Enzymatic Hydrolysis and Succinic Acid Fermentation from Steam-Exploded Corn Stalk at High Solid Concentration by Recombinant Escherichia coli

Steam-exploded corn stalk biomass was used as the substrate for succinic acid production via lignocellulose enzymatic hydrolysis and fermentation. Succinic acid fermentation was investigated in Escherichia coli strains overexpressing cyanobacterium Anabaena sp. 7120 ecaA gene encoding carbonic anhydrase (CA). For the washed steam-exploded corn stalk at 30 % substrate concentration, i.e., 30 % water-insoluble solids (WIS), enzymatic hydrolysis yielded 97.5 g/l glucose solution and a cellulose conversion of 73.6 %, thus a high succinic acid level up to 38.6 g/l. With the unwashed steam-exploded corn stalk, though a cellulose conversion of 71.2 % was obtained in hydrolysis at 30 % solid concentration (27.9 % WIS), its hydrolysate did not ferment at all, and the hydrolysate of 25 % solid loading containing 3.8 g/l acetic acid and 168.2 mg/l furfural exerted a strong inhibition on succinic acid production.     

Pretreatment of corn stover using wet oxidation to enhance enzymatic digestibility

Corn stover is an abundant, promising raw material for fuel ethanol production. Although it has a high cellulose content, without pretreatment it resists enzymatic hydrolysis, like most lignocellulosic materials. Wet oxidation (water, oxygen, mild alkali or acid, elevated temperature and pressure) was investigated to enhance the enzymatic digestibility of corn stover. Six different combinations of reaction temperature, time, and pH were applied. The best conditions (60g/L of corn stover, 195°C, 15 min, 12 bar O₂, 2 g/L of Na₂CO₃) increased the enzymatic conversion of corn stover four times, compared to untreated material. Under these conditions 60% of hemicellulose and 30% of lignin were solubilized, whereas 90% of cellulose remained in the solid fraction. After 24-h hydrolysis at 50°C using 25 filter paper units (FPU)/g of dry matter (DM) biomass, the achieved conversion of cellulose to glucose was about 85%. Decreasing the hydrolysis temperature to 40°C increased hydrolysis time from 24 to 72 h. Decreasing the enzyme loading to 5 FPU/g of DM biomass slightly decreased the enzymatic conversion from 83.4 to 71%. Thus, enzyme loading can be reduced without significantly affecting the efficiency of hydrolysis, an important economical aspect.     

Ethanol Production from Residual Wood Chips of Cellulose Industry: Acid Pretreatment Investigation,Hemicellulosic Hydrolysate Fermentation,and Remaining Solid Fraction Fermentation by SSF Process

Current research indicates the ethanol fuel production from lignocellulosic materials, such as residual wood chips from the cellulose industry, as new emerging technology. This work aimed at evaluating the ethanol production from hemicellulose of eucalyptus chips by diluted acid pretreatment and the subsequent fermentation of the generated hydrolysate by a flocculating strain of Pichia stipitis. The remaining solid fraction generated after pretreatment was subjected to enzymatic hydrolysis, which was carried out simultaneously with glucose fermentation [saccharification and fermentation (SSF) process] using a strain of Saccharomyces cerevisiae. The acid pretreatment was evaluated using a central composite design for sulfuric acid concentration (1.0–4.0 v/v) and solid to liquid ratio (1:2–1:4, grams to milliliter) as independent variables. A maximum xylose concentration of 50 g/L was obtained in the hemicellulosic hydrolysate. The fermentation of hemicellulosic hydrolysate and the SSF process were performed in bioreactors and the final ethanol concentrations of 15.3 g/L and 28.7 g/L were obtained, respectively.     

Determination of glucose and ethanol after enzymatic hydrolysis and fermentation of biomass using Raman spectroscopy

