Comparison of SO2 and H2SO4 impregnation of softwood prior to steam pretreatment on ethanol production

The pretreatment of softwood with sulfuric acid impregnation in the production of ethanol, based on enzymatic hydrolysis, has been investigated. The parameters investigated were: H₂SO₄ concentration (0.5 – 4.4% w/w liquid), temperature (180 – 240°C), and residence time (1-20 minutes). The combined severity (log Ro-pH) was used to combine the parameters into a single reaction ordinate. The highest yields of fermentable sugars, i.e., glucose and mannose, were obtained at a combined severity of 3. At this severity, however, the fermentability declined and the ethanol yield decreased. In a comparison with previous results, SO₂ impregnation was found to be preferable, since it resulted in approximately the same sugar yields, but better fermentability.     

Steam pretreatment of Salix with and without SO2 impregnation for production of bioethanol

In the wood-to-ethanol process, pretreatment of the material is necessary prior to enzymatic hydrolysis to obtain high overall yields of sugar and ethanol. Steam pretreatment of fast-growing Salix either with or without SO₂ impregnation has been investigated by varying different parameters. Overall glucose yields of above 90% and overall xylose yields higher than 80% were obtained both with and without impregnation. However, the most favorable pretreatment conditions for the separate yields of glucose and xylose differed to a lower degree using SO₂-impregnated wood chips, resulting in higher total sugar yield than that obtained with non-impregnated wood chips.     

Comparison of the fermentability of enzymatic hydrolyzates of sugarcane bagasse pretreated by steam explosion using different impregnating agents

Sugarcane bagasse is a potential lignocellulosic feedstock for ethanol production, since it is cheap, readily available, and has a high carbohydrate content. In this work, bagasse was subjected to steam explosion pretreatment with different impregnation conditions. Three parallel pretreatments were carried out, one without any impregnation, a second with sulfur dioxide, and a third with sulfuric acid as the impregnating agent. The pretreatments were performed at 205°C for 10 min. The pretreated material was then hydrolyzed using celluloytic enzymes. The chemical composition of the hydrolyzates was analyzed. The highest yields of xylose (16.2 g/100 g dry bagasse), arabinose (1.5 g/100 g), and total sugar (52.9 g/100 g) were obtained in the hydrolysis of the SO₂-impregnated bagasse. The H₂SO₄-impregnated bagasse gave the highest glucose yield (35.9 g/100 g) but the lowest total sugar yield (42.3 g/100 g) among the three methods. The low total sugar yield from the H₂SO₄-impregnated bagasse was largely due to by-product formation, as the dehydration of xylose to furfural. Sulfuric acid impregnation led to a three-fold increase in the concentration of the fermentation inhibitors furfural and 5-hydroxymethylfurfural (HMF) and a two-fold increase in the concentration of inhibitory aliphatic acids (formic, acetic, and levulinic acids) compared to the other two pretreatment methods. The total content of phenolic compounds was not strongly affected by the different pretreatment methods, but the quantities of separate phenolic compounds were widely different in the hydrolyzate from the H₂SO₄-impregnated bagasse compared with the other two hydrolyzates. No major differences in the content of inhibitors were observed in the hydrolyzates obtained from SO₂-impregnated and non-impregnated bagasse. The fermentability of all three hydrolyzates was tested with a xylose-utilizing Saccharomyces cerevisiae strain with and without nutrient supplementation. The hydrolyzates of SO₂-impregnated and nonimpregnated bagasse showed similar fermentability, whereas the hydrolyzate of H₂SO₄-impregnated bagasse fermented considerably poorer.     

Optimization of steam pretreatment of SO2-impregnated corn stover for fuel ethanol production

In this study, corn stover with a dry matter content of 20% was impregnated with SO₂ and then steam pretreated for various times at various temperatures. The pretreatment was evaluated by enzymatic hydrolysis of the solid material and analysis of the sugar content in the liquid. The maximum overall yield of glucose, 89% of the theoretical based on the glucan in the raw material, was achieved when the corn stover was pretreated at 200°C for 10 min. The maximum overall yield of xylose, 78%, was obtained with pretreatment at 190°C for 5 min.     

