Characterization of Normal and Waxy Corn Starch for Bioethanol

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Characterization of normal and waxy corn starch for bioethanol production Hanyu Yangcheng, Hongxin Jiang, Michael Blanco, and Jay-lin Jane J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/jf305100n • Publication Date (Web): 16 Dec 2012 Downloaded from http://pubs.acs.org on December 26, 2012

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Journal of Agricultural and Food Chemistry

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Characterization of Normal and Waxy Corn Starch for Bioethanol Production

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Hanyu Yangcheng,† Hongxin Jiang,† Michael Blanco, †,‡ Jay-lin Jane*,†

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†

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USA;

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‡

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University, Ames, IA 50011, USA

Department of Food Science and Human Nutrition, Iowa State University, Ames, IA 50011

USDA-ARS Plant Introduction Research Unit, and Department of Agronomy, Iowa State

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*Corresponding author:

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Tel: 01 515-294-9892; Fax: 01 515-294-8181;

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E-mail address: [email protected] (J.Jane)

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ACS Paragon Plus Environment


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ABSTRACT Objectives of this study were to compare ethanol production between normal and

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waxy corn using a cold-fermentation process and to understand effects of starch structures

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and properties on the ethanol production. Ethanol yields positively correlated (p37) of waxy corn starch negatively correlated with

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the starch-hydrolysis rate and the ethanol yield. These results indicated that starch structures

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and properties of the normal and waxy corn had significant effects on the ethanol yield using

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a cold-fermentation process.

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Keywords: Bioethanol; cold fermentation; starch-ethanol conversion efficiency; normal corn;

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waxy corn; starch.

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INTRODUCTION The United States of America is the largest petroleum consuming country in the

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world, and large portions of the petroleum, peaking in 2005 at over 60%, has been imported

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from foreign countries.1 The nation's heavy dependence on foreign oil supply raised concerns

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of its energy security, which led to a huge surge of interest in alternative liquid fuels produced

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from renewable sources. Ethanol is an attractive bio-renewable fuel because it can be

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produced from starchy or sugar-containing crops.2 Representing 10% of the nation's motor

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fuel supply in 2011, fuel ethanol has reduced the nation’s net import of foreign petroleum to

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45% in 2011.3

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Ethanol is produced almost exclusively from corn in the United States.2, 4 Production

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of ethanol from corn requires hydrolysis of starch to glucose, and glucose is then fermented

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by yeast to produce ethanol because yeast cannot utilize starch directly.5 A conventional

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process for ethanol production is to gelatinize starch in dry-grind grain, and the gelatinized

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starch is hydrolyzed to dextrin using thermal-stable α-amylases (liquefaction). The resulting

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dextrin dispersion is cooled to 60 °C and then saccharified with amyloglucosidase to produce

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glucose that is the major substrate for ethanol fermentation. In this process, energy used to

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cook starch increases the production cost and decreases the energy return of ethanol.6, 7 In

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addition, amylose-lipid complex and retrograded starch formed in the gelatinized starch are

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resistant to enzyme hydrolysis and reduce the amount of fermentable sugar production and,

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thus, decrease the ethanol yield.6, 8

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To reduce energy costs and improve the net energy yield, raw-starch (cold)

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fermentation has been developed.7, 9 In a cold-fermentation process, raw starch is hydrolyzed

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by granular-starch hydrolyzing enzymes to produce fermentable sugars without prior cooking

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and liquefaction, and the sugars are fermented simultaneously by yeast to produce ethanol.

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Compared with the conventional process, cold fermentation effectively decreases the energy

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input, simplifies the process, reduces osmotic stress to yeast, and minimizes occurrence of the

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Maillard reaction, amylose-lipid complex and retrograded-starch during and after the heating

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process.6, 9, 10 Furthermore, cold fermentation does not require a large capital investment and

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is more feasible for small-scale or on-farm ethanol production.7 Raw-starch hydrolysis of the

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cold-fermentation process provides another advantage of displaying a low viscosity of the

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slurry. Thus, it allows a greater solids-loading and a larger production-capacity of the

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equipment for fermentation.9 An industrial process of cold fermentation using raw-starch

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hydrolyzing enzymes was recently developed.10 This innovative technique for ethanol

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production showed an improved ethanol yield and produced a co-product, distiller's dry

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grains (DDG), with better qualities than the conventional process. The protein in the DDG

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produced using the cold fermentation is not denatured and, thus, retains good properties for

