Enzymatic Process for Corn Dry‐Grind High‐Solids Fermentation

A modified dry‐grind process that combined the use of conventional amylases (glucoamylase [GA]), phytase, and granular starch hydrolyzing enzymes (GSHE) to achieve low liquefaction viscosities and low glucose concentrations during simultaneous saccharification and fermentation (SSF) with a high slurry solids content (>33% w/w) was developed. Doses of GSHE and GA were optimized for the modified process. At 35% solids content, the modified process had 80% lower slurry viscosity, 24% lower peak glucose concentration, 7.5% higher final ethanol concentration, and 51% higher fermentation rate compared with the conventional dry‐grind process. At 40% solids content, the modified process had lower viscosities, lower peak and residual glucose concentrations, and higher ethanol concentrations than the conventional process; however, the results were in contrast to those for 35% solids content. At 40% solids content, SSF did not run to completion for conventional or modified processes, and more than 2.5% w/v of residual glucose was left in the fermentation broth. Final ethanol concentration achieved with the modified process at 40% solids content was 19.5% v/v, similar to the ethanol concentration achieved with the modified process at 35% solids content. At 35% slurry solids content, a GSHE level of 1.25 μL/g db of corn and a GA level of 0.25 μL/g db of corn were selected as optimum enzyme doses for the modified process.     

Comparison of Enzymatic (E‐Mill) and Conventional Dry‐Grind Corn Processes Using a Granular Starch Hydrolyzing Enzyme

A new low temperature liquefaction and saccharification enzyme STARGEN 001 (Genencor International, Palo Alto, CA) with high granular starch hydrolyzing activity was used in enzymatic dry‐grind corn process to improve recovery of germ and pericarp fiber before fermentation. Enzymatic dry‐grind corn process was compared with conventional dry‐grind corn process using STARGEN 001 with same process parameters of dry solid content, pH, temperature, enzyme and yeast usage, and time. Sugar, ethanol, glycerol and organic acid profiles, fermentation rate, ethanol and coproducts yields were investigated. Final ethanol concentration of enzymatic dry‐grind corn process was 15.5 ± 0.2% (v/v), which was 9.2% higher than conventional process. Fermentation rate was also higher for enzymatic dry‐grind corn process. Ethanol yields of enzymatic and conventional dry‐grind corn processes were 0.395 ± 0.006 and 0.417 ± 0.002 L/kg (2.65 ± 0.04 and 2.80 ± 0.01 gal/bu), respectively. Three additional coproducts, germ 8.0 ± 0.4% (db), pericarp fiber 7.7 ± 0.4% (db), and endosperm fiber 5.2 ± 0.6% (db) were produced in addition to DDGS with enzymatic dry‐grind corn process. DDGS generated from enzymatic dry‐grind corn process was 66% less than conventional process.     

Evaluation and Strategies to Improve Fermentation Characteristics of Modified Dry‐Grind Corn Processes

New corn fractionation technologies that produce higher value coproducts from dry‐grind processing have been developed. Wet fractionation technologies involve a short soaking of corn followed by milling to recover germ and pericarp fiber in an aqueous medium before fermentation of degermed defibered slurry. In dry fractionation technologies, a dry degerm defiber (3D) process (similar to conventional corn dry‐milling) is used to separate germ and pericarp fiber before fermentation of the endosperm fraction. The effect of dry and wet fractionation technologies on the fermentation rates and ethanol yields were studied and compared with the conventional dry‐grind process. The wet process had the highest fermentation rate. The endosperm fraction obtained from 3D process had lowest fermentation rate and highest residual sugars at the end of fermentation. Strategies to improve the fermentation characteristics of endosperm fraction from 3D process were evaluated using two saccharification and fermentation processes. The endosperm fraction obtained from 3D process was liquefied by enzymatic hydrolysis and fermented using either separate saccharification (SS) and fermentation or simultaneous saccharification and fermentation (SSF). Corn germ soak water and B‐vitamins were added during fermentation to study the effect of micronutrient addition. Ethanol and sugar profiles were measured using HPLC. The endosperm fraction fermented using SSF produced higher ethanol yields than SS. Addition of B‐vitamins and germ soak water during SSF improved fermentation of 3D process and resulted in 2.6 and 2.3% (v/v) higher ethanol concentrations and fermentation rates compared with 3D process treatment with no addition of micronutrients.     

