Simultaneous Saccharification and Fermentation (SSF) of High Digestible Grain Sorghum for Ethanol Production

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1 Simultaneous Saccharification and Fermentation (SSF) of High Digestible Grain Sorghum for Ethanol Production J. R. Hernandez, S. C. Capareda, O. Portilllo, D. B. Hays, W. L. Rooney* ABSTRACT. The potential of high digestible grain sorghum (HDGS) with a modified starch protein endosperm matrix as an alternative to corn in ethanol production was investigated using simultaneous saccharification and fermentation (SSF). Protein and starch digestibilities and glucose and ethanol yields of HDGS were compared with those of normal grain sorghum (NGS) and corn using commercially available Saccharomyces cerevisiae yeast and the enzymes α-amylase and glucoamylase. Results showed that HDGS yielded higher amounts of glucose and ethanol than NGS and corn, particularly in the early part of the saccharification and fermentation process. After 2.5 h of saccharification, a glucose yield of 89% was obtained for HDGS compared to 72% and 75% for NGS and corn, respectively. An ethanol yield of 94% was obtained from HDGS compared to 81% and 84% for NGS and corn, respectively, after 21 to 24 h of fermentation. These results suggest that HDGS also has higher starch digestibility, resulting in a faster rate and higher enzymatic conversion of starch to glucose and higher yield of ethanol during hydrolysis and fermentation. When used in the current corn ethanol system, HDGS is expected to have several potential advantages, such as improved productivity of ethanol and quality of co-product animal feed (dry distillers dried grains with soluble, DDGS), reduced energy consumption during gelatinization and liquefaction, and reduced amount of enzyme during operation. Keywords. Enzymatic hydrolysis, Ethanol, High digestible grain sorghum, Simultaneous saccharification and fermentation. I n recent years, world crude oil prices have risen dramatically because of dwindling petroleum supplies coupled with increasing demand (EIA, 2006). As of 2006 and 2007, liquid transportation fuels such as gasoline, diesel, and jet fuel accounted for approximately 70% of daily crude oil consumption (about 3.18 billion L, or 840 million gal) in the U.S. (Gray et al., 2006; EIA, 2008). In response, the U.S. government Submitted for review in September 2008 as manuscript number BE 7674; approved for publication by the Biological Engineering Division of ASABE in March Presented at the 2007 ASABE Annual Meeting as Paper No The authors are Joan Rollog Hernandez, Graduate Research Assistant, and Sergio Canzana Capareda, ASABE Member, Assistant Professor, Department of Biological and Agricultural Engineering; and Ostillo Portillo, Graduate Research Assistant, Dirk B. Hays, Assistant Professor, and William L. Rooney, Associate Professor, Department of Soil and Crop Sciences, Texas A&M University, College Station, Texas. Corresponding author: Sergio Canzana Capareda, Department of Biological and Agricultural Engineering, 303D Scoates Hall, MS 2117 BAEN Dept., Texas A&M University, College Station, TX 77843; phone: ; scapareda@tamu.edu. Biological Engineering Transactions 4 (1): ASABE ISSN

2 is constantly finding ways to reduce its dependence on non-renewable energy resources and minimize the environmental problems associated with fossil fuel combustion. Thus, more attention is now focused on the production of renewable and environmentally friendly fuels like bioethanol and biodiesel. Ethanol presents one of the most promising and fastest growing clean fuel substitutes. It is already used as a substitute for methyl tert-butyl ether (MTBE) to increase the octane rating of gasoline. It is also being mixed directly with gasoline in 10% (E10), 15% (E15), or even 95% (E95) ethanol blends, which can be easily utilized by the current internal combustion engine vehicles (ICEVs) without any modifications (Hamelinck et al., 2005). According to Hill et al. (2006), the production and combustion of ethanol reduces greenhouse gas emissions by 12% relative to the fossil fuels it displaces. In the U.S., ethanol production reached 34 billion L (9 billion gal) in 2008 (RFA, 2008). Production of 24.6 billion L (6.5 billion gal) was achieved in 2007, with an additional 22.7 billion L (6 billion gal) per year capacity ready