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Article
Comparison of Cassava Starch with Corn as a
Feedstock for Bioethanol Production
Sarocha Pradyawong 1 , Ankita Juneja 2 , Muhammad Bilal Sadiq 1 , Athapol Noomhorm 1 and
Vijay Singh 2, *
1 Department of Food Engineering and Bioprocess Technology, Asian Institute of Technology, Pathum Thani
12120, Thailand; sarocha.prad@gmail.com (S.P.); m.bilalsadiq@hotmail.com (M.B.S.); athapol@ait.asia (A.N.)
2 Department of Agricultural and Biological Engineering, University of Illinois at Urbana-Champaign,
Urbana, IL 61801, USA; ajuneja@illinois.edu
* Correspondence: vsingh@illinois.edu; Tel.: +1-217-333-9510

Received: 9 October 2018; Accepted: 10 December 2018; Published: 13 December 2018 

Abstract: Cassava is a high potential feedstock for bioethanol production in Asian countries,
primarily due to high yield of carbohydrate per unit land, and its ability to grow on marginal lands
with minimal agrochemical requirements. The objective of this study was to compare the bioethanol
production from cassava starch with corn starch using a conventional and a raw/granular starch
hydrolyzing process (GSH). The fermentation performance of cassava starch was compared with
three corn starch types with different amylose: Amylopectin ratios. The final ethanol concentration
with cassava starch was similar to that of two corn starch types, dent corn and waxy corn for both
processes. For the cassava starch, the ethanol concentration achieved with GSH process was 2.8%
higher than that in the conventional process. Cassava starch yielded the highest fermentation rates of
the four starches investigated, during the conventional process. Ethanol production and fermentation
profiles comparable with corn, a widely used feedstock, makes cassava starch an attractive substrate
for bioethanol production.

Keywords: bioethanol; corn; cassava starch; granular starch hydrolysis; fermentation

1. Introduction
Increased energy consumption and raising environmental concerns have led the world to look
for alternative energy sources, especially in the transportation sector. Bioethanol is considered as
one of the most promising renewable transportation fuels, and is already blended into gasoline in
several countries. The global production of bioethanol in 2017 was estimated as 27.05 billion gallons,
with the US contributing about 58% (15.8 billion gallons) [1]. Since the economics of bioethanol are
highly dependent on the feedstock price, there has been an ongoing effort to search for low cost and
easily available raw materials [2]. Most of the bioethanol in the United States is produced from corn.
However, some countries, particularly in continental Asia, the use of another starch crop, cassava,
for energy purposes is encouraged. Cassava or tapioca (Manihot esculenta), is a plant native to South
America, with high nutritional value due to its starchy tuberous roots. Global production of cassava is
about 281 million tons a year, with Asia contributing about one third of this production [3]. More than
half of this global supply is contributed by Africa, where cassava is a primarily used as a food source.
Brazil is third largest producer of cassava in the world [3]. One of the major advantages of cassava
is its drought-tolerance and capability of growing on marginal soils and degraded lands, and still
containing third highest yield of carbohydrate per hectare after sugarcane and sugar beet [4]. The low
agro-chemical requirements reduce the energy input for growth of cassava biomass making the use
of this crop energy-efficient. Another species of the same genus (Manihot glaziovii) is also considered