Dry‐Grind Processing of Corn with Endogenous Liquefaction Enzymes

An amylase corn has been developed that produces an α‐amylase enzyme that is activated in the presence of water at elevated temperatures (>70°C). Amylase corn in the dry‐grind process was evaluated and compared with the performance of exogenous amylases used in dry‐grind processing. Amylase corn (1–10% by weight) was added to dent corn (of the same genetic background as the amylase corn) as treatments and resulting samples were evaluated for dry‐grind ethanol fermentation using 150‐g and 3‐kg laboratory procedures. Ethanol concentrations during fermentation were compared with the control treatment (0% amylase corn addition or 100% dent corn) which was processed with a conventional amount of exogenous α‐amylase enzymes used in the dry‐grind corn process. The 1% amylase corn treatment (adding 1% amylase corn to dent corn) was sufficient to liquefy starch into dextrins. Following fermentation, ethanol concentrations from the 1% amylase corn treatment were similar to that of the control. Peak and breakdown viscosities of liquefied slurries for all amylase corn treatments were significantly higher than the control treatment. In contrast, final viscosities of liquefied slurries for all amylase corn treatments were lower than those of the control. Protein, fat, ash, and crude fiber contents of DDGS samples from the 3% amylase corn treatment and control were similar.     

Enzyme production by wood-rot and soft-rot fungi cultivated on corn fiber followed by simultaneous saccharification and fermentation

This research aims at developing a biorefinery platform to convert lignocellulosic corn fiber into fermentable sugars at a moderate temperature (37 °C) with minimal use of chemicals. White-rot (Phanerochaete chrysosporium), brown-rot (Gloeophyllum trabeum), and soft-rot (Trichoderma reesei) fungi were used for in situ enzyme production to hydrolyze cellulosic and hemicellulosic components of corn fiber into fermentable sugars. Solid-substrate fermentation of corn fiber by either white- or brown-rot fungi followed by simultaneous saccharification and fermentation (SSF) with coculture of Saccharomyces cerevisiae has shown a possibility of enhancing wood rot saccharification of corn fiber for ethanol fermentation. The laboratory-scale fungal saccharification and fermentation process incorporated in situ cellulolytic enzyme induction, which enhanced overall enzymatic hydrolysis of hemi/cellulose components of corn fiber into simple sugars (mono-, di-, and trisaccharides). The yeast fermentation of the hydrolyzate yielded 7.8, 8.6, and 4.9 g ethanol per 100 g corn fiber when saccharified with the white-, brown-, and soft-rot fungi, respectively. The highest ethanol yield (8.6 g ethanol per 100 g initial corn fiber) is equivalent to 35% of the theoretical ethanol yield from starch and cellulose in corn fiber. This research has significant commercial potential to increase net ethanol production per bushel of corn through the utilization of corn fiber. There is also a great research opportunity to evaluate the remaining biomass residue (enriched with fungal protein) as animal feed.     

Protease Treatment to Improve Ethanol Fermentation in Modified Dry Grind Corn Processes

To improve fractionation efficiency in modified dry grind corn processes, we evaluated the effectiveness of protease treatment in reducing residual starch in endosperm fiber. Three schemes of protease treatment were conducted in three processes: 1) enzymatic milling or E‐Mill, 2) dry fractionation with raw starch fermentation or dry RS, and 3) dry fractionation with conventional fermentation or dry conv. Kinetics of free amino nitrogen production were similar in both dry and wet fractionation (E‐Mill), indicating that proteolysis was effective in all three schemes. At the end of fermentation, endosperm fiber was recovered and its residual starch measured. Using protease treatment, residual starch in the endosperm fiber was reduced by 1.9% w/w (22% relative reduction) in dry conv and 1.7% w/w (8% relative reduction) in dry RS, while no reduction was observed in the E‐Mill process. Protease treatment increased ethanol production rates early in fermentation (≤24 hr) but final ethanol concentrations were unaffected in both dry RS and E‐Mill. In dry conv, the addition of protease resulted in a decline in final ethanol concentration by 0.3% v/v, as well as a higher variability in liquefaction product concentration (higher standard deviations in the glucose and maltose yields). Protease treatment can be used effectively to enhance modified dry grind processes.     