at the end of 2008 (EERE, 2007). Due to the ratification of the Energy Independence and Security Act (EISA) of 2007, around billion L (35 billion gal) annual production of ethanol is being projected for the year 2022 (EERE, 2007). Ethanol can be made synthetically from petroleum or biochemically through microbial fermentation of biomass materials (Badger, 2002). In the U.S., ethanol is mainly produced via biochemical conversion of starch from corn grains (Gray et al., 2006; Mojovic et al., 2006). Based on the net energy balance of ethanol production, Hill et al. (2006) estimated that ethanol yields 25% more energy than the energy invested in its production (including crop production, transportation, conversion, and purification). If all of the corn produced in the U.S. were used for fermentation, about 49.2 billion L (13 billion gal) of ethanol per year could be realized (Gray et al., 2006). But because corn is also utilized for food and feeds, the use of a less expensive grain such as sorghum is advantageous. Grain sorghum (Sorghum bicolor (L.) Moench) ranks third in total production among all the cereal crops in the U.S. (Zhan et al., 2006). It is primarily used as a feed grain for livestock in the U.S., but in many semi-arid and tropical areas of the world it serves as a staple food grain (Dicko et al., 2006). The feed value of grain sorghum is similar to that of corn in terms of its starch content (55% to 75% of starch by kernel weight), but its protein and starch are less digestible (Serna-Saldivar and Rooney, 1995; Zhan et al., 2003). Due to poor wet-milling property and lower starch digestibility of normal sorghum, it has been underutilized for bio-based products and bio-energy production (Zhan et al., 2003, 2006). Several hypotheses have been suggested to explain this low digestibility and the high energy requirements needed for gelatinization prior to liquefaction and saccharification. The predominant theory is that the starch being imbedded in the protein body (kafirin) matrices restricts gelatinization. During heating, the kafirins in the protein bodies form more highly networked matrices of kafirins bridged together via disulfide cysteine residues that surround the starch granules and restrict enzyme accessibility during liquefication and saccharification. However, this theory remains largely untested (Taylor et al., 1984; Chandrashekar and Kirlies, 1988). The sorghum breeding program in the Department of Soil and Crop Sciences, Texas A&M University, has developed and identified high digestible grain sorghum (HDGS) genotypes. The HDGS was derived from germplasm identified and described 4 Biological Engineering Transactions

3 by Weaver et al. (1998). These varieties with modified endosperm matrices lack kafirin protein body structures that surround the starch granules and restrict gelatinization. HDGS is hypothesized to have several benefits for production of ethanol and distillers dried grain solubles (DDGS) for animal feed. First, the modified endosperm matrices, lacking resistant protein body structures, will reduce the temperature and duration at elevated temperatures needed to solubilize the grain starch for hydrolytic enzyme access and conversion to fermentable sugars. Second, the grain protein present has improved bioavailability (i.e., it is more digestible) for food and feed uses, and the protein present has 60% higher lysine content, similar to high lysine corn lines (Weaver et al., 1998). Lysine, an essential amino acid, is present at very low levels in vegetable proteins and is frequently used as a nutrient supplement for herbivorous animals. This amino acid is commonly ingested as lysine or lysine-containing proteins in animal feed (Chen et al., 1996), thereby making HDGS more favorable as a feedstock for dry grind ethanol fermentation. The development of the HDGS genotype increases the potential of grain sorghum as a feedstock for ethanol production as less time and energy will be required in the conversion process. In the end, using HDGS could result in a more positive net energy balance and more economically competitive ethanol production. It will also provide distillers increased income and market share via the improved essential amino acid and nutrition quality of the DDGS feed product. This study investigated the simultaneous saccharification and fermentation (SSF) process