Industrial Crops & Products 111 (2018) 247–253

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Industrial Crops & Products

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Research Paper

Ethanol production from mixtures of sweet sorghum juice and sorghum MARK
starch using very high gravity fermentation with urea supplementation
⁎
Nana Baah Appiah-Nkansaha, Ke Zhanga, William Rooneyb, Donghai Wanga,
a
Department of Biological and Agricultural Engineering, Kansas State University, Manhattan, KS, 66506, United States
b
Department of Soil and Crop Science, Texas A & M University, College Station, TX, 77843, United States

A R T I C L E I N F O A B S T R A C T

Keywords: The objective of this research was to enhance the production of ethanol from grain sorghum flour and sweet
Very high gravity fermentation sorghum juice using very high gravity (VHG) fermentation. Various amounts of grain sorghum flour were
Grain sorghum combined with different concentrations of sweet sorghum juice to form (≈33 g/100 mL) mashes. Mashes were
Sweet sorghum juice fermented to produce ethanol with and without urea supplementation via simultaneous saccharification and
Ethanol concentration
fermentation process. Based on one-way ANOVA results, all treatment mixtures had a significant effect on
Free amino nitrogen
ethanol production and efficiency (P < 0.05). It is hypothesized that the combination of inoculation size
(109 cells/ml), fermentation temperature (30 °C) and 16 mM urea supplementation had a synergistic effect on
ethanol production. Free amino nitrogen consumption dynamics and distiller’s dried grains with soluble results
suggested that assimilable nitrogen was made available for yeast growth and metabolism. Results showed that
20.25% (v/v) of ethanol and up to 96% fermentation efficiency could be obtained from ≈ 33% (w/v) dissolved
solids. Results also showed that the optimum sugar ratio of grain sorghum and sweet sorghum juice (18% sugar)
for VHG fermentation is 1–1 (sugar to sugar).

1. Introduction to decreased starch-to-water ratios; and increased productivity and rate

Biofuel processing technologies capable of increasing ethanol pro-
duction, cost-effectiveness, energy saving, and water efficiency in the
current dry-grind ethanol processes would significantly contribute to
meeting the growing demand for fuel in commercial transportation.
Very high gravity (VHG) fermentation, an evolving fermentation pro-
cess, has shown to be environmentally friendly, high yield, and cost-
effective in both ethanol and beer production (Udeh and Kgatla, 2013;
Yu et al., 2012; Puligundla et al., 2011; Nuanpeng et al., 2010;
Laopiaboon et al., 2009). VHG is described as “evolving” because it is
progressively being explored in the dry-grind process around the world
(Russell, 2003). VHG fermentation produces ethanol from mashes
containing higher concentrations of dissolved solids. The following are
some examples of VHG substrate concentrations reported by previous
researchers: sugar cane molasses (47.6% (w/v)); wheat mash (35% (w/
v)); rye (32–34% (w/v)); rice (31% (w/v)); and cornstarch (34% (w/v))
(Li et al., 2017; Chu-Ky et al., 2016; Puligundla et al., 2011). The ad-
vantages of VHG fermentation include a 58% potential reduction in
process water requirement due to high solid mash preparation; low
risks of bacterial contamination due to the inability for bacteria to
thrive under higher osmotic conditions; higher enzymatic activities due
of fermentation (Nuanpeng et al., 2010; Puligundla et al., 2011). In-
dustrial application of VHG fermentation technology is known to re-
duce the energy cost associated with downstream processes such as
distillation and stillage evaporation by 4% as well as capital and labor
costs (Nuanpeng et al., 2010; Bai et al., 2008).
A major challenge associated with VHG fermentation, however, is
the long hours involved to complete the process. This is often referred
to as stuck or sluggish fermentation (Russell, 2003; Peralta-Contreras
et al., 2013). Sluggish fermentation may arise due to inadequate yeast
metabolic activity to completely catalyze sugars into alcohol. The high
initial sugar concentrations (without nutrients), deficient amino ni-
trogen, low water activity, and higher ethanol concentration result in
high osmotic pressure, which causes high intracellular ethanol accu-
mulation and creates stress to the yeast cells. This situation negatively
impacts yeast dynamics and physiological fitness and slows the fer-
mentation process (Kawa-Rygielska and Pietrzak, 2014; Udeh and
Kgatla 2013Russell, 2003; Thomas et al., 1994). Long VHG fermenta-
tion duration (12 days) for wheat mash containing 36.5 g of dissolved
solids per 100 mL at 17 °C using Saccharomyces cerevisiae has been re-
ported (Jones and Ingledew, 1994). In another study, less than 50% of
total sugars from wheat mash was fermented for 9 days (Thomas et al.,

