Feasibility of reed for biobutanol production hydrolyzed by crude cellulase

The aim of this study was to efficiently utilize reed for both cellulase and biobutanol production. The unprocessed cellulase blend produced under solid-state fermentation using reed as the substrate showed a similar reducing sugar yield using Whatman filter paper to the commercial enzyme blend (38.61%). Organosolv pretreatment method could efficiently reduce hemicellulose (29.3%–14.6%) and lignin (17.2%–14.1%) content and increase cellulose content (42.5%–62.3%) from reed. Enzymatic hydrolysis of organosolv-pretreated reed using the crude cellulase with enzyme loading of 25 FPU/g reed, 20% solid content at 50 °C and pH 5.5 resulted in a reed hydrolysate containing 40.01 g/L glucose and 3.55 g/L xylose after 72 h. Fermentation of the hydrolysate medium by Clostridium acetobutylicum produced 9.07 and 14.24 g/L of biobutanol and ABE with yield of 0.21 g/g and 0.33 g/g, respectively. This study proved that crude cellulase complex produced under solid state fermentation and organsolv pretreatment can efficiently provide reed hydrolysate that can be converted to biobutanol without any commercial cellulase usage.     

Developments in biobutanol production: New insights

Biobutanol will become an attractive, economic and sustainable fuel as petroleum oil leads towards expensive fuel due to diminishing oil reserves and an increase of green house gases in the atmosphere. The major challenges in biobutanol production are low butanol titer, availability of compatible feedstocks, and product inhibition. These hurdles are being resolved using several genetic engineering techniques, metabolic engineering strategies, and promising integrated continuous fermentation processes with efficient product recovery techniques (like gas stripping). Adequate success in utilizing renewable and cost-effective cellulosic materials as feedstocks has opened up novel grounds for the advancement in economic biobutanol production. In this direction, Clostridium beijerinckii is being explored as promising strain to produce biobutanol from cellulosic materials. Moreover, high biobutanol titer is being focused through genetic modifications of Clostridia and non-Clostridia organisms (e.g., Escherichia coli, Saccharomyces cerevisiae, Pseudomonas putida, and Bacillus subtilis) in both aerobic and anaerobic fermentation. Further, application of various novel genetic tools and genome sequencing of hyper-butanol-producing Clostridial organism will enhance the scope of genetic engineering for biobutanol production. Therefore, consolidation of academic and industrial research towards economic synthesis of biobutanol illustrates the possibility of substantial breakthrough in future. In this review, we focus on (i) selection of suitable bacterial strain (ii) availability of cheaper biomass to produce butanol (iii) metabolic engineering strategies of various microorganisms (iv) attempts at process development and (v) biobutanol recovery techniques that provide future direction of economical biobutanol fermentation.     

Utilization of palm kernel cake as a renewable feedstock for fermentative hydrogen production

Fermentative hydrogen generation was studied using palm kernel cake (PKC) as sustainable cellulosic biomass. PKC was subjected to an acid hydrolysis approach using dilute H₂SO₄ (7% v/v). PKC hydrolysate obtained was then diluted (70%) and used as a substrate for hydrogen generation. Chemical analysis showed that the main fermentable sugars in diluted PKC hydrolysate were glucose, xylose and mannose with the concentrations of 2.75 g/L, 2.60 g/L and 27.75 g/L, respectively. Hydrogen production was carried out by the cultivation of Clostridium acetobutylicum YM1 on PKC hydrolysate. The effect of incubation temperature, the initial pH of culture medium and microbial inoculum size on hydrogen production was studied using a statistical model. The analysis of the model generated showed that the initial pH value of the culture medium and inoculum size had significant effects on the hydrogen production. The study showed that the optimum conditions for the biohydrogen production were 30.57 °C temperature, pH 5.5 and 20% inoculum size. A verification experiment was performed in the optimum conditions determined. Experimental results of the verification test showed that a cumulative hydrogen volume of 1575 ml/L was generated with consuming 2.75 g/L glucose, 2.20 g/L xylose and 16.31 g/L mannose.     

