Biomass, Sugar, and Bioethanol Potential of Sweet Corn

GCB Bioenergy (2015) 7, 153–160, doi: 10.1111/gcbb.12136

TECHNICAL ADVANCE Biomass, sugar, and bioethanol potential of sweet corn

JAIME BARROS-RIOS*1 ,ALOIAROMANI´ † ,GILGARROTE† andBERNARDO ORDAS* *Mision Biologica de Galicia (CSIC), Apartado 28, Pontevedra 36080, Spain, †Department of Chemical Engineering, Faculty of Science, University of Vigo (Campus As Lagoas), Ourense 32004, Spain

Abstract Sweet corn is a widely distributed crop that generates agricultural waste without signiﬁcant commercial value. In this study, we show that sweet corn varieties produce large amounts of residual biomass (10 t ha 1) with high content of soluble sugars (25% of dry matter) in a short growing season (3 months). The potential ethanol production from structural and soluble sugars extracted from sweet corn stover reached up to 4400 l ha 1 in the most productive hybrids, 33% of which (1500 l ha 1) were obtained by direct fermentation of free sugars. We found wide genetic variation for biomass yield and soluble sugars content suggesting that those traits can be included as complementary traits in sweet corn breeding programs. Dual-purpose sweet corn hybrids can have an added value for the farmers contributing to energy generation without affecting food supply or the environment.

Keywords: agricultural residue, biofuel, ethanol, lignocellulosic biomass, maize, sugar crops

Received 21 April 2013; revised version received 2 August 2013 and accepted 10 September 2013

fermentation and saccharification, the conversion of lig- Introduction nocellulosic biomass to ethanol requires a mechanical, Manufacture of biofuels from plant sugars may provide physicochemical or biological pretreatment to make the a sustainable alternative to the problems derived from polymers accessible to hydrolytic enzymes (Eggleston the extensive utilization of fossil fuels due to their large et al., 2010; RenYong et al., 2010; Takamizawa et al., availability, renewability, and the possibility of reduc- 2010). These pretreatments have a relatively high cost tion of CO2-emissions (Tilman et al., 2009). Sugars are which has limited the commercialization of biomass eth- mainly present in plant tissues as soluble or free sugars anol (Mosier et al., 2008; Krishnan et al., 2010). Conse- (monosaccharides or disaccharides), reserve polysaccha- quently, the energy efficiency is highest in sugar crops, rides (starch), and structural polysaccharides (cellulose intermediate in starchy crops, and lowest in lignocellu- and hemicelluloses). Structural polysaccharides together losic crops (Hattori & Morita, 2010; Reijnders, 2010). with lignin are the main cell wall components that con- The use of food crops for first-generation bioethanol stitute what is called lignocellulosic biomass. According production generates a direct competition between to the raw material used to obtain ethanol, crops can be energy and food uses. Similarly, dedicated energy crops classified into sugar, starchy, and lignocellulosic crops like switchgrass or giant miscanthus could also compete (Fischer et al., 2010). The main sugar crops are sugar- indirectly with food production if they are grown on cane in tropical areas, and sugar beet and sweet sor- agricultural land (Walsh et al., 2003). Biofuels produc- ghum in temperate areas. Corn and other cereal crops tion using biomass residues of food crops seems an are being used for starch ethanol production because of appealing alternative because it does not compete with their high grain yield and starch accumulation in the food and does not require new inputs for cultivation. grain (Mabee et al., 2011). Any plant can be used to Sweet corn is a widely distributed crop in temperate obtain lignocellulosic ethanol because the vegetative and tropical zones. About one million of hectares are part is made of lignocellulosic biomass. Sugars fermen- annually harvested in the world (USDA, 2010). Sweet tation is required for the three types of crops, while the corn mutation occurs in field corn as a natural sponta- hydrolysis of the polymers (saccharification) is also neous mutation in the genes that control conversion of required for starchy and lignocellulosic crops. Besides sugar to starch inside the endosperm of the corn kernel. There are several mutations responsible for the various types of sweet corn. The sugary1 (su1) mutant has been 1 Present address: Department of Plant Physiology, Umeå Plant traditionally used since pre-Columbus times and con- å å Science Centre, Ume University, Ume 90187, Sweden. tains about 5–10% sugar by weight. The shrunken2 (sh2) Correspondence: Bernardo Ordas, tel. +34 986 854800, fax mutant replaced su1 in processed sweet corn because it +34 986 841362, e-mail: [email protected] has a longer harvest season that allows better response