⁎
Corresponding author.
E-mail address: dwang@ksu.edu (D. Wang).

http://dx.doi.org/10.1016/j.indcrop.2017.10.028
Received 19 April 2017; Received in revised form 16 October 2017; Accepted 17 October 2017
0926-6690/ © 2017 Published by Elsevier B.V.
N.B. Appiah-Nkansah et al.
1994). Wang et al. (1998) also reported VHG fermentation duration of
120–114 h for rye and triticale mashes containing 28.5 g dissolved so-
lids per 100 mL at 20 °C.
These long fermentation durations may be solved by mash supple-
mentation. In VHG bioethanol production from cereal, the grain is
ground and mixed with water to form an evenly suspended con-
centrated slurry, which is cooked in the presence of enzyme alpha-
amylase, saccharified with glucoamylase, and fermented with
Saccharomyces cerevisiae to produce ethanol. During the starch-to-sugar
conversion process, also known as mashing or liquefaction, complex
nitrogenous compounds (proteins) and nutrients are released into the
media due to the digestion of protein and starch (Wang et al., 2008; Xu
et al., 2007; Thomas and Ingledew, 1990). These nitrogenous materials
are digested into amino acids, peptides, and free amino nitrogen (FAN)
and then used by microorganisms (Peralta-Contreras et al., 2013;
Thomas and Ingledew, 1990). FAN is a soluble protein that has been
digested into free amino acids and small peptides, which are essential
for yeast development and metabolism (Djameh et al., 2015;
Goldammer, 2008). Therefore, supplementing mash with nitrogenous
nutrients such as urea, yeast extract, peptone, spent brewer’s yeast, corn
steep liquor, and ammonium salts may stimulate ethanol production by
promoting the growth of yeast cells (Li et al., 2017; Pradeep et al.,
2012). Besides nitrogenous nutrients, mineral elements such as po-
tassium, magnesium, zinc, calcium, and manganese, which are required
in very little amounts, are known to be vital for yeast growth (Udeh and
Kgatla, 2013). In particular, urea supplementation in VGH systems is
known to be relatively cost-effective and result in favorable ethanol
yields (Chang et al., 2011; Jones and Inglede, 1994).
In comparison to corn, sweet sorghum is considered a more efficient
and cost-effective source of energy because of the lower nitrogen input
and water requirement for its production (Pfeiffer et al., 2013). Sugar
content in the juice may range from 13.7 to 15.89% (Wu et al., 2010)
and can directly be fermented by yeast Saccharomyces cerevisiae into
ethanol (Appiah-Nkansah et al., 2015). The juice is normally obtained
by mechanically crushing harvested stalks using roller mills or screw
presses (Harrison and O’Hara, 2013). Extraction of fermentable sugars
and nonstructural carbohydrates from sweet sorghum biomass and
grains by the diffusion method has recently been proposed (Appiah-
Nkansah et al., 2016). Grain sorghum, a drought-resistant crop with
good fermentation characteristics such as high starch content
(62.9–73.3%, db) and fast fermentation, has also been recognized as a
potential feedstock for the production of biofuels (Wu et al., 2010).
Currently, grain sorghum is blended with corn in some commercial
ethanol plants in the U.S. (Nghiem et al., 2016). While VHG ethanol
fermentation studies have been conducted using sweet sorghum juice
(Deesuth et al., 2015; Deesuth et al., 2016; Khongsay et al., 2012;
Nuanpeng et al., 2010; Laopaiboon et al., 2009), sorghum grains
(Chang et al., 2011), sweet potato mash (Zhang et al., 2010), cassava
starch (Yingling et al., 2011), and finger millet mash (Pradeep et al.,
2010), very little is known about VHG co-fermenting grain sorghum
starch with sweet sorghum juice. Our previous work suggested that very
high gravity fermentation using sweet sorghum juice and grain sor-
ghum could be applied to boost ethanol yields (Appiah-Nkansah et al.,
2015). To increase ethanol titers and productivity, it is therefore im-
perative that VHG fermentation from sweet sorghum juice co-fermented
with grain sorghum starch is explored. Results obtained may be useful
for sorghum growers and the ethanol industry.
In this study, ethanol productivity is enhanced using simultaneous sac-
charification and fermentation from mixtures of sweet sorghum juice and
