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ISSN: 0738-8551 (print), 1549-7801 (electronic)

Crit Rev Biotechnol, 2016; 36(2): 368–388

! 2014 Informa Healthcare USA, Inc. DOI: 10.3109/07388551.2014.973014

REVIEW ARTICLE

Experimental design methods for bioengineering applications

_
Tuğba Keskin Gündoğdu, Irem Deniz, Gülizar Çalışkan, Erdem Sefa Şahin, and Nuri Azbar

Department of Bioengineering, Ege University, Bornova-Izmir, Turkey

Abstract Keywords
Experimental design is a form of process analysis in which certain factors are selected to obtain Bioprocess, Box–Behnken design, central
the desired responses of interest. It may also be used for the determination of the effects of composite design, full factorial design,
various independent factors on a dependent factor. The bioengineering discipline includes Placket–Burrman design, Taguchi design
many different areas of scientific interest, and each study area is affected and governed by
many different factors. Briefly analyzing the important factors and selecting an experimental History
design for optimization are very effective tools for the design of any bioprocess under question.
This review summarizes experimental design methods that can be used to investigate various Received 24 September 2013
factors relating to bioengineering processes. The experimental methods generally used in Revised 25 August 2014
bioengineering are as follows: full factorial design, fractional factorial design, Plackett–Burman Accepted 19 September 2014
design, Taguchi design, Box–Behnken design and central composite design. These design Published online 3 November 2014
methods are briefly introduced, and then the application of these design methods to study
different bioengineering processes is analyzed.