© 2014 John Wiley & Sons Ltd 153 154 J. BARROS-RIOS et al. to market demands (Marshall & Tracy, 2003). In contrast Five representative ears from each plot were sampled and to field corn that is harvested 150–180 days after plant- shelled to estimate wet grain yield which is one of the main ing (DAP), sweet corn is harvested when the concentra- traits in processing trades. After removing the ears, the stover tion of free sugars in the endosperm is highest (21 days samples consisted of stalks, leaf blades, and leaf sheaths of the five representative plants in each plot. The stover samples after flowering or, ca. 90 DAP) (Marshall & Tracy, 2003). were chopped with a yard waste chipper. Two subsamples of The content of soluble sugars in leaves and stalks of approximately 400 g were obtained from each entry. One sub- field corn has been shown to reach a maximum between sample was frozen, grounded in a knife mill, reground in a 60 and 90 DAP, decreasing sharply at later stages of cyclone-type mill to pass a 0.5-mm screen, and lyophilized for development (Hume & Campbell, 1972; Pordesimo compositional analysis. The second subsample was dried for et al., 2005); these results suggest that a shorter growing 5 days at 80 °C in order to correct the biomass yield to a dry season, like the season of sweet corn, might enable to matter (DM) basis. The biomass DM yield includes only stover use more efficiently the capacity of stover for fuel etha- because the grain is assumed to be used for food. nol production because a higher concentration of soluble sugars may be expected. The objective of this Soluble sugars analyses work was to assess the potential of the residual biomass (stalks and leaves) of sweet corn for bioethanol Sweet corn stover samples were Soxhlet extracted with distilled production. water (liquid to solid ratio of 50 g of distilled water/g of stover DM) for determination of soluble sugars (NREL/TP-510-42619, http://www.nrel.gov/biomass/pdfs/42619.pdf). Monosaccha- Materials and methods rides (glucose, fructose, xylose, mannose, galactose, and arabinose) and a disaccharide (sucrose) in liquors were determined Plant material and experimental design by HPLC-IR (1100 series Hewlett-Packard chromatograph equipped with a refractive index detector) using a CARBOsep Three super-sweet corn hybrids (homozygous for the allele CHO 682 column (Transgenomic, Glasgow, UK). The opera- sh2) and four sweet corn hybrids (homozygous for the allele tional conditions were: distilled water as mobile phase, flow su1) were evaluated at three different locations in 2010 and rate 0.4 ml min 1, and temperature 80 °C as has been previ- 2011. The su1 hybrids were Tablevee and Flavorvee, developed ously described (Rivas et al., 2012). Total soluble sugar concen- by Kerr (1979, 1981), and GH5704 and GH6462 currently com- tration was calculated as the sum of the concentrations of all mercialized by Syngenta AG (Basel, Switzerland). The sh2 monosaccharides and disaccharides. hybrids were GSS1477 and GSS2259 commercialized by Syn- genta AG and SF681 by Semillas Fito (Barcelona, Spain). The locations are located in Northwestern Spain with cool, wet Cell wall polysaccharide and lignin analyses springs and a short summer, conditions similar to those found Polysaccharides and lignin content in stover samples were in many areas of Western Europe. The average monthly tem- measured by means of quantitative acid hydrolysis with perate increased along the growing season from values ranging 72% w/w H SO following standard methods (NREL/ 12–18 °C across sites at sowing to values ranging 18–22 °Cat 2 4 TP-510-42618, http://www.nrel.gov/biomass/pdfs/42618.pdf). harvest. On the other hand, the total rainfall during the grow- Liquors from the quantitative acid hydrolysis were assayed for ing season ranged from 150 to 294 mm across sites. The soil of glucose, xylose, arabinose, and acetic acid by HPLC using a the locations is acid sandy loam with a high content of organic Refractive Index detector and a BioRad Aminex (Hercules, CA, matter. Land was prepared following conventional tillage as USA) HPX-87H column, eluted with 0.01 M H2SO4 at a flow described in Wisconsin Corn Agronomy (2012), while adequate rate of 0.6 ml min 1. The solid residue after hydrolysis was N-P-K fertilization was applied during land preparation considered as Klason lignin. according to soil test recommendations. The experiments were not irrigated. The experimental design was a randomized com- plete block design with three replicates. Each plot had two Potential ethanol production rows spaced 0.80 m apart, and each row consisted of 25 hills spaced 0.21 m apart. Hills were overplanted and thinned after The potential ethanol produced from soluble sugars was calcu- emergence to obtain a final plant density of ca. 70 000 plants lated as follows: 1 ha . We included a border row at the beginning and the end 184 92 92 1 Et ¼ sucrose þ glucose þ fructose of the blocks and discarded the first and last plant of each row PotSS 342 180 180 q for controlling competition. The ears and the stover of all 1 plants of each plot were harvested and weighted at the appro- where EtPotSS is the potential ethanol from soluble sugars (l kg priate time for sweet corn in the area (21 days after flowering). DM), 184/342, 92/180, and 92/180 are the stoichiometric factors Harvesting date varied among plots according to the particular for sucrose (g kg 1 DM), glucose (g kg 1 DM) and fructose flowering date of each plot. The number of commercial ears (g kg 1 DM) conversion into ethanol, respectively, and q is the with at least 16 straight rows and with the kernels filled to the density of ethanol (0.789 kg l 1). Because xylose, mannose, tip of a 20 cm ear was determined (Marshall & Tracy, 2003). galactose, and arabinose concentrations were low (<3gkg 1