ground sorghum grain under VHG conditions, both in the presence and
absence of urea supplementation. The microorganism used is Saccharomyces
cerevisiae. Saccharomyces cerevisiae is generally used for ethanol fermenta-
tion and baking purposes (Russell, 2003). The ideal ratio of the starch and
juice required for high ethanol yield and fermentation efficiency is estab-
lished. FAN consumption dynamics by the yeast and their effect on ethanol
levels and fermentation efficiency were also investigated.
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Industrial Crops & Products 111 (2018) 247–253
2. Materials and methods
2.1. Materials
Regular grain sorghum provided by Kansas State University
Agricultural Research Farm and sweet sorghum juice provided by Texas
A & M AgriLife Research Sorghum Breeding Program were used in this
study. The moisture content of the ground grain sorghum was de-
termined using American Association of Cereal Chemists (AACC) and
National Renewable Energy Laboratory (NREL) standard methods
(AACC, 2000; Sluiter et al., 2008). The grain flour was stored in sealed
plastic bags at room temperature. To obtain the juice, stalks were
harvested and crushed using a three-roller mill (Ampro Sugar Cane
Mill). Extracted juice was strained and immediately frozen at a tem-
perature of −23 °C. Prior to use, it was thawed at room temperature.
The juice was concentrated from about 11% sugars to 14%, 16%, and
18% sugar contents by a vacuum evaporation process at room tem-
perature using a Rotavapor (Büchi Labortechnik AG, Flawil, Switzer-
land). Urea (Certified ACS) was obtained from Fisher Scientific (Fair
Lawn, New Jersey, USA).
2.2. Starch and sugar content analysis
The total starch content of the materials was analyzed using a total
starch kit (Megazyme International) in adherence to the AACC standard
method (AACC, 2000). The sugar content of sweet sorghum juice was
analyzed using HPLC with a Rezex RCM Monosaccharide
(300 × 7.80 mm) column and a Refractive Index Detector
RID—G1362A (Agilent Technologies, Santa Clara, CA) following the
method described for ethanol in Section 2.9.
2.3. Protein content analysis
The crude protein analysis of the DDGS was completed using an
LECO TruMac N (St. Joseph, MI, USA) analyzer. Initially, the instru-
ment was prepared for operation as described in the TruMac operator’s
instruction manual. The system was then conditioned by analyzing 3–5
blanks. The calibration standard used was 0.5 g of EDTA. A crucible was
used to weigh 0.5 g of the sample, and the sample mass and sample
identification were entered into the software. The samples were run in
duplicate and the percent crude protein was recorded.
2.4. Free amino nitrogen (FAN)
FAN in all the mashes was analyzed by the ninhydrin-based method
as described in the modified International Ninhydrin Method (The
Brewery Analysis software LZV936, 2014). FAN is an important che-
mical property in starch fermentation that significantly correlates to
fermentation efficiency.
2.5. Mashing and liquefaction of sorghum with sweet sorghum juice
A detailed flowchart of the mashing, liquefaction, and sacchar-
ification process is shown in Fig. 1. For mashing, 18.14 g, 20.67 g,
23.2 g, and 41.32 g (db) grain sorghum flour were combined with
100 mL sweet sorghum juice of different sugar concentrations (14%,
16%, 18% and 0% (i.e., distilled water)) in 250 mL Erlenmeyer flasks to
form ≈ 33% (w/v) mashes. The flour was gently dispersed in preheated
juice (≈ 60–70 °C) in 100 mL Erlenmeyer flasks, and then 27.55 μL of
Liquozyme®SC DS (α—amylase 267 KNU/g, 1.266 g/mL; Novozyme
Inc., Franklinton, NC) and 0.14 g of KH2PO4 were added. The flasks
were carefully transferred into a 70 °C water-bath shaker and set to
operate at 180 rpm. The temperature of the water bath was gradually
increased from 70 °C to 90 °C in a 30 min period, kept at 90 °C for a few
minutes, and then lowered to 86 °C. Liquefaction continued for 60 min.
Flasks were then removed from the water bath, and materials sticking
N.B. Appiah-Nkansah et al. Industrial Crops & Products 111 (2018) 247–253

Fig. 1. Experimental procedure for mashing, lique-

faction, and SSF process.