Raman spectroscopy has been used for the quantitative determination of the conversion efficiency at each step in the production of ethanol from biomass. The method requires little sample preparation; therefore, it is suitable for screening large numbers of biomass samples and reaction conditions in a complex sample matrix. Dilute acid or ammonia-pretreated corn stover was used as a model biomass for these studies. Ammonia pretreatment was suitable for subsequent measurements with Raman spectroscopy, but dilute acid-pretreated corn stover generated a large background signal that surpassed the Raman signal. The background signal is attributed to lignin, which remains in the plant tissue after dilute acid pretreatment. A commercial enzyme mixture was used for the enzymatic hydrolysis of corn stover, and glucose levels were measured with a dispersive 785 nm Raman spectrometer. The glucose detection limit in hydrolysis liquor by Raman spectroscopy was 8 g L⁻¹. The mean hydrolysis efficiency for three replicate measurements obtained with Raman spectroscopy (86 ± 4%) was compared to the result obtained using an enzymatic reaction with UV-vis spectrophotometry detection (78 ± 8%). The results indicate good accuracy, as determined using a Student's t-test, and better precision for the Raman spectroscopy measurement relative to the enzymatic detection assay. The detection of glucose in hydrolysis broth by Raman spectroscopy showed no spectral interference, provided the sample was filtered to remove insoluble cellulose prior to analysis. The hydrolysate was further subjected to fermentation to yield ethanol. The detection limit for ethanol in fermentation broth by Raman spectroscopy was found to be 6 g L⁻¹. Comparison of the fermentation efficiencies measured by Raman spectroscopy (80 ± 10%) and gas chromatrography-mass spectrometry (87 ± 9%) were statistically the same. The work demonstrates the utility of Raman spectroscopy for screening the entire conversion process to generate lignocellulosic ethanol.     

Enhancing the Enzymatic Hydrolysis of Corn Stover by an Integrated Wet-milling and Alkali Pretreatment

An integrated wet-milling and alkali pretreatment was applied to corn stover prior to enzymatic hydrolysis. The effects of NaOH concentration in the pretreatment on crystalline structure, chemical composition, and reducing-sugar yield of corn stover were investigated, and the mechanism of increasing reducing-sugar yield by the pretreatment was discussed. The experimental results showed that the crystalline structure of corn stover was disrupted, and lignin was removed, while cellulose and hemicellulose were retained in corn stover by the pretreatment with 1% NaOH in 1 h. The reducing-sugar yield from the pretreated corn stovers increased from 20.2% to 46.7% when the NaOH concentration increased from 0% to 1%. The 1% NaOH pretreated corn stover had a holocellulose conversion of 55.1%. The increase in reducing-sugar yield was related to the crystalline structure disruption and delignification of corn stover. It was clarified that the pretreatment significantly enhanced the conversion of cellulose and hemicellulose in the corn stover to sugars.     

Optimization of steam pretreatment of corn stover to enhance enzymatic digestibility

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

Statistical Optimization of Recycled-Paper Enzymatic Hydrolysis for Simultaneous Saccharification and Fermentation Via Central Composite Design

A central composite design of the response surface methodology (RSM) was employed to study the effects of temperature, enzyme concentration, and stirring rate on recycled-paper enzymatic hydrolysis. Among the three variables, temperature and enzyme concentration significantly affected the conversion efficiency of substrate, whereas stirring rate was not effective. A quadratic polynomial equation was obtained for enzymatic hydrolysis by multiple regression analysis using RSM. The results of validation experiments were coincident with the predicted model. The optimum conditions for enzymatic hydrolysis were temperature, enzyme concentration, and stirring rate of 43.1 °C, 20 FPU g⁻¹ substrate, and 145 rpm, respectively. In the subsequent simultaneous saccharification and fermentation (SSF) experiment under the optimum conditions, the highest 28.7 g ethanol l⁻¹ was reached in the fed-batch SSF when 5% (w/v) substrate concentration was used initially, and another 5% added after 12 h fermentation. This ethanol output corresponded to 77.7% of the theoretical yield based on the glucose content in the raw material.     

Effects of temperature and moisture on dilute-acid steam explosion pretreatment of corn stover and cellulase enzyme digestibility

Corn stover is emerging as a viable feedstock for producing bioethanol from renewable resources. Dilute-acid pretreatment of corn stover can solubilize a significant portion of the hemicellulosic component and enhance the enzymatic digestibility of the remaining cellulose for fermentation into ethanol. In this study, dilute H₂SO₄ pretreatment of corn stover was performed in a steam explosion reactor at 160°C, 180°C, and 190°C, approx 1 wt% H₂SO₄, and 70-s to 840-s residence times. The combined severity (Log₁₀ [R _o ] - pH), an expression relating pH, temperature, and residence time of pretreatment, ranged from 1.8 to 2.4. Soluble xylose yields varied from 63 to 77% of theoretical from pretreatments of corn stover at 160 and 180°C. However, yields >90% of theoretical were found with dilute-acid pretreatments at 190°C. A narrower range of higher combined severities was required for pretreatment to obtain high soluble xylose yields when the moisture content of the acid-impregnated feedstock was increased from 55 to 63 wt%. Simultaneous saccharification and fermentation (SSF) of washed solids from corn stover pretreated at 190°C, using an enzyme loading of 15 filter paper units (FPU)/g of cellulose, gave ethanol yields in excess of 85%. Similar SSF ethanol yields were found using washed solid residues from 160 and 180°C pretreatments at similar combined severities but required a higher enzyme loading of approx 25 FPU/g of cellulose.     

