ENERGY EXPLORATION & EXPLOITATION · Volume 30 · Number 2 · 2012 pp.

193–205 193

Optimization of enzymatic hydrolysis of corn stover for

improved ethanol production

Vasudeo P. Zambare 1 and Lew P. Christopher 1,2*

1
Center for Bioprocessing Research & Development
2
Department of Chemical & Biological Engineering South Dakota
School of Mines & Technology 501 E St Joseph,
Rapid City 57701, South Dakota, USA
*
Author for corresponding. Email: lew.christopher@sdsmt.edu

(Received 22 June 2011; accepted 18 December 2011)

Abstract
Response surface methodology (RSM) was used to optimize the enzymatic
hydrolysis of corn stover (CS), an abundant agricultural residue in the USA. A
five-level, three-variable central composite design (CCD) was employed in a
total of 20 experiments to model and evaluate the impact of pH (4.1–6.0), solids
loadings (6.6–23.4%), and enzyme loadings (6.6−23.4 FPU g−1 DM) on glucose
yield from thermo-mechanically extruded CS. The extruded CS was first
hydrolyzed with the crude cellulase of Penicillium pinophilum ATCC 200401
and then fermented to ethanol with Saccharomyces cerevisiae ATCC 24860.
Although all three variables had a significant impact, the enzyme loadings
proved the most significant parameter for maximizing the glucose yield. A
partial cubic equation could accurately model the response surface of enzymatic
hydrolysis as the analysis of variance (ANOVA) showed a coefficient of
determination (R2) of 0.82. At the optimal conditions of pH of 4.5, solids
loadings of 10% and enzyme loadings of 20 FPU g−1 DM, the enzymatic
hydrolysis of pretreated CS produced a glucose yield of 57.6% of the glucose
maximum yield which was an increase of 10.4% over the non-optimized
controls at zero-level central points. The predicted results based on the RSM
regression model were in good agreement with the actual experimental values.
The model can present a rapid means for estimating lignocellulose conversion
yields within the selected ranges.

Keywords: Corn stover, Bioprocessing, Enzymatic hydrolysis, Solids loadings,

Ethanol, Optimization, Response surface methodology

1. INTRODUCTION
Corn has been traditionally number one crop in the United States. In 2009, the
country produced 13.15 billion bushels of corn on 86.48 million acres which
represents 41.9% of the world’s corn production (Jennings, 2010). Annually, nearly
194 Optimization of enzymatic hydrolysis of corn stover for improved ethanol production

545 million dry tons of corn stover (CS), the biomass residue from harvesting of corn
grain, are produced in the US (Aden et al., 2002). More than half the dry matter (DM)
in CS consists of polysaccharides such as glucan, xylan, galactan, arabinan and
mannan (Hames et al., 2003). This makes CS an attractive and abundant agricultural
waste of no food value which could however be utilized as inexpensive feedstock for
production of sugars, ethanol, organic acids, etc. The utilization of lignocellulosic
biomass for value-added biofuels and biochemicals involves a few bioprocessing
steps which determine the product yields and process efficiency such as biomass
pretreatment, enzymatic hydrolysis and fermentation (Ekinci et al., 2011).
The biomass pretreatment is intended to open up the lignocellulosic matrix and its
efficiency is evaluated based on factors affecting the enzymatic digestibility of
the lignocellulosic matrix such as accessible surface area (porosity), cellulose
crystallinity, lignin and hemicellulose content. The pretreatment process should be
efficient and economical, allowing the use of low enzyme loadings, and prevent
carbohydrate degradation and formation of products inhibitory to the subsequent
enzymatic hydrolysis and fermentation (Taherzadeh and Karimi, 2007). Pretreatments
of biomass employing alkali, dilute acid, steam, ammonia, ammonia fiber explosion,
lime, hot water, wet oxidation, etc. have been used (Galbe and Zachi, 2002;
Taherzadeh and Karimi, 2007; Balat, 2010). Among these, thermo-mechanical
extrusion was shown to improve substrate digestibility, destroy recalcitrant lignin-
carbohydrate complexes, and decrease relative viscosity (Karunanithy and
Muthukumarappan, 2010).
The pretreatment efficiency impacts on the following step of enzymatic
hydrolysis of pretreated cellulosic material using microbial cellulases. Several
reviews have summarized the microbial production of cellulases and their use for
saccharification of agricultural waste such as corn cobs and stover, saw dust, rice
husks, wheat bran, etc. (Rajoka, 2005; Hao et al., 2006; Ja’afaru and Fagade, 2007;
Folakemi et al., 2008; Milala et al., 2009; Lau et al., 2010; Reddy et al., 2010). The
production costs of cellulase have been estimated to account for up to 50% of the
total ethanol production costs (Himmel et al., 1997). Hence, in order to save costs,
the enzyme loadings need to be optimized for a substrate-specific process (Shen
and Agblevor 2008) taking into account factors such as solids loadings, hydrolysis
time, pH, enzyme inhibitions by end product or soluble components, etc. (Hodge et al.,
2008). The use of low enzyme loadings and high solids in hydrolysis of
lignocellulose to fermentable sugars has important economic implications for
reduced size of reactor vessels, increased throughput, reduced usage of water and
reduced costs of waste water treatment, all of that resulting in reduced capital and
operating costs of ethanol production (Kwiatkowski et al., 2006). Evidently, there
is an economic tradeoff between the capital and energy savings realized by
operation at low enzyme/high solids loadings, on one hand, and reduced yield of
fermentable sugars and ethanol, on the other. Hence, it is important to examine and
optimize these parameters to improve the bioconversion economics (Humbird et al.,
2010).
To date, optimization of cellulase production and hydrolysis have mostly
utilized the traditional one-factor-at-a-time optimization method. Although
ENERGY EXPLORATION & EXPLOITATION · Volume 30 · Number 2 · 2012 195