Potential for using enriched cultures and thermotolerant bacterial isolates for production of biohydrogen from oil palm sap and microbial community analysis

A limited number of bacteria can convert oil palm (Elaeis guineensis) sap to hydrogen with satisfactory yield and productivity. In this study, a total of 18 fermentative enriched cultures and 36 newly isolated thermotolerant bacterial strains were compared for hydrogen production from oil palm (OP) sap. The new isolates were obtained from hot springs, palm oil mill effluent and oil palm sap. The test was conducted in three steps: (i) a test for hydrogen production from mixed substrates (cellulose, starch, xylose, and glucose) and OP sap; (ii) a test for substrate concentration tolerance; and (iii) a test for thermotolerance. Five enriched candidates for each of the hydrogen producers were selected according to the criteria defined for the screening test. The hydrogen production of these selected bacterial strains from hot springs were cultivated in batch fermentation of oil palm sap at room temperature (30 ± 2 °C). Five enriched cultures, namely 81RN1, OPS, 85RN5, 89SR3-2 and 112YL1 were found to give high cumulative hydrogen formation of 1085, 1009, 994, 983 and 778 mL H₂/L-OP sap, respectively, with the hydrogen content of 29.8, 29.4, 28.7, 27.1 and 27.5%, respectively. PCR–DGGE profiling showed that all these five enriched cultures consisted of species closely related to the genus Clostridium sp. based on the 16S rRNA gene. For pure cultures, the top five hydrogen producers were the isolates encoded as PS-3, PS-4, PS-5, PS-7 and PS-8 exhibiting the hydrogen production of 1973, 1774, 1335, 1170 and 1070 mL H₂/L-OP sap, respectively, with the hydrogen content of 33.7, 29.6, 32.5, 31.5 and 26.4%, respectively. Identification of these high hydrogen producers using 16S rRNA sequence matching showed that the isolates PS-3 and PS-8 belonged to Clostridium beijerinckii, while the isolate PS-7 belonged to Clostridium acetobutylicum and the isolates PS-4 and PS-5 belonged to Klebsiella sp. and Klebsiella pneumoniae, respectively. Therefore, the pure culture C. beijerinckii PS-3 exhibited 1.8 folds higher hydrogen production (1973 mL H₂/L-OP sap) than the enriched cultures of 81RN1 (1085 mL H₂/L-OP sap).     

The production of biohydrogen by a novel strain Clostridium sp. YM1 in dark fermentation process

In view of increasing attempts for the production of renewable energy, the production of biohydrogen energy by a new mesophilic bacterium Clostridium sp. YM1 was performed for the first time in the dark fermentation. Experimental results showed that the fermentative hydrogen was successfully produced by Clostridium sp. YM1 with the highest cumulative hydrogen volume of 3821 ml/L with a hydrogen yield of 1.7 mol H₂/mol glucose consumed. Similar results revealed that optimum incubation temperature and pH value of culture medium were 37 °C and 6.5, respectively. The study of hydrogen production from glucose and xylose revealed that this strain was able to generate higher hydrogen from glucose compared to that from xylose. The profile of volatile fatty acids produced showed that hydrogen generation by Clostridium sp. YM1 was butyrate-type fermentation. Moreover, the findings of this study indicated that an increase in head space of fermentation culture positively enhanced hydrogen production.     

Comparative performance analysis of diesel engine fuelled with hydrogen enriched edible and non-edible biodiesel

In the present study, a comparative analysis of enrichment of hydrogen alongside diesel fuel and two different sources of biodiesel namely rice bran oil is an edible oil, and karanja oil being non-edible is tested. Hydrogen at a fixed flow rate of 7 lpm is inducted through the intake manifold. A total of six fuel samples are considered: diesel (D), hydrogen-enriched diesel (D + H₂), hydrogen-enriched 10, and 20% rice bran biodiesel blend (RB10 + H₂ and RB20 + H₂), and hydrogen-enriched 10 and 20% karanja biodiesel blend (KB10 + H₂ and KB20 + H₂). Results indicate that enrichment of hydrogen improves combustion and results in 2.5% and 1.6% increase in the brake thermal efficiency of diesel fuel and rice bran biodiesel, respectively. For karanja biodiesel the increment is negligible. Fuel consumption of the D + H? is 6.35% lower and for RB10 + H? and KB10 + H? it is decreased by 2.9% and 1.3%, respectively. The Presence of hydrogen shows the 4–38% lower CO emissions and 6–14% lower UHC emission due to better combustion. The blends RB10 + H? and KB10 + H? produce up to 6–13% higher NOx emission and that for the blends RB20 + H? and KB20 + H? it goes up to 25%. Overall rice bran oil is found to provide better performance than karanja biodiesel.     

