Dilute-sulfuric acid pretreatment of corn stover in pilot-scale reactor

Corn stover is a domestic feedstock that has potential to produce significant quantities of fuel ethanol and other bioenergy and biobased products. However, comprehensive yield and carbon mass balance information and validated kinetic models for dilute-sulfuric acid (H₂SO₄) pretreatment of corn stover have not been available. This has hindered the estimation of process economics and also limited the ability to perform technoeconomic modeling to guide research. To better characterize pretreatment and assess its kinetics, we pretreated corn stover in a continuous 1 t/d reactor. Corn stover was pretreated at 20% (w/w) solids concentration over a range of conditions encompassing residence times of 3–12 min, temperatures of 165–195°C, and H₂SO₄ concentrations of 0.5–1.4% (w/w). Xylan conversion yield and carbon mass balance data were collected at each run condition. Performance results were used to estimate kinetic model parameters assuming biphasic hemicellulose hydrolysis and a hydrolysis mechanism incorporating formation of intermediate xylo-oligomers. In addition, some of the pretreated solids were tested in a simultaneous saccharification and fermentation (SSF) process to measure the reactivity of their cellulose component to enzymatic digestion by cellulase enzymes. Monomeric xylose yields of 69–71% and total xylose yields (monomers and oligomers) of 70–77% were achieved with performance level depending on pretreatment severity. Cellulose conversion yields in SSF of 80–87% were obtained for some of the most digestible pretreated solids.     

Pretreatment of corn stover using wet oxidation to enhance enzymatic digestibility

Corn stover is an abundant, promising raw material for fuel ethanol production. Although it has a high cellulose content, without pretreatment it resists enzymatic hydrolysis, like most lignocellulosic materials. Wet oxidation (water, oxygen, mild alkali or acid, elevated temperature and pressure) was investigated to enhance the enzymatic digestibility of corn stover. Six different combinations of reaction temperature, time, and pH were applied. The best conditions (60g/L of corn stover, 195°C, 15 min, 12 bar O₂, 2 g/L of Na₂CO₃) increased the enzymatic conversion of corn stover four times, compared to untreated material. Under these conditions 60% of hemicellulose and 30% of lignin were solubilized, whereas 90% of cellulose remained in the solid fraction. After 24-h hydrolysis at 50°C using 25 filter paper units (FPU)/g of dry matter (DM) biomass, the achieved conversion of cellulose to glucose was about 85%. Decreasing the hydrolysis temperature to 40°C increased hydrolysis time from 24 to 72 h. Decreasing the enzyme loading to 5 FPU/g of DM biomass slightly decreased the enzymatic conversion from 83.4 to 71%. Thus, enzyme loading can be reduced without significantly affecting the efficiency of hydrolysis, an important economical aspect.     

Stagewise Dilute-Acid Pretreatment and Enzyme Hydrolysis of Distillers’ Grains and Corn Fiber

Distillers’ grains and corn fiber are the coproducts of the corn dry grind and wet milling industries, respectively. Availability of distillers’ grains and corn fiber at the ethanol plant and their high levels of lignocellulosic material make these coproducts attractive feedstocks for conversion to ethanol. In this study, dilute sulfuric acid hydrolysis of these coproducts was investigated in a multistage scheme. After the completion of each pretreatment stage, the liquid substrate was separated and reused in the succeeding pretreatment stage with a fresh substrate. The substrate from each stage was also subjected to enzyme hydrolysis in a separate experiment. The sulfuric acid concentration and the substrate loading were maintained at 1.0 vol% and 15.0 wt.%, respectively, and the temperature was maintained at 120 °C in all the experiments. Experiments were also performed to study the effect of removing oil from the samples prior to the pretreatment. The highest concentration of monomeric sugars (MS) was observed when three stages of pretreatment were followed by the enzyme reaction. The enzyme hydrolysis of the three-stage pretreated dried distillers’ grains and corn fiber yielded 122.6 ± 5.8 and 184.5 ± 4.1 mg/mL of MS, respectively. The formation of inhibitory products was also monitored.     

