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Fermentation of Soybean Meal Hydrolyzates with Saccharomyces cerevisiae

and Zymomonas mobilis for Ethanol Production: Fermentation of SBM
hydrolyzates…

Article  in  Journal of Food Science · May 2015

DOI: 10.1111/1750-3841.12907 · Source: PubMed

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 Fermentation of Soybean Meal Hydrolyzates with
Saccharomyces cerevisiae and Zymomonas mobilis
for Ethanol Production
Deivis E. Luján-Rhenals, Rubén O. Morawicki, Edward E. Gbur, and Steven C. Ricke

Abstract: 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
E: Food Engineering &

fermentation. These substrates were produced from the dilute-acid hydrolysis of soybean meal at 135 °C for 45 min with
Materials Science

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.

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

Introduction For this research, the capability of 2 microorganisms—

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 be-
coming 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 over-
looked is soybean meal (SBM), a coproduct of soybean oil pro-
duction. 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 car-
bohydrates, half are structural and the other half are comprised of
low-molecular weight sugars, oligosaccharides, and starch in rela-
tively small quantities (Karr-Lilienthal and others 2005). Around
17% of low-molecular-weight sugars consist of a mixture of glu-
cose, 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 Lujan-
Rhenals, 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 Córdoba, Programa de Ingenierı́a de Alimentos. Sede Berástegui. Km. 12
Via Cereté-Ciénaga de Oro, Córdoba, 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).
Saccharomyces cerevisiae and Zymomonas mobilis—to 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 hy-
drolyzates 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 treat-
ing 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 (Luján-Rhenals
2013).
Hydrolyzates were prepared by treating 50 g of SBM with
250 mL of H2 SO4 solution in 500-mL PYREX R
media stor-
age bottles with screw caps (Luján-Rhenals 2013). To avoid pres-
sure buildup inside the bottles during the heat treatment, caps
were not completely tightened. A Tuttnauer 2340E Steam Auto-
clave (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

C 2015 Institute of Food Technologists

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E1512 Journal of Food Science r Vol. 80, Nr. 7, 2015 doi: 10.1111/1750-3841.12907
Further reproduction without permission is prohibited
Fermentation of SBM hydrolyzates . . .

Table 1–Experimental design for the ethanolic fermentation of Cells reactivation and preparation of preinoculum
soybean meal hydrolyzates with S. cerevisiae NRRL Y-2233 and
Z. mobilis NRRL B-4286.
and inoculum
Both S. cerevisiae and Z. mobilis were reactivated in 5 mL of yeast
Standard order Run order Microorganism Broth malt (YM) broth (Dickinson and Company, Sparks, Md., U.S.A.)
1 7 S. cerevisiae SBMB0 and incubated at 30 °C for 24 h in an orbital shaker (Thermo
2 14 S. cerevisiae SBMB0 Scientific Max Q 4450, Dubuque, Iowa, U.S.A.) set at 150 rpm.
3 6 S. cerevisiae SBMB1 The preinoculum consisted of 5 mL of sterile YM broth at 30 °C
4 18 S. cerevisiae SBMB1 where both S. cerevisiae and Z. mobilis were inoculated separately
5 2 S. cerevisiae SBMB2
6 9 S. cerevisiae SBMB2 and grown overnight in the orbital shaker. For each respective
7 1 S. cerevisiae SBMB3 strain, 0.2 mL of the culture was plated out with a loop over the
8 12 S. cerevisiae SBMB3 surface of 2 petri dishes containing YM agar (20 g/L of glucose,
9 11 S. cerevisiae SBMB4 3 g/L of yeast extract, 5 g/L of peptone, and 3 g/L of malt extract)
10 16 S. cerevisiae SBMB4
11 3 Z. mobilis SBMB0
and grown overnight at 30 °C before adding to the final inoculum.
12 19 Z. mobilis SBMB0 The inoculum was prepared by adding all the colonies formed in
13 4 Z. mobilis SBMB1 both dishes to 80 mL of SBMB and allowing the cells to adapt by

E: Food Engineering &

14 17 Z. mobilis SBMB1 incubating overnight at 30 °C in the orbital shaker.