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other applications, such as biodegradable plastics.11

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Aiming to improve the ethanol yield, numerous studies have been conducted to

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evaluate the ethanol-fermentation performance using a variety of waxy and non-waxy

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cultivars.12-15 Ondas et al. reported that, using a conventional fermentation process with

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cooking, waxy wheat and corn showed greater starch-ethanol conversion efficiencies than

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non-waxy counterparts.12 Wu et al. reported a negative correlation between the amylose

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content of starch and starch-ethanol conversion efficiency using a conventional process.13

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Employing isolated maize starch with different amylose contents, Sharma et al. have shown

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that corn starch with 0% amylose produced ethanol at a higher yield than corn starch with

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30% or 70% amylose using both conventional and cold-fermentation process.14, 15 These

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studies showed that waxy cultivars, containing little amylose in the starch, had a better

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ethanol-fermentation performance than non-waxy cultivars. Nevertheless, there have been

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few studies to date comparing ethanol production of dry-grind waxy corn with normal corn

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using a cold-fermentation process and reporting effects of starch structures and properties,

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including amylopectin structures and starch thermal properties, on the ethanol yield. This study aimed to compare ethanol yields and starch-ethanol conversion efficiencies

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of uncooked waxy and normal corn, using a cold-fermentation process. Structures and

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properties of the waxy and normal corn starches were characterized to reveal effects of starch

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structures and properties on the ethanol yield. Results obtained from this study can be applied

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to improve ethanol production using cold fermentation by selecting corn varieties with starch

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of desirable structures and properties.

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MATERIALS AND METHODS Materials. Four normal corn lines (08GEM04701-4704) and eight waxy corn lines

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(08GEM05036-5040, 5042-5044) were developed by the USDA-ARS Germplasm

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Enhancement of Maize (GEM) Project. The inventory numbers are used throughout the

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manuscript, which indicate the seed sources used. The corn lines were selected to represent

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corn of racial diversities comprising germplasm from seven races and three tropical hybrids

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originated from seven countries. All the corn lines were grown at the North Central Regional

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Plant Introduction Station farm (Ames, IA) in both 2009 and 2010 crop seasons. Pedigree,

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racial background, and geographic origin of each line are included in Supporting Information.

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Ethanol Red dry yeast (>20×109 living cells/g) was obtained from Lesaffre yeast corporation

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(Milwaukee, WI). Lactrol (virginiamycin) was from Phibro Animal Health Co. (Ridgefield,

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NJ). Isotab (hop acid) was from Beta Tec Hop Products (Washington, D.C.). Novozyme 5009

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raw-starch hydrolyzing enzymes containing a mixture of fungal α-amylase and

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amyloglucosidase were from Novozyme (Franklinton, NC). Pseudomonas isoamylase (EC

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3.2.1.68, 1000U/mL) and total starch kit were from Megazyme International Ireland

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(Wicklow, Ireland). All other chemicals were reagent grade and were obtained from either

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Sigma-Aldrich Co. (St. Louis, MO) or Fisher Scientiﬁc (Pittsburgh, PA) and used without

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further purification. Dry grinding of corn kernels. Maize kernels of GEM lines were dried to

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approximately 12% moisture and were then ground using a Cyclone Mill (UDY Corp., Fort

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Collins, CO), screening with a steel sieve of 0.5mm pore size.

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Cold fermentation. Dry-grind corn (35g, dry base, db) was placed in a polypropylene

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bottle (125mL, prior autoclave-sterilized). Distilled water containing liquid urea (0.03%,

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w/w), lactrol (2ppm), isotab (40ppm) and acetate buffer (10mM, pH4.2) was added to the

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dry-grind corn to make a mash of 100g total weight (35% solid content). The mash was then

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mixed with 0.5g dry yeast and raw-starch hydrolyzing enzymes (0.46%, v/w of dry-grind

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corn as recommended by the manufacturer) (Novozyme). The samples were incubated in a

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shaker incubator at 29 °C and 160rpm for 96h. Aliquots (8.0mL) were taken from the

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fermentation broth at 96h fermentation time and centrifuged at 7,010g for 10min. The

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supernatant was filtered through a nylon membrane filter with 0.2µm pore size. The ethanol

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concentration was analyzed using a HPLC system consisting of a Prostar 210 pump (Varian,

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Walnut Creek, CA), an injection valve (model 7725i) (Rheodyne, Oak Harbor, WA), and a