Enhancing the enzymatic hydrolysis of cellulosic materials using simultaneous ball milling

One of the limiting factors restricting the effective and efficient bioconversion of softwood-derived lignocellulosic residues is the recalcitrance of the substrate following pretreatment. Consequently, the ensuing enzymatic process requires relatively high enzyme loadings to produce monomeric carbohydrates that are readily fermentable by ethanologenic microorganisms. In an attempt to circumvent the need for larger enzyme loadings, a simultaneous physical and enzymatic hydrolysis treatment was evaluated. A ball-mill reactor was used as the digestion vessel, and the extent and rate of hydrolysis were monitored. Concurrently, enzyme adsorption profiles and the rate of conversion during the course of hydrolysis were monitored. α-Cellulose, employed as a model substrate, and SO₂-impregnated steam-exploded Douglas-fir wood chips were assessed as the cellulosic substrates. The softwood-derived substrate was further posttreated with water and hot alkaline hydrogen peroxide to remove >90% of the original lignin. Experiments at different reaction conditions were evaluated, including substrate concentration, enzyme loading, reaction volumes, and number of ball beads employed during mechanical milling. It was apparent that the best conditions for the enzymatic hydrolysis of α-cellulose were attained using a higher number of beads, while the presence of air-liquid interface did not seem to affect the rate of saccharification. Similarly, when employing the lignocellulosic substrate, up to 100% hydrolysis could be achieved with a minimum enzyme loading (10 filter paper units/g of cellulose), at lower substrate concentrations and with a greater number of reaction beads during milling. It was apparent that the combined strategy of simultaneous ball milling and enzymatic hydrolysis could improve the rate of saccharification and/or reduce the enzyme loading required to attain total hydrolysis of the carbohydrate moieties.     

Ethanol Production from Cashew Apple Bagasse: Improvement of Enzymatic Hydrolysis by Microwave-Assisted Alkali Pretreatment

In this work, the potential of microwave-assisted alkali pretreatment in order to improve the rupture of the recalcitrant structures of the cashew able bagasse (CAB), lignocellulosic by-product in Brazil with no commercial value, is obtained from cashew apple process to juice production, was studied. First, biomass composition of CAB was determined, and the percentage of glucan and lignin was 20.54 ± 0.70% and 33.80 ± 1.30%, respectively. CAB content in terms of cellulose, hemicelluloses, and lignin, 19.21 ± 0.35%, 12.05 ± 0.37%, and 38.11 ± 0.08%, respectively, was also determined. Results showed that, after enzymatic hydrolysis, alkali concentration exerted influence on glucose formation, after pretreatment with 0.2 and 1.0 mo L⁻¹ of NaOH (372 ± 12 and 355 ± 37 mg g_glucan⁻¹) when 2% (w/v) of cashew apple bagasse pretreated by microwave-assisted alkali pretreatment (CAB-M) was used. On the other hand, pretreatment time (15–30 min) and microwave power (600–900 W) exerted no significant effect on hydrolysis. On enzymatic hydrolysis step, improvement on solid percentage (16% w/v) and enzyme load (30 FPU g_CAB-M⁻¹) increased glucose concentration to 15 g L⁻¹. The fermentation of the hydrolyzate by Saccharomyces cerevesiae resulted in ethanol concentration and productivity of 5.6 g L⁻¹ and 1.41 g L⁻¹ h⁻¹, respectively.     

Chemical pretreatments of corn stover for enhancing enzymatic digestibility

Corn stover, the most abundant agricultural residue in Hungary, is a potential raw material for the production of fuel ethanol as a result of its high content of carbohydrates, but a pretreatment is required for its efficient hydrolysis. In this article, we describe the results using various chemicals such as dilute H₂SO₄, HCl, and NaOH separately as well as consecutively under relative mild conditions (120°C, 1h). Pretreatment with 5% H₂SO₄ or 5% HCl solubilized 85% of the hemicellulose fraction, but the enzymatic conversion of pretreated materials increased only two times compared to the untreated corn stover. Applying acidic pretreatment following a 1-d soaking in base achieved enzymatic conversion that was nearly the theoretical maximum (95.7%). Pretreatment with 10% NaOH decreased the lignin fraction >95%, increased the enzymatic conversion more than four times, and gave a 79.4% enzymatic conversion. However, by increasing the reaction time, the enzymatic degradability could also be increased significantly, using a less concentrated base. When the time of pretreatment was increased three times (0.5% NaOH at 120°C), the amount of total released sugars was 47.9 g from 100 g (dry matter) of untreated corn stover.     