Characterization of a Trichoderma atroviride strain isolated from switchgrass bales and its use to saccharify ammonia-pretreated switchgrass for biobutanol production

The feedstock-specific enzyme systems for saccharification of biofuel feedstocks like switchgrass may potentially provide better enzymatic systems for production of second-generation biofuels. One strategy to develop these enzyme systems could be to harness the microorganisms growing naturally on specific feedstocks. This study presents the isolation and screening of fungal cultures from switchgrass bales for saccharification of ammonia-pretreated switchgrass for subsequent biobutanol production. The best performing fungal isolate during screening was identified through Sanger sequencing of its ITS region to be a unique strain of Trichoderma atroviride and further characterized for production of an enzyme system for saccharification of ammonia pretreated switchgrass. The maximum FPase, CMCase and xylanase activity produced by T. atroviride CUA1 were 0.25 fpu/mL, 0.18 IU/mL and 5.8 IU/mL, respectively. T. atroviride CUA1 also produced considerable amount of β-glucosidase activity. This isolate was used to produce an enzyme system to convert switchgrass to soluble sugars that were then fermented to butanol, ethanol, acetate and butyrate. Glucose was the major product of hydrolysis of ammonia-pretreated switchgrass performed using the enzyme system produced by the isolate. This fungus may be useful for the hydrolysis for the bioenergy crop of switchgrass to overcome this rate-limiting step in the overall conversion of biomass to biofuels.     

Direct bioconversion of sorghum extract sugars to free fatty acids using metabolically engineered Escherichia coli strains: Value addition to the sorghum bioenergy crop

The unprecedented challenge of meeting the energy demand of growing economies has increased the need to produce hydrocarbon fuels using renewable sources. Among different platforms being researched these days, hydrocarbon biofuel and chemical production from free fatty acids (FFAs) have gained a lot of attention. The current study reports on value addition to sorghum energy crop through utilization of sorghum extract as a renewable carbon source for FFA production using genetically engineered Escherichia coli. In order to reduce the production cost of FFAs, direct sucrose utilizing E. coli strains were developed. The E. coli strain MG1655 with fadD mutant (named as ML103) either carrying the plasmid bearing the gene for acyl-ACP thioesterase (TE) from Ricinus communis (pXZ18) or a plasmid bearing a combination of the TE and the native (3R)-hydroxyacyl-ACP dehydrase gene (fabZ) (pXZ18Z) was modified to utilize sucrose by incorporating the pUR400 plasmid. The newly created strains utilized a mixture of pure sugars and sorghum extract efficiently. The 24 h pH adjusted culture of ML103 pXZ18Z pUR400 produced a maximum of 6.18 ± 0.52 g l⁻¹ from 30 g l⁻¹ of pure sugar mixture and 2.95 ± 0.04 g l⁻¹ with sorghum extract (15 g l⁻¹ equivalent sugar concentration). These data suggest that modified E. coli strains were capable of directly utilizing sucrose and produce FFAs from it. Successful demonstration of direct bioconversion of sorghum extract to FFA was validated as a possible value addition of the sorghum bioenergy crop.     

Isolation and characterization of high hydrogen-producing strain Clostridium beijerinckii PS-3 from fermented oil palm sap

Felled oil palm trunk (OPT) (25 years old) is an abundant biomass in Southern Thailand. The OPT composition was 31.28–42.85% cellulose, 19.73–25.56% hemicellulose, 10.74–18.47% lignin, 1.63–2.25% protein, 1.60–1.83% fat, 1.12–1.35% ash and trace amount of minerals (0.01–0.40%). Oil palm sap extracted from OPT was found to contain 15.72 g/L glucose, 2.25 g/L xylose, and 0.086 g/L arabinose. A total of twenty samples from hot springs (45–75 °C and pH 6.5–8.4), oil palm sap and palm oil mill effluent were enriched for isolation of hydrogen-producing bacteria. The highest hydrogen-producing strain was isolated from oil palm sap and identified as Clostridium beijerinckii PS-3 using biochemical test and 16S rRNA gene analysis. Among various carbon sources tested, glucose, xylose, starch and cellulose were the preferred substrates for hydrogen production. The strain PS-3 could produce the maximum hydrogen yield of 140.9 ml H₂/g total sugar and the cumulative hydrogen production of 1973  ml/L-oil palm sap. Therefore, C. beijerinckii PS-3 is a potential candidate for fermentative hydrogen production from mixed sugars of the oil palm sap.     