Comparison of Different Pretreatment Strategies for Enzymatic Hydrolysis of Wheat and Barley Straw

In biomass-to-ethanol processes a physico-chemical pretreatment of the lignocellulosic biomass is a critical requirement for enhancing the accessibility of the cellulose substrate to enzymatic attack. This report evaluates the efficacy on barley and wheat straw of three different pretreatment procedures: acid or water impregnation followed by steam explosion versus hot water extraction. The pretreatments were compared after enzyme treatment using a cellulase enzyme system, Celluclast 1.5 L from Trichoderma reesei, and a beta-glucosidase, Novozyme 188 from Aspergillus niger. Barley straw generally produced higher glucose concentrations after enzymatic hydrolysis than wheat straw. Acid or water impregnation followed by steam explosion of barley straw was the best pretreatment in terms of resulting glucose concentration in the liquid hydrolysate after enzymatic hydrolysis. When the glucose concentrations obtained after enzymatic hydrolyses were related to the potential glucose present in the pretreated residues, the highest yield, approximately 48% (g g-1), was obtained with hot water extraction pretreatment of barley straw; this pretreatment also produced highest yields for wheat straw, producing a glucose yield of approximately 39% (g g-1). Addition of extra enzyme (Celluclast 1.5 L+Novozyme 188) during enzymatic hydrolysis resulted in the highest total glucose concentrations from barley straw, 32-39 g L-1, but the relative increases in glucose yields were higher on wheat straw than on barley straw. Maldi-TOF MS analyses of supernatants of pretreated barley and wheat straw samples subjected to acid and water impregnation, respectively, and steam explosion, revealed that the water impregnated + steam-exploded samples gave a wider range of pentose oligomers than the corresponding acid-impregnated samples.     

Enhanced Enzymatic Saccharification of Barley Straw Pretreated by Ethanosolv Technology

The fermentable sugars in lignocellulosic biomass are derived from cellulose and hemicellulose, which are not readily accessible to enzymatic saccharification because of their recalcitrance. An ethanosolv pretreatment method was applied for the enzymatic saccharification of barley straw with an inorganic acid. The effects of four process variables (temperature, time, catalyst dose, and ethanol concentration) on the barley straw pretreatment were analyzed over a broad range using a small composite design and a response surface methodology. The yield of the residual solid and composition of the solid fraction differed as ethanosolv conditions varied within the experimental range. A glucan recovery, xylan recovery, and delignification were 85%, 14%, and 69% at center point conditions (170°C, 60 min, 1.0% (w/w) H₂SO₄, and 50% (w/w) ethanol), respectively. Ethanosolv pretreatment removed lignin effectively. Additionally, the highest enzymatic digestibility of 85.3% was obtained after 72 h at center point conditions.     

Enzymatic Hydrolysis and Ethanol Fermentation of High Dry Matter Wet-Exploded Wheat Straw at Low Enzyme Loading

Wheat straw was pretreated by wet explosion using three different oxidizing agents (H₂O₂, O₂, and air). The effect of the pretreatment was evaluated based on glucose and xylose liberated during enzymatic hydrolysis. The results showed that pretreatment with the use of O₂ as oxidizing agent was the most efficient in enhancing overall convertibility of the raw material to sugars and minimizing generation of furfural as a by-product. For scale-up of the process, high dry matter (DM) concentrations of 15–20% will be necessary. However, high DM hydrolysis and fermentation are limited by high viscosity of the material, higher inhibition of the enzymes, and fermenting microorganism. The wet-explosion pretreatment method enabled relatively high yields from both enzymatic hydrolysis and simultaneous saccharification and fermentation (SSF) to be obtained when performed on unwashed slurry with 14% DM and a low enzyme loading of 10 FPU/g cellulose in an industrial acceptable time frame of 96 h. Cellulose and hemicellulose conversion from enzymatic hydrolysis were 70 and 68%, respectively, and an overall ethanol yield from SSF was 68%.     

Combined use of H2SO4 and SO2 impregnation for steam pretreatment of spruce in ethanol production

Fuel ethanol can be produced from softwood through hydrolysis in an enzymatic process. Prior to enzymatic hydrolysis of the softwood, pretreatment is necessary. In this study, two-step steam pretreatment employing dilute H₂SO₄ impregnation in the first step and SO₂ impregnation in the second step, to improve the overall sugar and ethanol yield, was investigated. The first pretreatment step was performed under conditions of low severity (180°C, 10 min, 0.5% H₂SO₄) to optimize the amount of hydrolyzed hemicellulose. In the second step, the washed solid material from the first pretreatment step was impregnated with SO₂ and pretreated under conditions of higher severity to make the cellulose more accessible to enzymatic attack, as well as to hydrolyze a portion of the cellulose. A wide range of conditions was used in the second step to determine the most favorable combination. The temperatures investigated were between 190 and 230°C, the residence times were 2, 5, and 10 min; and the SO₂ concentration was 3%. The effect of pretreatment was assessed by both enzymatic hydrolysis of the solids and by simultaneous saccharification and fermentation (SSF) of the whole slurry, after the second pretreatment step. For each set of pretreatment conditions, the liquid fraction was also fermented to determine any inhibitory effects. Ethanol yield using the SSF configuration reached 66% of the theoretical value for pretreatment conditions in the second step of 210°C and 5 min. The sugar yield using the separate hydrolysis and fermentation configuration reached 71% for pretreatment conditions of 220°C and 5 min.     