Influence of ionic-liquid incubation temperature on changes in cellulose structure,biomass composition,and enzymatic digestibility

Varying ionic liquid, 1-ethyl 3-methyl imidazolium acetate, pretreatment incubation temperature on lignocellulosic biomass substrates, corn stover, switchgrass and poplar, can have dramatic effects on the enzymatic digestibility of the resultant regenerated biomass. In order to delineate the chemical and physical changes resulting from the pretreatment process and correlate changes with enzymatic digestibility, X-ray powder and fiber diffraction, ¹³C cross polarization/magic angle spinning nuclear magnetic resonance spectroscopy, and compositional analysis was completed on poplar, corn stover and switchgrass samples. Optimal pretreatment incubation temperatures were most closely associated with the retention of amorphous substrates upon drying of regenerated biomass. Maximal glucan to glucose conversion for 24 h enzyme hydrolysis was observed for corn stover, switchgrass and poplar at ionic liquid incubation temperatures of 100, 110 and 120 °C, respectively. We hypothesize that effective pretreatment temperatures must attain lignin redistribution and retention of xylan for optimal enzyme digestibility.     

Effect of sulfuric and phosphoric acid pretreatments on enzymatic hydrolysis of corn stover

The pretreatment of corn stover with H₂SO₄ and H₃PO₄ was investigated. Pretreatments were carried out from 30 to 120 min in a batch reactor at 121°C, with acid concentrations ranging from 0 to 2% (w/v) at a solid concentration of 5% (w/v). Pretreated corn stover was washed with distilled water until the filtrate was adjusted to pH 7.0, followed by surfactant swelling of the cellulosic fraction in a 0–10% (w/v) solution of Tween-80 at room temperature for 12 h. The dilute acid treatment proved to be a very effective method in terms of hemicellulose recovery and cellulose digetibility. Hemicellulose recovery was 62–90%, and enzymatic digestibility of the cellulose that remained in the solid was >80% with 2% (w/v) acid. In all cases studied, the performance of H₂SO₄ pretreatment (hemicellulose recovery and cellulose digestibility) was significantly better than obtained with H₃PO₄. Enzymatic hydrolysis was more effective using surfactant than without it, producing 10–20% more sugar. Furthermore, digestibility was investigated as a function of hemicellulose removal. It was found that digestibility was more directly related to hemicellulose removal than to delignification.     

Hydrolytic Enzyme of Cellulose for Complex Formulation Applied Research

To improve the enzymatic hydrolytic efficiency and reduce the supplementation of enzymes, the mixture designed experimental approach was used to optimize the composition of enzyme mixture and promote the hydrolysis of ball-milled corn stover. From the experimental results, a synergistic effect was found when combinations of the three enzymes, two kinds of cellulases and a kind of xylanase, were used. The optimal hydrolysis of pretreated corn stover accorded with enzymes activity ration of FPU/CMCase/β-glucosidase/xylanase = 4.4:1:75:829, and the hydrolysis efficiency of corn stover increased significantly compared with using individual enzyme. The results indicated that the mixture design experiment could be an effective tool for optimized enzyme mixture for lignocellulose hydrolysis.     

Pretreatment of Corn Stover Silage with Fe(NO<Subscript>3</Subscript>)<Subscript>3</Subscript> for Fermentable Sugar Production

Corn stover silage is an attractive raw material for the production of biofuels and chemicals due to its high content of carbohydrates and easy degradability. The effects of Fe(NO₃)₃ pretreatment conditions on sugar yields were investigated for corn stover silage. In addition, a combined severity factor was used to evaluate the effect of pretreatment conditions on the concentration of total sugars and inhibitors. Optimum pretreatment condition was obtained at 150 °C for 10 min with 0.05 M Fe(NO₃)₃, at which the yields of soluble xylose and glucose in liquid achieved 91.80% of initial xylose, 96.74% of initial arabinose and 19.09% of initial glucose, respectively, meanwhile, 91.84% of initial xylose, 98.24% of initial arabinose, and 19.91% of initial glucose were removed. In addition, a severity analysis showed that the maximum sugar concentration of 33.48 g/l was achieved at combined severity parameter value of 0.62, while the inhibitor concentration was only 0.03 g/l. Fe(NO₃)₃ is an effective catalyst to enhance hemicellulose hydrolysis in corn stover silage, the yields of monomeric xylose in the liquid fraction reached as high as 91.06% of initial xylose and 96.22% of initial arabinose, respectively.     

