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Energy & Fuels 2006, 20, 2732-2736
Enzymatic Hydrolysis of Distiller’s Dry Grain and Solubles (DDGS) Using Ammonia Fiber Expansion Pretreatment Bryan Bals, Bruce Dale,* and Venkatesh Balan Department of Chemical Engineering and Materials Science, 2527 Engineering Building, Michigan State UniVersity, East Lansing, Michigan 48824 ReceiVed June 27, 2006. ReVised Manuscript ReceiVed August 23, 2006
Approximately 4 billion gallons of fuel ethanol was produced in 2005 in the United States, mostly from corn grain, and production is expected to continue to increase in the future. The major coproduct is distiller’s dry grain and solubles (DDGS), which is sold as an animal feed. The increase in DDGS supply is expected to drive its value down, and thus, further value addition is necessary. One approach is to convert the cellulose in DDGS into sugars for increased ethanol production, leaving a residue higher in protein content. In this work, the effect of ammonia fiber expansion (AFEX) pretreatment on the enzymatic hydrolysis of both wet and dry DDGS was studied. Hydrolysis of AFEX treated samples gave 190 g glucose/kg dry biomass, or virtually complete conversion of cellulose after 72 h. Optimal AFEX conditions for dry and wet DDGS were 70° C and 0.8 kg anhydrous NH3/kg dry biomass and 80° C and 0.6 kg NH3/kg dry biomass, respectively. Xylose yields were negligible.
Introduction There is considerable interest in using ethanol as an additive to gasoline and, ultimately, as a liquid fuel. Approximately 4 billion gallons of ethanol was produced in 2005, mostly from corn grain, an increase of 17% over the previous year and over twice as much as was produced in 2001.1 The Energy Policy Act, signed into law in August 2005, requires that 7.5 billion gal/year of biofuels, most notably ethanol, are to be mixed with gasoline by 2012.2 Several states in the Midwest have adopted or are considering more stringent renewable fuel standards. To meet this need, 29 new ethanol plants are under construction to add to the 95 plants operating as of January 2006.1 Thus, demand for ethanol in the United States is expected to continue its rapid growth in the near future. The dominant process for producing grain ethanol in the United States is the dry-grind process, which contributes about 79% of current ethanol production. During this process, distiller’s dry grain and solubles (DDGS) is created as a coproduct, and over 9 million metric tons of this material was produced in 2005.3 This residue has a high protein content and is currently used primarily as feed for the beef and dairy industry, while it is used only sparingly in the poultry and swine feed markets. However, as the supply of DDGS increases, its price is expected to decrease in relation to other feeds such as soybean meal. Thus, it may be necessary to increase the value of DDGS in order to keep dry-grind ethanol plants cost competitive. One possible approach is the enzymatic hydrolysis of the * Corresponding author. Phone: 1-517-353-6777. E-mail: bdale@ egr.msu.edu. (1) From Niche to Nation: Ethanol Industry Outlook 2006; Renewable Fuels Association: Washington, D.C., 2006; p 4. (2) Energy Policy Act of 2005, 109th Congress, 1st Session, Washington, D.C., 2005; Sec. 1501. (3) From Niche to Nation: Ethanol Industry Outlook 2006; Renewable Fuels Association: Washington, D.C., 2006; p 14.
