymyxa ZJ-9, which was capable of utilizing inulin without previous hydrolysis. The Jerusalem artichoke pomace (JAP) derived from the conversion of Jerusalem artichoke powder into inulin extract, which was usually used for biorefinery by submerged fermentation (SMF), was utilized in solid state fermentation (SSF) to produce R,R-2,3-BD. In this study, the fermentation parameters of SSF were optimized and determined in flasks. A novel bioreactor was designed and assembled for the laboratory scale-up of SSF, with a maximum yield of R,R-2,3-BD (67.90g/kg (JAP)). This result is a 36.3% improvement compared with the flasks. Based on the same bath of Jerusalem artichoke powder, the total output of R,R-2,3-BD increased by 38.8% for the SSF of JAP combined with the SMF of inulin extraction. Overall, the utilization of JAP for R,R-2,3-BD production was beneficial to the comprehensive utilization of Jerusalem artichoke tuber.

Design of solid state bioreactor for industrial applications: An overview to conventional bioreactors

Abstract Industrial fermentation is the process that has been used in the production of a variety of bio-products that have a broad area of applications. Among the processes, the Solid State Fermentation (SSF) is vital, and its utilization has been growing every day. It is used in many industries including fertilizers, pharma, and food, where it has gained its importance as a substitute for the Submerged Fermentation (SmF). One primary application of SSF is in the production of enzymes that can be used for therapeutic purposes, whose yield is low and cost is higher in case of conventional methods. The major advantage of using SSF is the fact that raw materials for this process are obtained from agricultural wastes and to an extent some industrially produced wastes can be utilized. The usage of SSF is limited because of its inability to be used in large-scale production and in the extraction of the required products. The primary requirement is to overcome any difficulties that are associated with the currently used industrial bioreactors. Important factors that have to be considered is that every reactor has its own significance, and they can give a much better result when compared to other bioreactors; an extensive study has to be done to determine which bioreactors have a better capability on any particular enzyme. The main aim of this review is to study the limitations (e.g. heat transfer) of the conventional bioreactors that are available and how to overcome these limitations shall be addressed in the review.

Effect of surfactants and inducers on increased uricase production under submerged fermentations by Bacillus cereus

ABSTRACTUricase is a clinical enzyme used for the oxidation of uric acid crystals in gout disease. The present study aimed to increase the suitable surfactant-mediated uricase production on induction by different concentrations of inducers. The efficiency of Bacillus cereus to produce extracellular uricase enzyme was studied in uric acid-containing agar plates. Among the studied inducers, uric acid is the potential inducer for uricase production under submerged fermentations (SMF), which induced 19.41 U/ml uricase in medium containing 2.0 g/L of uric acid, however further increase in the uric acid concentration decreased uricase production, which could be because of substrate inhibition. The physical parameters including agitation speed (rpm) and time duration (h) of uricase production were optimized and found to produce optimum uricase at 150 rpm in 26 h of SMF. Among the studied surfactants, nonionic surfactant, polyvinyl alcohol has shown a remarkable increase in the uricase production of 31.58 U/ml, which is a 61% increase under optimized conditions in SMF. The stability of produced uricase was found at pH 7.5 and temperature 30°C. Also the effects of various metal ions (1 mM) on the uricase activity were studied and observed to be inhibitory in nature in the descending order K+ > Ca2+ > Zn2+ > Fe3+ > Ni2+ > Mg2+ > Mn2+ > Cu2+.

