Fermentation of Soybean Meal Hydrolyzates with

Saccharomyces cerevisiae and Zymomonas mobilis

for Ethanol Production
Deivis E. Lujan-Rhenals, Ruben O. Morawicki, Edward E. Gbur, and Steven C. Ricke

Most of the ethanol currently produced by fermentation is derived from sugar cane, corn, or beets. However,
it makes good ecological and economic sense to use the carbohydrates contained in by-products and coproducts of the
food processing industry for ethanol production. Soybean meal, a co-product of the production of soybean oil, has a
relatively high carbohydrate content that could be a reasonable substrate for ethanol production after fermentable sugars
are released via hydrolysis. In this research, the capability of Saccharomyces cerevisiae NRRL Y-2233 and Zymomonas mobilis
subsp. mobilis NRRL B-4286 to produce ethanol was evaluated using soybean meal hydrolyzates as substrates for the
fermentation. These substrates were produced from the dilute-acid hydrolysis of soybean meal at 135 C for 45 min with
0, 0.5%, 1.25%, and 2% H2 SO4 and at 120 C for 30 min with 1.25% H2 SO4 . Kinetic parameters of the fermentation
were estimated using the logistic model. Ethanol production using S. cerevisiae was highest with the substrates obtained at
135 C, 45 min, and 0.5% H2 SO4 and fermented for 8 h, 8 g/L (4 g ethanol/100 g fresh SBM), while Z. mobilis reached
its maximum ethanol production, 9.2 g/L (4.6 g ethanol/100 g fresh SBM) in the first 20 h of fermentation with the
same hydrolyzate.
Abstract:

E: Food Engineering &

Materials Science

Keywords: ethanol, fermentation, hydrolysate, soybean meal, Saccharomyces cerevisiae, Zymomonas mobilis

Introduction

Ethanol is 1 of the only 2 transportation fuels on the market

that is used primarily as part of a mixture with petroleum-based
gasoline. Currently, most ethanol is produced by fermentation of
sugars derived from cereals or sugar crops, which raises concerns
about the use of food crops for the biofuel industry. Therefore,
production of bioethanol from nonhuman food sources is becoming attractive, especially from coproducts associated with food
production (Neureiter and others 2002; Karmee and Lin 2014).
A potential source of fermentable sugars that has been overlooked is soybean meal (SBM), a coproduct of soybean oil production. Soybean meal, a premium protein source for animal feed,
contains approximately 42% total carbohydrates and 55% protein
(Baker and Stein 2009; Da Silva and others 2009). Of the total carbohydrates, half are structural and the other half are comprised of
low-molecular weight sugars, oligosaccharides, and starch in relatively small quantities (Karr-Lilienthal and others 2005). Around
17% of low-molecular-weight sugars consist of a mixture of glucose, arabinose, galactose, fructose, and sucrose (Grieshop and
others 2003). An additional merit of SBM as a potential source of
fermentable sugars for ethanol production is that after sugars are
selectively extracted, a high-protein meal is left behind which can
be used for animal feed. Before SBM can be used as a substrate,
fermentable sugars need to be released from the solid matrix, by
hydrolysis with dilute acid or other methods.

MS 20150149 Submitted 1/26/2015, Accepted 4/17/2015. Authors LujanRhenals, Morawicki, and Ricke are with Food Science Dept., 2650 N. Young
Ave., Univ. of Arkansas, Fayetteville, AR 72704. Author Lujan-Rhenals is also with
Univ. de Cordoba, Programa de Ingeniera de Alimentos. Sede Berastegui. Km. 12
Via Cerete-Cienaga de Oro, Cordoba, Colombia. Author Gbur is with Agricultural
Statistics Laboratory, Univ. of Arkansas Div. of Agriculture, W. Maple St., Fayetteville,
AR 72701. Author Ricke is also with Univ. of Arkansas Center for Food Safety,
2650 N. Young Ave., Fayetteville, AR 72704. Direct inquiries to author Morawicki
(E-mail: rmorawic@uark.edu).

E1512

Journal of Food Science r Vol. 80, Nr. 7, 2015

For this research, the capability of 2 microorganisms

Saccharomyces cerevisiae and Zymomonas mobilisto ferment SBM
hydrolyzates was tested. S. cerevisiae is one of the most common
organisms used in industrial ethanolic fermentations. It is highly
robust, resistant to toxic inhibitors, and highly tolerant of low pH
levels, which minimizes the risk of contamination (Weber and
others 2010). Z. mobilis, a gram-negative bacterium, is attractive
for ethanol production due to its high productivity. A downside
is that Z. mobilis has low resistance to toxic inhibitors and can
ferment only glucose, fructose, and sucrose (Rogers and others
1982; Doran 1997; Weber and other 2010).
The objective of this research was to evaluate the ability of
S. cerevisiae and Z. mobilis to produce ethanol using acid hydrolyzates of SBM as substrates. The kinetic parameters of the
fermentation were determined using the logistic model.

