Use of tropical maize for bioethanol production. Ming-Hsu Chen, Prabhjot Kaur, Bruce Dien, Frederick Below, Michael L. Vincent & Vijay Singh

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1 Use of tropical maize for bioethanol production Ming-Hsu Chen, Prabhjot Kaur, Bruce Dien, Frederick Below, Michael L. Vincent & Vijay Singh World Journal of Microbiology and Biotechnology ISSN DOI /s

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3 DOI /s ORIGINAL PAPER Use of tropical maize for bioethanol production Ming-Hsu Chen Prabhjot Kaur Bruce Dien Frederick Below Michael L. Vincent Vijay Singh Received: 20 December 2012 / Accepted: 11 March 2013 Ó Springer Science+Business Media Dordrecht 2013 Abstract Tropical maize is an alternative energy crop being considered as a feedstock for bioethanol production in the North Central and Midwest United States. Tropical maize is advantageous because it produces large amounts of soluble sugars in its stalks, creates a large amount of biomass, and requires lower inputs (e.g. nitrogen) than grain corn. Soluble sugars, including sucrose, glucose and fructose were extracted by pressing the stalks at dough stage (R4). The initial extracted syrup fermented faster than the control culture grown on a yeast extract/phosphate/sucrose medium. The syrup was subsequently concentrated times, supplemented with urea, and fermented using Saccharomyces cerevisiae for up to 96 h. The final ethanol concentrations obtained were 8.1 % (v/v) to 15.6 % (v/v), equivalent to % of the theoretical yields. However, fermentation productivity decreased with sugar concentration, suggesting that the yeast might be osmotically stressed at the increased sugar concentrations. These results provide in-depth information for utilizing tropical maize syrup for bioethanol production that will help in tropical maize breeding and development for use as another feedstock for the biofuel industry. M.-H. Chen P. Kaur V. Singh (&) Department of Agricultural and Biological Engineering, University of Illinois at Urbana-Champaign, Urbana, IL, USA vsingh@illinois.edu B. Dien BioEnergy Research Unit, Agricultural Research Service, United States Department of Agriculture, National Center for Agricultural Utilization Research, Peoria, IL, USA F. Below M. L. Vincent Department of Crop Sciences, University of Illinois at Urbana-Champaign, Urbana, IL, USA Keywords Introduction Tropical maize Fermentation Ethanol The United States consumed 4.9 billion bushels of corn for the bioethanol industry that produced approximately 13.2 billion gallons of ethanol (EIA 2010). Grain corn is the major feedstock for ethanol production in the United States (Wang et al. 2005). In contrast, ethanol within Brazil is produced largely from sugar cane. Ethanol originating from sugar cane is less expensive in part because soluble sugars are directly extracted from the stalks in contrast to corn where the starch needs to be liquefied and hydrolyzed prior to fermentation by Saccharomyces cerevisiae yeast (Quintero et al. 2008). S. cerevisiae hydrolyzes sucrose, from sugar cane, to glucose and fructose for fermentation by producing an extracellular invertase (Novick and Schekman 1979). While sugar cane production is unsuitable for temperate climates, recent studies have focused on sweet sorghum (Laopaiboon and Laopaiboon 2012; Liu and Shen 2008), which also accumulates soluble sugars in its stalks, as an alternative to grain corn for ethanol production. Tropical maize is a hybrid bred by crossing tropical and temperate-adapted cultivars (White et al. 2011). When grown in middle latitudes, it accumulates large amounts of extractable sugars (sucrose, glucose, fructose), produces larger amounts of plant cell wall biomass, and generates little or no grain. From its temperate parent, it gains the ability to grow in the US Corn Belt, as well as accessing all the genetic benefits of modern commercial corn seed, including a higher stress tolerance and lower susceptibility to diseases and insect pests. Compared with commercial feed corn hybrids, tropical maize requires 50 % less