successfully used, this approach is tedious and time consuming as it normally takes
between 4 to 12 months for complete optimization, and unfortunately overlooks the
combined interactions between physical and nutritional factors affecting
fermentation. The above disadvantages of the one-factor-at-a-time approach
however can be overcome using statistical methods such as response surface
methodology (RSM) (Krishna and Chowdary, 2000; Kim et al., 2008; Jeya et al.,
2009; Lee et al., 2009; Elumalai and Thangavelu, 2010). RSM is a standard, well
established mathematical optimization procedure, which has recently gained
attention bioprocess optimization (Ferreira et al., 2009; Ko et al., 2009; Zhang et al.,
2009). For instance, Ma et al. (2009) evaluated the effects of microwave
pretreatment conditions on enzymatic saccharification of rice straw using Box-
Behnken design and RSM. Fang et al. (2010) employed Plackett–Burman design
and central composite design (CCD) to improve the glucose yield from steam
exploded corn stover using cellulases from a mixed culture of Trichoderma reesei
and Aspergillus niger.
This study aims at statistical optimization of enzymatic saccharification of corn
stover with Penicillium pinophilum cellulases for improved glucose and ethanol
yields. It also demonstrates the advantages of statistical methods for optimization that
can save time and costs from the reduced number of individual experiments and can
improve the accuracy and interpretation of research findings.

2. MATERIALS AND METHODS

2.1. Materials and chemicals
Pre-milled CS was obtained from South Dakota State University (SDSU), Brookings,
SD, USA. It was air-dried to 9.9% moisture content (DM) and stored at 4°C until
further use. Analytical grade dinitrosalicylic acid (DNS) and glucose were purchased
from Fisher Scientific (Pittsburgh, PA, USA).

2.2. Extrusion pretreatment

CS at a moisture content of 20% (wet basis) was thermo-mechanically pretreated using
a single screw extruder (Brabender Plasti-corder Extruder Model PL2000,
Hackensack, NJ, USA). During extrusion, the screw speed of the extruder and
temperature of barrel were maintained at 100 rpm and 100°C, respectively. The
chemical composition of CS before and after pretreatment was analyzed by Olson
Biochemistry Laboratories, SDSU, Brookings, SD, USA using standard
chromatography methods.

2.3. Enzyme assay

The Filter Paperase (FPase) activity was assayed according to the procedure described
by Ghose (1987). The reaction mixture contained 1 mL of 50 mM acetate buffer
(pH 5), 50 mg Whatman filter paper No. 1 and 0.5 mL enzyme solution. After 10 min
incubation at 45°C, a DNS reagent (3 mL) was added and the reducing sugars were
measured according to Miller (1959). One unit of filter paperase activity (FPU) was
defined as the amount of enzyme that released 1 µmol of glucose per minute under the
assay conditions.
196 Optimization of enzymatic hydrolysis of corn stover for improved ethanol production