Materials and Methods

Substrates for fermentation
Five hydrolyzates of commercial SBM were used as substrates
for the fermentations. The hydrolyzates were obtained by treating soybean meals with dilute sulfuric acid under the following
conditions: 135 C for 45 min at 4 acid concentrations (0.0%,
0.5%, 1.25%, and 2.0% H2 SO4 ) and 120 C, 1.25% H2 SO4 for 30
min. In previous research, these conditions have proven to produce
the highest concentrations of fermentable sugars (Lujan-Rhenals
2013).
Hydrolyzates were prepared by treating 50 g of SBM with
R
250 mL of H2 SO4 solution in 500-mL PYREX
media storage bottles with screw caps (Lujan-Rhenals 2013). To avoid pressure buildup inside the bottles during the heat treatment, caps
were not completely tightened. A Tuttnauer 2340E Steam Autoclave (Tuttnauer U.S.A, Delran, N.J., U.S.A.) operated according
to the preset program #1 (fast exhaust without drying) was used
for the hydrolysis. Transient heating was not considered in the
R

C 2015 Institute of Food Technologists

doi: 10.1111/1750-3841.12907
Further reproduction without permission is prohibited

Table 1Experimental design for the ethanolic fermentation of

soybean meal hydrolyzates with S. cerevisiae NRRL Y-2233 and
Z. mobilis NRRL B-4286.
Standard order
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20

Run order

Microorganism

Broth

7
14
6
18
2
9
1
12
11
16
3
19
4
17
8
10
5
13
15
20

S. cerevisiae
S. cerevisiae
S. cerevisiae
S. cerevisiae
S. cerevisiae
S. cerevisiae
S. cerevisiae
S. cerevisiae
S. cerevisiae
S. cerevisiae
Z. mobilis
Z. mobilis
Z. mobilis
Z. mobilis
Z. mobilis
Z. mobilis
Z. mobilis
Z. mobilis
Z. mobilis
Z. mobilis

SBMB0
SBMB0
SBMB1
SBMB1
SBMB2
SBMB2
SBMB3
SBMB3
SBMB4
SBMB4
SBMB0
SBMB0
SBMB1
SBMB1
SBMB2
SBMB2
SBMB3
SBMB3
SBMB4
SBMB4

SBMB0: 135C, 0% H2 SO4 , 45 min.

SBMB1: 135C, 0.5% H2 SO4 , 45 min.
SBMB2: 135C, 1.25% H2 SO4 , 45 min.
SBMB3: 135C, 2% H2 SO4 , 45 min.
SBMB4: 120C, 1.25% H2 SO4 , 30 min.

analysis. After the heat treatment, the reaction was stopped by

cooling the mix in an ice-water bath followed by the modification
of pH to a range between 5.0 and 5.5 with NaOH pellets. Solids
and liquid were then separated by centrifugation at 3900 g for
35 min at 10 C in an Allegra X-22R centrifuge with an SX4250
Rotor (Beckman Coulter Inc., Brea, Calif., U.S.A.). Afterwards,
the supernatants were filtered through Whatman #4 filter paper
(Whatman Plc., Maidstone, Kent, U.K.), analyzed for the concentration of fermentable sugars, and used as substrates for the
fermentation. From this point forward, these substrates will be
referred to as soybean meal broth (SBMB) as listed in Table 1.

Cells reactivation and preparation of preinoculum

and inoculum
Both S. cerevisiae and Z. mobilis were reactivated in 5 mL of yeast
malt (YM) broth (Dickinson and Company, Sparks, Md., U.S.A.)
and incubated at 30 C for 24 h in an orbital shaker (Thermo
Scientific Max Q 4450, Dubuque, Iowa, U.S.A.) set at 150 rpm.
The preinoculum consisted of 5 mL of sterile YM broth at 30 C
where both S. cerevisiae and Z. mobilis were inoculated separately
and grown overnight in the orbital shaker. For each respective
strain, 0.2 mL of the culture was plated out with a loop over the
surface of 2 petri dishes containing YM agar (20 g/L of glucose,
3 g/L of yeast extract, 5 g/L of peptone, and 3 g/L of malt extract)
and grown overnight at 30 C before adding to the final inoculum.
The inoculum was prepared by adding all the colonies formed in
both dishes to 80 mL of SBMB and allowing the cells to adapt by
incubating overnight at 30 C in the orbital shaker.
Fermentation
Fermentations were conducted at 30 C for 36 h with an initial pH of 5 to 5.5, dissolved oxygen (%DO) less than 1% after
stabilization, and an initial biomass concentration between 7106
and 1107 cells/mL (Mullins and Nesmith 1987; Siqueira and
others 2008; Laopaiboon and others 2009). The fermentor was a
1.3-LBioflo/CellinGen 115 Benchtop Fermentor & Bioreactor
(New Brunswick Scientific, Edison, N.J., U.S.A.) with control systems for temperature, pH, %DO, agitation, and foam level. During
fermentation, samples were taken every 4 h to estimate fermentation kinetic parameters maximum specific growth rate (max),
biomass yield from sugar (Yx/s), and ethanol yield from sugar
(Yp/s)along with volumetric ethanol productivity, rp (g/L/h).
Cell density (nr. of cells/L) was determined with a hemacytometer
(Double Neubauer Counting Chamber, 3110, Hausser Scientific,
Horsham, Pa., U.S.A.) and a phase contrast microscope (Nikon
Eclipse E400, Nikon Corporation, Tokyo, Japan). The cell density was correlated with a calibration curve of cell dry weight
obtained by drying aqueous solutions of known concentrations of
cells at 80 C for 20 to 24 h (Alfenore and others 2002; Buhner
and Agblevor 2004). All kinetic parameters were calculated using
the following equations:

Microbial strains
Strains of S. cerevisiae NRRL Y-2233 and Z. mobilis subsp.
dx
Yx/s =
Biomass yield (g biomass/g sugars)
(1)
mobilis NRRL B-4286 were provided as lyophilized powders from
ds
the culture collection of the United States Dept. of Agriculture
(USDA), Agriculture Research Service (Peoria, Ill., U.S.A.).
where x and s represent concentrations of cellular biomass (g/L)
and substrate (g/L), respectively.
Fermentable sugar analysis
dp
Fermentable sugars in the hydrolyzates were analyzed with Y p/s =
Ethanol yield from substrate (g ethanol/g sugars)
ds
High-Performance Size Exclusion Chromatography and Refractive Index (HPSEC-RI) detection. The chromatograph was a Wa(2)
ters, (Milford, Mass., U.S.A.) with a 515 HPLC pump, a manual
injector with a 50-L sample loop, and a 2410 refractive in- where P represents the concentration of ethanol (g/L)
dex detector set at 40 C. Columns used for the separation of
sugars were a Shodex OH Pack SB-804 HQ (300 8 mm) fol- r p = (q p)x Volumetric ethanol productivity (g ethanol/Lh)
lowed by Shodex OH Pack SB-802 HQ (300 8 mm) (Showa
(3)
Denko America, Inc., New York, N.Y., U.S.A.) connected in
series. Columns were preceded by a Shodex OH pack SB-G
dp 1
(50 6 mm) guard column. Columns and guard column were
qp =
Specific rate of product formation
(4)
dt x
maintained at 55 C in a column heater. A solution of 0.1 M
NaNO3 with 0.2% NaN3 run isocratically at a flow rate of
0.4 mL/min was used as the mobile phase. Sugars were quan(r s ) r x/Yxs r p/Yps
Maintenance coefficient
ms =
tified using 6-point calibration curves with fructose and glucose
x
(g sugar/g cell h)
(5)
as standards.
Vol. 80, Nr. 7, 2015 r Journal of Food Science E1513

E: Food Engineering &

Materials Science

Fermentation of SBM hydrolyzates . . .

Fermentation of SBM hydrolyzates . . .

E: Food Engineering &

Materials Science

(CH3 COONa) which is the sodium salt of acetic acid formed durwhere rs = g sugar consumed/L, rx = g cell produced/L h, and ing the reaction and sodium sulfate (Na2 SO4 ) formed during the
rp = g ethanol produced/L h.
neutralization of the sulfuric acid with sodium hydroxide. (Yang
and others 2010) demonstrated that the combination of elevated
Growth kinetics and model development for the ethanol
Na+ and acetate ions led to a synergistic inhibitory effect on strain
fermentation
ZM4.
The kinetic parameters of biomass production were determined
with the logistic model (Eq (6)) (Wang and others 2004).
Kinetics of substrate consumption during fermentation