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.     

Ethanol production from steam-explosion pretreated wheat straw

Bioconversion of cereal straw to bioethanol is becoming an attractive alternative to conventional fuel ethanol production from grains. In this work, the best operational conditions for steam-explosion pretreatment of wheat straw for ethanol production by a simultaneous saccharification and fermentation process were studied, using diluted acid [H₂SO₄ 0.9% (w/w)] and water as preimpregnation agents. Acid-or water-impregnated biomass was steam-exploded at different temperatures (160–200°C) and residence times (5, 10, and 20 min). Composition of solid and filtrate obtained after pretreatment, enzymatic digestibility and ethanol production of pretreated wheat straw at different experimental conditions was analyzed. The best pretreatment conditions to obtain high conversion yield to ethanol (approx 80% of theoretical) of cellulose-rich residue after steam-explosion were 190°C and 10 min or 200°C and 5 min, in acid-impregnated straw. However, 180°C for 10 min in acid-impregnated biomass provided the highest ethanol yield referred to raw material (140 L/t wheat straw), and sugars recovery yield in the filtrate (300 g/kg wheat straw).     

Pretreatment and enzymatic saccharification of corn fiber

Corn fiber consists of about 20% starch, 14% cellulose, and 35% hemicellulose, and has the potential to serve as a low-cost feedstock for production of fuel ethanol. Several pretreatments (hot water, alkali, and dilute, acid) and enzymatic saccharification procedures were evaluated for the conversion of corn fiber starch, cellulose, and hemicellulose to monomeric sugars. Hot water pretreatment (121°C, 1 h) facilitated the enzymatic sacch arification of starch and cellulose but not hemicellulose. Hydrolysis of corn fiber pretreated with alkali un dersimilar conditions by enzymatic means gave similar results. Hemicellulose and starch components were converted to monomeric sugars by dilute H₂SO₄ pretreatment (0.5–1.0%, v/v) at 121°C. Based on these findings, a method for pretreatment and enzymatic saccharification of corn fiber is presented. It in volves the pretreatment of corn fiber (15% solid, w/v) with dilute acid (0.5% H₂SO₄, v/v) at 121°C for 1 h, neutralization to pH 5.0, then saccharification of the pretreated corn fiber material with commercial cellulase and β-glucosidase preparations The yield of monomeric sugars from corn fiber was typically 85–100% of the theoretical yield. Names are necessary to report factually on available data; however, the USDA neither guarantees nor warrants the standard of the product, and the use of the name by USDA implies no approval of the product to the exclusion of others that may also be suitable.     

Optimizing Phosphoric Acid plus Hydrogen Peroxide (PHP) Pretreatment on Wheat Straw by Response Surface Method for Enzymatic Saccharification

Wheat straw was pretreated by phosphoric acid plus hydrogen peroxide (PHP), in which temperature, time, and H₃PO₄ proportion for pretreatment were investigated by using response surface method. Results indicated that hemicellulose and lignin removal positively responded to the increase of pretreatment temperature, H₃PO₄ proportion, and time. H₃PO₄ proportion was the most important variable to control cellulose recovery, followed by pretreatment temperature and time. Moreover, these three variables all negatively related to cellulose recovery. Increasing H₃PO₄ proportion can improve enzymatic hydrolysis; however, reduction on cellulose recovery results in decrease of glucose yield. Extra high temperature or long time for pretreatment was not beneficial to enzymatic hydrolysis and glucose yield. Based on the criterion for minimizing H₃PO₄ usage and maximizing glucose yield, the optimized pretreatment conditions was 40 °C, 2.0 h, and H₃PO₄ proportion of 70.2 % (H₂O₂ proportion of 5.2 %), by which glucose yielded 299 mg/g wheat straw (946.2 mg/g cellulose) after 72-h enzymatic hydrolysis.     

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.     