Introduction Taguchi design, Box–Behnken design and central composite

In an experiment, one or more process variables (or factors)
are deliberately changed to observe the effect on one or more
response variables. Statistical design of experiments is an
efficient procedure for planning experiments and analyzing
the results such that objective conclusions can be drawn.
Determining the objectives of an experiment is the first step
of an experimental design. Selecting the process factors and
responses to be analyzed is the second important step in
optimizing the important variables in a process.
In general, experimental design is the design of a system to
obtain information using various experimental techniques.
Experimental design can be used to evaluate physical objects,
chemical formulations, structures, components and materials.
Bioengineering studies various areas of production including
enzyme production, biological water treatment, tissue culture,
nanoparticles, biofuel production, the isolation of micro-
organisms and the industrial biological production of com-
pounds such as proteins, lipids and aromatic compounds.
Every process is affected by different factors. Optimizing
these parameters is an important step in obtaining maximum
production with minimum costs. Developing an experimental
design is the most effective means of process optimization.
This review summarizes the experimental design methods that
are used to investigate the effects of various factors on various
bioengineering processes including: Full factorial design,
fractional factorial design, Plackett–Burman (PB) design,
Address for correspondence: Nuri Azbar, Department of Bioengineering,
Ege University, 35100, Bornova-Izmir, Turkey. Tel: +90 232 3884955
(31). Fax: + 90 232 3884955. E-mail: nuri.azbar@ege.edu.tr
design. Each design method is briefly introduced, the
advantages and disadvantages of each are briefly discussed
and various processes are analyzed.
Experimental design is a process in which important
factors are selected and controlled using different designs to
obtain important responses and is generally followed by
statistical analysis.
The literature contains a vast number of papers on
statistical design, this review however specifically focuses
on the experimental design applications commonly used in
bioengineering studies, discussing the latest published papers.
Experimental design applications in bioengineering
Full factorial design
In statistics, a design that consists of two or more factors in
which every factor interacts with each other at different levels
is termed full factorial design. Full factorial design studies the
effect of factors on response variables as well as the
interaction between them.
Factorial design was first used in the nineteenth century by
John Bennet Lawes and Joseph Henry Gilbert. Ronald Fisher
argued in 1926 that complex designs were more efficient than
those in which one factor was studied at a time. The term
‘‘factorial’’ was first used by Fisher in his book ‘‘The Design
of Experiment’’ (Fisher, 1935).
It is practical to study the effect of many factors
simultaneously at different levels. The resulting design is a
combination of different variables at various levels, enabling
the design to depict the interactions of different factors; this
characteristic makes the design more efficient while dealing
DOI: 10.3109/07388551.2014.973014
with a large number of variables. Factorial design can be
generally divided into full factorial design and fractional
factorial design.
A uniform framework is provided by single coded factor
levels in an attempt to determine the effect of a particular
factor in an experimental context. Factorial design is usually
used in coded factor levels. The actual factor level corres-
ponding to the factor level of a factorial design can be
assigned very clearly while using it. After completing all the
experiments and obtaining results, the analysis can be
performed using either coded factors or actual factors
(Wang & Wan, 2009). Using coded factors in the analysis
has some advantages. For example, in coded factor level
analysis, the coefficients of the model are dimensionless and
directly comparable. Thus, this method is very effective for
determining the real effects. After performing an analysis
using coded factors, the results can be summarized using
actual factors (Montgomery, 2008).
In a full factorial design, the major aim is to determine the
effect of each term and the interaction between these terms
using a model. If there are three factors to be analyzed
(a  b  c), then the first factor is tested at a levels, the second
factor is tested at b levels and the third factor is tested at
c levels. The number of runs of n factors at a levels is an.
Two-level designs are the most commonly used designs that
can be denoted as 2n (Wang & Wan, 2009). Various
bioengineering processes with different full factorial analyses
are summarized briefly below.
Mendes et al. (2012) investigated that the daily relative
growth rates of the red macroalgae Gralicaria domingensis in
synthetic seawater. The effect of the combined influence
of five factors [light (L), temperature (T) and nitrate (N),
phosphate (P) and molybdate (M) concentrations] was
determined using a full factorial design. The ranges of the
experimental cultivation conditions used were as follows:
T, 18–26  C; L, 74–162 mmol photons m2 s1; N,
40–80 mmol/L; P, 8–16 mmol/L and M, 1–5 nmol/L. The
results were analyzed using the analysis of variance
(ANOVA) test method. According to the ANOVA test, the
factors N (p ¼ 0.01327) and T (p ¼ 0.00025) are highly
significant at the a ¼ 0.05 level (Mendes et al., 2012).
The production of poly-3-hydroxybutyrate (PHB) by
Methylocytis hirsute from natural gas was first studied in
two different media by Rahnama et al. (2012). After selecting
the medium, the effects of methane-to-air ratio (20/80–50/50–
80/20) and nitrogen content (0–50–100 mg/L) on PHB
production were determined in a bubble column using a 32
full factorial design. Both of these factors were found to be
significant, and a maximum accumulation of 42.5% w/w of
cell dry weight was achieved (Rahnama et al., 2012).
Nalakath et al. (2012) investigated the effect of four factors
on the bioconversion of carbon monoxide to ethanol and
acetic acid by Clostridium autsethogenaum using a 24 full
factorial design. The four studied factors were initial pH
(4.75–5.75), initial total pressure (0.8–1.6 bar), cysteine-
HClH2O concentration (0.5–1.2 g/L) and yeast extract (YE)
concentration (0.6–1.6 g/L). The maximum ethanol produc-
tion was enhanced by up to 200% at conditions pH 4.75 and a
YE concentration of 1.6 g/L. All the main effects and the
interaction effects were found to be statistically significant
Experimental design methods for bioengineering applications 369
(p50.05). Main effect plots indicated that increasing the
initial pH and using higher YE concentrations negatively
affected ethanol production, whereas increasing the initial
pressure and the cysteine-HClH2O concentration had a
positive effect. An ethanol production of 0.065 g/L was
achieved using the following values of the factors: pH (4.75),
pressure (1.6 bar), cysteine-HClH2O (1.2 g/L) and YE con-
centration (1.6 g/L; Nalakath et al., 2012).
The optimal conditions for microwave-assisted enzymatic
biodiesel synthesis were investigated using a 22 full factorial
design. The important factors affecting the production of
biodiesel were the ethanol-to-beef tallow ratio (X1, 6–12) and
the reaction temperature (X2, 40–50  C). The transesterifica-
tion yield (Y) was selected as the response. The reactions
were catalyzed by lipase from Burkholderia cepacia, which
was immobilized on silica, and were performed using 8–15 W
of microwave radiation. High conversions were achieved
using lower molar ratios of ethanol-to-beef tallow, and the
effect of temperature was observed to be not significant based
on statistical analysis. Under optimized conditions (a 1:6
molar ratio of beef tallow to ethanol at 50  C), the fatty acids
in the original beef tallow were almost completely converted
into ethyl esters in 8 h of reaction time, and a productivity of
92 mg ethyl esters/g h was achieved (a 6-fold increase; Da Rós
et al., 2012).
Virological testing of bottled water is another bioengin-
eering application. A study was conducted to choose the
best tool for detecting viruses in bottled water. Different
approaches were examined; for example, the recovery of viral
RNA was measured following the in situ lysis of virus
particles in the aqueous phase. A second method detected the
recovery of viral RNA following the lysis of virus particles,
and the third detection method generated the lowest genome
recovery, regardless of water and virus type. Two methods
were compared because they were considered viruses. Using a
344121 full factorial design, viral RNA recovery was
determined. The independent variables used were three
types of water with 3 different mineral compositions; four
viruses (poliovirus, hepatitis A virus, Norovirus and MS2
phage); three incubation times (1, 10 and 20 days) and two
methods (A and B). Every factor except incubation time was
found to be significant. Method A provided the best results,
suggesting that this method should be used to detect the
hepatitis A virus (Huguet et al., 2012).
A study of the metabolic responses of a new neuronal
human cell line, AGE1HN, to various substrate values showed
that reduced substrate and pyruvate load improves metabolic
efficiency leading to improved growth and a1 antitrypsin
(A1AT) production. A 32 full factorial design was used to
analyze the adaptation of metabolism to various pyruvate and
glutamate concentrations. The concentrations of pyruvate
tested were 2, 5 and 9 mM and the concentrations of
glutamate tested were 5, 7.5 and 10 mM. The most important
result was that higher pyruvate concentrations in the medium
decreased cell proliferation and reduced the efficiency of
substrate use. However, the highest viable cell density and
A1AT concentration (167% of the batch) could be achieved
without adding pyruvate (Niklas et al., 2012).
To study the individual and combined effects of osmo-
lality on the phases of cell growth and virus production, a
370 T. K. Gündoğdu et al.
52 full factorial design was studied. A statistical design was
used to investigate the effect of osmotic stress on cell
physiology. Two factors were chosen for study: the osmo-
lality of the cell growth (250, 290, 330, 370 and 410 nm)
and the osmolality of the virus produced (250, 290, 330,
370 and 410 nm). Statistical analysis indicated that the
growth of the cells under hyperosmotic conditions induced
favorable physiological states for viral production. An 11-
fold increase in virus productivity was achieved (Shen &
Kamen, 2012).
A 33 full factorial design was used to investigate the effects
of pH, temperature and agitation rate on bacterial growth and
bacteriosin production. Statistical analysis showed that pH
was the most important physical factor for bacterial growth
and bacteriosin production (Martı́nez-Cardeñas et al., 2012).
The effect of different doses of surfactant and buffer were
tested using a 41 31 full factorial experimental design in a
lipase production by solid-state fermentation experiments.
Four buffer-to-wet solids substrate ratios and three surfactant
percentages in aqueous extracts were tested to determine the
best conditions for lypolytic activity. For solid samples, the
lypolytic activity reached 120 000 UA/g of dry matter (Santis-
Navarro et al., 2011).
Aspergillus japonicus URM5620 can produce b-glucosi-
dase (BG), total cellulase (FPase) and endogluconase
(CMCase). To determine the effects of substrate amount,
initial moisture, pH and temperature on the production of
these enzymes, a 24 full factorial design was used. All the
factors were found to be significant, and the optimum
conditions for all the studied enzymes were as follows: 5 g
substrate, 15% initial moisture, 25  C temperature and pH 6.
After a 120-h fermentation under these conditions, the
maximal activities for BG, FPase, CMCase were found to
be 88.3, 953.4 and 191.64 U/g of dry substrate, respectively
(Herculano et al., 2011).
Wang & Wan (2011) used a 42 full factorial design to
determine the combined effects of temperature and pH on
bio-hydrogen production. Temperature and pH were found to
interactively affect both hydrogen yield and the average
hydrogen production rate. The maximum yield and hydrogen
production rates were predicted as 308 mL H2/g of substrate
and 11.5 mL/h, respectively (Wang & Wan, 2011).
Hendry et al. (2011) designed a novel continuous super-
critical water gasification reactor to investigate glucose
gasification in supercritical water at high temperatures and
low residence times. A 23 full factorial experiment was
performed to investigate the effects of feed concentration,
temperature and residence time on glucose gasification. The
responses studied were the overall gasification efficiency
(GE), carbon efficiency (CE), H2 yield, CH4 yield and
gasification rates. The most important outcomes of the design
were as follows: first the interaction between feed concentra-
tion and temperature has an important effect on both GE and
CE. Second, temperature and residence time affected CE
(Hendry et al., 2011).
Inga edulis is a flavonoid-rich plant from Amazonia. To
optimize the drying of its leaves by a forced convection dryer,
a 32 full factorial design was used to determine the effects of
temperature and airflow velocity (factors) on the drying time
(response). Both factors were found to be significant for
Crit Rev Biotechnol, 2016; 36(2): 368–388
achieving a moisture content lower than 5.86/100 g during
40 min of drying time (Silva et al., 2011).
Bioethanol can be produced by Pichia stipitis NRRL-
Y7124 using rice straw hemicellulosic hydrolysate as a
fermentation medium. To detect the effects of agitation and
aeration rates on the bioconversion of xylose into bioethanol,
a 22 full factorial experimental design was used. The factorial
design assay showed strong interactive effects of the factors
on ethanol production yields. The best results (YP/S ¼ 0.37 g/g
and 75% process efficiency) were found using a 2.5 Vflask/
Vmedium ratio and 200 rpm agitation (Silva et al., 2010).
Freshly harvested, air-dried rice straw can be pre-treated by
two methods – using either NaOH or Ca(OH)2. Full factorial
experiments were designed to measure the effects of alkaline
loading and pre-treatment time on the delignification and
sugar yield after enzymatic hydrolysis. Alkaline loading and
reaction time both had significant effects on delignification,
but only alkaline loading had a significant effect on enzymatic
hydrolysis (Cheng et al., 2010).
Testing of different species showed that Candida bijinensis
exhibited the highest extracellular proteolytic activity
response. Enzyme production using this yeast was optimized
using a 23 full factorial test to test the effects of temperature,
pH and soybean flour concentration. The highest production
of extracellular proteases (573 U/mL) was achieved at 35  C,
pH 6.5 and 0.5% w/w soybean concentration (de Araújo Viana
et al., 2010).
The effects of strain, media and agitation rate on traditional
yoghurt production were investigated on the lactic acid and
b-galactosidase yields. Based on ANOVA tests that were
performed after the experiments, all the linear and interaction
terms were found to be significant for lactic acid formation
(Tari et al., 2010).
A 24 full factorial design was used to investigate the effect
of nitrogen source (YE, urea, ammonium sulfate and peptone)
on the maximum variation of surface tension (DST) and
emulsification index (EI) to determine the efficiency of bio-
surfactant production by Yorrowina lipolytica IMUFRJ
50682. The optimal result was 73.1% EI and 20.9 m Nm1
of DST (Fontes et al., 2010).
Aeration and the agitation are the two main factors
affecting the oxygen mass transfer coefficient, Marques
et al. (2009) designed a 22 full factorial experiment to study
these factors. Both variables showed statistically significant
effects on kLa, and the highest values obtained were 70.3 s1
(extractive fermentation) and 241 s1 (simple fermentation)
using a 5 vvm aeration rate and 1200 rpm agitation (Marques
et al., 2009).
An immobilization method for the inclusion of viable cells
of Candida guillermondii into a polyvinyl alcohol (PVA)
hydrogel using a freeze-thaw method was applied by de
Cunha et al. (2009). Entrapment experiments were performed
based on a 23 full factorial experimental design that tested
PVA concentration, freezing temperature and the number of
freezing–thawing circles as factors and using the xylose to
xylitol bioconversion rate as the response. At the end of the
experiments, a maximum yield of 0.49 g xylose/xylitol was
achieved (da Cunha et al., 2009). Several experimental sets of
full factorial design studies used in bioengineering are listed
in Table 1.
Table 1. Full-factorial design applications in bioengineering.
Process
Microalgae production
(Gralicaria domingensis)
Polyhydroxy butyrate pro-
duction by Methylocytis
hirsute
Ethanol production by
Clostridium
autsethogenaum
Biodiesel production
Virus detection in bottled
water
Analyze the adaptation of
metabolism to different
substrates
Virus productivity
Bacterial growth and bac-
teriosin production
Lipase production
b-Glucosidase, total cellu-
lose (FPase) and endo-
gluconase (CMCase)
Full factorial
design model Software Factors studied
25 STATISTICA 9.1 (StatSoft Inc., Temperature (18–26  C), Light
2010) (74–162 mmol photons m2 s1),
Nitrogen (40–80 mmol/L), phosphorus
(8–16 mmol/L), M (1–5 nmol/L)
(T and N are significant).
32 MINITAB Release 15 (Minitab Methane to air ratio (20/80–50/50–80/20),
Inc. State College, PA) nitrogen content (0–50–100 mg/L)
(All factors are significant)
24 Minitab 16 (Minitab Inc. State Initial pH (4.75–5.75), initial total pres-
College, PA) sure (0.8–1.6 bar), cystein-HCl.H2O
concentration (0.5–1.2 g/L), yeast
extract concentration (0.6–1.6 g/L)
(All factors are significant)
22 Statistica 7.0 (StatSoft Co.) Ethanol to beef tallow ratio (6–12),
reaction temperature (40–50  C)
(ethanol to beef tallow ratio is
significant)
34 41 21 Minitab 14 (Minitab Inc. State Three waters with three different mineral
College, PA) composition; four viruses, poliovirus–
hepatitis A virus–Norovirus–MS2
phage; 3 incubations times, 1–10–20
days and two methods, A–B
(All of the factors are significant except
incubation times)
32 NA Pyruvate (2–5–9 mM), glutamate
(5–7.5–10 mM)
(Pyruvate is significant)
52 (DOE++ software, ReliaSoft Osmolality of cell growth (250–290–330–
Corporation, Tucson, AZ) 370–410), osmolality of virus pro-
duced (250–290–330–370–410)
(both factors are significant)
33 STATGRAPHICS Plus version 5 pH (6.0, 7.2, 8.0), temperature (26, 28,
(Statistical Graphic Corp., 30  C), agitation (150, 180, 210 rpm)
Warrenton, VA) (pH is the significant factor)
41 31 Microsoft Office Excel Solver Buffer to wet solid substrate ratio
tool. (15–90–200–500 mL/g), the surfactant
percentages in aqueous extract
(2–6–10% v/v)
(both factors are significant)
24 Statistica 8.0 Substrate amount (5–15 g/L), initial
moisture (15–35%), temperature
(25–35  C), pH (4–6)
(All factors are significant)
Response References
Maximum growth rate of Mendes et al. (2012)
6.4% day1
DOI: 10.3109/07388551.2014.973014
Polyhydroxy butyrate accumu- Rahnama et al. (2012)
lation 42.5% w/w of cell
dry weight
0.065 g/L ethanol production Nalakath et al. (2012)
92 mg ethyl esters/g h Da Rós et al. (2012)
production
Method A should be used to Huguet et al. (2012)
detect hepatitis A virus
Highest viable cell density and Niklas et al. (2012)
A1AT concentration (167%
of batch) can be reached
without pyruvate feeding
11-fold increase in virus Shen & Kamen (2012)
productivity
Highest bacteriosin activity Martı́nez-Cardeñas et al. (2012)
was found with LBIT 269
with induced B.
thuringiensis
The lypolytic activity reached Santis-Navarro et al. (2011)
up to 120 000 UA/g or dry
matter
Conditions the best activities Herculano et al. (2011)
for b-glucosidase, FPase,
Experimental design methods for bioengineering applications
CMCase were found as;
88.3, 953.4, 191.64 U/g
dry substrate, respectively
371
(continued )