Model-based fed-batch for high-solids enzymatic cellulose hydrolysis

While many kinetic models have been developed for the enzymatic hydrolysis of cellulose, few have been extensively applied for process design, optimization, or control. High-solids operation of the enzymatic hydrolysis of lignocellulose is motivated by both its operation decreasing capital costs and increasing product concentration and hence separation costs. This work utilizes both insights obtained from experimental work and kinetic modeling to develop an optimization strategy for cellulose saccharification at insoluble solids levels greater than 15% (w/w), where mixing in stirred tank reactors (STRs) becomes problematic. A previously developed model for batch enzymatic hydrolysis of cellulose was modified to consider the effects of feeding in the context of fed-batch operation. By solving the set of model differential equations, a feeding profile was developed to maintain the insoluble solids concentration at a constant or manageable level throughout the course of the reaction. Using this approach, a stream of relatively concentrated solids (and cellulase enzymes) can be used to increase the final sugar concentration within the reactor without requiring the high initial levels of insoluble solids that would be required if the operation were performed in batch mode. Experimental application in bench-scale STRs using a feed stream of dilute acid-pretreated corn stover solids and cellulase enzymes resulted in similar cellulose conversion profiles to those achieved in batch shake-flask reactors where temperature control issues are mitigated. Final cellulose conversions reached approximately 80% of theoretical for fed-batch STRs fed to reach a cumulative solids level of 25% (w/w) initial insoluble solids.     

Kinetics of Enzymatic High-Solid Hydrolysis of Lignocellulosic Biomass Studied by Calorimetry

Enzymatic hydrolysis of high-solid biomass (>10% w/w dry mass) has become increasingly important as a key step in the production of second-generation bioethanol. To this end, development of quantitative real-time assays is desirable both for empirical optimization and for detailed kinetic analysis. In the current work, we have investigated the application of isothermal calorimetry to study the kinetics of enzymatic hydrolysis of two substrates (pretreated corn stover and Avicel) at high-solid contents (up to 29% w/w). It was found that the calorimetric heat flow provided a true measure of the hydrolysis rate with a detection limit of about 500 pmol glucose s⁻¹. Hence, calorimetry is shown to be a highly sensitive real-time method, applicable for high solids, and independent on the complexity of the substrate. Dose–response experiments with a typical cellulase cocktail enabled a multidimensional analysis of the interrelationships of enzyme load and the rate, time, and extent of the reaction. The results suggest that the hydrolysis rate of pretreated corn stover is limited initially by available attack points on the substrate surface (<10% conversion) but becomes proportional to enzyme dosage (excess of attack points) at later stages (>10% conversion). This kinetic profile is interpreted as an increase in polymer end concentration (substrate for CBH) as the hydrolysis progresses, probably due to EG activity in the enzyme cocktail. Finally, irreversible enzyme inactivation did not appear to be the source of reduced hydrolysis rate over time.     

Process Evaluation of Enzymatic Hydrolysis with Filtrate Recycle for the Production of High Concentration Sugars