Optimization for simultaneous enhancement of biobutanol and biohydrogen production

Acetone-butanol-ethanol (ABE) fermentation guarantees a sustainable route for biohydrogen and biobutanol production. This research work is committed towards the enhancement of biohydrogen and biobutanol production by single and multi-parameter optimization for the improvement of substrate energy recovery using C. saccharoperbutylacetonicum. Single parameters optimization (SPO) manifested that headspace of 60% (v/v) and butyric acid supplementation of 9 g/L and temperatures of 30 °C and 37 °C were suitable for obtaining maximum biohydrogen and biobutanol production, respectively. The interaction between these parameters was further evaluated by implementing a 5-level 3-factor Central Composite Design (CCD). In the present study, a central composite design was employed to enhance the biohydrogen and biobutanol production. In addition, the experimental results were analyzed by response surface methodology (RSM) and artificial intelligence (AI) techniques such as artificial neural network (ANN). The prediction capability of RSM was further compared with ANN for predicting the optimum parameters that would lead to maximum biohydrogen and biobutanol production. ANN yielded higher values of biohydrogen and biobutanol. ANN was found to be superior as compared to RSM in terms of prediction accuracy for both biohydrogen and biobutanol because of its higher coefficient of determination (R²) and lower root mean square error (RMSE) value. Process temperature (32.65 °C), headspace (58.21% (v/v)) and butyric acid supplementation (9.16 g/L) led to maximum substrate energy recovery of 78% with biohydrogen and biobutanol production of 5.9 L/L and 16.75 g/L, respectively. Process parameter optimization led to a significant increase in substrate energy recovery from Biphasic fermentation.     

Simultaneous biohydrogen (H2) and bioplastic (poly-β-hydroxybutyrate-PHB) productions under dark,photo, and subsequent dark and photo fermentation utilizing various wastes

The present study is focused on bio hydrogen (H₂) and bioplastic (i.e., poly-β-hydroxybutyrate; PHB) productions utilizing various wastes under dark fermentation, photo fermentation and subsequent dark-photo fermentation. Potential bio H₂ and PHB producing microbes were enriched and isolated. The effects of substrate (rice husk hydrolysate, rice straw hydrolysate, dairy industry wastewater, and rice mill wastewater) concentration (10–100%) and pH (5.5–8.0) were examined in the batch mode under the dark and photo fermentation conditions. Using 100% rice straw hydrolysate at pH 7, the maximum bio H₂ (1.53 ± 0.04 mol H₂/mol glucose) and PHB (9.8 ± 0.14 g/L) were produced under dark fermentation condition by Bacillus cereus. In the subsequent dark-photo fermentation, the highest amounts of bio H₂ and PHB were recorded utilizing 100% rice straw hydrolysate (1.82 ± 0.01 mol H₂/mol glucose and 19.15 ± 0.25 g/L PHB) at a pH of 7.0 using Bacillus cereus (KR809374) and Rhodopseudomonas rutila. The subsequent dark-photo fermentative bio H₂ and PHB productions obtained using renewable biomass (i.e., rice husk hydrolysate and rice straw hydrolysate) can be considered with respect to the sustainable management of global energy sources and environmental issues.     

Physical properties of rice husk and bran briquettes under low pressure densification for rural applications

Agriculture generates large amount of by-products that could be used to produce energy and reduce the amount of fuelwood required to meet the daily cooking needs, especially in developing countries. Rice is a major crop grown in West Africa and rice husk is a by-product of the milling process. The goal of this study was to develop a low cost system to produce biomass briquettes from rice husks in the context of a rural village. A manual press generating a pressure of 4.2 MPa was developed and used. The influence of the briquette formulation (type of binder, binder content, water addition, and bran content) was studied. The binders investigated were cassava wastewater, rice dust, and okra stem gum. The physical properties (density, moisture content, calorific value, durability, and compressive strength) were tested to identify the briquettes with the highest quality, i.e. greatest physical integrity. The briquettes made with rice dust had the highest durability (91.9%) and compressive strength (2.54 kN), while the briquettes made with cassava starch wastewater had the greatest density (441.18 kg m⁻³). Water added to the rice husk before densification positively influenced the briquette quality while bran seemed to mostly increase the density, but not necessarily the briquette quality. The briquette formulation did not significantly influence the calorific value. With a higher heating value of 16.08 MJ kg⁻¹ dry basis, rice husk briquettes represent an interesting alternative to fuelwood.     