Energies 2018, 11, 3476; doi:10.3390/en11123476 www.mdpi.com/journal/energies


Energies 2018, 11, 3476 2 of 11
Energies 2018, 11, x FOR PEER REVIEW 2 of 11

considered
as a bioethanol as a bioethanol
source for its source forto
ability itsgrow
ability ontonon-arable
grow on non-arable
lands [5]. lands
Due to[5]. Due
high to high and
drought drought
heat
and heat tolerance, high yield of starch, and its capability of growing
tolerance, high yield of starch, and its capability of growing on poor soils without high maintenance, on poor soils without high
maintenance,
cassava is thuscassava
gainingisattention
thus gainingas a attention
bioethanolassource a bioethanol
all oversource all over the world.
the world.
The
The conventional method employed for ethanol production from cassava
conventional method employed for ethanol production from cassava roots
roots usually
usually requires
requires
gelatinization of starch followed by liquefaction and saccharification
gelatinization of starch followed by liquefaction and saccharification and consequently fermenting and consequently fermenting the
the sugars formed with yeast or bacteria [6,7]. Apart from the conventional
sugars formed with yeast or bacteria [6,7]. Apart from the conventional conversion method, raw starch conversion method, raw
starch hydrolysis
hydrolysis has alsohasbeenalso been
used forused for production
production of ethanol of from
ethanol fromstarch
cassava cassava starch
using mixedusing mixed
culture of
culture of microorganisms [8,9]. Both processes (conventional and raw
microorganisms [8,9]. Both processes (conventional and raw starch hydrolysis) are used to convert starch hydrolysis) are used to
convert
starch tostarch
ethanol to for
ethanol for both
both corn and corn
cassava.and Since
cassava.cornSince corn is a well-established
is a well-established feedstock forfeedstock
ethanolfor in
ethanol in the United States [10], it is imperative to know the practical yields
the United States [10], it is imperative to know the practical yields of cassava in comparison to corn of cassava in comparison
to corntostarch
starch to understand
understand the industrial
the industrial scale feasibility
scale feasibility of cassavaof ascassava
a feedstockas afor
feedstock
bioethanolfor production.
bioethanol
production. The cost of cassava ethanol is theoretically estimated
The cost of cassava ethanol is theoretically estimated as $0.237/L [11]. The theoretical ethanol as $0.237/L [11]. The theoretical
yield
ethanol yield (kg/ha/year) from cassava has been estimated as three
(kg/ha/year) from cassava has been estimated as three times that of corn [12]. However, there times that of corn [12]. However,
is a lack
there is a lack
of studies of studies
comparing comparing
the ethanol the ethanol
production production from
experimentally experimentally from these
these two ethanol two ethanol
producing starch
producing starch crops. Bioethanol production from cassava starch
crops. Bioethanol production from cassava starch has been attempted with various mixed strains has been attempted with various of
mixed strains of bacteria and yeast [13,14], however, there are limited
bacteria and yeast [13,14], however, there are limited studies employing process similar to corn dry studies employing process
similar to corn
grind, which is dry
widelygrind,
used which is widely
process used process
for bioethanol for bioethanol
production production
in US. Although in US. Although
both cassava and corn
both cassava and corn share the traits of starchy crops, there are some
share the traits of starchy crops, there are some structural differences in both the starches, which structural differences in both
are
the starches, which are shown in Table 1, major one being the ratio
shown in Table 1, major one being the ratio of amylose to amylopectin. Amylose is a linear chain of amylose to amylopectin.
Amylose is a linear
polysaccharide chain polysaccharide
of glucose molecules connected of glucose molecules
by α-1,4 connected
glycosidic bonds,by α-1,4 glycosidic
whereas amylopectin bonds,
is a
whereas amylopectin is a water soluble, highly branched polymer,
water soluble, highly branched polymer, where glucose moelecules are connected with α-1,4 glycosidicwhere glucose moelecules are
connected
bonds and with α-1,4 glycosidic
the branching bonds
takes place withandα-1,6
the branching takes place
glycosidic bonds, with α-1,6
occurring everyglycosidic bonds,
24 to 630 glucose
occurring every 24 to 630 glucose units (Figure 1). The extraction of corn
units (Figure 1). The extraction of corn starch is complex due to the need for steeping to loosen starch is complex due to the
the
need for steeping to loosen the protein matrices surrounding the starch,
protein matrices surrounding the starch, whereas cassava starch is easier to extract due to low quantity whereas cassava starch is
easier to extract due to low
of proteins and fats in the tuber [15].quantity of proteins and fats in the tuber [15].

Figure 1.
Figure Structures of
1. Structures of amylose
amylose and
and amylopectin.
amylopectin.

The objective of this study was to compare the fermentation profile and ethanol production of
The objective of this study was to compare the fermentation profile and ethanol production of
cassava starch with corn starch, using two approaches: the conventional and the granular starch
cassava starch with corn starch, using two approaches: the conventional and the granular starch
hydrolysis (GSH) process (Figure 2). For a broader comparison, three commercially used corn types
hydrolysis (GSH) process (Figure 2). For a broader comparison, three commercially used corn types
with different amylose content (dent corn, waxy corn and high amylose corn) were used. Regular
dent corn starch contains about 20 to 30% amylose and 70 to 80% amylopectin, whereas waxy corn
has <5% and high amylose corn has >35% amylose content [16].

Table 1. Comparison of normal corn starch and cassava starch [17].

Corn Starch Cassava Starch


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Content of starch (% db) 64 to 78 30 to 35
Average granular diameter (μm) 10 15
Amylose content (% of total starch) 20 to 30
with different amylose content (dent corn, waxy corn and high amylose corn) 17were used. Regular dent
Degree of polymerization (DPn) 3000 800
corn starch contains about 20 to 30% amylose and 70 to 80% amylopectin, whereas waxy corn has <5%
Gelatinization temperature range (°C) 75 to 80 65 to 70
and high amylose corn has >35% amylose content [16].

Figure 2. Schematics of the laboratory-scale conventional dry grind and GSH (granular starch
Figure 2. Schematics
hydrolysis) process for of the laboratory-scale
ethanol production. conventional dry grind and GSH (granular starch
hydrolysis) process for ethanol production.
Table 1. Comparison of normal corn starch and cassava starch [17].
2. Materials and Methods
Corn Starch Cassava Starch