DM), the potential ethanol values for these monosaccharides sources of variation were: environments, blocks (environ- could not be accurately determined, and were not included in ments), mutations, varieties (mutations), the interactions the calculation. The potential ethanol obtained from structural between factors, and the experimental error. Environments and polysaccharides was calculated as follows: blocks were considered random effects, while mutations and varieties were considered fixed effects. Environments were con- ¼ð Þ 180 92 þð þ Þ 150 þ 92 1 EtPotP glucan xylan arabinan sidered a combination of locations and years. Least square 162 180 132 180 q means and SE were estimated and provided in figures as where EtPotP is the potential ethanol from polysaccharides appropriate. The difference between sweet corn mutants and (l kg 1 DM), 180/162 is the stoichiometric factor for glucan varieties was evaluated by Fischer protected LDS’s test at (g kg 1 DM) conversion into glucose, 92/180 is the stoichiome- P < 0.05 level. tric factor for glucose conversion into ethanol, 150/132 is the stoichiometric factor for xylan or arabinan (g kg 1) conversion into xylose or arabinose, 92/180 is the stoichiometric factor for Results and Discussion xylose or arabinose conversion into ethanol and q is the density of ethanol (g l 1). Total potential bioethanol conversion (l t 1) Biomass production was calculated as the sum of ethanol obtained from both solu- Stover DM yield ranged from 6.4 to 9.8 t ha 1, while ble sugars and structural polysaccharides fractions. Potential the average height was higher than 2 m for all hybrids bioethanol yield (l ha 1) was calculated for each genotype on 1 (Fig. 1a). The highest yielding hybrid (GSS2259) the basis of the corresponding biomass DM yield value (t ha ). exceeded the residual biomass yield of 49 elite cultivars of field corn (i.e., corn without a sweet corn mutant) Inoculum and fermentation of soluble sugars evaluated for ethanol production (Lorenz et al., 2009). The strain used in the fermentations was Saccharomyces cerevisi- This is particularly interesting because some of the culti- ae CECT-1170 obtained from the Spanish Collection of Type vars evaluated by Lorenz et al. (2009) had been selected Cultures (Valencia, Spain). Cell cultures were grown for 24 h at for their forage yield. The residual biomass of GSS2259 32 °C with shaking at 200 rpm. for 24 h in a medium contain- (9.8 t ha 1) was in the range of the whole-plant biomass ing 10 g glucose l 1 5 g peptone l 1, 3 g malt extract l 1, and observed in some sugar crops as sugar beet (10 t ha 1) 3 g yeast extract l 1. Biomass concentrations in the media were (FAO, 2012), or sweet sorghum (5–20 t ha 1) (Soileau & measured on a dry cell weight basis. The fermentation experi- Bradford, 1985; Tamang et al., 2011). The length of the ments were carried out in 100 ml Erlenmeyer flasks placed in growing season of the sweet corn hybrids (3 months, = ° an orbital shaker at 100 rpm (pH 5) and 32 C. The fermenta- Fig. 1b) was much lower than the length of the grow- tion media were prepared by mixing 36 ml of the Soxhlet ing season of sugar beet (6–10 months) and sugarcane extracted liquid with soluble sugars extracted from stover (8–24 months) (Reijnders, 2010). The short period from (sucrose, glucose, and fructose), 2 ml of nutrients (5 g peptone l 1, 3 g malt extract l 1 and 3 g yeast extract l 1) and 2 ml of sowing to harvest observed in sweet corn allows the inocula (leading to an initial yeast cell concentration of design of rotation systems with either other vegetables 0.535 g l 1). The experiments were started when the inocula to increase the efficiency of land use, or with legumes was added. The extracted liquor was sterilized by ultrafiltration that fix nitrogen to reduce the use of fertilizers. using Steritop filter units (Millipore, Billerica, MA, USA). The nutrients were autoclaved (121 °C, 20 min) separately from extracted sugars. At given fermentation times (0, 0.5, 1, 1.5, 2, 3, Stover composition 4, and 6.5 h) samples (0.5 ml) were withdrawn and centrifuged The concentration of soluble sugars of the sweet corn (3400 g, 10 min). Monosaccharides and disaccharides in liquors hybrids (186–261 g kg 1 DM, Fig. 2a) was in the range were determined by HPLC-IR as previously described. Ethanol of the sweet sorghum varieties evaluated by Zhao et al. was determined by a BioRad Aminex HPX-87H column, eluted ° 1 (2009), but lower than the concentration of commercial with 0.01 M H2SO4,T50 C, and at a flow rate of 0.6 ml min . The ethanol conversion efficiency was calculated as follows: sugarcane or sugar beet varieties (Eggleston et al., 2010). In contrast to sugarcane, which can only grow in CE ¼ 100 [ethanol]. tropical or subtropical areas, sweet corn can be culti- EtPotSS vated in a wide range of environments. In addition, where CE is the ethanol conversion efficiency (%), [ethanol] is the highest ethanol concentration (l kg 1) achieved in the sweet corn is very adaptable to diverse soil conditions, while sugar beet requires a pH above 6.5 for an experiment and EtPotSS is the potential ethanol from soluble sugars (l kg 1). adequate performance (Eggleston et al., 2010). The concentration of glucan (200–280 g kg 1 of DM), xylan (130–150 g kg 1 of DM), and arabinan(19 g kg 1 of Statistical analysis DM) in sweet corn stover (Fig. 2b) was lower than the Combined analyses of variance over locations were computed concentration of those elements in field corn stover using the MIXED procedure of SAS (SAS Institute, 2008). The harvested at grain physiological maturity (about

