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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 High-Performance
Size Exclusion hromatography and Refractive Index (HPSEC-RI) detection. The
chromatograph was aWaters, (Milford, Mass., U.S.A.) with a 5 15 HPLC pump, a
manual injector with a 50-L sample loop, and a 2410 refractive index detector
set at 40 C. Columns used for the separation of sugars were a Shodex OH Pack
SB-804 HQ (300 8 mm) followed 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 quantified using
6-point calibration curves with fructose and glucose as standards. 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 sterileYMbroth 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:
Growth kinetics and model development for the ethanol fermentation
The kinetic parameters of biomass production were determined with the
logistic model (Eq (6)) (Wang and others 2004).
(6)
where dx/dt is the rate of biomass during the time of fermentation (g cell/ h),
max is the maximum specific growth rate (h-1), x is the biomass
concentration (g/L), and xmax is the maximum biomass concentration (g/L).
Considering the following boundary conditions: t = 0, x = xo, S = So, and P = 0,
Eq (6) was integrated and Eq (7) was obtained as a result, which relates
biomass production (X) and fermentation time (t) and was used to fit the
experimental data.
(7)
For the calculation of the kinetics parameters, only glucose and fructose were
considered as growth-limiting-substrates because these sugars were consumed
in the highest and most consistent quantities by the microorganisms according
to the HPLC analysis.
Experimental design
For the fermentation, the experimental design was a complete randomized
block design with microorganism type and broth as factors (Table 1). At each
level, experiments were run twice.
Statistical analysis
Data was analyzed with SAS Version 9.2 software (SAS Inst. Inc., Cary, N.C.,
U.S.A.) and the kinetic parameters max, xmax, and xo determined. Analysis of
covariance (ANCOVA) was used to fit the data to the logistic model using the
Gauss-Newton nonlinear regression method and comparisons of each
regression coefficient across the treatments. For the minimum inhibitor
concentrations and kinetics parameters, data were analyzed with JMPR version
9.0.0 (SAS Inst. Inc.). To analyze the kinetic parameters (Yp/s, Yx/s, ms, and
qp), an analysis of variance (ANOVA) was used and differences in the mean of
the kinetic parameters were analyzed using the TukeyKramer test ( = 0.05).
Results and Discussion
Ethanol fermentation with S. cerevisiae and Z. mobilis S. cerevisiae exhibited
optimal responses for the production of ethanol, sugars consumption, and cell
growth in all the SBM broths used in this study (Figure 1). However, in most
fermentation broths, Z. mobilis was not as efficient (Figure 2). This bacterium
exhibited no growth with the hydrolyzate SBMB3 (135 C, 2% H2SO4, 45 min),
but with the SBMB1 (135 C, 0.5% H2SO4, 45 min) displayed its maximum
ethanol production. S. cerevisiae, on the other hand, demonstrated rapid sugar
consumption, likely because it is a robust microorganism that was not critically
affected by the inhibitors present in most of the hydrolyzate SBMB (LujanRhenals and others 2014). Zymomonas mobilis was affected by inhibitory
compounds of the SBMB ((Lujan-Rhenals and others 2014) and also by the
presence of salts coming from the hydrolyzate, such as sodium acetate
(CH3COONa) which is the sodium salt of acetic acid formed during the reaction
and sodium sulfate (Na2SO4) formed during the neutralization of the sulfuric
acid with sodium hydroxide. (Yang and others 2010) demonstrated that the
combination of elevated Na+ and acetate ions led to a synergistic inhibitory
effect on strain ZM4.
Kinetics of substrate consumption during fermentation
S. cerevisiae exhibited its most rapid sugar consumption of 15.6 g/L during the
initial 12 h of fermentation with the SBMB4 (120 C, 1.25% H2SO4, 30 min) and
15.1 g/L during the 1st 12 h of fermentation with the SBMB1 (135 C, 0.5%
H2SO4, 45 min) (Figure 1e). On the other hand, the SBM broths from the most
severe hydrolysis treatments appeared to result in a low rate of sugar use from
the fermentation broth by S. cerevisiae (Figure 1e). This low rate of sugar
consumption occurred due to toxic compounds formed during the hydrolysis
process that reduces the yeast growth (Lujan-Rhenals and others 2014). Z.
mobilis also expressed its most rapid sugar uptake of 15.0 g/L per 16 h of
fermentation with the SBMB1 (135 C, 0.5% H2SO4, 45 min) (Figure 2b). Similar
to S. cerevisiae, Z. mobilis had low rates of apparent sugar consumption during
growth in the broths (Figure 2c and 2d) resulting from the most severe
pretreatments, particularly SBMB2 (135 C, 1.25% H2SO4, 45 min), which is
probably due to the inhibitory effect of acetic acid and salts as previously
discussed.
