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Response surface methodology in media optimization for production of β-

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DOI: 10.1007/s11240-008-9350-8

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Plant Cell Tiss Organ Cult (2008) 93:123–132
DOI 10.1007/s11240-008-9350-8
ORIGINAL PAPER
Response surface methodology in media optimization
for production of b-carotene from Daucus carota
V. M. Hanchinal Æ S. A. Survase Æ
S. K. Sawant Æ U. S. Annapure
Received: 18 July 2007 / Accepted: 11 February 2008 / Published online: 5 March 2008
Ó Springer Science+Business Media B.V. 2008
Abstract Plants are a valuable source of a vast array
of chemical compounds including fragrances, flavours,
food additives, colours, natural sweeteners, industrial
feedstocks, anti-microbials and pharmaceuticals. The
present study reports on application of Response
Surface Methodology (RSM) in media optimization
for suspension culture for the production of
b-carotene. Growth kinetics of carrot cells in suspen-
sion culture has been carried out to understand the
relationship between growth and b-carotene forma-
tion. The maximum production of b-carotene obtained
using the optimized medium was 13.614 lg/g dry
weight cell mass. The l (specific growth rate) and td
(doubling time) were found to be higher for 20 g DW/l
inoculum size.
Keywords b-Carotene
Response surface
methodology
Plant tissue culture
Abbreviations
RSM Response surface methodology
DW Dry weight
2,4-D 2,4 Dichloro phenoxy acetic acid
V. M. Hanchinal
S. A. Survase
S. K. Sawant

U. S. Annapure (&)
Food Engineering and Technology Department, Institute
of Chemical Technology, University of Mumbai,
Matunga, Mumbai 400 019, India
e-mail: usa@udct.org
MS Murashige and Skoog
B5 Gamborg’s B5 media
BA 6- Benzyl amino purine
DNSA Di-nitro salicylic acid
D/W Distilled water
Introduction
The evolving commercial importance of the second-
ary metabolites has in recent years resulted in a great
interest to alter the production of bioactive plant
metabolites by means of cell culture technology,
which can provide continuous large-scale and reliable
source of plant pharmaceuticals (Mulabagal and
Hsin-Sheng 2004). Carotenoids are a group of
pigments with colours varying from red to yellow
and the most common carotenoid is the yellow-
orange pigment of the carrot (Daucus carota), the
b-carotene. It is a typical unoxygenated carotenoid,
which is present in almost all plant tissues, especially
in carrots. It has special importance in the mamma-
lian diet in that it is converted by the liver to retinol,
which is subsequently oxidized to retinal (vitamin A),
the prosthetic group of optical receptor protein opsin.
The supplementation of b-carotene significantly
reduces the progression of cardiovascular disease,
certain type of cancers, risk of cataracts and light
sensitivity disorders, and enhances immune markers
in HIV infected patients (Cinar 2004).
The single dimensional approach such as that used
in ‘‘one factor-at-a-time’’ is laborious and time
123
124
consuming, especially for large number of variables,
and frequently does not guarantee the determination
of optimal conditions (Xu et al. 2003). These
limitations of a single factor optimization process
can be eliminated by optimizing all the affecting
parameters collectively by statistical experimental
design using response surface methodology (RSM).
RSM can be used to evaluate the relative significance
of several affecting factors even in the presence of
complex interactions. Combinatorial interactions of
medium components with the cell metabolism and
the production of the desired compound are numerous
and the optimum processes may be developed using
an effective experimental design procedure. The
application of statistical techniques in fermentation
process development can result in improved product
yields, reduced process variability, closer confirma-
tion of the output response (product yield or
productivity) to nominal and target requirements
and reduced development and overall costs (Rao et al.
2000).
Central composite design is the most widely used
response surface design. Although rotatability is a
desirable property of a central composite design
where there is a difficulty in extending the star points
beyond the experimental region defined by the upper
and lower limits of each factor, a face-centered
design can be used (Tsapatsaris and Kotzekidou
2004).
To the best of our knowledge there are no reports
documenting use of response surface methodology
for the optimization of media components in suspen-
sion culture for the production of b-carotene. The
concentrations of media components were optimized
for the maximum production of b-carotene.
Materials and methods
Materials
Benzyl adenine, kinetin, 2,4-dichlorophenoxy acetic
acid, agar, di-sodium EDTA were procured from
HIMEDIA Laboratories Pvt. Ltd. Mumbai. CaCl2