Improvement of Dry‐Fractionation Ethanol Fermentation by Partial Germ Supplementation

Ethanol fermentation of dry‐fractionated grits (corn endosperm pieces) containing different levels of germ was studied with the dry‐grind process. Partial removal of the germ fraction allows for marketing the germ fraction and potentially more efficient fermentation. Grits obtained from a dry‐milling plant were mixed with different amounts of germ (2, 5, 7, and 10% germ of the total sample) and compared with control grits (0% germ). Fermentation rates of germ‐supplemented grits (2, 5, 7, and 10% germ) were faster than control grits (0% germ). Addition of 2% germ was sufficient to achieve a high ethanol concentration (19.06% v/v) compared with control grits (18.18% v/v). Fermentation of dry‐fractionated grits (92, 95, and 97% grits) obtained from a commercial facility was also compared with ground whole corn (control). Fermentation rates were slower and final ethanol concentrations were lower for commercial grits than the control sample. However, in a final experiment, commercial grits were subjected to raw starch hydrolyzing (RSH) enzyme, resulting in higher ethanol concentrations (20.22, 19.90, and 19.49% v/v for 92, 95, and 97% grits, respectively) compared with the whole corn control (18.64% v/v). Therefore, high ethanol concentrations can be achieved with dry‐fractionated grits provided the inclusion of a certain amount of germ and the use of RSH enzyme for controlled starch hydrolysis.     

Use of Pigmented Maize in Both Conventional Dry‐Grind and Modified Processes Using Granular Starch Hydrolyzing Enzyme

In the dry‐grind ethanol process, distillers dried grains with solubles (DDGS) is the main coproduct, which is primarily used as an ingredient in ruminant animal diets. Increasing the value of DDGS will improve the profitability of the dry‐grind ethanol process. One way to increase DDGS value is to use pigmented maize as the feedstock for ethanol production. Pigmented maize is rich in anthocyanin content, and the anthocyanin imparts red, blue, and purple color to the grain. It is reported that anthocyanin would be absorbed by yeast cell walls during the fermentation process. The effects of anthocyanin on fermentation characteristics in the dry‐grind process are not known. In this study, the effects of anthocyanin in conventional (conventional starch hydrolyzing enzymes) and modified (granular starch hydrolyzing enzymes [GSHE]) dry‐grind processes were evaluated. The modified process using GSHE replaced high‐temperature liquefaction. The ethanol conversion efficiencies of pigmented maize were comparable to that of yellow dent corn in both conventional (78.4 ± 0.5% for blue maize, 74.3 ± 0.4% for red maize, 81.2 ± 1.0% for purple maize, and 75.1 ± 0.2% for yellow dent corn) and modified dry‐grind processes using GSHE (83.8 ± 0.8% for blue maize, 81.1 ± 0.3% for red maize, 93.5 ± 0.8% for purple maize, and 85.6 ± 0.1% for yellow dent corn). Total anthocyanin content in DDGS from the modified process was 1.4, 1.9, and 2.4 times of that from the conventional process for purple, red, and blue maize samples, respectively. These results indicated that pigmented maize rich in anthocyanin did not negatively affect the fermentation characteristics of the dry‐grind process and that there was a potential to use pigmented maize in the dry‐grind process, especially when using GSHE.     

Use of Phytases in Ethanol Production from E‐Mill Corn Processing

Effects of phytase addition, germ, and pericarp fiber recovery were evaluated for the E‐Mill dry grind corn process. In the E‐Mill process, corn was soaked in water followed by incubation with starch hydrolyzing enzymes. For each phytase treatment, an additional phytase incubation step was performed before incubation with starch hydrolyzing enzymes. Germ and pericarp fiber were recovered after incubation with starch hydrolyzing enzymes. Preliminary studies on phytase addition resulted in germ with higher oil (40.9%), protein (20.0%), and lower residual starch (12.2%) contents compared to oil (39.1%), protein (19.2%), and starch (18.1%) in germ from the E‐Mill process without phytase addition. Phytase treatment resulted in lower residual starch contents in pericarp fiber (19.9%) compared to pericarp fiber without phytase addition (27.4%). Results obtained led to further investigation of effects of phytase on final ethanol concentrations, germ, pericarp fiber, and DDGS recovery. Final ethanol concentrations were higher in E‐Mill processing with phytase addition (17.4% v/v) than without addition of phytase (16.6% v/v). Incubation with phytases resulted in germ with 4.3% higher oil and 2.5% lower residual starch content compared to control process. Phytase treatment also resulted in lower residual starch and higher protein contents (6.58 and 36.5%, respectively) in DDGS compared to DDGS without phytase incubations (8.14 and 34.2%, respectively). Phytase incubation in E‐Mill processing may assist in increasing coproduct values as well as lead to increased ethanol concentrations.     