2.4. Maximum glucose yield

Pretreated corn stover (PCS) was acid hydrolyzed as described in TAPPI Test
Methods (1984). Samples of 0.35 g were treated with 3 mL of 72% sulfuric acid (v/v),
stirred and placed in a 30°C water bath for 1 h with occasional stirring. The content
was washed with 84 mL of water in a 250 mL capacity flask. The whole content was
autoclaved at 103°C (15 psi) for 1 h. After autoclaving, samples were cooled to room
temperature using an ice bath and neutralized with alkali. The filtered hydrolyzates
were analyzed for glucose with a 2700 Biochemistry Analyzer (YSI Life Sciences,
Yellow Spring, Ohio, USA) according to the manufacturer’s instructions. Following
acid hydrolysis, a glucose yield of 0.2580 g glucose/ g biomass was obtained and
assumed as the maximum glucose yield of 100%.

2.5. Enzymatic hydrolysis

Samples of extruded CS corresponding to solids loadings of 6.6, 10.0, 15.0, 20.0 and
23.4% were weighed out and added together with 10 mL of 100 mM acetate buffer
to 25 mL capacity screw cap bottles. The bottles containing solids and buffer
(without enzyme) were sterilized in an autoclave (121°C, 20 min). Aliquots of filter-
sterilized enzyme was then aseptically added to the bottles to obtain final enzyme
loadings of 6.6, 10.0, 15.0, 20.0 and 23.4 FPU g−1 DM and the whole reaction
mixture of enzyme and substrate was incubated at 45oC under shaking condition
(150 rpm). Samples were withdrawn from each bottle after 96 h of enzymatic
hydrolysis and centrifuged at 5,000 rpm for 10 min. The clear supernatant from the
enzymatic hydrolyzates was used to determine the glucose concentration with the
2700 Biochemistry Analyzer. The glucose yields from the enzymatic hydrolysis of
pretreated CS were expressed as % of the maximum glucose yield.

2.6. Experimental design for enzymatic hydrolysis

A 23 full factorial design was used in the optimization of enzymatic hydrolysis of
pretreated corn stover (PCS) for ethanol production. Solids loadings (X1, % DM),
cellulase loadings (X2, FPU g−1 DM), and pH (X3) were chosen as independent
variables. The glucose yield (%) was used as an dependent output variable. A total of
20 experiments that included 8 cube points (runs 1–8), 6 star points (runs 9–14) and 6
replicas of the central points (runs 15–20) were performed to fit a second order
polynomial model. The values of test variables at different pH (4.1–6), solids
(6.6–23.4%) and enzyme loadings (6.6–23.4 FPU g−1 DM) coded from −1.68 to + 1.68
and their interaction according to CCD are shown in Table 1. The ranges of variables
used in this work were selected based on literature and manufacturer’s
recommendations. For instance, 10–25 FPU g−1 DM is the recommended dosage range
for cellulose hydrolysis (Kaar and Holtzapple, 2000; Lindedam et al., 2010) whereas
10 to 15% is the most commonly used range for solids loadings (Cara et al., 2007).

2.7. Ethanol fermentation

Saccharomyces cerevisiae ATCC 24860 was used in the ethanol fermentation
studies. This strain has been reported to possess a high stress tolerance against pH,
inhibitors, xylose, temperature and ethanol (Geng et al., 2010). It was preserved at
ENERGY EXPLORATION & EXPLOITATION · Volume 30 · Number 2 · 2012 197

Table 1. Central composite design with observed and predicted response of

glucose and ethanol yields.

X1a X2b X3c

Actual Actual Actual Glucose yield (%) Ethanol yield
Experiment (coded) (coded) (coded) Observed Predicted (%)
1 10(−1) 10(−1) 4.5(−1) 48.1 47.5 76.7
2 20(1) 10(−1) 4.5(−1) 43.8 44.2 67.2
3 10(−1) 20(1) 4.5(−1) 55.7 59.9 78.9
4 20(1) 20(1) 4.5(−1) 45.7 45.6 73.0
5 10(−1) 10(−1) 5.6(1) 35.4 36.4 58.3
6 20(1) 10(−1) 5.6(1) 47.2 44.0 61.6
7 10(−1) 20(1) 5.6(1) 47.5 48.0 98.5
8 20(1) 20(1) 5.6(1) 42.9 44.5 77.7
9 6.6 (−1.68) 15(0) 5.1(0) 53.4 49.1 90.6
10 23.4 (1.68) 15(0) 5.1(0) 44.0 43.4 68.2
11 15(0) 6.6 (−1.68) 5.1(0) 36.7 40.8 83.7
12 15(0) 23.4(1.68) 5.1(0) 52.6 51.7 77.8
13 15(0) 15(0) 4.1(−1.68) 50.7 51.3 77.2
14 15(0) 15(0) 6(1.68) 38.2 41.2 87.8
15 15(0) 15(0) 5.1(0) 48.5 46.3 77.5
16 15(0) 15(0) 5.1(0) 47.0 46.3 48.0
17 15(0) 15(0) 5.1(0) 45.5 46.3 82.4
18 15(0) 15(0) 5.1(0) 47.4 46.3 78.7
19 15(0) 15(0) 5.1(0) 47.6 46.3 77.3
20 15(0) 15(0) 5.1(0) 47.1 46.3 78.9
a
X1, Coded value of solids loadings;
b
X2, Coded value of enzyme loadings;
c
X3, Coded value of pH.