on the inner surface of the flasks were pushed back into the mashes Ethanol yield% (v/v) was calculated according to the following equa-
with a spatula. The spatula and inner surface of the flasks were rinsed tion;
with 3–5 mL of distilled water. pH of the mashes was adjusted to ≈ 4.2
g
by 2 molar HCl after cooling to room temperature. ⎡
Ethanol yield %(v / v ) = ⎢mass of CO2 evolved (g )x
(
MMEthol 46
mol) ⎛
x⎜
1
⎤
⎥
⎢ g g ⎥
⎢
⎣
MMCO2 (44
mol) ⎜ρ
⎝ Ethol(0.789
ml ) ⎥
⎦
2.6. Microorganism and inoculum preparation
where MMEthol and MMCO2 are molecular masses of ethanol and CO2
The microorganism used in this study was Saccharomyces cerevisiae respectively and ρEthol is the density of ethanol at 20 °C.
(Lesaffre Yeast Corporation, Milwaukee, WI). Dry yeast was activated Ethanol fermentation efficiency was calculated as;
by dispersing 1.0 g of active dry yeast in 19 mL of pre-cultured broth Actual Ethanol yeild
(containing 20 g of glucose, 5.0 g of peptone, 3.0 g of yeast extracts, Ethanol fermentation efficiency = x 100(%)
Theoretical Ethanol yield
1.0 g of KH2PO4, and 0.5 g of MgSO4·7H2O per liter) and incubated at
38 °C for 30 min in a 12400 Incubator Shaker (New Brunswick
Scientific Co. Inc., Edison, NJ) operating at 200 rpm. The activated 2.8. Distillation
yeast culture had a cell concentration of about 1 × 109 viable cells/mL.
After 72 h of fermentation, the finished mash was transferred to a
2.7. Simultaneous saccharification and fermentation (SSF) 500 mL distillation flask. The Erlenmeyer flask was washed several
times with 100 mL of distilled water. Two drops of an antifoam agent,
Following enzymatic hydrolysis of substrates, 1.38 mL of the acti- Antifoam 24, at a concentration of 1.01 g/cm3 at 25 °C (Sigma-Aldrich
vated yeast culture, 137.7 μL of Spirizyme, (750 AGU/g, about 1.15 g/ Co., St. Louis, MO) were added to the distillation flask before the flask
mL) (Novozymes, Franklinton, NC), and 0.41 g of yeast extract were was placed on a heating unit to prevent foaming during distillation.
measured into mashes in each flask. Flasks were sealed with an S-air- Distillates were collected into a 100 mL volumetric flask immersed in
lock with mineral oil. Another batch of samples was prepared in the ice water. When distillates in the volumetric flask approached the
same procedure, this time with the addition of 0.96 g of urea to form 100 mL mark (about 99 mL), the volumetric flask was removed from
16 mM urea solution per 100 mL in each flask. In this study, 16 mM the distillation unit.
urea supplementation was used due to its potential effect on ethanol
production under very high gravity conditions (Chang et al., 2011; 2.9. Ethanol, glycerol, and organic acid analysis
Wang et al., 1998; Jones and Ingledew, 1993). Fermentation was con-
ducted at 30 °C for 72 h in an Incubator Shaker (New Brunswick Sci- Ethanol and glycerol concentrations were analyzed by high-pressure
entific Co. Inc., Edison, NJ) operating at 150 rpm. Fermentation per- liquid chromatography (HPLC) with a Rezex RCM Monosaccharide
formance was monitored by weighing the fermentation flasks over the (300 × 7.80 mm) column and a Refractive Index Detector RID-G1362A
3-day incubation period at 4, 8, 16, 24, 32, 40, 48, 56, 64, and 72 h of (Agilent Technologies, Santa Clara, CA). Twenty microliters of the
fermentation, respectively. Weight loss occurred due to the evolution of sample are injected into the mobile phase, which is HPLC-grade deio-
CO2 during the fermentation process (C6H12O6 → 2C2H6O + 2CO2↑). nized water. The mobile phase with the analyte was pumped at a high

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pressure of ≈33–42 bar into a column packed with monosaccharides

calcium ions. The elution rate was maintained at 0.6 mL/min, and
column temperature was maintained at 80 °C. The components (i.e.,
ethanol or glycerol) separated in the column were detected by the re-
fractive index detector and quantified. The theoretical ethanol yield
was determined using the total starch contents in the samples, assuming
0.511 g ethanol from 1 g of starch (Thomas et al., 1996). Fermentation
efficiencies were calculated as the actual ethanol yield divided by the
theoretical ethanol yield.
Organic acid concentrations were analyzed by HPLC with a Rezec
ROA-Organic acid H+ column using 5 mM H2SO4 solution as the mobile
phase.