Overall, the maintenance coefficient ms (g sugar consumed/ g cell h) for both
microorganisms did not yield statistically significant differences except for S.
cerevisiae with SBMB2 and SBMB3, which gave higher values (2.7 and 2.8 g
sugar/g cell h, respectively) than the other coefficients (Table 2). However,
numerically, all ms coefficients for Z. mobilis were lower than the ms
coefficients calculated for S. cerevisiae which means that the bacteria
apparently required less carbon substrate per cell than the yeast. This result
has also been previously reported by (Dien and others 2003). Therefore, Z.
mobilis utilizes less substrate per gram of cells produced than S. cerevisiae and
has more carbon available for the production of ethanol (Dien and others
2003).
Kinetics of ethanol production
S. cerevisiae exhibited its maximum ethanol production of 8 g/L during the 1st
8 h of fermentation of the SBMB1 (135 C, 0.5% H2SO4, 45 min) and SBMB4
(120 C, 1.25% H2SO4, 30 min). In contrast, the lowest ethanol production
(5.67 g/L ethanol) occurred with the SBMB3 (135 C, 2% H2SO4, 45 min) after
28 h of fermentation, where possibly some inhibitor compounds (salts, acetate
from acetic acid, and other unidentified toxic substances) could have reduced
the ethanol productivity. Ethanol production by Z. mobilis peaked at 9.2 g/L
ethanol after 20 h of fermentation with the SBMB1 (135 C, 0.5% H2SO4, 45
min). The SBMB0 (135 C, 0% H2SO4, 45 min) allowed a maximum ethanol rate
of 3.9 g/L ethanol after 20 h of fermentation. However, the lowest ethanol
production levels1 g/L at 20 hwere attained with the SBMB2 (135 C, 1.25%
H2SO4, 45 min) and SBMB4 (120 C, 1.25% H2SO4, 30 min), which is likely
caused by inhibitors (acetate from acetic acid, other salts, and unknown
compounds). There have been previous reports that ethanol production with Z.
mobilis and its growth can be reduced substantially even with low
concentrations (2 to 8 g/L) of acetic acid (Wang 2008).
The ethanol yield (Yp/s, g ethanol/g of sugars) reported in Table 2 did not show
significant statistical differences between the 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 CunhaPereira 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). 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/Lhwith 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 of acetic acid, salts, and other toxic unknown compounds formed
during the acid hydrolysis in the broths obtained under severe treatments.
Kinetics of biomass growth and model development for the fermentation
Data reported in Figure 1a indicate that S. cerevisiae yielded its maximum
biomass concentration (2.3 g/L) after 12 h of anaerobic fermentation when
exposed to the SBM0 (135 C, 0% H2SO4, 45 min) followed by enzymatic
treatment with cellulase. Likewise, this maximum concentration of biomass (2.3
g/L) was also reached with SBMB4 (120 C, 1.25% H2SO4, 30 min) at 24 h.
However, SBMB3 (135 C, 2% H2SO4, 45 min) exhibited a major inhibition of
yeast growth since the biomass yielded no more 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.
Z. mobilis exhibited its highest biomass concentration, 3.9 g/L, after 20 h of
fermentation (Figure 7.2a) with the SBMB0 (135 C, 0%, H2SO4, 45 min),
followed by enzymatic treatment with cellulase.
Likewise, the biomass produced (3.3 g/L) when fermenting the SBMB1 (135 C,
0.5% H2SO4, 45 min) was very close to the former but required 24 h to reach
this level. In contrast, SBM2 (135 C, 2% H2SO4, 45 min) exhibited the primary
inhibitory effects on Z. mobilis growth due to higher concentrations of salts,
acetic acid, and other inhibitors in the broth that restricted its growth (Doran
1997; Yang and others 2010). In general, Z. mobilis also showed growthassociated 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 andWang 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 maximumconcentration 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% H2SO4, 45 min and the
one at 120 C, 1.25% H2SO4, 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%H2SO4, 45
min.