2H2O, MgSO4
7H2O, (NH4)2
SO4, Ca(NO3)2
4H2O,
NaH2PO4
H2O, MnSO4
H2O, CuSO4
5H2O, Fe2S-
O4
7H2O, ZnSO4
7H2O, KI, KH2PO4, MgSO4

7H2O, FeCl3
6H2O, giberillic acid, inositol, sucrose,
glucose, nicotinic acid, pyridoxine, glycine thiamine,
123
Plant Cell Tiss Organ Cult (2008) 93:123–132
ethanol, butylatedhydroxy toluene were procured
from S. D. Fine chemicals Ltd. Mumbai. KNO3,
HCl, CoCl2
6H2O, NH4NO3, H3BO3, Na2MoO4

2H2O, Na2SO4, sodium potassium tartarate, ammo-
nium molybdate, ascorbic acid, sodium hypochlorite,
benzylkonium chloride, beta-carotene were procured
from E. Merck—Mumbai.
Daucus carota (orange variety, Indam 459) was
obtained from the local market Matunga, Mumbai,
India. Hitachi UV/visible spectrophotometer was
used to estimate b-carotene
Methods
Sterilization of explants
Two types of sterilization techniques were tested for
callus initiation process (CIP). In first technique (A),
carrot pieces were suspended in 1% Teepol (1 h),
washed in running tap water (½ h) and finally with
distilled water. Further work was done in laminar
airflow. They were treated with 70% ethanol (30 s),
soaked in 10% benzylkonium chloride solution
(2 min) and then treated with 100% sodium hypo-
chlorite solution (5 min). To neutralize the effect of
excess alkali they were treated with 0.01 N HCl
(3 min) followed by washing with sterile distilled
water.
In second technique (B), carrot pieces were treated
with 1% Teepol and washed thoroughly with water.
They were subjected to 0.01 N HCl (5 min) treatment
and then with 1% Tween 80 (30 min) and finally
washed with distilled water. Subsequent treatments
were carried in the laminar airflow, wherein; they
were subjected to 70% alcohol (30 s) followed by
distilled water washing (2 min). Further, treatment
with freshly prepared mixture of 0.1% HgCl2 in 1%
Tween 80 was carried out (10 min). Finally, distilled
water washing was given (5 min).
Callus initiation from different parts of root
Roots of D. carota after sterilization were excised
and the inner cambium portion was selected as the
explant. Such explants were excised from both root
head and root tip of the carrot. They were inoculated
on MS and B5 basal media supplemented with
vitamins, and plant growth regulators as 2,
4-D + BA and 2,4-D + Kinetin. The effect of the
Plant Cell Tiss Organ Cult (2008) 93:123–132 125