4°C and maintained on yeast and mold (YM) agar medium which contained (g L−1):
10, yeast extract; 10, peptone; 20, glucose; 20, agar (pH 6). Inoculum was grown in
Erlenmeyer flasks containing 100 mL of YM broth on an orbital shaker at 30°C and
150 rpm. The enzymatic hydrolyzates were supplemented with 0.1% pre-autoclaved
yeast extract, inoculated with 1 mL of 24 h-grown S. cerevisiae (107 cells mL−1) and
incubated at 30°C and 150 rpm. Samples were withdrawn after 72 h of fermentation
and centrifuged at 5,000 rpm for 10 min. The clear supernatant was used to determine
the ethanol yield using the 2700 Biochemistry Analyzer. The ethanol yields obtained
from the enzymatic hydrolyzates were calculated as % of the ethanol theoretical
maximum yield using the following formula: Ethanol yield (%) = [g-ethanol
g-glucose−1/ 0.51] × 100.

2.8. Statistical analysis

The second-degree polynomials were calculated with Design-Expert Version 8 software
(StatEase Inc., MN, USA) to estimate the response of the dependent variables (Eq. 1).
198 Optimization of enzymatic hydrolysis of corn stover for improved ethanol production

Y = b0 + ∑ bi X i + ∑ bij X ij + ∑ bi 2 X i 2 (1)

where Y is the predicted response for glucose yield in %, b0 is the intercept, bi is the
coefficient for linear direct effect, bij is the coefficient for interaction effect; bi2 is
the coefficient for quadratic effect (a positive or negative significant value implies
possible interaction between the medium constituents); Xi, Xij, and Xi2 are the
independent variables. The quality of fit for the second order equation was
expressed by the coefficient of determination (R2) and its statistical significance was
determined by the F-test. The effect of each independent variable and their
interaction effects were determined. The number of parameters that were chosen to
be included for each model were determined based on the significance (α = 0.05) of
each model parameter using the F-test. To maximize the sugar recovery from
pretreated CS, numerical optimization was used for determination of the optimal
levels of the four variables.

2.9. Model validation

An experimental run of enzymatic hydrolysis was conducted under the optimal conditions
established from the experimental work as described in “Results and Discussion”:
enzyme loadings of 20 FPU g−1DM, solids loading of 10%, pH of 4.5, temperature of
45°C and hydrolysis time of 96 h. The ethanol fermentation was performed using S.
cerevisae at 30°C, 150 rpm and pH 5.6 for 72 h. All experiments were performed in
triplicate and standard deviations were calculated from the mean of the triplicate analyses.

3. RESULTS AND DISCUSSION

3.1. Pretreatment
The pretreatment of CS in a single screw extruder prior to enzymatic hydrolysis was
shown to be effective in fractionating the hemicellulose and lignin components with a
minimal yield loss (Karunanithy and Muthukumarappan, 2011). Our work confirmed
previous findings that extrusion did not significantly change the cellulose, hemicellulose
and lignin content of CS (Table 2). Similar observations were also found in reports for
switchgrass and prairie cord grass by Karunanithy and Muthukumarappan (2010).

3.2. Statistical analysis of enzymatic hydrolysis

The significance of each coefficient (coded and uncoded) was determined by the
Fisher F- and p-values (Table 3). The second order or quadratic main effects of solids

Table 2. Compositional analysis of corn stover before and after thermo-

mechanical extrusion.

