Partial flow of compressed-hot water through corn stover to enhance hemicellulose sugar recovery and enzymatic digestibility of cellulose

Flowthrough pretreatment with compressed-hot water can significantly increase the removal of xylan and lignin and enhance xylose sugar yields and cellulose digestibility, especially at high flow rates. However, continuous flowthrough operations that realize these benefits suffer from a large amount of water consumption that leads to high energy requirements for pretreatment and downstream processing. Because high flow rates are particularly effective early in hemicellulose hydrolysis and less effective later, flow with compressed-hot water was applied at selected intervals, and performance was compared with that of batch and flowthrough operations for corn stover pretreated with compressed-hot water at 200 degrees C. Partial flow reduced water consumption by 60% compared with continuous flowthrough operation but still achieved higher xylose sugar yields (84-89%) compared to batch pretreatment (46.6%). In addition, corn stover cellulose pretreated by partial flow had higher enzymatic digestibility (88-90%) than batch operations (approximately 85%) at otherwise identical conditions, apparently due to much higher lignin removal for the former (40-45% vs 10-12%). Partial flow also reduced degradation, with recovery of xylose and glucose in the solids and hydrolyzate increased to 90-92% vs only about 76% for batch operation. The partial flow approach could be further improved by optimizing the operating strategy and reaction conditions, suggesting that this novel pretreatment could lead to advanced biomass pretreatment technology.     

Influence of physico-chemical changes on enzymatic digestibility of ionic liquid and AFEX pretreated corn stover

Ionic liquid (IL) and ammonia fiber expansion (AFEX) pretreatments were studied to develop the first direct side-by-side comparative assessment on their respective impacts on biomass structure, composition, process mass balance, and enzymatic saccharification efficiency. AFEX pretreatment completely preserves plant carbohydrates, whereas IL pretreatment extracts 76% of hemicellulose. In contrast to AFEX, the native crystal structure of the recovered corn stover from IL pretreatment was significantly disrupted. For both techniques, more than 70% of the theoretical sugar yield was attained after 48 h of hydrolysis using commercial enzyme cocktails. IL pretreatment requires less enzyme loading and a shorter hydrolysis time to reach 90% yields. Hemicellulase addition led to significant improvements in the yields of glucose and xylose for AFEX pretreated corn stover, but not for IL pretreated stover. These results provide new insights into the mechanisms of IL and AFEX pretreatment, as well as the advantages and disadvantages of each.     

Effect of structural features on enzyme digestibility of corn stover

Corn stover was pretreated with excess calcium hydroxide (0.5 g Ca(OH)2/g raw biomass) in non-oxidative and oxidative conditions at 25, 35, 45, and 55 degrees C. The enzymatic digestibility of lime-treated corn stover was affected by the change of structural features (acetylation, lignification, and crystallization) resulting from the treatment. Extensive delignification required oxidative treatment and additional consumption of lime (up to 0.17 g Ca(OH)2/g biomass). Deacetylation reached a plateau within 1 week and there were no significant differences between non-oxidative and oxidative conditions at 55 degrees C; both conditions removed approximately 90% of the acetyl groups in 1 week at all temperatures studied. Delignification highly depended on temperature and the presence of oxygen. Lignin and hemicellulose were selectively removed (or solubilized), but cellulose was not affected by lime pretreatment in mild temperatures (25-55 degrees C), even though corn stover was contacted with alkali for a long time, 16 weeks. The degree of crystallinity slightly increased from 43% to 60% with delignification because amorphous components (lignin, hemicellulose) were removed. However, the increased crystallinity did not negatively affect the 3-d sugar yield of enzymatic hydrolysis. Oxidative lime pretreatment lowered the acetyl and lignin contents to obtain high digestibility, regardless of crystallinity. The non-linear models for 3-d hydrolysis yields of glucan (Y(g)), xylan (Y(x)), and holocellulose (Y(gx)) were empirically established as a function of the residual lignin (L) for the corn stover pretreated with lime and air.     