Effect of Sorghum Decortication and Use of Protease Before Liquefaction with Thermoresistant α‐Amylase on Efficiency of Bioethanol Production

The aim was to study the dual effect of sorghum decortication and protease treatment before liquefaction with α‐amylase on the performance of subsequent steps of saccharification and fermentation. A bifactorial experiment with a level of confidence of P < 0.05 was designed to study differences among grains (maize, whole, and decorticated sorghum) and the effectiveness of the protease before liquefaction. Sorghum was decorticated to remove most of the pericarp and part of the germ and increase starch concentration of the feedstock. The decorticated sorghum had significantly higher starch hydrolysis during liquefaction compared with the whole kernel. These hydrolyzates contained ≈50% more reducing sugars than the untreated counterparts. At the end of saccharification, the final glucose concentration in hydrolyzates treated without protease was the highest for maize (180 mg/mL), followed by decorticated sorghum (165 mg/mL), and whole sorghum (145 mg/mL). Decortication and protease treatment had a significant effect on fermentation times. In decorticated sorghum mash treated with and without protease, fermentation times were 22 and 60 hr, respectively. The decorticated sorghum treated with protease yielded similar amounts of ethanol compared with maize and 44% more ethanol compared with the untreated whole sorghum. Both sorghum decortication and protease treatments before hydrolysis with α‐amylase are recommended to increase ethanol yields, lower yields of distilled grains, and save liquefaction, saccharification, and fermentation times.     

Ethanol Fermentation of Starch from Field Peas

Field peas (Pisum sativum) 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 73.7% wt and 77.8% wt starch, respectively, a nearly two‐fold enrichment compared with whole peas. The fractions were liquefied and saccharified using industrial α‐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 ethanol/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.     

Effects of Protease and Urea on a Granular Starch Hydrolyzing Process for Corn Ethanol Production

The dry grind process using granular starch hydrolyzing enzymes (GSHE) saves energy. The amount of GSHE used is an important factor affecting dry grind process economics. Proteases can weaken protein matrix to aid starch release and may reduce GSHE doses. Two specific proteases, an exoprotease and an endoprotease, were evaluated in the dry grind process using GSHE (GSH process). The effect of protease and urea addition on GSH process was also evaluated. Addition of these proteases resulted in higher ethanol concentrations (mean increase of 0.3–1.8 v/v) and lower distillers' dried grains with solubles (DDGS) yields (mean decrease of 1.3–8.0% db) compared with the control (no protease addition). As protease levels and GSHE increased, ethanol concentrations increased and DDGS yields decreased. Protease addition reduced the required GSHE dose. Final mean ethanol concentrations without urea (15.2% v/v) were higher than with urea (15.0% v/v) in GSH process across all protease treatments.     

Fate of Bt Protein and Influence of Corn Hybrid on Ethanol Production

Corn hybrids were compared to determine the fate of recombinant Bt protein (CRY1Ab from Bacillus thuringiensis) in coproducts from dry grind and wet‐milled corn during production of fuel ethanol. Two pairs of Bt and non‐Bt hybrids were wet milled, and each fraction was examined for the presence of the Bt protein. Bt protein was found in the germ, gluten, and fiber fractions of Bt hybrids. In addition, one set of Bt and non‐Bt hybrids were treated by the dry‐grind ethanol process and Bt protein was monitored during each step of the process. The Bt protein was not detected after liquefaction. Subsequent experiments determined that the Bt protein is rapidly denatured at liquefaction temperatures. Finally, five hybrids were compared for ethanol yield after dry grinding. Analysis of fermentation data with an F‐test revealed the percent of total starch available for conversion into ethanol varied significantly among the hybrids (P < 0.002), indicating ethanol yield is not exclusively dependent on starch content. No difference, however, was observed between Bt and non‐Bt corn hybrids for either ethanol productivity or yield.     