English

Protease has gained a very important position in many industries such as food, pharmaceutical, chemical and leather industries. In this research, protease was obtained from bacteria. The bacterial strain was obtained from soil which was collected from different areas of Lahore, Pakistan. Fermentation medium (by using sub-merged fermentation technique) was incubated for 48 h at 37&deg;C temperature and agitation speed of 200 rpm. The protease was partially purified with 70% ammonium sulphate. Four different supports were used for the immobilization of the bacterial protease by physical adsorption method. When partially purified protease was immobilized on Amberlite (XAD 761), its production amount was 371.42%&nbsp; (52 U/ml/g support) with strain T5; 208.33% (25 U/ml/g support) with strain T3 and 342.24% (64 U/ml/g support) with strain H3; when it was immobilized on Duolite (A568), its amount of production was 314.28% (44 U/ml/g support) with strain T5; 225% (27 U/ml/g support) with strain T3 and 395.72% (74 U/ml/g support) with strain H3; when it was immobilized on Lewatit (VPOC 1600), the amount of production was 450% (63 U/ml/g support) with strain T5; 541.66% (65 U/ml/g support) with strain T3 and 320.85% (60 U/ml/g support) with strain H3. But, when it was immobilized on Pentynyl Dextran (NT4L360), its amount of production was 271.42% (38 U/ml/g support) with strain T5, 3483.33% (418 U/ml/g support) with strain T3 and 304.81% (57 U/ml/g support) with strain H3. Immobilized protease from bacterial strain T3 had the highest immobilizing activity of 3483.33% (418 U/ml/g support). This immobilized protease with the highest activity could be used in food, pharmaceutical and leather industries. Key words: Protease, bacterial strain, immobilizing activity.

Biochemical Characterization of Extracellular Cellulase from Tuber maculatum Mycelium Produced Under Submerged Fermentation

Interaction of truffle mycelium with the host plant involves the excretion of extracellular enzymes. The ability of Tuber maculatum mycelium to produce an extracellular cellulase during submerged fermentation was demonstrated for the first time. T. maculatum mycelia were isolated and tested for extracellular cellulase production at variable pH on solid agar medium, and the highest activity was observed at pH7.0. Furthermore, T. maculatum was subjected to submerged fermentation in basal salt medium for cellulase production. Under optimized conditions using sodium carboxymethyl cellulose (0.5% w/v) as carbon source and an initial pH of 7.0, the enzyme production yielded 1.70U/mL of cellulase in the cell-free supernatant after 7days of incubation time. The optimum of the obtained cellulase's activity was at pH5.0 and a temperature of 50°C. The enzyme showed good thermostability at 50°C by retaining 99% of its maximal activity over an incubation time of 100min. The cellulase activity was inhibited by Fe2+ and slightly activated by Mn2+ and Cu2+ at 1mM concentration. The results indicated that truffle mycelium is utilizing cellulosic energy source in the root system, and the optimal conditions are those existing in the acidic Finnish soil.

Bioactivity of a Novel Glycolipid Produced by a Halophilic Buttiauxella sp. and Improving Submerged Fermentation Using a Response Surface Method.

An antimicrobial glycolipid biosurfactant (GBS), extracted and identified from a marine bacterium, was studied to inhibit pathogenic microorganisms. Production of the GBS was optimized using a statistical method, a response surface method (RSM) with a central composite design (CCD) for obtaining maximum yields on a cost-effective substrate, molasses. The GBS-producing bacterium was identified as Buttiauxella Species in terms of biochemical and molecular characteristics. This compound showed a desirable antimicrobial activity against some pathogens such as E. coli, Bacillus subtilis, Bacillus cereus, Candida albicans, Aspergilus niger, Salmonella enterica. The rheological studies described the stability of the GBS at high values in a range of pH (7–8), temperature (20–60) and salinity (0%–3%). The statistical optimization of GBS fermentation was found to be pH 7, temperature 33 °C, Peptone 1%, NaCl 1% and molasses 1%. The potency of the GBS as an effective antimicrobial agent provides evidence for its use against food and human pathogens. Moreover, favorable production of the GBS in the presence of molasses as a cheap substrate and the feasibility of pilot scale fermentation using an RSM method could expand its uses in food, pharmaceutical products and oil industries.

