Ultrasound pretreatment of cassava chip slurry to enhance sugar release for subsequent ethanol production

The use of ultrasound pretreatment to enhance liquefaction and saccharification of cassava chips was investigated. Cassava chip slurry samples were subjected to sonication for 10-40 s at three power levels of low (2 W/mL), medium (5 W/mL), and high (8 W/mL). The samples were simultaneously exposed to enzymes to convert starch into glucose. The cassava particle size declined nearly 40-fold following ultrasonic pretreatment at high power input. Scanning electron micrographs of both unsonicated (control) and sonicated samples showed disruption of fibrous material in cassava chips but did not affect the granular structure of starch. Reducing sugar release improved in direct proportion to the power input and sonication time. The reducing sugar increase was as much as 180% with respect to the control groups. The slurry samples with enzyme addition during sonication resulted in better reducing sugar release than the samples with enzyme addition after sonication. The heat generated during sonication below starch gelatinization temperature apparently had no effect on the reducing sugar release. The reducing sugar yield and energy efficiency of ultrasound pretreated samples increased with total solids (TS) contents. The highest reducing sugar yield of 22 g/100 g of sample and efficiency of 323% were obtained for cassava slurry with 25% TS at high power. The reducing sugar yield at the completion of reaction (R(infinity)) were over twofold higher compared to the control groups. The integration of ultrasound into a cassava-based ethanol plant may significantly improve the overall ethanol yield.     

Simultaneous saccharification and fermentation and economic evaluation of ultrasonic and jet cooking pretreatment of corn slurry

The potential of ultrasonics to replace hydrocooking in corn-to-ethanol plants was examined in this study. Batch and continuous experiments were conducted on corn slurry with sonication at a frequency of 20 kHz. Batch mode used a catenoidal horn operated at an amplitude of 144 μm peak-to-peak (p–p) for 90 s. Continuous experiments used a donut horn operating at inner radius amplitude of 12 μm p–p. Jet-cooked samples from the same ethanol plant were compared with ultrasonicated samples. The highest starch-to-ethanol conversion was obtained by the jet-cooked samples with a yield of 74% of the theoretical yield. Batch and continuous sonication achieved 71.2% and 68% conversion, respectively, however, statistical analysis showed no significant difference between the jet cooking and ultrasonication. On the basis of the similar performance, an economic analysis was conducted comparing jet cooking and ultrasonic pretreatment. The analysis showed that the capital cost for the ultrasonics system was ~10 times higher compared to the capital cost of a hydrocooker. However,due to the large energy requirements of hydrocookers, the analysis showed lower total overall costs for continuous ultrasonication than that for jet cooking, assuming the current energy prices. Because of the high utility cost calculated for jet cooking, it is concluded that ultrasonication poses as a more economical option than jet cooking. Overall, the study shows that ultrasonics is a technically and economically viable alternative to jet cooking in dry-grind corn ethanol plant.     

Microscopic examination of changes of plant cell structure in corn stover due to hot water pretreatment and enzymatic hydrolysis

Particle size associated with accessible surface area has a significant impact on the saccharification of plant cell walls by cellulolytic enzymes. Small particle sizes of untreated cellulosic substrate are more readily hydrolyzed than large ones because of higher specific surface area. Pretreatment enlarges accessible and susceptible surface area leading to enhanced cellulose hydrolysis. These hypotheses were tested using ground corn stover in the size ranges of 425-710 and 53-75 microm. Ultrastructural changes in these particles were imaged after treatment with cellulolytic enzymes before and after liquid hot water pretreatment. The smaller 53-75 microm corn stover particles are 1.5x more susceptible to hydrolysis than 425-710 microm corn stover particles. This difference between the two particle size ranges is eliminated when the stover is pretreated with liquid hot water pretreatment at 190 degrees C for 15 min, at pH between 4.3 and 6.2. This pretreatment causes ultrastructural changes and formation of micron-sized pores that make the cellulose more accessible to hydrolytic enzymes.     