media was studied in terms of initiation and growth optimum nutrient concentrations, for the production
of the explants. The dry weight (DW) of callus was of b–carotene. A central composite rotatable exper-
measured after a month and b-carotene content was imental design (CCRD) for three independent
estimated spectrophotometrically. variables was used to obtain the combination of
values that optimizes the response within the region
Optimization of plant growth regulators for callus of three dimensional observation spaces, which
initiation, growth and b-carotene production allows one to design a minimal number of experi-
ments. The experiments were designed using the
Sterilized carrot explants were aseptically transferred software, Design Expert Version 6.0.10 trial version
into jars with MS and B5 medium supplemented with (State Ease, Minneapolis, MN). The medium com-
vitamins and varying combinations of plant growth ponents selected for the optimization were sucrose
regulators: 2,4-D + kinetin and 2,4-D + BA. The (carbon source), potassium nitrate and ammonium
media combinations are coded with numbers as sulphate (nitrogen source) and sodium di-hydrogen
shown in Table 1. The dry weight (DW) of callus phosphate (phosphate source). The concentration of
was measured after a month and b-carotene content minor nutrients, vitamins and plant growth hormones
was estimated spectrophotometrically. were kept constant. pH was adjusted to 5.8 ± 0.1.
The different liquid media combinations were auto-
Establishment of cell suspension culture claved and used for inoculation. Callus maintained on
solid B5 1 (B5 + 0.1 2,4-D + 0.1 BA) media was
One month old callus of D. carota grown on solid MS aseptically transferred to liquid B5 1(B5 + 0.1 2,4-
and B5 media were aseptically excised into pieces D + 0.1 BA). The fully grown suspension culture
and transferred into liquid MS and B5 media. The was harvested for b-carotene extraction after 20 days.
cultures were maintained by weekly subculture. Regression analysis was performed on the data
obtained from the design experiments.
Optimization of major nutrients for b-carotene Decoding of the variables was done according to
production in suspension culture of D. carota cells the Eq. 1.
using RSM Xi  Xcp
xi ¼ i ¼ 1; 2; 3; . . .; k ð1Þ
DXi
RSM is an empirical statistical modeling technique
employed for multiple regression analysis using where: xi, dimensionless value of an independent
quantitative data obtained from properly designed variable; Xi, real value of an independent variable;
experiments to solve multivariable equations simul- Xcp, real value of an independent variable at the
taneously (Rao 2000). RSM is used to determine the center point; and DXi, step change of real value of the
variable i corresponding to a variation of a unit for
Table 1 Media MS and B5 with plant growth regulators desig- the dimensionless value of the variable i.
nated with numbers The experiments were carried out at least in
Media B5 Media MS duplicate, which was necessary to estimate the vari-
2,4-D + BA 2,4-D + BA ability of measurements, i.e. the repeatability of the
phenomenon. Replicates at the center of the domain in
(1) 0.1 + 0.1 (9) 0.1 + 0.1
three blocks permit the checking of the absence of bias
(2) 0.1 + 1.0 (10) 0.1 + 1.0
between several sets of experiments. The relationship
(3) 1.0 + 0.1 (11) 1.0 + 0.1
of the independent variables and the response was
(4) 1.0 + 1.0 (12) 1.0 + 1.0
calculated by the second order polynomial equation:
2,4-D + K 2,4-D + K X k X k
Y ¼ b0 þ bi Xi þ bii Xi Xj
(5) 0.1 + 0.1 (13) 0.1 + 0.1
X X i¼1 i¼1
(6) 0.1 + 1.0 (14) 0.1 + 1.0 þ i jbij Xi Xj ð2Þ
(7) 1.0 + 0.1 (15) 1.0 + 0.1 i\j
(8) 1.0 + 1.0 (16) 1.0 + 1.0
Y is the predicted response; b0 a constant; bi the linear

123
126
coefficient; bii the squared coefficient; and bij the
cross-product coefficient, k is number of factors.
The second order polynomial coefficients were
calculated using the software package Design Expert
Version 6.0.10 to estimate the responses of the
dependent variable. Response surface plots were also
obtained using Design Expert Version 6.0.10.
Amongst the nutritional factors, sucrose (or glucose)
may be of special significance in secondary product
formation. Concentrations above 2% w/v have been
found to enhance the yield of different compounds not
only by stimulating cell growth but also by increasing
the rate of product synthesis (Stafford et al. 1986).
Nitrogen is supplied as NH4+ or NO3- or as a
combination of both in most standard culture media
for plant cell culture (Panda et al. 1992). B5 media
contains KNO3 and (NH4)2SO4 as nitrogen source with
a ratio of NH4+:NO3- as 1:12.5. The concentration of
total nitrogen was varied from 8 to 92 mM.
Phosphate: the concentration of phosphate in cell
culture medium is generally kept between 0.05 and
3 mM and has been reported to have a profound
effect on the secondary metabolism of plant cells
(Panda et al. 1992). B5 media contains NaH2PO4
H2O
as the source of phosphate. Its concentration was
varied from 0.16 to 1.84 mM.
Studying growth kinetics
1-month-old callus grown on solid B5 1 media was
aseptically transferred to liquid B5 1 media. B5 1 was
selected as it gave maximum callus growth. 7 flasks were
inoculated with an inoculum size of 0.4 g DW in 50 ml
media. The FW, DW, b-carotene content and sugar
consumption were estimated after every 7 days till day
49. Similar procedure was carried out for inoculum size
of 1 g and 4 g DW to evaluate optimum inoculum size.
The specific growth rate, l (Eq. 3) was calculated
from the slope of the straight line obtained by plotting
ln x vs. time t during exponential growth phase. The
doubling time, td was calculated using Eq. 4:


1 dx
l¼ ð3Þ
x dt
0:693
td ¼ ð4Þ
l a boiling water bath for 10 min. after which they
The overall maximum biomass, maximum
b-carotene production, % increase in biomass and
123
Plant Cell Tiss Organ Cult (2008) 93:123–132
b-carotene were calculated for each inoculum size
and results were compared.
Analytical determination
Estimation of fresh and dry cell weight in cell
suspension culture (Pollard 1984)
Whatmann No.1 filter paper disks (diameter-70 mm)
were placed in the oven (60°C) at least 24 h before
use. The dried filter paper (W1) disk was weighed
directly after removal from the oven (1 disk/sample
aliquot). The disk was placed on Buchner filter
(Diameter-70 mm) under vacuum. The disk was
wetted with distilled water (about 3 ml), under
vacuum for a constant period of time (10 s). The
wet filter (W2) disk was reweighed immediately. The
disk on the filter bed was replaced and positioned on
the filter top centrally. Known volume (20 ml) of
properly mixed culture was filtered. When the cells
appeared ‘‘dry’’, vacuum was allowed to operate, for
the same period of time as above. The wet disk was
reweighed (W3) immediately. The wet disk contain-
ing cells placed in a petri dish were acetone dried for
2 h. After drying weight (W4) of cells was noted
down. Fresh weight was calculated by using the
formula W3 - W2 and dry weight calculated by
using the formula W4 - W1.
Estimation of total sugars in cell suspension culture
Total sugar content of the cell suspension culture was
estimated using DNSA method as developed by
Miller (1959). Samples from the cell suspension
culture were withdrawn after every 7 days for
49 days and estimated for the changes in the sucrose
concentration as follows. About 10 ml of the sus-
pension medium was taken and diluted with water to
50 ml this was then subjected to hydrolysis using
6 ml of 6.34 N HCl at 60°C for 45 min. The solution
was cooled and then neutralized using 40% NaOH.
The volume was then made upto 100 ml. About 1 ml
of this neutralized solution was taken in a test tube to
which 1 ml of DNSA reagent was added (1.6 g
NaOH + 1 g DNSA + 30 g sodium potassium tart-
arate in 100 ml water). The tubes were then placed in
were cooled and 10 ml D/W was added to make up
the volume to 12 ml. The red colored complex
Plant Cell Tiss Organ Cult (2008) 93:123–132
formed by the reaction of nitro group of DNSA with
reducing sugars was read at 540 nm using Hitachi
UV/vis spectrophotometer.
Estimation of sugars in cell suspension culture
The reducing sugars of the suspension culture were
determined by DNSA, a method developed by Miller
(1959). The difference in the values of total sugars
and reducing sugars of the same sample accounted for
the amount of sucrose (Panda et al. 1990).
Estimation of b-carotene
b-carotene was estimated spectrophotometrically
using Hitachi UV/Vis spectrophotometer. Standard
graph of b-carotene was plotted for concentration
varying from 1 to 5 lg/ml. b-carotene extracted in
petroleum ether gives maximum absorbance at
450 nm. Concentration of b-carotene was estimated
using standard graph.
Results and discussion
The sterilization method (A) proved to be more effective
for the callus initiation of D. carota. The reason for
inefficiency of (B) may be due to harsh treatment by
HgCl2. Therefore sterilization method (A) was selected
for the subsequent experiments. The media coded as B5
1 (0.1 2, 4-D + 0.1 BA) gave the maximum cell mass
whereas B5 7 (1.0 2, 4-D + 0.1 K) given the maximum
production of b-carotene.
Optimization of major nutrients using RSM
To examine the combined effect of three different
medium components (independent variables), on
b-carotene production, a central composite factorial
design of 23 = 8, plus 6 centre points and
(2 9 3 = 6) star points leading to a total of
20 experiments were performed. Eq. 5 represents
b-carotene lg=g DW ¼ 13:502 þ ð0:814  SucroseÞ þ ð0:728  NitrogenÞ  ð1:523  Sucrose2 Þ
 ð3:351  Nitrogen2 Þ  ð2:353  Phosphate2 Þ þ ð1:176  Sucrose  PhosphateÞ
 ð1:335  Nitrogen  PhosphateÞ
127
the mathematical model relating the production of
b-carotene with the independent process variables, Xi
and the second order polynomial coefficient for each
term of the equation determined through multiple
regression analysis using the Design Expert. The
experimental and predicted values of yields of
b-carotene are also given in Table 2. The coded
values of independent variables are given in Table 2.
The results were analyzed using ANOVA i.e.
analysis of variance suitable for the experimental
design used and cited in Table 3. The ANOVA of
the quadratic regression model indicates that the
model is significant. The Model F-value of 27.14
implies that the model is significant. Model F-value
is calculated as ratio of mean square regression and
mean square residual. Model P-value (Prob [ F) is
very low (0.0001). This signifies that the model is
significant.
The P values were used as a tool to check the
significance of each of the coefficients, which, in
turn, are necessary to understand the pattern of the
mutual interactions between the test variables. The t
ratio and the corresponding P values, along with the
coefficient estimate, are given in Table 3. The
smaller the magnitude of the P, the more significant
is the corresponding coefficient. Values of P less than
0.0500 indicate model terms are significant. The
coefficient estimates and the corresponding P values
suggests that, among the test variables used in
the study, X1 (sucrose), X2 (nitrogen), X3 (phos-
phate), X1 9 X3 (sucrose 9 phosphate) and X2 9
X3 (nitrogen 9 phosphate) are significant model
terms. Sucrose (P \ 0.0185) has the largest effect
on b-carotene production, followed by nitrogen
(P \ 0.0306). The mutual interaction between
sucrose and phosphate (P \ 0.0111) and nitrogen
and phosphate (P \ 0.0055) were also found to be
important. Other interactions were found to be
insignificant.
The corresponding second-order response model
for Eq. 2 that was found after analysis for the
regression was
ð5Þ
123
128 Plant Cell Tiss Organ Cult (2008) 93:123–132

