Germ soak water as nutrient source to improve fermentation of corn grits from modified corn dry grind process
© The Author(s) 2017
Received: 19 June 2017
Accepted: 17 August 2017
Published: 23 August 2017
Corn fractionation in modified dry grind processes results in low fermentation efficiency of corn grits because of nutrient deficiency. This study investigated the use of nutrient-rich water from germ soaking to improve grits fermentation in the conventional dry grind and granular starch hydrolysis (GSH) processes. Comparison of germ soak water with the use of protease and external B-vitamin addition in improving grits fermentation was conducted. Use of water from optimum soaking conditions (12 h at 30 °C) resulted in complete fermentation with 29 and 8% higher final ethanol yields compared to that of control in conventional and GSH process, respectively. Fermentation rate (4–24 h) of corn grits with germ soak water (0.492 v/v-h) was more than double than that of control (0.208 v/v-h) in case of conventional dry grind process. The soaking process also increased the oil concentration in the germ by about 36%, which would enhance its economic value.
KeywordsEthanol Dry grind Granular starch hydrolysis Yeast nutrition Fermentation
Bioethanol is considered as one of the most promising renewable alternatives to petroleum-based transportation fuel. In the conventional dry grind process, corn starch is liquefied to dextrins at high temperature and pressure, which are further converted to glucose during the saccharification process. Glucose is simultaneously fermented to ethanol by yeast, and this combined process is known as simultaneous saccharification and fermentation (SSF). In an alternate approach, granular starch hydrolyzing enzymes (GSHE) can directly hydrolyze the raw granular starch into glucose at low temperatures, without the need of liquefaction step. At the end of both processes, remaining non-fermentable components (germ, fiber, protein, and residual starch) are recovered as DDGS (distillers dried grains with solubles), a coproduct primarily used as ruminant animal food. Fractionation of corn to recover germ and pericarp, prior to hydrolysis, is one way to generate valued coproducts and simultaneously improve nutritional value of DDGS (low fiber due to removal of pericarp) (Murthy et al. 2006a, b). Germ and pericarp obtained from the modified process can be refined to obtain valuable products including corn oil from corn germ and corn fiber oil from pericarp fiber. Corn fiber oil has very high economic value because its constituents have nutraceutical properties (Moreau et al. 1996; Murthy et al. 2006b). Grits obtained after germ and pericarp removal contain relatively high amount of starch, which would produce higher ethanol concentrations compared to whole corn at same solid loadings.
However, removal of germ during corn fractionation also removes the soluble proteins and micronutrients present in the germ that are essential for yeast during the fermentation process. Also the lipids, present in germ and the aleurone layer below the pericarp, are essential to maintain membrane integrity and yeast activity, especially during high glucose and ethanol concentrations. Murthy et al. (2006a) reported that both initial rate of fermentation and final ethanol concentrations were low for endosperm obtained from 3D process compared to those from wet fractionation (E-milling). One way to address this problem to some extent is addition of protease enzymes. Addition of proteases causes hydrolysis of the protein matrix surrounding the starch granules, which produces free amino nitrogen (FAN) as well as improve accessibility of starch to enzymes. Fermentation efficiency can also be improved by adding external nutrition, such as yeast extract, lipid supplementation, and B-vitamin complex. However, both protease enzymes and external nutrient add up to the cost of the process and counter the benefits of fractionation. One potential cost-effective approach could be the extraction of these nutrients from the recovered germ, as suggested by Murthy et al. (2006a). The study reported that the water obtained after soaking of fractionated germ (2 h soaking) resulted in increase of final ethanol concentrations from 12.3 to 14.7% (v/v) during conventional dry grind processing of corn grits.
This study aims to investigate this approach in detail and optimize the process conditions (germ soaking time and amount) to maximize the fermentation rate and final ethanol concentrations of corn grits during conventional dry grind as well as GSH process. Germ water was obtained from two soaking conditions and its effect on fermentation performance of corn grits was compared to those from control, protease addition, and B-vitamin addition. Combination of germ water and B-vitamins was also investigated to determine the maximum achievable ethanol efficiency. Composition of raw germ and germ after soaking was also evaluated to determine the changes in oil concentrations.
Flaking grits and germ samples were obtained from a commercial corn dry-milling plant (Bunge, Danville, IL, USA). Samples were stored in refrigerator at 4 °C till analysis. All enzymes including conventional α-amylase (Spezyme RSL with reported activity of 20,100 NLC/g), conventional glucoamylase [distillase SSF, with reported activity of 380 GAU/g (GAU: glucoamylase unit)], GSHE (Stargen 002), and Protease (Fermgen) are commonly used commercial enzymes and were generously donated by DuPont Industrial Biosciences (Palo Alto). GSHE contained α-amylase from A. kawachi expressed in T. reesei and glucoamylase from T. reesei, and had an activity of >570 GAU/g. Protease enzyme contained fungal protease obtained from genetically modified selected strain of T. reesei, with an activity of >1000 SAPU/g (SAPU is spectrophotometric acid protease units). Conventional active dry yeast (ethanol red) was obtained from the Fermentis-Lesaffre Yeast Corporation (Milwaukee, Wisconsin).
