Ethanol production from alkaline peroxide pretreated enzymatically saccharified wheat straw

Wheat straw used in this study contained 44.24 +/- 0.28% cellulose and 25.23 +/- 0.11% hemicellulose. Alkaline H(2)O(2) pretreatment and enzymatic saccharification were evaluated for conversion of wheat straw cellulose and hemicellulose to fermentable sugars. The maximum yield of monomeric sugars from wheat straw (8.6%, w/v) by alkaline peroxide pretreatment (2.15% H(2)O(2), v/v; pH 11.5; 35 degrees C; 24 h) and enzymatic saccharification (45 degrees C, pH 5.0, 120 h) by three commercial enzyme preparations (cellulase, beta-glucosidase, and xylanase) using 0.16 mL of each enzyme preparation per g of straw was 672 +/- 4 mg/g (96.7% yield). During the pretreatment, no measurable quantities of furfural and hydroxymethyl furfural were produced. The concentration of ethanol (per L) from alkaline peroxide pretreated enzyme saccharified wheat straw (66.0 g) hydrolyzate by recombinant Escherichia coli strain FBR5 at pH 6.5 and 37 degrees C in 48 h was 18.9 +/- 0.9 g with a yield of 0.46 g per g of available sugars (0.29 g/g straw). The ethanol concentration (per L) was 15.1 +/- 0.1 g with a yield of 0.23 g/g of straw in the case of simultaneous saccharification and fermentation by the E. coli strain at pH 6.0 and 37 degrees C in 48 h.     

Optimization of Dilute Acid Pretreatment and Enzymatic Hydrolysis of <Emphasis Type="Italic">Phalaris aquatica</Emphasis> L. Lignocellulosic Biomass in Batch and Fed-Batch Processes

Phalaris aquatica L., a rich in holocellulose (69.80 %) and deficient in lignin (6.70 %) herbaceous, perennial grass species, was utilized in a two-step (biomass pretreatment-enzymatic hydrolysis) saccharification process for sugars recovery. The Taguchi methodology was employed to determine the dilute acid pretreatment and enzymatic hydrolysis conditions that optimized hemicellulose conversion (75.04 %), minimized the production of inhibitory compounds (1.41 g/L), and maximized the cellulose to glucose yield (69.69 %) of mixed particulate biomass (particles <1000 μm) under batch conditions. The effect of biomass particle size on saccharification process efficiency was also investigated. It was found that small-size biomass particles (53–106 μm) resulted in maximum hemicellulose conversion (81.12 %) and cellulose to glucose yield (93.24 %). The determined optimal conditions were then applied to a combined batch pretreatment process followed by a fed-batch enzymatic hydrolysis process that maximized glucose concentration (62.24 g/L) and yield (92.48 %). The overall efficiency of the saccharification process was 88.13 %.     

Dilute acid pretreatment, enzymatic saccharification, and fermentation of rice hulls to ethanol

Rice hulls, a complex lignocellulosic material with high lignin (15.38 +/- 0.2%) and ash (18.71 +/- 0.01%) content, contain 35.62 +/- 0.12% cellulose and 11.96 +/- 0.73% hemicellulose and has the potential to serve as a low-cost feedstock for production of ethanol. Dilute H2SO4 pretreatments at varied temperature (120-190 degrees C) and enzymatic saccharification (45 degrees C, pH 5.0) were evaluated for conversion of rice hull cellulose and hemicellulose to monomeric sugars. The maximum yield of monomeric sugars from rice hulls (15%, w/v) by dilute H2SO4 (1.0%, v/v) pretreatment and enzymatic saccharification (45 degrees C, pH 5.0, 72 h) using cellulase, beta-glucosidase, xylanase, esterase, and Tween 20 was 287 +/- 3 mg/g (60% yield based on total carbohydrate content). Under this condition, no furfural and hydroxymethyl furfural were produced. The yield of ethanol per L by the mixed sugar utilizing recombinant Escherichia colistrain FBR 5 from rice hull hydrolyzate containing 43.6 +/- 3.0 g fermentable sugars (glucose, 18.2 +/- 1.4 g; xylose, 21.4 +/- 1.1 g; arabinose, 2.4 +/- 0.3 g; galactose, 1.6 +/- 0.2 g) was 18.7 +/- 0.6 g (0.43 +/- 0.02 g/g sugars obtained; 0.13 +/- 0.01 g/g rice hulls) at pH 6.5 and 35 degrees C. Detoxification of the acid- and enzyme-treated rice hull hydrolyzate by overliming (pH 10.5, 90 degrees C, 30 min) reduced the time required for maximum ethanol production (17 +/- 0.2 g from 42.0 +/- 0.7 g sugars per L) by the E. coli strain from 64 to 39 h in the case of separate hydrolysis and fermentation and increased the maximum ethanol yield (per L) from 7.1 +/- 2.3 g in 140 h to 9.1 +/- 0.7 g in 112 h in the case of simultaneous saccharification and fermentation.     