15 8 Z. mobilis SBMB2
16 10 Z. mobilis SBMB2
17 5 Z. mobilis SBMB3
18 13 Z. mobilis SBMB3
19 15 Z. mobilis SBMB4
20 20 Z. mobilis SBMB4
SBMB0: 135˚C, 0% H2 SO4 , 45 min.
SBMB1: 135˚C, 0.5% H2 SO4 , 45 min.
SBMB2: 135˚C, 1.25% H2 SO4 , 45 min.
SBMB3: 135˚C, 2% H2 SO4 , 45 min.
SBMB4: 120˚C, 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 con-
centration 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.
Microbial strains
Strains of S. cerevisiae NRRL Y-2233 and Z. mobilis subsp.
mobilis NRRL B-4286 were provided as lyophilized powders from
the culture collection of the United States Dept. of Agriculture
(USDA), Agriculture Research Service (Peoria, Ill., U.S.A.).
Fermentable sugar analysis
Fermentable sugars in the hydrolyzates were analyzed with Y p/s = −
High-Performance Size Exclusion Chromatography and Refrac-
tive Index (HPSEC-RI) detection. The chromatograph was a Wa-
ters, (Milford, Mass., U.S.A.) with a 515 HPLC pump, a manual
injector with a 50-μL 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
Denko America, Inc., New York, N.Y., U.S.A.) connected in
series. Columns were preceded by a Shodex OH pack SB-G
(50 × 6 mm) guard column. Columns and guard column were
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-
tified using 6-point calibration curves with fructose and glucose
as standards.
Materials Science
Fermentation
Fermentations were conducted at 30 °C for 36 h with an ini-
tial pH of 5 to 5.5, dissolved oxygen (%DO) less than 1% after
stabilization, and an initial biomass concentration between 7×106
and 1×107 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 sys-
tems for temperature, pH, %DO, agitation, and foam level. During
fermentation, samples were taken every 4 h to estimate fermen-
tation kinetic parameters maximum specific growth rate (μmax),
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 den-
sity 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:
dx
Yx/s = − Biomass yield (g biomass/g sugars) (1)
ds
where x and s represent concentrations of cellular biomass (g/L)
and substrate (g/L), respectively.
dp
Ethanol yield from substrate (g ethanol/g sugars)
ds
(2)
(3)
dp 1
qp = Specific rate of product formation (4)
dt x
(r s ) − r x/Yxs − r p/Yps
ms = Maintenance coefficient
x
(g sugar/g cell h) (5)

Vol. 80, Nr. 7, 2015 r Journal of Food Science E1513

 Fermentation of SBM hydrolyzates . . .

(CH3 COONa) which is the sodium salt of acetic acid formed dur-
where 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 = μmax 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), μmax 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).
E: Food Engineering &

low rate of sugar consumption occurred due to toxic compounds

Considering the following boundary conditions: t = 0, x = xo ,
Materials Science

formed during the hydrolysis process that reduces the yeast growth
S = So, and P = 0, Eq (6) was integrated and Eq (7) was obtained
(Luján-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 ,
xo xmax e μ max t 45 min) (Figure 2b). Similar to S. cerevisiae, Z. mobilis had low
x= (7) rates of apparent sugar consumption during growth in the broths
xmax − xo + xo e μ 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.
quantities by the microorganisms according to the HPLC analysis. Overall, the maintenance coefficient ms (g sugar consumed/
g cell h) for both microorganisms did not yield statistically signifi-
Experimental 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
factors (Table 1). At each level, experiments were run twice. coefficients for Z. mobilis were lower than the ms coefficients cal-
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). There-
Cary, N.C., U.S.A.) and the kinetic parameters μmax , 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
and kinetics parameters, data were analyzed with JMP R
version 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 Tukey–Kramer test (α = 0.05). ethanol) occurred with the SBMB3 (135 °C, 2% H2 SO4 , 45 min)
Results and Discussion after 28 h of fermentation, where possibly some inhibitor com-
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.
S. cerevisiae exhibited optimal responses for the production of Ethanol production by Z. mobilis peaked at 9.2 g/L ethanol af-
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 fermen-
bacterium exhibited no growth with the hydrolyzate SBMB3 tation. However, the lowest ethanol production levels—1 g/L at
(135 °C, 2% H2 SO4 , 45 min), but with the SBMB1 20 h—were 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 (Luján-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 ((Luján-Rhenals and others 2014) and also by the pres- The 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 . . .