2.10. Statistical analysis

Statistical analysis was performed using Microsoft® Excel® 2013.

Analysis of variance was performed using the SAS 9.4 service pack 4 for
Windows (SAS Institute Inc., Cary, NC). All experiments were con-
ducted at least in duplicate. Means of the duplicated experiments were Fig. 2. Effect of the ratios of grain sorghum flour and sweet sorghum juice on ethanol
yield (with and without urea supplementation) during VHG fermentation (33% mashes,
reported. Pairwise comparisons were performed for the means using the
v/v).
Tukey adjustment at a significance level of 0.05.

3. Results and discussion

3.1. Effects of urea supplementation and of grain starch and juice ratios on
ethanol production

All results are means of duplicate experiments. The chemical com-

position of the materials used in this study is represented in Table 1. All
samples contained mashes made up of sugar concentrations of about
33 g of dissolved carbohydrates per 100 mL. The ethanol fermentation
profile and fermentation efficiencies of mashes with and without urea
supplementation are represented in Figs. 2 and 3, respectively. In
general, urea supplemented slurries achieved increased ethanol fer-
mentation production. Ethanol levels from supplemented mashes were
generally different than their non-supplemented counterparts based on
pairwise comparisons at 5% level of significance. The addition of urea
as a source of usable nitrogen source most likely played an important
role in increasing the overall metabolic heat generation while pro- Fig. 3. Effect of the ratios of grain sorghum flour and sweet sorghum juice on ethanol
fermentation efficiency (with and without urea supplementation) during VHG fermen-
tecting the yeast from osmotic stress due to higher substrate con-
tation (33% mashes, v/v).
centrations (Theerarattananoon et al., 2008). This might be the fun-
damental reason for the enhanced ethanol production. The plateau of
the fermentation performance curve (Fig. 2) suggests that ethanol The high efficiency (Fig. 3) obtained in this work suggested that more
production of all the supplemented mashes were completed within the sugars were consumed in a shorter period. The accelerated sugar uptake
first 40 h. Ethanol concentrations after the first 48 h ranged from 19.61 within 48 h agrees with previous results obtained from rye and triticale
to 19.90% (v/v). under VHG conditions (Wang et al., 1998). We can conclude that for
Based on one-way ANOVA results, different ratios of grain sorghum maximum ethanol production from VHG grain sorghum and sweet
and sweet sorghum juice had a significant effect on ethanol production sorghum juice, a ratio of 50% grain sorghum to 50% sweet sorghum
and efficiency (P < 0.05). Supplemented mash containing 18% juice juice (18% sugar) should be considered. The sweet sorghum juice may
produced 3.7 g/L/h ethanol after the first 40 h. This sample produced be concentrated by the addition of syrup as suggested by Nghiem et al.
the highest ethanol concentration of 20.25% (v/v) with about 96% (2016).
fermentation efficiency (Fig. 3) after 72 h. After 48 h of fermentation, The yeast cell is generally considered to be the heart of ethanol
98% of the total ethanol was produced by a supplemented sample fermentation (Russell, 2003), and it is affected by the following con-
containing 18% juice sugars. Also, ethanol level from the 18% juice ditions: environment, nutrition, and gene type (Udeh and Kgatla,
slurry was nearly 13% higher than that from a non-supplemented 2013). Yeast cells will, therefore, struggle to maintain their viability
slurry. Besides higher ethanol yields, supplemented mashes also took and metabolic activity when excessively exposed to any of these factors
less time to complete fermentation due to the rapid substrate con- (Udeh and Kgatla, 2013). VHG mashes without urea supplementation,
sumption. Maximum ethanol yields ranged from 19.93 to 20.25% (v/v). in this work, generally displayed slow fermentation rates with the final

Table 1
Initial characteristics of grain sorghum starch and sweet sorghum juice.