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(b)

Fig. 1 Mean comparisons for plant height, wet grain and biomass yield (a), days from planting to harvest, and commercial ears per hectare (b) among six sweet corn hybrids grown at three locations and 2 years in Spain. Error bars indicate the standard error of the mean. Means within a trait not sharing a common letter (a–c) are signiﬁcantly different (P < 0.05).

300–350, 170–200 and 26–29 g kg 1 of DM) (Lorenz quality. The concentration of Klason lignin of the sweet et al., 2009; Lorenzana et al., 2010).The biomass collec- corn hybrids (about 150 g kg 1 of DM) was lower than tion at early stages of development could explain the the concentration found in normal field corn hybrids high concentration of soluble sugars observed in sweet (about 170 g kg 1 of DM) (Lorenzana et al., 2010). The corn stover which later on will be probably polymer- lignification of the cell wall was probably not completed ized into carbohydrates (Jung, 2003; Pordesimo et al., when the biomass of sweet corn hybrids was collected 2005). At difference of field corn, the biomass may be (Jung, 2003). Although a reduced lignin content could harvested early in sweet corn because the grain is suppose a detrimental impact on plant (Pedersen et al., adequate for human consumption at 21–23 days after 2005; Lorenz et al., 2009), sweet corn hybrids escape flowering. from this problem because biomass is collected when Lignin limits enzyme accessibility to cellulose increas- the plants are still green. ing the cost of the pretreatment which is the single most expensive process step in making biomass ethanol Bioethanol production (Wyman, 2003). For this reason, obtaining genotypes with a low concentration of lignin is one of the main tar- Bioethanol production from free or soluble sugars gets for the genetic improvement of the stover quality requires less processing and is more efficient than bio- for lignocellulosic ethanol (Lewis et al., 2010; Lorenz ethanol production from polysaccharides. The potential et al., 2010). However, the reduction of lignin might ethanol that can be obtained from the soluble sugars of have undesirable agronomic effects such as an increase the sweet corn hybrids was estimated to range from in lodging and drought susceptibility which have to be 0.123 to 0.172 l kg 1 (Fig. 3a), and the ethanol conver- taken into account in the genetic improvement of stover sion efficiency (CE) of the most productive sweet corn