Combined sugar yields for dilute sulfuric acid pretreatment of corn stover followed by enzymatic hydrolysis of the remaining solids

A number of previous studies determined dilute acid pretreatment conditions that maximize xylose yields from pretreatment or glucose yields from subsequent digestion of the pretreated cellulose, but our emphasis was on identifying conditions to realize the highest yields of both sugars from both stages. Thus, individual xylose and glucose yields are reported as a percentage of the total potential yield of both sugars over a range of sulfuric acid concentrations of 0.22%, 0.49% and 0.98% w/w at 140, 160, 180 and 200 degrees C. Up to 15% of the total potential sugar in the substrate could be released as glucose during pretreatment and between 15% and 90+% of the xylose remaining in the solid residue could be recovered in subsequent enzymatic hydrolysis, depending on the enzyme loading. Glucose yields increased from as high as 56% of total maximum potential glucose plus xylose for just enzymatic digestion to 60% when glucose released in pretreatment was included. Xylose yields similarly increased from as high as 34% of total potential sugars for pretreatment alone to between 35% and 37% when credit was taken for xylose released in digestion. Yields were shown to be much lower if no acid was used. Conditions that maximized individual sugar yields were often not the same as those that maximized total sugar yields, demonstrating the importance of clearly defining pretreatment goals when optimizing the process. Overall, up to about 92.5% of the total sugars originally available in the corn stover used could be recovered for coupled dilute acid pretreatment and enzymatic hydrolysis. These results also suggest that enhanced hemicellulase activity could further improve xylose yields, particularly for low cellulase loadings.     

Development and validation of a kinetic model for enzymatic saccharification of lignocellulosic biomass

A multireaction kinetic model was developed for closed-system enzymatic hydrolysis of lignocellulosic biomass such as corn stover. Three hydrolysis reactions were modeled, two heterogeneous reactions for cellulose breakdown to cellobiose and glucose and one homogeneous reaction for hydrolyzing cellobiose to glucose. Cellulase adsorption onto pretreated lignocellulose was modeled via a Langmuir-type isotherm. The sugar products of cellulose hydrolysis, cellobiose and glucose, as well as xylose, the dominant sugar prevalent in most hemicellulose hydrolyzates, were assumed to competitively inhibit the enzymatic hydrolysis reactions. Model parameters were estimated from experimental data generated using dilute acid pretreated corn stover as the substrate. The model performed well in predicting cellulose hydrolysis trends at experimental conditions both inside and outside the design space used for parameter estimation and can be used for in silico process optimization.     

Novel Single-step Pretreatment of Steam Explosion and Choline Chloride to De-lignify Corn Stover for Enhancing Enzymatic Edibility

It is important to develop efficient and economically feasible pretreatment methods for lignocellulosic biomass, to increase annual biomass production. A number of pretreatment methods were introduced to promote subsequent enzymatic hydrolysis of biomass for green energy processes. Pretreatment with steam explosion removes the only xylan at high severity but increases lignin content. In this study, corn stover soaked in choline chloride solution before the steam explosion is economically feasible as it reduced cost. Enzymatic hydrolysis of de-lignified corn stover is enhanced by combinatorial pretreatments of steam explosion and choline chloride. Corn stover pretreated with choline chloride at the ratio of 1:2.2 (w/w), 1.0 MPa, 184 °C, for 15 min efficiently expelled 84.7% lignin and 78.9% xylan. The residual solid comprised of 74.59% glucan and 7.51% xylan was changed to 84.2% glucose and 78.3% xylose with enzyme stacking of 10FPU/g. This single-step pretreatment had ∼ 4.5 and 6.4 times higher glucose yield than SE-pretreated and untreated corn stover, respectively. Furthermore, SEM, XRD and FTIR indicated the porosity, crystalline changes, methoxy bond-cleavage respectively due to the lignin and hemicellulose expulsion. Thus, the released acetic acid during this process introduced this novel strategy, which significantly builds the viability of biomass in short pretreatment time.     