cellulose into glucose for fermentation into ethanol. The remaining grain would have a higher protein content, thereby increasing its value and possibly allowing it to expand more fully into the swine and poultry markets. The increase in ethanol production due to cellulose hydrolysis would also provide additional economic benefit. Tucker et al. reported ethanol yields of 73% of the theoretical value using dilute-acid pretreatment and also demonstrated that the resulting hydrolyzed distiller’s grain could be included in turkey feed at up to 10% total feed.4 Cellulose is resistant to enzymatic hydrolysis due to its densely packed and rigid crystalline structure. A novel pretreatment method to improve the efficiency of enzymatic hydrolysis is the ammonia fiber expansion (AFEX) process. Liquid ammonia binds to the cellulose under high pressure (200-500 psi) and moderate temperatures (70-100 °C). The pressure is then quickly released. Virtually all of the ammonia can be recovered. This process decrystallizes the cellulose, hydrolyzes hemicellulose, removes and depolymerizes lignin, and increases the surface area available for hydrolysis, thereby significantly increasing the rate of enzymatic hydrolysis.5 Previous work has shown this process to give near theoretical yields of glucose on many different types of biomass.6-9 AFEX also compares favorably with other pretreatment methods, producing high (4) Tucker, M.; Nagle, N.; Jennings, E.; Ibsen, K.; Aden, A.; Nguyem, Q.; Kim K.; Noll, S. Appl. Biochem. Biotechnol. 2004, 113-116, 11391157. (5) Mosier, N.; Wyman, C.; Dale, B.; Elander, R.; Lee, Y.; Holtzapple, M.; Ladisch, M. Bioresour. Technol. 2005, 96, 673-686. (6) Mosier, N.; Wyman, C.; Dale, B.; Elander, R.; Holtzapple, M.; Ladisch, M.; Lee, Y. Bioresour. Technol. 2005, 96, 2026-2032. (7) Holtzapple, M.; Jun, J.; Ashok, G.; Patibandla, S.; Dale, B. Appl. Biochem. Biotechnol. 1991, 28-29, 59-74. (8) Sulbaran-de-Ferrer, B.; Aristiguieta, M.; Dale, B.; Ferrer, A.; Ojedade-Rodriguez, G. Appl. Biochem. Biotechnol. 2003, 105-108, 155-164. (9) Teymouri, F.; Laureano-Perez, L.; Alizadeh, H.; Dale, B. Bioresour. Technol. 2005, 96, 2014-2018.
10.1021/ef060299s CCC: $33.50 © 2006 American Chemical Society Published on Web 10/04/2006
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yields of glucose and xylose at low enzyme loadings despite not removing any lignin or hemicellulose from the process.10 The goal of this study, then, is to determine the optimal conditions in terms of temperature and ammonia loading for the AFEX process on DDGS to provide maximum glucose yields. The effect of adding xylanase and amylase to hydrolyze xylose and starch, respectively, was also determined. Experimental Section Material Source. The DDGS was obtained from Big River Resources (West Burlington, IA) and dried to approximately 11.5% moisture content (total weight basis). Analysis done at Purdue University revealed an estimated composition of 16.0% ( 6.6% cellulose, 8.2% ( 3.3% xylan, 5.3% ( 0.7% arabinan, 5.2% starch, and 26.4% protein. Errors represent 95% confidence intervals. Residual cellulose and xylan remaining in the grain after hydrolysis were determined by acid hydrolysis using the method described in the LAP-002 protocol from the National Renewable Energy Laboratory.11 Lipids, protein, and ash were analyzed by Dairy One Forage Laboratory (Ithaca, NY). Biomass Pretreatment. The AFEX pretreatment process was done in a 300 mL stainless steel pressure vessel. Water was mixed with the DDGS to adjust to the desired moisture content and then loaded into the vessel. Glass spheres were added to minimize void space, thereby reducing the amount of ammonia in the vapor phase within the reactor. The lid was bolted shut, and a sample cylinder loaded with the proper amount of liquid anhydrous ammonia was connected, allowing the ammonia to be charged into the vessel. The reactor was heated using a 400 W PARR heating mantle and allowed to stand at the desired temperature ((1 °C) for five minutes. The pressure was explosively released by rapidly turning the exhaust valve. The treated samples were removed and were placed in a fume hood overnight to remove any residual ammonia. Enzymatic Hydrolysis. The enzymatic hydrolysis procedure was based upon the LAP-009 protocol from the National Renewable Energy Laboratory.11 An amount of biomass equal to 0.15 g of cellulose was placed in a vial and brought to a total volume of 15 mL with autoclaved water. The solution was buffered to pH 4.8 by 0.75 mL of 1 M citrate buffer. Spezyme CP (Genencor, Palo Alto, CA) cellulase was loaded at 16.5 FPU/g glucan (31 mg protein/g glucan), and β-glucosidase (Novozyme 188, Bagsvaerd, Denmark), at 56 pNPGU/g glucan. In one experiment, Multifect Xylanase (Genencor) was used at 10-50% of the cellulase mass loading in addition to the other enzymes. One experiment involved the amylase Stargen (Genencor) added along with the cellulases. All samples were incubated at 50 °C with 75 rpm rotation. Samples were collected at 24, 72, and 168 h for analysis. Analytical Methods. Sugar analysis was done using a Waters high performance liquid chromatograph (HPLC) system equipped with a Bio-Rad (Richmond, CA) Aminex HPX-87P carbohydrate analysis column. Degassed HPLC water with a flow rate of 0.6 mL/min was used as the mobile phase, while the temperature in the column was kept constant at 85 °C. A Waters 410 differential refractometer was used to measure the peaks obtained. Images of the surface of pretreated and untreated DDGS were taken at magnifications from 150× to 1500× using an environmental scanning electron microscope (ESEM). A Spectrum One Fourier transform infrared (FTIR) system with a universal attenuated total reflection accessory was used to obtain spectra readings from 600 to 4000 cm-1 of both AFEX treated and untreated grain.