Depolymerization of Chitosan with a Crude Cellulase Preparation from <Emphasis Type="Italic">Aspergillus Niger</Emphasis>

A crude cellulase preparation from Aspergillus niger was used to depolymerize chitosan. The depolymerization process was followed by measuring the apparent viscocity and the intrinsic viscosity. The optimum conditions for enzymatic hydrolysis were investigated. On the selected optimum conditions (pH 5.0, temperature 50 °C, and an enzyme to substrate ratio of 1:5), chitosan was hydrolyzed for 1, 4, 8, and 24 h, its viscosity-average molecular weights were 3.49 × 10⁴, 1.18 × 10⁴, 5.83 × 10³, and 1.13 × 10³, respectively. Compared with chitosan having viscosity-average molecular weight of 5.18 × 10⁵ before enzymatic hydrolysis, the crude cellulase preparation had rather apparent effect on depolymerization of chitosan. Through the comparison of different origin of cellulases, the prepared cellulase has good ability of enzymatic hydrolysis. The reproducibility and reversibility for enzymatic hydrolysis was appraised. The data are of value for the production of low-molecular weight chitosans and chitooligomers of medical and biotechnological interest.     

Enhancing the Enzymatic Hydrolysis of Corn Stover by an Integrated Wet-milling and Alkali Pretreatment

An integrated wet-milling and alkali pretreatment was applied to corn stover prior to enzymatic hydrolysis. The effects of NaOH concentration in the pretreatment on crystalline structure, chemical composition, and reducing-sugar yield of corn stover were investigated, and the mechanism of increasing reducing-sugar yield by the pretreatment was discussed. The experimental results showed that the crystalline structure of corn stover was disrupted, and lignin was removed, while cellulose and hemicellulose were retained in corn stover by the pretreatment with 1% NaOH in 1 h. The reducing-sugar yield from the pretreated corn stovers increased from 20.2% to 46.7% when the NaOH concentration increased from 0% to 1%. The 1% NaOH pretreated corn stover had a holocellulose conversion of 55.1%. The increase in reducing-sugar yield was related to the crystalline structure disruption and delignification of corn stover. It was clarified that the pretreatment significantly enhanced the conversion of cellulose and hemicellulose in the corn stover to sugars.     

Cellulose solvent-based pretreatment for corn stover and avicel: concentrated phosphoric acid versus ionic liquid [BMIM]Cl

Since cellulose accessibility has become more recognized as the major substrate characteristic limiting hydrolysis rates and glucan digestibilities, cellulose solvent-based lignocellulose pretreatments have gained attention. In this study, we employed cellulose solvent- and organic solvent-based lignocellulose fractionation using two cellulose solvents: concentrated phosphoric acid [~85?% (w/w) H₃PO₄] and an ionic liquid Butyl-3-methylimidazolium chloride ([BMIM]Cl). Enzymatic glucan digestibilities of concentrated phosphoric acid- and [BMIM]Cl-pretreated corn stover were 96 and 55?% after 72?h at five filter paper units of cellulase per gram of glucan, respectively. Regenerated amorphous cellulose by concentrated phosphoric acid and [BMIM]Cl had digestibilities of 100 and 92?%, respectively. Our results suggested that differences in enzymatic glucan digestibilities of concentrated phosphoric acid- and [BMIM]Cl-pretreated corn stover were attributed to combinatory factors. These results provide insights into mechanisms of cellulose solvent-based pretreatment and effects of residual cellulose solvents and lignin on enzymatic cellulose hydrolysis.     

Pretreatment of corn stover with acidic electrolyzed water and FeCl3 leads to enhanced enzymatic hydrolysis

Pretreatment has been viewed as the most efficient strategy for lignocellulosic biomass-to-fermentable sugars conversion. In this study a novel pretreatment with acidic electrolyzed water (AEW) and FeCl₃ was proposed and tested to deconstruct the recalcitrance of corn stover and enhance its subsequent cellulose-to-sugar conversion. The effects of AEW pH and FeCl₃ concentration on hemicellulose degradation were investigated, and the results showed the highest hemicellulose removal (93.40 %) and recovery (93.04 %) were achieved at AEW pH 2.30 and FeCl₃ concentration 0.05 mol/L. Further research on the properties of AEW solutions with FeCl₃, including their pH, ORP, and DO revealed the synergistic effects of strong acidity and high oxidizing capacity of the solution could boost hemicellulose breakup and enhance the enzymatic hydrolysis of cellulose (92.00 %) by removing most of hemicellulose and increasing the accessibility and digestibility of cellulose. Therefore, these studies prove AEW coupled with FeCl₃ pretreatment is an effective and promising approach in biomass-to-biofuel process.     