Biomimetic catalysis for hemicellulose hydrolysis in corn stover

Efficient and economical hydrolysis of plant cell wall polysaccharides into monomeric sugars is a significant technical hurdle in biomass processing for renewable fuels and chemicals. One possible approach to overcoming this hurdle is a biomimetic approach with dicarboxylic acid catalyst mimicking the catalytic core microenvironment in natural enzymes. This paper reports developments in the use of a dicarboxylic acid catalyst, maleic acid, for hemicellulose hydrolysis in corn stover. Hemicellulose hydrolysis and xylose degradation kinetics in the presence of maleic acid was compared to sulfuric acid. At optimized reaction conditions for each acid, maleic acid hydrolysis results in minimal xylose degradation, whereas sulfuric acid causes 3-10 times more xylose degradation. These results formed the basis for optimizing the hydrolysis of hemicellulose from corn stover using maleic acid. At 40 g/L dry corn stover solid-loading, both acid catalysts can achieve near-quantitative monomeric xylose yield. At higher solids loadings (150-200 g dry stover per liter), sulfuric acid catalyzed hydrolysis results in more than 30% degradation of the xylose, even under the previously reported optimal condition. However, as a result of minimized xylose degradation, optimized biomimetic hydrolysis of hemicellulose by maleic acid can reach approximately 95% monomeric xylose yields with trace amounts of furfural. Fermentation of the resulting unconditioned hydrolysate by recombinant S. cerevisiae results in 87% of theoretical ethanol yield. Enzyme digestibility experiments on the residual corn stover solids show that >90% yields of glucose can be produced in 160 h from the remaining cellulose with cellulases (15 FPU/g-glucan).     

Lime pretreatment and enzymatic hydrolysis of corn stover

Corn stover was pretreated with an excess of calcium hydroxide (0.5 g Ca(OH)2/g raw biomass) in non-oxidative and oxidative conditions at 25, 35, 45, and 55 degrees C. The optimal condition is 55 degrees C for 4 weeks with aeration. Glucan (91.3%) and xylan (51.8%) were converted to glucose and xylose respectively, when the treated corn stover was enzymatically hydrolyzed with 15 FPU/g cellulose. Only 0.073 g Ca(OH)2 was consumed per g of raw corn stover. Of the initial lignin, 87.5% was maximally removed. Almost all acetyl groups were removed. After 4 weeks at 55 degrees C with aeration, some cellulose and hemicellulose were solubilized as monomers and oligomers in the pretreatment liquor. When considering the dissolved fragments of glucan and xylan in the pretreatment liquor, the overall yields of glucose and xylose were 93.2% and 79.5% at 15 FPU/g cellulose. The pretreatment liquor has no inhibitory effect on ethanol fermentation.     

Enzymatic digestion of liquid hot water pretreated hybrid poplar

Liquid hot (LHW) water pretreatment (LHW) of lignocellulosic material enhances enzymatic conversion of cellulose to glucose by solubilizing hemicellulose fraction of the biomass, while leaving the cellulose more reactive and accessible to cellulase enzymes. Within the range of pretreatment conditions tested in this study, the optimized LHW pretreatment conditions for a 15% (wt/vol) slurry of hybrid poplar were found to be 200^oC, 10 min, which resulted in the highest fermentable sugar yield with minimal formation of sugar decomposition products during the pretreatment. The LHW pretreatment solubilized 62% of hemicellulose as soluble oligomers. Hot‐washing of the pretreated poplar slurry increased the efficiency of hydrolysis by doubling the yield of glucose for a given enzyme dose. The 15% (wt/vol) slurry of hybrid poplar, pretreated at the optimal conditions and hot‐washed, resulted in 54% glucose yield by 15 FPU cellulase per gram glucan after 120 h. The hydrolysate contained 56 g/L glucose and 12 g/L xylose. The effect of cellulase loading on the enzymatic digestibility of the pretreated poplar is also reported. Total monomeric sugar yield (glucose and xylose) reached 67% after 72 h of hydrolysis when 40 FPU cellulase per gram glucan were used. An overall mass balance of the poplar‐to‐ethanol process was established based on the experimentally determined composition and hydrolysis efficiencies of the liquid hot water pretreated poplar. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

BSA treatment to enhance enzymatic hydrolysis of cellulose in lignin containing substrates

