Cellulosic Ethanol Research Articles

Production of the Cellulase Enzyme System by Locally Isolated Trichoderma and Aspergillus Species Cultivated on Banana Pseudostem during Solid-State Fermentation

The production cost of cellulases is regarded as a limiting factor in the cellulosic ethanol production chain. Trichoderma and Aspergillus species were used to produce cellulases through solid-state fermentation (SSF) utilizing banana pseudostem (BPS) as a carbon source. The production of cellulases was investigated at various substrate moisture contents (65–80%), incubation temperatures (30–40 °C), substrate pre-treatment methods (3% w/v NaOH, 5% v/v H2SO4, and water), and with different co-culturing of microorganisms. Trichoderma longibrachiatum LMLSAUL 14-1 produced the maximum total cellulase (75 FPU/g d.s), endoglucanase (11.35 U/g d.s), and β-glucosidase (235.83 U/g d.s) activities at a 75% moisture content of the untreated BPS at 30 °C in static culture. Pre-treatment of BPS improved the production of specific enzymes. Aspergillus fumigatus LMLPS 13-4 produced more β-glucosidase (259.8 U/g d.s) when grown on acid-pre-treated BPS, whereas T. harzianum LMLBP07 13-5 produced the highest β-glucosidase activity (319.5 U/g d.s) on alkali-pre-treated BPS. Co-culturing of T. harzianum LMLBP07 13-5 and A. fumigatus LMLPS 13-4 improved the production of endoglucanase. These results suggest that banana pseudostem, a waste product of the banana industry, could be a potentially cheaper and abundant substrate for the production of the cellulase enzymes.

Process simulation of cassava cellulosic ethanol production with low to medium-pressure steam heat integration

Energy demand tends to increase by 80% in 2050 due to the rapid growth of the global population. However, the energy resources rapidly decrease for responding to the global need. Many previous researchers suggest the ideas for solving this situation and the most interesting solution is the heat integration which intends to produce cost-effective energy with minimal impact on the environment. Energy integration thus should be noticeable for the industry process to minimize the loss along the industrial process. The ethanol production process is interested in this study because it can add value to agricultural products like cassava which is an industrial crop in several countries and produces ethanol products that can decrease the usage of gasoline. Here, the heat integration model was developed for the ethanol production process by using low to medium-pressure steam. For the simulation model, the ethanol production process used the cellulose content as the main inlet component to be easy for applicable corresponding to feed casava cellulosic substances. The process contained three sections including enzymatic hydrolysis, simultaneous saccharification fermentation (SSF), and fractional distillation. The quality and quantity of low to medium-pressure steam were investigated for maintaining the operation of the enzymatic hydrolysis reactor by varying steam pressure between 1–2 atm and steam temperature between 100–105 °C. The results achieved the methodology to find optimum conditions by the ratio between steam flow rate and product quantity. The best case could reduce 6.49% of the excessive usage of low to medium-pressure steam which pointed out the way to use the energy efficiently.

Semi-Simultaneous Saccharification and Fermentation Improved by Lignin and Extractives Removal from Sugarcane Bagasse

Lignocellulosic biomass and agro-industrial residues are a source of fermentable sugars; however, pretreatments are needed to overcome biomass recalcitrance. This study evaluated the effect of sugarcane bagasse hydrolysis and fermentation in response to dilute acid pretreatment. In natura bagasse, extractive-free bagasse, partially delignified bagasse, and bagasse with added butylated hydroxytoluene antioxidant were pretreated with diluted acid and investigated in semi-simultaneous saccharification and fermentation (S-SSF). The effect of butylated hydroxytoluene (BHT) resulted in lower yields of inhibitors in the liquid fraction of the acid pretreatment (0.01 g L−1 of furfural, 0.01 g L−1 of 5-hydroxymethylfurfural, and 0.68 g L−1 of acetic acid). Partially delignified material and material with BHT resulted in biomass with low hemicellulose and lignin contents, indicating that BHT influenced lignin removal. Extractives removal showed benefits for the acid pretreatment, decreasing the dioxane-soluble material, and a higher yield of glucose and ethanol via S-SSF for the partially delignified material. Enzymatic saccharification of partially delignified material showed 87% of cellulose conversion (24 h with 15 FPU/g), and after 48 h of S-SSF (25 FPU/g), residual 7.06 g L−1 of glucose and production of 15.17 g L−1 of ethanol were observed. The low content of extractives, lignin, and dioxane soluble material resulted in better cellulose accessibility and ethanol yield. Chemical compounds can help remove lignin from biomass favoring ethanol production by S-SSF.

