Fiber Separated from Distillers Dried Grains with Solubles as a Feedstock for Ethanol Production 1
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Radhakrishnan Srinivasan, Bruce S. Dien, Kent D. Rausch, M. E. Tumbleson,3 and Vijay Singh ABSTRACT
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Cereal Chem. 84(6):563–566
In the dry-grind process, corn starch is converted into sugars that are fermented into ethanol. The remaining corn components (protein, fiber, fat, and ash) form a coproduct, distillers dried grains with solubles (DDGS). In a previous study, the combination of sieving and elutriation (air classification), known as the elusieve process, was effective in separating fiber from DDGS. In this study, elusieve fiber was evaluated for ethanol production and results were compared with those reported in other studies for fiber from different corn processing techniques. Fiber samples were pretreated using acid hydrolysis followed by enzymatic treatment. The hydrolyzate was fermented using Escherichia coli FBR5 strain. Efficiency of ethanol production from elusieve fiber was 89–91%, similar to that for
pericarp fiber from wet-milling and quick fiber processes (86–90%). Ethanol yields from elusieve fiber were 0.23–0.25 L/kg (0.027–0.030 gal/lb); similar to ethanol yields from wet-milling pericarp fiber and quick fiber. Fermentations were completed within 50 hr. Elusieve fiber conversion could result in 1.2–2.7% increase in ethanol production from dry-grind plants. It could be economically feasible to use elusieve fiber along with other feedstock in a plant producing ethanol from cellulosic feedstocks. Due to the small scale of operation and the stage of technology development for cellulosic conversion to ethanol, implementation of elusieve fiber conversion to ethanol within a dry-grind plant may not be currently economically feasible.
Fuel ethanol production from dry-grind corn plants is increasing rapidly (RFA 2006). In dry-grind processing, corn starch is converted into sugars that are fermented into ethanol. The remaining corn components (protein, fiber, fat, and ash) form the coproduct, distillers dried grains with solubles (DDGS). Fiber in the corn contains cellulose and hemicellulose that are not converted into sugars in the dry-grind process. In corn wet-milling, pericarp fiber is a coproduct from fractionation of corn. Singh et al (1999) developed the quick fiber process to remove pericarp fiber (called quick fiber) using floatation of fiber in the slurry before fermentation in the dry-grind corn process. The elusieve process separated fiber from DDGS using sieving and elutriation (air flow) (Srinivasan et al 2005). Dien et al (2004) converted corn fiber into ethanol by pretreatment and simultaneous saccharification and fermentation (SSF). Pretreatment is an initial step that prepares cellulose for enzymatic conversion: cellulase converts cellulose into glucose. Hydrolysis of hemicellulose releases sugars such as arabinose and xylose. Wild type Escherichia coli strains can ferment pentose sugars, but they also produce organic acids along with ethanol. A recombinant ethanologenic E. coli strain, FBR5, was engineered to produce ethanol from sugar mixtures such as those found in corn fiber hydrolyzates (Dien et al 2004). Corn fiber has been used as feedstock in a pilot-plant facility for ethanol production (Schell et al 2004). Dien et al (2004) and Singh et al (2004) evaluated wet-milling pericarp fiber and quick fiber for ethanol production. Efficiency of ethanol production, based on carbohydrate content, was 89– 91%. Ethanol yield from quick fiber was 0.15 L/kg (0.018 gal/lb) to 0.27 L/kg (0.032 gal/lb) of dry fiber. Ethanol yield from wetmilling pericarp fiber was 0.19 L/kg (0.023 gal/lb). Tucker et al (2004) converted fiber from distillers wet grains (DWG), a process
stream in dry-grind plants, into ethanol by pretreating whole DWG and fermenting the sugars into ethanol using S. cerivisiae D5 A . The residual material had higher protein content and lower fiber content. Fiber separated using the elusieve process can be used to produce ethanol in the dry-grind plants and thereby increase revenues. The objective of this study was to evaluate ethanol production from elusieve fiber.
1 Former
graduate student, Agricultural and Biological Engineering, University of Illinois at Urbana-Champaign, Urbana, IL 61801. Presently, assistant research professor, Agricultural and Biological Engineering, Mississippi State University, Mississippi State, MS 39762. 2 Lead scientist, National Center for Agricultural Utilization Research, ARS, USDA, Peoria, IL 61604. 3 Associate professor, professor emeritus, and associate professor, Agricultural and Biological Engineering, University of Illinois at Urbana-Champaign, Urbana, IL 61801. 4 Corresponding author. Phone: 217-333-9510. Fax: 217-244-0323. E-mail address:
[email protected] doi:10.1094 / CCHEM-84-6-0563 © 2007 AACC International, Inc.