Impact of recycling stillage on conversion of dilute sulfuric acid pretreated corn stover to ethanol

Both the current corn starch to ethanol industry and the emerging lignocellulosic biofuels industry view recycling of spent fermentation broth or stillage as a method to reduce fresh water use. The objective of this study was to understand the impact of recycling stillage on conversion of corn stover to ethanol. Sugars in a dilute‐acid pretreated corn stover hydrolysate were fermented to ethanol by the glucose–xylose fermenting bacteria Zymomonas mobilis 8b. Three serial fermentations were performed at two different initial sugar concentrations using either 10% or 25% of the stillage as makeup water for the next fermentation in the series. Serial fermentations were performed to achieve near steady state concentration of inhibitors and other compounds in the corn stover hydrolysate. Little impact on ethanol yields was seen at sugar concentrations equivalent to pretreated corn stover slurry at 15% (w/w) with 10% recycle of the stillage. However, ethanol yields became progressively poorer as the sugar concentration increased and fraction of the stillage recycled increased. At an equivalent corn stover slurry concentration of 20% with 25% recycled stillage the ethanol yield was only 5%. For this microorganism with dilute‐acid pretreated corn stover, recycling a large fraction of the stillage had a significant negative impact on fermentation performance. Although this finding is of concern for biochemical‐based lignocellulose conversion processes, other microorganism/pretreatment technology combinations will likely perform differently. Biotechnol. Bioeng. 2010;105: 992–996. © 2009 Wiley Periodicals, Inc.     

Simultaneous saccharification and co-fermentation of glucose and xylose in steam-pretreated corn stover at high fiber content with Saccharomyces cerevisiae TMB3400

The two main sugars in the agricultural by-product corn stover are glucose and xylose. Co-fermentation of glucose and xylose at high content of water-insoluble solids (WIS) without detoxification is a prerequisite to obtain high ethanol concentration and to reduce production costs. A recombinant strain of Saccharomyces cerevisiae, TMB3400, was used in simultaneous saccharification and fermentation (SSF) of whole pretreated slurry of corn stover at high WIS. TMB3400 co-fermented glucose and xylose with relatively high ethanol yields giving high final ethanol concentration. The ethanol productivity increased with increasing concentration of pretreatment hydrolysate in the yeast production medium and when SSF was performed in a fed-batch mode.     

High solid simultaneous saccharification and fermentation of wet oxidized corn stover to ethanol

In this study ethanol was produced from corn stover pretreated by alkaline and acidic wet oxidation (WO) (195 degrees C, 15 min, 12 bar oxygen) followed by nonisothermal simultaneous saccharification and fermentation (SSF). In the first step of the SSF, small amounts of cellulases were added at 50 degrees C, the optimal temperature of enzymes, in order to obtain better mixing condition due to some liquefaction. In the second step more cellulases were added in combination with dried baker's yeast (Saccharomyces cerevisiae) at 30 degrees C. The phenols (0.4-0.5 g/L) and carboxylic acids (4.6-5.9 g/L) were present in the hemicellulose rich hydrolyzate at subinhibitory levels, thus no detoxification was needed prior to SSF of the whole slurry. Based on the cellulose available in the WO corn stover 83% of the theoretical ethanol yield was obtained under optimized SSF conditions. This was achieved with a substrate concentration of 12% dry matter (DM) acidic WO corn stover at 30 FPU/g DM (43.5 FPU/g cellulose) enzyme loading. Even with 20 and 15 FPU/g DM (corresponding to 29 and 22 FPU/g cellulose) enzyme loading, ethanol yields of 76 and 73%, respectively, were obtained. After 120 h of SSF the highest ethanol concentration of 52 g/L (6 vol.%) was achieved, which exceeds the technical and economical limit of the industrial-scale alcohol distillation. The SSF results showed that the cellulose in pretreated corn stover can be efficiently fermented to ethanol with up to 15% DM concentration. A further increase of substrate concentration reduced the ethanol yield significant as a result of insufficient mass transfer. It was also shown that the fermentation could be followed with an easy monitoring system based on the weight loss of the produced CO2.     

Detecting cellulase penetration into corn stover cell walls by immuno‐electron microscopy