CUTTING FREQUENCY IN CAYMAN GRASS (Urochloa HYBRID) ON THE CALORIFIC POWER OF THE MEXICAN WET TROPIC

The objective of this study was to evaluate the effect of cutting frequency in Cayman grass (Urochloa HYBRID) on biomass yield, moisture, ash, ethereal extract, neutral detergent fiber (FDN), acid detergent fiber (FDA), acid detergent lignin (LDA), crude protein (PC), calorific value, and theoretical bioethanol yield. Four cutting frequencies were established as treatments: 30, 60, 90, and 120 d, arranged in a completely randomized block design with three replications. Data were analyzed with GLM (SAS), and means were compared with the Tukey test (p ≤ 0.05). The highest biomass production (11.9 Mg ha-1 year-1), calorific value (15.1 MJ kg-1), and LDA (5.7 %) were obtained at the 120 d cutting frequency. The concentration of FDN (61.8 %), FDA (43.6 %), cellulose (38.1 %), and theoretical bioethanol production (218.4 L Mg-1 MS) were statistically different at the cutting frequency of 90 d. The values of hemicellulose (18. 7 %) and ethereal extract (1.8 %) were statistically different at the 60 d-cutting frequency; while PC (9.7 %) and ash (11.8 %) showed significant differences at the 30-d cutting frequency. Based on the biomass yield and calorific value of Cayman grass, it can be considered as a potential plant material for cellulosic ethanol production.

Thirteen‐year stover harvest and tillage effects on soil compaction in Iowa

AbstractCorn (Zea mays L.) stover is an abundant biomass source with multiple end‐uses including cellulosic biofuel production. However, stover removal may increase soil compaction by reducing organic matter inputs and increasing vehicle loads during harvest. While numerous studies have reported stover removal impacts on soil physical quality, few have assessed the role played by traffic compaction. Our objective was to quantify subsurface soil compaction after 13 years of chisel plow versus no‐till management and no, moderate (3.5 ± 1.1 Mg ha−1 year−1), or high (5.0 ± 1.7 Mg ha−1 year−1) stover harvest rates. Penetration resistance was measured in most‐ and least‐trafficked interrow spaces. Chisel plowed plots with moderate and high levels of stover removal had higher penetration resistance in trafficked areas relative to least‐trafficked areas, whereas there was no evidence of traffic compaction when stover was retained. Traffic compaction did not negatively impact yields, which were greater with high levels of stover removal compared to no removal. The no‐till practice led to very small increases in penetration resistance with wheel traffic and had no evidence of increased compaction with residue removal. This lack of traffic compaction indicated soils under no‐till practice have a higher load‐bearing capacity than soils under chisel plow practice. Overall, there were no yield‐limiting effects of tillage practice or stover removal, and no evidence of soil compaction below the plow layer, suggesting stover removal with both tillage practices can be effectively employed without detrimental effects on plant or soil health.

Effect of Production Factors on the Bioethanol Yield of Tropical Sawdust

Bioethanol production from cellulosic materials is important in mitigating the concomitant displacement and exploitation of primary food crops for biofuel production and reducing carbon emissions which exacerbate climate change. The problem of reduced yield in the production and availability of yeast locally poses a barrier to market adoption and penetration of bioethanol. The study examined the effect of particle size and different yeast strains on the yield of bioethanol from waste sawdust that was sourced from a local timber processing centre. The samples of yeast were prepared from baker’s yeast (Saccharomyces cerevisiae) and palm wine yeast (Saccharomyces chevalieri). The sawdust was reduced to 212 μm, 300 μm, and 500 μm particle sizes. The samples of each particle size were pretreated and hydrolyzed with H2SO4 and fermented with S. cerevisiae or S. chevalieri yeast. The results obtained show that the weight, pH, density, viscosity, flash point, and heating value of the produced bioethanol ranged between 221.67 and 322.64 g, 6.2 and 6.6, 0.821 and 0.878 g/mL, 1.073 and 1.193, 14 and 16°C, and 20.5 and 23.1 MJ/kg, respectively, while the alcohol content of each of the samples was 69%. Furthermore, the bioethanol yield from Saccharomyces cerevisiae yeast was 213.9 mL, 193.2 mL, and 186.3 mL, for the 212 μm, 300 μm, and 500 μm particles, while for Saccharomyces chevalieri yeast, the yield was 289.8 mL, 255.3 mL, and 220.8 mL for the 212 μm, 300 μm, and 500 μm, respectively. An ANOVA on the effect of particle size on ethanol yield shows a significant difference at 5% level of significance. The study demonstrated that the use of locally produced yeast and increasing the surface area of sawdust increase bioethanol yield. Hence, it was concluded that better yeast strain use and biomass particle size reduction to a level that allows the optimal surface area for the reaction improve the yield of bioethanol. The study outcome can help in ameliorating the continued dependence on fossil fuels and the food security problems arising from displacing or utilizing food for fuel and could also encourage commercial-scale cellulosic ethanol production from waste.