of HDGS, a grain sorghum with improved protein digestibility for ethanol production, using commercially available α-amylase, glucoamylase, and Saccharomyces cerevisiae yeast. Specifically, it compared HDGS starch digestibility with that of corn and of low protein digestibility grain sorghum (normal grain sorghum, or NGS) using enzymatic hydrolysis and saccharification. The efficiency of starch conversion to glucose and ethanol during SSF of the HDGS, NGS, and corn substrates was also evaluated. Materials and Methods Substrates Dry-milled samples of HDGS, NGS, and corn grains were obtained from the Sorghum Breeding Center in the Department of Soil and Crop Sciences, Texas A&M University, and ground using a Jay Bee 1647 hammer mill (Jay Bee Mfg., Inc., Tyler, Tex.) to pass through a sieve of 1 mm opening diameter. The samples were then oven dried at 105 C to constant mass for moisture determination. The grain sorghum samples used were a recombinant inbred line from the cross of BTx635 (high mold resistant grain sorghum cultivar) P (high lysine grain sorghum cultivar). The starch content of the samples was determined using a commercially available kit (Megazyme International, Wicklow, Ireland), while in vitro protein digestibility was analyzed using the modified method of Mertz et al. (1984). The protein digestibility method involved three stages (fig. 1): protein digestion using pepsin, protein extraction, and turbidity assay. The turbidity assay provided a measure of protein digestibility; absorbance is directly proportional to the protein concentration in the extraction buffer. The absorbance was measured using a UV/VIS scanning spectrophotometer (Cole Parmer, Vernon Hills, Ill.). The turbid solutions were read at 562 nm (Aboubacar et al., 2003). 4(1):

4 Figure 1. Schematic diagram of the protein digestibility assay. Microorganism and Culture Media An industrial strain of Saccharomyces cerevisiae from the Home Brewery (Ozark, Mo.) was used for the fermentation. It was isolated from a commercially available Super Yeast dry brewer s yeast that can produce and tolerate up to 20% ethanol. A stock culture was maintained in a mm Petri dish with yeast peptone dextrose (YPD) medium containing 5 g L -1 yeast extract, 10 g L -1 peptone, 20 g L -1 glucose, and 20 g L -1 agar at ph 5.5 and stored at 4 C. Pre-cultures were prepared by inoculating a loopful of yeast from an isolated colony of the stock culture into 400 ml of autoclaved yeast malt (YM) broth in a 500 ml Erlenmeyer flask with a cotton plug. The yeast cells were aerobically propagated in YM broth consisting of 3 g L -1 yeast extract, 2 g L -1 malt extract, 5 g L -1 peptone, and 10 g L -1 glucose at ph 5.5 and 32 C in a rotary shaker with a speed of 150 rpm for 48 h. An inoculum concentration of 10% v/v was used in the entire fermentation experiment. Enzymes The enzymes used in this study, namely SPEZYME EXTRA and G-ZYME 480 Ethanol, were samples provided by Genencor International (Rochester, N.Y.). SPEZYME EXTRA enzyme, derived from a genetically modified strain of Bacillus licheniformis, was used to liquefy the grain samples. This thermostable starchhydrolyzing α-amylase can tolerate liquefaction temperatures greater than 85 C (185 F) and is very stable at liquefaction ph as low as 5.4. According to Genencor International s standard method for the determination of α-amylase activity, one alpha amylase unit (AAU) of bacterial α-amylase is the amount of enzyme required to hydrolyze 10 mg of starch per minute under specified conditions. The typical enzyme activity of SPEZYME EXTRA is 14,000 AAU per gram, and its typical density is 1.14 g ml -1. The G-ZYME 480 Ethanol enzyme, which is an optimized blend of extracellular enzymes from selected strains of Aspergillus niger, Rhizopus oryzae, and a genetically modified strain of Bacillus licheniformis, was used to produce glucose from the liquefied mash for ethanol fermentation. The typical density of the G-ZYME 480 Ethanol saccharifying enzyme is 1.13 to 1.15 g ml -1, and its minimum enzyme activity is 380 gluco-amylase units (GAU) per gram. One GAU is the amount of enzyme needed to release 1 g of glucose per hour from soluble starch substrates under the conditions of the assay set by Genencor International. The optimal temperature range for G-ZYME 480 Ethanol is 58 C to 65 C (137 F to 149 F), and it has excellent stability up to 65 C. 6 Biological Engineering Transactions