4 nitrogen fertilizer and preserves the benefits of a broad cultivation range, possibilities of further genetic improvement, and an established production system (White et al. 2011). Owing to its high sugar content, this creates the possibility of using tropical maize in the US to emulate the sugar cane to ethanol process of Brazil. Tropical maize also produces 20 metric tons of biomass per hectare on a dry weight basis, two times greater than grain corn. This production yield also compares favorably with the reported yields (15 tons/ha) for switchgrass (Panicum virgatum) (Parrish and Fike 2005) and miscanthus (Miscanthus 9 giganteus) (20? dry tons per hectare) (Lewandowskia et al. 2000). An advantage of tropical maize is that a great number of farmers are also experienced and equipped for cultivating corn. Although the costeffectiveness of cellulosic bioethanol is still several years away, the combined theoretical ethanol yield of sugar and lignocellulosic bioethanol of tropical maize is 25 % greater than for commercial corn (White et al. 2011). The energy balance ratio (energy invested vs. energy returned) for corn grain ethanol is 2.3 (Shapouri et al. 2008), but the energy balance ratio of tropical maize approaches ten (White et al. 2011), which is similar to sugarcane and sweet sorghum (Goldemberg 2007). For farmers, tropical maize represents a viable cropping alternative that will fit into most Midwest cropping rotations and provides both low risk and high potential economic return, depending upon markets (White et al. 2011). There is negligible information on fermentation of tropical maize juice to bioethanol. In this study, our objective was to determine the ethanol fermentation potential and properties of various pretreatments of juice obtained from pressed tropical maize. Materials and methods Raw materials Tropical maize (Zea mays L.) plants were grown in 2010 on the experimental farm of the Department of Crop Sciences, University of Illinois at Urbana-Champaign. The plants were harvested at dough stage (R4) and the juice in the corn stalk was extracted using a UnIcorn press which was custom made to extract stalk juice for sugar analysis and fermentation studies. The basic design of the unit includes four pairs of rollers (20.3 cm in diameter and 40.6 cm in length) that progressively squeeze the stalks and drag them through the machine; the juice is collected in a series of drip pans. A hydraulic jack and a leaf spring system apply pressure to the stalks; 82,737 kpa was found to be optimum for extracting juices from stalks. The machine is equipped with a 17.9 kw Honda engine (GX670, Honda, Alpharetta, GA) and can simultaneously press 8 stalks. The syrup was filtered through no. 2 Whatman filter paper (Whatman, Buckinghamshire, UK) and stored at 4 C. Media preparation and fermentation condition A subset of the initial filtered syrup was autoclaved at 121 C for 15 min at kpa, and then adjusted to a ph of 4.5 using 2 N HCl. The three different syrup treatments (1) initial syrup, (2) autoclaved syrup, and (3) autoclaved and ph adjusted syrup were tested for fermentation rates by comparing them to a standard yeast extract/phosphate/ sucrose (YEPS) medium which had similar sugar composition (containing 10 % sucrose, 2.5 % glucose, 0.75 % fructose, 1.0 % yeast extract, and 0.2 % KH 2 PO 4 at ph 4.5). The sugar contents in YEPS were selected based on HPLC quantification of soluble sugars in initial maize syrup. The original formula of YEPS was reported in Bulawayo et al. (1996). The autoclaved syrup was then concentrated 1.25, 1.5, 1.75 and 2.25 times using a lyophilizer (Freezone 6, Labconco, Kansas city, MO) in order to promote increased final ethanol concentration. The fermentation reaction was conducted at 32 C and ph 5.7. Thirty ml aliquots of syrup were fermented in 50 ml Erlenmeyer flasks using urea as nitrogen source (0.5 % w/v). Commercial ethanol red yeast (Lesaffre yeast corporation, Milwaukee, WI) was used for ethanol production. Ethanol red yeast is a Saccharomyces cerevisiae strain which is specially developed for fuel ethanol industry (Offman et al. 2008). According to manufacturer s specification this strain is suited for high sugar and gravity mediums and displays good tolerance to ethanol and temperature changes. The yeast culture was prepared by incubating 5 g of ethanol red yeast added to 25 ml of deionized water in a 32 C shaking water bath (120 rpms) for 25 min. Syrup was fermented by adding yeast culture at a concentration of 0.5 ml/100 ml syrup. Fermentation was conducted in a shaking water bath for 96 h at 120 rpm and sampled periodically for ethanol and other metabolites. Sugar, ethanol and metabolites analysis At each sampling point, 1 ml aliquots were removed from each of the fermentation treatments and filtered through a 0.2 lm membrane (Syringe filter, 13 mm Nylon, Waters Corporation, Ann Arbor, MI). The aliquots were then analyzed for ethanol, sucrose, glucose, fructose, glycerol, lactic acid and acetic acid using high performance liquid chromatography (HPLC) fitted with two different ion exclusion columns (Biorad Aminex HPX-87H & HPX-87P, Biorad, Hercules, CA) coupled with a refractive index detector (Model 2414, Waters Corporation, Milford, MA).