Methods for Characterization of Residual Starch in Distiller's Dried Grains with Solubles (DDGS)

The objective of this study was to establish methods for determining the content and components of residual starch in distiller's dried grains with solubles (DDGS), a coproduct from dry‐grind corn ethanol production. Four DDGS prepared in our laboratory and one DDGS obtained from a commercial ethanol manufacturer were used for the study. Quantitative analysis of total residual sugar (TRS) in DDGS was performed by determining d ‐glucose produced by enzymatic hydrolysis of oligosaccharides and residual starch remaining in hexane‐defatted DDGS after being dispersed in 90% DMSO. The TRS consisted of free glucose, oligosaccharides, and residual starch. The commercial manufacturer's DDGS contained more TRS (15.8%, w/w db) than the laboratory‐processed DDGS (2.4–2.9%, w/w db). The content of residual starch remaining in the commercial DDGS (5.5% w/w db) was also larger than the laboratory‐processed DDGS (1.9–2.5% w/w db). Analyses of molecular weight distribution showed that the residual starch in DDGS consisted of short‐chain amylose and amylopectin, respectively, as the major and minor components. The short‐chain amylose molecules constituted 86.5–94.1% of the residual starch. The major population of the short‐chain amyloses had an average degree of polymerization (DP) of 85, closely resembling the length of enzyme‐resistant fragments of amylose‐lipid complexes.     

Hydrolysis and Fermentation of Pericarp and Endosperm Fibers Recovered from Enzymatic Corn Dry‐Grind Process

A modified dry‐grind corn process has been developed that allows recovery of both pericarp and endosperm fibers as coproducts at the front end of the process before fermentation. The modified process is called enzymatic milling (E‐Mill) dry‐grind process. In a conventional dry‐grind corn process, only the starch component of the corn kernel is converted into ethanol. Additional ethanol can be produced from corn if the fiber component can also be converted into ethanol. In this study, pericarp and endosperm fibers recovered in the E‐Mill dry‐grind process were evaluated as a potential ethanol feedstock. Both fractions were tested for fermentability and potential ethanol yield. Total ethanol yield recovered from corn by fermenting starch, pericarp, and endosperm fibers was also determined. Results show that endosperm fiber produced 20.5% more ethanol than pericarp fiber on a g/100 g of fiber basis. Total ethanol yield obtained by fermenting starch and both fiber fractions was 0.370 L/kg compared with ethanol yield of 0.334 L/kg obtained by fermenting starch alone.     

Resistant Starch Escaped from Ethanol Production: Evidence from Confocal Laser Scanning Microscopy of Distiller's Dried Grains with Solubles (DDGS)

The origin of resistant starch (RS) in distiller's dried grains with solubles (DDGS) of triticale, wheat, barley, and corn from dry‐grind ethanol production was studied. A considerable portion of starch (up to 18% in DDGS) escaped from either granular starch hydrolysis or conventional jet‐cooking and fermentation processes. Confocal laser scanning microscopy revealed that some starch granules were still encapsulated in cells of grain kernel or embedded in protein matrix after milling and were thus physically inaccessible to amylases (type RS1). The crystalline structures of native starch granules were not completely degraded by amylases, retaining the skeletal structures in residual starch during granular starch hydrolysis or leaving residue granules and fragments with layered structures after jet‐cooking followed by the liquefaction and saccharification process, indicating the presence of RS2. Moreover, retrograded starch molecules (mainly amylose) as RS3, complexes of starch with other nonfermentable components as RS4, and starch–lipid complexes as RS5 were also present in DDGS. In general, the RS that escaped from the granular starch hydrolysis process was mainly RS1 and RS2, whereas that from the jet‐cooking process contained all types of RS (RS1 to RS5). Thus, the starch conversion efficiency and ethanol yield could be potentially affected by the presence of various RS in DDGS.     

Optimization of simultaneous saccharification and fermentation for the production of ethanol from lignocellulosic biomass

Simultaneous saccharification and fermentation (SSF) of alkaline hydrogen peroxide pretreated Antigonum leptopus (Linn) leaves to ethanol was optimized using cellulase from Trichoderma reesei QM-9414 (Celluclast from Novo) and Saccharomyces cerevisiae NRRL-Y-132 cells. Response surface methodology (RSM) and a three-level four-variable design were employed to evaluate the effects of SSF process variables such as cellulase concentration (20-100 FPU/g of substrate), substrate concentration (5-15% w/v), incubation time (24-72 h), and temperature (35-45 degrees C) on ethanol production efficiency. Cellulase and substrate concentrations were found to be the most significant variables. The optimum conditions arrived at are as follows: cellulase = 100 FPU/g of substrate, substrate = 15% (w/v), incubation time = 57.2 h, and temperature = 38.5 degrees C. At these conditions, the predicted ethanol yield was 3.02% (w/v) and the actual experimental value was 3.0% (w/v).     