Optimization of steam pretreatment of corn stover to enhance enzymatic digestibility

Among the available agricultural byproducts, corn stover, with its yearly production of 10 million t (dry basis), is the most abundant promising raw material for fuel ethanol production in Hungary. In the United States, more than 216 million to fcorn stover is produced annually, of which a portion also could possibly be collected for conversion to ethanol. However, a network of lignin and hemicellulose protects cellulose, which is the major source of fermentable sugars in corn stover (approx 40% of the dry matter [DM]). Steam pretreatment removes the major part of the hemicellulose from the solid material and makes the cellulose more susceptible to enzymatic digestion. We studied 12 different combinations of reaction temperature, time, and pH during steam pretreatment. The best conditions (200°C, 5 min, 2% H₂SO₄) increased the enzymatic conversion (from cellulose to glucose) of corn stover more then four times, compared to untreated material. However, steam pretreatment at 190°C for 5 min with 2% sulfuric acid resulted in the highest overall yield of sugars, 56.1 g from 100 g of untreated material (DM), corresponding to 73% of the theoretical. The liquor following steam explosion was fermented using Saccharomyces cerevisiae to investigate the inhibitory effect of the pretreatment. The achieved ethanol yield was slightly higher than that obtained with a reference sugar solution. This demonstrates that baker's yeast could adapt to the pretreated liquor and ferment the glucose to ethanol efficiently.     

Production of Astaxanthin from Cellulosic Biomass Sugars by Mutants of the Yeast <Emphasis Type="Italic">Phaffia rhodozyma</Emphasis>

Astaxanthin is a potential high-value coproduct in an ethanol biorefinery. Three mutant strains of the astaxanthin-producing yeast Phaffia rhodozyma, which were derived from the parent strain ATCC 24202 (UCD 67-210) and designated JTM166, JTM185, and SSM19, were tested for their capability of utilizing the major sugars that can be generated from cellulosic biomass, including glucose, xylose, and arabinose, for astaxanthin production. While all three strains were capable of metabolizing these sugars, individually and in mixtures, JTM185 demonstrated the greatest sugar utilization and astaxanthin production. Astaxanthin yield by this strain (milligrams astaxanthin per gram of sugar consumed) was highest for xylose, followed by arabinose and then glucose. The kinetics of sugar utilization by strain JTM185 was studied in fermenters using mixtures of glucose, xylose, and arabinose at varied concentrations. It was found that glucose was utilized preferentially, followed by xylose, and lastly, arabinose. Astaxanthin yield was significantly affected by sugar concentrations. Highest yields were observed with sugar mixtures containing the highest concentrations of xylose and arabinose. Hydrolysates produced from sugarcane bagasse and barley straw pretreated by the soaking in aqueous ammonia method and hydrolyzed with the commercial cellulase preparation, Accellerase™ 1000, were used for astaxanthin production by the mutant strain JTM185. The organism was capable of metabolizing all of the sugars present in the hydrolysates from both biomass sources and produced similar amounts of astaxanthin from both hydrolysates, although these amounts were lower when compared to yields obtained with reagent grade sugars.     

Two-step steam pretreatment of softwood with SO2 impregnation for ethanol production

Two-step steam pretreatment of softwood was investigated with the aim of improving the enzymatic digestibility for ethanol production. In the first step, softwood was impregnated with SO₂ and steam pretreated at different severities. The first step was performed at low severity to hydrolyze the hemicellulose and release the sugars into the solution. The combination of time and temperature that yielded the highest amount of hemicellulosic sugars in the solution was determined. In the second step, the washed solid material from the optimized first step was impregnated once more with SO₂ and steam pretreated under more severe conditions to enhance the enzymatic digestibility. The investigated temperature range was between 180 and 220°C, and the residence times were 2, 5 and 10 min. The effectiveness of pretreatment was assessed by both enzymatic hydrolysis of the solids and simultaneous saccharification and fermentation (SSF) of the whole slurry after the second pretreatment step, in the presence of antibiotics. For each pretreatment combination, the liquid fraction was fermented to determine any inhibiting effects. At low severity in the second pretreatment step, a high conversion of cellulose was obtained in the enzymatic hydrolysis step, and at a high severity a high conversion of cellulose was obtained in the second pretreatment step. This resulted in an overall yield of sugars that was nearly constant over a wide range of severity. Compared with the one-step steam pretreatment, the two-step steam pretreatment resulted in a higher yield of sugar and in a slightly higher yield of ethanol. The overall sugar yield, when assessed by enzymatic hydrolysis, reached 80%. In the SSF configuration, an overall ethanol yield of 69% was attained.     