MATERIALS AND METHODS Fiber Separation from DDGS DDGS material (75 kg) was divided into three batches of 25 kg each. Each batch was sieved using a vibratory sifter (model ZS30S6666, SWECO Vibro-Energy Separator, Florence, KY) using five screens US7 (2800 μm), 24T (869 μm), 34T (582 μm), 48T (389 μm), and 62T (295 μm). Material (2 kg) was sieved on each screen for 1 hr. Material passing through the sieve with a larger opening was collected and placed on the next smaller sieve size. Only one sieve was used at a time. The four intermediate sieve categories, material retained on 24T (869 μm), 34T (582 μm), 48T (389 μm), and 62T (295 μm) screens, were subjected to elutriation using an apparatus described by Srinivasan et al (2005); elutriation column diameter was 155 mm. Material carried by air to the top of the elutriation column was the lighter fraction; material that settled in the bottom was the heavier fraction. The apparatus consisted of an elutriation column, an air blower for supplying air to the elutriation column, a surge box mechanism for controlling airflow, a vibratory feeder for feeding material into the column, and collection vessels for receiving the lighter and heavier fractions. Due to low fiber content, elutriation was not performed for the largest and the smallest sieve categories. Material was fed into the elutriation column at a rate of 5 g/min. Elutriation was performed at two air velocities to obtain lighter fraction yields of 15 and 25%. Three passes of elutriation were performed at each individual air velocity by recycling the heavier fraction into the column to ensure complete elutriation. Sample order within each batch was randomized. Fiber (lighter) fractions, at 15% yield (lower air velocities), from elutriation of four sieve categories were combined into a single fiber sample, F1. Fiber (lighter) fractions, at 25% yield (higher air velocities), from elutriation of four sieve categories were combined into a single fiber sample, F2. From three batches of DDGS, a total of six samples were available for ethanol studies. Vol. 84, No. 6, 2007
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Sugar Compositional Analysis Fiber sugar composition was determined using the procedure described in Dien et al (1997). Fiber samples were hydrolyzed with 2N trifluoroacetic acid (TFA) for 1 hr at 100°C. Sugars were analyzed using high-performance liquid chromatography (HPLC) (Thermo Finnigan Spectra System, San Jose, CA) with an HPX87C column (300 × 7.8 mm, BioRad, Hercules, CA) at 85°C. The column was eluted with distilled water at a flow rate of 0.6 mL/min. The detector was a 410 differential refractometer (Waters, Milford, MA). Cellulose content was determined using the method outlined in Dien et al (1997). Fiber sugar composition was determined in duplicate for each sample. Moisture content was determined using ASTM method E1756-01 by determining weight loss after drying the sample for 18 hr at 105°C. Moisture content was determined in duplicate for each sample. Ethanol Production Two 10-g replicates of each fiber-enriched sample were prepared. Samples were treated with 100 mL of H2SO4 (1% w/v) for 1 hr in an autoclave (121°C, 2 atm). Samples were adjusted to pH 4.5 by adding 2.7N Ca(OH)2. Enzymes (60 filter paper units/g of GC220 Cellulase, Genencor, Rochester, NY, and 40 units/g of Novozyme 188 Cellobiase, Novozymes, Franklinton, NC) were added. Samples were saccharified for 18 hr at 50°C. The samples were adjusted to pH 7.0 by adding 2.7N Ca(OH)2 and prepared for fermentation by adding 12.5 mL of 10× buffered basal medium Lysogeny broth (LB) (1M 3-(N-morpholino) propanesulfonic acid (MOPS), pH 7.0, 50 g/L of yeast extract, and 10 g/L of tryptone). The hydrolyzate was inoculated with ethanologenic E. coli strain FBR5 (5% v/v) that had been grown overnight (Dien et al 2004). Fermentations incubated six days at 35°C were sampled daily. A control sample with glucose (2.2% w/v), xylose (2.6% w/v), and arabinose (1.1% w/v) in the same basal medium was fermented using test samples. Deionized water (100 mL) was added and the mixture was autoclaved for 15 min at 121°C and 2 atm. Like the hydrolyzates, sugar solutions were supplemented with 12.5 mL of LB and MOPS solution, inoculated with E. coli FBR5, and fermented for six days.
TABLE I Composition (% wb) of Carbohydrates in Fibers Obtained by Elusieve Processing of DDGS-1 and Control Samplea,b Sample
Glucose
Xylosec
Arabinose
Total Carb
18.4a 17.8b 28.8 24.2
20.6a 16.4b 19.8 17.7
9.4a 7.9b 9.9 9.2
48.4a 42.0b 58.5 51.1
F1 F2 Quick fiberd Wet-milling pericarp fibere a
Values are reported as means of three batches and two replicates from each batch. b Values in a column followed by the same letter are not different (P < 0.05). c Xylose values also include a minor amount of galactose; both sugars coelute in the HPLC column. d Values reported by Dien et al (2004). e Values reported by Singh et al (2004).
