Ethanol Fermentation of Starch from Field Peas

Cereal Chem. 82(5):554-558 Field peas (Pisum salivum) were evaluated as a potential feedstock for ethanol production. Ground peas were dry-milled and separated into starch, protein, and fibrous fractions by air classification. Starch-enriched fractions prepared from whole peas and dehulled peas contained 73.717c wt and 77.8% wt starch, respectively, a nearly two-fold enrichment compared with whole peas. The fractions were liquefied and saccharified Field peas (Pisum satjvum, dry peas) are consumed as a source of protein for human diets around the world and are used as feed for animals including swine and ruminants (Hickling 2003). Field peas are legumes containing 46% starch, 23% crude protein, and 1.4% oil. The crop is grown in Europe, Asia, and increasingly in North America (Hickling 2003). In the United States and Canada, 502,000 (Larry White, North Dakota Dry Pea and Lentil Office, personal communication) and 3.3 million (Statistics Canada 2004) metric tons, respectively, of field peas were harvested in 2004. Although field peas are mostly fed whole, it is possible to separate the protein fraction, which is most valuable from a feed perspective, from the starch (Vose et al 1976: Tyler et al 1981; Wu and Nichols 2005). Much of the research aimed at utilization of field peas in food and feed applications has targeted the protein and fibrous fractions (Satin 1980: Klein and Raidl 1986; Madsen and Buechbjerg 1987; Viacroze 1987: Richardson and Nickel 1988; Case 2003). However, economics suggest that the starch should also be used. Coproducts that utilize the starch are needed, and some research toward use of field pea starch in industrial and food applications has been conducted (Johnson 1979; Vasanthan et al 1998; Ratnayake et al 2001; Ratnayake et al 2002; Hoover and Zhou 2003). In the United States, production of ethanol for use as a renewable fuel generates a commodity-scale market for starch. Currently, most ethanol in the United States is produced from corn starch by yeast fermentation. However, production of ethanol is expected to increase over the next decade (MacDonald et al 2003) and alternative feedstocks may be useful or necessary (Wisner and Baumel 2004). Field peas are a potential new source of starch for ethanol production. Here, we determined the feasibility of fermenting the starch from field peas to ethanol. MATERIALS AND METHODS Reagents Field peas (P sativum. cv. Eclipse) were grown in 2003 in Ogle County in northern Illinois. Enzymes were supplied by Genencor International (Beloit, WI) and included ct-amylase (Spezyme Ethyl). glucoamylase (Optidex L-400), glucoamylase plus pullulanase Fermentation Biotechnology Research Unit, National Center for Agricultural Utilization Research, ARS-USDA, Peoria, IL 61604. Names are necessary to report factually on available data: however, the USDA neither guarantees nor warrants the standard of the product, and the use of the name by the USDA implies no approval of the product to the exclusion of others that may also be suitable. 2 Corresponding author. Phone: 309-681-6271. E-mail: [email protected] New Crops and Processing Technology Research Unit, National Center for Agricultural Utilization Research, ARS-USDA, Peoria. IL 61604. DOl: 10.1094/CC-82-0554 This article is in the public domain and not copyrightable. It may be freely reprinted with Customary crediting of the source. AACC International Inc., 2005. 554 CEREAL CHEMISTRY using industrial a-amylase and glucoamylase at recommended enzyme loadings. A final ethanol concentration of 11.0% (w/v) was obtained in 48-52 hr. with yields of 0.43-0.48 g of elhanol/g of glucose. Starch present in whole ground peas was also saccharified and fermented, with 97% of the starch fermented when an autoclaving step was included in the liquefaction stage. mixture (Optimax 4060 VHP), protease (GC 106), and cell ulase/xylanase (GC220). Enzyme units are as reported by the supplier. Separation of Field Pea Fractions Field peas were ground in an pin mill (Alpine model 160Z, Augsburg, Germany) at 14,000. 