S. cerevisiae exhibited its most rapid sugar consumption of
x
d x/d t = max x 1
(6) 15.6 g/L during the initial 12 h of fermentation with the SBMB4
xmax
(120 C, 1.25% H2 SO4 , 30 min) and 15.1 g/L during the 1st 12 h
of fermentation with the SBMB1 (135 C, 0.5% H2 SO4 , 45 min)
where dx/dt is the rate of biomass during the time of fermentation
(Figure 1e). On the other hand, the SBM broths from the most se(g cell/ h), max is the maximum specific growth rate (h-1 ), x is the
vere hydrolysis treatments appeared to result in a low rate of sugar
biomass concentration (g/L), and xmax is the maximum biomass
use from the fermentation broth by S. cerevisiae (Figure 1e). This
concentration (g/L).
low rate of sugar consumption occurred due to toxic compounds
Considering the following boundary conditions: t = 0, x = xo ,
formed during the hydrolysis process that reduces the yeast growth
S = So, and P = 0, Eq (6) was integrated and Eq (7) was obtained
(Lujan-Rhenals and others 2014).
as a result, which relates biomass production (X) and fermentation
Z. mobilis also expressed its most rapid sugar uptake of 15.0 g/L
time (t) and was used to fit the experimental data.
per 16 h of fermentation with the SBMB1 (135 C, 0.5% H2 SO4 ,
45 min) (Figure 2b). Similar to S. cerevisiae, Z. mobilis had low
xo xmax e max t
(7)
x=
rates
of apparent sugar consumption during growth in the broths
xmax xo + xo e max t
(Figure 2c and 2d) resulting from the most severe pretreatments,
For the calculation of the kinetics parameters, only glucose and particularly SBMB2 (135 C, 1.25% H2 SO4 , 45 min), which is
fructose were considered as growth-limiting-substrates because probably due to the inhibitory effect of acetic acid and salts as
these sugars were consumed in the highest and most consistent previously discussed.
Overall, the maintenance coefficient ms (g sugar consumed/
quantities by the microorganisms according to the HPLC analysis.
g cell h) for both microorganisms did not yield statistically signifiExperimental design
cant differences except for S. cerevisiae with SBMB2 and SBMB3,
For the fermentation, the experimental design was a complete which gave higher values (2.7 and 2.8 g sugar/g cell h, respectively)
randomized block design with microorganism type and broth as than the other coefficients (Table 2). However, numerically, all ms
coefficients for Z. mobilis were lower than the ms coefficients calfactors (Table 1). At each level, experiments were run twice.
culated for S. cerevisiae which means that the bacteria apparently
Statistical analysis
required less carbon substrate per cell than the yeast. This result has
Data was analyzed with SAS Version 9.2 software (SAS Inst. Inc., also been previously reported by (Dien and others 2003). ThereCary, N.C., U.S.A.) and the kinetic parameters max , xmax, and xo fore, Z. mobilis utilizes less substrate per gram of cells produced
determined. Analysis of covariance (ANCOVA) was used to fit than S. cerevisiae and has more carbon available for the production
the data to the logistic model using the Gauss-Newton nonlinear of ethanol (Dien and others 2003).
regression method and comparisons of each regression coefficient
across the treatments. For the minimum inhibitor concentrations Kinetics of ethanol production
R
version
and kinetics parameters, data were analyzed with JMP
S. cerevisiae exhibited its maximum ethanol production of
9.0.0 (SAS Inst. Inc.). To analyze the kinetic parameters (Yp/s, 8 g/L during the 1st 8 h of fermentation of the SBMB1 (135 C,
Yx/s, ms, and qp), an analysis of variance (ANOVA) was used and 0.5% H2 SO4 , 45 min) and SBMB4 (120 C, 1.25% H2 SO4 ,
differences in the mean of the kinetic parameters were analyzed 30 min). In contrast, the lowest ethanol production (5.67 g/L
using the TukeyKramer test ( = 0.05).
ethanol) occurred with the SBMB3 (135 C, 2% H2 SO4 , 45 min)
after 28 h of fermentation, where possibly some inhibitor comResults and Discussion
pounds (salts, acetate from acetic acid, and other unidentified toxic
Ethanol fermentation with S. cerevisiae and Z. mobilis
substances) could have reduced the ethanol productivity.
Ethanol production by Z. mobilis peaked at 9.2 g/L ethanol afS. cerevisiae exhibited optimal responses for the production of
ethanol, sugars consumption, and cell growth in all the SBM ter 20 h of fermentation with the SBMB1 (135 C, 0.5% H2 SO4 ,
broths used in this study (Figure 1). However, in most fermen- 45 min). The SBMB0 (135 C, 0% H2 SO4 , 45 min) allowed a
tation broths, Z. mobilis was not as efficient (Figure 2). This maximum ethanol rate of 3.9 g/L ethanol after 20 h of fermenbacterium exhibited no growth with the hydrolyzate SBMB3 tation. However, the lowest ethanol production levels1 g/L at
(135 C, 2% H2 SO4 , 45 min), but with the SBMB1 20 hwere attained with the SBMB2 (135 C, 1.25% H2 SO4 ,
(135 C, 0.5% H2 SO4 , 45 min) displayed its maximum ethanol 45 min) and SBMB4 (120 C, 1.25% H2 SO4 , 30 min), which
production. S. cerevisiae, on the other hand, demonstrated rapid is likely caused by inhibitors (acetate from acetic acid, other salts,
sugar consumption, likely because it is a robust microorganism and unknown compounds). There have been previous reports that
that was not critically affected by the inhibitors present in most ethanol production with Z. mobilis and its growth can be reduced
of the hydrolyzate SBMB (Lujan-Rhenals and others 2014). Zy- substantially even with low concentrations (2 to 8 g/L) of acetic
momonas mobilis was affected by inhibitory compounds of the acid (Wang 2008).
SBMB ((Lujan-Rhenals and others 2014) and also by the presThe ethanol yield (Yp/s, g ethanol/g of sugars) reported in
ence of salts coming from the hydrolyzate, such as sodium acetate Table 2 did not show significant statistical differences between the
E1514 Journal of Food Science r Vol. 80, Nr. 7, 2015

Fermentation of SBM hydrolyzates . . .

