Ethanol production from hydrothermal pretreated corn stover with a loop reactor

Hydrothermal pretreatment on raw corn stover (RCS) with a loop reactor was investigated at 195 °C for different times varying between 10 min and 30 min. After pretreatment, the slurry was separated into water-insoluble solid (WIS) and liquid phase. Glucan and xylan were found in the both phases. The pretreatment condition showed a significant impact on xylan recovery. As the pretreatment time prolonged from 10 min to 30 min, the xylan recovery from liquid phase changed between 39.5% and 45.6% and the total xylan recoveries decreased from 84.7% to 61.6%. While the glucan recovery seemed not sensitive to the different pretreatment times. The glucan recovered from liquid was from 4.9% to 5.6% and the total glucan recoveries from all the pretreatments were higher than 98%. Besides HMF and furfural, acetic, lactic, formic and glycolic acids were also found in the liquid phase. All the concentrations of these potential inhibitors were lower enough not to affect the activity of the Saccharomyces cerevisiae (S. cerevisiae). Compared with the ethanol production of 32.4% from the RCS with S. cerevisiae, all the WISs gave higher ethanol productions ranging between 61.2% and 71.2%. When the xylan was taken into consideration, the best pretreatment condition would be 195 °C, 15 min and the estimated total ethanol production was 201 g kg^?1 RCS by assuming the fermentation of both C-6 and C-5 with the ethanol yield of 0.51 g g^?1 and 0.47 g g^?1, respectively.     

Optimization of the medium composition for the improvement of hydrogen and butanol production using Clostridium saccharoperbutylacetonicum DSM 14923

The dark fermentation process promises a sustainable route for biohydrogen production. But the low substrate conversion efficiency hinders its commercial feasibility. The present study aims towards simultaneous hydrogen and butanol production to enhance the net energy recovery. Process parameters optimization revealed that pH of 6.5, temperature of 37 °C and inoculum size of 7% (v/v) were suitable for obtaining maximum hydrogen and butanol yields by C. saccharoperbutylacetonicum. Starch and xylan were observed to be preferred carbon sources suggesting effective utilization of C₅ and C₆ sugars. The maximum hydrogen yield of 264.3 mL g⁻¹ starch and 216 mL g⁻¹ xylan and butanol yield of 0.27 g g⁻¹ starch and 0.24 g g⁻¹ xylan were obtained with overall energy recovery of 85.61% (starch) and 75.22% (xylan), respectively. The present research work indicates the potency of bi-phasic fermentation to boost energy recovery from starch/xylan based feedstock.     

Development of an integrated pretreatment fractionation process for fermentable sugars and lignin: Application to almond (Prunus dulcis) shell

An environmentally friendly pretreatment process was developed to fractionate cellulose, hemicellulose and lignin from almond (Prunus dulcis) shells, consisting of hot water pretreatment (HWP) coupled with organic solvent (organosolv) pretreatment of water/ethanol (OWEP). This integrated pretreatment process proved more effective on the basis of yield of fermentable sugar and lignin separation compared with HWP alone, dilute acid pretreatment (DAP), ammonia pretreatment (AP), lime pretreatment LP, organosolv water/ethanol pretreatment (OWEP), and organosolv water/acetone pretreatment (OWAP). In the coupled hot water-organosolv process, hemicellulose sugars were recovered in the first residual liquid while varying amounts of cellulose was retained in the residual solid. The lignin fraction was obtained by simply adjusting the pH from the second liquid. The optimal two-stage process consisted of first HWP stage at 195 °C for 30 min, resulting in w_glucose = 95.4% glucose recovery yield and w_xylose = 92.2% xylose removal. The second organosolv OWEP stage was operated at 195 °C for 20 min, in ethanol in water mixtures of <phi>_ethanol = 50% and resulted in nearly w_glucose = 100% glucose recovery yield, w_xylose = 90% xylose and w_lignin = 61% lignin removal. After enzymatic hydrolysis, glucose yield was up to w_glucose = 95%, compared to 61% yield from untreated almond. Images obtained via scanning electron microscopy (SEM) highlighted the differences in almond structure from the varying pretreatment methods during biomass fractionation.     