Table 2 Central composite

Run Media componentsa b-carotene (lg/g DW)
rotatable design (CCRD)
matrix of independent Sucrose (%) Nitrogen (mM) Phosphorus (mM) Experimental Predicted
variables and their
corresponding experimental 1 2 (-1) 25 (-1) 0.5 (-1) 4.76 5.06
and predicted values of 2 4 (1) 25 (-1) 0.5 (-1) 4.00 4.16
b-carotene
3 2 (-1) 75 (1) 0.5 (-1) 10.12 9.01
4 4 (1) 75 (1) 0.5 (-1) 9.48 8.47
5 2 (-1) 25 (-1) 1.5 (1) 4.08 4.57
6 4 (1) 25 (-1) 1.5 (1) 7.77 8.37
7 2 (-1) 75 (1) 1.5 (1) 3.85 3.17
8 4 (1) 75 (1) 1.5 (1) 8.17 7.34
9 1.32 (-1.68) 50 (0) 1 (0) 7.48 7.82
10 4.68 (1.68) 50 (0) 1 (0) 10.17 10.56
11 3 (0) 8 (-1.68) 1 (0) 3.97 2.79
12 3 (0) 92 (1.68) 1 (0) 3.34 5.24
13 3 (0) 50 (0) 0.16 (-1.68) 6.79 7.52
14 3 (0) 50 (0) 1.84 (1.68) 6.16 6.16
15 3 (0) 50 (0) 1 (0) 12.41 13.50
16 3 (0) 50 (0) 1 (0) 13.64 13.50
17 3 (0) 50 (0) 1 (0) 13.74 13.50
18 3 (0) 50 (0) 1 (0) 13.88 13.50
19 3 (0) 50 (0) 1 (0) 13.81 13.50
a
Values in parenthesis are 20 3 (0) 50 (0) 1 (0) 13.63 13.50
coded values of variables