Composition (%) Before extrusion After extrusion

Cellulose 32.6 32.3
Hemicellulose 24.1 23.8
Lignin 19.0 18.3
ENERGY EXPLORATION & EXPLOITATION · Volume 30 · Number 2 · 2012 199

Table 3. Analysis of variance (ANOVA) of glucose yield from pretreated corn

stover as function of solids loadings (X1), enzyme loadings (X2) and pH (X3).

Source Sum of squares DF Mean square F–value p–value (Prob > F)

Model 425.44 6.00 70.91 10.37 0.0003*
X1-Solid loadings 38.32 1.00 38.32 5.61 0.0341*
X2-Enzyme loadings 141.59 1.00 141.59 20.71 0.0005*
X3-pH 125.45 1.00 125.45 18.35 0.0009*
X12 61.45 1.00 61.45 8.99 0.0103*
X13 58.27 1.00 58.27 8.52 0.0119*
X23 0.36 1.00 0.36 0.05 0.8222
*
Significant variable. Coefficient of determination (R2), 0.82. Adjusted coefficient of determination (R2Adj),
0.74. Coefficient of variation (CV), 5.65%. Adequate precision ratio, 15.15. DF, degree of freedom.

loadings, enzyme loadings and pH proved significant, as evident from their respective
p values (pX1 = 0.0341 < 0.05, pX2 = 0.0005, pX3 = 0.0009 < 0.01, pX12 = 0.0103 < 0.05
and pX13 = 0.0119 < 0.05). The model indicated that the enzyme loadings have a major
effect on enzymatic hydrolysis.

y = 46.25 − 1.67X1 + 3.22X2 − 3.03X3 − 2.77X12 + 2.70X13 − 0.21X23 (2)

where y is the predicted response for % glucose yield during hydrolysis, and X1, X2
and X3 are the coded values for solids loadings, enzyme loadings and pH,
respectively.
The statistical significance of Eq. (2) was controlled by the F- and p-values (Table 3).
The significant response for coefficients was found to be mainly dependant on
the F-value and the resultant low p-value. Therefore, the greater the F-value and lower
the p-value, the more significant the corresponding coefficient. The Fisher F-test (Long et
al., 2009) with a very low probability value demonstrated a very high significance for
the regression model. The goodness of the model fit was verified by the coefficient of
determination (R2 = 0.82) which indicated that only 18% of the total variations could
not be explained by the model. The value of the adjusted coefficient of determination
was also high (R2Adj = 0.74), thus suggesting a high significance (p-value < 0.01) of
the model. The improved precision and reliability of the conducted experiments was
evident from the relatively low value of the coefficient of variation (CV = 5.65%;
Table 3). The adequate precision measures the signal to noise ratio and a ratio greater
than 4 is desirable (Lu et al., 2009). In this work, a ratio of 15.15 was obtained which
indicates an adequate signal (Table 3). Therefore, the model can be used to navigate
the design space.
The response surface plots presented as a function of two variables at a time, while
maintaining the third variable at a fixed zero level (coded units), were helpful in
understanding both the main and the interaction effect of these two variables. The
response surfaces suggested that the glucose yield could be enhanced by increasing the
enzyme loadings (Fig. 1a, c) while decreasing pH (Fig. 1b, c) and moderate solids
200 Optimization of enzymatic hydrolysis of corn stover for improved ethanol production

(a)
54
52
Glucose yield (%) 50
48
46
44
42
40

20.00 20.00
18.00 18.00
16.00 16.00
14.00 14.00
−1
Enzyme loadings (FPU g DM) 12.00 12.00 Solids loadings (%)
10.00 10.00

(b)
54
52
Glucose yield (%)

50
48
46
44
42
40

5.60 20.00
5.38 18.00
5.16 16.00
4.94 14.00
pH 4.72 12.00 Solids loadings (%)
4.50 10.00

(c)
54
52
50
Glucose yield (%)

48
46
44
42
40

5.60 20.00
5.38 18.00
5.16 16.00
4.94 14.00
pH 4.72 12.00 Enzyme loadings (FPU g−1DM)
4.50 10.00