Process
Biohydrogen production
Supercritical water
gasification
Drying of Inga edulis leaves
by forced convection
dryer
Bioethanol production by
Pichia stipitis NRRL-
Y7124
Bioethanol production from
rice straw
Extracellular proteolytic
activity by Candida
Bijinensis species
Lactic acid production
Biosurfactant production
kLa determination in a fer-
mentation system
Polyvinyl alcohol (PVA)-
gel immobilization of
Candida guillermondii
Table 1. Continued
Full factorial
design model Software Factors studied
2
4 NA pH (6–7–8–9), temperature
(30–35–40–45  C)
(both factors are significant)
23 NA Feed concentration (10–15%), tempera-
ture (750–800  C), average residence
time (4–6.5 s)
(feed concentration and temperature
interaction have an effect on both GE
and CE; temperature and residence
time had an effect on CE)
32 STATISTICA Kernel Release 7.1 Temperature (40–55–70  C), airflow
(StatSoft Inc., 2006, Tulsa, OK) velocities of (0.6–1.2–1.8 m/s)
for Windows XP
22 Statistica 6.0 Agitation (100–200 rpm), aeration
(V flask/V medium ratio
of 2.5–5.0)
32 JMP IN version 7 (SAS Institute, Alkaline loading and reaction time (Both
Cary, NC) alkaline loading and reaction times had
significant effects on delignification
but only alkaline loading had signifi-
cant effect on enzymatic hydrolysis)
23 Statistica 8.0 (StatSoft, Tulsa, Soybean flour concentration
OK). (0.1–0.5% w/w), pH (6.5–8.5), tem-
perature (25–35  C)
26 ‘‘Design Expert’’ software version Temperature, pH, speed of agitation,
7.1.3 (Stat-Ease, Inc., enzyme concentration, buffer concen-
Minneapolis, MN) tration, surfactant type, surfactant
concentration
(all the linear and the interaction terms
were found to be significant)
24 Statistica 7.0 Peptone (0–12.8 mg/L), yeast extract
(5–15 mg/L), ammonium sulfate
(0–10 mg/L), urea (0–0.2 mg/L)
(ammonium sulfate and yeast extract
had significantly influenced both
dependent variables, DST and EI. On
the other hand, urea did not signifi-
cantly influence DST nor EI in the
range studied)
22 Statistic 6.0 Agitation (700–1200 rpm), aeration
(2.0–5.0 vvm)
(Both variables showed statistically
significant effects on kLa)
23 Statgraphics 6.0 PVA concentration (80–120 g/L), the
freezing temperature (10 to 20  C),
the number of freezing–thawing
cycles (1–5)
372
Response References
The maximum yield and Wang & Wan (2011)
hydrogen production rates
were predicted as 308 mL
H2/g substrate and
11.5 mL/h, respectively
Increasing reaction temperature Hendry et al. (2011)
T. K. Gündoğdu et al.
from 750 to 800  C showed
large increases in efficien-
cies (+29% for GE), yields
and gasification rate
(+8 g/L s)
5.86/100 g moisture content Silva et al. (2011)
lower than 40 min drying
time
Product yield of 0.37 g/g with a Silva et al. (2010)
process efficiency of 75%
27% delignification for lime Cheng et al. (2010)
pre-treatment and 23.1 %
delignification for NaOH
pre-treatment
Extracellular protease produc- de Araújo Viana et al. (2010)
tion of 573 U/mL
Lb22b strain in skim milk Tari et al. (2010)
media under static condition
produced 2.30 times more
lactic acid than in whey
20.9 m Nm1 for DST and Fontes et al. (2010)
73.1% for EI by using 0.5 g/
L yeast extract
70.3 s1 by extractive fermen- Marques et al. (2009)
tation and 241 s1 by simple
fermentation
Maximum yield of 0.49 g da Cunha et al. (2009)
xylose/xylitol
Crit Rev Biotechnol, 2016; 36(2): 368–388
DOI: 10.3109/07388551.2014.973014
Plackett–Burman design
PB designs, also known as ‘‘Hadamard Matrix Designs’’, are
two-level fractional factorial designs developed by Plackett
and Burman; this design type has been widely used to
determine important factors in bioengineering systems (Wang
& Wan, 2009). The number of runs for a PB design is set to
multiple of 4 (e.g., 12, 16, 20 or 24). If the number of factors
(‘‘real’’ factors) are less than n ¼ N  1, a subset of PB design
for N runs can be used (John, 1996). In some cases, repetitions
can be used to determine the experimental errors (Wang &
Wan, 2009).
Plackett–Burman design is widely used in several scientific
areas because it presents several advantages; for example, it
holds ‘‘hidden projection properties’’, which means that
taking the projections of a design can result in a desirable
experimental result that is not immediately apparent. One
such hidden projection property of these designs is that
resolution V designs can be constructed from the projections
of their factors (Briggs, 2011).
For the experimental results, the effects of factors are
defined using a first-order polynomial equation model
[Equation (1)] for the PB design;
X
k 13–76 that overexpresses FMN1 gene coding for riboflavin
y ¼ 0 þ t xt ð1Þ
t¼1
where y is the response,  0 is a constant, t is a linear
coefficient, and xi is the coded factor level.
For the case of two levels (L ¼ 2), orthogonal matrices can
be generated as 1 or 1 (Hadamard matrices). This method
could be used to find such matrices of size N which is equal
to a multiple of 4. It worked for all such N up to 100 except
N ¼ 92. PB designs are mostly used when n is a multiple of 4
not a power of 2. If there are more than 2 cases, Plackett and
Burman give specifics for designs having a number of
experiments equal to the number of levels L to some integer
power, for L ¼ 3, 4, 5 or 7 (Wang & Wan, 2009).
Plackett–Burman statistical design is widely used to
determine the effective parameters of processes, especially
bioprocesses, because biological materials do not have stable
structures and are easily affected by the environment. In
addition, biochemical and physicochemical variations occur
in bioprocesses; therefore, the effective parameters must be
determined. Prakash & Srivastava (2005) used the PB method
to determine the effective parameters in azadirachtin produc-
tion from Azadirachta indica. The authors studied the factors
of glucose, nitrate, phosphate, magnesium, calcium and
inoculum concentration. The Ñ value helped the authors to
decide which factors are the most effective. All nutrients
except phosphate and MgSO47H2O concentration yielded
positive Ñ values. Inoculum level exhibited the numerically
highest value, indicating that this factor had the greatest
positive influence on biomass production. The next highest
values were for nitrate and glucose, which were the main
components of the microbial growth (Prakash & Srivastava,
2005).
Gao et al. (2009) used the PB method to optimize the
medium for the production of avermectin B1a using
Streptomyces avermitilis. Corn starch, a-amylase, soya
Experimental design methods for bioengineering applications 373
flour, YE, Na2MoO42H2O, MnSO4H2O, (NH4)2SO4,
CoCl2 and CaCO3 were studied as factors. At the end of the
study, researchers presented a Pareto chart. In the chart, corn
starch, YE, CaCO3, CoCl2, MnSO4H2O, soya flour,
(NH4)2SO4 and a-amylase, Na2MoO42H2O had the greatest
effect on B1a production using Streptomyces avermitilis (Gao
et al., 2009).
Plackett–Burman design is widely used to determine
effective factors in bioengineering applications. These factors
may have chemical, physical and environmental effects on the
catalytic activity of enzymes (Table 2). Singh & Bishnoi
(2012) used the PB method to optimize the enzymatic
hydrolysis of microwave/alkali-pretreated wheat straw and
ethanol production by yeast. The authors investigated the
effects of substrate concentration, Tween-80 concentration,
BGL dose, cellulase concentration, hydrolysis time and
agitation on enzymatic activity. At the end of the study, all
the factors presented negative Ñ values except for BGL dose
and hydrolysis time. Substrate concentration, hydrolysis time,
BGL and cellulase dosage were significant at a confidence
level of greater than 95%, and the remaining factors were non-
significant (Singh & Bishnoi, 2012). Yatsyshyn et al. (2010)
studied on the recombinant strain of the yeast Candida famata
kinase, accumulates significant amounts of flavin mononu-
cleotide (FMN) in the cultural liquid. After medium compos-
ition optimization in shake flask cultures, the two-level PB
design was performed to screen medium components that
significantly influence the FMN production. Fifteen variables
tested and KH2PO4, CaCl2, (NH4)6Mo7O24, CuSO4 and YE,
were identified as the most significant factors for FMN
production (confidence levels above 95%; Yatsyshyn et al.,
2010). Another study was on the production optimization of
a-amylase from Aspergillus oryzae CBS 819.72 fungus, using
a by-product of wheat grinding (gruel) as sole carbon source.
The screening of 19 nutrients for their influence on a-amylase
production was performed using a PB design and KH2PO4,
urea, glycerol, (NH4)2SO4, CoCl2, casein hydrolysate, soy-
bean meal hydrolysate, MgSO4 were selected based on their
positive influence on enzyme formation (Kammoun et al.,
2008). Poly-"-lysine ("-PL) is a non-toxic biopolymer with
antimicrobial properties. The "-PL production by
Treptomyces noursei NRRL 5126 in shake-flask cultures
was optimized by identifying the most significant medium
components (glycerol, proteose peptone and ammonium
sulfate) by Placket–Burman design and glycerol and glucose
was decided to be the most effective components of the
medium (Bankar & Singhal, 2010). For the production of
laccase by Pleurotus florida NCIM 1243, 11 components
were screened for their significant effect PB factorial design.
At the end of the statistical analysis glucose (carbon source),
asparagine (nitrogen source), CuSO4 (inducer) and incubation
period were found to have highest positive influence on the
laccase production (Palvannan & Sathishkumar, 2010).
Taguchi design
The history of the Taguchi method is based in Japan. The
engineer Dr. Genichi Taguchi conducted extensive research
on the design of statistical experiment techniques at the
 374

Table 2. Plackett–Burrman design applications in bioengineering.