Ethanol Concentration Measurement Ethanol concentrations were determined using the procedure described by Dien et al (1997). Ethanol concentrations were measured by gas-liquid chromatography using a gas chromatograph (5890A Hewlett Packard, Wilmington, NJ) equipped with an 80/100 Porapak Q column (6 ft × 1.8 in., Supelco, Bellefonte, PA) and a flameionization detector (5890 Hewlett Packard, Palo Alto, CA). Ethanol Yield Calculation Ethanol yields (L/kg of dry fiber) were calculated as final ethanol concentration (% w/v) / (0.79 kg ethanol/L of ethanol) dry fiber initial concentration (% w/v)
Statistical Analyses Analysis of variance (ANOVA) and Tukey’s test (SAS Institute, Cary, NC) were used to compare means of compositions and yields from elusieve processing of three batches. Statistical significance level was 5% (P < 0.05). RESULTS AND DISCUSSION Xylose and arabinose contents were similar for elusieve fiber, wet-milling pericarp, and quick fiber (Table I). Glucose contents were higher for wet-milling pericarp and quick fiber (24.2–28.8%) than for elusieve fiber (17.8–8.4%). Elusieve fiber had lower glucose content probably because starch conversion to ethanol results in low starch content in DDGS. Total carbohydrate content of elusieve fiber was 42.0–48.4%, which was lower than for wetmilling pericarp and quick fiber (51.1 and 58.5%, respectively) because of lower glucose content. Individual carbohydrate content was higher in F1 than in F2 as expected, because F1 (low-yield elusieve fiber) has lower nonfiber content due to lower elutriation air velocities. Repeatability of carbohydrate determination was good (coefficient of variation [COV] ± 5%). F1 and F2 moisture content were 8.0 and 7.5%, respectively (COV ± 5%). Glucose, xylose, and arabinose concentrations in the saccharified sample and ethanol concentration were higher for F1 than for F2, which corresponded to carbohydrate contents in fiber samples (Tables I and II). Repeatability of sugar and ethanol concentration determination was good (COV ± 5%). The maximum ethanol yield using ethanologenic E. coli strains was 51 g of ethanol/100 g of monosaccharides (Dien et al 2005). Based on total carbohydrate contents in F1 and F2, the efficiency of ethanol production was 89–91%. Dien et al (2004) and Singh et al (2004) reported similar efficiencies (86–91%) for ethanol production from wet-milling pericarp and quick fiber. For elusieve fiber, ethanol production efficiency based on sugars in the saccharified slurry was 100– 103% and was similar to efficiency of the control (100%). Fermentations were completed within 50 hr; final ethanol concentrations for F1 and F2 were 17 and 18 g/L, respectively (Fig. 1). Glucose fermented the quickest; concentrations decreased to zero within 24 hr for all fermentations (Fig. 2). There were decreases in xylose and arabinose concentrations as fermentation progressed (Figs. 3 and 4). An important difference between the control and fiber hydrolyzate fermentations was a small amount
TABLE II Composition (wb) of Sugars in Saccharified Slurry, Ethanol Concentration at End of Fermentation, and Efficiency of Fermentation of Fibera Expected Ethanol Conc (% w/v) Based on Sugars in Sample
Glucose
Xylose
Arabinose
Ethanol Conc (% w/v)
Original Sample
Saccharified Slurry
F1 F2 Control
1.39b 1.31c 1.98a
1.50b 1.16c 2.48a
0.73b 0.64c 0.99a
1.84b 1.64c 2.67a
2.07 1.82 2.67
1.84 1.59 na
a
Efficiency (%) Based on Sugars in Original Sample 88.9 90.6 na
Saccharified Slurry 99.9 103.5 99.6
Values are reported as means of three batches and two replicates per batch. Values in the same column followed by same letter are not different (P < 0.05).