Effect of Maize Biomass Composition on the Optimization of Dilute-Acid Pretreatments and Enzymatic Saccharification

At the core of cellulosic ethanol research are innovations leading to reductions in the chemical and energetic stringency of thermochemical pretreatments and enzymatic saccharification. In this study, key compositional features of maize cell walls influencing the enzymatic conversion of biomass into fermentable sugars were identified. Stem samples from eight contrasting genotypes were subjected to a series of thermal dilute-acid pretreatments of increasing severity and evaluated for glucose release after enzymatic saccharification. The biochemically diverse set of genotypes displayed significant differences in glucose yields at all processing conditions evaluated. The results revealed that mechanisms controlling biomass conversion efficiency vary in relation to pretreatment severity. At highly severe pretreatments, cellulose conversion efficiency was primarily influenced by the inherent efficacy of the thermochemical process, and maximum glucose yields were obtained from cellulosic feedstocks harboring the highest cellulose contents per dry gram of biomass. When mild dilute-acid pretreatments were applied, however, maximum bioconversion efficiency and glucose yields were observed for genotypes combining high stem cellulose contents, reduced cell wall lignin and highly substituted hemicelluloses. For the best-performing genotype, glucose yields under sub-optimal processing regimes were only 10 % lower than the genotype-set mean at the most stringent processing conditions evaluated, while furfural production was reduced by approximately 95 %. Our results ultimately established that cellulosic feedstocks with tailored cell wall compositions can help reduce the chemical and energetic intensity of pretreatments used in the industry and improve the commercial and environmental performance of biomass-to-ethanol conversion technologies.     

Pretreatment of Siam weed stem by several chemical methods for increasing the enzymatic digestibility

Siam weed [Chromolaena odorata (L.) King & Robinson], an invasive exotic weed in China, was proposed as a feedstock for bioethanol production. This would be a promising way of using for an invasive weed that needs management and control. It was found that the glucan content of the weed stem was similar to that of sugarcane bagasse, but higher than those of corn stover and wheat straw. Several chemical pretreatment methods were applied to the weed stem to increase its enzymatic digestibility. Mild sulfuric acid (<120°C) or alkali pretreatment did not markedly increase the enzymatic digestibility. However, peracetic acid (PAA) pretreatment dramatically enhanced the enzymatic hydrolysis of the weed stem. Compared to some other common agricultural residues, the weed stem was more difficult to pretreat and digest by cellulase. Fourier transform infrared (FTIR) spectra analysis indicated that the cellulose-related bands became more intensive after pretreatment, especially for PAA-pretreated samples. According to X-ray diffraction spectra, the biomass solids had higher crystallinity indices after pretreatment, although these indices were similar for all of the pretreated samples.     

Effects of preheating on briquetting and subsequent hydrothermal pretreatment for enzymatic saccharification of wheat straw

Briquetting of plant biomass with low bulk density is an advantage for handling, transport, and storage of the material, and heating of the biomass prior to the briquetting facilitates the densification process and improves the physical properties of the briquettes. This study investigates the effects of preheating prior to briquetting of wheat straw (WS) on subsequent hydrothermal pretreatment and enzymatic conversion to fermentable sugars. WS (11% moisture content) was densified to briquettes under different conditions; without preheating or with preheating at 75 or 125°C for either 5 or 10 min. Subsequent hydrothermal pretreatment was done for both un-briquetted WS and for briquettes. Enzymatic saccharification was afterwards performed for all samples. The results showed that as expected, nonpretreated WS briquettes gave very low sugar yields (22–29% of the cellulose content), even though preheating at 125°C prior to briquetting (without pretreatment) improved sugar yields somewhat. When combined with pretreatment, briquetting with preheating showed neutral or negative effects on sugar yield. This result suggests that moderate preheating (75°C for 5 min) before briquetting improved bulk density and compressive resistance of briquettes without impeding subsequent enzymatic conversion. However, excessive preheating (75 or 125°C for 10 min) before briquetting may result in irreversible structural modifications that hinder the interaction between biomass and water during pretreatment, thereby decreasing the accessibility of cellulose to enzymatic saccharification.     