Effects of discharge characteristics and evaluation of pressure of shock wave on underwater discharge as pretreatment for enzymatic saccharification

Cellulosic bioethanol produced from wood is a candidate for alternative fuel to fossil fuels. In the cellulosic ethanol production process, pretreatment before enzymatic saccharification should be introduced for acceleration of the process. We proposed a new pretreatment technique for the cleavage of inter-cellulose hydrogen bonds by underwater discharge shock waves. In this study, we quantitatively analyzed the relationship among discharge conditions, shock wave properties, and treatment efficiency to estimate the optimal treatment conditions. The shock wave pressure was measured at several MPa, and optical microscopy results suggested wood flour fragmentation. The relationship between the discharge characteristics and the treatment effect was investigated by measuring the water retention value as an indicator of hydrogen bond cleavage and the amount of glucose as bioethanol intermediate products. The discharge energy of 0.85–1.16 J/pulse did not affect the treatment effect, while an increased discharge frequency in the range of 3.3–7.3 Hz did. The independence of treatment effect on the discharge energy was investigated to arise from the saturation in the discharge-current rising rate and shock-wave pressure.

Developing Supportive Bioeconomy Policies

Developing Supportive Bioeconomy Policies

Target integration of an exogenous β-glucosidase enhances cellulose degradation and ethanol production in Clostridium cellulolyticum

The bacteria Clostridium cellulolyticum is a promising candidate for consolidated bioprocessing (CBP). However, genetic engineering is necessary to improve this organism’s cellulose degradation and bioconversion efficiencies to meet standard industrial requirements. In this study, CRISPR-Cas9n was used to integrate an efficient β-glucosidase into the genome of C. cellulolyticum, disrupting lactate dehydrogenase (ldh) expression and reducing lactate production. The engineered strain showed a 7.4-fold increase in β-glucosidase activity, a 70% decrease in ldh expression, a 12% increase in cellulose degradation, and a 32% increase in ethanol production compared to wild type. Additionally, ldh was identified as a potential site for heterologous expression. These results demonstrate that simultaneous β-glucosidase integration and lactate dehydrogenase disruption is an effective strategy for increasing cellulose to ethanol bioconversion rates in C. cellulolyticum.

Which crop has the highest bioethanol yield in the United States?

Annual U.S. production of bioethanol, primarily produced from corn starch in the U.S. Midwest, rose to 57 billion liters in 2021, which fulfilled the required conventional biofuel target set forth by the Energy Independence and Security Act (EISA) of 2007. At the same time, the U.S. fell short of the cellulosic or advanced biofuel target of 79 billion liters. The growth of bioenergy grasses (e.g., Miscanthus and switchgrass) across the Central and Eastern U.S. has the potential to feed enhanced cellulosic bioethanol production and, if successful, increase renewable fuel volumes. However, water consumption and climate change and its extremes are critical concerns in corn and bioenergy grass productivity. These concerns are compounded by the demands on potentially productive land areas and water devoted to producing biofuels. This is a fundamental Food-Energy-Water System (FEWS) nexus challenge. We apply a computational framework to estimate potential bioenergy yield and conversion to bioethanol yield across the U.S., based on crop field studies and conversion technology analysis for three crops—corn, Miscanthus, and two cultivars of switchgrass (Cave-in-Rock and Alamo). The current study identifies regions where each crop has its highest yield across the Center and Eastern U.S. While growing bioenergy grasses requires more water than corn, one advantage they have as a source of bioethanol is that they control nitrogen leaching relative to corn. Bioenergy grasses also maintain steadily high productivity under extreme climate conditions, such as drought and heatwaves in the year 2012 over the U.S. Midwest, because the perennial growing season and the deeper and denser roots can ameliorate the soil water stress. While the potential ethanol yield could be enhanced using energy grasses, their practical success in becoming a potential source of ethanol yield remains limited by socio-economic and operational constraints and concerns regarding competition with food production.