Biomass (g/L)

1.5

1.0

0.5
0.0

2
0

10

20

30

40

2.0

15

1.5

10

1.0

0.5
0.0

10

30

1.0

10

0.5

0.0

30

40

35
30
25

1.5

20

1.0

15
10

0.5
0.0

5
0

10

20

30

40

Time (h)

Time (h)
Biomass (g/L)

Sugars (g/L)

2.0

Biomass (g/L)

Biomass (g/L)

15

20

Sugars and Ethanol (g/L)

20

1.5

10

40

D SBMB3

2.5

Sugars and Ethanol (g/L)

25

2.0

30

Biomass (g/L)

Ethanol (g/L)

Sugars (g/L)

C SBMB2

2.5

20

Time (h)

Time (h)
Biomass (g/L)

20

Sugars and Ethanol (g/L)

10

2.0

B SBMB1

2.5

Sugars and Ethanol (g/L)

12

Biomass (g/L)

A SBMB0

2.5

These values for S. cerevisiae are comparable to, and some higher
than those reported by other researchers (Da Cunha-Pereira and
others 2011). However, Z. mobilis reached a maximum ethanol
volumetric productivity (rp) of 0.61 g ethanol/Lh only with the
SBM1, which is lower than the values, 1.4 to 1.8 g ethanol/Lh with
Z. mobilis during the production of ethanol using SBM molasses
(Letti and others 2012). Also, it is lower than 2.8 g ethanol/Lh
obtained for the production of ethanol with Z. mobilis from
sweet potato (Zhang and Feng 2010); however, it is higher than
0.59 g/Lh using lignocellulosic material as substrate (Mohagheghi
and others 2002). These results demonstrate that the application
of Z. mobilis for ethanol fermentation is not feasible when the
substrate is SBM broth obtained under the conditions applied in
these experiments. This is probably due to the high concentration

Sugars (g/L)

Ethanol (g/L)

Biomass (g/L)

Sugars (g/L)

Ethanol (g/L)

463

2.5

25

2.0

20

1.5

15

1.0

10

0.5

0.0

10

20

30

40

Sugars and Ethanol (g/L)

Biomass (g/L)

E SBMB4

Time (h)
Biomass (g/L)

Sugars (g/L)

Ethanol (g/L)

Figure 1Fermentation profiles of SBM broths with S. cerevisiae NRRL Y-2233. Acronyms defined in Table 1.
Vol. 80, Nr. 7, 2015 r Journal of Food Science E1515

E: Food Engineering &

Materials Science

2 microorganisms and the respective SBM broths used. However,

numerically both microorganisms showed similar ethanol yields
to the theoretical values, which is 100% for 0.51 g ethanol/g of
sugars (Doran 1997). Fermentations in the SBM0 and SBMB1
gave the highest ethanol yields, 96% of the theoretical yield for
both microorganisms, which are comparable or even higher than
values reported in previous research (Siqueira and others 2008;
Zhang and Feng 2010; Da Cunha-Pereira and others 2011; Letti
and others 2012; Romao and others 2012). Additionally, the
highest ethanol volumetric productivity (rp) for S. cerevisiae was
achieved with the SBM1 (1.01g ethanol/L h) and SBMB4 (1.00
g ethanol/L h), with no significant difference between the 2, but
differed with respect to the other broths used in this research
(Table 2).

Fermentation of SBM hydrolyzates . . .

Table 2Means of kinetic parameters of ethanol fermentation in batch culture with S. cerevisiae NRRL Y-2233 and Z. mobilis
subspecies mobilis NRRL B-4286 in hydrolyzed soybean meal broths SE. Acronyms defined in Table 1.
SBM broth

Microorganism

Yp/s
(g ethanol/g sugar)

Yx/s
(g cell/g sugar)

Ms
(g sugar/g cell h)

Rp
(g ethanol/Lh)

S. cerevisiae
Z. mobilis
S. cerevisiae
Z. mobilis
S. cerevisiae
Z. mobilis
S. cerevisiae
S. cerevisiae
Z. mobilis

0.49 0.021 a
0.49 0.007 a
0.49 0.014 a
0.48 0.028 a
0.46 0.007 a
0.26 0.042 a
0.40 0.057 a
0.49 0.007 a
0.29 0.113 a

0.23 0.014 abc

0.23 0.057 ab
0.10 0.014 cd
0.08 0.014 d
0.04 0.007 d
0.15 0.021 bcd
0.02 0.007 d
0.11 0.007 bcd
0.32 0.014 a

0.69 b
0.44 b
1.19 b
0.97 b
2.73 a
0.65 b
2.78 a
1.13 b
0.17 b

0.67 0.028 b
0.37 0.007 cd
1.00 0.028 a
0.61 0.007 bc
0.61 0.113 bc
0.11 0.007 de
0.29 0.092 de
1.01 0.021 a
0.07 0.007 e

SBMB0
SBMB0
SBMB1
SBMB1
SBMB2
SBMB2
SBMB3
SBMB4
SBMB4

Means followed by the same letters in each column did not have significant differences. (P = 0.05). Z. mobilis exhibited no growth with the SBMB3.