Table 3 Analysis of
Factora Coefficient Sum of Standard d.f.b F value Pc
variance (ANOVA) for the
estimate squares error
experimental results of the
central-composite design Intercept or 13.50 280.23 0.44 9 27.14 \0.0001*
(Quadratic Model) model
X1 0.81 9.05 0.29 1 7.89 0.0185*
X2 0.73 7.26 0.29 1 6.33 0.0306*
X3 -0.41 2.25 0.29 1 1.97 0.1912
X12 -1.52 33.47 0.28 1 29.17 0.0003*
X22 -3.35 161.92 0.28 1 141.14 \0.0001*
a
X1, sucrose; X2, nitrogen; X32 -2.35 79.80 0.28 1 69.56 \0.0001*
X3, phosphate X1 9 X2 0.092 0.067 0.38 1 0.059 0.8137
b
Degree of freedom X1 9 X3 1.18 11.08 0.38 1 9.66 0.0111*
c
* P \ 0.05 are significant, X2 9 X3 -1.34 14.27 0.38 1 12.44 0.0055*
R2 = 0.96

The fit of the model was also expressed by the
coefficient of determination R2, which was found to
be 0.96, indicating that 96.0 % of the variability in
the response could be explained by the model. This is
also evident from the fact that the plot of predicted
versus experimental b-carotene content in Fig. 1 is
close to y = x showing that the prediction of
experimental data is quite satisfactory.
By substituting the corresponding coded concen-
tration levels of the factors into the regression
equation, the maximum predictable response for
b-carotene production was calculated and was exper-
imentally verified. The maximum production of
b-carotene obtained using the optimized medium
was 13.614 lg/g, which is in correlation with the
predicted value of 13.660 lg/g.

123
Plant Cell Tiss Organ Cult (2008) 93:123–132 129

point levels. Graphs are given here to highlight the

roles played by various factors (Fig. 2). From the
central point of the contour plot or from the bump of
the 3D plot the optimal composition of medium
components was identified.
The optimal concentrations for the three compo-
nents as obtained from the maximum point of the
model were calculated to be as 3.25%, 53 and
0.97 mM for sucrose, nitrogen and phosphate,
respectively (Table 4).

Studies in growth kinetics of cell suspension

culture of D. carota

Figure 3 shows the growth profile of carrot cells for

Fig. 1 Predicted Vs experimental values of b-carotene pro-
duction by D. carota cell suspension
Accordingly, three-dimensional graphs were gen-
erated for the pair-wise combination of the three
factors, while keeping the other two at their center
inoculum size of 0.4 g in 50 ml B5 1 media
(inoculum size 8 g DW/l), depicting lag phase
(7 days), log/exponential phase (14 days), stationary
phase (14 days) and death phase (14 days). Rate of
consumption of sucrose is also shown. Initially,
sucrose is converted to glucose and fructose due to
which there is rise in the level of glucose during the

Fig. 2 Surface response

plot for b-carotene
production: (a) Effect of
sucrose and nitrogen when
other variables are held at
zero level. (b) Effect of
sucrose and phosphate when
other variables are held at
zero level. (c) Effect of
nitrogen and phosphate
when other variables are
held at zero level

123
130 Plant Cell Tiss Organ Cult (2008) 93:123–132

Table 4 Optimized medium composition for b-carotene production by D. carota cell suspension
No Media component concentration b-Carotene (lg/g DW)
Sucrose (g/100 ml) Nitrogen (mM) Phosphate (mM)

1a 2 25 1.094 9.631 ± 0.25

2b 3 50 1 13.501 ± 0.48
3c 3.25 53 0.97 13.614 ± 0.37
4d 3.25 53 0.97 13.660 ± 0.54
a
The values before optimization
b
The composition of center points in Table 2
c
The optimized values derived from RSM regression and b-carotene production in this study
d
The predicted optimum values and predicted maximum b-carotene production derived from RSM regression in this study
e
Results are mean ± SD of three determinations

12 inoculum size 8 (g DW/L) 30 inoculum size 20 (g DW/L)

12
β-carotene (µg/g)

10 25
10

β-carotene (µg/g)
Glucose(g/L)
Sucrose(g/L)

8 20
DW (g)

TS(g/L)

DW (g) 8
6 15
6
4 10
4
2 5
2
0 0
0 10 20 30 40 50 0
Time (days) 0 10 20 30 40 50
DW (g) β-carotene (µg/g) Time (days)
TS (g/L) Glucose (g/L)
DW (g) β-carotene (µg/g)
Sucrose (g/L)