Corn grits and germ composition
Composition analysis of corn grits, raw germ, and soaked germ was performed as per American Association of Cereal Chemists International (AACCI) standard procedures. The moisture content of corn grits was determined by drying the samples in hot air oven at 135 °C for 2 h (AACC international approved method 44-19.01). Corn grits and germ (before and after soaking) were analyzed for crude protein content (method 990.03), oil (method 920.39), and neutral detergent fiber (method 2002.04) in a commercial analytical laboratory (Illinois crop improvement association, Champaign, IL, USA). All analyses were conducted in duplicates. Starch content in the ground corn grits was determined using acid hydrolysis method (Vidal et al. 2009). Briefly, about 1 g of ground corn samples (~1 g) were mixed with 50 mL dilute HCl (0.4 N) in 100 mL autoclavable bottles, and the slurry was autoclaved at 126 °C for 1 h (Napco Model 9000D, Thermo Fisher 157 Scientific, Waltham, MA). Pure glucose and starch samples were used to determine glucose recovery factors. After cooling, 1 mL aliquot samples was withdrawn and centrifuged at 1500×g for 5 min (Model 5415 D, Brinkmann–Eppendorf, Hamburg, Germany). The supernatants were analyzed in the HPLC for glucose determination.
Dry grind process
Description of treatments investigated in processing corn grits using conventional dry grind and GSH process
DI water (% of liquid in slurry)
Germ soak water (% of liquid in slurry)
Germ soaking conditions
Control with protease
Partial germ water
Partial germ water—long time
Full germ water
Partial germ water and B-vitamin
Germ soak water was obtained by soaking the germ under two conditions: (i) 30 °C for 2 h and (ii) 30 °C for 12 h (Table 1). In each case, 25 g of germ was mixed in 250 mL of deionized (DI) water in 500 mL flasks and was incubated as per conditions mentioned in Table 1, with continuous shaking at 125 rpm. After soaking, the liquid was vacuum-filtered through Whatman No. 4 filter paper and used to make slurry as described in Table 1. Two dosages of germ water were investigated: (1) one-third (33.33%) of total liquid in slurry, referred as partial germ water (treatments T3, T4, T7 in Table 1), (2) 100% of liquid in slurry, referred as full germ water (treatment T5 in Table 1) in the article. The first case (partial germ water) represents the water obtained from the soaking of germ proportional (10%) to corn grits used in the experiment. In the current study, 62.5 mL of germ water was added in total 250 mL slurry (62.5 g corn grits and 187.5 mL liquid). Full germ water case was investigated to determine the effect of adding excess nutrients on the fermentation efficiency. Other than germ soak water, two additional set of treatments (T6 and T7) were performed by addition of B-vitamins. In treatment T6, conditions were similar to that of control, except excess of vitamin B12 and B-complex were added at the start of SSF process. In the case of treatment T7, combined effect of germ soak water and B-vitamins was investigated and excess of vitamin B12 and B-complex was added in addition to germ soak water (Table 1).
The front-end operations (cleaning, grinding, and slurry making) were similar to that of conventional dry grind process described above (Fig. 1). Liquefaction step is not required in this process. The slurry prepared was adjusted to a pH of 4.1 using 10 N sulfuric acid, and GSHE (0.234 mL), urea (0.4 μL of 50% w/v solution) and yeast inoculum (2 mL) were added. Yeast inoculum was prepared as described in the previous section. The slurry was incubated at 32 °C for 72 h in an automatic incubator with continuous agitation (150 rpm), and 2 mL of samples were drawn at 4, 8, 12, 24, 48, and 72 h to monitor the fermentation. This process was also investigated for all conditions presented in Table 1.
Samples collected were centrifuged at 9729 g (5415 D, Brinkmann Eppendorf, Hamburg, Germany) for 10 min, and clear liquid was passed through 0.2 µm syringe filters (nylon Acrodisc WAT200834, Pall Life Sciences, Port Washington, NY) into 150 µL HPLC vials. The vials were immediately stored at −20 °C until analysis. The filtrate was then analyzed using HPLC with an ion-exclusion column (Aminex HPX-87H, Bio-Rad, Hercules, CA, USA). The mobile phase was 0.005 M sulfuric acid at 50 °C at a flow rate of 0.6 mL/min. The amounts of sugars, alcohols, and organic acids were quantified using a refractive index detector and multiple standards.
Fermentation rate and ethanol yield
Analysis of variance (1-way ANOVA) and Fisher’s least significant difference (LSD) tests were used to compare the glucose and ethanol concentrations (SAS version 9.3). The level selected to show the statistical significance in all cases was 5% (P < 0.05).