Process simulation and lab trials were carried out to demonstrate and confirm the efficiency of the concept that recycling hydrolysate at low total solid enzymatic hydrolysis is one of the options to increase the sugar concentration without mixing problems. Higher sugar concentration can reduce the capital cost for fermentation and distillation because of smaller retention volume. Meanwhile, operation cost will also decrease for less operating volume and less energy required for distillation. With the computer simulation, time and efforts can be saved to achieve the steady state of recycling process, which is the scenario for industrial production. This paper, to the best of our knowledge, is the first paper discussing steady-state saccharification with recycling of the filtrate form enzymatic hydrolysis to increase sugar concentration. Recycled enzymes in the filtrate (15–30% of the original enzyme loading) resulted in 5–10% higher carbohydrate conversion compared to the case in which recycled enzymes were denatured. The recycled hydrolysate yielded 10% higher carbohydrate conversion compared to pure sugar simulated hydrolysate at the same enzyme loading, which indicated hydrolysis by-products could boost enzymatic hydrolysis. The high sugar concentration (pure sugar simulated) showed inhibition effect, since about 15% decrease in carbohydrate conversion was observed compared with the case with no sugar added. The overall effect of hydrolysate recycling at WinGEMS simulated steady-state conditions with 5% total solids was increasing the sugar concentration from 35 to 141 g/l, while the carbohydrate conversion was 2% higher for recycling at steady state (87%) compared with no recycling strategy (85%). Ten percent and 15% total solid processes were also evaluated in this study.     

Effect of surfactants and zeolites on simultaneous saccharification and fermentation of steam-exploded poplar biomass to ethanol

In this work, the effect of the addition of different concentrations of Tween-80 and three different zeolite-like products on enzymatic hydrolysis, ethanol fermentation, and simultaneous saccharification and fermentation (SSF) process has been investigated. The ability of these products to enhance the effectiveness of the SSF process to ethanol of steam-exploded poplar biomass using the thermotolerant strainKluyveromyces marxianus EMS-26 has been tested. Tween-80 (0.4 g/L) increased enzymatic hydrolysis yield by 20% when compared to results obtained in hydrolysis in absence of the additive. Zeolite-like products (ZESEP-56 and ZECER-56) (2.5 g/L) improved rates of conversion and ethanol yields in the fermentation of liquid fraction recovered from steam-exploded poplar. The periods required for the completion of fermentation were approx 10 h in the presence of zeolite-like products and 24 h in the absence of additives. The probable mode of action is through lowered levels of inhibitory substances because of adsorption by the additive.     

Ethanol Production from H<Subscript>2</Subscript>SO<Subscript>3</Subscript>-Steam-Pretreated Fresh Sweet Sorghum Stem by Simultaneous Saccharification and Fermentation

The present work presents an alternative approach to ethanol production from sweet sorghum: without detoxification, acid-impregnated fresh sweet sorghum stem which contains soluble (glucose and sucrose) and insoluble carbohydrates (cellulose and hemicellulose) was steam pretreated under mild temperature of 100 °C. Simultaneous saccharification and fermentation experiments were performed on the pretreated slurries using Saccharomyces cerevisiae. Experimentally, ground fresh sweet sorghum stem was combined with H₂SO₃ at dosages of 0.25, 0.50, and 0.75 g/g dry matter (DM) and steam pretreated by varying the residence time (60, 120, or 240 min). According to enzymatic hydrolysis results and ethanol yields, H₂SO₃ was a powerful and mild acid for improving enzymatic digestibility of sorghum stem. At a solid loading of 10% (w/v) and acid dosage of 0.25 g/g DM H₂SO₃ at 100 °C for 120 min, 44.5 g/L ethanol was obtained after 48 ± 4 h of simultaneous saccharification and fermentation. This corresponded to an overall ethanol yield of 110% of the theoretical one, based on the soluble carbohydrates in the fresh sweet sorghum stem. The concentrations of hydroxymethylfurfural and furfural of the sulfurous acid pretreated samples were below 0.4 g/L. Ethanol would not inhibit the cellulase activity, at least under the concentration of 34 g/L.     

Optimization of SO2-catalyzed steam pretreatment of corn fiber for ethanol production

A batch reactor was employed to steam explode corn fiber at various degrees of severity to evaluate the potential of using this feedstock as part of an enzymatically mediated cellulose-to-ethanol process. Severity was controlled by altering temperature (150–230°C), residence time (1–9 min), and SO₂ concentration (0–6% [w/w] dry matter). The effects of varying the different parameters were assessed by response surface modeling. The results indicated that maximum sugar yields (hemicellulose-derived water soluble, and cellulose-derived following enzymatic hydrolysis) were recovered from corn fiber pretreated at 190°C for 5 minutes after exposure to 3% SO₂. Sequential SO₂-catalyzed steam explosion and enzymatic hydrolysis resulted in a conversion efficiency of 81% of the combined original hemicellulose and cellulose in the corn fiber to monomeric sugars. An additional posthydrolysis step performed on water soluble hemicellulose stream increased the concentration of sugars available for fermentation by 10%, resulting in the high conversion efficiency of 91%. Saccharomyces cerevisiae was able to ferment the resultant corn fiber hydrolysates, perhydrolysate, and liquid fraction from the posthydrolysis steps to 89, 94, and 85% of theoretical ethanol conversion, respectively. It was apparent that all of the parameters investigated during the steam explosion pretreatment had a significant effect on sugar recovery, inhibitory formation, enzymatic conversion efficiency, and fermentation capacity of the yeast.     