Fig. 4 Production profile of b-carotene by cell suspension

Fig. 3 Production profile of b-carotene by cell suspension
culture for inoculum size of 20 g DW/l (DW indicates dry
culture of D. carota (TS is total sugars and DW indicates dry
weight of cells)
weight of cells)

lag phase. During the exponential phase, it is

consumed for the growth of cells and production of
b-carotene. b-carotene production increases on day inoculum size 40 (g DW/L)
16
and reaches to a maximum. This content of cells
β-carotene (µg/g)

decreases when they enter the stationary phase. This 12

DW (g)

may be due to unstability of b-carotene. Table 5

8
shows amount of sucrose consumed for b-carotene
production and growth of cells. This is represented in
4
the form of yield factors (YX/S and YP/S).
0
Table 5 Sucrose consumption, b-carotene production and 0 10 20 30 40 50
yield factors for inoculum size of 8 g DW/l Time (days)
Sucrose Sucrose Max. Max. b- Yield Yield DW (g) β-carotene (µg/g)
(g/l) utilization DCW carotene factor factor
(%) (g/l) (mg/l) YX/S YP/S
Fig. 5 Production profile of b-carotene by cell suspension
20 96.25 153.24 1.566 0.358 0.355 culture for inoculum size of 40 g DW/l (DW indicates dry
weight of cells)

123
Plant Cell Tiss Organ Cult (2008) 93:123–132 131

20 inoculum size 80 (g DW/L) 50 ml media (80 g DW/l) is as shown in Figs. 4, 5,

6 respectively. In all the four cases, b-carotene
β-carotene (µg/g)

16 reaches to a maximum of approximately 10 lg/g

DW during exponential phase and ten declines.
DW (g)

12
This was in accordance with the reports by
8 Shimizu et al. (1979), who reported that carote-
noids were synthesized in the early logarithmic
4 phase and synthesis rate sharply delined as the
0 culture aged.
0 10 20 30 40 50 60 The Fig. 7 shows comparison of growth profile for
Time (days) the different inoculum size. The exponential phase is
DW (g) β-carotene (µg/g) steeper and between shorter time period in the case of
20 g DW/l inoculum size when compared to the other
Fig. 6 Production profile of b-carotene by cell suspension inoculum sizes. The exponential phase for 80 g DW/l
culture for inoculum size of 80 g DW/l (DW indicates dry is very long. The lag period increased with increasing
weight of cells) inoculum size.
Kinetic parameters like specific growth rate (l)
and doubling time (td) were estimated and compar-
The growth pattern and b-carotene production
ison is shown in Table 6. The l and td were found to
for inoculum size 1 g DW/50 ml media (20 g DW/l),
be higher for 20 g DW/l inoculum size. The td
2 g DW/50 ml media (40 g DW/l) and 4 g DW/
increased rapidly from 40 g DW/l inoculum size. An
inoculum size beyond the optimum value led to
18 competence between the cells for intake of nutrients.
Table 6 shows yield factor, YP/X for the four different
15
inoculum sizes.
12
DW (g)

9
6
Conclusion
3
0 Response surface methodology could be used for the
0 10 20 30 40 50 60
Time (days) optimization of media components for the maximum
production of b-carotene. RSM resulted in the
8 g DW/L 20 g DW/L
40 g DW/L 80 g DW/L
maximum production of b-carotene as 13.61 lg/g
DW compared to 9.63 lg/g DW before optimization.
Fig. 7 Comparison of growth profiles of different inoculum Specific growth rate and doubling time were found to
size (DW indicates dry weight of cells) be higher for 20 g DW/l inoculum size.

Table 6 Kinetic parameters for the different inoculum size

Inoculum lmax td Max. cell DW % Increase in Max. b-carotene conc. b-carotene content of cells Yield factors,
(g/l) (day-1) (h) (g/l) DW (mg/l) (lg/g DW) Yp/x

8 0.259 64 153.24 18.155 1.566 (24 days) 10.371 0.817

20 0.277 60 210.8 9.54 2.136 (26 days) 10.831 0.841
40 0.123 135 284.2 6.105 2.340 (26 days) 9.873 0.452
80 0.074 224 328.28 3.1035 2.321 (26 days) 9.83 0.811

123
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