5 The HPLC samples were eluted with M H 2 SO 4 at 50 C with a flow rate of 0.6 ml/min through the HPX-87H column then eluted with B-pure purified water (Barnstead Thermolyne Corp., Dubuque, IA) at 85 C with a flow rate of 0.6 ml/min through the HPX-87P column. Experimental design and statistical analysis All experiments were performed in duplicates and expressed as mean ± standard deviation. Statistical analysis was done to compare means using Fisher LSD procedure with a = 0.05 using SAS software (version 9.3, SAS, Cary, NC). Results and discussion Fermentation from maize syrup and YEPS medium The initial freshly extracted syrup from the tropical maize plant was found to be composed of 90.2 g/l sucrose, 22.5 g/l glucose and 18.2 g/l fructose (Fig. 1). The initial syrup was fermented at three conditions as extracted, autoclaved, and autoclaved with ph adjustment (Fig. 2). Compared with the YEPS standard medium, formulated to a similar sugar concentration, the tropical maize syrup, regardless of pretreatment, had two times faster fermentation rates than YEPS. This increased fermentation rate of the maize stalk syrup suggests that it contained additional nutritional factors which promoted fermentation (Fig. 2). The autoclaved syrup had a ph value 5.7, which is more neutral than typical yeast fermentations, but acidifying the syrup to ph to 4.5 prior to fermentation had no effect on rate or yield (Fig. 2). The untreated initial syrup culture fermented slower than the other cultures but eventually achieved the same final ethanol concentration. An absence of residual sugars in any of the cultures suggests that syrup is readily fermented by yeast (Figs. 4, 5, 6). Wang and Blascheck (2011) reported that tropical maize syrup contains many minerals and nutrients (in g/l, N: 0.64; P: 0.13; K: 1.01; Na: ; Ca: 0.16; Mg: 0.27; Fe: 0.042; Mn: 0.011; S: 0.034) that may promote yeast growth and help in increasing rate of fermentation. Wu et al. (2010) reported that heating sweet sorghum juice in an autoclave caused nutrient loss, consequently resulting in reduced fermentation efficiency. However, no effect of autoclaving was observed on fermentation of tropical maize syrup. Using unpasteurized syrup for fermentation might negatively impact ethanol yield due to bacteria contamination. In this study, 1.0 g/l of lactic acid was observed in the untreated syrup following 72 h of fermentation, suggesting bacterial activity (data not shown). However, the presence of contaminants did not affect the final ethanol yield as indicated by Fig. 2. Processing methods have been shown to affect bacterial contamination in sugar crops. Rein et al. (1989) reported reduced fermentation efficiency of % for fresh sweet sorghum juice, probably due to bacterial contamination; however, Wu et al. (2010) reportedincreased fermentation efficiency of 94.6 % on similar samples as a result of using hand harvesting and leaf stripping. Ethanol profile In order to determine if higher ethanol titers could be achieved by concentrating the syrup prior to fermentation, syrup was concentrated 1.25, 1.5, 1.75 and 2.25 times. Higher ethanol titers were achieved with greater syrup concentration Fig. 1 HPLC profile of the initial tropical maize syrup Fig. 2 The ethanol production from maize syrup and YEPS medium. At 72 h of fermentation, ethanol concentrations are YEPS: 8.04 ± 0.49 (% v/v); corn syrup (autoclaved): 7.55 ± 0.10 (% v/v); corn syrup (autoclaved; ph4.5): 7.58 (% v/v); corn syrup (initial): 7.44 (% v/v)