In general, pretreatments are designed to enhance the accessibility of cellulose to enzymes, allowing for more efficient conversion. In this study, we have detected the penetration of major cellulases present in a commercial enzyme preparation (Spezyme CP) into corn stem cell walls following mild‐, moderate‐ and high‐severity dilute sulfuric acid pretreatments. The Trichoderma reesei enzymes, Cel7A (CBH I) and Cel7B (EG I), as well as the cell wall matrix components xylan and lignin were visualized within digested corn stover cell walls by immuno transmission electron microscopy (TEM) using enzyme‐ and polymer‐specific antibodies. Low severity dilute‐acid pretreatment (20 min at 100°C) enabled <1% of the thickness of secondary cell walls to be penetrated by enzyme, moderate severity pretreatment at (20 min at 120°C) allowed the enzymes to penetrate ～20% of the cell wall, and the high severity (20 min pretreatment at 150°C) allowed 100% penetration of even the thickest cell walls. These data allow direct visualization of the dramatic effect dilute‐acid pretreatment has on altering the condensed ultrastructure of biomass cell walls. Loosening of plant cell wall structure due to pretreatment and the subsequently improved access by cellulases has been hypothesized by the biomass conversion community for over two decades, and for the first time, this study provides direct visual evidence to verify this hypothesis. Further, the high‐resolution enzyme penetration studies presented here provide insight into the mechanisms of cell wall deconstruction by cellulolytic enzymes. Biotechnol. Bioeng. 2009;103: 480–489. © 2009 Wiley Periodicals, Inc.     

Thermodynamics of the Corn-Ethanol Biofuel Cycle

This article defines sustainability and sustainable cyclic processes, and quantifies the degree of non-renewability of a major biofuel: ethanol produced from industrially grown corn. It demonstrates that more fossil energy is used to produce ethanol from corn than the ethanol's calorific value. Analysis of the carbon cycle shows that all leftovers from ethanol production must be returned back to the fields to limit the irreversible mining of soil humus. Thus, production of ethanol from whole plants is unsustainable. In 2004, ethanol production from corn will generate 8 million tons of incremental CO₂, over and above the amount of CO₂ generated by burning gasoline with 115% of the calorific value of this ethanol. It next calculates the cumulative exergy (available free energy) consumed in corn farming and ethanol production, and estimates the minimum amount of work necessary to restore the key non-renewable resources consumed by the industrial corn-ethanol cycle. This amount of work is compared with the maximum useful work obtained from the industrial corn-ethanol cycle. It appears that if the corn-ethanol exergy is used to power a car engine, the minimum restoration work is about 6 times the maximum useful work from the cycle. This ratio drops down to 2 if an ideal fuel cell is used to process the ethanol. The article estimates the U.S. taxpayer subsidies of the industrial corn-ethanol cycle at $3.8 billion in 2004. The parallel subsidies by the environment are estimated at $1.8 billion in 2004. The latter estimate will increase manifold when the restoration costs of aquifers, streams, and rivers, and the Gulf of Mexico are also included. Finally, the article estimates that (per year and unit area) the inefficient solar cells produce ～ 100 times more electricity than corn ethanol. There is a need for more reliance on sunlight, the only source of renewable energy on the earth.     

Microscopic analysis of corn fiber using starch- and cellulose-specific molecular probes

Ethanol is the primary liquid transportation fuel produced from renewable feedstocks in the United States today. The majority of corn grain, the primary feedstock for ethanol production, has been historically processed in wet mills yielding products such as gluten feed, gluten meal, starch, and germ. Starch extracted from the grain is used to produce ethanol in saccharification and fermentation steps; however the extraction of starch is not 100% efficient. To better understand starch extraction during the wet milling process, we have developed fluorescent probes that can be used to visually localize starch and cellulose in samples using confocal microscopy. These probes are based on the binding specificities of two types of carbohydrate binding modules (CBMs), which are small substrate-specific protein domains derived from carbohydrate degrading enzymes. CBMs were fused, using molecular cloning techniques, to a green fluorescent protein (GFP) or to the red fluorescent protein DsRed (RFP). Using these engineered probes, we found that the binding of the starch-specific probe correlates with starch content in corn fiber samples. We also demonstrate that there is starch internally localized in the endosperm that may contribute to the high starch content in corn fiber. We also surprisingly found that the cellulose-specific probe did not bind to most corn fiber samples, but only to corn fiber that had been hydrolyzed using a thermochemical process that removes the residual starch and much of the hemicellulose. Our findings should be of interest to those working to increase the efficiency of the corn grain to ethanol process.     