Cellulase and bovine serum albumin (BSA) were added to Avicel cellulose and solids containing 56% cellulose and 28% lignin from dilute sulfuric acid pretreatment of corn stover. Little BSA was adsorbed on Avicel cellulose, while pretreated corn stover solids adsorbed considerable amounts of this protein. On the other hand, cellulase was highly adsorbed on both substrates. Adding a 1% concentration of BSA to dilute acid pretreated corn stover prior to enzyme addition at 15 FPU/g cellulose enhanced filter paper activity in solution by about a factor of 2 and beta-glucosidase activity in solution by about a factor of 14. Overall, these results suggested that BSA treatment reduced adsorption of cellulase and particularly beta-glucosidase on lignin. Of particular note, BSA treatment of pretreated corn stover solids prior to enzymatic hydrolysis increased 72 h glucose yields from about 82% to about 92% at a cellulase loading of 15 FPU/g cellulose or achieved about the same yield at a loading of 7.5 FPU/g cellulose. Similar improvements were also observed for enzymatic hydrolysis of ammonia fiber explosion (AFEX) pretreated corn stover and Douglas fir treated by SO(2) steam explosion and for simultaneous saccharification and fermentation (SSF) of BSA pretreated corn stover. In addition, BSA treatment prior to hydrolysis reduced the need for beta-glucosidase supplementation of SSF. The results are consistent with non-specific competitive, irreversible adsorption of BSA on lignin and identify promising strategies to reduce enzyme requirements for cellulose hydrolysis.     

Dilute sulfuric acid cycle spray flow-through pretreatment of corn stover for enhancement of sugar recovery

A cycle spray flow-through reactor was designed and used to pretreat corn stover in dilute sulfuric acid medium. The dilute sulfuric acid cycle spray flow-through (DCF) process enhanced xylose sugar yields and cellulose digestibility while increasing the removal of lignin. Within the DCF system, the xylose sugar yields of 90–93% could be achieved for corn stover pretreated with 2% (w/v) dilute sulfuric acid at 95 °C during the optimal reaction time (90 min). The remaining solid residue exhibited enzymatic digestibility of 90–95% with cellulase loading of 60 FPU/g glucan that was due to the effective lignin removal (70–75%) in this process. Compared with flow-through and compress-hot water pretreatment process, the DCF method produces a higher sugar concentration and higher xylose monomer yield. The novel DCF process provides a feasible approach for lignocellulosic material pretreatment.     

Production of ethanol from corn stover hemicellulose hydrolyzate using <Emphasis Type="Italic">Pichia stipitis</Emphasis>

Hemicellulose liquid hydrolyzate from dilute acid pretreated corn stover was fermented to ethanol using Pichia stipitis CBS 6054. The fermentation rate increased with aeration but the pH also increased due to consumption of acetic acid by Pichia stipitis. Hemicellulose hydrolyzate containing 34 g/L xylose, 8 g/L glucose, 8 g/L Acetic acid, 0.73 g/L furfural, and 1 g/L hydroxymethyl furfural was fermented to 15 g/L ethanol in 72 h. The yield in all the hemicellulose hydrolyzates was 0.37–0.44 g ethanol/g (glucose + xylose). Nondetoxified hemicellulose hydrolyzate from dilute acid pretreated corn stover was fermented to ethanol with high yields, and this has the potential to improve the economics of the biomass to ethanol process.     

Enhanced enzymatic hydrolysis and structural features of corn stover by FeCl3 pretreatment

Corn stover was pretreated with FeCl₃ to remove almost all of the hemicellulose present and then hydrolyzed with cellulase and β-glucosidase to produce glucose. Enzymatic hydrolysis of corn stover that had been pretreated with FeCl₃ at 160 °C for 20 min resulted in an optimum yield of 98.0%. This yield was significantly higher than that of untreated corn stover (22.8%). FeCl₃ pretreatment apparently damaged the surface of corn stover and significantly increased the enzymatic digestibility, as evidenced by SEM and XRD analysis data. FTIR analysis indicated that FeCl₃ pretreatment could disrupt almost all the ether linkages and some ester linkages between lignin and carbohydrates but had no effect on delignification. The FeCl₃ pretreatment technique, as a novel pretreatment method, enhances enzymatic hydrolysis of lignocellulosic biomass by destructing chemical composition and altering structural features.     