Combination of dilute acid and ionic liquid pretreatments of sugarcane bagasse for glucose by enzymatic hydrolysis

Loss of hemicellulose and inability to effectively decrystallize cellulose, result in low yield and high cost of sugars derived from biomass. In this work, dilute sulfuric acid pretreatment could easily remove most of hemicellulose as sugars. The sugars were successfully used for 2,3-butanediol production with relative high yield (36.1%). Then, the remained solid residue after acid-pretreatment was further pretreated by ionic liquid (IL) to decrease its crystallinity for subsequent enzymatic saccharification. The combination of dilute acid- and IL-pretreatments resulted in significant higher glucose yield (95.5%) in enzymatic saccharification, which was more effective than using dilute acid- or IL-pretreatment alone. This strategy seems a promising route to achieve high yield of sugars from both hemicellulose and cellulose for biorefinery.     

Correlating physical changes and enhanced enzymatic saccharification of pine flour pretreated by N-methylmorpholine-N-oxide

Pretreatment of lignocellulosic biomass by N-methylmorpholine-N-oxide (NMMO), a solvent used in the textile industry to dissolve cellulose for production of regenerated cellulose fibers, was observed to enhance significantly enzymatic saccharification and fermentation. The enhancement was speculated to have been caused by reduced cellulose crystallinity after dissolution and precipitation processes. This work focused on assessing several physical changes and their correlations to enzymatic saccharification of pine flour after NMMO pretreatment. Results from microstructure, surface chemical composition, and cellulose accessibility complementarily illustrated the enrichment of cellulose on pine flour surface after NMMO pretreatment. Cellulose accessibility was highly correlated to the overall glucan conversion rate. Changes in crystallinity were correlated to the initial hydrolysis rate but not overall glucan conversion rate. Findings from this work may contribute to lignocellulosic bioenergy from development of novel pretreatment technologies utilizing NMMO.     

Optimal conditions for pretreatment of rice straw with n-butylamine for enzymatic solubilization

Summary Three different pretreatment methods with n-butylamine (n-BA) were used to obtain fermentable sugars in a high yield from rice straw. The optimal conditions of each method were as follows: treating at boiling point for 1 h under refluxing in 10 w/w% n-BA with the weight ratio of n-BA to original rice straw more than 1.0, autoclaving at 120°C for 1 h in 1 w/w% n-BA with the weight ratio more than 0.1, and wetting for 2 h with the circulating condensate of the vapour evaporated from 2.5 w/w% n-BA with the weight ratio more than 0.8. Soaking rice straw with n-BA before the above pretreatments was not needed. For the circulation pretreatment, the overall cumulative yield of total sugars (70% of cellulose and hemicellulose in original rice straw) was best for both pretreatment and enzymatic solubilization steps, because there was no decomposition of monosaccharides during the pretreatment. Furthermore, the optimal degree of delignification for enzymatic solubilization of the pretreated rice straw was approximately 60% of lignin in the original.     

Efficient conversion of sugarcane stalks into ethanol employing low temperature alkali pretreatment method

An alternative route for bio-ethanol production from sugarcane stalks (juice and bagasse) featuring a previously reported low temperature alkali pretreatment method was evaluated. Test-tube scale pretreatment-saccharification experiments were carried out to determine optimal LTA pretreatment conditions for sugarcane bagasse with regard to the efficiency of enzymatic hydrolysis of the cellulose. Free fermentable sugars and bagasse recovered from 2 kg of sugarcane stalks were jointly converted into ethanol via separate enzymatic hydrolysis and fermentation (SHF). Results showed that 98% of the cellulose present in the optimally pretreated bagasse was hydrolyzed into glucose after 72-h enzymatic saccharification using commercially available cellulase and β-glucosidase preparations at relatively low enzyme loading. The fermentable sugars in the mixture of the sugar juice and the bagasse hydrolysate were readily converted into 193.5 mL of ethanol by Saccharomyces cerevisiae within 12h, achieving 88% of the theoretical yield from the sugars and cellulose.     