Optimizing Dilute-Acid Pretreatment of Rapeseed Straw for Extraction of Hemicellulose

Biological conversion of biomass into fuels and chemicals requires hydrolysis of the polysaccharide fraction into monomeric sugars prior to fermentation. Hydrolysis can be performed enzymatically or with mineral acids. In this study, dilute sulfuric acid was used as a catalyst for the pretreatment of rapeseed straw. The purpose of this study is to optimize the pretreatment process in a 15-mL bomb tube reactor and investigate the effects of the acid concentration, temperature, and reaction time. These parameters influence hemicellulose removal and production of sugars (xylose, glucose, and arabinose) in the hydrolyzate as well as the formation of by-products (furfural, 5-hydroxymethylfurfural, and acetic acid). Statistical analysis was based on a model composition corresponding to a 3³ orthogonal factorial design and employed the response surface methodology to optimize the pretreatment conditions, aiming to attain maximum xylan, mannan, and galactan (XMG) extraction from hemicellulose of rapeseed straw. The obtained optimum conditions were: H₂SO₄ concentration of 1.76% and temperature of 152.6 °C with a reaction time of 21 min. Under these optimal conditions, 85.5% of the total sugar was recovered after acid hydrolysis (78.9% XMG and 6.6% glucan). The hydrolyzate contained 1.60 g/L glucose, 0.61 g/L arabinose, 10.49 g/L xylose, mannose, and galactose, 0.39 g/L cellobiose, 0.94 g/L fructose, 0.02 g/L 1,6-anhydro-glucose, 1.17 g/L formic acid, 2.94 g/L acetic acid, 0.04 g/L levulinic acid, 0.04 g/L 5-hydroxymethylfurfural, and 0.98 g/L furfural.     

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.     

Consolidated Conversion of Hulled Barley into Fermentable Sugars Using Chemical,Thermal, and Enzymatic (CTE) Treatment

A novel process using chemical, thermal, and enzymatic treatment for conversion of hulled barley into fermentable sugars was developed. The purpose of this process is to convert both lignocellulosic polysaccharides and starch in hulled barley grains into fermentable sugars simultaneously without a need for grinding and hull separation. In this study, hulled barley grains were treated with 0.1 and 1.0 wt.-% sulfuric acid at various temperatures ranging from 110 to 170 °C in a 63-ml flow-through packed-bed stainless steel reactor. After sulfuric acid pretreatment, simultaneous conversion of lignocellulose and starch in the barley grains into fermentable sugars was performed using an enzyme cocktail, which included α-amylase, glucoamylase, cellulase, and β-glucosidase. Both starch and non-starch polysaccharides in the pre-treated barley grains were readily converted to fermentable sugars. The treated hulled barley grains, including their hull, were completely hydrolyzed to fermentable sugars with recovery of almost 100% of the available glucose and xylose. The pretreatment conditions of this chemical, thermal, and enzymatic (CTE) process for achieving maximum yield of fermentable sugars were 1.0 wt.% sulfuric acid and 110 °C. In addition to starch, the acid pretreatment also retained most of the available proteins in solid form, which is essential for subsequent production of fuel ethanol and high protein distiller’s dried grains with solubles co-product.     

Hybrid SSF/SHF Processing of SO<Subscript>2</Subscript> Pretreated Wheat Straw—Tuning Co-fermentation by Yeast Inoculum Size and Hydrolysis Time

Wheat straw is one of the main agricultural residues of interest for bioethanol production. This work examines conversion of steam-pretreated wheat straw (using SO₂ as a catalyst) in a hybrid process consisting of a short enzymatic prehydrolysis step and a subsequent simultaneous saccharification and fermentation (SSF) step with a xylose-fermenting strain of Saccharomyces cerevisiae. A successful process requires a balanced design of reaction time and temperature in the prehydrolysis step and yeast inoculum size and temperature in the SSF step. The pretreated material obtained after steam pretreatment at 210 °C for 5 min using 2.5 % SO₂ (based on moisture content) showed a very good enzymatic digestibility at 45 °C but clearly lower at 30 °C. Furthermore, the pretreatment liquid was found to be rather inhibitory to the yeast, partly due to a furfural content of more than 3 g/L. The effect of varying the yeast inoculum size in this medium was assessed, and at a yeast inoculum size of 4 g/L, a complete conversion of glucose and a 90 % conversion of xylose were obtained within 50 h. An ethanol yield (based on the glucan and xylan in the pretreated material) of 0.39 g/g was achieved for a process with this yeast inoculum size in a hybrid process (10 % water-insoluble solid (WIS)) with 4 h prehydrolysis time and a total process time of 96 h. The obtained xylose conversion was 95 %. A longer prehydrolysis time or a lower yeast inoculum size resulted in incomplete xylose conversion.