5 Figure 2. Simultaneous saccharification and fermentation flowchart. Starch Hydrolysis and Saccharification Figure 2 shows a flowchart of the SSF process. Hydrolysis was performed in four 2000 ml Erlenmeyer flasks heated on a temperature-controlled hot plate (Fisher Scientific) with a magnetic stirrer at an agitation speed of 150 rpm. Split dosing of SPEZYME EXTRA enzyme was done for liquefaction of dry-milled grains of HDGS, NGS, and corn meal. The initial dose of enzyme (0.02% w/w of dry substrate) was added during the gelatinization stage of the starch to reduce the viscosity while cooking. Erlenmeyer flasks with 1000 ml of a mixture containing 220 g dry grain substrate, 3 g peptone, 1 g KH 2 PO 4, and 1 g NH 4 Cl at 5.5 ph were heated up to 100 C for 1 h. The second dosing of SPEZYME EXTRA enzyme (0.02% w/w of dry substrate) was done when the temperature reached 85 C, and the ph was adjusted to 5.5 using 1 N H 2 SO 4. Liquefaction was continued for 30 min at 80 C, and the solution was then cooled for another 30 min until the temperature reached 65 C. G-ZYME 480 Ethanol enzyme (0.1% w/w of dry solid) was added after adjusting the ph to 4.5 using 1 N H 2 SO 4 at 65 C. Saccharification with G-ZYME 480 Ethanol was done for 30 min at 60 C, and the solution was then cooled to 35 C. Fermentation When the hydrolyzates reached 35 C, they were transferred into a 2000 ml polyethylene bottle with screw cap that was sterilized using boiling water at 100 C. For each type of substrate, three containers were inoculated with 48 h yeast culture (10% 4(1):

6 v/v), and the remaining container served as the control. All containers were incubated in a rotary shaker at 150 rpm and 32 C for 72 h. Samples were collected after the first 3 h of inoculation and then every 10 to 12 h thereafter. After sampling, about 1 ml was immediately plated for microbial analysis, and approximately 15 ml was centrifuged at 3000 rpm for 10 min. The supernatant was placed in a 20 ml scintillation vial and stored at -4 C until it was analyzed for sugar and ethanol content. Fermentation for each grain sample was done in triplicate while an additional setup that was not inoculated with yeast served as the control for complete glucose conversion. Anaerobic conditions were maintained to maximize the glucose-to-ethanol conversion pathway during fermentation (Ingledew, 1999), except when the screw cap was removed during sampling times and venting out of CO 2. Analysis For microbial analysis, samples were serially diluted using peptone saline diluent (1 g L -1 peptone and 8.5 g L -1 NaCl) and plated using plate count agar (PCA), which contained 1 g L -1 glucose, 2.5 g L -1 yeast extract, 5 g L -1 tryptone, and 15g L -1 agar. Sugar and ethanol concentrations were measured using high-performance liquid chromatography (HPLC) with an autosampler, Shodex SP0810 packed column, and a refractive index (RI) detector (Waters Corp., Milford, Mass.). The column temperature was maintained at 78 C. Each sample was analyzed for 20 min using HPLC water as the eluent at a flow rate of 0.8 ml min -1. SPSS statistical software was used to analyze the data. One-way analysis of variance (ANOVA) and paired t-test were used to determine significant differences of the means. Least significant difference (LSD) was performed for multiple comparison of three replicates in each treatment at α = Results and Discussion Protein Digestibility and Starch Content of the Grain Sorghum The starch and protein digestibility analysis of the grain sorghum used in this study is reported in table 1. Results showed that the starch content of NGS was significantly higher (p < ) than that of HDGS by about 3%. However, by calculating the percentage difference of absorbance in the turbidity assay, NGS was found to have 34.15% less digestible protein compared to HDGS. Apparently, during protein digestion with pepsin (first stage extraction), less protein is discarded when using NGS compared to HDGS. Thus, more undigested protein is left in the NGS grains and subsequently in the trichloroacetic acid (TCA) extraction buffer. After the TCA buffer was added to the washed and digested sorghum flour during the second stage of protein extraction, rapid turbidity of the TCA mixture was observed. A significantly [a] [b] Table 1. Absorbance reading of extraction buffer and starch content of the sorghum grain samples. Analysis NGS [a] HDGS [a] Difference (%) [b] Absorbance reading of extraction buffer after 1 h incubation 0.41 ± ± Starch content (% d.b.) ± ± Means of three replicates. Difference = (NGS - HDGS) / NGS Biological Engineering Transactions