Process Software Factors studied Response References

Media optimization for cell growth
and azadirachtin production in
Azadirachta indica (A. Juss)
suspension cultures
Enzymatic hydrolysis optimization
Design-Expert Version 5.0.9 (Stat-
Ease Corporation, USA)
Design Expert (Statease 6.1 trial
Glucose (15–60 g/L), nitrate (15–90 mM),
phosphate (0.5–2.5 mM),
MgSO47H2O (0.18–0.74 g/L),
inoculum level (0.22–0.88 g/L)
Substrate concentration (10–50 g/L),
Maximum dry cell weight and
azadirachtin production were
21.2 g/L and 2.65 mg/g,
respectively
Substrate concentration, hydrolysis
Prakash & Srivastava
(2005)
Singh & Bishnoi (2012)
T. K. Gündoğdu et al.

of wheat straw and ethanol
production
Production of flavin mononucleo-
tide by the recombinant strain
of the Candida famata
Production of avermectin B1a by
Streptomyces avermitilis
14-12A
a-Amylase production by
Aspergillus oryzae CBS 819.72
version)
Statistica 6.0 (Stat Soft, Inc.)
Minitab 15.0
SPSS (Version 11.0.12001, LEAD
Technologies, Inc., USA)
Tween 80 concentration (0.05–0.1%), time, b-glucosidase and cellu-
b-glucosidase dose (50–200 IU/g), lase dosage were significant at
cellulase concentration (5–15 FPU/g), confidence level of more than
hydrolysis time (12–48 h), agitation 95% on enzymatic hydrolysis
(100–150 rpm)
Sucrose (20–30 g), urea (1–3 g), yeast Maximum flavin mononucleotide Yatsyshyn et al. (2010)
extract (0.5–2 g), KH2PO4 (0.5–2 g) production was identified as
MgSO47H2O (0.2–1.2 g), CaCl24H2O 133.7 mg/L
(0.2–1.2 g), (NH4)6Mo74H2O
(0.0012–0.1 g), ZnSO47H2O
(0.000308–0.1 g), CuSO45H2O
(0.000039–0.001 g), CoCl26H2O
(0–0.005 g), Fe(NH4)2(SO4)26H2O
(0–0.001 g), C6H5O7Na3NH2O
(0–1 g), BeSO45H2O (0–0.124 g),
NaF (0–0.042 g)
(KH2PO4, CaCl24H2O,
(NH4)6Mo74H2O, BeSO45H2O yeast
extract have strong effect on flavin
mononucleotide production)
Corn starch (112–168 g/L), a-amylase Maximum avermectin B1a pro- Gao et al. (2009)
(0.08–0.12 g/L), soya flour (22.4– duction is observed with
33.6 g/L), yeast extract (8–12 g/L), 168 g/L corn starch and
Na2MoO42H2O (0.0176–0.0264 g/L), 9.2 g/L yeast extract
MnSO4H2O (0.00184–0.00276 g/L),
(NH4)2SO4 (0.2–0.3 g/L), CoCl2
(0.016–0.024 g/L), CaCO3
(0.64–0.96 g/L)
(Corn starch has positive and also yeast
extract has negative effect on aver-
mectin B1a production)
K2HPO4 (0.1–0.2 g/g), KH2PO4 KH2PO4, urea, glycerol, Kammoun et al. (2008)
(0.1–0.2 g/g), NaCl (0.05–0.1 g/g), (NH4)2SO4, CoCl2, casein
MgSO4 (0.05–0.1 g/g), yeast extract hydrolysate, soybean meal
(0.1–0.2 g/g), corn step liquor (0.1– hydrolysate, MgSO4 had posi-
0.2 g/g), soyabean meal hydrolysate tive influence on enzyme
(0.1–0.2 g/g), urea (0.1–0.2 g/g), casein formation
acid hydrolysate (0.1–0.2 g/g), glycerol
(0.1–0.2 g/g), (NH4)2SO4 (0.1–0.2 g/g),
Crit Rev Biotechnol, 2016; 36(2): 368–388
DOI: 10.3109/07388551.2014.973014 Experimental design methods for bioengineering applications 375

Palvannan & Sathishkumar

Electronic Control Laboratory at the end of the 1940s. His

Bankar & Singhal (2010)

fundamental goal was to achieve user-friendly experimental
design. Taguchi developed several new approaches for
experimental design in several scientific areas that are
termed ‘‘Taguchi Methods’’. Taguchi design is type of

(2010)
fractional factorial design that uses an orthogonal array. The
method enables experiments to be studied using a compara-
tively small number of runs while the effects of many factors
on a response are studied at two or more levels. A method was
developed by Taguchi for experimental design to investigate
the affect of mean and variance of different parameters on
incubation period have positive
effect on poly-"-lysine produc-

process. It involves the usage of orthogonal arrays (OAs) to

tion level which are 41.81 ±
Glycerol and glucose has more

effect on laccase production

0.66 and 37.43 ± 0.18 mg/L,

asparagine, KNO3, CuSO4,

organize the parameters. Instead of testing all combinations

Glucose, sucrose, raffinose,

like factorial design Taguchi method uses pairs of combin-

ations. The Taguchi method can be effectively used when the
number of variables is between 3 and 50. Taguchi arrays can
respectively

be drawn out manually or found online. The arrays can be

selected by the number of parameters and number of levels.
Optimization of the performance can be done by Analysis of
Variance, Fisher exact test or Chi-squared test (Briggs, 2011;
John, 1996; Rao et al., 2008; Wang & Wan, 2009).
Taguchi methods have some advantages over other
methods. Taguchi methods take advantage of two-, three-
CoCl2 (0.1–0.2 g/g), NH3 (0.1–0.2 g/g),

raffinose (5–15 g/L), starch (5–15 g/L),

asparagine (2–10 g/L), urea (2–10 g/L),
0.1 g/g), FeCl3 (0.05–0.1 g/g), MnSO4

2 mM), CuSO4 (50–500 mM), alcohol

and multi-level factor analysis. Taguchi designs are similar

Glucose (5–15 g/L), sucrose (5–15 g/L),
(0.2–0.6%), (NH4)2SO4 (0.4–1.2%),
CaCl2 (0.05–0.1 g/g), ZnCl2 (0.05–

to familiar fractional factorial designs. However, Taguchi

KNO3 (2–10 g/L), xylidine (0.2–
(0.05–0.1 g/g), Tween 80 (0.05–

Glycerol (3–7%), proteose peptone

FeSO4 (0.001–0.005%), ZnSO4

designs are widely used in scientific studies and industrial

(0.2–2 mM), incubation time

engineering because they have been used to establish several

(0.002–0.006%), MgSO4

well-known experimental methods. Especially, Taguchi

methods can rapidly, accurately and precisely provide tech-
nical information regarding experiments that are used to
(0.003–0.07%)

design and produce low cost, highly reliable products and

(120–240 h)

processes. Applications that are used to produce Taguchi

0.1 g/g)

designs allow engineers to create flexible technology for the

design and production of high quality products and to
dramatically reduce research, development and delivery
time (Rao et al., 2008).
The Taguchi method has been successfully applied to the
optimization of microbial fermentation medium components
in bioengineering experiments and has been used to determine
the major component involved or the quantity of a component.
Different OAs are used in different optimization experiments.
Several studies that are arranged using different OAs are
shown in Table 3.
Several requirements are necessary for microbial activities;
macro- and micro-nutrients are important for biomass
production, which is directly related to the valued output of
a process. If any one of these nutrients is below the normal
NA

NA

limit, microbial growth and product quality is directly

affected. Therefore, the optimization of medium components
has found increasing importance in bioengineering applica-
Optimization of poly-"-lysine pro-
duction by Streptomyces nour-

tions. Teng & Xu (2008) used the Taguchi method and RSM
Pleurotus florida NCIM 1243

for optimize lipase production. The authors used an L18

Production of laccase from

(21  37) orthogonal array and studied many factors simul-

taneously. At the end of study, the authors recorded whole-cell
lipase activity that was two times higher than a non-optimized
NA: Not available.
sei NRRL 5126

condition using their method. The Taguchi method has the

important advantage of requiring fewer runs than one-
factor-at-a-time (OFAT) experimental methods. The results
show that the optimum conditions for lipase production are
as follows: agitation 200 rpm, inoculum 3.3–108 spores/L,