6 Table 1 Ethanol concentrations and fermentation rates for tropical maize syrup Concentration fold Initial sugar concentration (g/l) Sucrose Glucose Fructose Ethanol 96 h (% v/v) Ethanol yield (% of theoretical) Fermentation Rates at 12 h (% v/v/h) ± 0.02 A 92.2 A 0.45 A ± 0.01 B 90.3 B 0.45 A ± 0.05 C 91.8 AB 0.42 B ± 0.20 D 91.0 AB 0.38 C ± 0.01 E 76.4 C 0.25 D The following theoretical ethanol yield values have been used for the calculation (g of ethanol/100 g of sugar): sucrose, 53.8; glucose, 51.1; fructose, The results of the Fisher s least significant difference (LSD) at a = 0.05 were shown in superscript symbols. Columns with same superscript letters indicate that there were not any significant differences between results (Table 1). The sugar syrup had the highest ethanol yield (15.6 %) followed by sugar syrup (14.5 %), sugar syrup (12.7 %), sugar syrup (10.0 %) and sugar syrup (8.1 %). Initial fermentation rates, measured at 12 h, decreased with increasing initial sugar concentration (Table 1). Declining ethanol productivity is indicative of microbial stress factors (Gibson et al. 2007). There are several possible microbial stressors including sugar concentration, organic acid concentration, and ethanol concentration, all of which would act in a synergistic manner (Zhao and Bai 2009). The increased sugar fermentations also took longer to reach completion. Fermentations were complete at 24 h for the 1 and syrup concentrations; at 48 h for 1.5 and syrup concentrations; and remained incomplete at 96 h for syrup concentration (Fig. 3). Depending upon the treatments, final ethanol yields were approximately % of the theoretical value among 1 9 to treatments; whereas treatment had lower efficiency at 76.4 %, mostly because of unfermented sugars (Figs. 5, 6). According to a prior study (Zhao and Bai 2009), sugar concentrations greater than 250 g/l (25 %) osmotically stress yeast and reduce fermentation rates. Jones et al. (1994) performed very high gravity (VHG) fermentation on sugarcane juice and obtained a final ethanol concentration of 14.0 % (v/v). When the sugarcane juice in the above experiment was nutritionally fortified using molasses or wheat hydrolysate, the ethanol titers increased to 15.1 and 16.2 % (v/v), respectively. Bulawayo et al. (1996) used different yeast strains in sorghum juice fermentation and reached the highest ethanol yield of % (w/v). Laopaiboon et al. (2007) reported the ethanol yield of 10 % (w/v) from sorghum juice in batch fermentation. Wu et al. (2010) observed a maximum ethanol titer of 13 % (w/v) which is 16.6 % (v/v) for sorghum juice, and is comparable to the maximum achieved in this study (Fig. 3). Sugar profiles In contrast to corn grain fermentations, three sugars are present in maize stalk syrup: sucrose, glucose, and fructose. Fig. 3 Ethanol production during fermentation. At 96 h of fermentation, ethanol concentrations are 1.009: 8.09 ± 0.02 (% v/v); 1.259: ± 0.01 (% v/v); 1.509: ± 0.05 (% v/v); 1.759: ± 0.20 (% v/v); 2.259: ± 0.01 (% v/v) Fig. 4 Sucrose concentration during fermentation. At 96 h of fermentation, sucrose concentrations are 1.009: 0 ± 0 (% w/v); 1.259: 0 ± 0 (% w/v); 1.509: 0 ± 0 (% w/v); 1.759: 0 ± 0 (% w/v); 2.259: 0.01 ± 0.01 (% w/v)

7 In order to determine which sugar might be limiting the rate of fermentation, we monitored the concentration of each sugar during the fermentation reaction. Sucrose The sucrose concentration decreased rapidly during fermentation. The sucrose in 19 to treatments was depleted within 24 h and with the treatment it was depleted within 36 h (Fig. 4). Sucrose is not directly transported into the yeast cell. Instead, an externally exported invertase hydrolyzes the sucrose in the solution to glucose and fructose, which are transported across the cell membrane (Koschwanez et al. 2011). Glucose Accumulation of glucose was observed in the beginning of the fermentation (up to 9 h) as a result of