7 higher absorbance reading (p = 0.009) was obtained in the turbidity assay for the NGS sample compared to the HDGS sample after 1 h of TCA incubation, indicating that NGS has a higher amount of extracted protein. Starch Hydrolysis and Saccharification Figure 3 shows the HPLC chromatograms of the NGS, HDGS, and corn substrates during the processes of gelatinization (A), liquefaction (B), and initial saccharification (C). The two major starch polymer components, i.e., amylose, a mostly linear α-d-(1-4)-glucan, and amylopectin, a branched α-d-(1-4)-glucan that has α-d-(1-6) linkages at the branch points, are originally water insoluble and partially crystalline in form (Shuler and Kargi, 2002). Starch granules are initially gelatinized by heating the starch suspension to make them more accessible during enzymatic hydrolysis. Rapid and high-temperature cooking of starch weakens the inter- and intra-molecular hydrogen bonding. This makes the granules swell and absorb water, resulting in increased viscosity of the starch solution (Nichols et al., 2008). As shown in chromatogram A, there was hydrolysis into various smaller units (particularly dextrin and small amounts of glucose). The peak representing dextrin was lowest for HDGS, followed by corn and NGS, while the glucose peak was observed to be highest for HDGS, followed by corn and NGS, after 1 h of gelatinization with 0.01% of SPEZYME EXTRA at 100 C. Addition of heat-stable α-amylase during gelatinization helped in the partial thinning of the mash while cooking. The endo-amylase in SPEZYME EXTRA randomly breaks α-1-4-glycosidic bonds to quickly reduce the viscosity of gelatinized starch, producing soluble dextrin and oligosaccharides (Shuler and Kargi, 2002). During liquefaction, thinning of the gelatinized starch becomes more noticeable. Upon extension of the second enzyme dosing action for another 1 h at 80 C (fig. 3, chromatogram B), peaks representing shorter chains of glucose increased in height due to further conversion of the long chains of glucose into dextrin, maltriose, maltose, and glucose. G-ZYME 480 Ethanol, which contains fungal glucoamylase, catalyzes the release of successive glucose units from the non-reducing ends of soluble dextrin by hydrolyzing both linear and branched glycosidic linkages. As shown in figure 3, chromatogram C, there was a drastic lowering of the dextrin peak and rise of the glucose peak NGS HDGS Corn 2 Figure 3. HPLC Chromatograms of NGS, HDGS, and corn: (A) gelatinization with 0.01% SPEZYME EXTRA at 100 C for 1 h, (B) liquefaction with 0.02% SPEZYME EXTRA α-amylase enzyme at 80 C for 1 h, and (C) saccharification with 0.1% G-ZYME 480 Ethanol glucoamylase enzyme at 60 C for 1 h and while cooling down to 35 C (peaks: 1 = dextrin, 2 = maltose, and 3 = glucose). 4(1):

8 for all three substrates after 2 h of saccharification with G-ZYME 480. Another distinctive observation is the absence of the maltose peak in the HDGS chromatogram and the presence of this peak in both the NGS and corn chromatograms. This also shows that NGS, HDGS, and corn have differences in starch digestibility and accessibility during enzymatic hydrolysis and saccharification. Figure 4 shows the amounts of glucose produced from the NGS, HDGS, and corn control samples during hydrolysis and saccharification using an initial substrate concentration of 22% (w/v). Utilizing the data from table 1 for grain sorghum and the literature value of 73.7% (d.b.) starch content for commercial corn (Mojovic et al., 2006), the efficiency (% yield) of starch-to-glucose conversion after cooking and liquefaction, and simultaneous saccharification, after 2.5 h and 57 h were calculated. Percentage yields are reported in table 2. The paired t-test (α = 0.05) of glucose conversion in the entire hydrolysis and saccharification process showed that corn had significantly higher glucose yield compared to NGS (p < ) but was not significantly different when compared to HDGS (p = 0.930). After 2.5 h of saccharification, HDGS had the highest efficiency (89%) and