2 microorganisms and the respective SBM broths used. However, These values for S. cerevisiae are comparable to, and some higher
numerically both microorganisms showed similar ethanol yields than those reported by other researchers (Da Cunha-Pereira and
to the theoretical values, which is 100% for 0.51 g ethanol/g of others 2011). However, Z. mobilis reached a maximum ethanol
sugars (Doran 1997). Fermentations in the SBM0 and SBMB1 volumetric productivity (rp) of 0.61 g ethanol/Lh only with the
gave the highest ethanol yields, 96% of the theoretical yield for SBM1, which is lower than the values, 1.4 to 1.8 g ethanol/Lh with
both microorganisms, which are comparable or even higher than Z. mobilis during the production of ethanol using SBM molasses
values reported in previous research (Siqueira and others 2008; (Letti and others 2012). Also, it is lower than 2.8 g ethanol/Lh
Zhang and Feng 2010; Da Cunha-Pereira and others 2011; Letti obtained for the production of ethanol with Z. mobilis from
and others 2012; Romao and others 2012). Additionally, the sweet potato (Zhang and Feng 2010); however, it is higher than
highest ethanol volumetric productivity (rp) for S. cerevisiae was 0.59 g/Lh using lignocellulosic material as substrate (Mohagheghi
achieved with the SBM1 (1.01g ethanol/L h) and SBMB4 (1.00 and others 2002). These results demonstrate that the application
g ethanol/L h), with no significant difference between the 2, but of Z. mobilis for ethanol fermentation is not feasible when the
differed with respect to the other broths used in this research substrate is SBM broth obtained under the conditions applied in
(Table 2). these experiments. This is probably due to the high concentration

E: Food Engineering &

Materials Science
A SBMB0 2.5 B SBMB1 20
2.5 12

Sugars and Ethanol (g/L)

Sugars and Ethanol (g/L)
2.0
2.0 10 15

Biomass (g/L)
Biomass (g/L)

8 1.5
1.5 10
6 1.0
1.0
4 5
0.5
0.5 2

0.0 0 0.0 0
0 10 20 30 40 0 10 20 30 40

Time (h) Time (h)

Biomass (g/L) Sugars (g/L) Ethanol (g/L) Biomass (g/L) Sugars (g/L)

2.5
C SBMB2 30 2.5
D SBMB3 35
30

Sugars and Ethanol (g/L)

25
Sugars and Ethanol (g/L)

2.0 2.0
25
Biomass (g/L)

20
Biomass (g/L)

1.5 1.5 20
15
1.0 1.0 15
10
10
0.5 0.5
5 5

0.0 0 0.0 0
0 10 20 30 40 0 10 20 30 40
Time (h) Time (h)
Biomass (g/L) Sugars (g/L) Ethanol (g/L)
Biomass (g/L) Sugars (g/L) Ethanol (g/L)
463
E SBMB4
2.5 25
Sugars and Ethanol (g/L)

2.0 20
Biomass (g/L)

1.5 15

1.0 10

0.5 5

0.0 0
0 10 20 30 40
Time (h)
Biomass (g/L) Sugars (g/L) Ethanol (g/L)

Figure 1–Fermentation 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

 Fermentation of SBM hydrolyzates . . .

Table 2–Means 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.

Yp/s Yx/s Ms Rp
SBM broth Microorganism (g ethanol/g sugar) (g cell/g sugar) (g sugar/g cell h) (g ethanol/Lh)
SBMB0 S. cerevisiae 0.49 ± 0.021 a 0.23 ± 0.014 abc 0.69 b 0.67 ± 0.028 b
SBMB0 Z. mobilis 0.49 ± 0.007 a 0.23 ± 0.057 ab 0.44 b 0.37 ± 0.007 cd
SBMB1 S. cerevisiae 0.49 ± 0.014 a 0.10 ± 0.014 cd 1.19 b 1.00 ± 0.028 a
SBMB1 Z. mobilis 0.48 ± 0.028 a 0.08 ± 0.014 d 0.97 b 0.61 ± 0.007 bc
SBMB2 S. cerevisiae 0.46 ± 0.007 a 0.04 ± 0.007 d 2.73 a 0.61 ± 0.113 bc
SBMB2 Z. mobilis 0.26 ± 0.042 a 0.15 ± 0.021 bcd 0.65 b 0.11 ± 0.007 de
SBMB3 S. cerevisiae 0.40 ± 0.057 a 0.02 ± 0.007 d 2.78 a 0.29 ± 0.092 de
SBMB4 S. cerevisiae 0.49 ± 0.007 a 0.11 ± 0.007 bcd 1.13 b 1.01 ± 0.021 a
SBMB4 Z. mobilis 0.29 ± 0.113 a 0.32 ± 0.014 a 0.17 b 0.07 ± 0.007 e
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.

of acetic acid, salts, and other toxic unknown compounds formed However, SBMB3 (135 °C, 2% H2 SO4 , 45 min) exhibited a ma-
E: Food Engineering &
Materials Science

during 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 cel-
maximum 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,