Effect of Aflatoxin B1 on Dry‐Grind Ethanol Process

Aflatoxins, like all mycotoxins, are toxic fungal metabolites that can have adverse health effects on animals and human beings. Aflatoxins are a major concern for the dry‐grind corn processing industry as it is believed that aflatoxins affect yeast and reduce its efficacy in producing ethanol. In the present study, aflatoxin B1 (100, 200, 350, or 775 ppb) was added to mycotoxin‐free corn and laboratory‐scale fermentations were conducted. No effect of aflatoxin B1 was observed on the fermentation rates or final ethanol concentrations. Mean ethanol concentration in the fermenter was 14.01–14.51% (v/v) at 60 hr for all the treatments. In the dry‐grind ethanol process, 55% of aflatoxin B1 was detected in wet grains and 45% in thin stillage.     

Solid-substrate fermentation of corn fiber by Phanerochaete chrysosporium and subsequent fermentation of hydrolysate into ethanol

The goal of this study was to develop a fungal process for ethanol production from corn fiber. Laboratory-scale solid-substrate fermentation was performed using the white-rot fungus Phanerochaete chrysosporium in 1 L polypropylene bottles as reactors via incubation at 37 degrees C for up to 3 days. Extracellular enzymes produced in situ by P. chrysosporium degraded lignin and enhanced saccharification of polysaccharides in corn fiber. The percentage biomass weight loss and Klason lignin reduction were 34 and 41%, respectively. Anaerobic incubation at 37 degrees C following 2 day incubation reduced the fungal sugar consumption and enhanced the in situ cellulolytic enzyme activities. Two days of aerobic solid-substrate fermentation of corn fiber with P. chrysosporium, followed by anaerobic static submerged-culture fermentation resulted in 1.7 g of ethanol/100 g of corn fiber in 6 days, whereas yeast ( Saccharomyces cerevisiae) cocultured with P. chrysosporium demonstrated enhanced ethanol production of 3 g of ethanol/100 g of corn fiber. Specific enzyme activity assays suggested starch and hemi/cellulose contribution of fermentable sugar.     

Ethanol Production from Modified and Conventional Dry‐Grind Processes Using Different Corn Types

Different corn types were used to compare ethanol production from the conventional dry‐grind process to wet or dry fractionation processes. High oil, dent corn with high starch extractability, dent corn with low starch extractability and waxy corn were selected. In the conventional process, corn was ground using a hammer mill; water was added to produce slurry which was fermented. In the wet fractionation process, corn was soaked in water; germ and pericarp fiber were removed before fermentation. In the dry fractionation process, corn was tempered, degerminated, and passed through a roller mill. Germ and pericarp fiber were separated from the endosperm. Due to removal of germ and pericarp fiber in the fractionation methods, more corn was used in the wet (10%) and dry (15%) fractionation processes than in the conventional process. Water was added to endosperm and the resulting slurry was fermented. Oil, protein, and residual starch in germ were analyzed. Pericarp fiber was analyzed for residual starch and neutral detergent fiber (NDF) content. Analysis of variance and Fisher's least significant difference test were used to compare means of final ethanol concentrations as well as germ and pericarp fiber yields. The wet fractionation process had the highest final ethanol concentrations (15.7% v/v) compared with dry fractionation (15.0% v/v) and conventional process (14.1% v/v). Higher ethanol concentrations were observed in fractionation processes compared to the conventional process due to higher fermentable substrate per batch available as a result of germ and pericarp fiber removal. Germ and pericarp yields were 7.47 and 6.03% for the wet fractionation process and 7.19 and 6.22% for the dry fractionation process, respectively. Germ obtained from the wet fractionation process had higher oil content (34% db) compared with the dry fractionation method (11% db). Residual starch content in the germ fraction was 16% for wet fractionation and 44% for dry fractionation. Residual starch in the pericarp fiber fraction was lower for the wet fractionation process (19.9%) compared with dry fractionation (23.7%).