Effect of magnesium chloride on fractionation and enzymatic digestibility of cattails

The effect of magnesium chloride (a Lewis acid) on the fractionation of aquatic plant cattail was studied using a Dionex accelerated solvent extractor varying salt concentration (0–0.4 M), treatment temperature (140–180°C), and residence time (5–15 min). MgCl₂ treatment enhanced xylan degradation and delignification, and up to 33.8% of total xylan was directly hydrolyzed to the xylose monomer. By removing xylan and lignin, the MgCl₂ treatment effectively increased enzymatic digestibility of cattail cellulose and xylan. When cattails were treated with the 0.4 M MgCl₂ at 180°C for 15 min and subsequently hydrolyzed for 48 h with a cellulase loading of 15 FPU/g glucan, the highest glucose yield of 61.7% and the highest total xylose yield of 90.6% were reached, respectively. Application of Lewis acids as a novel biomass fractionation approach will require lower xylanase loading for hydrolysis, and have lower corrosion to equipment compared to other acid pretreatment processes.     

Hydrogen production from soft-drink wastewater in an upflow anaerobic packed-bed reactor

The production of hydrogen from soft-drink wastewater in two upflow anaerobic packed-bed reactors was evaluated. The results show that soft-drink wastewater is a good source for hydrogen generation. Data from both reactors indicate that the reactor without medium containing macro- and micronutrients (R2) provided a higher hydrogen yield (3.5 mol H₂ mol⁻¹ of sucrose) as compared to the reactor (R1) with a nutrient-containing medium (3.3 mol H₂ mol⁻¹ of sucrose). Reactor R2 continuously produced hydrogen, whereas reactor R1 exhibited a short period of production and produced lower amounts of hydrogen. Better hydrogen production rates and percentages of biogas were also observed for reactor R2, which produced 0.4 L h⁻¹ L⁻¹ and 15.8% of H₂, compared to reactor R1, which produced 0.2 L h⁻¹ L⁻¹ and 2.6% of H₂. The difference in performance between the reactors was likely due to changes in the metabolic pathway for hydrogen production and decreases in bed porosity as a result of excessive biomass growth in reactor R1. Molecular biological analyses of samples from reactors R1 and R2 indicated the presence of several microorganisms, including Clostridium (91% similarity), Enterobacter (93% similarity) and Klebsiella (97% similarity).     

Effectiveness of dilute oxalic acid pretreatment of Miscanthus × giganteus biomass for ethanol production

In the present study the effect of temperature, reaction time and dilute oxalic acid (OA) concentration during steam-pretreatment of Miscanthus × gigantueus has been evaluated using the combined severity factor (CS). At the highest CS glucan and lignin content in the water insoluble fraction (WIF) increased, while xylan content decreased. While glucose recovery in the water soluble fraction (WSF) was found at low concentration when mild CS were used (≤5.0 g L⁻¹ at CS ≤ 2.17), xylose and arabinose concentrations were higher at low-mild CS (1.58–2.17) with a concentration peak at CS 2.03 (39.9 and 3.2 g L⁻¹ for xylose and arabinose, respectively). The decrease in pentoses coincided with inhibitory formation in the WSF, namely acetic acid, furfural, HMF and phenolic compounds. Glucan conversion rose from 46.1% at CS 1.54 to 91.2% at CS 2.76. Likewise, maximum ethanol concentration was achieved at CS 2.76, corresponding to 20.2 g L⁻¹ and a volumetric ethanol productivity of 0.28 g L⁻¹ h⁻¹. Negative correlations have been found between xylan vs. glucan conversion and xylan vs. ethanol production, suggesting that decreasing the xylan content in WIF increases both saccharification rate and ethanol concentration (R² 0.91 and R² 0.93, respectively). On the other hand, a positive correlation was found between ethanol production and glucan conversion (R² 0.93). Fermentation of WSF by Scheffersomyces (Pichia) stipitis CBS 6054 at CS 1.54 produced 12.1 g L⁻¹ of ethanol after 96 h incubation with a volumetric ethanol productivity of 0.13 g L⁻¹ h⁻¹.     