Arrowroot as a novel substrate for ethanol production by solid state simultaneous saccharification and fermentation

Ethanol production from Canna edulis Ker was successfully carried out by solid state simultaneous saccharification and fermentation. The enzymatic hydrolysis conditions of C. edulis were optimized by Plackett–Burman design. The effect of inert carrier (corncob and rice bran) on ethanol fermentation and the kinetics of solid state simultaneous saccharification and fermentation was investigated. It was found that C. edulis was an alternative substrate for ethanol production, 10.1% (v/v) of ethanol concentration can attained when 40 g corncob and 10 g rice bran per 100 g C. edulis powder were added for ethanol fermentation. No shortage of fermentable sugars was observed during solid state simultaneous saccharification and fermentation. There was no wastewater produced in the process of ethanol production from C. edulis with solid state simultaneous saccharification and fermentation and the ethanol yield of more than 0.28 tonne per one tonne feedstock was achieved. This is first report for ethanol production from C. edulis powder.     

Effect of carbon sources on the photobiological production of hydrogen using Rhodobacter sphaeroides RV

Rhodobacter sphaeroides RV was employed to produce hydrogen for the photo-fermentation of sole (acetate, propionate, butyrate, lactate, malate, succinate, ethanol, glucose, citrate and sodium carbonate) and compound carbon sources (malate and succinate, lactate and succinate). The concentrations of sole carbon sources on hydrogen production were investigated in batch assays at 0.8 g/L sodium glutamate and the maximum hydrogen yield was 424 mmol H₂/mol-substrate obtained at 0.8 g/L sodium propionate. The maximum hydrogen yield reached 794 mmol H₂/mol-substrate for 2.02 g lactate and 2.0 g succinate as the compound carbon source. The results showed hydrogen production for the compound carbon source was better than the sole carbon source.     

Investigation of biomass gasification hydrogen and electricity co-production with carbon dioxide capture and storage

Biomass is one of the renewable energy resources which can be used instead of fossil fuels to diminish environment pollution and emission of greenhouse gases. Hydrogen as a biomass is considered as an alternative fuel which can be derived from a variety of domestically available primary sources. In this paper, a hydrogen and electricity co-generation plant with rice husk is proposed. Rice husk with water vapor and oxygen produces syngas in gasifier. In this design, electricity is generated by using two Rankine cycles. The Results show that the net electric efficiency and hydrogen production efficiency are 1.5% and 40.0%, respectively. Hydrogen production is 1.316 kg/s in case which carbon dioxide is gathered and stored. The electricity generation is 5.923 MW_e. The main propose of implementing Rankine cycle is to eliminate hydrogen combustion for generating electricity and to reduce NO_x production. Furthermore, three kinds of membranes are studied in this paper.     

The atmospheric steam engine as energy converter for low and medium temperature thermal energy

Many industrial processes and renewable energy sources produce thermal energy with temperatures below 100 °C. The cost-effective generation of mechanical energy from this thermal energy still constitutes an engineering problem. The atmospheric steam engine is a very simple machine which employs the steam generated by boiling water at atmospheric pressures. Its main disadvantage is the low theoretical efficiency of 0.064. In this article, first the theory of the atmospheric steam engine is extended to show that operation for temperatures between 60 °C and 100 °C is possible although efficiencies are further reduced. Second, the addition of a forced expansion stroke, where the steam volume is increased using external energy, is shown to lead to significantly increased overall efficiencies ranging from 0.084 for a boiler temperature of T₀ = 60 °C to 0.25 for T₀ = 100 °C. The simplicity of the machine indicates cost-effectiveness. The theoretical work shows that the atmospheric steam engine still has development potential.     

Effects of rice husk particle size on biohydrogen production under solid state fermentation

As a renewable energy source bio-hydrogen production from lignocellulosic wastes is a promising approach which can produce clean fuel with no CO₂ emissions. Utilization of agro-industrial residues in solid state fermentation (SSF) is offering a solution to solid wastes disposal and providing an economical process of value-added products such as hydrogen.In this study three different particle size of rice husk (<2000 μm, <300 μm, <74 μm) was subjected to batch SSF with a Clostridium termitidis: Clostridium intestinale ratio of 5:1. C. termitidis is a cellulolytic microorganism that has the ability to hydrolyze cellulosic substances and C. intestinale is able to grow on glucose having a potential of enhancing hydrogen production when used in the co-culture. 5 g dw rice husk with 75% humidity was used as substrate in SSF under mesophilic conditions. The highest HF Volume (29.26 mL) and the highest yield (5.9 mL H₂ g⁻¹ substrate) were obtained with the smallest particle size (<74 μm). The main metabolites obtained from the fermentation media were acetic, butyric, propionic and lactic acids. The second best production yield (3.99 mL H₂ g⁻¹ substrate) was obtained with the middle particle size (<300 μm) rice husk with a HF of 19.71 mL.     