A SBMB0

B SBMB1
10

3.0

2.5

2.0
1.5

1.0

0.5
0.0

10

20

30

40

20

3.5

Biomass (g/L)

Biomass (g/L)

3.5

4.0
3.0

15

2.5
2.0

10

1.5
1.0

0.5

0.0

10

20

Time (h)
Biomass (g/L)

Sugars (g/L)

Biomass (g/L)

Ethanol (g/L)

C SBMB2

2.0
10

1.5
1.0

0.5
20

30

40

20

3.0

Biomass (g/L)

15

25

3.5
2.5

15

2.0
10

1.5
1.0

0.5
0.0

10

Time (h)
Biomass (g/L)

Sugars (g/L)

20

40

Time (h)
Ethanol (g/L)

Biomass (g/L)

Sugars (g/L)

Figure 2Fermentation profiles of SBM broths with Z. mobilis NRRL B-4286. Acronyms defined in Table 1.

E1516 Journal of Food Science r Vol. 80, Nr. 7, 2015

30

Sugars and Ethanol (g/L)

2.5

Sugars and Ethanol (g/L)

20

3.0

10

Ethanol (g/L)

4.0

25

3.5

Time (h)
Sugars (g/L)

40

D SBMB4

4.0

0.0

30

Sugars and Ethanol (g/L)

12

Sugars and Ethanol (g/L)

4.0

Biomass (g/L)

E: Food Engineering &

Materials Science

of acetic acid, salts, and other toxic unknown compounds formed However, SBMB3 (135 C, 2% H2 SO4 , 45 min) exhibited a maduring the acid hydrolysis in the broths obtained under severe jor inhibition of yeast growth since the biomass yielded no more
treatments.
than 0.5 g/L after 32 h of fermentation. Overall, production of
biomass by S. cerevisiae appeared to be directly associated with
ethanol production.
Kinetics of biomass growth and model development
Z. mobilis exhibited its highest biomass concentration, 3.9 g/L,
for the fermentation
after 20 h of fermentation (Figure 7.2a) with the SBMB0 (135 C,
Data reported in Figure 1a indicate that S. cerevisiae yielded its 0%, H2 SO4 , 45 min), followed by enzymatic treatment with celmaximum biomass concentration (2.3 g/L) after 12 h of anaerobic lulase. Likewise, the biomass produced (3.3 g/L) when fermenting
fermentation when exposed to the SBM0 (135 C, 0% H2 SO4 , the SBMB1 (135 C, 0.5% H2 SO4 , 45 min) was very close to the
45 min) followed by enzymatic treatment with cellulase. Like- former but required 24 h to reach this level. In contrast, SBM2
wise, this maximum concentration of biomass (2.3 g/L) was also (135 C, 2% H2 SO4 , 45 min) exhibited the primary inhibitory
reached with SBMB4 (120 C, 1.25% H2 SO4 , 30 min) at 24 h. effects on Z. mobilis growth due to higher concentrations of salts,

Ethanol (g/L)

Fermentation of SBM hydrolyzates . . .

Table 3Means of logistic model parameters of ethanol fermentation in batch culture with S. cerevisiae NRRL Y-2233 and Z. mobilis
subspecies mobilis NRRL B-4286 in hydrolyzed soybean meal broths SE. Acronyms defined in Table 1.
SBM broth
SBMB0
SBMB0
SBMB1
SBMB1
SBMB2
SBMB2
SBMB3
SBMB4
SBMB4

Microorganism

max (h-1 )

xo (g biomass/L)

xmax (h-1 )

S. cerevisiae
Z. mobilis
S. cerevisiae
Z. mobilis
S. cerevisiae
Z. mobilis
S. cerevisiae
S. cerevisiae
Z. mobilis

0.63 0.12 a
0.55 0.05 a
0.45 0.09 afgh
0.16 0.02 bk
0.21 0.11 bdfi
0.45 0.25 aijkl
0.19 0.15 begj
0.60 0.11 a
0.29 0.03 cdehl

0.17 0.08 a
0.18 0.05 a
0.32 0.09 a
0.18 0.04a
0.16 0.08 a
0.18 0.1 a
0.14 0.08 a
0.24 0.09 a
0.26 0.06 a

2.28 0.05 a
3.79 0.06 f l
1.81 0.05 b
5.1 0.92 g l
0.66 0.10 cej
0.65 0.05 hjk
0.49 0.12 dek
2.23 0.05 a
2.97 0.09 i