starch-to-glucose conversion (126.3 g L - 1 ) among the three substrates, whereas after 57 h of saccharification at 32 C, glucose concentrations of 136.4, 138.9, and g L -1 were produced from NGS, HDGS, and corn, respectively. The dip in the NGS curve may be attributed to improper mixing or non-uniform distribution of the starch granule particles in the reactors during the initial stage of fermentation. However, once the fermentation had progressed, the glucose concentration continued to increase. Figure 4. Glucose concentration of the control during starch hydrolysis and saccharification. Conditions: cooking time = 1 h, hydrolysis time at 80 C = 1.5 h, saccharification time at 60 C = 2.5 h, saccharification time at 32 C = 72 h. 10 Biological Engineering Transactions

9 [a] [b] [c] [d] Table 2. Glucose yield during starch hydrolysis and saccharification of the control samples of NGS, HDGS, and corn. After 2.5 h [a] After 5 h [b] After 62 h [c] Substrate Glucose (g L -1 ) Yield (%) [d] Glucose (g L -1 ) Yield (%) [d] Glucose (g L -1 ) NGS HDGS Corn Total yield (%) [d] 1 h of cooking at 100 C and 1h hydrolysis at 80 C with SPEZYME EXTRA enzyme. 1 h saccharification with G-ZYME 480 Ethanol enzyme at 60 C and another 1.5 h while cooling until 35 C. 57 h saccharification with G-ZYME 480 Ethanol enzyme at 32 C. Yield = glucose converted (g L -1 ) / theoretical glucose (g L -1 ) 100. Although HDGS has significantly lower starch content than NGS and corn, the higher concentration of glucose obtained from HDGS indicates that the starch in HDGS is more digestible than NGS starch, resulting in a faster rate of glucose conversion and shorter time for complete saccharification. This implies that the protein digestibility of grain sorghum affects its starch digestibility. Studies by Zhan et al. (2006) and Hamaker (2004) have in fact confirmed that increasing the sorghum protein digestibility significantly increased its starch digestibility. Therefore, development of a grain sorghum variety with improved protein digestibility should benefit the ethanol fermentation process of sorghum. When used in the current corn ethanol system, HDGS is expected to have several potential advantages, including improved productivity of ethanol, improved quality of co-product animal feed (DDGS), reduced energy consumption during gelatinization and liquefaction, and reduced amount of enzyme during operation to save on capital expenses. Fermentation, Microbial Count, and Ethanol Production Figure 5 shows the microbial counts and glucose concentrations for each substrate within 72 h fermentation. During sample collection every 10 to 12 h, the 1 L headspace in the fermentation setup allowed a constant supply of oxygen into the mixture. This small amount of oxygen was able to maintain the number of viable cells throughout the experiment. An approximate tenfold increase in microbial count was observed during the first 3 h of fermentation in all of the substrates, while loss of cell viability was observed after 40 h of fermentation. In addition, the paired t-test (α = 0.05) of yeast viability was significantly higher for HDGS compared to NGS (p = 0.026) but not significantly different with corn (p = 0.084) during the entire SSF process. Glucose was continuously produced but was not enough to sustain the growth of yeast during the production of ethanol. After 24 h of fermentation, the concentration of glucose was constantly low (1 to 3 g L -1 ) for all of the substrates. The paired t-test (α = 0.05) showed that there was no significant difference in glucose concentration during the entire 72 h SSF using NGS, HDGS, and corn. Further study is therefore necessary to evaluate the conditions that will favor an increased and steady production of ethanol. The combination of substrate, enzymes, and yeast concentration should be optimized during hydrolysis and fermentation. The ethanol production curves for HDGS, NGS, and corn using simultaneous saccharification and fermentation are shown in figure 6. Among the three substrates, 4(1):