Table 3. Taguchi design applications in bioengineering.
Process
a-Amylase production by
Aspergillus oryzae CBS
819.72
Whole-cell lipase produc-
tion in submerged fer-
mentation by Rhizopus
chinensis using statis-
tical method
Griseofulvin production in
a batch bioreactor
Biodegradation of 4-chlor-
ophenol by Candida
tropicalis PHB5
Hydrogen production by
anaerobic fermentation
of cow manure
Laccase production by
Pleurotus ostreatus 1804
Taguchi design model Software Factors studied
1 7
L18 (2  3 ) SPSS Version 11.0.1 (2001, MgSO4 (0–0.1 g/g), KH2PO4
LEAD Technologies, (0–0.1–0.2 g/g), urea (0–0.25–0.5 g/g),
Inc., USA) soybean meal hydrolysate
(0–0.25–0.25 g/g), caseinic acid
hydrolysate (0–0.25–0.5 g/g), glycerol
(0–0.25–0.5 g/g), (NH4)2SO4 (0–0.05–
0.1 g/g), CoCl2 (0–0.05–0.1 g/g)
L18 (21  37) Qualitek-4 (Nutek Inc., Agitation (150–200 rpm), inoculum
Bloomfield Hills, MI) (3.3  106–3.3  107–3.3  108 spores/
L), maltose (0.5–1.5–2.5% w/v), olive
oil (0.5–1.5–2.5% w/v), peptone (4–6–
8%), pH (5–6–7), K2HPO4 (0.1–0.2–
0.3%), fermentation volume (20–30–
40 mL/250 mL)
L8 (27) NA Sucrose (37.5–50 g/L), K2HPO4
(1.25–1.5 g/L), NaNO3
(2.25–3.75 g/L), FeSO45H2O (0.005–
0.0125 g/L), pH (5.5–7.5), agitation
(150–250 rpm), aeration rate (4–6
vvm)
L27 (310) Qualitek-4 (Nutek Inc., MgSO47H2O (0.1–0.2–0.3 g/L), phos-
Bloomfield Hills, MI) phate ion (0.4–0.5–0.6 g/L), NaCl
(0.04–0.05–0.06 g/L), yeast extract
(0.5–1–1.5 g/L), CaCl22H2O (0.01–
0.02–0.03 g/L), temperature (28–30–
32  C), pH (5–6–7), inoculum size
(1–2–3 % v/v), incubation time
(50–55–60 h), agitation (120–150–
180 rpm), trace element solution
(1–2–3 % v/v)
L18 (21  37) Statistica 8.0 (StatSoft Agitation (150–250 rpm), pH (5–6–7),
Software, Inc.) temperature (50–60–70  C), substrate
concentration (22.15–30.86–
39.57 g/L), K2HPO4 (3–4–5 g/L),
ultrasound (20–30–40 W)
L18 (21  37) Qualitek-4 (Nutek Inc., pH (5.0–5.5), glucose (1–1.5–2 g/L),
Bloomfield Hills, MI) wheat bran (1–2–3 g/L), urea (0.5–
0.75–1 g/L), inoculum (5–7–10 discs),
yeast extract (1–1.5–2 % w/v), inducer
(0.5–1–1.5 mM), K2HPO4 (0.15–0.2–
0.25 % w/v)
376
Response References
After optimization of stu- (Kammoun et al., 2008)
died factors, a -amylase
production was increased
T. K. Gündoğdu et al.
from 40.1 to 151.1 U/mL
Inoculum level, fermenta- Teng & Xu (2008)
tion volume, olive oil
and peptone had positive
effect on lipase produc-
tion and increased lipase
production upon to two
times
Maximum griseofulvin Venkata-Dasu et al. (2003)
production was identi-
fied as 1.19 g/L
Maximum biomass and Basak et al. (2013)
residual 4-chlorophenol
concentration were
defined as 752.5 mg/L,
501.03 respectively
Temperature and pH were Wang et al. (2012)
identified as significant
parameters for hydrogen
production
Maximum laccase activity Prasad et al. (2005)
was determined as
716.9 U/mL
Crit Rev Biotechnol, 2016; 36(2): 368–388
Hydrogen production and NA Qualitek-4 (Nutek Inc., Feed composition, organic loading rate Maximum hydrogen pro- Mohan et al. (2009)
wastewater treatment Bloomfield Hills, MI) (0.863–1.986–2.560–3.843–5.892 kg ductions were 1.92 mmol
processes COD/m3day), inlet pH (5.5–7), nature H2/day, 0.52 mol H2/kg
of anaerobic mixed inoculums (anaer- COD-day whereas sub-
obic mixed culture treating distillery strate degradation rate
DOI: 10.3109/07388551.2014.973014

wastewater, anaerobic mixed culture was 4.56 kg COD/

treating chemical wastewater), nature m3-day
of pre-treatment (heat-shock, chemical,
untreated, combined pre-treatment of
heat-shock, pH and chemical)
Polyhydroxy alkanoates L18 (21  37) Qualitek-4 (Nutek Inc., FeCl3 (yes–no, 50 g/L), glucose (0–6– Microenvironment, pH and Mohan & Venkateswar-
(PHA) synthesis by Bloomfield Hills, MI) 12 g/L), volatile fatty acid (3–6–9 g/L), glucose concentration Reddy (2012)
mixed culture a:p:b (70:10:20–25:50:25–20:10:70 had strong effect on bio-
%), nh4cl (100–200–300 mg/L), plastic production
K2HPO4 (50–100–150 mg/L), pH
(5–7–8), microenvironment (aerobic–
microaerophilic–anaerobic)
Ferrous bio-oxidation rate L16 (45) Qualitek-4 Version 4.82.0 Temperature (50–55–60–65  C), initial Maximum Fe2+ bio-oxida- Mousavi et al. (2007)
in a packed bed (Nutek Inc., Bloomfield pH (1–1.5–2–2.5), dilution rate tion rate was determined
bioreactor Hills, MI) (0.1–0.2–0.3–0.4 h1), initial Fe3+ as 7.8 g/L/h.
concentration (1–3–5–7 g/L), aeration
rate (100–150–200–250 mL/min)
Optimization of lipase-cat- L27 (313) Design Expert Version 6.07 Reaction time (7–18–24 h), temperature Maximum xylitol yield was Adnani et al. (2010)
alyzed synthesis of xyli- (State-Ease, Inc, statistic (30–50–60  C), enzyme amount 97.4%
tol ester made Easy Minneapolis, (0.05–0.12–0.3 g), amount of molecu-
MN) lar sieve (1–2.5–4 g), substrate molar
ratio (0.33–0.5–1), xylitol concentra-
tion (0.015–0.008–0.005 g/mL)
Tannase production by L18 (21  35) Qualitek-4 (Nutek Inc., Temperature (30–35–40  C), pH (4–5), Maximum tannase pro- Das Mohapatra et al. (2009)
Bacillus licheniformis Bloomfield Hills, MI) tannic acid concentration (0.5–1–1.5 g/ duced was 0.43 U/mL
KBR6 in submerged 100 mL), phosphate concentration
culture (0.15–0.30–0.45 g/100 mL), nitrogen
concentration (0.35–0.7–1.05 g/
100 mL), Mg2+ ion concentration
(0.05–0.1–0.15 g/100 mL

NA: Not available

Experimental design methods for bioengineering applications
377
378 T. K. Gündoğdu et al.
maltose 0.5% (w/v), olive oil 1.5% (w/v), peptone 4% (w/v),
K2HPO4 0.3% (w/v), fermentation volume 20 mL/250 mL
flask and pH 5.5 (Teng & Xu, 2008). Taguchi design
commonly used for optimization of enzyme production such
as lipase (Adnani et al., 2010), tannase (Das Mohapatra et al.,
2009), a-amylase (Kammoun et al., 2008) and laccase (Prasad
et al., 2005).
Candida tropicalis PHB5 is able to grow on 4-chlorophe-
nol and metabolize this substrate. The Taguchi orthogonal
array design methodology was used to improve the
4-chlorophenol biodegradation ability of strain PHB5.
Experiments were undertaken to study the effects of eleven
decided that biomass yield on 4-chlorophenol could be
increased from 409.7 mg/L to 1046.9 mg/L and the amount
factors affecting the growth of C. tropicalis on 4-chlorophenol
and its degradation and it has been of 4-chlorophenol
degraded could be increased from 314.5 mg/L to 949.7 mg/L
(Basak et al., 2013).
Venkata-Dasu et al. (2003) optimized griseofulvin pro-
duction in a batch reactor with 8 runs using L8 (27). The
authors determined the values of significant parameters for
production. Sucrose, K2HPO4, NaNO3, FeSO45H2O, pH,
agitation and aeration rate were tested as factors (Venkata-
Dasu et al., 2003).
Wang et al. (2012) used Taguchi Design to determine the
most effective parameter values for the production of
biohydrogen from wastewater using an L18 (21  37) orthog-
onal array; the factors tested were agitation, pH, temperature,
substrate concentration, K2HPO4, concentration and ultra-
sound. At the end of the study, the authors reported that the
most important factors were temperature, pH, substrate
concentration, ultrasound, K2HPO4 concentration and agita-
tion (Wang et al., 2012). Combined process efficiency with
respect to fermentative hydrogen (H2) production and waste-
water treatment was also evaluated in a series of batch
experiments to detect the role of selected factors such as
origin of inoculum, pre-treatment, inlet pH and feed com-
position under anaerobic microenvironment using mixed
culture (Mohan et al., 2009). Another study based on
polyhydroxy alkanoates (PHA) synthesis by mixed culture
using L18 (21  37) Taguchi design showed that microenvir-
onment, pH and glucose concentration had strong effect on
bio-plastic production (Mohan & Venkateswar-Reddy, 2012).
Mousavi et al. (2007) studied on process parameter
optimization of the ferrous bio-oxidation rate by immobiliza-
tion of a native Sulfobacillus species on the surface of low
density polyethylene particles in a packed-bed bioreactor
using Taguchi method. Five control factors, including tem-
perature, initial pH of feed solution, dilution rate, initial
concentration of Fe and aeration rate in four levels are
considered in Taguchi technique. L16 orthogonal array has
been used to determine the signal to noise (S/N) ratio
(Mousavi et al., 2007).
Response surface methodology
Response surface methodology (RSM) is used for developing,
improving and optimizing processes based on various statis-
tical and mathematical techniques. The main objective is to
identify the conditions yielding the highest production using a
Crit Rev Biotechnol, 2016; 36(2): 368–388
small number of experiments that provide a large amount of
information (Erkucuk et al., 2009). Several RSM designs
exist. In this review, Box–Behnken design (BBD) and central
composite design (CCD), and their application to bioengin-
eering processes, are discussed in detail.
Box–Behnken design
A BBD is a three-level fractional factorial design developed
by Box and Behnken that is applied to determine the nature
of the response surface in an experimental region (Majumder
et al., 2009; Wang & Wan, 2009). The design is a combination
of a two-level factorial design with an incomplete block
design and in each block, a certain number of factors are put
through all combinations for the design, while other factors
are kept at the central levels. This design has several
advantages, such as having three levels that can be coded as
1 (low), 0 (middle) and +1 (high; Majumder et al., 2009),
creating an independent quadratic design (Das et al., 2012)
and providing an easier way to arrange and to interpret the
results. The design includes an incomplete block design, and
each block consists of the maximum and minimum values,
factorial design values and the central values of the factors
(Majumder et al., 2009).
Box–Behnken design also provides a time-consuming (Yin
et al., 2011) and an economical alternative to CCD, because it
has fewer factor levels and does not contain extremely high or
low levels. BBD is widely used in bioengineering processes as
it decreases the number of experimental sets (Table 4). For
example, Coban et al. (2012) studied the effects of three
factors (inoculum ratio, reducing sugar concentration and
fermentation time) on bioethanol fermentation yield response
using a BBD and 15 runs whereas Gharibzahedi et al. (2012)
studied the optimization of canthaxanthin production using
CCD with 20 runs.
The correlation between the variables and responses can be
determined by fitting to a second-order polynomial equation
[Equation (2); Majumder et al., 2009].
X X X
Y ¼ 0 þ  i Xi þ ii Xi2 þ ij Xi Xi ð2Þ
where Y is the predicted response variable; Xi and Xj (i ¼ 1, 3;
j ¼ 1, 3, i 6¼ j) represent the independent variables and o,
 i, ii and ij are constant regression coefficients of the
model.
The BBD also has the important advantage of requiring
fewer runs than OFAT experimental methods. This design
provides enough data to fit quadratic models for bioengin-
eering applications. For example, Chavalparit & Ongwandee
(2009) studied the optimization of an electrocoagulation
process for the treatment of biodiesel wastewater using
quadratic regression models. The authors used a BBD for
three factors (initial pH, applied voltage and reaction time),
involving three blocks and a set of 15 experimental runs to
optimize the operating conditions. The results showed that
chemical oxygen demand (COD), oil and grease and
suspended solids (SS) were effectively reduced (Table 4) by
electrocoagulation under the following optimum conditions:
pH 6.06, applied voltage 18.2 V, and reaction time 23.5 min
(Chavalparit & Ongwandee, 2009).
Table 4. Box–Behnken design applications in bioengineering.