Results and Discussion The effect of reaction temperature on glucose conversion at 1:1 kg ammonia/kg biomass ammonia loading is seen in Figure (10) Alizadeh, H.; Teymouri, F.; Gilbert, T.; Dale, B. Appl. Biochem. Biotechnol. 2005, 121-124, 1133-1141. (11) NREL, Chemical Analysis and Testing (CAT) Standard Procedures; National Renewable Energy Laboratory: Golden, CO, 2004.
Figure 1. Effect of temperature on the theoretical glucose conversion of AFEX treated DDGS. The moisture content was held constant at 11.5% total weight basis (twb) and 1:1 kg/kg ammonia loading. The 100% theoretical yield is based upon a cellulose content of 16.0% dry weight basis (dwb). All runs were done in duplicate, and error bars represent the maximum and minimum values.
Figure 2. Effect of ammonia loading on the theoretical glucose conversion of AFEX treated DDGS. The moisture content and temperature were held constant at 11.5% twb and 70 °C, respectively. The 100% theoretical yield is based upon a cellulose content of 16.0% dwb. All runs were done in duplicate and error bars represent the maximum and minimum values.
1. Numbers are given as a percent of the theoretical maximum yields, which is defined as all of the cellulose present being converted to glucose. The glucose yields increase sharply from 60 to 70 °C, while further temperature increase has a statistically insignificant (p < 0.05) effect. At 100 °C, the grain appeared to be burnt, although this did not affect glucose yields. In all cases from 70 to 100 °C, greater than 100% of the theoretical glucose yield can be obtained after 72 h of hydrolysis. Here, 100% yield is defined to be complete conversion of the cellulose, assumed to be 16.0% of the DDGS, into glucose. Thus, there are two possible explanations for obtaining yields in excess of 100%: either the measured cellulose composition is lower than its actual value or there is some starch hydrolysis occurring. There is a fairly large error associated with the cellulose value due to the inherent error associated with this method, especially for samples such as DDGS that have been dried at high temperatures.11 Adding the cellulase enzymes to pure starch does release some glucose (data not shown), making it likely that these enzymes are hydrolyzing the residual starch in the DDGS as well. Figure 2 shows the effect of the ammonia-to-biomass ratio on the glucose yields during enzymatic hydrolysis of DDGS, with a fixed temperature of 70°C and no added moisture content. The yield increases as more ammonia is loaded up to a 0.8:1 kg NH3/kg biomass ratio, while higher levels of ammonia decreased the resulting glucose yield. Similar trends have been
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Figure 3. Effect of temperature on the theoretical glucose conversion of AFEX treated wet (60% twb) DDGS. Ammonia loading was held constant at 1:1 kg/kg. The 100% theoretical yield is based upon a cellulose content of 16.0% dwb. All runs were done in duplicate, and error bars represent the maximum and minimum values.