Statistics-Based Optimization of Cellulase and Xylanase Production by the Endophytic Fungus Talaromyces Funiculosus using Agricultural Waste Materials

Lignocellulosic biomass can be utilized as a low-cost, renewable, and sustainable feedstock for obtaining non-fossil energy sources with low CO2 emission. One of the most promising technologies for producing 2G biofuels is the saccharification of agricultural waste materials with the help of cellulolytic enzymes, followed by yeast fermentation of sugars into cellulosic ethanol. Cellulases are multi-component enzymes involved in the degradation of cellulose, which can synergistically degrade cellulose and includes three major categories: endoglucanase (EC 3.2.1.4), exoglucanase or cellobiohydrolase (EC 3.2.1.91), and β-glucosidase (EC 3.2.1.21). The core enzyme used for the degradation of the xylan skeleton of hemicellulose is endo-β-1,4-xylanase (EC 3.2.1.8). The high cost of enzymes synthesized by fungi is a bottleneck for the production of cellulosic ethanol. Optimization of the nutrient medium composition is an important factor in increasing the production of enzymes and the efficiency of lignocellulosic biomass hydrolysis. The aim of the current study was to optimize the production of cellulolytic and xylanolytic enzymes through cultivation of filamentous fungus Talaromyces funiculosus on low-cost nutrient media with non-pretreated agricultural waste materials. Methods. Filamentous fungus Talaromyces funiculosus was grown on potato-dextrose agar for 10—14 days at 26±2 °С. To obtain the culture filtrate, the fungus was cultivated under submerged conditions in an Erlenmeyer flask for 4 days. The nutrient medium composition was varied according to the factor experiment design. A two-step optimization of the nutrient medium composition was used. A screening experiment with the Plackett-Burman fractional factorial design and response surface methodology with the Box-Behnken design were used to optimize cellulase production. The enzymatic activity was determined by measuring the reduced sugar production after the enzymes hydrolysis with specific substrates: exoglucanase with filter paper, endoglucanase with carboxymethylcellulose, and xylanase with beech wood xylan, using the colorimetric DNS method with glucose or xylose as a standard. The activity of β-glucosidase was determined by the hydrolysis reaction of p-nitrophenyl-β-D-glucopyranoside, which results in the formation of p-nitrophenol, quantified at 410 nm. Results. As a result of experiments with using agricultural waste, including wheat straw, corn stalk, and corn cob as carbon sources of the culture medium, it was shown that T. funiculosus is able to grow and produce cellulase and xylanase on all non-pretreated substrates studied. The two-step sequential optimization of the nutrient medium composition for T. funiculosus cultivation according to the Plackett-Berman and Box-Behnken designs made it possible to increase the activity of cellulolytic and xylanolytic enzymes by 2.4—2.6 times. The optimized cultivation medium does not contain such expensive components as Avicel, peptone, and yeast extract and has the following composition, g/L: corn stalks — 50.0; urea — 0.86; NaNO3 — 1.0; KH2PO4 — 6.0; KCl — 0.25; MgSO4 — 0.25; FeSO4 — 0.01. Conclusions. The studied strain of T. funiculosus produces a lignocellulosic enzyme complex with a high level of β-glucosidase activity when cultivated on an optimized nutrient medium with untreated agricultural waste and is promising for the conversion of lignocellulosic biomass into fermentable sugars.