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Fig. 2 Mean comparisons for soluble sugars (a), and structural polysaccharides and lignin (b) in the stover of six sweet corn hybrids grown at three locations and 2 years in Spain. Error bars indicate the standard error of the mean. Means within a trait not sharing a common letter (a–c) are significantly different (P < 0.05). hybrids GSS2259 and Flavorvee was 99 and 100%, production from lignocellulosic biomass of the sweet respectively. All sweet corn hybrids evaluated showed corn hybrids was about 0.3 l kg 1 of DM (Fig. 3a) or CEs >93% (Fig. 3b) indicating that real bioethanol pro- 2500 l ha 1. The overall bioethanol production obtained duction using the conditions of the experiments indi- from both soluble sugars and structural polysaccharides cated in the section Inoculum and fermentation of fractions in the most productive sweet corn hybrids soluble sugars was close to potential estimations. Con- (GSS2259 and Flavorvee) was estimated to be, ca. centrations of ethanol close to the maximum were 4600 l ha 1 (Fig. 3a) which is in the range of the total achieved at 3 h of fermentation for all genotypes, except bioethanol production from structural sugars in the top Flavorvee (4 h), probably due to the high concentration field corn hybrids (Lorenz et al., 2009). of sucrose of this genotype. Our results suggest that it is Some properties of sweet corn biomass are compara- possible to obtain about 1500 l of ethanol per hectare by ble to those found in feedstock used or proposed for simple fermentation of soluble sugars of the sweet corn sugar and/or lignocellulosic ethanol production. Tech- stover. Utilization of soluble sugars involved about 25% nical challenges of using sugar crops, as sweet sorghum, of stover biomass DM yield meaning that 75% of total for biofuels, are the fast sugar degradation during stor- biomass remained unused. This lignocellulosic biomass age and the short harvest period (Wu et al., 2010). Some can be burnt in boilers for steam and electricity genera- procedures to increase the storage stability have been tion or used to produce bioethanol increasing the over- proposed: producing thickened juice by evaporation, all efficiency of the process. The potential bioethanol applying formic acid under anaerobic conditions, and

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Fig. 3 Mean comparisons for potential bioethanol conversion (l kg 1) obtained from soluble sugars and structural polysaccharides, and total potential bioethanol yield (l ha 1) of six sweet corn hybrids grown at three locations and 2 years in Spain (a). Error bars indicate the SE of the mean. Means within a trait not sharing a common letter (a–c) are significantly different (P < 0.05). Fermentation kinetics for bioethanol conversion from soluble sugars by sweet corn mutant type is shown (b); CE is the ethanol conversion efficiency. air-dried until constant weight at 85 °C (Matsakas & (Cardona et al., 2010). Alternatives to the expensive Christakopoulos, 2013; Rohowsky et al., 2013). On the detoxification procedures could be the development of other hand, the length of the processing season of a bio- microorganisms that tolerate the inhibitors (Tomas-Pej o energy factory could be lengthened by integrating the et al., 2010) or thermo-mechanical extrusion pretreat- residuals of sweet corn in the same factory with residu- ment (Heredia-Olea et al., 2013). als of other sugar crops as sugarcane or sugar beet The underutilized residuals that are indirectly gener- (Eggleston et al., 2010). In the sugar crops the soluble ated during the cultivation of sweet corn with the aim sugars and the structural sugars, after being separated, of grain consumption can produce biofuels without are usually subjected to independent processes for bio- threatening food supplies or the environment since sto- ethanol production (Dias et al., 2009; Shen et al., 2012) ver biomass can be used once the grain has been har- which adds complexity and cost to processing. Matsa- vested for food production, and so two commercial kas & Christakopoulos (2013) propose the optimization products are obtained with the same agricultural of ethanol production from sweet sorghum without first inputs. However, the stover removal has a detrimental extracting soluble sugars by including an enzymatic liq- effect on soil organic matter and erosion (Mann et al., uefaction step. Fermentation and hydrolysis inhibitors, 2002) that has to be considered when assessing the such as furfural and acetic acid, can be generated dur- exploitation of the stover for bioenergy. Blanco-Canqui ing the pretreatment of lignocellulosics. Several detoxifi- & Lal (2009a), based on several studies, conclude that cation methods have been suggested: neutralization, the amount of crop residue that can be removed with- overliming, adsorption with activated charcoal, etc. out causing serious impact on soil is site-specific.

Changes in crop production practices would be Acknowledgements required to, at least partially, offset the negative impacts This study was supported by funds from the Xunta de Galicia of stover removal on soil characteristics (Archer & (project 09MRU033403PR). The authors thank Syngenta SA and Johnson, 2012). In addition, the removal of the stover Semillas Fito for providing seed of sweet corn hybrids. We reduces the return of mineral matters to soil which thank Javier Betran and Serge Dubos from Syngenta SA for obliges to increase chemical fertilization (Blanco-Canqui their help in the development of the work. J. Barros-Rios & Lal, 2009b). acknowledges a grant from the Ministry of Science and Innova- tion (Spain). B Ordas acknowledge a contract from the Xunta de Galicia (Program Parga Pondal). 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