10 (a) (b) Glucose concentration versus fermentation time Figure 5. Changes in (a) viable yeast cells and (b) glucose concentration within 72 h of the grain fermentation. Hydrolysis conditions prior to inoculation at 35 C: cooking time = 1 h, liquefaction time = 1.5 h, and saccharification time = 2.5 h. HDGS obtained the highest ethanol yield throughout almost all the 72 h of fermentation. The difference was most noticeable after 20 h of fermentation. As shown in table 3, the calculated theoretical ethanol yield and ethanol concentration were also significantly higher for HDGS compared to NGS and corn (p = and 0.009, respectively) after 21 to 24h of SSF. An ethanol yield of 94% was obtained from HDGS, compared to 81% and 84% for NGS and corn, respectively. Although there were no significant differences in ethanol concentrations after 72 h of SSF using NGS, HDGS, and corn, early completion of HDGS fermentation suggests that the altered protein matrix in the genetically modified variety of grain sorghum improved the sorghum 12 Biological Engineering Transactions

11 Figure 6. Ethanol concentration during 72 h of simultaneous hydrolysis and ethanol fermentation. [a] [b] [c] Table 3. Ethanol yield during simultaneous saccharification and fermentation of grain substrates. After 21 to 24 h SSF After 72 h of SSF Overall v/v Ethanol Yield v/v Ethanol Yield [c] Y P/S Substrate % [a] (g L -1 ) (%) [b] % [a] (g L -1 ) (%) [b] (g g -1 ) HDGS NGS Corn Means of three replicates. Yield = ethanol converted (g L -1 ) / theoretical ethanol (g L -1 ) 100. Ethanol yield based on amount of starch (g ethanol converted / g theoretical glucose). starch digestibility during enzymatic hydrolysis, which then contributed to the faster and higher starch conversion to glucose and ultimately to ethanol. It is to be noted that the same conditions for enzymes, yeast, and substrates during hydrolysis and fermentation were maintained in all tests such that the only factor affecting ethanol yield would be starch digestibility. The findings of this study can be summarized as follows: No significant change in ethanol concentration was observed from 40 h to 72 h of SSF using NGS. No significant change in ethanol concentration was observed from 21 h to 72 h of SSF using HDGS. No significant change in ethanol concentration was observed from 45 h to 72 h of SSF using corn. The efficiencies of the substrates for ethanol fermentation can be ranked as follows: NGS < corn < HDGS. Conclusion The high digestible grain sorghum (HDGS) variety gave the highest ethanol yield compared to normal grain sorghum (NGS) and corn throughout almost all the 72 h of 4(1):

12 simultaneous saccharification and fermentation (SSF). The higher protein digestibility of HDGS resulted in higher starch digestibility, allowing a more rapid starch conversion to glucose and ethanol and higher yield during enzymatic hydrolysis and fermentation. Paired t-tests (α = 0.05) of glucose conversion in the entire hydrolysis and saccharification process showed that corn had significantly higher glucose yield compared to NGS (p < ) but was not significantly different compared to HDGS (p = 0.930). However, only HDGS achieved early completion of glucose conversion after 48 h and ethanol production after 24 h of SSF. These results suggest that using a grain sorghum variety with improved protein digestibility results in a better ethanol fermentation process of sorghum. Sorghum is primarily used as animal feed in the U.S. and can be produced in drier climates that cannot support corn production because uses less water than corn for cultivation (Smith and Mullins, 2001). The use of the newly developed HDGS may provide a more efficient alternative to corn for ethanol production via SSF. Since grain sorghum is a less expensive grain than corn, the use of grain sorghum with enhanced starch digestibility may further reduce both material and processing costs during fermentation. Possible process improvements could be realized through reducing enzyme dosages and shortening of liquefaction and fermentation times. Further cost reduction can also be achieved by optimizing the combination of substrates, enzymes, and yeast during hydrolysis and fermentation. Moreover, the use of enzymes with higher specific activities that could provide more efficient starch conversion to glucose at a shorter time than the current commercial enzymes could also be explored. Acknowledgements The authors would like to thank the following for providing assistance, materials, and/or financial support for this research study: federal grants from US-DOT-Sun Grant South Central Region and USA-AID-INTSORMIL (D. B. Hays); the Biochemistry