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Prostar 355 refractive index detector (Varian, Walnut Creek, CA), following the procedures

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previously reported.16

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Ethanol conversion efficiency was calculated using the equation: conversion

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efficiency (%) = 100% × ethanol yield (w/w)/theoretical yield of ethanol. The theoretical

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yield of ethanol is 56.73 g ethanol/100 g starch, which is calculated on the basis of 1 g of

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starch being hydrolyzed into 1.11 g of glucose, and 1 mol of glucose fermented to produce 2

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mol of ethanol.4

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Starch content assay. The starch content of the corn grain was analyzed using a total

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starch kit (Megazyme International, Wicklow, Ireland), following the AACC 76.13 method.17

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Starch isolation by wet-milling. Starch was isolated from corn kernels using a wet-

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milling method.18

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Starch hydrolysis of uncooked isolated-starch and dry-grind grain. Dry-grind

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corn containing 200mg starch (db) or isolated starch (200mg, db) suspended in a sodium

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acetate buffer (20mL, 10mM, pH4.2) was pre-incubated at 29 °C for 30min. Novozyme 5009

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raw-starch hydrolyzing enzymes (0.67%, v/w of starch) were then added, and the incubation

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continued at 29 °C with constant shaking at 160rpm for 96h. Aliquots (0.1mL) of the

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hydrolysate were withdrawn at different time intervals and were mixed with 1mL of 66%

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ethanol (v/v). The mixture was centrifuged at 6,600g for 5min, and the supernatant was

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collected. The glucose content in the supernatant was determined using a glucose

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oxidase/peroxidase (GOPOD) assay (Megazyme International, Wicklow, Ireland).

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Amylose content of starch. Amylose contents of waxy and normal corn starches

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were determined using Sepharose CL-2B gel-permeation chromatography (GPC) followed by

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a total carbohydrate analysis and using an iodine potentiometric-titration method.18, 19

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Branch-chain-length distributions of amylopectin. Amylopectin of normal corn

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starch was separated from amylose and collected using a GPC column packed with Sepharose

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CL-2B gel (Pharmacia Inc., Piscataway, NJ).18 The amylopectin was debranched using

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isoamylase (Megazyme International Irelands, Wicklow, Ireland) following the methods

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previously reported.20 The waxy corn starch was debranched without separation of the

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amylopectin from amylose by GPC because there was no detectable amylose in the GPC

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profile. The debranched sample was labeled with 8-amino-1,3,6-pyrenetrisulfonic acid

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(APTS) and analyzed using a fluorophore-assisted capillary electrophoresis (P/ACE MDQ)

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(Beckman Coulter, Fullerton, CA) following the methods previously reported.21, 22

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Thermal properties of starch. Thermal properties of the isolated starch were

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analyzed using a Diamond Differential Scanning Calorimeter (Perkin-Elmer, Norwalk, CT).19

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Starch gelatinization onset (To), peak (Tp), and conclusion temperatures (Tc), and enthalpy

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change (∆H) were obtained using the Pyris software (Perkin-Elmer). The gelatinized starch

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samples were stored at 4 °C for 7 days and then analyzed using the same parameters for

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measuring their percentages retrogradation. The percentage retrogradation was calculated

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using the equation: Retrogradation (%) = 100% × ∆H of dissociation of retrograded

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starch/∆H of starch gelatinization.

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Statistical analysis. The experimental design of this study was a randomized

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complete block design with the corn variety (normal or waxy) as a fixed effect and the crop

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season (year 2009 or 2010) as a random block effect. The model statement for the analyses

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was: Yjk = µ + Vj + Sk + εjk, where Yjk = the dependent variable, µ = overall mean, Vj = corn

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variety (j= normal or waxy), Sk = crop season (k= year 2009 or 2010), and εjk = residual error.

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Data was analyzed using the PROC MIXED procedure of SAS 9.2 (SAS Institute, Inc., Cary,

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NC). Average values or means of values presented in the text are least squares means (LSM)

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if without further indication. Differences of LSM between normal and waxy corn lines were

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analyzed using Tukey’s procedure. The normality of data for each variable was investigated

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using the PROC UNIVARIATE procedure. Because all the variables except data of total

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starch content were not normally distributed (data not shown), Spearman correlation test was

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used to analyze correlations between the ethanol yield, total starch content, and

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physicochemical properties of the starch. Statistical significance was declared at p

Characterization of Normal and Waxy Corn Starch for Bioethanol

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