Samples Sugar (%) Starch (%) Crude Protein (%) Nitrogen FAN mg/L Moisture Content (%)

Grain sorghum 72 13.07 2.09 83.71 6

Sweet sorghum juice 13

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N.B. Appiah-Nkansah et al.
Table 2
DDGS nitrogen and protein contents (%) with and without urea supplementation.
Samples With supplement
Nitrogen (%)
14% sugar + 23.2 g (db) 5.77 ± 0.07
16% sugar + 20.67 g (db) 5.65 ± 0.05
18% sugar + 18.14 g (db) 5.50 ± 0.05
41.32 g (db) (No juice) 6.17 ± 0.13
Values are means ± SD (standard deviation).
ethanol concentration ranging from16.59–19.47% (v/v) after 72 h. The
sluggish fermentation performance could be attributed to the inhibition
of yeast growth and the development of osmotic stress conditions due to
higher sugar levels (≈33 g/100 mL). The ethanol fermentation profile
curves suggested that the process would have continued after 72 h.
Among the unsupplemented mashes, those contained no juice yielded
the highest amount of ethanol (19.47% (v/v)). The high yield obtained
from the grain sorghum flour mash could only be attributed to the grain
protein digestibility as further explained in Section 3.3. It has been
established that besides starch content, protein digestibility positively
affects ethanol production from grain sorghum (Wang et al., 2008; Wu
et al., 2007).
3.2. Effect of additional factors on ethanol production
Several factors such as inoculation size, temperature, FAN, pH,
fermentation time, substrate concentration, and agitation rate all affect
ethanol production (Zabed et al., 2014). Although all experiments in
this study were conducted under specified operation conditions
(109 cells/ml, 30 °C, 16 mM urea), we theorized that the combination of
these factors had a synergistic effect on ethanol production and effi-
ciency based on the following reasons. First, increased initial inocula-
tion size of 109 cells/mL may have contributed to the shortened ethanol
fermentation duration of 16 mM urea-supplemented mashes by facil-
itating the rapid consumption of sugars (Fig. 1). Second, the fermen-
tation temperature in this study was maintained at 30 °C, which is
known to be the optimum temperature for S. cerevisiae (Zabed et al.,
2014). Previous researchers have reported low ethanol yields with
lower temperatures (Jones et al., 1994; Thomas and Ingledew, 1990;
Thomas et al., 1994; Thomas et al., 1993). Additionally, higher tem-
peratures could denature the tertiary structures of the enzymes and
nucleic acids that regulate microbial activity and the fermentation
process (Zabed et al., 2014; Russell, 2003).
Industrial Crops & Products 111 (2018) 247–253
Without supplement
Protein (%) Nitrogen (%) Protein (%)
36.06 ± 0.46 4.97 ± 0.08 31.07 ± 0.5
35.32 ± 0.28 4.45 ± 0.10 27.82 ± 0.62
34.39 ± 0.28 4.37 ± 0.07 27.33 ± 0.42
38.59 ± 0.79 5.94 ± 0.18 37.12 ± 1.13
3.3. Free amino nitrogen
In general, the amount of usable nitrogen in media is a function of
FAN levels. It has been suggested that the required FAN levels for VHG
fermentation are 150 g/mL (Dlamini et al., 2015; Thomas and
Ingledew, 1990). A wide FAN variation of 31–139 mg/L in the wort of
different sorghum grain has also been reported (Dlamini et al., 2015).
In the present work, a separate experiment was conducted to study the
production and the consumption kinetics of FAN, and the results are
represented in Fig. 5a–b. It must be noted that FAN analysis was done
following the liquefaction stage (i.e., 0 h before SSF) at 24, 48 and 72 h
of the fermentation. With the exception of the mash containing 0%
sugars, FAN uptake for other unsupplemented mashes occurred in the
first 50 h into the fermentation (Fig. 5a). The crude protein and ni-
trogen contents of the raw ground grain in this study were 13.07% and
2.09%, respectively, and those for the DDGS were 37.12% and 5.94%,
respectively. The nitrogen content after the liquefaction and SSF pro-
cess increased by about 184%. Therefore, it is clear that the initial FAN
level (83.71 mg/L) and the rapid decline of the FAN profile of the 0%
sugars unsupplemented mash is due to grain sorghum protein digest-
ibility and the release of usable nitrogen into the media during lique-
faction and fermentation. This facilitated yeast metabolism and led to
higher fermentation efficiency by 91% and higher yield by 19.47% (v/
v). This is consistent with previous findings that high protein digest-
ibility resulted in higher levels of FAN for yeast metabolism (Nghiem
et al., 2016).
With regard to the supplemented mashes, most of the FAN uptake
occurred during the first 30 h into the fermentation (Fig. 5b) followed
by a gradual rise in the production of FAN after the first 30 h. This could
be attributed to the secretion of peptide enzyme from yeast cells into
the media due to partial autolysis of cells (Peralta-Contreras et al.,
2013; Wang et al., 1998; Thomas et al., 1993) or the release of FAN due
to protein degradation inside the cell with urea supplementation (Wang
et al., 1998). Additionally, the increased nitrogen and protein contents
Fig. 4. Glycerol production during VHG fermenta-
tion of grain sorghum flour and sweet sorghum juice
at various ratios.
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of the DDGS (Table 2) from the initial 13.07% and 2.09% suggested
that the release of nitrogen into the media resulted in enhanced yeast
activities and rapid glucose uptake, shortened fermentation time, and
improved fermentation yields and efficiencies. Similar findings were
also reported by Peralta-Contreras et al. (2013).