Preliminary evaluation of organosolv pre-treatment of sugar cane bagasse for glucose production: Application of 2 experimental design

Sugar cane bagasse was submitted to ethanol organosolv pre-treatment using a 50 L pilot scale reactor. The influence of catalyst type (H₂SO₄ or NaOH), catalyst concentration (1.25–1.50% w/w on dry fiber) and process time (60–90 min) on total solid recovery and solid composition (glucan, xylan and lignin contents) was evaluated by performing a 2³ full factorial experimental design. Pretreated sugar cane bagasse was further submitted to enzymatic hydrolysis using a commercial enzyme complex formed by cellulases and β-glucosidases. Glucose concentration in the hydrolysates and glucose yield referred to initial raw material (g glucose/100 g sugar cane bagasse) were used to select the best operational conditions. Concerning the enzymatic hydrolysis, the resulting glucose concentration was found to be dependent on xylan contents of the pretreated material. The modelling equations for glucose concentration and glucose yield as a function of the pre-treatment variables and the statistical analysis are also discussed in this work.     

Electricity generation by microbial fuel cells fuelled with wheat straw hydrolysate

Electricity production from microbial fuel cells fueled with hydrolysate produced by hydrothermal treatment of wheat straw can achieve both energy production and domestic wastewater purification. The hydrolysate contained mainly xylan, carboxylic acids, and phenolic compounds. Power generation and substrate utilization from the hydrolysate was compared with the ones obtained by defined synthetic substrates. The power density increased from 47 mW m⁻² to 148 mW m⁻² with the hydrolysate:wastewater ratio (R_HW in m³ m⁻³) increasing from 0 to 0.06 (corresponding to 0-0.7 g dm⁻³ of carbohydrates). The power density with the hydrolysate was higher than the one with only xylan (120 mW m⁻²) and carboxylic acids as fuel. The higher power density can be caused by the presence of phenolic compounds in the hydrolysates, which could mediate electron transport. Electricity generation with the hydrolysate resulted in 95% degradation of the xylan and glucan. The study demonstrates that lignocellulosic hydrolysate can be used for co-treatment with domestic wastewater for power generation in microbial fuel cells.     

Biohydrogen production by Thermoanaerobacterium thermosaccharolyticum KKU-ED1: Culture conditions optimization using xylan as the substrate

Thermophilic hydrogen production from xylan by Thermoanaerobacterium thermosaccharolyticum KKU-ED1 isolated from elephant dung was investigated using batch fermentation. The optimum conditions for hydrogen production from xylan by the strain KKU-ED1 were an initial pH of 7.0, temperature of 55 °C and xylan concentration of 15 g/L. Under the optimum conditions, the hydrogen yield (HY), hydrogen production rate (HPR) and xylanase activity were 120.05 ± 15.07 mL H₂/g xylan, 11.53 ± 0.19 mL H₂/L h and 0.41 units/mL, respectively. The optimum conditions were then used to produce hydrogen from 62.5 g/L sugarcane bagasse (SCB) (equivalent to 15 g/L xylan) in which the HY and HPR of 1.39 ± 0.10 mL H₂/g SCB (5.77 ± 0.41 mL H₂/g xylan) and 0.66 ± 0.04 mL H₂/L h, respectively, were achieved. In comparison to the other strains, the HY of the strain KKU-ED1 (120.05 ± 15.07 mL H₂/g xylan) was close to that of Clostridium sp. strain X53 (125.40 mL H₂/g xylan) and Clostridium butyricum CGS5 (90.70 mL H₂/g xylan hydrolysate).     

One-stage H2 and CH4 and two-stage H2 + CH4 production from grass silage and from solid and liquid fractions of NaOH pre-treated grass silage

In the present study, mesophilic CH₄ production from grass silage in a one-stage process was compared with the combined thermophilic H₂ and mesophilic CH₄ production in a two-stage process. In addition, solid and liquid fractions separated from NaOH pre-treated grass silage were also used as substrates. Results showed that higher CH₄ yield was obtained from grass silage in a two-stage process (467 ml g⁻¹ volatile solids (VS)_original) compared with a one-stage process (431 ml g⁻¹ VS_original). Similarly, CH₄ yield from solid fraction increased from 252 to 413 ml g⁻¹ VS_original whereas CH₄ yield from liquid fraction decreased from 82 to 60 ml g⁻¹ VS_original in a two-stage compared to a one-stage process. NaOH pre-treatment increased combined H₂ yield by 15% (from 5.54 to 6.46 ml g⁻¹ VS_original). In contrast, NaOH pre-treatment decreased the combined CH₄ yield by 23%. Compared to the energy value of CH₄ yield obtained, the energy value of H₂ yield remained low. According to this study, highest CH₄ yield (495 ml g⁻¹ VS_original) could be obtained, if grass silage was first pre-treated with NaOH, and the separated solid fraction was digested in a two-stage (thermophilic H₂ and mesophilic CH₄) process while the liquid fraction could be treated directly in a one-stage CH₄ process.     