Influences of carbon sources on the biomass,production and compositions of exopolysaccharides from Paecilomyces hepiali HN1

The parasitic fungus, Paecilomyces hepiali, is used to produce Cordyceps materials as succedaneum of natural Cordyceps sinensis in China. The purpose of this research was to investigate the effects of glucose, mannose, sucrose, lactose as solo carbon source and sucrose + lactose or mannose + sucrose as synthetic carbon source on the growth of mycelium and production, chemical composition, molecular weight distribution and monosaccharide composition of exopolysaccharides from P. hepiali HN1 (PHEPS). The maximum mycelium biomass of 12.16 kg m⁻³ and PHEPS yield of 4.57 kg m⁻³ were achieved from the culture with sucrose (50 kg m⁻³) as carbon source. The resulting PHEPS was characterized by analyses of chemical composition, size-exclusion chromatography and high performance liquid chromatography with 1-phenyl-3-methyl-5-pyrazolone pre-column derivatization. It was found that the chemical compositions and monosaccharide ratios in PHEPS were significantly affected by the carbon sources used. Glucose or mannose as carbon source enhanced the biosynthesis of PHEPS with higher-molecular weight (>1000 kD), but solo carbon source of lactose or synthetic carbon source of mannose + lactose did not increase the ratio of galactose in PHEPS. The metabolism kinetics of carbon sources demonstrated the correlation between PHEPS synthesis and the utilization of carbon sources. These findings will be useful for further works on the production, structure and function of PHEPS.     

Characteristics of hydrogen production by an aciduric transposon-mutagenized strain of Pantoea agglomerans BH18

Based on the generation of Pantoea agglomerans BH18 library using Tn7 transposon system, mutant screens were conducted for improvement the ability of hydrogen production of this strain. These mutants were used to test for ability of hydrogen production in the initial pH of 5.0. In contrast to wild type strain BH18, a transposon mutant, named as strain TB108, was screened for high hydrogen-producing capability and acid tolerance in the initial pH of 5.0. The factors required for hydrogen production of the aciduric transposon-mutagenized strain TB108 were determined. The mutant strain TB108 similar as wild type strain BH18 was able to produce hydrogen over a wide range of salt concentration from 0.4% to 6%. Under the marine conditions with the initial pH of 5.0 and glucose concentration of 10 g/L, the total hydrogen production of the mutant TB108 was (1.36 ± 0.04) mol H₂/mol glucose (mean ± S.E.), increasing by 55% compared with wild type. In addition, the mutant strain TB108 could produce hydrogen using many carbon sources such as fructose, glucose, sucrose, sorbitol and so on. This result demonstrated that the mutant strain with high acid tolerance is beneficial for improvement of hydrogen production.     

Hydrolysis of pretreated rice straw by an enzyme cocktail comprising acidic xylanase from Aspergillus sp. for bioethanol production

The most crucial enzyme involved in xylan hydrolysis is endoxylanase which cleaves the internal glycosidic bonds of xylan. The aim of this work was to study the production of extracellular xylanase by a locally isolated strain of Aspergillus sp. under solid-state fermentation (SSF) and to evaluate the potential of the enzyme in enzymatic hydrolysis of pretreated rice straw. Xylanase production reached maximum with incubation period (96 h), moisture level (80%), inoculum size (3 × 10⁶ spores/mL), pH (4.8), temperature (25 °C), carbon source (wheat bran) and nitrogen source (yeast extract). Under optimized conditions, xylanase production reached to 5059 IU/gds. Crude xylanase was used for supplementing the enzyme cocktail comprising cellulases (Zytex, India), β-glucosidase (In-house) and xylanase (In-house) for the saccharification of alkali-pretreated rice straw to get the maximum reducing sugar production. The cocktail containing the three enzymes resulted a maximum of 574.8 mg/g of total reducing sugars in comparison to 430.2 mg/g sugars by the cocktail without xylanase. These results proved that the crude xylanase preparation from Aspergillus sp. could be a potent candidate for the enzyme cocktail preparation for biomass hydrolysis in lignocellulosic bioethanol program.