Number
Response of runs R2 Variables Optimized conditions Biocatalyst/substrate References
Bioethanol fermentation yield 15 NA Inoculum (S. cerevisiae) ratio 1.25% S. cerevisiae Coban et al. (2012)
Reducing sugar concentration 20 g/L
Fermentation time 2 days
Bacterial cellulose production 27 98 Carbohydrate of maple syrup 30 (g/L) Acetobacter xylinum BPR Zeng et al. (2011)
DOI: 10.3109/07388551.2014.973014

2001
Incubation period 30 days
Size of inoculum 6%
Agitation 135 rpm
Extracellular 5-aminolevulinic 15 94–98 Succinate 10 g/L Rhodopseudomonas Choorit et al. (2011)
acid (ALA) yield palustris KG31
Glycine 5 g/L
LA 15 g/L
Activity of heterotrophic nitrifica- 27 90 Initial pH 8.46 Agrobacterium sp. LAD9 Chen & Ni (2012)
tion–aerobic de-nitrification
C/N ratio 8.28
Temperature 27.9  C
Agitation 150 rpm
Polysaccharides production 29 92 Extraction temperature 61.8  C Tricholoma matsutake Yin et al. (2011)
Extraction pH 4.14
Extraction time 3.2 h
Complex enzyme amount 2.1% (w/v)
Enzyme activity (Laccase, Lip, 17 94.5–99.4 Moisture content Optimum level for all Phlebia floridensis Sharma & Arora (2010)
MnP, Xylanase, CMCase) enzymes was different
Malt extract
NH4Cl
Xylanase activity 10 97 Yeast extract Not in the domain of the Melanocarpus albomyces Narang et al. (2001)
experiment IIS68
Surfactant
Urea
Initial moisture
Inoculum age
Harvest time
Removal of chromium (VI) % 29 92.71 Initial chromium concentration 50–60 mg/L Nostoc linckia Mona et al. (2011)
pH 2
Cyanobacterial dose 1.05 g
Temperature 25  C
Xylanase production 17 97 Concentration of urea 1.27 Melanocarpus albomyces Biswas et al. (2010)
IITD3A
Inoculum size 5.0% (v/v)
pH of the medium 7.4
Synthesis dipeptide derivative, N- 15 98.3 Reaction time 92.3 min a-chymotrypsin Ju et al. (2012)
Ac-Phe-Gly-NH2, catalyzed by
immobilized a-chymotrypsin in
a stirred tank bioreactor
Reaction temperature 36.2  C
Experimental design methods for bioengineering applications

pH 8.7
(continued )
379
 Table 4. Continued
380

Number
Response of runs R2 Variables Optimized conditions Biocatalyst/substrate References
2
Hydrogen production yield 15 97 Light intensity 175 W/m Rhodobacter capsulatus Ghosh et al. (2012)
JP91
Glucose 35 mM
Glutamate 4.5 mM
Hydrogen production yield 13 93.2 Initial pH 5.15 Mixed anaerobic Ray et al. (2010)
mesophilic culture
T. K. Gündoğdu et al.

Glucose 3.49 mol

Linoleic acid 1.9 g/L
Hydrogen production yield 12 98 Temperature 60  C Thermophilic mixed culture Roy et al. (2012)
pH 6.5
Substrate concentration 10 g/L
Microalgae cell dry weight 15 99 Bacteriological peptone 1 g/L Chlorella saccharophila Isleten-Hosoglu et al.
(2012)
Glucose 20 g/L
Glycerol 5 g/L
Ethanol productivity 29 99.7 pH 5.5 Saccharomyces cerevisiae Singh & Bishnoi (2013)
Temperature 30  C
Inoculum level 3.3%
TRS concentration 6.5%
Glucan production 15 98.65 Sucrose 5.95% Leuconostoc dextranicum Majumder et al. (2009)
NRRL B-1146
Peptone 0.52%
Yeast extract 2.9%
Spirulina biomass productivity, 15 NA Blend concentration 0.40 g/L Spirulina platensis Radmann et al. (2007)
Specific growth rate, Doubling
time
Renewal rate 40%
Zarrouk medium 20%
HLC III synthesis 15 96.09 Induction time 3.2 h Escherichia coli Zhang et al. (2010)
Inoculum age 12.6 h
pH 6.7
Methane yield 17 96 Ca(OH)2 concentration 50% Rice straw (Song et al., 2012)
Pretreatment time 3d
Inoculum 50%
Removal efficiencies of Chemical 15 NA Initial pH 6.06 With using electrocoagula- Chavalparit & Ongwandee
oxygen demand (COD) and oil tion (EC) system for (2009)
and grease (O&G), suspended treatment
solid (SS)
Applied voltage 18.2 V
Reaction time 23.5 min
CMCase activity, b-glucosidase 30 96 Fermentation time 86–88 h Aspergillus fumigatus Das et al. (2012)
activity, FPase activity, ABK9
Xylanase activity
pH of the medium 6.1–6.2
Substrate ratio (WB:RS) 1.1
Substrate amount 10.5 g
Yield of starcholeate 24 93 Reaction temperature 44  C Horchani et al. (2010)
Crit Rev Biotechnol, 2016; 36(2): 368–388
DOI: 10.3109/07388551.2014.973014 Experimental design methods for bioengineering applications 381

BBD is also useful for optimizing ethanol fermentation

(Coban et al., 2012; Singh & Bishnoi, 2013); biological

Erkucuk et al. (2009)

Haddar et al. (2012)

Akgun et al. (2011)

compounds (Akgun et al., 2011; Choorit et al., 2011; Erkucuk
et al., 2009; Horchani et al., 2010; Ju et al., 2012; Majumder
et al., 2009; Yin et al., 2011; Zhang et al., 2010); enzymes
(Biswas et al., 2010; Das et al., 2012; Haddar et al., 2012;
Narang et al., 2001; Sharma & Arora, 2010); bacterial
cellulose (BC) production (Zeng et al., 2011); heterotrophic
nitrification and aerobic denitrification (Chen & Ni, 2012);
chromium removal (Mona et al., 2011); COD, O&G and SS
(Chavalparit & Ongwandee, 2009); hydrogen (Ghosh et al.,
Bacillus mojavensis A21
Staphylococcus aureus

2012; Ray et al., 2010; Roy et al., 2012) and microalgae

production (Isleten-Hosoglu et al., 2012; Radmann et al.,
Steviare baudiana

Alkanna tinctoria
2007) and methane yield (Song et al., 2012). As an example,
Ghosh et al. (2012) studied the effects of glucose, glutamate
(SAL3)

concentration and light intensity on hydrogen production from

glucose via single-stage photofermentation using a Box–
Behnken design. The authors concluded that under optimal
conditions with a light intensity of 175 W/m2, 35 mM glucose
and 4.5 mM glutamate, a maximum hydrogen yield of 5.5
(±0.15) mol H2/mol glucose was obtained (Ghosh et al.,
2012). Another study used a Box–Benkhen design and several
initial pH conditions in a mixed batch anaerobic mesophilic
culture fed with glucose and linoleic acid (LA) to optimize
hydrogen production yield. The optimal values of initial pH,
18.66 g/L

176 rpm
1.04 g/L

211 bar

175 bar

glucose and LA concentration for maximum hydrogen

5 g/min
34.08 h
386 IU

17.4%
80  C

80  C
0.18

production yield were obtained as 5.15, 3.49 mol and

1.9 g L1, respectively (Ray et al., 2010).
Sharma & Arora (2010) applied BBD to investigate the
effects of moisture content, inorganic nitrogen source
Concentration of ethanol–water

(NH4Cl) and malt extract on lignocellulolytic enzymes

Amount of immobilized lipase
Starch/oleic acid molar ratio

during solid state fermentation. Optimum manganese perox-

idase production was attained (50–55 mg/g of WS) with
increased moisture content and laccase production. These
supplements also significantly (p50.05) enhanced the pro-
Cultivation time

duction of CMCase and xylanase (Sharma & Arora, 2010).