Effect of Laccase Detoxification on Bioethanol Production from Liquid Fraction of Steam-Pretreated Olive Tree Pruning

During lignocellulosic bioethanol production, the whole slurry obtained by steam explosion is filtered, generating a water-insoluble fraction rich in cellulose which is used for saccharification and ethanol fermentation, as well as a liquid fraction containing solubilised glucose and xylose but also some inhibitory by-products (furan derivatives, weak acids and phenols), which limits its use for this purpose. Since utilization of this liquid fraction to ethanol is essential for an economically feasible cellulosic ethanol process, this work studied a laccase from Myceliophthora thermophila to detoxify the liquid fraction obtained from steam-pretreated olive tree pruning (OTP) and to overcome the effects of these inhibitors. Then, the fermentation of laccase-treated liquid fraction was evaluated on ethanol production by different Saccharomyces cerevisiae strains, including the Ethanol Red, with the capacity to ferment glucose but not xylose, and the xylose-fermenting recombinant strain F12. Laccase treatment reduced total phenols content by 87% from OTP liquid fraction, not affecting furan derivatives and weak acids concentration. Consequently, the fermentative behavior of both Ethanol Red and F12 strains was improved, and ethanol production and yields were increased. Moreover, F12 strain was capable of utilizing some xylose, which increased ethanol production (10.1 g/L) compared to Ethanol Red strain (8.6 g/L).

Full-Chain FeCl3 Catalyzation Is Sufficient to Boost Cellulase Secretion and Cellulosic Ethanol along with Valorized Supercapacitor and Biosorbent Using Desirable Corn Stalk

Cellulosic ethanol is regarded as a perfect additive for petrol fuels for global carbon neutralization. As bioethanol conversion requires strong biomass pretreatment and overpriced enzymatic hydrolysis, it is increasingly considered in the exploration of biomass processes with fewer chemicals for cost-effective biofuels and value-added bioproducts. In this study, we performed optimal liquid-hot-water pretreatment (190 °C for 10 min) co-supplied with 4% FeCl3 to achieve the near-complete biomass enzymatic saccharification of desirable corn stalk for high bioethanol production, and all the enzyme-undigestible lignocellulose residues were then examined as active biosorbents for high Cd adsorption. Furthermore, by incubating Trichoderma reesei with the desired corn stalk co-supplied with 0.05% FeCl3 for the secretion of lignocellulose-degradation enzymes in vivo, we examined five secreted enzyme activities elevated by 1.3-3.0-fold in vitro, compared to the control without FeCl3 supplementation. After further supplying 1:2 (w/w) FeCl3 into the T. reesei-undigested lignocellulose residue for the thermal-carbonization process, we generated highly porous carbon with specific electroconductivity raised by 3-12-fold for the supercapacitor. Therefore, this work demonstrates that FeCl3 can act as a universal catalyst for the full-chain enhancement of biological, biochemical, and chemical conversions of lignocellulose substrates, providing a green-like strategy for low-cost biofuels and high-value bioproducts.

Catalytic co-pyrolysis of cellulosic ethanol–processing residue with high-density polyethylene over biomass bottom ash catalyst

Catalytic co-pyrolysis of cellulosic ethanol–processing residue with high-density polyethylene over biomass bottom ash catalyst

Cellulosic Ethanol Production from Weed Biomass Hydrolysate of Vietnamosasa pusilla

Lignocellulosic biomass can be used as a renewable and sustainable energy source to help reduce the consequences of global warming. In the new energy age, the bioconversion of lignocellulosic biomass into green and clean energy displays remarkable potential and makes efficient use of waste. Bioethanol is a biofuel that can diminish reliance on fossil fuels while minimizing carbon emissions and increasing energy efficiency. Various lignocellulosic materials and weed biomass species have been selected as potential alternative energy sources. Vietnamosasa pusilla, a weed belonging to the Poaceae family, contains more than 40% glucan. However, research on the applications of this material is limited. Thus, here we aimed to achieve maximum fermentable glucose recovery and bioethanol production from weed biomass (V. pusilla). To this end, V. pusilla feedstocks were treated with varying concentrations of H3PO4 and then subjected to enzymatic hydrolysis. The results indicated that after pretreatment with different concentrations of H3PO4, the glucose recovery and digestibility at each concentration were markedly enhanced. Moreover, 87.5% of cellulosic ethanol was obtained from V. pusilla biomass hydrolysate medium without detoxification. Overall, our findings reveal that V. pusilla biomass can be introduced into sugar-based biorefineries to produce biofuels and other valuable chemicals.