2.1. Materials Content of starch (% db) 64 to 78 30 to 35


Average granular diameter (µm) 10 15
Amylose
Dent corn starchcontent (% of~30%
(Melojel, total starch)
amylose), waxy20corn
to 30 starch (Amioca,17~1% amylose), high
amylose corn Degree
starch of polymerization
(Hylon V, ~65%(DPn) 3000
amylose) and cassava 800 3600, Ingredion,
starch (Novation
Gelatinization temperature range (◦ C) 75 to 80 65 to 70
Indianapolis, IN, USA) were obtained from Ingredion Incorporated (Westchester, IL, USA). Enzymes,
including conventional α-amylase (Spezyme® CL, with reported activity of 15,225 AAU/g; AAU:
2. Materials
alpha amylaseandunits),
Methods
conventional glucoamylase (Distillase® SSF, with reported activity of 380
GAU/g; GAU: glucoamylase unit), and GSH enzyme (StargenTM 002, with an activity of 570 GAU/g;
2.1. Materials
GAU: glycoamylase unit), were provided by DuPont Industrial Biosciences (Palo Alto, CA, USA).
Dent cornactive
Conventional starchdry
(Melojel, ~30%obtained
yeast was amylose),
fromwaxy
the corn starch (Amioca,
Fermentis-Lesaffre Yeast~1% amylose),
Corporation
high amylose corn starch (Hylon V, ~65% amylose) and cassava starch (Novation 3600,
(Milwaukee, WI, USA). All starch samples and enzymes were stored at 4 °C until use. All the Ingredion,
Indianapolis,
chemicals used IN,were
USA)ofwere obtained
analytical fromand
grade Ingredion Incorporated
were purchased from(Westchester, IL, USA).
Sigma Aldrich Enzymes,
(St. Louis, MO,
including conventional α-amylase (Spezyme ® CL, with reported activity of 15,225 AAU/g; AAU:
USA).
alpha amylase units), conventional glucoamylase (Distillase® SSF, with reported activity of 380 GAU/g;
GAU: glucoamylase
2.2. Conventional unit), and GSH enzyme (StargenTM 002, with an activity of 570 GAU/g;
Process
GAU: glycoamylase unit), were provided by DuPont Industrial Biosciences (Palo Alto, CA, USA).
Conventional active dry yeast was obtained from the Fermentis-Lesaffre Yeast Corporation (Milwaukee,
WI, USA). All starch samples and enzymes were stored at 4 ◦ C until use. All the chemicals used were
of analytical grade and were purchased from Sigma Aldrich (St. Louis, MO, USA).
Energies 2018, 11, 3476 4 of 11

2.2. Conventional Process


For liquefaction and simultaneous saccharification and fermentation (SSF), a conventional dry
grind process as described previously in literature [18,19] was used, with slight modifications (Figure 2).
In summary, all four starch (30 g) samples were mixed with distilled water to make a slurry with
25% solids on dry basis. All the experiments were performed in triplicate. The pH of the slurry
was adjusted to 5.7 using 2 N sulfuric acid, and 10 µL of Spezyme® CL was added for liquefaction.
The liquefaction was performed in shaker water bath at 85 ◦ C for 2 h with continuous agitation at
100 rpm. The liquefied slurry was then transferred in 1-L flask and cooled down to 32 ◦ C and pH
was adjusted to 4.2 using 2 N sulfuric acid. Distillase® SSF (22.56 µL), urea (0.4 g/100 g of starch),
yeast malt broth (4 g/100 g of starch), and 5 mL yeast culture (1.8 × 108 cells/mL) were then added
for SSF. Yeast culture was prepared by dispersing 11 g of active dry yeast and 1 g yeast malt broth in
89 mL of distilled water and agitated at 50 rpm and 30 ◦ C for 20 min. The broth was fermented at 32 ◦ C
for 72 h with continuous agitation at 100 rpm in shaking water bath. Fermentation was monitored by
drawing 1-mL sample from the fermentation slurry at 0, 2, 4, 6, 8, 20, 24, 48, and 72 h of SSF.

2.3. Granular Starch Hydrolysis (GSH)


GSH of starch slurry (25% dry solids content) was carried out by the method previously described
in thecliterature [18,19], with slight modifications (Figure 2). To summarize, the pH of slurry was
adjusted to 4.2 by using 2 N sulfuric acid and 37.7 µL StargenTM 002 was added to slurry and
temperature was maintained at 48 ◦ C for 2 h with continuous agitation at 100 rpm. The slurry was
then cooled down to 32 ◦ C followed by pH adjustment to 4.2 using 2 N sulfuric acid. StargenTM
002 (37.7 µL), urea (0.4 g/100 g of starch), yeast malt broth (4 g/100 g of starch), and 5 mL yeast
(1.8 × 108 cells/mL) were then added to carry out fermentation. Yeast culture was prepared similarly
as described above. The broth was fermented at 32 ◦ C for 72 h with continuous agitation at 100 rpm in
shaking water bath. Fermentation was monitored by drawing 1-mL sample from the fermentation
slurry at 0, 2, 4, 6, 8, 20, 24, 48, and 72 h of SSF.

2.4. HPLC analysis


Samples collected during both conventional and GSH process, were centrifuged (model 5415 D,
Eppendorf, Westbury, NY, USA) at 11,000 rpm for 3 min. The supernatant was then filtered through
0.2-µm syringe filter into 1-mL HPLC vials. The vials were immediately stored at −20 ◦ C until analysis.
The filtrate was analyzed using HPLC with an ion-exclusion column (Aminex HPX-87H, Bio-Rad,
Hercules, CA) and refractive index detector (model 2414, Waters Corporation, Milford, MA, USA).
The mobile phase was 0.005 M sulfuric acid at 50 ◦ C at a flow rate of 0.6 mL/min. Each sample
was analyzed to determine the concentrations of ethanol, glucose, maltose, maltotriose, and glycerol.
The HPLC was calibrated with standards and each sample was injected twice for analysis.