Pipeline transport and simultaneous saccharification of corn stover

Pipeline transport of corn stover delivered by truck from the field is evaluated against a range of truck transport costs. Corn stover transported by pipeline at 20% solids concentration (wet basis) or higher could directly enter an ethanol fermentation plant, and hence the investment in the pipeline inlet end processing facilities displaces comparable investment in the plant. At 20% solids, pipeline transport of corn stover costs less than trucking at capacities in excess of 1.4 M drytonnes/yr when compared to a mid range of truck transport cost (excluding any credit for economies of scale achieved in the ethanol fermentation plant from larger scale due to multiple pipelines). Pipelining of corn stover gives the opportunity to conduct simultaneous transport and saccharification (STS). If current enzymes are used, this would require elevated temperature. Heating of the slurry for STS, which in a fermentation plant is achieved from waste heat, is a significant cost element (more than 5 cents/l of ethanol) if done at the pipeline inlet unless waste heat is available, for example from an electric power plant located adjacent to the pipeline inlet. Heat loss in a 1.26 m pipeline carrying 2 M drytonnes/yr is about 5 degrees C at a distance of 400 km in typical prairie clay soils, and would not likely require insulation; smaller pipelines or different soil conditions might require insulation for STS. Saccharification in the pipeline would reduce the need for investment in the fermentation plant, saving about 0.2 cents/l of ethanol. Transport of corn stover in multiple pipelines offers the opportunity to develop a large ethanol fermentation plant, avoiding some of the diseconomies of scale that arise from smaller plants whose capacities are limited by issues of truck congestion.     

Enzymatic hydrolysis of biomass from wood

Current research and development in cellulosic ethanol production has been focused mainly on agricultural residues and dedicated energy crops such as corn stover and switchgrass; however, woody biomass remains a very important feedstock for ethanol production. The precise composition of hemicellulose in the wood is strongly dependent on the plant species, therefore different types of enzymes are needed based on hemicellulose complexity and type of pretreatment. In general, hardwood species have much lower recalcitrance to enzymes than softwood. For hardwood, xylanases, beta‐xylosidases and xyloglucanases are the main hemicellulases involved in degradation of the hemicellulose backbone, while for softwood the effect of mannanases and beta‐mannosidases is more relevant. Furthermore, there are different key accessory enzymes involved in removing the hemicellulosic fraction and increasing accessibility of cellulases to the cellulose fibres improving the hydrolysis process. A diversity of enzymatic cocktails has been tested using from low to high densities of biomass (2–20% total solids) and a broad range of results has been obtained. The performance of recently developed commercial cocktails on hardwoods and softwoods will enable a further step for the commercialization of fuel ethanol from wood.     

Allocation procedure in ethanol production system from corn grain i. system expansion

We investigated the system expansion approach to net energy analysis for ethanol production from domestic corn grain. Production systems included in this study are ethanol production from corn dry milling and corn wet milling, corn grain production (the agricultural system), soybean products from soybean milling (i.e. soybean oil and soybean meal) and urea production to determine the net energy associated with ethanol derived from corn grain. These five product systems are mutually interdependent. That is, all these systems generate products which compete with or displace all other comparable products in the market place. The displacement ratios between products compare the equivalence of their marketplace functions. The net energy, including transportation to consumers, is 0.56 MJ_net/MJ of ethanol from corn grain regardless of the ethanol production technology employed. Using ethanol as a liquid transportation fuel could reduce domestic use of fossil fuels, particularly petroleum. Sensitivity analyses show that the choice of allocation procedures has the greatest impact on fuel ethanol net energy. Process energy associated with wet milling, dry milling and the corn agricultural process also significantly influences the net energy due to the wide ranges of available process energy values. The system expansion approach can completely eliminate allocation procedures in the foreground system of ethanol production from corn grain.     

Effect of polyphenol content on the hydrolysis and fermentation of grain sorghum starch

The high polyphenol content of birdproff grain sorghum has been associated with impaired nutritional quality of the grain and with reduced brewing value of birdproof grain sorghum malt due to enzyme inhibition. In this investigation, high polyphenol grain sorghum was evaluated as a feedstock for fermentation ethanol production using NaOH pretreatment to inactivate the polyphenolic compounds prior to hydrolysis with commercial amylases. The polyphenolic inhibition of starch hydrolysis was minimal at a grain sorghum slurry concentration of 20% dry solids, but became pronounced at slurry concentrations of 28% and higher. At these high slurry concentrations the liquefaction and subsequent saccharification and fermentation were markedly improved by alkaline pretreatment. The highest ethanol concentration (12·3%, vol/vol), coupled with the best starch conversion efficiency to ethanol (83·5%), was obtained with a 28% grain sorghum slurry using a partial simultaneous saccharification and fermentation procedure. The residual fermented solids had a crude protein content of 45·4%. Tannic acid decreased yeast cell viability in synthetic media, but had no effect on the hydrolysis or fermentation of grain sorghum starch.     