seen with other biomass species as well as DDGS at 80 °C (data not shown). Cellulose conversion was nearly 86% after 24 h and over 108% after 72 h. After an additional 96 h, however, the increase in glucose conversion was minimal. This is a major improvement over untreated DDGS, where only 83% of the theoretical glucose yield can be obtained. The rate of hydrolysis also increased greatly, as only 66% of theoretical cellulose was converted in untreated DDGS after 24 h. Thus, optimal AFEX conditions for DDGS were determined to be 70 °C and 0.8:1 kg/kg ammonia loading. These are fairly mild compared to the optimal conditions for other types of biomass, most likely due to the relatively low lignin and cellulose content as well as the effect of the dry-grind ethanol process.9-10 Under these conditions, approximately 108% of the theoretical glucose conversion is achieved after 72 h, or 190 g glucose/kg dry biomass. This represents a 30% increase over untreated DDGS, which produces 146 g glucose/kg dry biomass. Approximately 59% of the DDGS was solubilized during the hydrolysis (i.e., the original dry mass of the sample was reduced by approximately three-fifths). In the dry-grind process, the solid residue remaining after distillation exists at about 60% moisture total weight basis (twb) and is then combined with the stillage and dried to form DDGS.12 However, drying can be an energy intensive and expensive process, so it may be advantageous to process the material without this step. However, previous work on other biomass has shown that the moisture content of the biomass during AFEX can significantly affect the efficacy of the pretreatment.10 Thus, optimal conditions for wet DDGS are also needed. The effect of reaction temperature at 1:1 ammonia loading is seen in Figure 3. As with the dry DDGS, the glucose yield increased between 60 to 70 °C and remained constant at higher temperatures. The effect of ammonia loading on wet DDGS at a constant temperature of 80 °C is seen in Figure 4. Here, yields remained constant until 1.2:1 kg NH3/kg dry biomass. Because ammonia is a significant cost factor in this pretreatment process, the lowest loading should be chosen. Yields at 70 °C were lower than those at 80 °C at all ammonia loadings except for 1:1 kg/ kg (data not shown). Under these conditions, the glucose yield after 72 h is over 103% of the theoretical yield. Although yields may decrease slightly, it appears to be unnecessary to dry the DDGS prior to the AFEX pretreatment step. (12) National Corn Grower’s Association. Ethanol and Co-Products. http://www.ncga.com/ethanol/co_products/definition_production.htm (accessed June 2006).
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Figure 4. Effect of ammonia loading on the theoretical glucose conversion of AFEX treated wet (60% twb) DDGS. The reactor temperature was held constant at 80 °C. The 100% theoretical yield is based upon a cellulose content of 16.0% dwb. All runs were done in duplicate, and error bars represent the maximum and minimum values.
Figure 5. Effect of amylase on glucose yields for both AFEX treated and untreated DDGS. Cellulase loading was 16.5 FPU/g glucan, while amylase was added at 14 mg enzyme/g glucan. β-Glucosidase was added at 56 pNPGU/g glucan in all cases. The 100% theoretical yield is based upon a cellulose content of 16.0% dwb.
In all cases described above, the xylose yields were negligible. Multifect Xylanase was added at up to 50% of the cellulase loading. However, no xylose was detected at any level of xylanase loading after 168 h, meaning less than 20% of the xylan was hydrolyzed. The hemicellulose in corn grain is a complex arabinoxylan structure, consisting of a xylan backbone with several branching and cross-linked chains.13-14 Effectively hydrolyzing this structure requires breaking several different bonds and, thus, requires more enzymes than simply xylanase. Work done at the USDA National Center for Agricultural Utilization Research has shown that a combination of pectinase and ferulic esterase in addition to the cellulases can give yields in excess of 90% of the theoretical value for both xylose and arabinose after AFEX pretreatment.15 As the dry-grind process did not obtain complete conversion to ethanol, this sample of DDGS contains some residual starch. Thus, attempts were also made to increase the glucose yields by the addition of amylase. However, as seen in Figure 5, adding Stargen amylase at 14 mg enzyme/g glucan to the cellulase cocktail did not significantly (p < 0.05) affect the overall glucose yield. As stated previously, a portion of the starch may already be being broken down by the cellulase enzymes, thus reducing the need for additional amylase. Moreover, the amylase (13) Koukiekolo, R.; Cho, H.; Kosugi, A.; Inui, M.; Yukawa, H.; Doi, R. Appl. EnViron. Microbiol. 2005, 71 (7), 3504-3511. (14) Leathers, T. FEMS Yeast Res. 2003, 3, 133-140. (15) Dien, B. Private correspondence, 2006.
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Table 1. Composition Analysis of Untreated DDGS as well as the Hydrolyzed DDGSa hydrolyzed DDGS composition
DDGS (%)
%
g/100 g DDGS
crude protein cellulose xylan fat ash
26.4 16.0 8.2 10.4 3.4
49.4 nd 13.8 14.6 3.0
20.25 nd 5.64 5.99 1.24
a Here, 41 g of hydrolyzed grain are obtained for every 100 g of DDGS. Two values are given for the hydrolyzed DDGS: the percent composition of the hydrolyzed grain and that on the basis of the original DDGS. Also, nd ) not detected. Values for DDGS were obtained by Purdue University except for the ash content.