Structural comparison and enhanced enzymatic hydrolysis of eucalyptus cellulose via pretreatment with different ionic liquids and catalysts

Eucalyptus was fractionated with mild alkaline process, and the obtained cellulose fraction was pretreated with various ionic liquids (ILs) to enhance the enzymatic saccharification. The results showed that the ILs used was efficient for the hydrolysis of cellulose, with the maximum total reducing sugars (TRS) yield over 80% at 50 °C. The regenerated cellulose substrate exhibited a significant improvement about 4.4–6.4 folds enhancement on saccharification rate during the first 4 h reaction. The crystallinity index (CrI) of cellulose via 1-ally-3-methylimidazolium ([AMIM]Cl) pretreatment was significantly decreased from 70.2% to 31.2%, resulting in structural change from cellulose I to cellulose II, which enabled the cellulase enzymes easier access to hydrolyze cellulose. However, 1-butyl-3methylimidazolium acesulfamate ([BMIM]Ace) pretreatment had no large effect on the CrI although a high conversion yield in glucose was obtained. The surface morphologies of the regenerated substrate which was pretreated via 1-butyl-3-methylimidazolium chloride ([BMIM]Cl) and 1-ethyl-3-methylimidazolium acetate ([EMIM]Ac) showed more porous and incompact network of cellulose when compared with the untreated substrate. This result indicated a better accessibility by cellulases to the cellulose surface. Besides, a certain amount of catalysts such as MgCl₂ and H₂SO₄ could improve the rate of enzymatic saccharification.     

Enzymatic saccharification of woody biomass micro/nanofibrillated by continuous extrusion process I--effect of additives with cellulose affinity

Mechanical micro/nanofibrillation of Douglas fir was performed by a continuous extrusion process in an attempt to develop a cost-effective pretreatment method for enzymatic saccharification. Additives with cellulose affinity (ethylene glycol, glycerol, and dimethyl sulfoxide) were used to effectively fibrillate the wood cell wall into submicron- or nano-scale, thus opening up the cell wall structure for improving enzymatic accessibility, and lower the extrusion torque. Morphological characterization showed that ethylene glycol was the most effective additive for fibrillation. The fibrillated products were converted into glucose with a high yield by enzymatic saccharification. The maximum cellulose-to-glucose conversion was achieved when ethylene glycol was used; the value was 62.4%. The glucose yield was approximately 6 times higher than that of the untreated raw material.     

Deposition of lignin droplets produced during dilute acid pretreatment of maize stems retards enzymatic hydrolysis of cellulose

Electron microscopy of lignocellulosic biomass following high-temperature pretreatment revealed the presence of spherical formations on the surface of the residual biomass. The hypothesis that these droplet formations are composed of lignins and possible lignin carbohydrate complexes is being explored. Experiments were conducted to better understand the formation of these "lignin" droplets and the possible implications they might have on the enzymatic saccharification of pretreated biomass. It was demonstrated that these droplets are produced from corn stover during pretreatment under neutral and acidic pH at and above 130 degrees C, and that they can deposit back onto the surface of residual biomass. The deposition of droplets produced under certain pretreatment conditions (acidic pH; T > 150 degrees C) and captured onto pure cellulose was shown to have a negative effect (5-20%) on the enzymatic saccharification of this substrate. It was noted that droplet density (per unit area) was greater and droplet size more variable under conditions where the greatest impact on enzymatic cellulose conversion was observed. These results indicate that this phenomenon has the potential to adversely affect the efficiency of enzymatic conversion in a lignocellulosic biorefinery.     

Hemicelluloses negatively affect lignocellulose crystallinity for high biomass digestibility under NaOH and H2SO4 pretreatments in Miscanthus