acetic acid, and other inhibitors in the broth that restricted its
growth (Doran 1997; Yang and others 2010). In general, Z. mobilis also showed growth-associated ethanol production with the
exception of those broths where the bacteria were considerably
inhibited.
Data of cell growth responses fitted to a logistic model during
the 1st 24 h of fermentation are shown in Table 3. The maximum specific growth rates (max ) shows that in the SBM0 and
SBMB4, max for S. cerevisiae (0.63 and 0.60 h-1 , respectively)
were higher but not significantly different than max for Z. mobilis,
0.55 h-1 , in the substrate T3C0t3. Wang and others (2004), also
using the logistic model, reported a max of 0.1 h-1 for the ethanolic fermentation of apple juice with S. cerevisiae ( Huang and Wang
2010) obtained a max of 5.2 h1 using Saccharomyces diastaticus
with mixed sugars as a substrate. Mohagheghi and others (2002)
reported for Z. mobilis a max of 0.34 h-1 for ethanolic fermentation of hydrolyzed lignocellulosic biomass. In general, max for S.
cerevisiae was higher for substrates obtained with pretreatments of
less severity; Z. mobilis max did not show the same pattern due to
the effect of inhibitors present in some of the SBM broths.
The initial concentration of biomass (x0 ) was not significantly
different among all the broths for both microorganisms, which
is an indication that the inoculum was homogeneously prepared
and introduced to each SBM broth. The other kinetic parameter
derived by fitting the logistic model to the 1st 24 h of fermentation
was the maximum concentration of biomass (xmax ). For S. cerevisiae,
xmax was 2.28 and 2.23 g/L for SBMB0 and SBMB4, respectively,
while the maximum xmax for Z. mobilis was 3.79 g/L and 5.1 g/L
for the SBMB0 and SBMB1, respectively.
The biomass yield from substrate (Yx/s) listed in Table 2 was
significantly higher (P = 0.05) for S. cerevisiae (0.1 g cell/g sugar
consumed) than Z. mobilis (0.08 g cell/ g sugar consumed) when
fermenting the SBMB1. Higher values with S. cerevisiae than
Z. mobilis have also been reported by other researchers (Dien and
others 2003, Aitabdelkader and Baratti 1993). This is a function of
the better resistance of the yeast to the inhibitors present in these
substrates (Weber and others 2010). However, with SBM2 the effect was the opposite, the value for Z. mobilis was 0.15 g cell/g
sugar and for S. cerevisiae was 0.04 g cell/g sugar. Nonetheless, most
of the values for S. cerevisiae were higher than the previous reports
(Siqueira and others 2008) for the production of bioethanol from
soybean molasses and similar to values (0.14 g/g) reported in other
work (Ahmad and others 2011).

Conclusions
S. cerevisiae exhibited good qualities in the production of
ethanol, sugars consumption, and cell growth for all acidhydrolyzed soybean meal broths tested without any additional

nutrients, while Z. mobilis had a lower performance for most

broths. The highest ethanol production of S. cerevisiae, 8 g
ethanol/L, (4 g ethanol/100 g fresh SBM) was reached during
the 1st 8 h of fermentation with the broth obtained at 135 C,
0.5% H2 SO4 , 45 min and the one at 120 C, 1.25% H2 SO4 , and
30 min. However, Z. mobilis yielded maximum ethanol production, 9.2 g/L ethanol, (4.6 g ethanol/100 g fresh SBM) only after
the 1st 20 h with the hydrolyzate obtained at 135 C, 0.5% H2 SO4 ,
45 min.

Acknowledgments
This research was partially supported by the Arkansas Soybean
Promotion Board. Deivis E. Lujan-Rhenals was supported in
part by COLCIENCIAS of Colombia, LASPAU, and Univ. de
Cordoba (Colombia).

Author Contributions
Deivis Lujan-Rhenals performed the experiments, analyzed the
data, and wrote the paper. Ruben Morawicki designed the experiments, analyzed the data, and wrote the paper. Edward Gbur
provided technical details in the experimental design and statistical analysis. Steven C. Ricke provided technical support with the
fermentation.

References
Ahmad F, Jameel AT, Kamarudin MH, Mel M. 2011. Study of growth kinetic and modeling of
ethanol production by Saccharomyces cerevisae. Afr J Biotechnol 10:188426.
Aitabdelkader N, Baratti JC. 1993. Estimation of kinetic and yield parametersethanol fermentation of a glucose and fructose mixture using Zymomona mobilis. Biotechnol Tech 7:32934.
Alfenore S, Molina-Jouve C, Guillouet SE, Uribelarrea JL, Goma G, Benbadis L. 2002. Improving ethanol production and viability of Saccharomyces cerevisiae by a vitamin feeding strategy
during fed-batch process. Appl Microbiol Biotechnol 60:6772.
Baker KM, Stein HH. 2009. Amino acid digestibility and concentration of digestible and metabolizable energy in soybean meal produced from high protein or low oligosaccharide varieties
of soybeans and fed to growing pigs. J Anim Sci 87:228290.
Buhner J, Agblevor FA. 2004. Effect of detoxification of dilute-acid corn fiber hydrolysate on
xylitol production. Appl Biochem Biotechnol 119:1330.
Da Cunha-Pereira F, Hickert LR, Sehnem NT, deSouza-Cruz PB, Rosa CA, Zachia Ayub
MA. 2011. Conversion of sugars present in rice hull hydrolysates into ethanol by Spathaspora
arborariae, Saccharomyces cerevisiae, and their co-fermentations. Bioresour Technol 102:421825.
Da Silva FM, DeAndrade M, Guim A, Silva R, Dos Santos LE, Vieira JC. 2009. Replacement
of soybean meal by cottonseed meal in diets based on spineless cactus for lactating cows. Rev
Braz Zootec 38:19952000.
Dien B, Cotta M, Jeffries T. 2003. Bacteria engineered for fuel ethanol production: current
status. Appl Microbiol Biotechnol 3:25866.
Doran PM. 1997. Bioprocess engineering principles, solutions manual.1st ed. Academic Press
Limited. London, UK. 137p.
Grieshop CM, Kadzere CT, Clapper GM, Flickinger EA, Bauer LL, Frazier RL, Fahey Jr GC.
2003. Chemical and nutritional characteristics of United States soybeans and soybean meals.
J Agric Food Chem 51:768491.
Huang W, Wang F. 2010. Kinetic modeling of batch fermentation for mixed-sugar to ethanol
production. J Taiwan Inst Chem Eng 41:43439.
Karmee SK, Lin CSK. 2014. Valorization of food waste to biofuel: current trends and technological challenges. Sustain Chem Process 2:14.
Karr-Lilienthal LK, Kadzere CT, Grieshop CM, Fahey Jr GC. 2005. Chemical and nutritional
properties of soybean carbohydrates as related to nonruminants: a review. Livest Prod Sci
97:112.