Conversion of rice straw to sugars by dilute-acid hydrolysis

Hydrolysis of rice straw by dilute sulfuric acid at high temperature and pressure was investigated in one and two stages. The hydrolyses were carried out in a 10-l reactor, where the hydrolysis retention time (3–10 min), pressure (10–35 bar) and acid concentration (0–1%) were examined. Optimization of first stage hydrolysis is desirable to achieve the highest yield of the sugars from hemicellulose and also as a pretreatment for enzymatic hydrolysis. The results show the ability of first stage hydrolysis to depolymerize xylan to xylose with a maximum yield of 80.8% at hydrolysis pressure of 15 bar, 10 min retention time and 0.5% acid concentration. However, the yield of glucose from glucan was relatively low in first stage hydrolysis at a maximum of 25.8%. The solid residuals were subjected to further dilute-acid hydrolysis in this study. This second-stage hydrolysis without addition of the acid could not increase the yield of glucose from glucan beyond 26.6%. On the other hand, the best results of the hydrolysis were achieved, when 0.5% sulfuric acid was added prior to each stage in two-stage hydrolysis. The best results of the second stage of the hydrolysis were achieved at the hydrolysis pressure and the retention time of 30 bar and 3 min in the second stage hydrolysis, where a total of 78.9% of xylan and 46.6% of glucan were converted to xylose and glucose, respectively in the two stages. Formation of furfural and HMF were functions of the hydrolysis pressure, acid concentration, and retention time, whereas the concentration of acetic acid was almost constant at pressure of higher than 10 bar and a total retention time of 10 min.     

Bioreactor for glycerol conversion into H2 by bacterium Enterobacter aerogenes

Glycerol was used as a substrate for H₂ production by bacterium Enterobacter aerogenes in the test tubes and bioreactor. A BioFlo/CelliGen 115 bioreactor (10 L working volume) was utilized to conduct the experiments for conversion of glycerol into H₂ by E. aerogenes cells. The highest H₂ production rate was observed under 2% glycerol in the culture medium. The glycerol uptake efficiency by bacteria in the bioreactor was found to be 65% during the 6 day period, matching glycerol uptake efficiency observed in the test tubes experiment (65%).Hydrogen production from glycerol (2% glycerol, v/v) by E. aerogenes in the bioreactor and test tubes was measured over the 6 days, showing the maximal H₂ rate at 650 mL g⁻¹ dry weight h⁻¹. The yield of H₂ production from glycerol at 0.89 mol/mol in the bioreactor was high, corresponding to the theoretical yield of 1 mol of H₂ per 1 mol of glycerol.     

Reutilization of carbon sources through sugar recovery from waste rice straw

Rice straw was utilized for the cultivation of Phanerochaete chrysoporium to produce cellobiose dehydrogenase. The composition of the rice straw after fermentation was found to be 28.77% glucan, 19.05% xylan and 54.81% other lignin containing sugars. The glucan and xylan content decreased due to the consumption of glucan and xylan by P. chrysoporium. After fermentation, the rice straw waste was subjected to chemical pretreatment to remove lignin. The effect of dilute acid pretreatment was not notable because of the glucose loss. However, when the rice straw after fermentation was treated with aqueous ammonia, the composition changed to 44.73% glucan, 25.43% xylan and 29.52% other lignin containing sugars. The aqueous ammonia pretreatment was optimized and an ammonia concentration, reaction time and temperature of 20%, 6 h and 60 °C, respectively, were determined to be the optimal pretreatment conditions After removal of lignin, the initial reaction rate was increased to 0.009583 g/L s, which was about 3 fold higher than the rice straw after fermentation. X-ray diffractometry was performed to investigate the crystallinity index, and the XRD results showed that biological treatment and the combination of both biological treatment and chemical pretreatment decreased the crystallinity index.     