Optimization of Culture Conditions for Production of Amylase by Bacillus Amyloliquifaciens SH8 Using Response Surface Methodology

In the present study Response surface methodology (RSM) was used to investigate the combined effect of relevant process variables to maximize the production of amylase in submerge fermentation by Bacillus amyloliquifaciens SH8. The process variables include pH temperature, inoculums size, incubation day, substrate concentration. A 54 factorial central composite design (CCD) using response surface methodology (RSM) was employed to obtain interaction between the process variables and optimizing these variables. Total 54 experiments were carried out in shake flask and a three dimensional response surface was generated to determine the effect of process variables on amylase production. The optimal calculated values of tested variables for maximal production of amylase were: pH 5, temperature 45°C, inoculums size 5%, incubation day of 5 and substrate concentration of 0.60% with 16.07 IU/ml of amylase activity.

Secretomic Insight into Glucose Metabolism of Aspergillus brasiliensis in Solid-State Fermentation.

The genus Aspergillus is ubiquitous in nature and includes various species extensively exploited industrially due to their ability to produce and secrete a variety of enzymes and metabolites. Most processes are performed in submerged fermentation (SmF); however, solid-state fermentation (SSF) offers several advantages, including lower catabolite repression and substrate inhibition and higher productivity and stability of the enzymes produced. This study aimed to explain the improved metabolic behavior of A. brasiliensis ATCC9642 in SSF at high glucose concentrations through a proteomic approach. Online respirometric analysis provided reproducible samples for secretomic studies when the maximum CO2 production rate occurred, ensuring consistent physiological states. Extracellular extracts from SSF cultures were treated by SDS-PAGE, digested with trypsin, and analyzed by LC-MS/MS. Of 531 sequences identified, 207 proteins were analyzed. Twenty-five were identified as the most abundant unregulated proteins; 87 were found to be up-regulated and 95 were down-regulated with increasing glucose concentration. Of the regulated proteins, 120 were enzymes, most involved in the metabolism of carbohydrates (51), amino acids (23), and nucleotides (9). This study shows the high protein secretory activity of A. brasiliensis under SSF conditions. High glucose concentration favors catabolic activities, while some stress-related proteins and those involved in proteolysis are down-regulated.

Phytase production by solid-state fermentation of groundnut oil cake by Aspergillus niger: A bioprocess optimization study for animal feedstock applications

ABSTRACTThis investigation deals with the use of agro-industrial waste, namely groundnut oil cake (GOC), for phytase production by the fungi Aspergillus niger NCIM 563. Plackett–Burman design (PBD) was used to evaluate the effect of 11 process variables and studies here showed that phytase production was significantly influenced by glucose, dextrin, distilled water, and MgSO4 · 7H2O. The use of response surface methodology (RSM) by Box–Behnken design (BBD) of experiments further enhanced the production by a remarkable 36.67-fold from the original finding of 15 IU/gds (grams of dry substrate) to 550 IU/gds. This is the highest solid-state fermentation (SSF) phytase production reported when compared to other microorganisms and in fact betters the best known by a factor of 2. Experiments carried out using dried fermented koji for phosphorus and mineral release and also thermal stability have shown the phytase to be as efficient as the liquid enzyme extract. Also, the enzyme, while exhibiting optimal activity under acidic conditions, was found to have significant activity in a broad range of pH values (1.5–6.5). The studies suggest the suitability of the koji supplemented with phytase produced in an SSF process by the “generally regarded as safe” (GRAS) microorganism A. niger as a cost-effective value-added livestock feed when compared to that obtained by submerged fermentation (SmF).