sucrose hydrolysis (Fig. 5). The highest peak of glucose for treatments was observed at 3 9 h at 2.93, 3.52, 4.14, 4.81 and 5.94 % (w/v), respectively. Most of the glucose was depleted by 24 h, except the treatment had residual glucose of 0.21 % (w/v) was observed at the end of fermentation (Fig. 5). Fructose Fructose was co-fermented with glucose and also accumulated in the early part of the fermentation because of sucrose hydrolysis (Fig. 6). The highest peaks of fructose that occurred at treatments were observed at 12 h at 4.91, 6.32, 7.14 and 7.16 % (w/v), respectively. All fructose (w/v) was depleted before 60 h of fermentation time. The highest fructose peak of treatment was observed at 24 h, fructose increased up to 9.83 % (w/v) and at the end of fermentation 5.55 % (w/v) residual fructose was left in fermentation medium (Fig. 6). The tropical maize syrup is a mixture of several fermentable sugars; therefore, the preference of the yeast to a specific sugar influences the fermentation efficiency. According to a prior study (D Amore et al. 1989), glucose and fructose are taken up at the same rate when cultured separately with Saccharomyces cerevisiae. However, when presented in equal amounts, glucose was utilized twice as fast as fructose, which was also observed in this study. This explains why the treatment ended with 5.55 % fructose and only 0.21 % glucose. Greater residual fructose was also observed in a sweet sorghum syrup fermentation study (Wu et al. 2010). Sugar uptake kinetics is also different in tropical maize syrup fermentation. When yeast is grown in glucose and fructose syrup, sugar flux mainly governs the sugar uptake and diffusion through cell membrane. In case of tropical maize syrup fermentation, sugar flux is also affected by sucrose diffusion to cell wall, the hydrolysis rate at the cell wall and breakdown and release of sugars. The released monosaccharides generally diffuse away from its original cell but could be utilized by neighboring cells. Recent studies have reported microorganisms like budding yeast (Saccharomyces cerevisiae), which colonize in multicelled groups, were better able to utilized sugar mixtures than yeasts that grow as single cells (Koschwanez et al. 2011). Glycerol and organic acids profile Glycerol is the major by-product produced in yeast fermentations (Nordström 1966). Glycerol serves the dual Fig. 5 Glucose concentration during fermentation. At 96 h of fermentation, glucose concentrations are 1.009: 0 ± 0 (% w/v); 1.259: 0 ± 0 (% w/v); 1.509: 0 ± 0 (% w/v); 1.759: 0 ± 0 (% w/v); 2.259: 0.21 ± 0.01 (% w/v) Fig. 6 Fructose concentration during fermentation. At 96 h of fermentation, fructose concentrations are 1.009: 0.03 ± 0.01 (% w/v); 1.259: 0.03 ± 0.00 (% w/v); 1.509: 0.08 ± 0.00 (% w/v); 1.759: 0.15 ± 0.01 (% w/v); 2.259: 5.55 ± 0.12 (% w/v)

8 Fig. 7 Glycerol production during fermentation. At 96 h of fermentation, glycerol concentrations are 1.009: 0.66 ± 0.00 (% w/v); 1.259: 0.80 ± 0.01 (% w/v); 1.509: 0.96 ± 0.03 (% w/v); 1.759: 1.07 ± 0.02 (% w/v); 2.259: 1.43 ± 0.02 (% w/v) function of maintaining cell redox balance and acting as an osmotic protector (Shen et al. 1999). Glycerol production varied from 0.66 to 1.43 % (v/v) for the 19 to concentrated sugar solutions (Fig. 7). Glycerol production for the corn stalk extract in this study is similar to that reported for a corn grain ethanol process, at 1.2 % (w/v) (Russel 2003). The production of acetic acid and lactic acid are indicators of bacterial contamination during fermentation. Concentrations of acetic acid greater than % (w/v) and lactic acid greater than % (w/v) cause yeast stress (Narendranath et al. 2001). While no lactic acid was detected during fermentation