Effects of temperature and moisture on dilute-acid steam explosion pretreatment of corn stover and cellulase enzyme digestibility

Corn stover is emerging as a viable feedstock for producing bioethanol from renewable resources. Dilute-acid pretreatment of corn stover can solubilize a significant portion of the hemicellulosic component and enhance the enzymatic digestibility of the remaining cellulose for fermentation into ethanol. In this study, dilute H₂SO₄ pretreatment of corn stover was performed in a steam explosion reactor at 160°C, 180°C, and 190°C, approx 1 wt% H₂SO₄, and 70-s to 840-s residence times. The combined severity (Log₁₀ [R _o ] - pH), an expression relating pH, temperature, and residence time of pretreatment, ranged from 1.8 to 2.4. Soluble xylose yields varied from 63 to 77% of theoretical from pretreatments of corn stover at 160 and 180°C. However, yields >90% of theoretical were found with dilute-acid pretreatments at 190°C. A narrower range of higher combined severities was required for pretreatment to obtain high soluble xylose yields when the moisture content of the acid-impregnated feedstock was increased from 55 to 63 wt%. Simultaneous saccharification and fermentation (SSF) of washed solids from corn stover pretreated at 190°C, using an enzyme loading of 15 filter paper units (FPU)/g of cellulose, gave ethanol yields in excess of 85%. Similar SSF ethanol yields were found using washed solid residues from 160 and 180°C pretreatments at similar combined severities but required a higher enzyme loading of approx 25 FPU/g of cellulose.     

Determination of glucose and ethanol after enzymatic hydrolysis and fermentation of biomass using Raman spectroscopy

Raman spectroscopy has been used for the quantitative determination of the conversion efficiency at each step in the production of ethanol from biomass. The method requires little sample preparation; therefore, it is suitable for screening large numbers of biomass samples and reaction conditions in a complex sample matrix. Dilute acid or ammonia-pretreated corn stover was used as a model biomass for these studies. Ammonia pretreatment was suitable for subsequent measurements with Raman spectroscopy, but dilute acid-pretreated corn stover generated a large background signal that surpassed the Raman signal. The background signal is attributed to lignin, which remains in the plant tissue after dilute acid pretreatment. A commercial enzyme mixture was used for the enzymatic hydrolysis of corn stover, and glucose levels were measured with a dispersive 785 nm Raman spectrometer. The glucose detection limit in hydrolysis liquor by Raman spectroscopy was 8 g L⁻¹. The mean hydrolysis efficiency for three replicate measurements obtained with Raman spectroscopy (86 ± 4%) was compared to the result obtained using an enzymatic reaction with UV-vis spectrophotometry detection (78 ± 8%). The results indicate good accuracy, as determined using a Student's t-test, and better precision for the Raman spectroscopy measurement relative to the enzymatic detection assay. The detection of glucose in hydrolysis broth by Raman spectroscopy showed no spectral interference, provided the sample was filtered to remove insoluble cellulose prior to analysis. The hydrolysate was further subjected to fermentation to yield ethanol. The detection limit for ethanol in fermentation broth by Raman spectroscopy was found to be 6 g L⁻¹. Comparison of the fermentation efficiencies measured by Raman spectroscopy (80 ± 10%) and gas chromatrography-mass spectrometry (87 ± 9%) were statistically the same. The work demonstrates the utility of Raman spectroscopy for screening the entire conversion process to generate lignocellulosic ethanol.     

Summary of findings from the Biomass Refining Consortium for Applied Fundamentals and Innovation (CAFI): corn stover pretreatment

The Biomass Refining Consortium for Applied Fundamentals and Innovation, with members from Auburn University, Dartmouth College, Michigan State University, the National Renewable Energy Laboratory, Purdue University, Texas A&M University, the University of British Columbia, and the University of California at Riverside, has developed comparative data on the conversion of corn stover to sugars by several leading pretreatment technologies. These technologies include ammonia fiber expansion pretreatment, ammonia recycle percolation pretreatment, dilute sulfuric acid pretreatment, flowthrough pretreatment (hot water or dilute acid), lime pretreatment, controlled pH hot water pretreatment, and sulfur dioxide steam explosion pretreatment. Over the course of two separate USDA- and DOE-funded projects, these pretreatment technologies were applied to two different corn stover batches, followed by enzymatic hydrolysis of the remaining solids from each pretreatment technology using identical enzyme preparations, enzyme loadings, and enzymatic hydrolysis assays. Identical analytical methods and a consistent material balance methodology were employed to develop comparative sugar yield data for each pretreatment and subsequent enzymatic hydrolysis. Although there were differences in the profiles of sugar release, with the more acidic pretreatments releasing more xylose directly in the pretreatment step than the alkaline pretreatments, the overall glucose and xylose yields (monomers + oligomers) from combined pretreatment and enzymatic hydrolysis process steps were very similar for all of these leading pretreatment technologies. Some of the water-only and alkaline pretreatment technologies resulted in significant amounts of residual xylose oligomers still remaining after enzymatic hydrolysis that may require specialized enzyme preparations to fully convert xylose oligomers to monomers.     