2.5. Fermentation Rates and Ethanol Yields


Ethanol concentration was measured and was plotted against time. Fermentation rates were
calculated during the initial linear phase of fermentation (up to 8 h) by calculating the slope of the plot.
Starch-to-ethanol conversion efficiencies were calculated as the ratio of actual ethanol yields with the
theoretical ethanol yield (Equation (1)):

EEtOH
ηEtOH = ·100 (1)
ETh_EtOH

where ETh_EtOH is theoretical ethanol yield, L/kg dry starch; EEtOH is the actual ethanol yield, L/kg
dry starch.
Energies 2018, 11, 3476 5 of 11

Energies 2018, 11, x FOR PEER REVIEW 5 of 11


Theoretical yields were estimated based on the starch content, assuming complete starch
conversionFinal
andethanol concentration and
100% fermentation fermentation
efficiency. Actualrate for all yields
ethanol four starches was analyzed
were calculated by analysis
based on methods
of variance (1-way ANOVA)
described elsewhere [20]. and Fisher’s least significant difference (LSD) tests (SAS version 9.4,
Cary, NC, USA). The level selected to show the statistical significance in all cases was 5% (p < 0.05).
All the experiments
2.6. Statistical Analysis were performed in triplicates.
Final ethanol concentration and fermentation rate for all four starches was analyzed by analysis
3. Results
of variance (1-way ANOVA) and Fisher’s least significant difference (LSD) tests (SAS version 9.4, Cary,
3.1. Conventional
NC, USA). The levelDry Grind to
selected Process
show the statistical significance in all cases was 5% (p < 0.05). All the
experimentsThe were performed
fermentation in triplicates.
profiles for all starches during the conventional dry grind process are shown
in Figure 3. The final ethanol concentration for dent corn starch (133.2 g/L) was the highest among
3. Results
the four starches analyzed. However, statistically there was no significant difference in the final
ethanol concentrations of the three starches—dent corn, waxy corn and cassava starch—which
3.1. Conventional
indicated theDry Grind Process
suitability of cassava starch to be used as a comparable starch crop for bioethanol
production
The in Asianprofiles
fermentation countries.
forThe ethanol yield
all starches duringwiththehigh amylose corn
conventional drystarch
grindwas lower are
process thanshown
the in
other three starches. This was expected due to the high amylose:amylopectin ratio in the high
Figure 3. The final ethanol concentration for dent corn starch (133.2 g/L) was the highest among the
amylose corn starch, which has previously been reported to decrease the ethanol yield [21]. Since
four starches analyzed. However, statistically there was no significant difference in the final ethanol
amylose is a linear chain polymer, it aligns itself with strong hydrogen bonds, making it difficult for
concentrations
the enzymes ofto
the three
act, starches—dent
resulting in reduced corn, waxyofcorn
hydrolysis and cassava
amylose [21]. High starch—which indicated the
temperatures provided
suitability
duringofliquefaction
cassava starch to beinused
of starches as a comparable
conventional dry grindstarch
processcrop forinbioethanol
assist breaking down production
these in
Asianhydrogen
countries. The
bonds andethanol
make theyield withavailable
amylose high amylose cornThe
to enzymes. starch
finalwas lower
ethanol than the
produced other three
by cassava
starches.
(124.4This was expected
g/L) starch was higher due
thantothat
thereported
high amylose:amylopectin
previously in literatureratio in the
(~71 g/L) [22].high amylose corn
About 5 g/L and 1 g/L, glucose was left unconverted in case
starch, which has previously been reported to decrease the ethanol yield [21]. Since of dent cornamylose
and cassava,
is a linear
respectively; whereas complete conversion was observed for waxy corn and high
chain polymer, it aligns itself with strong hydrogen bonds, making it difficult for the enzymes amylose corn starchto act,
(Figure
resulting 3). However,
in reduced glucoseofreleased
hydrolysis amylose in[21].
the first
High8 temperatures
h is highest for cassava during
provided starch (119.5 g/L), of
liquefaction
indicating better breakdown of starch in to polysaccharides during liquefaction. In contrast, high
starches in conventional dry grind process assist in breaking down these hydrogen bonds and make
amylose corn starch released least glucose during fermentation, which can be attributed to the
the amylose available to enzymes. The final ethanol produced by cassava (124.4 g/L) starch was higher
inefficient hydrolysis due to high amylose content. Similar trend of low glucose formation with high
than amylose
that reported
contentpreviously
was observed in literature (~71
in an earlier g/L)
study [22].
[21].

Figure
Figure 3. Comparison
3. Comparison of ethanol
of ethanol and and glucose
glucose profiles
profiles of dent
of dent corncorn (DC),
(DC), waxywaxy
corncorn
(WC),(WC),
highhigh
amylose
corn amylose
(HAC), corn
and (HAC),
cassavaand cassava
starch (CS)starch (CS)conventional
during during conventional fermentation
fermentation at 30at◦30
(SSF (SSF °C for
C for 72 72
h with
h with glucoamylase
glucoamylase and yeast).and yeast).