A SBMB0 B SBMB1
4.0 12 4.0 20

Sugars and Ethanol (g/L)

Sugars and Ethanol (g/L)

3.5 3.5
10
3.0 3.0 15
Biomass (g/L)

Biomass (g/L)

8
2.5 2.5
2.0 6 2.0 10
1.5 1.5
4
1.0 1.0 5
2
0.5 0.5
0.0 0 0.0 0
0 10 20 30 40 0 10 20 30 40
Time (h) Time (h)
Biomass (g/L) Sugars (g/L) Ethanol (g/L)
Biomass (g/L) Sugars (g/L) Ethanol (g/L)

C SBMB2 D SBMB4

4.0 25 4.0 25
Sugars and Ethanol (g/L)

3.5 3.5
Sugars and Ethanol (g/L)

20 20
3.0 3.0
Biomass (g/L)
Biomass (g/L)

2.5 2.5 15
15
2.0 2.0
10 1.5 10
1.5
1.0 1.0
5 5
0.5 0.5
0.0 0 0.0 0
0 10 20 30 40 0 10 20 30 40
Time (h) Time (h)
Biomass (g/L) Sugars (g/L) Ethanol (g/L) Biomass (g/L) Sugars (g/L) Ethanol (g/L)

Figure 2–Fermentation 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

Fermentation of SBM hydrolyzates . . .
Table 3–Means 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 Microorganism
SBMB0 S. cerevisiae
SBMB0 Z. mobilis
SBMB1 S. cerevisiae
SBMB1 Z. mobilis
SBMB2 S. cerevisiae
SBMB2 Z. mobilis
SBMB3 S. cerevisiae
SBMB4 S. cerevisiae
SBMB4 Z. mobilis
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.
acetic acid, and other inhibitors in the broth that restricted its
growth (Doran 1997; Yang and others 2010). In general, Z. mo-
bilis 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 maxi-
mum specific growth rates (μmax ) shows that in the SBM0 and
SBMB4, μmax for S. cerevisiae (0.63 and 0.60 h-1 , respectively)
were higher but not significantly different than μmax for Z. mobilis,
0.55 h-1 , in the substrate T3C0t3. Wang and others (2004), also
using the logistic model, reported a μmax of 0.1 h-1 for the ethano-
lic fermentation of apple juice with S. cerevisiae ( Huang and Wang
2010) obtained a μmax of 5.2 h–1 using Saccharomyces diastaticus
with mixed sugars as a substrate. Mohagheghi and others (2002)
reported for Z. mobilis a μmax of 0.34 h-1 for ethanolic fermenta-
tion of hydrolyzed lignocellulosic biomass. In general, μmax for S.
cerevisiae was higher for substrates obtained with pretreatments of
less severity; Z. mobilis μmax 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 ef-
fect 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 acid-
hydrolyzed soybean meal broths tested without any additional
µmax (h-1 ) xo (g biomass/L) xmax (h-1 )
0.63 ± 0.12 a 0.17 ± 0.08 a 2.28 ± 0.05 a
0.55 ± 0.05 a 0.18 ± 0.05 a 3.79 ± 0.06 f l
0.45 ± 0.09 afgh 0.32 ± 0.09 a 1.81 ± 0.05 b
0.16 ± 0.02 bk 0.18 ± 0.04a 5.1 ± 0.92 g l
0.21 ± 0.11 bdfi 0.16 ± 0.08 a 0.66 ± 0.10 cej
0.45 ± 0.25 aijkl 0.18 ± 0.1 a 0.65 ± 0.05 hjk
0.19 ± 0.15 begj 0.14 ± 0.08 a 0.49 ± 0.12 dek
0.60 ± 0.11 a 0.24 ± 0.09 a 2.23 ± 0.05 a
0.29 ± 0.03 cdehl 0.26 ± 0.06 a 2.97 ± 0.09 i
nutrients, while Z. mobilis had a lower performance for most
E: Food Engineering &
broths. The highest ethanol production of S. cerevisiae, 8 g
Materials Science
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 produc-
tion, 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. Luján-Rhenals was supported in
part by COLCIENCIAS of Colombia, LASPAU, and Univ. de
Córdoba (Colombia).
Author Contributions
Deivis Luján-Rhenals performed the experiments, analyzed the
data, and wrote the paper. Ruben Morawicki designed the exper-
iments, analyzed the data, and wrote the paper. Edward Gbur
provided technical details in the experimental design and statisti-
cal analysis. Steven C. Ricke provided technical support with the
fermentation.
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