Ethanol Production from Residual Wood Chips of Cellulose Industry: Acid Pretreatment Investigation,Hemicellulosic Hydrolysate Fermentation,and Remaining Solid Fraction Fermentation by SSF Process

Current research indicates the ethanol fuel production from lignocellulosic materials, such as residual wood chips from the cellulose industry, as new emerging technology. This work aimed at evaluating the ethanol production from hemicellulose of eucalyptus chips by diluted acid pretreatment and the subsequent fermentation of the generated hydrolysate by a flocculating strain of Pichia stipitis. The remaining solid fraction generated after pretreatment was subjected to enzymatic hydrolysis, which was carried out simultaneously with glucose fermentation [saccharification and fermentation (SSF) process] using a strain of Saccharomyces cerevisiae. The acid pretreatment was evaluated using a central composite design for sulfuric acid concentration (1.0–4.0 v/v) and solid to liquid ratio (1:2–1:4, grams to milliliter) as independent variables. A maximum xylose concentration of 50 g/L was obtained in the hemicellulosic hydrolysate. The fermentation of hemicellulosic hydrolysate and the SSF process were performed in bioreactors and the final ethanol concentrations of 15.3 g/L and 28.7 g/L were obtained, respectively.     

Influence of High Solid Concentration on Enzymatic Hydrolysis and Fermentation of Steam-Exploded Corn Stover Biomass

Steam-exploded corn stover biomass was used as the substrate for fed-batch separate enzymatic hydrolysis and fermentation (SHF) to investigate the solid concentration ranging from 10% to 30% (w/w) on the lignocellulose enzymatic hydrolysis and fermentation. The treatment of washing the steam-exploded material was also evaluated by experiments. The results showed that cellulose conversion changed little with increasing solid concentration, and fermentation by Saccharomyces cerevisiae revealed a nearly same ethanol yield with the water-washed steam-exploded corn stover. For the washed material at 30% substrate concentration, i.e., 30% water insoluble solids (WIS), enzymatic hydrolysis yielded 103.3 g/l glucose solution and a cellulose conversion of 72.5%, thus a high ethanol level up to 49.5 g/l. With the unwashed steam-exploded corn stover, though a cellulose conversion of 70.9% was obtained in hydrolysis at 30% solid concentration (27.9% WIS), its hydrolysate did not ferment at all, and the hydrolysate of 20% solid loading containing 3.3 g/l acetic acid and 145 mg/l furfural already exerted a strong inhibition on the fermentation and ethanol production.     

Pretreatment of corn stover by soaking in aqueous ammonia

Soaking in aqueous ammonia (SAA) was investigated as a pretreatment method for corn stover. In this method, the feedstock was soaked in aqueous ammonia over an extended period (10–60 d) at room temperature. It was done without agitation at atmospheric pressure. SAA treatment removed 55–74% of the lignin, but retained nearly 100% of the glucan and 85% of the xylan. The xylan remaining in the corn stover after SAA treatment was hydrolyzed along with the glucan by xylanase present in the Spezyme CP enzyme. In the simultaneous saccharification and fermentation (SSF) test of SAA-treated corn stover, using S. cerevisiae (D₅A), an ethanol yield of 73% of theoretical maximum was obtained on the basis of the glucan content in the treated corn stover. The accumulation of xylose in the SSF appears to inhibit the cellulase activity on glucan hydrolysis, which limits the yield of ethanol. In the simultaneous saccharification and co-fermentation (SSCF) test, using recombinant E. coli (KO11), both the glucan and xylose were effectively utilized, resulting in on overall ethanol yield of 77% based on the glucan and xylan content of the substrate. When the SSCF process is used, the fact that the xylan fraction is retained during pretreatment is a desirable feature since the overall bioconversion can be carried out in a single step without separate recovery of xylose from the pretreatment liquid.