Lab under Dr. Karthikeyan in the Department of Biological and Agricultural Engineering (BAEN), Texas A&M University; the Department of Chemical Engineering, Texas A&M University; Texas AgriLife Research (AgriLife); Genencor International, Inc.; Dr. Amado Maglinao, Sr.; and BAEN student workers. References Aboubacar, A., J. D. Axtell, L. Nduulu, and B. R. Hamaker Turbidity assay for rapid and efficient identification of high protein digestibility sorghum lines. Cereal Chem. 80(1): Badger, P. C Ethanol from cellulose: A general review. In Trends in New Crops and New Uses, J. Janick and A. Whipkey, eds. Alexandria, Va.: ASHS Press. Chandrashekar, A., and A. W. Kirlies Influence of protein on starch gelatinization in sorghum. Cereal Chem. 65(6): Chen, R. L., M. H. Lee, and K. S. Matsumoto Selective biosensing of L-lysine by a lowtemperature flow-injection technique using an immobilized lysine oxidase reactor. Analytical Sci. 12(1): Dicko, M. H., H. Gruppen, A. S. Traoré, A. G. J. Voragen, and W. J. H. van Berkel Sorghum grain as human food in Africa: Relevance of content of starch and amylase activities. African J. Biotech. 5(5): EERE Biomass multi-year program. Washington, D.C.: U.S. Department of Energy, Energy Efficiency and Renewable Energy. Available at: www1.eere.energy.gov/biomass/ pdfs/biomass_mypp_november2010.pdf. Accessed 29 March Biological Engineering Transactions

13 EIA A primer on gasoline prices. Washington, D.C.: U.S. Department of Energy, Energy Information Administration. Available at: gasolinepricesprimer/eia1_2005primerm.html and petroleum/analysis_publications/primer_on_gasoline_prices/html/printerversion.pdf. Accessed 29 March EIA Energy basics 101. Washington, D.C.: U.S. Department of Energy, Energy Information Administration. Available at: Accessed 7 March Gray, K. A., L. Zhao, and M. Emptage Bioethanol. Current Opinion in Chem. Biol. 10(2): Hamaker, B. R., Crop utilization and marketing: Chemical and physical aspects of food and nutritional quality of sorghum and millet. Annual report of INTSORMIL. West Lafayette, Ind.: Purdue University, INTSORMIL Collaborative Research Support Program (CRSP). Available at Accessed 5 May Hamelinck, C. A., G. V. Hooijdonk, and A. P. C. Faaij Ethanol from lignocellulosic biomass: Techno-economic performance in short, middle, and long term. Biomass and Bioenergy 28(4): Hill, J., E. Nelson, D. Tilman, S. Polasky, and D. Tiffany Environmental, economic, and energetic costs and benefits of biodiesel and ethanol biofuels. PNAS 103(30): Ingledew, W. M Alcohol production by Saccharomyces cerevisiae: A yeast primer. In The Alcohol Textbook, rd ed. Nottingham, U.K.: Nottingham University Press. Mertz, E. T., M. M. Hassen, C. Cairns-Whittern, A. W. Kirleis, L Tu, and J. D. Axtell Pepsin digestibility of proteins in sorghum and other major cereals. Proc. Natl. Acad. Sci. 81(1): 1-2. Mojovic, L., S. Nikolic, M. Rakin, and M. Vukasinovic Production of bioethanol from corn meal hydrolyzates. Fuel 85(12-13): Nichols, N. N., D. A. Monceaux, B. S. Dien, and R. J. Bothast Production of ethanol from corn and sugarcane. In Bioenergy, Washington, D.C.: ASM Press. RFA Annual industry outlook 2008: Historic U.S. fuel ethanol production. Washington, D.C.: Renewable Fuel Association. Available at: Accessed 26 February Serna-Saldivar, S. O., and L. W. Rooney Structure and chemistry of sorghum and millets. In Sorghum and Millets Chemistry and Technology, D. A. V. Dendy, ed. St. Paul, Minn.: American Association of Cereal Chemists. Shuler, M. L., and F. Kargi Bioprocess Engineering: Basic Concepts. 2nd ed. Upper Saddle River, N.J.: Prentice Hall International Series. Smith, K. A., and C. E. Mullins Soil and Environmental Analysis: Physical Methods. 2nd ed. New York, N.Y.: Marcel Dekker. Taylor, J. R. N., L. Novellie, and N. V. D. W. Liedenberg Sorghum protein body composition and ultrastructure. Cereal Chem. 61(1): Weaver C. A., B. R. Hamaker, and J. D. Axtell Discovery of grain sorghum germplasm with high uncooked and cooked in vitro protein digestibility. Cereal Chem. 75(5): Zhan, X., D. Wang, X. S. Sun, S. Kim, and D. Y. C. Fung Lactic acid production using extrusion-cooked grain sorghum. Trans. ASAE 46(2): Zhan, X., D. Wang, S. R. Bean, X. Mo, X. S. Sun, and D. Boyle Ethanol production from supercritical-fluid-extrusion cooked sorghum. Ind. Crops and Products 23(3): (1):