is likely inhibiting the cellulase by binding to cellulose sites, thus decreasing cellulose conversion and offsetting any increase in starch hydrolysis. Increasing amylase loading did not improve the glucose yields (data not shown). The compositional and feed characteristics of the hydrolyzed distiller’s grain (HDG) are shown in Table 1. This residue would likely be used as a protein supplement in animal feed unless further value can be added to it. No residual cellulose was detected, consistent with the high conversion obtained earlier. Xylan content increased, although not all of the xylan is recovered in the solid residue. Thus, there may be some xylose oligosaccharides present in the hydrolysate. This represents a substantial amount of fiber still present in the HDG, although as stated earlier work is underway to completely hydrolyze the hemicellulose. The protein content increased from 26% to 49%, comparable to 48% soybean meal commonly used as a protein source. This is equivalent to 77% of the original protein in DDGS, although it is likely that some cellulases are still bound to the solids. The protein remaining in the hydrolysate can be used as a protein source for the subsequent sugar fermentation. It may also be possible to collect these proteins and add them to the HDG, further increasing its protein content. Fat content in the HDG also increased, which along with the increased protein leads to the high digestible energy content. The lipids remaining in the hydrolysate may be converted to biodiesel or used as a feed source during fermentation. The amount of ash remained constant, meaning that it is unlikely that it will affect the nutritional value of the HDG in comparison to the original DDGS. Feed trials are required to determine if these hydrolyzed distiller’s grains are suitable for the various feed markets. Visually, the only change in the appearance of DDGS after AFEX pretreatment is that the grain becomes darker. An ESEM image of the grain magnified 500× is shown in Figure 6. There does not appear to be any significant difference between the structures of pretreated and untreated grain. This may be due to the low cellulose content, making it difficult to see if any decrystalization occurred. Infrared spectroscopy appears to confirm that chemical changes did occur, however. As seen in Figure 7, bands associated with lignin decreased after AFEX pretreatment, thus implying that delignification occurred. Furthermore, the decrease in C-H bonds implies depolymerization as well.16 However, no evidence of hemicellulose hydrolysis exists, as there is no increase in acetyl groups. This is consistent with the negligible xylose yields obtained during hydrolysis. It is unclear why the AFEX process is unable to break down the hemicellulose in this case, although the complex nature of corn grain’s hemicellulose may be a factor. (16) Laureano-Perez, L.; Teymouri, F.; Alizadeh, H.; Dale, B. Appl. Biochem. Biotechnol. 2005, 121-124, 1081-1100.
Figure 6. ESEM image of DDGS magnified at 500× both before (a) and after (b) AFEX pretreatment (70 °C, 11.5% MC, 1:1 kg NH3/kg dry biomass).
Figure 7. FTIR spectra of DDGS both before and after AFEX pretreatment (70 °C, 11.5% MC, 1:1 kg NH3/kg dry biomass). Peaks to note are the following: (a) O-H stretch, (b) C-H stretch, (c) carbonyl, (d) lignin.
Conclusion Our experimental results show that the AFEX process is an effective pretreatment for the enzymatic hydrolysis of DDGS. Under mild conditions, glucose yields increased from 83% to 108% of the theoretical value after 72 h of hydrolysis, or 190
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g glucose/kg dry biomass. Furthermore, yields only decreased slightly at higher moisture content, indicating that this process may be effective without a costly drying step. Xylose yields were negligible even after the addition of xylanases. The low xylose conversion is consistent with FTIR analysis, as no evidence of hemicellulose hydrolysis is seen. Adding amylase did not improve glucan hydrolysis, most likely due to the fact that the cellulase cocktail used is already effective at breaking down starch. These are encouraging results, indicating that hydrolysis of the fiber in DDGS may be economically feasible. This would not only increase the ethanol production in the plant but would also help offset the expected decline in value of DDGS by increasing its protein content. The remaining grain could potentially be sold as a higher value animal feed due to this increase in protein. Further studies concerning adding additional value to the hydrolyzed distiller’s grain are needed, as well as
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increasing xylan and arabinan conversion. In addition, process economic modeling and life cycle analysis are required to optimize pretreatment and hydrolysis parameters. Acknowledgment. The material in this work was supported by the Midwest Consortium for Sustainable Biobased Products and Bioenergy through DOE contract DE-FG36-04GO14220. The Midwest Consortium is making a concerted effort to provide core capabilities and technologies for a biobased chemical industry anchored in the rural communities of the Midwest. This collaboration involves four major Land-Grant Universities and three federal laboratories that have worked together since 1999: Purdue University, the University of Illinois, Michigan State University, Iowa State University, Argonne National Laboratory, Ames Laboratory, and the USDA National Center for Agricultural Utilization Research. EF060299S