Dilute-sulfuric acid pretreatment of corn stover in pilot-scale reactor

Corn stover is a domestic feedstock that has potential to produce significant quantities of fuel ethanol and other bioenergy and biobased products. However, comprehensive yield and carbon mass balance information and validated kinetic models for dilute-sulfuric acid (H₂SO₄) pretreatment of corn stover have not been available. This has hindered the estimation of process economics and also limited the ability to perform technoeconomic modeling to guide research. To better characterize pretreatment and assess its kinetics, we pretreated corn stover in a continuous 1 t/d reactor. Corn stover was pretreated at 20% (w/w) solids concentration over a range of conditions encompassing residence times of 3–12 min, temperatures of 165–195°C, and H₂SO₄ concentrations of 0.5–1.4% (w/w). Xylan conversion yield and carbon mass balance data were collected at each run condition. Performance results were used to estimate kinetic model parameters assuming biphasic hemicellulose hydrolysis and a hydrolysis mechanism incorporating formation of intermediate xylo-oligomers. In addition, some of the pretreated solids were tested in a simultaneous saccharification and fermentation (SSF) process to measure the reactivity of their cellulose component to enzymatic digestion by cellulase enzymes. Monomeric xylose yields of 69–71% and total xylose yields (monomers and oligomers) of 70–77% were achieved with performance level depending on pretreatment severity. Cellulose conversion yields in SSF of 80–87% were obtained for some of the most digestible pretreated solids.     

Ball Milling Pretreatment of Corn Stover for Enhancing the Efficiency of Enzymatic Hydrolysis

Ethanol can be produced from lignocellulosic biomass with the usage of ball milling pretreatment followed by enzymatic hydrolysis and fermentation. The sugar yields from lignocellulosic feed stocks are critical parameters for ethanol production process. The research results from this paper indicated that the yields of glucose and xylose were improved by adding any of the following dilute chemical reagents: H₂SO₄, HCl, HNO₃, CH₃COOH, HCOOH, H₃PO₄, and NaOH, KOH, Ca(OH)₂, NH₃·H₂O in the ball milling pretreatment of corn stover. The optimal enzymatic hydrolysis efficiencies were obtained under the conditions of ball milling in the alkali medium that was due to delignification. The data also demonstrated that ball milling pretreatment was a robust process. From the microscope image of ball milling-pretreated corn stover, it could be observed that the particle size of material was decreased and the fiber structure was more loosely organized. Meanwhile, the results indicate that the treatment effect of wet milling is better than that of dry milling. The optimum parameters for the milling process were ball speed of 350 r/min, solid/liquid ratio of 1:10, raw material particle size with 0.5 mm, and number of balls of 20 (steel ball, Φ = 10 mm), grinding for 30 min. In comparison with water milling process, alkaline milling treatment could increase the enzymatic hydrolysis efficiency of corn stover by 110%; and through the digestion process with the combination of xylanase and cellulase mixture, the hydrolysis efficiency could increase by 160%.     

Effect of sodium dodecyl sulfate and cetyltrimethylammonium bromide catanionic surfactant on the enzymatic hydrolysis of Avicel and corn stover

Nonionic surfactants could effectively improve the enzymatic hydrolysis efficiency of lignocellulose, while small molecule anionic and cationic surfactants usually inhibited the enzymatic hydrolysis. The results showed that the anionic surfactant sodium dodecyl sulfate (SDS) could improve the enzymatic hydrolysis efficiency of Avicel at the concentration range of 0.1–1 mM, but it did inhibit enzymatic hydrolysis at higher concentration. Cationic surfactant cetyltrimethylammonium bromide (CTAB) was used to regulate the surface charge of SDS; thereby catanionic surfactant SDS-CTAB was formed. The effect of SDS-CTAB catanionic surfactant with varied molar ratios on the enzymatic hydrolysis of pure cellulose and corn stover at various enzymatic hydrolysis environments was investigated. SDS-CTAB could increase the enzymatic hydrolysis of corn stover at high solid loading from 33.3 to 42.4%. Using SDS-CTAB could reduce about 58% of the cellulase dosage to achieve 80% of the enzymatic hydrolysis of corn stover. SDS-CTAB catanionic surfactant could regulate the surface charge of cellulase in the hydrolyzate and reduce the non-productive adsorption of cellulase on the lignin, thereby improving the enzymatic hydrolysis efficiency of lignocellulose.     