3 x 14,000, or 9 x 14.000 rpm and fractionated in a laboratory model air classifier (Pillsbury. Minneapolis, MN) according to particle size. The classifier was first set at a 15-pm cutpoint to obtain a coarse and a fine fraction. The coarse fraction was then classified successively with 18-, 24-. and 30-l.Lm cutpotnts to obtain four fine fractions (Fractions 1-4) and a coarse residue. Cutpoints were chosen to yield the best enrichment of protein and starch. Fraction 4 has the highest starch content with particle size of 24-30 pm. A complete description of pin-milling and air-classification of field peas was presented earlier (Wu and Nichols 2005). Simultaneous Saccharification and Fermentation Simultaneous saccharitication and fermentation (SSF) of pea starch from whole or dehulled field peas was done in 250-mL Erlenmeyer flasks containing 100 mL of distilled water and 25 g (or in one experiment, up to 40 g) of a starch-enriched field pea fraction (Table I). The sample was adjusted to pH 6.0-6.5 with Ca(OH)2, and cx-amylase (60 U/g of starch) was added. The flasks were covered with foil, heated to 90°C in a water bath, and maintained at that temperature for 60 inin with occasional mixing. The mash was allowed to cool slightly before adjusting to pH 4.0-4.5 with phosphoric acid. Glucoamylase (0.5 U/g of starch) was added along with 0.05% (w/v) (NH4 ) 2SO4, 0.04% Antifoam 289 (Sigma, St. Louis, MO) and corn steep liquor (1% solids final concentration, Grain Processing Corp., Muscatine, IA). In one experiment. cellulase (9 U of cellulase/g of starch plus unspecified xylanase activity) was added in addition to glucoarnylase, or a glucoamylase plus pullulanase mixture (0.6 U of glucoamylase and 1.0 U of pullulanase/g of starch) was added instead of glucoamylase alone. Yeast inoculum (5%, v/v) was added from a preculture of Saccharornvces cerevisiae Y-2034 (ARS Culture Collection, Peoria, IL) grown overnight in a liquid medium containing 5 gIL of yeast extract, 10 g/L of peptone (both from Becton, Dickinson and Co., Sparks, MD), and 50 gIL of glucose. The flasks containing mash, enzymes, and yeast were capped with butyl rubber stoppers, vented with a 22-gauge needle, and incubated at 32°C with gentle shaking. Fermentations were monitored by measuring weight loss reflective of CO2 production, and ethanol concentrations were determined by HPLC analysis of culture supernatants at the end of the fermentations (usually 72 hr). In one time-course experiment, ethanol production was calculated from weight loss and then corrected based on final HPLC-determjned ethanol values; correction was necessary because calculation based on weight loss tends to overestimate ethanol yield somewhat due to loss of other volatiles. All the experiments were conducted in duplicate. Fermentation residuals from each flask were dried at 60°C and ground for compositional analysis. Fermentation of Whole Ground Peas Field peas were ground by processing through a plate mill (model 4E. Straub Co., Croydon. PA) and the whole flour was collected with larger hull pieces included. SSF was conducted as described above for starch-enriched fractions using 500-mL Pyrex bottles instead of flasks. The mixture (25 g of flour in 100 mL of water) was adjusted to pH 6.0-6.5 with Ca(OH) 2, heated to 90°C. and mixed with 200 U of cc-amylase (17 U/g of starch). The bottles were capped and either maintained at 90°C or autoclaved (121°C and 18 psi) for 15 mm, followed in both cases by an additional 400 U of a-amylase (34 U/g of starch) and 60 min of incubation at 90°C. Next, the mash was adjusted to pH 4.0-4.5 with phosphoric acid and 0.05% (w/v) (NH 4) 2SO4 and 0.04% Antifoam 289 were added. Glucoamylase (0.4 U/g of flour or 0.87 U/g of starch) and 5% yeast inoculum were added to initiate ethanol fermentation. In some cases, a glucoamylase/pullulanase mixture was added instead of glucoamylase, along with cellulase (6.2 U/g of flour or 13.5 U/g of starch) or acid-stable protease (1.0 U/g of flour or 2.2 U/g of starch). The threaded caps were replaced with rubber stoppers vented with a 22-gauge needle, and the fermentations were monitored during 72 hr of incubation at 32°C as described above. Residual material was dried at 60°C and ground for compositional analysis. Analytical Procedures Moisture was measured by drying samples at 105°C until samples reached a stable weight. Glucose and ethanol were measured by high-pressure liquid chromatography using an Aminex HPC87H column (Bio-Rad. Richmond, CA) and a refractive index detector. Samples were run at 65°C and eluted at 0.6 mL/min with 5 mM sulfuric acid. Starch content in whole and fractionated field peas was measured enzymatically (Trinder 1969) and free sugars were determined by Official Method 2000.17 (AOAC 2000). Starch in fermentation solids was calculated from glucose liberated by trifiuoroacetic acid treatment (Dien et al 1997). Nitrogen, oil, phosphorous, amino acid, and free sugar content were determined in a commercial laboratory. Protein was calculated from N x 6.25 using AOAC Official Method 976.06 for nitrogen determination. Methods 920.39 and 968.08 were used to determine oil and phosphorous content, respectively (AOAC 2000). Amino acid composition was determined by the method of Gerhke et al (1987). All samples were analyzed in duplicate.