AboutThe initial
5 g/L andplot of ethanol
1 g/L, vs time
glucose was (up
left to 8 h) provides
unconverted inacase
linear
of trend (Figure
dent corn and4a), slope ofrespectively;
cassava, which
was used to calculate the fermentation rate. All starches for conventional process showed good linear
whereas complete conversion was observed for waxy corn and high amylose corn starch (Figure 3).
However, glucose released in the first 8 h is highest for cassava starch (119.5 g/L), indicating better
breakdown of starch in to polysaccharides during liquefaction. In contrast, high amylose corn starch
Energies 2018, 11, 3476 6 of 11

released least glucose during fermentation, which can be attributed to the inefficient hydrolysis due to
high amylose content. Similar trend of low glucose formation with high amylose content was observed
in an earlier study [21].
The initial plot of ethanol vs time (up to 8 h) provides a linear trend (Figure 4a), slope of which
wasEnergies
used 2018,
to calculate the fermentation
11, x FOR PEER REVIEW rate. All starches for conventional process showed good 6 of 11linear
fit for first 8 h of fermentation. For GSH process, high amylose corn was the only starch that did not
fit for first 8 h of fermentation. For GSH process, high amylose corn was the only starch that did not
fit the linear plot (R2 = 0.47), where other fits could be investigated. The fermentation rates of all
fit the linear plot (R2 = 0.47), where other fits could be investigated. The fermentation rates of all four
four starches are shown in Table 2. Highest fermentation rate was observed for cassava starch (0.965),
starches are shown in Table 2. Highest fermentation rate was observed for cassava starch (0.965),
followed by high amylose corn starch (0.931). During the initial hours of fermentation, the fermentation
followed by high amylose corn starch (0.931). During the initial hours of fermentation, the
ratefermentation
of cassava is observed
rate to is
of cassava beobserved
statistically
to behigher than higher
statistically that ofthan
dentthat
corn. Despite
of dent corn.low finallow
Despite ethanol
concentration,
final ethanolhigh fermentation
concentration, highrate for high amylose
fermentation rate for corn
high starch
amylosecancorn
be due to can
starch the better
be duefunctioning
to the
of the yeast in presence of low glucose concentrations.
better functioning of the yeast in presence of low glucose concentrations.

Figure
Figure 4. Fermentation
4. Fermentation ratescalculated
rates calculatedasasslopes
slopesof
ofethanol
ethanol concentration
concentration plotted
plottedwith
withtime
timeduring
during the
first 8 h of fermentation during (a) conventional and (b) GSH (granular starch hydrolysis)process.
the first 8 h of fermentation during (a) conventional and (b) GSH (granular starch hydrolysis) process.

Table 2. Fermentation rates for ethanol production during the conventional and GSH process.

Starch Type Conventional GSH


Energies 2018, 11, 3476 7 of 11

Table 2. Fermentation rates for ethanol production during the conventional and GSH process.

Energies 2018, 11, x FOR PEER REVIEW


Starch Type Conventional GSH 7 of 11
Dent corn 0.7415 b 0.9355 a
Dent corn
Waxy corn 0.7415c b
0.649 0.9355 a0.876 b
Waxy
High amylose corn
corn 0.649ac
0.931 0.876 b0.102 c
High amylose corn
Cassava 0.931aa
0.965 0.102 c 0.928 a
Cassava 0.965 a 0.928 a
Similar letters represent statistically similar values at p < 0.05.
Similar letters represent statistically similar values at p < 0.05.

The fermentation
The fermentationefficiencies of starch
efficiencies to ethanol
of starch to ethanol conversion
conversion inin
conventional
conventionalprocess
processranged
ranged from
62.8%from
to 81.9%, highest being for dent corn starch and lowest for high amylose
62.8% to 81.9%, highest being for dent corn starch and lowest for high amylose corn starchcorn starch (Figure 5).
The efficiency
(Figure 5).of dent
The corn starch
efficiency was
of dent higher
corn than
starch waspreviously
higher thandetermined efficiency of
previously determined dent corn
efficiency of grits
dent
(62.4%) cornThe
[23]. grits (62.4%) [23]. efficiency
fermentation The fermentation efficiency
of cassava starchofby
cassava starch bymethod
conventional conventional method as
was calculated
74.5%,was calculated
which as 74.5%,
was higher that which
earlier was higherresults
reported that earlier
of 68%reported
efficiencyresults
with of 68% efficiency
Saccharomyces with [8],
cerevisiae
Saccharomyces cerevisiae [8], but lower than another reported value of 82.88%
but lower than another reported value of 82.88% [11]. In case of GSH process, the efficiency of dent[11]. In case of GSH
process, the efficiency of dent corn starch was 74.1%, comparable to previously reported in literature
corn starch was 74.1%, comparable to previously reported in literature (76.7%) [23]. The fermentation
(76.7%) [23]. The fermentation efficiency of cassava starch was calculated as 77%, which was 3.7%
efficiency of cassava starch was calculated as 77%, which was 3.7% higher than that with conventional
higher than that with conventional process. Previously reported literature has shown fermentation
process. Previously
efficiency reported
of cassava rangingliterature has shown
from 82–99%, based onfermentation efficiency
the variety of cassava andof cassava ranging
microorganism used from
82–99%, based
[9,11]. on the variety of cassava and microorganism used [9,11].