Cepas microbianas
Las cepas de S. cerevisiae NRRL Y-2233 y Z. mobilis subsp. mobilis NRRL B4286 se ofrece como polvos liofilizados de la coleccin de cultivos del
Departamento de Agricultura de Estados Unidos (USDA), Servicio de
Investigacin Agrcola (Peoria, Ill., EE.UU.).
Anlisis azcar fermentable
Azcares fermentables en los hidrolizados fueron analizados con de alto
rendimiento hromatography Tamao exclusin y deteccin de ndice de
refraccin (HPSEC-RI). El cromatgrafo fue aWaters, (Milford, Mass., EE.UU.) con
una bomba de 5 15 HPLC, un inyector manual con un bucle de muestra de 50 l,
y un detector de ndice de refraccin 2410 fijado en 40 C. Columnas utilizadas
para la separacin de azcares eran un Pack OH HQ SB-804 Shodex (300 8
mm), seguido de Shodex OH Paquete HQ SB-802 (300 8 mm) (Showa Denko
America, Inc., Nueva York, NY, EE.UU.) conectado en serie. Las columnas fueron
precedidos por una manada Shodex OH SB-G (50 6 mm) columna de
seguridad. Columnas y columna de seguridad se mantuvieron a 55 C en un
calentador de columna. Una solucin de 0,1 M NaNO3 con 0,2% NaN3 funcionar
isocrticamente a un caudal de 0,4 ml / min fue utilizado como la fase mvil.
Los azcares se cuantificaron utilizando curvas de calibracin de 6 puntos con
fructosa y glucosa como estndares. Las clulas reactivacin y preparacin de
preinculo y inculo Tanto S. cerevisiae y Z. mobilis se reactivaron en 5 ml de
malta levadura (YM) caldo (Dickinson and Company, Sparks, Md., EE.UU.) y se
incubaron a 30 C durante 24 h en un agitador orbital (Thermo Scientific Max
Q 4450, Dubuque, Iowa, EE.UU.) fij en 150 rpm. El preinculo consista en 5 ml
de sterileYMbroth a 30 C, donde tanto S. cerevisiae y Z. mobilis se inocularon
por separado y se cultivaron durante la noche en el agitador orbital. Para cada
cepa respectiva, 0,2 ml del cultivo se sembr a cabo con un bucle sobre la
superficie de 2 platos de petri que contienen agar YM (20 g / L de glucosa, 3 g /
L de extracto de levadura, 5 g / L de peptona, y 3 g / L de extracto de malta) y
se cultivaron durante la noche a 30 C antes de aadir al inculo final. El
inculo se prepar aadiendo todas las colonias formadas en ambos platos a
80 ml de SBMB y permitiendo que las clulas se adaptan mediante la
incubacin durante la noche a 30 C en el agitador orbital.
Fermentacin
Las fermentaciones se llevaron a cabo a 30 C durante 36 h con un pH inicial
de 5 a 5,5, oxgeno disuelto (DO%) de menos de 1% despus de la
estabilizacin, y una concentracin de biomasa inicial entre 7 106 y 1 x 107
clulas / ml (Mullins y Nesmith 1987; Siqueira y otros 2008; Laopaiboon y otros
2009). El fermentador fue un sobremesa fermentador y biorreactor (New
Brunswick Scientific, Edison, NJ, EE.UU.) con sistemas de control de
Conclusiones
S. cerevisiae mostraba buenas cualidades en la produccin de etanol, el
consumo de azcares, y el crecimiento celular para todos los caldos de la
harina de soja acidhydrolyzed probados sin ningn tipo de nutrientes
adicionales, mientras Z. mobilis tena un rendimiento inferior para la mayora
de caldos. Se alcanz la mayor produccin de etanol de S. cerevisiae, 8 g de
etanol / L, (4 g de etanol / 100 g SBM fresco) durante la primera 8 h de
fermentacin con el caldo obtenido a 135 C, 0,5% de H2SO4, 45 min y el de
120 C, 1,25% de H2SO4, y 30 min. Sin embargo, Z. mobilis produjo la mxima
produccin de etanol, 9,2 g / L de etanol, (4,6 g de etanol / 100 g SBM fresco)
slo despus de la primera 20 h con el hidrolizado obtenido a 135 C, 0,5% de
H2SO4, 45 min.