Enhancement in hydrogen production by co-cultures of Bacillus and Enterobacter

Defined co-cultures of hydrogen (H₂) producers belonging to Citrobacter, Enterobacter, Klebsiella and Bacillus were used for enhancing the efficiency of biological H₂ production. Out of 11 co-cultures consisting of 2–4 strains, two co-cultures composed of Bacillus cereus EGU43, Enterobacter cloacae HPC123, and Klebsiella sp. HPC793 resulted in H₂ yield up to 3.0 mol mol⁻¹ of glucose. Up-scaling of the reactor by 16-fold resulted in a corresponding increase in H₂ production with an actual evolution of 7.44 L of H₂. It constituted 58.2% of the total biogas. Continuous culture evolution of H₂ by co-cultures (B. cereus EGU43 and E. cloacae HPC123) immobilized on ligno-cellulosic materials resulted in 6.4-fold improvement in H₂ yield compared to free floating bacteria. This synergistic influence of B. cereus and E. cloacae can offer a better strategy for H₂ production than undefined or mixed cultures.     

Optimization of photosynthetic hydrogen yield from platinized photosystem I complexes using response surface methodology

Light-dependent hydrogen production by platinized Photosystem I isolated from the cyanobacterium Thermosynechococcus elongatus BP-1 was optimized using response surface methodology (RSM). The process parameters studied included temperature, light intensity and wavelength, and platinum salt concentration. Application of RSM generated a model that agrees well with the data for H₂ yield (R² = 0.99 and p < 0.001). Significant effects on the total H₂ yield were seen when the platinum salt concentration and temperature were varied during platinization. However, light intensity during platinization had a minimal effect on the total H₂ yield within the region studied. The values of the parameters used during the platinization that optimized the production of H₂ were light intensity of 240 μE m⁻² s⁻¹, platinum salt concentration of 636 μM and temperature of 31 °C. A subsequent validation experiment at the predicted conditions for optimal process yield gave the maximum H₂ yield measured in the study, which was 8.02 μmol H₂ per mg chlorophyll.     

Valorization of Leucaena leucocephala for energy and chemicals from autohydrolysis

In this work, Leucaena leucocephala K366 was characterized chemical and energy terms, and assessed its potential as a lignocellulosic raw material and energetic and industrial crop specie, and its integral fractionation by autohydrolysis by evaluating its calorific value, holocellulose, glucan, xylan, arabinan, lignin and oligomers and monomers contents in autohydrolysis liquor and solid phase. Also, this paper will consider the influence of the temperature and time of autohydrolysis process from L. leucocephala K366 to obtain valuable liquor and a suitable solid phase to produce energy by combustion.Valuable liquor was obtained from the autohydrolysis of L. leucocephala by simultaneously using operating temperatures and times in the medium-high ranges studied, namely: 172-184 °C and 15-30 min. The optimum processing conditions provided an acceptable yield (16-26%), and high xylose and xylo-oligomer contents in the liquor (10.0 and 58.6%, respectively, of the amounts present in the starting raw material when operating at 184 °C for 30 min) in comparison with other raw materials. The arabinan fraction was extracted virtually completely —only 8.3% remained in the solid fraction—, and the acetyl group fraction was recovered in full. In addition, these conditions reduced the glucose content of the liquor to 2.9% of the amount present in the raw material while largely preserving the integrity of cellulose fibers.Klason lignin was scarcely dissolved under the operating conditions of the autohydrolysis process. This increased the calorific value of the solid phase by 9% (under the most drastic operating conditions) with respect to the starting raw material.     

Two-stage anaerobic dry digestion of blue mussel and reed

Blue mussels and reeds were explored as a new biomass type in the Kalmar County of Sweden to improve renewable transport fuel production in the form of biogas. Anaerobic digestion of blue mussels and reeds was performed at a laboratory-scale to evaluate biogas production in a two-stage dry digestion system. The two-stage system consisted of a leach bed reactor and an upflow anaerobic sludge blanket (UASB) reactor. The two-stage system was efficient for the digestion of blue mussels, including shells, and a methane yield of 0.33 m³/kg volatile solids (VS) was obtained. The meat fraction of blue mussels was easily solubilised in the leach bed reactor and the soluble organic materials were rapidly converted in the UASB reactor from which 68% of the methane was produced. However, the digestion of mussels including shells gave low production capacity, which may result in a less economically viable biogas process. A low methane potential, 0.22 m³/kg VS, was obtained in the anaerobic two-stage digestion of reeds after 107 days; however, it was comparable to similar types of biomass, such as straw. About 80% of the methane was produced in the leach bed reactor. Hence, only a leach bed reactor (dry digestion) may be needed to digest reed. The two-stage anaerobic digestion of blue mussels and reeds resulted in an energy potential of 16.6 and 10.7 GWh/year, respectively, from the estimated harvest amounts. Two-stage anaerobic digestion of new organic materials such as blue mussels and reeds can be a promising biomass resource as land-based biomass start to be limited and conflict with food resources can be avoided.     