DC WC HAC CS
100
90 81.86
Fermentation Efficiency (%)

76.97
80 75.56 74.49 74.16 76.81
70 62.84
60
50
40
30
20 11.76
10
0
Conventional GSH

Figure
Figure 5. Fermentation
5. Fermentation efficienciesofoffour
efficiencies fourstarches
starches (dent
(dentcorn
corn(DC),
(DC),waxy
waxycorn (WC),
corn highhigh
(WC), amylase
amylase
corn corn
(HAC)(HAC)
andand cassava
cassava starch
starch (CS)with
(CS) withconventional
conventional and
andGSH
GSHmethod.
method.

Concentrations
Concentrations of of other
other sugarsand
sugars and sugar
sugar alcohol
alcohol produced
producedin in thethe
conventional
conventional process are are
process
presented in Table 3. Final glycerol production was highest among all sugars and sugar alcohols for
presented in Table 3. Final glycerol production was highest among all sugars and sugar alcohols for
all four starch types. Glycerol formation by Saccharomyces cerevisiae under anaerobic conditions is
all four starch types. Glycerol formation by Saccharomyces cerevisiae under anaerobic conditions is
caused by the need for reoxidation of NADH [24]. Highest glycerol concentration was observed in
caused by the need for reoxidation of NADH [24]. Highest glycerol concentration was observed in
dent corn (8.02 g/L), which was statistically similar to that produced with cassava, whereas waxy
dent corn
cornstarch
(8.02produced
g/L), which was statistically
the lowest glycerol (5.30similar to that produced
g/L). Maltotriose with
concentration at cassava, whereas
the beginning of SSFwaxy
corn was
starch produced
in range the lowest
of 22.08–33.94 glycerol
g/L, similar (5.30 g/L).
to maltose Maltotriose
concentration, concentration
except for high amyloseat the
cornbeginning
starch, of
SSF was
wherein no
range of 22.08–33.94
maltose was detectedg/L, similarfermentation.
throughout to maltose concentration,
At the end of the except for highnoamylose
fermentation, maltose corn
was
starch, detected
where nofor waxy corn
maltose wasstarch, and both
detected dent cornfermentation.
throughout and cassava starchAt had
the less
endthan 1 g/Lfermentation,
of the maltose
concentration. Similarly, maltotriose concentration in all four starch
no maltose was detected for waxy corn starch, and both dent corn and cassava starch hadvarieties at the end ofless
the than
fermentation was below 5 g/L. Concentration of DP4+ at the beginning of SSF was
1 g/L maltose concentration. Similarly, maltotriose concentration in all four starch varieties at the observed to be end
of theabout 250 g/L for all starches except high amylose corn starch, where the starting concentration was
fermentation was below 5 g/L. Concentration of DP4+ at the beginning of SSF was observed to
184 g/L. However, the concentration of DP4+ at the end of fermentation was less than 1 g/L for all
be about 250 g/L for all starches except high amylose corn starch, where the starting concentration
starches.
was 184 g/L. However, the concentration of DP4+ at the end of fermentation was less than 1 g/L for
all starches. Table 3. Concentrations of other sugars in conventional process.
Energies 2018, 11, 3476 8 of 11

Table 3. Concentrations of other sugars in conventional process.

Time (hours) Maltotriose (g/L) Maltose (g/L) Glycerol (g/L)