ABSTRACT: BACKGROUND: Lignocellulose is the most abundant biomass on earth. However, biomass recalcitrance has become a major factor affecting biofuel production. Although cellulose crystallinity significantly influences biomass saccharification, little is known about the impact of three major wall polymers on cellulose crystallization. In this study, we selected six typical pairs of Miscanthus samples that presented different cell wall compositions, and then compared their cellulose crystallinity and biomass digestibility after various chemical pretreatments. RESULTS: A Miscanthus sample with a high hemicelluloses level was determined to have a relatively low cellulose crystallinity index (CrI) and enhanced biomass digestibility at similar rates after pretreatments of NaOH and H2SO4 with three concentrations. By contrast, a Miscanthus sample with a high cellulose or lignin level showed increased CrI and low biomass saccharification, particularly after H2SO4 pretreatment. Correlation analysis revealed that the cellulose CrI negatively affected biomass digestion. Increased hemicelluloses level by 25% or decreased cellulose and lignin contents by 31% and 37% were also found to result in increased hexose yields by 1.3-times to 2.2-times released from enzymatic hydrolysis after NaOH or H2SO4 pretreatments. The findings indicated that hemicelluloses were the dominant and positive factor, whereas cellulose and lignin had synergistic and negative effects on biomass digestibility. CONCLUSIONS: Using six pairs of Miscanthus samples with different cell wall compositions, hemicelluloses were revealed to be the dominant factor that positively determined biomass digestibility after pretreatments with NaOH or H2SO4 by negatively affecting cellulose crystallinity. The results suggested potential approaches to the genetic modifications of bioenergy crops.     

Dilute acid pretreatment, enzymatic saccharification and fermentation of wheat straw to ethanol

Wheat straw consists of 48.57 ± 0.30% cellulose and 27.70 ± 0.12% hemicellulose on dry solid (DS) basis and has the potential to serve as a low cost feedstock for production of ethanol. Dilute acid pretreatment at varied temperature and enzymatic saccharification were evaluated for conversion of wheat straw cellulose and hemicellulose to monomeric sugars. The maximum yield of monomeric sugars from wheat straw (7.83%, w/v, DS) by dilute H₂SO₄ (0.75%, v/v) pretreatment and enzymatic saccharification (45 °C, pH 5.0, 72 h) using cellulase, β-glucosidase, xylanase and esterase was 565 ± 10 mg/g. Under this condition, no measurable quantities of furfural and hydroxymethyl furfural were produced. The yield of ethanol (per litre) from acid pretreated enzyme saccharified wheat straw (78.3 g) hydrolyzate by recombinant Escherichia coli strain FBR5 was 19 ± 1 g with a yield of 0.24 g/g DS. Detoxification of the acid and enzyme treated wheat straw hydrolyzate by overliming reduced the fermentation time from 118 to 39 h in the case of separate hydrolysis and fermentation (35 °C, pH 6.5), and increased the ethanol yield from 13 ± 2 to 17 ± 0 g/l and decreased the fermentation time from 136 to 112 h in the case of simultaneous saccharification and fermentation (35 °C, pH 6.0).     

Effect of dilute acid pretreatment on the saccharification and fermentation of rye straw

This research shows the effect of dilute acid pretreatment with various sulfuric acid concentrations (0.5–2.0% [wt/vol]) on enzymatic saccharification and fermentation yield of rye straw. After pretreatment, solids of rye straw were suspended in Na citrate buffer or post-pretreatment liquids (prehydrolysates) containing sugars liberated after hemicellulose hydrolysis. Saccharification was conducted using enzymes dosage of 15 or 25 FPU/g cellulose. Cellulose saccharification rate after rye straw pretreatment was enhanced by performing enzymatic hydrolysis in sodium citrate buffer in comparison with hemicellulose prehydrolysate. The maximum cellulose saccharification rate (69%) was reached in sodium citrate buffer (biomass pretreated with 2.0% [wt/vol] H₂SO₄). Lignocellulosic complex of rye straw after pretreatment was subjected to separate hydrolysis and fermentation (SHF) or separate hydrolysis and co-fermentation (SHCF). The SHF processes conducted in the sodium citrate buffer using monoculture of Saccharomyces cerevisiae (Ethanol Red) were more efficient compared to hemicellulose prehydrolysate in respect with ethanol yields. Maximum fermentation efficiency of SHF processes obtained after rye straw pretreatment at 1.5% [wt/vol] H₂SO₄ and saccharification using enzymes dosage of 25 FPU/g in sodium citrate buffer, achieving 40.6% of theoretical yield. However, SHCF process using cocultures of pentose-fermenting yeast, after pretreatment of raw material at 1.5% [wt/vol] H₂SO₄ and hydrolysis using enzymes dosage of 25 FPU/g, resulted in the highest ethanol yield among studied methods, achieving 9.4 g/L of ethanol, corresponding to 55% of theoretical yield.     