3.4. Co-products of yeast metabolism

Glycerol, an important co-product of yeast alcoholic fermentation,

helps in the maintenance of cell redox balance and also serves as an
osmoprotectant; therefore, it helps to maintain the high viability of
yeast cells under VHG conditions and is usually produced in small
amounts (Russell, 2003). The glycerol (Fig. 4) and acetic acid produced
in this study ranged from 0.05 to 0.07 mg/mL and 0.00–0.16 mg/mL.
The production of the co-products could most likely be attributed to the
yeast response mechanism, which is triggered by exposure to high
substrate concentration. In general, it can be observed that supple-
mented mashes produced slightly higher amounts of co-products than
non-supplemented ones. Thus, more glycogen was hydrolyzed and
made available to sustain the supplemented media. The production of
more co-products could be due to high osmotic pressure due to in-
creased sugar concentration when supplemented with urea. Kawa-
Rygielska and Pietrzak (2014) described that during ethanol fermen-
tation of VHG maize mashes supplemented with spent brewer’s yeast,
more glycerol and organic acids were produced. Similar results with the
production of glycerol and organic acids were observed during the VHG
fermentation of wine musts supplemented with ammonium sulfate
(Bely et al., 2003). In this work, small concentrations of acetic acid
(0.13–17 mg/mL) were observed at the end of the fermentation of
studied mashes.

3.5. Mass balance

A mass balance was developed for the conversion of 18.48 g grain

sorghum starch and 18% sugar content in sweet sorghum juice with
urea supplementation as shown in Fig. 6. 18.48 g grain sorghum and
18% sugar content of sweet sorghum juice produced 15.98 g ethanol
Fig. 5. FAN content changes as a function of fermentation time during VHG fermentation,
(20.25% (v/v)), 15.44 g CO2, 8.68 g DDGS, 0.16 mg/mL acetic acid,
a-with supplementation, and b-without supplementation.
and 0.06 mg/mL glycerol.

4. Conclusion

In this work, ethanol yield and ethanol fermentation efficiency of

Fig. 6. Mass balance of VHG fermentation using substrates contain-

ing18.48 g (db) grain sorghum and 100 mL sweet sorghum (18%
sugar) with supplementation.

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VHG mashes using ground sorghum grain and sweet sorghum juice at
various ratios were investigated. On average, fermentation of the sup-
plemented mashes was completed within the first 40 h. Maximum
ethanol titers (19.93–20.25% (v/v)) were obtained in less than two
days of fermentation with high ethanol efficiency (93.17–96.26%). The
18% juice concentration ratio produced the highest ethanol level
(20.25% (v/v)) and ethanol productivity of 3.7 g/L/h. In other words,
using VHG grain sorghum prepared with sweet sorghum juice at a ratio
of 50% grain sorghum to 50% sweet sorghum juice (18% sugars) would
result in higher ethanol production. Additionally, factors such as in-
oculation size, temperature, and FAN in combination with nutrient
supplementation influenced ethanol yield and ethanol fermentation
efficiency. Improving the production process of bioethanol to make it
more efficient using sweet sorghum juice and grain sorghum will result
in better utilization of the feedstock. This will translate into cost re-
duction, which will make the sorghum industry more profitable and
more attractive.
Acknowledgment
This work was supported by United Sorghum Checkoff Program
with grant number of RN004-14. This publication is contribution no.
17-151-J from the Kansas Agricultural Experiment Station, Manhattan,
KS.
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