of tropical maize syrup, concentrations of acetic acid detected was: 0.29 % acetic acid in the 19 syrup, 0.33 % in the syrup, 0.43 % in the 1.59 syrup, 0.50 % in the syrup and 0.69 % in the syrup at the end of fermentation (data not shown). We suspect the acetic acid was generated by the yeast because it was only detected 9 h into the fermentation and there was no other sign of bacterial fermentation. Yeast can generate acetic acid from ethanol through acetaldehyde to provide energy. Duggan (1964) demonstrated the ability of baker s yeast (S. cerevisiae) to utilize ethanol as a carbon source. Conclusions Tropical maize stalk syrup was evaluated for ethanol production. The extracted syrup had 9.02 % sucrose, 2.25 % glucose and 1.82 % fructose. The syrup culture fermented faster than a synthetic sugar control supplemented with yeast extract. Heat sterilizing the syrup improved the rate but not the final yield of fermentation. Adjusting the ph of the syrup prior to fermentation was found to be unnecessary. The syrup was subsequently concentrated in an effort to produce greater ethanol titers. The ethanol titer increased from 8.1 to 15.6 % by concentrating the initial syrup However, a concentration of the syrup was more favorable for ethanol production, because at concentration, glucose and fructose residual concentrations were 0.21 and 5.55 %. With the exception of the concentration, the ethanol conversion efficiencies compared favorably with those achieved commercially for corn grain, from 90.3 to 92.2 %. Osmotic stress at the higher sugar concentrations was evidenced by slower ethanol productivities and greater glycerol production. This study showed tropical maize syrup as an alternative feedstock source for bioethanol production. References Bulawayo B, Bvochora JM, Muzondo MI, Zvauya R (1996) Ethanol production by fermentation of sweet stem sorghum juice using various yeast strains. 12: D Amore T, Russell I, Stewart GG (1989) Sugar utilization by yeast during fermentation. J Ind Microbiol 4: Duggan PF (1964) Acetate and ethanol oxidation by yeast. Ir J Med Sci 39:19 30 EIA (2010) Annual energy review. Energy Information Association. Available online at showtext.cfm?t=ptb1003. Washington, DC. Accessed 27 Sept 2012 Gibson BR, Lawrence SJ, Leclaire JP, Powell CD, Smart KA (2007) Yeast responses to stresses associated with industrial brewery handling. FEMS Microbiol Rev 31: Goldemberg J (2007) Ethanol for an energy sustainable future. Science 315: Jones AM, Thomas KC, Ingledew WM (1994) Ethanolic fermentation of blackstrap molasses and sugarcane juice using very high gravity technology. J Agric Food Chem 42: Koschwanez JH, Foster KR, Murray AW (2011) Sucrose utilization in budding yeast as a model for the origin of undifferentiated multicellularity. PLoS Biol 9:1 10 Laopaiboon L, Laopaiboon P (2012) Ethanol production from sweet sorghum juice in repeated-batch fermentation by Saccharomyces cerevisiae immobilized on corncob. 28: Laopaiboon L, Thanonkeo P, Jaisil P, Laopaiboon P (2007) Ethanol production from sweet sorghum juice in batch and fed-batch fermentations by Saccharomyces cerevisiae. World J Microbiol Biotechnol 23: Lewandowskia I, Clifton-Brownb JC, Scurlockc JMO, Huismand W (2000) Miscanthus: European experience with a novel energy crop. Biomass Bioenerg 19: Liu R, Shen F (2008) Impacts of main factors on bioethanol fermentation from stalk juice of sweet sorghum by immobilized Saccharomyces cerevisiae (CICC 1308). Bioresour Technol 99: Narendranath NV, Thomas KC, Ingledew WM (2001) Effects of acetic acid and lactic acid on growth of Saccharomyces cerevisiae in a minimal medium. J Ind Microbiol Biotechnol 26:

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