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of residual xylose present following fermentation of fiber hydrolyzates. Ethanol yields from elusieve fiber were 0.23–0.25 L/kg of dry fiber (0.027–0.030 gal/lb); similar to ethanol yield from quick fiber 0.15–0.27 L/kg (0.018–0.032 gal/lb) (Dien et al 2004) of dry fiber, and ethanol yield from pericarp fiber of 0.19 L/kg (0.023 gal/lb) (Singh et al 2004) of dry fiber. For every 25.4 kg (1 bu) of corn processed by a dry-grind plant, 10.6 L (2.8 gal) of ethanol is produced and 6.8 kg (15 lb) of DDGS is produced. For every 1 kg of DDGS produced, 0.11 kg of F1 fiber or 0.22 kg of F2 fiber can be separated using the elusieve process. Based on ethanol yields of 0.23 L/kg of F1 and 0.25 L/kg of F2 (dry basis), cellulosic ethanol produced from 25.4 kg (1 bu) corn would be 0.16 L (0.03 gal) and 0.34 L (0.07 gal) for F1 and F2, respectively. This would amount to a 1.2–2.7% increase in ethanol production from the dry-grind plants. Due to the small scale of operation and the stage of technology development for lignocellulosic conversion to ethanol, we expect
that implementation of elusieve fiber conversion to ethanol within a dry-grind plant may not be currently economically feasible. It could be economically feasible to use elusieve fiber along with other feedstock in a plant producing ethanol from cellulosic feedstocks. Revenue obtained by producing ethanol from elusive fiber would be 11¢/kg ($99/ton) (based on average moisture content of 8%, conservative ethanol yield of 0.23L/kg of dry elusieve fiber, and ethanol price of 53¢/L ($2.00/gal). CONCLUSIONS Efficiency of ethanol production from elusieve fiber was similar to that for corn fiber from wet-milling and quick fiber processes. The ethanol yield from elusieve fiber was similar to ethanol yield from quick fiber.
Fig. 1. Fermentation profiles for saccharified fiber.
Fig. 3. Xylose fermentation profiles for saccharified fiber.
Fig. 2. Glucose fermentation profiles for saccharified fiber.
Fig. 4. Arabinose fermentation profiles for saccharified fiber. Vol. 84, No. 6, 2007
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Conversion efficiencies to ethanol were 89–91% and fermentations were completed within 50 hr. Elusieve fiber conversion could result in 1.2–2.7% increase in ethanol production from drygrind plants. Due to the small scale of operation and the stage of technology development for lignocellulosic conversion to ethanol, we expect that implementation of elusieve fiber conversion to ethanol within a dry grind plant may not be currently economically feasible. It could be economically feasible to use elusieve fiber along with other feedstocks in a plant producing ethanol from cellulosic feedstocks. ACKNOWLEDGMENTS Funded in part by Illinois Council for Food and Agricultural Research (C-FAR), Grant Number: IDA CF 04E-072-1. Thanks to Patricia J. O’Bryan, National Center for Agricultural Utilization Research, ARS, USDA, Peoria, IL, for technical assistance in composition analyses and fermentation of fiber samples. LITERATURE CITED Dien, B. S., Hespell, R. B., Ingram, L. O., and Bothast, R. J. 1997. Conversion of corn milling fibrous coproducts into ethanol by recombinant Escherichia coli strains K011 and SL40. World J. Microbiol. Biotechnol. 13:619-625. Dien, B. S., Nagle, N., Hicks, K. B., Singh, V., Moreau, R. A., Tucker, M. P., Nichols, N. N., Johnston, D. B., Cotta, M. A., Nguyen, Q., and
Bothast, R. J. 2004. Fermentation of “quick fiber” produced from a modified corn-milling process into ethanol and recovery of corn fiber oil. Appl. Biochem. Biotechnol. 115:937-949. Dien, B. S., Johnston, D. B., Hicks, K. B., Cotta, M. A., and Singh, V. 2005. Hydrolysis and fermentation of pericarp and endosperm fibers recovered from enzymatic corn dry grind process. Cereal Chem. 82:616620. RFA. 2006. U.S. fuel ethanol production capacity. www. ethanolrfa.org/ eth_prod_fac.html. Renewable Fuels Association: Washington, DC. Schell, D. J., Riley, C. J., Dowe, N., Farmer, J., Ibsen, K. N., Ruth, M. F., Toon, S. T., and Lumpkin, R. E. 2004. A bioethanol process development unit: Initial operating experiences and results with a corn fiber feedstock. Biores. Technol. 91:179-188. Singh, V., Moreau, R. A., Doner, L. W., Eckhoff, S. R., and Hicks, K. B. 1999. Recovery of fiber in the corn dry-grind ethanol process: A feedstock for valuable coproducts. Cereal Chem. 76:868-872. Singh, V., Dien, B. S., Johnston, D. B., Hicks, K. B., and Cotta, M. A. 2004. A comparison between conversion of pericarp and endosperm fiber from corn into ethanol. Proc. ASAE Annual Intnl. Mtg. Paper No. 046056. ASAE: St. Joseph, MI. Srinivasan, R., Moreau, R. A., Rausch, K. D., Belyea, R. L., Tumbleson, M. E., and Singh, V. 2005. Separation of fiber from distillers dried grains with solubles (DDGS) using sieving and elutriation. Cereal Chem. 82:528-533. Tucker, N. J., Jennings, E. W., Ibsen, K. N., Nguyen, Q. A., Kim, K. H., and Noll, S. L. 2004. Conversion of distillers grain into fuel alcohol and a higher-value animal feed by dilute acid pretreatment. Appl. Biochem. Biotechnol. 115:1139-1159.
[Received February 21, 2007. Accepted May 25, 2007.]
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