Production of 2,3- butanediol from pretreated corn cob byKlebsiella oxytoca in the presence of fungal cellulase

A simple and effective method of treatment of lignocellulosic material was used for the preparation of corn cob for the production of 2,3-butanediol byKlebsiella oxytoca ATCC 8724 in a simultaneous saccharification and fermentation process. During the treatment, lignin, and alkaline extractives were solubilized and separated from cellulose and hemicellulose fractions by dilute ammonia (10%) steeping. Hemicellulose was then hydrolyzed by dilute hydrochloric acid (1%, wJv) hydrolysis at 100°C at atmospheric pressure and separated from cellulose fraction. The remaining solid, with 90% of cellulose, was then used as the substrate. A butanediol concentration of 25 g/L and an ethanol concentration of 7 g/L were produced byK. oxytoca from 80 g/L of corn cob cellulose with a cellulase dosage of 8.5 IFPU/g corn cob cellulose after 72 h of SSF. With only dilute acid hydrolysis, a butanediol production rate of 0.21 g/L/h was obtained that is much lower than the case in which corn cob was treated with ammonia steeping prior to acid hydrolysis. The butanediol production rate for the latter was 0.36 g/L/h.     

Effect of Nutrients on Fermentation of Pretreated Wheat Straw at very High Dry Matter Content by Saccharomyces cerevisiae

Wheat straw hydrolysate produced by enzymatic hydrolysis of hydrothermal pretreated wheat straw at a very high solids concentration of 30% dry matter (w/w) was used for testing the effect of nutrients on their ability to improve fermentation performance of Saccharomyces cerevisiae. The nutrients tested were MgSO₄ and nitrogen sources; (NH₄)₂SO₄, urea, yeast extract, peptone and corn steep liquor. The fermentation was tested in a separate hydrolysis and fermentation process using a low amount of inoculum (0.33 g kg^?1) and a non-adapted baker’s yeast strain. A factorial screening design revealed that yeast extract, peptone, corn steep liquor and MgSO₄ were the most significant factors in obtaining a high fermentation rate, high ethanol yield and low glycerol formation. The highest volumetric ethanol productivity was 1.16 g kg^?1 h^?1 and with an ethanol yield close to maximum theoretical. The use of urea or (NH₄)₂SO₄ separately, together or in combination with MgSO₄ or vitamins did not improve fermentation rate and resulted in increased glycerol formation compared to the use of yeast extract. Yeast extract was the single best component in improving fermentation performance and a concentration of 3.5 g kg^?1 resulted in high ethanol yield and a volumetric productivity of 0.6 g kg^?1 h^?1.     

Soybean Hulls Pretreated Using Thermo-Mechanical Extrusion—Hydrolysis Efficiency,Fermentation Inhibitors,and Ethanol Yield

Soybean hulls were subjected to thermo-mechanical extrusion pretreatment at various in-barrel moisture contents and screw speeds. Extrusion degraded the lignocellulosic structure and enhanced enzymatic hydrolysis of soybean hulls, with up to 155% increase in glucose yield as compared to untreated substrate. Greater glucose yields were observed at higher in-barrel moistures (45% and 50%) and lower screw speed (280 and 350 rpm). Maximum 74% cellulose to glucose conversion resulted from using a two-enzyme cocktail consisting of cellulase and β-glucosidase. Conversion increased to 87% when a three-enzyme cocktail having a cell wall degrading enzyme complex was used for hydrolysis. Fermentation inhibitors, such as furfural, 5-(hydroxymethyl)-2-furaldehyde (HMF), and acetic acid, were found in the extrusion pretreated soybean hulls and hydrolysate. However, their concentrations were below the known thresholds for inhibition. Fermentation of hydrolysate by Saccharomyces cerevisiae led to high yields of ethanol, with concentration ranging from 13.04 to 15.44 g/L.