Temperature

Temperature
Barleybran

mixture

Bacterial cellulose (BC, a polymer of glucose units) is

CO2 flow
Agitation

Pressure

Pressure

produced by the bacterium Acetobacter xylinum BPR 2001.

NaCl

Zeng et al. (2011) optimized the factors of maple syrup

carbohydrate (30 g carbohydrate/l), incubation period (30
days), size of inoculum (6%) and rotation speed (135 rpm)
using a three-level, four-factor (27 run) BBD to increase BC
production (Zeng et al., 2011).
96.65
91.5
89.9
98

A Box–Behnken design was used to optimize environ-

mental factors such as methane yield, nitrification–denitrifi-
cation and the removal of COD. Chen & Ni (2012)
investigated the effects of initial pH, C/N ratio, temperature
and shaking speed on the activity of heterotrophic nitrifica-
29

15

15

tion–aerobic denitrification using the strain LAD9. It was

concluded that the maximum removal of ammonium occurred
under the following conditions: initial pH, 8.46; C/N ratio,
Stevioside and rebaudioside A

8.28; temperature, 27.9  C and shaking speed, 150 rpm;

temperature and shaking speed were found to have the largest
Yield of total alkannin

effect (Chen & Ni, 2012).

Mona et al. (2011) also used BBD to analyze the
NA: Not available.
Xylanase activity

effectiveness of cyanobacterial biomass (Nostoc linckia) for

the biosorption of Cr(VI) from aqueous waste using a
laboratory-scale batch fermentor. The process parameters
yields

used in this study were as follows: initial metal concentration

(10–100 mg/L), pH (2–6), temperature (25–45  C) and
382 T. K. Gündoğdu et al.
cyanobacterial dose (0.1–2.0 g). Maximum Cr(VI) biosorp-
tion was obtained at pH 2–4 with an initial metal concen-
tration of 10–55 mg/L in the aqueous solution (Mona et al.,
2011). Song et al. (2012) aimed to maximize methane yield
using BBD. The effect and interactions of Ca(OH)2
concentration (LC), pre-treatment time (PT) and inoculum
amount (IA) on biogas production rates were evaluated at
mesophilic temperature. It was concluded using 17 runs that
optimum methane yield was achieved under the following
conditions: LC, 50%; PT, 3 days; and IA, 50% (Song et al.,
2012).
Ju et al. (2012) studied the effects of various parameters on
the synthesis of a derivatized dipeptide, N-Ac-Phe-Gly-NH2,
which was catalyzed by immobilized a-chymotrypsin in a
stirred tank bioreactor. A 3-factor, 3-level BBD predicted
using 15 runs that the optimum conditions were as follows:
reaction time, 92.3 min; reaction temperature, 36.2  C; and
pH, 8.7. R2 and synthesis yield were 98.3 and 82.26%,
respectively. Reaction time and pH significantly affected the
yield of N-Ac-Phe-Gly-NH2, and the a-chymotrypsin–Fe3O4–
CS nanoparticles could be used to synthesize the dipeptide
(Ju et al., 2012).
Repeated batch cultivation of S. platensis was studied in
open raceway ponds at 30  C by Radmann et al. (2007). Using
a light intensity of 3000 lux and a 12-h photoperiod for a total
cultivation time of 60 days, three different dilutions were
optimized. It was found that the productivity of S. platensis
was 0.028–0.046 g L1 day1, and the maximum specific
growth rate (mmax) changed from 0.038 to 0.138 day1
(Radmann et al., 2007).
A study conducted by Das et al., (2012) investigated the
influence of four parameters [fermentation time (86–88 h),
medium pH (6.1–6.2), substrate amount (10.0–10.5 g) and
substrate ratio (wheat bran:rice straw) (1.1)] on enzyme
production. Endoglucanase, BG, FPase (filter-paper degrad-
ing activity) and xylanase activities of 826.2, 255.16, 102.5
and 1130.4 U/g, respectively, were achieved. It was concluded
that all factors were important, and optimal combinations
were at the midpoints of the edges as well as at the center of
the cubical design region (Das et al., 2012; Haddar et al.,
2012).
Stevia rebaudiana extracts are used as a low-calorie
sweetener and as a medicinal plant, and also for the
production of stevioside and rebaudioside A. To determine
the effects of pressure (150–350 bar), temperature (40–80  C)
and ethanol percentage (0, 10 and 20% in water) on the
glycoside composition of Stevia rebaudiana, a 15-run BBD
was applied. The optimum conditions were determined to be
211 bar, 80  C and 17.4% ethanol, and these conditions
yielded 36.66 mg/g stevioside and 17.79 mg/g rebaudioside A
(Erkucuk et al., 2009).
Central composite design
A CCD is defined as a five-level fractional factorial design
that uses external points and one point at the center of the
experimental region (Tarley et al., 2009). This central point is
supposed to have several properties, such as rotatability or
orthogonality, to fit quadratic polynomials (Boey et al., 2011;
Ruiz et al., 2011).
Crit Rev Biotechnol, 2016; 36(2): 368–388
The CCD is an important alternative to the full factorial,
three-level design as it requires fewer experiments while
giving comparable results. Therefore, CCD is the most
accepted experimental design for second-order models.
CCD is superior to the OFAT method because it is possible
to reveal corresponding interactions among the factors with
CCD using a smaller number of experiments. In addition, the
use of statistical approaches may expose the importance of
individual factors and the effect of each factor on the
response. Zain et al. (2007) identified the optimum carbon
and nitrogen concentrations in a feed medium for obtaining
maximum CGTase production using CCD; in addition, the
interaction between the C/N ratio and the biomass produc-
tion was evaluated as a secondary response. The authors
determined the optimum concentrations of carbon and
nitrogen sources to be 3.30% (w/v) and 0.13% (w/v),
respectively, and these values led to a maximum CGTase
activity of 80.12 ± 1.43 U/mL (Zain et al., 2007).
CCD is also useful for bioprocesses that optimize the
culture conditions for bioethanol (Dagnino et al., 2013; Harun
& Danquah, 2011), biodiesel (Lee et al., 2011; Vicente et al.,
1998; Yücel, 2012), enzyme (Chen et al., 2002; Donzelli
et al., 2005; Fakhfakh-Zouari et al., 2010; Heck et al., 2005;
Sathya et al., 1998; Sifour et al., 2010), biological compounds
(Corrêa et al., 2012; Hollebeeck et al., 2012) and protein
(Larentis et al., 2011; Qu et al., 2010) production and for
COD removal (Ahmadi et al., 2005; Muthukumar et al., 2004)
via microbial activities and cell culture techniques for
intelligent bioprocessing (Domingues et al., 2010; Lim
et al., 2007; Table 5). Gharibzahedi et al. (2012) also used
CCD to evaluate the effect of three nutrient components,
namely, D-glucose content (15–25 g/L), mannose content (10–
25 g/L) and Fe3+ concentration (20–40 ppm), on the growth of
the D. natronolimnaea HS-1 mutant; biomass dry weight,
total carotenoids and canthaxanthin production were the
studied responses. The optimum fermentation medium com-
position was 25 g/L D-glucose, 15.12 g/L mannose and
36.77 ppm of Fe3+; there was no significant difference
between the observed and predicted values (p50.05;
Gharibzahedi et al., 2012). The results indicate that CCD is
a key tool for optimizing the proportion of nutrient medium
components and leads to the desired response variable goals.
Xu & Ting (2004) determined optimal bioleaching condi-
tions to achieve the maximum metal leaching efficiency for
selected metals with 31 runs; four variables were studied,
namely, sucrose concentration, inoculum spore concentration,
fly ash pulp density and the time of addition of fly ash to the
fungus Aspergillus niger. It was concluded that sucrose
concentration and pulp density were more important factors
than spore concentration or the time of addition of the fly ash
(Xu & Ting, 2004). Bajpai et al. (2012) also used the RSM
approach for predictive model building and the optimization
of chromium adsorption onto activated carbon obtained from
tamarind wood. As limited studies have been performed on
chromium adsorption using the RSM approach using indi-
vidual and combined effects, four process parameters [contact
time, initial pH, initial Cr(VI) concentration and resin dose]
were optimized with 30 runs to maximize the removal of
hexavalent chromium. It was concluded that individual
process parameters were more significant than the combined
Table 5. Central composite design applications in bioengineering.