Bioethanol production from non-starch carbohydrate residues in process streams from a dry-mill ethanol plant

Slurries obtained from process streams in a starch-to-ethanol plant, Agroetanol AB in Norrk?ping, Sweden, were used to assess the potential increase in bioethanol yield if heat treatment followed by enzymatic hydrolysis were applied to the residual starch-free cellulose and hemicellulose fractions. The effects of different pretreatment conditions on flour (the raw material), the stream after saccharification of starch, before fermentation, and after fermentation were studied. The conditions resulting in the highest concentration of glucose and xylose in all streams were heat treatment at 130 degrees C for 40 min with 1% H(2)SO(4). Mass-balance calculations over the fermentation showed that approximately 64%, 54%, 75% and 67% of the glucan, xylan, galactan and arabinan, respectively, in the flour remained water insoluble in the process stream after fermentation without any additional treatment. Utilizing only the starch in the flour would theoretically yield 425 L ethanol per ton flour. By applying heat pretreatment to the water-insoluble material prior to enzymatic hydrolysis, the ethanol yield could be increased by 59 L per ton flour, i.e. a 14% increase compared with starch-only utilization, assuming fermentation of the additional pentose and hexose sugars liberated.     

Low temperature alkali pretreatment for improving enzymatic digestibility of sweet sorghum bagasse for ethanol production

A low temperature alkali pretreatment method was proposed for improving the enzymatic hydrolysis efficiency of lignocellulosic biomass for ethanol production. The effects of the pretreatment on the composition, structure and enzymatic digestibility of sweet sorghum bagasse were investigated. The mechanisms involved in the digestibility improvement were discussed with regard to the major factors contributing to the biomass recalcitrance. The pretreatment caused slight glucan loss but significantly reduced the lignin and xylan contents of the bagasse. Changes in cellulose crystal structure occurred under certain treatment conditions. The pretreated bagasse exhibited greatly improved enzymatic digestibility, with 24-h glucan saccharification yield reaching as high as 98% using commercially available cellulase and β-glucosidase. The digestibility improvement was largely attributed to the disruption of the lignin-carbohydrate matrix. The bagasse from a brown midrib (BMR) mutant was more susceptible to the pretreatment than a non-BMR variety tested, and consequently gave higher efficiency of enzymatic hydrolysis.     

CO2 enrichment and its relationship to bioconversion of cellulosic biomass of sweet potato (Ipomoea batatas L.) into fermentable sugars

Sweet potatoes (Ipomoea batatas L. (Lam) ‘Georgia-Jet’) were grown in open field plots and open top chambers at CO₂ concentrations of 354, 431, 506, and 659 μl liter⁻¹ for 90 days. The leaves and stems after the harvest were used as substrates for the production of fermentable sugars. Elevated CO₂ concentrations increased the cellulose content of stems, being most pronounced at 506 μl liter⁻¹. Hemicellulose content of leaves and stems as well as lignin content of stems decreased as a result of CO₂ enriched environment resulted in increased conversion of cellulose into fermentable sugars. The saccharification was greater in stems than in leaves. It was also found that chemical pretreatment of stems and leaves enhanced the enzymatic hydrolysis and the yields of glucose were higher than those from untreated stems and leaves.     

Chemical characteristics and ethanol fermentation of the cellulose component in autohydrolyzed bagasse

The chemical characteristics, enzymatic saccharification, and ethanol fermentation of autohydrolyzed lignocellulosic material that was exposed to steam explosion were investigated using bagasse as the sample. The effects of the steam explosion on the change in pH, organic acids production, degrees of polymerization and crystallinity of the cellulose component, and the amount of extractive components in the autohydrolyzated bagasse were examined. The steam explosion decreased the degree of polymerzation up to about 700 but increased the degree of crystallinity and the micelle width of the cellulose component in the bagasse. The steam explosion, at a pressure of 2.55 MPa for 3 mins, was the most effective for the delignification of bagasse. 40 g/L of glucose and 20 g/L of xylose were produced from 100 g/L of the autohydrolyzed bagasse by the enzymatic saccharification using mixed cellulases, acucelase and meicelase. The maximum ethanol concentration, 20 g/L, was obtained from the enzymatic hydrolyzate of 100 g/L of the autohydrolyzed bagasse by the ethanol fermentation usingPichia stipitis CBS 5773; the ethanol yield from sugars was 0.33 g/g sugars.