Vol. 80, Nr. 7, 2015 r Journal of Food Science E1517

E: Food Engineering &

Materials Science

Means followed by the same letters in each column did not have significant differences (p = 0.05). Z. mobilis exhibited no growth with the SBMB3.

Fermentation of SBM hydrolyzates . . .

E: Food Engineering &

Materials Science

Laopaiboon L, Nuanpeng S, Srinophakun P, Klanrit P, Laopaiboon P. 2009. Ethanol production

from sweet sorghum juice using very high gravity technology: effects of carbon and nitrogen
supplementations. Bioresour Technol 100:417682.
Letti LAJ, Karp SG, Woiciechowski AL, Soccol CR. 2012. Ethanol production from soybean
molasses by Zymomonas mobilis. Biomass Bioenerg 44:806.
Lujan-Rhenals D. 2013. Production of bioethanol by fermentation of sugars released by diluteacid and enzymatic hydrolysis of soybean meal. Doctoral thesis. Food Science Program. Univ.
of Arkansas, Fayetteville, U.S.A. p 4465.
Lujan-Rhenals D, Morawicki R, Ricke S. 2014. Tolerance of S. cerevisiae and Z. mobilis to
inhibitors produced during dilute acid hydrolysis of soybean meal. J. Environ. Sci. Health.,
Part B 49:30511.
Mohagheghi A, Evans K, Chou Y, Zhang M. 2002. Cofermentation of glucose, xylose, and
arabinose by genomic DNA-integrated xylose/arabinose fermenting strain of Zymomonas
mobilis AX101. Appl Biochem Biotechnol 98:88598.
Mullins JT, Nesmith CC. 1987. Acceleration of the rate of ethanol fermentation by addition of
nitrogen in high tannin grain-sorghum. Biotechnol Bioeng 30:10736.
Neureiter M, Danner H, Thomasser C, Saidi B, Braun R. 2002. Dilute-acid hydrolysis of
sugarcane bagasse at varying conditions. Appl Biochem Biotechnol 98:4958.
Rogers PLK, Lee J, Skotnicki ML, Tribe DE. 1982. Ethanol production by Zymomonas mobilis.
Adv Biochem Eng 23:3784.
Romao BB, Da Silva FB, DeResende MM, Cardoso VL. 2012. Ethanol production from
hydrolyzed soybean molasses. Energy Fuels 26:23106.

E1518 Journal of Food Science r Vol. 80, Nr. 7, 2015

Siqueira PF, Karp SG, Carvalho JC, Sturm W, Rodriguez-Leon JA, Tholozan JL, Singhania
RR, Pandey A, Soccol CR. 2008. Production of bio-ethanol from soybean molasses by
Saccharomyces cerevisiae at laboratory, pilot, and industrial scales. Bioresour.Technol 99:8156
63.
Wang D, Xu Y, Hu J, Zhao G. 2004. Fermentation kinetics of different sugars by apple wine
yeast Saccharomyces cerevisiae. J Inst Brew 110:3406.
Wang Y. 2008. Development of acetic-acid tolerant Zymomonas mobilis strains through adaptation. Master thesis in Chemical and Biomolecular Engineering. School of Chemical and
Biomolecular Engineering. Georgia Inst. of Technology. 23p.
Weber C, Farwick A, Benisch F, Brat D, Dietz H, Subtil T, Boles E. 2010. Trends and challenges
in the microbial production of lignocellulosic bioalcohol fuels. Appl Microbiol Biotechnol
87:130315.
Yang S, Land ML, Klingeman DM, Pelletier DA, Lu YS, Martin SL, Guo HB, Smith JC, Brown
SD. 2010. Paradigm for industrial strain improvement identifies sodium acetate tolerance loci
in Zymomonas mobilis and Saccharomyces cerevisiae. Proc Natl Acad Sci, U.S.A. June 8 2010.
107:10395400.
Zhang K, Feng H. 2010. Fermentation potentials of Zymomonas mobilis and its application in ethanol production from low-cost raw sweet potato. Afr J Biotechno 9:
61228.