Figure 1. Response surface plots, described by Eq. 3, representing the effect of solids
and enzyme loadings (a), solids loadings and pH (b), enzyme loadings and pH
(c), and their mutual effects on enzymatic hydrolysis of pretreated corn stover. Eq. 3:
y = 46.25−1.67X1 + 3.22X2 −3.03X3−2.77X12 + 2.70X13 −0.21X23 (y, predicted glucose
yield; X1, coded value of solids loadings; X2, coded value of enzyme loadings; X3,
coded value of pH).
ENERGY EXPLORATION & EXPLOITATION · Volume 30 · Number 2 · 2012 201

loadings (Fig. 1a, b). Hence, based on the RSM model from Eq. 2, the recommended
optimum values for the enzymatic hydrolysis to attain a predicted maximal glucose
yield of 59.9% were: 10% PCS solids loadings; 20 FPU g−1 DM; pH 4.5 (Fig. 1). At
the same time, a glucose yield of 46.2% was obtained (Table 1) at the zero-level
central points (15% PCG solids loadings; 15 FPU g−1 DM; pH 5.1), which represents
a deviation of 29% from the predicted optimum glucose yield (55.7%). Hence, the
RSM model showed a satisfactory performance and offered a stable response in
predicting the combined interactions of the three independent variables (pH, solids and
enzyme loadings) with respect to the glucose yields attained.
Literature suggests that the experimental results can be greatly enhanced with the
help of RSM compared to the conventional optimization methods (Mahat et al., 2004).
Using RSM, Mahat et al., (2004) and Tanyildizi et al., (2006) reported an increase in
the production yields of cyclodextrin glucanotransferase (CGTase) and α-amylase by
34% and 15%, respectively.

3.3. Ethanol fermentation

The enzyme hydrolyzates of PCS were fermented to ethanol with S. cerevisiae
(Table 1). It was observed that with the increase in the solids loadings, the ethanol
yields decreased despite the increase in the ethanol concentrations. For instance, at the
same enzyme loadings at ph (20 FPU g−1DM and pH 4.5), the ethanol yield was 78.9%
at solids 10% loadings (experimental run 3, Table 1) and 73.0%, at 20% solids
loadings (experimental run 4, Table 1). The increase in the pH from 4.5 to 5.6 had a
positive effect on the ethanol yield which peaked at 98.5% from the theoretical
maximum (experimental run 7, Table 1). Hence, to improve the ethanol yields from
CS it is recommended to carry out the hydrolysis with the P. pinophilum cellulases at
pH 4.5 and the ethanol fermentation with S. cerevisiae at pH 5.6.

3.4. Model validation

To validate the model developed in this work, an experiment under the optimal
condition conditions for enzymatic hydrolysis of pretreated CS (20 FPU g−1DM, 10%
solids loading, pH 4.5, 45°C, 96 h) produced a glucose yield of 57.6% which was in a
fairly good agreement with the predicted value of 61.0%. The subsequent fermentation
of the enzymatic hydrolyzate yielded 94.9% glucose to ethanol fermentation
efficiency. In comparison, we have previously reported a switchgrass-derived glucose
to ethanol fermentation efficiency of 92% (Zambare et al., 2011).

4. CONCLUSIONS
In this work, modeling of enzymatic hydrolysis of pretreated CS with cellulase
enzymes from Penicillium pinophilum was successfully performed using central
composite design and response surface methodology. The effects of solids loadings,
enzyme loadings and pH as independent variables were investigated in a total of
20 experiments. All three variables showed significant impact on enzymatic
hydrolysis with the solids loadings most significantly affecting the glucose yield from
CS. The coefficient of determination R2 of 0.82 and ANOVA implied that the model
satisfactorily represented the real relationship of the three parameters and the
response for enzymatic hydrolysis. The optimum levels obtained by the model were
202 Optimization of enzymatic hydrolysis of corn stover for improved ethanol production

successfully validated. Under the optimum enzymatic hydrolysis conditions (pH 4.5,
solids loadings of 10% and enzyme loadings of 20 FPU g−1 DM), the model
validation experiment produced a maximum glucose experimental yield of 57.6%
which represents a yield increase of 10.4% over the non-optimized controls at central
points (47.2% on average). Therefore, the use of RSM for optimization of process
parameters and their interactions could be very useful in improving product yields,
proves as both time and cost saving and maximizes the amount of information that
can be obtained while limiting the number of individual experiments. Furthermore,
the RSM predicts the interaction between the independent variables which results in
improved accuracy and precision of the research data and facilitates their
interpretation.

ACKNOWLEDGMENT
Financial support by the Center for Bioprocessing Research & Development (CBRD)
at the South Dakota School of Mines & Technology (SDSM&T), the South Dakota
Board of Reagents (SD BOR) and the South Dakota Governor’s Office for Economic
Development (GOED) is gratefully acknowledged.

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