Enzymatic digestibility and pretreatment degradation products of AFEX‐treated hardwoods (Populus nigra)

There is a growing need to find alternatives to crude oil as the primary feed stock for the chemicals and fuel industry and ethanol has been demonstrated to be a viable alternative. Among the various feed stocks for producing ethanol, poplar (Populus nigra × Populus maximowiczii) is considered to have great potential as a biorefinery feedstock in the United States, due to their widespread availability and good productivity in several parts of the country. We have optimized AFEX pretreatment conditions (180°C, 2:1 ammonia to biomass loading, 233% moisture, 30 minutes residence time) and by using various combinations of enzymes (commercical celluloses and xylanases) to achieve high glucan and xylan conversion (93 and 65%, respectively). We have also identified and quantified several important degradation products formed during AFEX using liquid chromatography followed by mass spectrometry (LC‐MS/MS). As a part of degradation product analysis, we have also quantified oligosaccharides in the AFEX water wash extracts by acid hydrolysis. It is interesting to note that corn stover (C4 grass) can be pretreated effectively using mild AFEX pretreatment conditions, while on the other hand hardwood poplar requires much harsher AFEX conditions to obtain equivalent sugar yields upon enzymatic hydrolysis. Comparing corn stover and poplar, we conclude that pretreatment severity and enzymatic hydrolysis efficiency are dictated to a large extent by lignin carbohydrate complexes and arabinoxylan cross‐linkages for AFEX. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Aboveground productivity and soil carbon storage of biofuel crops in Ohio

Biofuel crops may help achieve the goals of energy‐efficient renewable ethanol production and greenhouse gas (GHG) mitigation through carbon (C) storage. The objective of this study was to compare the aboveground biomass yields and soil organic C (SOC) stocks under four crops (no‐till corn, switchgrass, indiangrass, and willow) 7 years since establishment at three sites in Ohio to determine if high‐yielding biofuel crops are also capable of high levels of C storage. Corn grain had the highest potential ethanol yields, with an average of more than 4100 L ha^?1, and ethanol yields increased if both corn grain and stover were converted to biofuel, while willow had the lowest yields. The SOC concentration in soils under biofuels was generally unaffected by crop type; at one site, soil in the top 10 cm under willow contained nearly 13 Mg C ha^?1 more SOC (or 29% more) than did soils under switchgrass or corn. Crop type affected SOC content of macroaggregates in the top 10 cm of soil, where macroaggregates in soil under corn had lower C, N and C : N ratios than those under perennial grasses or trees. Overall, the results suggest that no‐till corn is capable of high ethanol yields and equivalent SOC stocks to 40 cm depth. Long‐term monitoring and measurement of SOC stocks at depth are required to determine whether this trend remains. In addition, ecological, energy, and GHG assessments should be made to estimate the C footprint of each feedstock.     

Maximizing the liquid fuel yield in a biorefining process

Biorefining strives to recover the maximum value from each fraction, at minimum energy cost. In order to seek an unbiased and thorough assessment of the alleged opportunity offered by biomass fuels, the direct conversion of various lignocellulosic biomass was studied: aspen pulp wood (Populus tremuloides), aspen wood pretreated with dilute acid, aspen lignin, aspen logging residues, corn stalk, corn spathe, corn cob, corn stover, corn stover pellet, corn stover pretreated with dilute acid, and lignin extracted from corn stover. Besides the heating rate, the yield of liquid products was found to be dependent on the final liquefaction temperature and the length of liquefaction time. The major compounds of the liquid products from various origins were identified by GC-MS. The lignin was found to be a good candidate for the liquefaction process, and biomass fractionation was necessary to maximize the yield of the liquid bio-fuel. The results suggest a biorefinery process accompanying pretreatment, fermentation to ethanol, liquefaction to bio-crude oil, and other thermo-conversion technologies, such as gasification. Other biorefinery options, including supercritical water gasification and the effectual utilization of the bio-crude oil, are also addressed.     