Energies 2018, 11, x FOR PEER REVIEW 8 of 11
DCS WCS HACS CS DCS WCS HASC CS DCS WCS HACS CS
0 Time (hours)
30.2 22.08 33.94 (g/L)29.11
Maltotriose 32.36 28.67 0.00
Maltose (g/L) 31.89 0.00Glycerol
0.00(g/L) 0.00 0.00
2 36.81 DCS16.10WCS 1.66 HACS 10.65 CS 72.96DCS 82.55
WCS HASC 0.00 94.03 DCS0.90WCS 0.00
CS HACS 0.00 CS 0.81
4 0 21.8 30.22.96 22.08 0.57
33.94 2.98 90.93 85.22
29.11 32.36 28.67 0.00
0.00 93.50
31.89 0.001.960.00 0.000.00 1.98
0.00 1.75
6 2 11.12 0.64 16.10 0.361.66 1.13
36.81 10.65 80.59
72.96 82.78
82.55 0.00 94.03
0.00 92.94 0.902.960.00 0.000.00 3.10
0.81 2.86
8 4 9.305 21.80.36 2.96 0.270.57 0.56 2.98 96.27
90.93 76.32
85.22 0.00 93.50
0.00 86.50 1.963.930.00 0.001.98 3.51.75 4.00
20 6 0.45 0.95 0.64 0.810.36 0.80
11.12 1.13 47.65
80.59 30.95
82.78 0.00 92.94
0.00 43.20 2.965.750.00 4.553.10 4.36
2.86 5.85
24 8 0.68 1.42 0.36 0.000.27 1.09
9.305 0.56 35.64
96.27 19.59
76.32 0.00 86.50
0.00 30.23 3.936.080.00 4.623.5 4.564.00 5.99
48 20 1.63 0.451.58 0.95 0.470.81 2.15 0.80 1.77 0.95
47.65 30.95 0.00 43.20
0.00 1.20 5.757.704.55 5.204.36 5.61
5.85 6.92
72 24 0.51 0.680.35 1.42 0.310.00 0.40 1.09 0.85 0.00
35.64 19.59 0.00 30.23
0.00 0.42 6.088.024.62 5.304.56 5.84
5.99 7.12
48
DCS: Dent Corn1.63
Starch;1.58
WCS: Waxy0.47 Corn 2.15 1.77
Starch; 0.95
HACS: 0.00 1.20
High Amylose 7.70
Corn Starch; 5.20Cassava
CS: 5.61 Starch.
6.92
72 0.51 0.35 0.31 0.40 0.85 0.00 0.00 0.42 8.02 5.30 5.84 7.12
DCS: Dent Corn Starch; WCS: Waxy Corn Starch; HACS: High Amylose Corn Starch; CS: Cassava
3.2. GSH Starch.
Process
The ethanol
3.2. GSH and glucose profiles for all four starches during GSH process are shown in Figure 6.
Process
Similar toThetheethanol
conventional process,
and glucose thefor
profiles final ethanol
all four concentrations
starches for cassava
during GSH process starch
are shown in (127.9
Figure g/L)
with GSH
6. Similar to the conventional process, the final ethanol concentrations for cassava starch (127.9 g/L) corn
process was similar to dent corn (122.6 g/L) and waxy corn (126.9 g/L). High amylose
starchwith
wasGSHfound to yield
process was lowest
similar ethanol concentrations
to dent corn (122.6 g/L) and(22.6
waxyg/L),
corndue to the
(126.9 g/L).inefficiency
High amylose of corn
enzymes
to cleave
starchthe bonds
was foundoftolong
yieldpolymeric chain
lowest ethanol of amylose.(22.6
concentrations Similar
g/L), final
due toethanol concentration
the inefficiency for high
of enzymes
to cleave
amylose the bonds
corn starch of g/L)
(17.3 long polymeric chain by
were observed of amylose.
Sharma Similar finalThe
et.al. [21]. ethanol concentration
ethanol for high
concentration for high
amylose corn during GSH process was found to be about 79% lower than that for conventional for
amylose corn starch (17.3 g/L) were observed by Sharma et.al. [21]. The ethanol concentration process.
highbe
This can amylose corn during
attributed GSHthat
to the fact process
the was found toinbethis
hydrolysis about
case79%waslower than thatUnlike
inefficient. for conventional
conventional
process. This can be attributed to the fact that the hydrolysis in this case was inefficient. Unlike
process, GSH process is carried out at low temperatures, which is not sufficient to break the hydrogen
conventional process, GSH process is carried out at low temperatures, which is not sufficient to break
bonds. Since the enzymes need a certain length of amylose/amylopectin polymer to attach and cleave
the hydrogen bonds. Since the enzymes need a certain length of amylose/amylopectin polymer to
the bonds,
attach the
and amylose
cleave theinbonds,
this case was inaccessible
the amylose in this casetowas
theinaccessible
enzymes. to the enzymes.

FigureFigure 6. Comparison
6. Comparison of ethanol
of ethanol and glucose
and glucose profiles
profiles of dent
of dent corncorn
(DC),(DC),
waxywaxy
corncorn
(WC),(WC),
highhigh
amylose
amylose corn (HAC), and cassava starch (CS) during granular starch hydrolysis (GSH) fermentation
corn (HAC), and cassava starch (CS) during granular starch hydrolysis (GSH) fermentation (SSF at
(SSF at 30 °C for 72 h with GSH enzyme and yeast).
30 ◦ C for 72 h with GSH enzyme and yeast).
Statistically, the final ethanol concentrations for dent corn, waxy corn and cassava starch were
Statistically, the final ethanol concentrations for dent corn, waxy corn and cassava starch were
similar. For cassava starch, the final ethanol concentrations with GSH process was 2.8% higher than
similar. For cassava starch, the final ethanol concentrations with GSH process was 2.8% higher than
that from conventional process, which suggests that with this starch source, the GSH process can be
that from
used conventional process,
to obtain higher ethanol which
yields. suggests thatoccurs
GSH process with this starch
at lower source, thethan
temperatures GSH process can be
conventional
used process,
to obtain higher
and ethanol
eliminates the yields. GSHstep,
liquefaction process occurs
thereby at lower
reducing temperatures
the energy and cost than conventional
for bioethanol
Energies 2018, 11, 3476 9 of 11