Improved ethanol yield and reduced Minimum Ethanol Selling Price (MESP) by modifying low severity dilute acid pretreatment with deacetylation and mechanical refining: 1) Experimental

ABSTRACT: BACKGROUND: Historically, acid pretreatment technology for the production of bio-ethanol from corn stover has required severe conditions to overcome biomass recalcitrance. However, the high usage of acid and steam at severe pretreatment conditions hinders the economic feasibility of the ethanol production from biomass. In addition, the amount of acetate and furfural produced during harsh pretreatment is in the range that strongly inhibits cell growth and impedes ethanol fermentation. The current work addresses these issues through pretreatment with lower acid concentrations and temperatures incorporated with deacetylation and mechanical refining. RESULTS: The results showed that deacetylation with 0.1 M NaOH before acid pretreatment improved the monomeric xylose yield in pretreatment by up to 20 % while keeping the furfural yield under 2 %. Deacetylation also improved the glucose yield by 10 % and the xylose yield by 20 % during low solids enzymatic hydrolysis. Mechanical refining using a PFI mill further improved sugar yields during both low- and high-solids enzymatic hydrolysis. Mechanical refining also allowed enzyme loadings to be reduced while maintaining high yields. Deacetylation and mechanical refining are shown to assist in achieving 90 % cellulose yield in high-solids (20 %) enzymatic hydrolysis. When fermentations were performed under pH control to evaluate the effect of deacetylation and mechanical refining on the ethanol yields, glucose and xylose utilizations over 90 % and ethanol yields over 90 % were achieved. Overall ethanol yields were calculated based on experimental results for the base case and modified cases. One modified case that integrated deacetylation, mechanical refining, and washing was estimated to produce 88 gallons of ethanol per ton of biomass. CONCLUSION: The current work developed a novel bio-ethanol process that features pretreatment with lower acid concentrations and temperatures incorporated with deacetylation and mechanical refining. The new process shows improved overall ethanol yields compared to traditional dilute acid pretreatment. The experimental results from this work support the techno-economic analysis and calculation of Minimum Ethanol Selling Price (MESP) detailed in our companion paper.     

Effect of biological pretreatment with Trametes hirsuta yj9 on enzymatic hydrolysis of corn stover

In this study, a newly isolated Trametes hirsuta yj9 was used to pretreat corn stover in order to enhance enzymatic digestibility. T. hirsuta yj9 preferentially degraded lignin to be as high as 71.49% after 42-day pretreatment. Laccase and xylanase was the major ligninolytic and hydrolytic enzyme, respectively and filter paper activity (FPA) increased gradually with prolonged pretreatment time. Sugar yields increased significantly after pretreatment with T. hirsuta yj9, reaching an enzymatic digestibility of 73.99% after 42 days of pretreatment. Scanning electron microscopy (SEM) showed significant structural changes in pretreated corn stover, the surface of pretreated corn stover became increasingly coarse, the gaps between cellulose fibers were visible, and many pores were developed. Correlation analysis showed that sugar yields were inversely proportional to the lignin contents, less related to cellulose and hemicellulose contents.     

固定化纤维素酶对预处理纤维素原料水解效果的影响

以壳聚糖为载体用交联法制备固定化纤维素酶,考察固定化纤维素酶对蒸爆、球磨、超声波、喷淋、高温预处理玉米秸秆纤维素原料的酶解效果.结果表明:物料经蒸爆预处理后酶水解效率最高可以达到95%,球磨预处理水解效率次之,达到60%.用电镜和FT-IR对处理前后秸秆结构进行表征分析,证明预处理对物料的物理结构及化学组成有一定的影响.蒸爆法和球磨法可以使物料致密的天然结构彻底破坏,从而增加物料的比表面积;蒸爆预处理可以使纤维素内部氢键和官能团改变,使物料更易于酶解.     

The pretreatment of corn stover with Gloeophyllum trabeum KU-41 for enzymatic hydrolysis

Background

Pretreatment is an essential step in the enzymatic hydrolysis of biomass for bio-ethanol production. The dominant concern in this step is how to decrease the high cost of pretreatment while achieving a high sugar yield. Fungal pretreatment of biomass was previously reported to be effective, with the advantage of having a low energy requirement and requiring no application of additional chemicals. In this work, Gloeophyllum trabeum KU-41 was chosen for corn stover pretreatment through screening with 40 strains of wood-rot fungi. The objective of the current work is to find out which characteristics of corn stover pretreated with G. trabeum KU-41 determine the pretreatment method to be successful and worthwhile to apply. This will be done by determining the lignin content, structural carbohydrate, cellulose crystallinity, initial adsorption capacity of cellulase and specific surface area of pretreated corn stover.