Conversion of corn milling fibrous co-products into ethanol by recombinant Escherichia coli strains K011 and SL40

Corn hulls and corn germ meal were both evaluated as feedstocks for production of ethanol for biofuel. Currently, these fibrous co-products are combined with corn steep liquor and the fermentation bottoms (if available) and marketed as cattle feed. Samples were obtained from wet and dry corn mills. The corn hulls and germ meal were evaluated for starch and hemicellulose compositions. Starch contents were 12 to 32% w/w and hemicellulose (arabinoxylans) contents were 23 to 64% w/w. Corn fibrous samples were hydrolysed, using dilute sulphuric acid, into mixed sugar streams containing arabinose, glucose and xylose. Total sugar concentrations in the hydrolysate varied from 8.4 to 10.8% w/v. The hydrolysates were fermented to ethanol using recombinant E. coli strains K011 and SL40. Ethanol yields were 0.38 to 0.41g ethanol produced/g total sugars consumed and fermentations were completed in 60h or less. However, residual xylose was detected for each hydrolysate fermentation and was especially significant for fermentations using strain SL40. Strain K011 was a superior ethanologenic strain compared with strain SL40 in terms of both ethanol yield and maximum productivity.     

Low Temperature and Long Residence Time AFEX Pretreatment of Corn Stover

Low temperature and long residence time pretreatments have been proposed as an alternative to conventional pretreatments within a centralized biorefinery, allowing for a decentralized pretreatment without high energy costs. Ammonia fiber expansion (AFEX?) pretreatment may be uniquely suitable for decentralized pretreatment, and this study considers the possibility of decreasing the temperature in AFEX pretreatment of corn stover. AFEX pretreatment at 40°C and 8?h produced comparable sugar and ethanol yields as conventional AFEX pretreatment at high temperatures and short residence time during subsequent hydrolysis and fermentation. Increasing the ammonia loading at these temperatures tends to increase digestibility, although the moisture content of the reaction has little effect. This study suggests a greater flexibility in AFEX pretreatment conditions than previously thought, allowing for an alternative approach for decentralized facilities if the economic conditions are appropriate.     

A complete enzymatic recovery of ferulic acid from corn residues with extracellular enzymes from Neosartorya spinosa NRRL185

An economic ferulic acid recovery from biomass via biological methods is of interest for a number of reasons. Ferulic acid is a precursor to vanillin synthesis. It is also a known antioxidant with potential food and medical applications. Despite its universal presence in all plant cell wall material, the complex structure of the plant cell wall makes ferulic acid recovery from biomass a challenging bioprocess. Previously, without pretreatment, very low (3-13%) recovery of ferulic acid from corn residues was achieved. We report here the discovery of a filamentous fungus Neosartorya spinosa NRRL185 capable of producing a full complement of enzymes to release ferulic acid and the development of an enzymatic process for a complete recovery of ferulic acid from corn bran and corn fibers. A partial characterization of the extracellular proteome of the microbe revealed the presence of at least seven cellulases and hemicellulases activities, including multiple iso-forms of xylanase and ferulic acid esterase. The recovered ferulic acid was bio-converted to vanillin, demonstrating its potential application in natural vanillin synthesis. The enzymatic ferulic acid recovery accompanied a significant release of reducing sugars (76-100%), suggesting much broader applications of the enzymes and enzyme mixtures from this organism.     

Optimization of pH controlled liquid hot water pretreatment of corn stover

Controlled pH, liquid hot water pretreatment of corn stover has been optimized for enzyme digestibility with respect to processing temperature and time. This processing technology does not require the addition of chemicals such as sulfuric acid, lime, or ammonia that add cost to the process because these chemicals must be neutralized or recovered in addition to the significant expense of the chemicals themselves. Second, an optimized controlled pH, liquid hot water pretreatment process maximizes the solubilization of the hemicellulose fraction as liquid soluble oligosaccharides while minimizing the formation of monomeric sugars. The optimized conditions for controlled pH, liquid hot water pretreatment of a 16% slurry of corn stover in water was found to be 190 degrees C for 15 min. At the optimal conditions, 90% of the cellulose was hydrolyzed to glucose by 15FPU of cellulase per gram of glucan. When the resulting pretreated slurry, in undiluted form, was hydrolyzed by 11FPU of cellulase per gram of glucan, a hydrolyzate containing 32.5 g/L glucose and 18 g/L xylose was formed. Both the xylose and the glucose in this undiluted hydrolyzate were shown to be fermented by recombinant yeast 424A(LNH-ST) to ethanol at 88% of theoretical yield.