Number
Response of runs R2 value Variables Optimized conditions Biocatalyst/substrate References
Clavulanic acid production 12 0.9078 Glycerol 70 mM Streptomyces clavuligerus Domingues et al. (2010)
ATCC 27064
Ornithine 1.7 mM
Polymethyl galacturonase 14 0.9 pH 2.3 Aspergillus niger NCIM Sathya et al. (1998)
production Temperature 23  C 548
Biodiesel yield 10 NA Catalyst amount 4.9 wt.% Waste cockle shell Boey et al. (2011)
DOI: 10.3109/07388551.2014.973014

MeOH/oil mass ratio 0.54:1

Ethanol yield 18 0.924 Temperature 30  C Saccharomyces cerevisiae Ruiz et al. (2011)
Substrate 2% CA11
Enzyme loading 30 FPU
Cellulase-free xylanase 11 0.8319 Temperature 60  C Bacillus coagulans BL69 Heck et al. (2005)
activity pH 7
Glucans and xylose for 10 0.92 H2SO4 0.3% (w/v) Rice hulls Dagnino et al. (2013)
bioethanol production Time 33 min
Elastase production 20 0.9112 Glucose Not in the domain of the Bacillus sp. EL31410 Chen et al., (2002)
experiment
Casein Corn steep flour
K2HPO4
MgSO4.7H2O
pH
b-1,3-Glucanase 29 0.930 Glucose 0.1% Trichoderma atroviride Donzelli et al. (2005)
strain P1
Ammonium ion 25 mM
Chitin 0.8%
Scleroglucan 0.2%
Inoculum 107 spores/run
Peptide activity 36 0.9933 Substrate 1.5% Porphyra yezoensis Qu et al. (2010)
Alcalase 5%
pH 9.0
Temperature 50  C
Hydrolysis time 60 min
Biodiesel production 30 0.9899 Methanol/oil molar ratio 38.67 Jatropha curcas Lee et al. (2011)
Reaction time 3.44 h
Catalyst amount 3.70 wt.%
Reaction temperature 115.87  C
Biodiesel production 30 0.9367 Reaction temperature 40  C T. lanuginosus Yücel (2012)
Molar ratio of methanol 5.3:1
to oil
Biocatalyst content 5.8% w/w
Reaction time 24 h
Pneumococcal surface 12 NA Inducer concentration 0.1 mM recEscherichia coli Larentis et al. (2011)
adhesin A yield Temperature 25  C
Time 16 h
Keratinases production 32 0.856 Feather meal 30 g/L Bacillus pumilus A1 Fakhfakh-Zouari et al.
(2010)
Soy petone 3.151 g/L
Experimental design methods for bioengineering applications

NaCl 2 g/L
KCl 0.5 g/L
383

(continued )
 Table 5. Continued
384

Number
Response of runs R2 value Variables Optimized conditions Biocatalyst/substrate References
KH2PO4 0.5 g/L
Bioethanol production 25 0.93 Acid concentration 1% (v/v) Saccharomyces cerevisiae Harun & Danquah (2011)
Temperature 15 g/L
Microalgae loading 140  C
Pre-treatment time 30 min
Oleic acid epoxide 11 0.98 Temperature 55  C PSCI Amano lipase Corrêa et al. (2012)
T. K. Gündoğdu et al.

production
Enzyme load 10%
Hydrogen peroxide load 0.2%
Reaction time 3h
Glycerol-inducible lipase 26 0.98 Glycerol 2.24% Geobacillus stearothermo- Sifour et al. (2010)
production philus strain-5
Tween 80 0.76%
Glucose 0.76%
K2HPO4 0.38%
Cyclodextrin glucanotrans- 17 0.9534 Carbon concentration 3.30% (w/v) Bacillus sp. TS1-1 Zain et al. (2007)
ferase production
Nitrogen concentration 0.13% (w/v)
Canthaxanthin production 20 0.996 D-glucose content 25 g/L Dietzia natronolimnaea Gharibzahedi et al. (2012)
HS-1
Mannose content 15.12 g/L
Fe3+ concentration 36.77 ppm
Gluconic acid production 31 0.997 Sucrose concentration 156 g/L Aspergillus niger Xu & Ting (2004)
via bioleaching of fly
ash
Spore concentration 0.6  107
Pulp density % 2.7
Time of addition 0 days
COD removal of acid dye 20 0.94 Salt concentration 5 g/L Acid Red 88 dye Muthukumar et al. (2004)
effluent
pH Alkaline
Time 195 s
Fenton’s peroxidation on 15 0.988 H2O2-to-Fe(II) ratio 8.33 Olive oil mill wastewater Ahmadi et al. (2005)
the removal of COD
from olive oil mill was-
tewater (OMW)
pH 4
OMW concentration 70%
Removal of chromium (VI) 30 0.88 Contact time 62.5 min Amberlite IRA 96 resin Bajpai et al. (2012)
from aqueous solution
using weakly anionic
resin
Initial pH 1.96
Initial Cr (VI) concentration 145.4 mg/L
Resin dose 8.51 g/L
Baicalin-loaded nanoparti- 13 0.9976 Lipid 0.69% (w/v) Baicalin-loaded Hao et al. (2012)
cles carrier system nanoparticles
Drug/lipid ratio 26.64% (w/w)
Crit Rev Biotechnol, 2016; 36(2): 368–388

NA: Not available.

DOI: 10.3109/07388551.2014.973014
model of these parameters. Initial solution pH was the most
significant parameter for chromium removal, followed by the
resin dose. The optimum process conditions for chromium
removal at 30  C using IRA 96 resin were as follows: contact
time, 62.56 min; pH, 1.96; initial Cr(VI) concentration,
145.4 mg/L and resin dose, 8.51 g/L; a maximal removal of
93.26% was obtained (Bajpai et al., 2012). However,
compared to CCD, OFAT is a classical approach and is
time consuming, requires many experiments and is not able to
detect interactive effects between factors. Moreover, the
optimized process conditions obtained using this method are
not reliable.
CCD supported by statistical software and is a well-
established approach for pharmaceutical formulation
development and the optimization of few well-designed
experiments; it is also suitable for pharmaceutical blending
problems because it enables investigation with the least
number of experiments (Gonzalez-Mira et al., 2010; Hao
et al., 2012; Jin et al., 2008).
The comparison of all experimental design methods
For optimization studies in bioengineering applications, the
complex polynomial function is solved to determine the
interactions amongst the variables investigated. In addition,
a minimum number of experimental runs is required to
evaluate the interaction in the model with minimum time
consumption and operational cost. Choosing the best experi-
mental design depends on the number of the factors in the
process (Table 6).
Factorial design is the most common design for N-way
ANOVA applications. In a factorial design, observations are
made for each combination of the levels of each factor. Using
a two-way design interaction, the effect of independent
variables on dependent variables can be estimated. Thereby,
interactions on every different level are revealed which cannot
be observed via one way analysis. Main advantage of the
factorial design is that it can lead more powerful test by
reducing the error variance which is the main problem in
t-tests. The disadvantage of the full factorial design is when
the number of factors is 5 or greater, a large number of runs is
required and is not very efficient (Fisher, 1935).
CCD contains an imbedded factorial design with center
points and is augmented with a group of axial points. One of
the advantages of CCD is to reduce the number of experi-
ments in the studies with a large number of factors and levels,
while giving comparable results and corresponding inter-
actions among the factors. In addition, CCD can provide high
Table 6. Summary table for choosing an experimental design
(NIST/SEMATECH, e-handbook).
Factors Comparison Screening Optimization
1 One factor at
a time
2–4 Randomized Full or fractional Central composite
design factorial design or Box–Behnken
design
5 or more Randomized Taguchi or Plackett–
design Burrman Design
Experimental design methods for bioengineering applications 385
quality predictions in studying linear, quadratic and inter-
action effect factors which influence a system. Also, the CCD
is robust and results in a model with high predictability, even
if the data points are outside the tested domain. Compared to
BBD, the R2 value from CCD generally shows higher value in
case where both designs fit well into the linear polynomial
model. Furthermore, CCD usually yields lower residual
standard error value than BBD. Thus, CCD predicts more
accurate data comparing to the actual experiment and is
considered as the most accepted experimental design for
second-order models.
BBD method presents an alternative way to CCDs. They
are a class of rotatable or nearly rotatable second-order
designs based on three-level incomplete factorial designs
(Ferreira et al., 2007). BBD has two main advantages over
CCDs: (i) the first one is that they only require factors to be
varied over three levels. This may decrease the cost of
experiment if actual prototypes are being set in experimen-
tation. (ii) The second one is that they usually (except for the
five-factor case) require less total runs than the CCD
(Lawson, 2010). BBD is a combination of a two-level
factorial design with an incomplete block design and in
each block, a certain number of factors are put through all
combinations for the design, while other factors are kept at
the central levels. This design has several advantages, such as
having three levels that can be coded as 1 (low), 0 (middle)
and +1 (high; Majumder et al., 2009), creating an independ-
ent quadratic design (Das et al., 2012) and providing an easier
way to arrange and to interpret the results. The design
includes an incomplete block design, and each block consists
of the maximum and minimum values, factorial design values
and the central values of the factors (Majumder et al., 2009).
It also provides a time-consuming (Yin et al., 2011) and an
economical alternative to CCD, because it has fewer factor
levels and does not contain extremely high or low levels. The
BBD has the important advantage of requiring fewer runs
than OFAT experimental methods. This design provides
enough data to fit quadratic models for many applications.
The disadvantage of the BBD is having limited capability for
orthogonal blocking compared to the CCD.
Placket–Burman design is required when a large number of
factors need to be screened for their effects at two levels.
Various methods have been proposed to limit the number of
candidate terms for experimental design model under study
(Lawson, 2010). The PB factorial design can identify main
factors from a large number of suspected contributor param-
eters for the desired response variables. Thus, these designs
are enormously useful in preliminary studies where the aim is
to identify variables that can be fixed or eliminated in
further investigation (Vatanara et al., 2007). On the other
hand, this design is that it allows a quick monitoring of all the
factors through limited number of experiments. The weakness
of the design is that replicates of design points are not
included to permit the calculation of error terms (Li &
Rasmussen, 2003).
Experimental design using the Taguchi method is a
powerful and effective approach to assist engineers to study
and understand how several process parameters affect the
process output without too much budget and time consump-
tion (Antony, 1997). OAs employed in the Taguchi method
386 T. K. Gündoğdu et al.
provide mainly three advantages to the users by: (i) reducing
the number of experiments significantly, (ii) fostering the
robustness of the process and (iii) minimizing the time
consumption and operational cost (Hong, 2012; Chowdhurya
et al., 2014). Last but not least, using the S/N ratio, to analyze
the results helps to decrease the sensitivity of the system to
sources of variation which provides better performance
(Zolgharneina et al., 2014).
Declaration of interest
The authors report no conflicts of interest. The authors alone
are responsible for the content and writing of this article.
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