Results

The content of xylan in pretreated corn stover was decreased by 43% in comparison to the untreated corn stover. The initial cellulase adsorption capacity and the specific surface area of corn stover pretreated with G. trabeum were increased by 7.0- and 2.5-fold, respectively. Also there was little increase in the cellulose crystallinity of pretreated corn stover.

Conclusion

G. trabeum has an efficient degradation system, and the results indicated that the conversion of cellulose to glucose increases as the accessibility of cellulose increases due to the partial removal of xylan and the structure breakage of the cell wall. This pretreatment method can be further explored as an alternative to the thermochemical pretreatment method.     

Kinetic modeling analysis of maleic acid-catalyzed hemicellulose hydrolysis in corn stover

Maleic acid-catalyzed hemicellulose hydrolysis reaction in corn stover was analyzed by kinetic modeling. Kinetic constants for Saeman and biphasic hydrolysis models were analyzed by an Arrhenius-type expansion which include activation energy and catalyst concentration factors. The activation energy for hemicellulose hydrolysis by maleic acid was determined to be 83.3 +/- 10.3 kJ/mol, which is significantly lower than the reported E(a) values for sulfuric acid catalyzed hemicellulose hydrolysis reaction. Model analysis suggest that increasing maleic acid concentrations from 0.05 to 0.2 M facilitate improvement in xylose yields from 40% to 85%, while the extent of improvement flattens to near-quantitative by increasing catalyst loading from 0.2 to 1 M. The model was confirmed for the hydrolysis of corn stover at 1 M maleic acid concentrations at 150 degrees C, resulting in a xylose yield of 96% of theoretical. The refined Saeman model was used to evaluate the optimal condition for monomeric xylose yield in the maleic acid-catalyzed reaction: low temperature reaction conditions were suggested, however, experimental results indicated that bi-phasic behavior dominated at low temperatures, which may be due to the insufficient removal of acetyl groups. A combination of experimental data and model analysis suggests that around 80-90% xylose yields can be achieved at reaction temperatures between 100 and 150 degrees C with 0.2 M maleic acid.     

Cellulase adsorption and relationship to features of corn stover solids produced by leading pretreatments

Although essential to enzymatic hydrolysis of cellulosic biomass to sugars for fermentation to ethanol or other products, enzyme adsorption and its relationship to substrate features has received limited attention, and little data and insight have been developed on cellulase adsorption for promising pretreatment options, with almost no data available to facilitate comparisons. Therefore, adsorption of cellulase on Avicel, and of cellulase and xylanase on corn stover solids resulting from ammonia fiber expansion (AFEX), ammonia recycled percolation (ARP), controlled pH, dilute acid, lime, and sulfur dioxide (SO₂) pretreatments were measured at 4°C. Langmuir adsorption parameters were then estimated by non‐linear regression using Polymath software, and cellulase accessibility to cellulose was estimated based on adsorption data for pretreated solids and lignin left after carbohydrate digestion. To determine the impact of delignification and deacetylation on cellulose accessibility, purified CBHI (Cel7A) adsorption at 4°C and hydrolysis with whole cellulase were followed for untreated (UT) corn stover. In all cases, cellulase attained equilibrium in less than 2 h, and upon dilution, solids pretreated by controlled pH technology showed the greatest desorption followed by solids from dilute acid and SO₂ pretreatments. Surprisingly, the lowest desorption was measured for Avicel glucan followed by solids from AFEX pretreatment. The higher cellulose accessibility for AFEX and lime pretreated solids could account for the good digestion reported in the literature for these approaches. Lime pretreated solids had the greatest xylanase capacity and AFEX solids the least, showing pretreatment pH did not seem to be controlling. The 24 h glucan hydrolysis rate data had a strong relationship to cellulase adsorption capacities, while 24 h xylan hydrolysis rate data showed no relationship to xylanase adsorption capacities. Furthermore, delignification greatly enhanced enzyme effectiveness but had a limited effect on cellulose accessibility. And because delignification enhanced release of xylose more than glucose, it appears that lignin did not directly control cellulose accessibility but restricted xylan accessibility which in turn controlled access to cellulose. Reducing the acetyl content in corn stover solids significantly improved both cellulose accessibility and enzyme effectiveness. Biotechnol. Bioeng. 2009;103: 252–267. © 2009 Wiley Periodicals, Inc.     

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.