Energies 2018, 11, x FOR PEER REVIEW 9 of 11


process, and eliminates the liquefaction step, thereby reducing the energy and cost for bioethanol
production,
production, whichwhich cancan be be an
an added
added benefit
benefit for
for using
using GSHGSH process
process for for bioethanol
bioethanol production
production from from
cassava.
cassava. In the current study, the final ethanol concentration with cassava starch (127.9 g/L) was
In the current study, the final ethanol concentration with cassava starch (127.9 g/L) was
higher
higher than
than previously
previously reported
reported in in literature
literature [8,19].
[8,19].
In
In the
the case
case of
of the
the GSH
GSH process,
process, the the initial
initial 88 hh ofof fermentation
fermentation had had aa linear
linear trend
trend ofof ethanol
ethanol
production with time for all starches, except high amylose corn
production with time for all starches, except high amylose corn starch (Figure 4b). This couldstarch (Figure 4b). This couldbe
be attributed to the fact that no glucose was detected after 4 h in this case,
attributed to the fact that no glucose was detected after 4 h in this case, and subsequently, no ethanol and subsequently, no
ethanol
change was change was observed.
observed. High amylose
High amylose corn therefore
corn starch, starch, therefore
had thehad the lowest
lowest fermentation
fermentation rate
rate (Table
(Table 2). Similar to conventional process, cassava had the highest initial
2). Similar to conventional process, cassava had the highest initial fermentation rates, which were fermentation rates, which
were statistically
statistically similarsimilar
to that toof
that
dentof dent
corn.corn.
In contrast to
In contrast toconventional
conventionalprocess,process,higher
higher sugars
sugars suchsuch as maltose,
as maltose, maltotriose
maltotriose and DP4+
and DP4+ were
were negligible in case of GSH process, due to simultaneous
negligible in case of GSH process, due to simultaneous consumption of these sugars. Glycerol consumption of these sugars.
Glycerol
production, production,
however, however, was observed
was observed in GSHinprocess
GSH process
(Figure(Figure 7). Concentration
7). Concentration of glycerol
of glycerol at theatend
the
end of the
of the fermentation
fermentation waswas highest
highest and and statistically
statistically similarfor
similar forwaxy
waxycorn
cornandand cassava.
cassava. High
High amylose
amylose
corn
corn starch produced lowest glycerol, primarily because of incomplete hydrolysis and amounts
starch produced lowest glycerol, primarily because of incomplete hydrolysis and reduced reduced
of sugars of
amounts released
sugarsfor yeast consumption.
released With inadequate
for yeast consumption. yeast growth
With inadequate yeastdue to lack
growth of to
due substrate,
lack of
glycerol production is reduced [24].
substrate, glycerol production is reduced [24].

9
Glycerol
8
7
Concentration (g/L)

6
5
4
3
2
1
0
DC WC HAC CS
Starch variety

Figure 7. Final concentration of glycerol with four starch varieties (dent corn (DC), waxy corn (WC),
Figure 7.
high amylose
amylose corn
corn (HAC),
(HAC), and
and cassava
cassava starch
starch (CS))
(CS)) for
for GSH
GSH (granular
(granular starch
starchhydrolysis)
hydrolysis)process.
process.

4.
4. Conclusions
Conclusions
This
This study
study investigated
investigated the
the bioethanol
bioethanol production
production potential
potential of
of cassava
cassava in
in comparison
comparison with with the
the
most
most widely used bioethanol substrate, corn. Both conventional and GSH process were used to
widely used bioethanol substrate, corn. Both conventional and GSH process were used to
evaluate
evaluate the
the fermentation
fermentation profile
profile of
of cassava
cassava starch
starch along
along with
with three
three corn
corn varieties
varieties with
with varying
varying
amylose: amylopectinratio.
amylose: amylopectin ratio.The
The final
final ethanol
ethanol concentration
concentration with
with cassava
cassava starch
starch waswas similar
similar to
to two
two
corncorn starch
starch varieties,
varieties, dentdent corn
corn andand waxy
waxy corn
corn starch,for
starch, forboth
bothconventional
conventionaland and GSH
GSH process.
process.
Ethanol concentration with cassava starch for GSH process was 2.8% higher
Ethanol concentration with cassava starch for GSH process was 2.8% higher than that for than that for conventional
process. For both
conventional conventional
process. For bothand GSH process,
conventional andthe fermentation
GSH ratefermentation
process, the for the initialrate
8 h of
forconventional
the initial 8
process fermentation
h of conventional was higher
process for cassava
fermentation starch than
was higher any of starch
for cassava the corn starch
than any varieties.
of the corn Overall,
starch
the fermentation profile of cassava starch, in terms of ethanol production and formation
varieties. Overall, the fermentation profile of cassava starch, in terms of ethanol production and of glycerol,
was similaroftoglycerol,
formation that of dent
wascorn and to
similar waxy
thatcorn starch,
of dent which
corn and suggests
waxy corn thestarch,
potential of cassava
which starch
suggests the
use for commercial production of bioethanol.
potential of cassava starch use for commercial production of bioethanol.
Author Contributions: S.P. carried out all the experiments, M.B.S. helped design the experiments, A.J. analyzed
the results and wrote the manuscript, A.N. and V.S. supervised the study, VS. edited the manuscript. All authors
read and approved the final manuscript.
Energies 2018, 11, 3476 10 of 11

Author Contributions: S.P. carried out all the experiments, M.B.S. helped design the experiments, A.J. analyzed
the results and wrote the manuscript, A.N. and V.S. supervised the study, VS. edited the manuscript. All authors
read and approved the final manuscript.
Funding: This research was funded by a grant from National Institute of Food and Agriculture (NIFA) as a part
of the NIFA (Hatch) Project No. ILLU-741-325—“Processing Corn and Cellulosic Biomass for Food, Fuel and
Industrial Products”.
Conflicts of Interest: The authors declare no conflict of interest.

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