Results and discussion
Composition of corn grits
Starch content in the corn grits was estimated as 86.5% on dry basis. Crude protein, oil, and neutral detergent fiber (NDF) were 6.1, 0.6, and 0.9% (dry basis), respectively. Based on this composition, the theoretical ethanol yield was calculated 0.62 L/kg dry corn grits (4.17 gal/bu).
Conventional dry grind process
Effect of germ soak water
About 4.5% glucose remained unconverted in the case of control, which along with high glycerol production resulted in very low starch-to-ethanol conversion efficiency (62.4%). Efficient fermentation with germ water (30 °C and 12 h) addition led to an increase in conversion efficiency to 82.7%, which was 12% (in relative terms) higher than that in the case of addition of 2-h germ-soaked water (73.8%). Since the glucose released in the first 8 h is similar for all three conditions (Fig. 2), it can be stated that the increased rate of fermentation in the 12-h germ-soaked water is due to the better functioning of the yeast in the presence of micronutrients and free amino acids present in germ soak water. These results indicate that longer soaking resulted in leaching out more nutrients that improved the yeast performance and led to high ethanol yields and fermentation rates. Due to the lack of these micronutrients in the control, fermentation was observed to be slowest among all treatments. Ethanol yields from control, treatment with 2-h germ water and treatment with 12-h germ water were estimated as 0.39, 0.46, and 0.51 L/kg dry grits (2.6, 3.1, and 3.5 gal/bu) respectively.
Germ water vs. protease addition
Effect of water amount
Effect of using partial vs. full germ water on the glucose and ethanol concentrations of corn grits during conventional dry grind process
Ethanol (% v/v)
Glucose (% w/v)
Effect of B-vitamins
Ethanol yields and conversion efficiencies for all treatments in dry grind process
Final ethanol concentration (%)
Ethanol conversion efficiency (%)
Fermentation rates (% v/v/h)
16.20 a b
82.83 a b c
Germ soak water 2 h 30 °C
Germ soak water 12 h 30 °C-PS
16.14 a b
82.67 a b c
Germ soak water 12 h 30 °C-FS
82.14 b c
B-vitamins + Germ soak watera
Final ethanol concentrations, fermentation rates (4–24 h), and ethanol conversion efficiencies for all treatment have been compiled in Table 3. Except for control and germ water from soaking at 30 °C and 2 h, the ethanol conversion efficiency was more than 80% in all treatments. Although conversion efficiency is similar in all other cases, the fermentation rate (4–24 h) was maximum for full germ slurry and treatment using both germ water and B-vitamins.
Granular starch hydrolysis process
Considering the advantages (low energy use and low glucose inhibition) and increasing trend of granular starch hydrolysis process in corn ethanol industry, it was important to investigate the effect of germ soak water on yeast performance in GSH process also. Performance of germ water for all conditions listed in Table 1 was studied and compared with control, protease addition, and B-vitamin addition.
Effect of germ soak water
Ethanol yields and conversion efficiencies for all treatments in GSH process
Final average ethanol concentration (%)
Average conversion efficiency (%)
Average fermentation rates (% v/v/h)
Germ soak water 2 h 30 °C
Germ soak water 12 h 30 °C-PS
Germ soak water 12 h 30 °C-FS
B-vitamins + germ soak watera
Effect of water amount
Effect of B-vitamins
Composition of germ
This study investigated and optimized the use of nutrient-rich water from corn germ soaking to improve fermentation of corn grits in comparison to through the use of protease enzymes or B-vitamin additions. Optimum soaking time and amount of germ water required was determined corresponding to maximum ethanol yield in conventional dry grind and granular starch hydrolysis process. The addition of germ water from soaking conditions of 30 °C for 12 h resulted in complete fermentation for both conventional and GSH processes, compared to significant residual sugars for control. Final ethanol yields were 29 and 8% higher than that of control in case of conventional and GSH process, respectively. GHS enzymes have previously reported to work better than conventional dry grind enzymes. However, the addition of germ water resulted in similar fermentation performance both GSHE and conventional enzymes. Initial ethanol production rates for samples supplemented with germ soak water were higher than that of samples supplemented with protease and similar to that from supplementation of B-vitamins for both processes. Due to leaching of micronutrients and soluble proteins, soaking process improved the oil concentrations in the germ, which would enhance its economic value. Overall, the use of germ water from optimum soaking conditions can potentially eliminate the need for protease enzymes or expensive nutrients addition for efficient fermentation, and provide other advantages of higher oil concentrations in germ, and potential of acid use reduction in the process.
AJ, DK, and VS designed the study. AJ and DK conducted experiments, analyzed data, and prepared the manuscript. VS reviewed the results, helped in data analysis, and edited the manuscript. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
Availability of data and materials
The all data generated and analyzed during this study are included in this within the manuscript in form of graphs and tables. The authors promise to provide the data used in graphs if required.
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