English

α-Amylases are enzymes responsible for breaking the α-1,4 bond in polysaccharides with three or more glucose units, occupying the second place in the most widely employed enzymes in industry in the world. The objective of this study was to compare the yields of α-amylase produced by the endophytic fungus, Penicillium digitatum , strain D1-FB, isolated from Baccharis dracunculifolia D.C. (Asteraceae), through the solid state fermentation (SSM) and submerged fermentation (SmF) processes, in addition to characterizing the produced enzyme. The two fermentations were conducted for 120 h, taking samples every 24 h to obtain the peaks of production. The enzymes were characterized according to their optimal pH and temperature for performance and stability regarding the incubation in the presence of ions, variations in pH and temperature. The maximum yield of the enzyme was observed with SSF, using rice bran as substrate after 72 h of fermentation, with 1,625 U/mL. The α-amylase had an optimal pH at 6.5 and optimal temperature at 37°C. All the ions resulted in a decrease in the activity of α- amylase in the concentration of 5 mM. The enzyme proved to be quite stable in a pH range of 6.0 to 7.5 and up to the temperature of 37°C, but it presented great drops in activity with temperatures above 45°C and in the presence of ions at the concentration of 5 mM. Keywords: Penicillium digitatum , α-amylase, starch, enzymes, endophytic

Development of a Cost Effective Medium for Enhanced Production of Bacillus thuringiensis ?-endotoxin

The present study was carried out to develop a sustainable production medium using locally available cheap raw materials for biopesticide production by Bacillus thuringiensis subsp. kurstaki (Btk) HD-73. In submerged fermentation (SmF) condition, the conventional Luria-Bertani (LB) medium which was enriched with nitrogen source (10% defatted soybean meal) supported 28.57% sporulation and 125% endotoxin increase over LB (alone). The effect of cystine on sporulation and endotoxin synthesis was highly pronounced in LB-soybean medium (LBS) with a range of 19.54% and 131.35% higher endotoxin yield respectively in SmF condition. While basal salts supplemented in soybean-cystine (SMc) medium, it resulted in 7.65% endotoxin yield compared to LB-soybean-cystine (LBSc) medium. Addition of molasses balanced the C: N ratio in the SMc medium thus helping 84.85% higher endotoxin synthesis after 24 hours fermentation. Substitution of basal salts with cost effective sea water yielded about 19.27% less endotoxin. The optimum medium thus obtained consisting of soybean extract-molasses-cystine with sea water was used in 3.0 L bioreactor cultivation for endotoxin synthesis by Btk HD-73 under 30% saturation of dO2 through cascade of agitation and aeration. The production rate obtained was 1.67 fold higher in bioreactor than in shake flask culture.Bangladesh J Microbiol, Volume 32, Number 1-2,June-Dec 2015, pp 1-6

Cellulase production using natural medium and its application on enzymatic hydrolysis of thermo chemically pretreated biomass.

Lignocellulosic bioethanol is an important renewable fuel for transportation purpose. Commercial production of lignocellulosic bioethanol mainly depends on cost of cellulase production, efficient pretreatment and enzymatic hydrolysis process. In the present study cellulase production from Aspergillus niger under submerged fermentation (SmF) was optimized using coconut water as natural medium. Maximum cellulase production (0.53 IU/mL) was achieved within 3 days of incubation using 8 % (w/v) waste paper and 0.07 % (w/v) glucose. The produced cellulase was applied for enzymatic hydrolysis of thermo chemically (dilute acid and alkaline) pretreated biomass (equal mixture of wheat straw and cotton stalk). Optimization of dilute acid and dilute alkaline pretreatment showed dilute alkaline pretreatment was more effective for higher reducing sugar production. Maximum reducing sugar yield of 398.0 mg/g dry biomass was obtained from dilute alkaline pretreated biomass (using 0.5 M sodium hydroxide, 8 % substrate concentration, 120 °C temperature and 20 min of incubation time). The presence of difference sugars (glucose, xylose, mannose, maltose) in the saccharified sample was confirmed by thin layer chromatographic analysis. The effectiveness of dilute alkaline pretreatment was further confirmed by biochemical composition (cellulose, hemicelluloses and lignin) and structural (furrier transformed infrared spectroscopic and scanning electron microscopic) analysis. The above result can be useful for commercial production of lignocellulosic bioethanol.