Evaluation of continuous biohydrogen production from enzymatically treated cornstalk hydrolysate

Enzymatically treated cornstalk hydrolysate was tested as substrate for H₂ production by Thermoanaerobacterium thermosaccharolyticum W16 in a continuous stirred tank reactor. The performance of strain W16 to ferment the main components of hydrolysate, mixture of glucose and xylose, in continuous culture was conducted at first, and then T. thermosaccharolyticum W16 was evaluated to ferment fully enzymatically hydrolysed cornstalk to produce H₂ in continuous operation mode. At the dilution rate of 0.020 h⁻¹, the H₂ yield and production rate reached a maximum of 1.9 mol H₂ mol⁻¹ sugars and 8.4 mmol H₂ L⁻¹ h⁻¹, respectively, accompanied with the maximum glucose and xylose utilizations of 86.3% and 77.6%. Continuous H₂ production from enzymatically treated cornstalk hydrolysate in this research provides a new direction for economic, efficient, and harmless H₂ production.     

Corn stover ethanol yield as affected by grain yield,Bt trait,and environment

Literature values for glucose release from corn stover are highly variable which would likely result in tremendous variability in bio-refinery ethanol yield from corn stover feedstock. A relatively recent change in United States corn genetics is the inclusion of the Bacillus thuringiensis (Bt) trait, which now accounts for three-fourths of all US planted corn acreage. The objective of this study was to evaluate the effect of corn grain yield, inclusion of the Bt trait, and location environment on corn stover quality for subsequent ethanol conversion. Two hybrid pairs (each having a Bt and non-Bt near-isoline) were analyzed giving a total of 4 hybrids. In 2010 and 2011, field plots were located in Michigan at four latitudinal differing locations in four replicated plots at each location. Stover composition and enzymatic digestibility was analyzed and estimated ethanol yield (g g⁻¹) was calculated based on hydrolyzable glucan and xylan levels. Analysis showed that there were no significant differences in total glucose or xylose levels nor in enzymatically hydrolyzable glucan and xylan concentrations between Bt corn stover and the non-Bt stover isolines. Regression analyses between corn grain yield (Mg ha⁻¹) and corn stover ethanol yield (g g⁻¹) showed an inverse relationship indicative of a photosynthate source-sink relationship. Nevertheless, the quantity of stover produced was found to be more critical than the quality of stover produced in maximizing potential stover ethanol yield on a land area basis.     

Optimization of alkaline pretreatment on corn stover for enhanced production of 1.3-propanediol and 2,3-butanediol by Klebsiella pneumoniae AJ4

Corn stover is one of the most promising lignocellulosic biomass that can be utilized for producing 1,3-propanediol and 2,3-butanediol. The pretreatment and enzymatic hydrolysis steps are essential for the bioconversion of lignocellulosic biomass to diols. For optimizing the pretreatment step, temperature, time, and NaOH concentration were evaluated based on total sugar recovery. Enzymatic hydrolysis for cellulose and hemicellulose were investigated at different solid-to-liquid ratios. The optimum conditions were found to be alkaline pretreatment with 0.25 mol dm⁻³ NaOH for 1 h at 60 °C followed by enzymatic hydrolysis at 50 °C for 48 h, with a solid slurry concentration of 100 g dm⁻³. Under these conditions, conversion rates of 92.55% and 78.82% were obtained from glucan and xylan, respectively. Diol production from fermentable sugars was 14.8 g dm⁻³, with a conversion yield and productivity of 0.46 g g⁻¹, and 0.98 g dm⁻³ h⁻¹, respectively. Our results are similar for diol production obtained using pure sugars under the same conditions. Therefore, mild alkaline pretreatment of corn stover facilitates delignification, significantly improving the rate of enzymatic saccharification and sugar recovery.