High bioethanol titer and yield from phosphoric acid plus hydrogen peroxide pretreated paper mulberry wood through optimization of simultaneous saccharification and fermentation

The optimization of key simultaneous saccharification and fermentation (SSF) parameters for bioethanol production from phosphoric acid plus hydrogen peroxide pretreated paper mulberry wood was carried out under two isothermal scenarios; the yeast optimum and trade-off temperatures of 35 and 38 °C, respectively. The optimal conditions established for SSF at 35 °C (solid loading: 16%; enzyme dosage: 9.8 mg protein/g glucan; and yeast concentration: 6.5 g/L) achieved high ethanol titer and yield of 77.34 g/L and 84.60% (0.432 g/g), respectively. These corresponded to 1.2 and 1.3-folds increases, compared to the results of the optimal SSF at a relatively higher temperature of 38 °C. The information from this study would prove beneficial in reducing process energy demands to some extent, while also helping to achieve high levels of both ethanol concentration and yield that are desired in cellulosic ethanol production.

Cellulose hydrolysis and bioethanol production from various types of lignocellulosic biomass after microwave-assisted hydrotropic pretreatment

This study examined the impact of microwave-assisted hydrotropic pretreatment using sodium cumene sulfonate on the enzymatic hydrolysis of cellulose contained in pine chips, beech chips and wheat straw biomasses as well as the effectiveness of the production of bioethanol from the obtained hydrolysates. The effectiveness of enzymatic hydrolysis of cellulose contained in beech chips and wheat straw subjected to pretreatment in optimised process conditions amounted to ca. 55–60%. As a result of this, hydrolysates containing glucose in the concentration of 76–84 g/L were acquired, which is a significant achievement in the case of cellulosic hydrolysates. A lower efficiency of the process was recorded when cellulose from pine chips was used (maximum hydrolysis efficiency was 17.21 ± 0.09%), which confirms a higher resistance of softwood biomass to the process of biodegradation. The highest concentration of ethanol, at the level of 41.44 ± 0.55 g/L, was achieved through the fermentation of the medium being a hydrolysate of wheat straw after microwave-assisted hydrotropic pretreatment. The developed raw material and hydrolysate preparation method indicates the possibility of using wheat straw after microwave-assisted hydrotropic pretreatment for efficient cellulosic ethanol production using high-gravity (HG) technology.

Biomass pretreatment method affects the physicochemical properties of biochar prepared from residues of lignocellulosic ethanol production

Biomass pretreatment method affects the physicochemical properties of biochar prepared from residues of lignocellulosic ethanol production

Evolving Robust and Interpretable Enzymes for the Bioethanol Industry

AbstractObtaining a robust and applicable enzyme for bioethanol production is a dream for biorefinery engineers. Herein, we describe a general method to evolve an all‐round and interpretable enzyme that can be directly employed in the bioethanol industry. By integrating the transferable protein evolution strategy InSiReP 2.0 (In Silico guided Recombination Process), enzymatic characterization for actual production, and computational molecular understanding, the model cellulase PvCel5A (endoglucanase II Cel5A from Penicillium verruculosum) was successfully evolved to overcome the remaining challenges of low ethanol and temperature tolerance, which primarily limited biomass transformation and bioethanol yield. Remarkably, application of the PvCel5A variants in both first‐ and second‐generation bioethanol production processes (i. Conventional corn ethanol fermentation combined with the in situ pretreatment process; ii. cellulosic ethanol fermentation process) resulted in a 5.7–10.1 % increase in the ethanol yield, which was unlikely to be achieved by other optimization techniques.

Evolving Robust and Interpretable Enzymes for the Bioethanol Industry.

Obtaining a robust and applicable enzyme for bioethanol production is a dream for biorefinery engineers. Herein, we describe a general method to evolve an all-round and interpretable enzyme that can be directly employed in the bioethanol industry. By integrating the transferable protein evolution strategy InSiReP 2.0 (In Silico guided Recombination Process), enzymatic characterization for actual production, and computational molecular understanding, the model cellulase PvCel5A (endoglucanase II Cel5A from Penicillium verruculosum) was successfully evolved to overcome the remaining challenges of low ethanol and temperature tolerance, which primarily limited biomass transformation and bioethanol yield. Remarkably, application of the PvCel5A variants in both first- and second-generation bioethanol production processes (i. Conventional corn ethanol fermentation combined with the in situ pretreatment process; ii. cellulosic ethanol fermentation process) resulted in a 5.7-10.1 % increase in the ethanol yield, which was unlikely to be achieved by other optimization techniques.