Plant Cell Tiss Organ Cult (2008) 93:123132 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 S. A. Survase S. K. Sawant U. S. Annapure

Received: 18 July 2007 / Accepted: 11 February 2008 / Published online: 5 March 2008 Springer Science+Business Media B.V. 2008

Abstract Plants are a valuable source of a vast array of chemical compounds including fragrances, avours, 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 suspension culture has been carried out to understand the relationship between growth and b-carotene formation. The maximum production of b-carotene obtained using the optimized medium was 13.614 lg/g dry weight cell mass. The l (specic 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

MS B5 BA DNSA D/W

Murashige and Skoog Gamborgs B5 media 6- Benzyl amino purine Di-nitro salicylic acid Distilled water

Introduction The evolving commercial importance of the secondary 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 yelloworange 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 mammalian 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 signicantly 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

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

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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 signicance 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 conrmation 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 difculty in extending the star points beyond the experimental region dened 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 suspension culture for the production of b-carotene. The concentrations of media components were optimized for the maximum production of b-carotene.

ethanol, butylatedhydroxy toluene were procured from S. D. Fine chemicals Ltd. Mumbai. KNO3, HCl, CoCl26H2O, NH4NO3, H3BO3, Na2MoO4 2H2O, Na2SO4, sodium potassium tartarate, ammonium molybdate, ascorbic acid, sodium hypochlorite, benzylkonium chloride, beta-carotene were procured from E. MerckMumbai. 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 rst technique (A), carrot pieces were suspended in 1% Teepol (1 h), washed in running tap water ( h) and nally with distilled water. Further work was done in laminar airow. They were treated with 70% ethanol (30 s), soaked in 10% benzylkonium chloride solution (2 min) and then treated with 100% sodium hypochlorite 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 nally washed with distilled water. Subsequent treatments were carried in the laminar airow, 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

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, MgSO47H2O, (NH4)2SO4, Ca(NO3)24H2O, NaH2PO4H2O, MnSO4H2O, CuSO45H2O, Fe2SO47H2O, ZnSO47H2O, KI, KH2PO4, MgSO4 7H2O, FeCl36H2O, giberillic acid, inositol, sucrose, glucose, nicotinic acid, pyridoxine, glycine thiamine,

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

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media was studied in terms of initiation and growth of the explants. The dry weight (DW) of callus was measured after a month and b-carotene content was estimated spectrophotometrically. Optimization of plant growth regulators for callus initiation, growth and b-carotene production Sterilized carrot explants were aseptically transferred into jars with MS and B5 medium supplemented with vitamins and varying combinations of plant growth regulators: 2,4-D + kinetin and 2,4-D + BA. The media combinations are coded with numbers as shown in Table 1. The dry weight (DW) of callus was measured after a month and b-carotene content was estimated spectrophotometrically. Establishment of cell suspension culture One month old callus of D. carota grown on solid MS and B5 media were aseptically excised into pieces and transferred into liquid MS and B5 media. The cultures were maintained by weekly subculture. Optimization of major nutrients for b-carotene production in suspension culture of D. carota cells using RSM RSM is an empirical statistical modeling technique employed for multiple regression analysis using quantitative data obtained from properly designed experiments to solve multivariable equations simultaneously (Rao 2000). RSM is used to determine the
Table 1 Media MS and B5 with plant growth regulators designated with numbers Media B5 2,4-D + BA (1) (2) (3) (4) 0.1 + 0.1 0.1 + 1.0 1.0 + 0.1 1.0 + 1.0 2,4-D + K (5) (6) (7) (8) 0.1 + 0.1 0.1 + 1.0 1.0 + 0.1 1.0 + 1.0 (13) (14) (15) (16) (9) (10) (11) (12) Media MS 2,4-D + BA 0.1 + 0.1 0.1 + 1.0 1.0 + 0.1 1.0 + 1.0 2,4-D + K 0.1 + 0.1 0.1 + 1.0 1.0 + 0.1 1.0 + 1.0

optimum nutrient concentrations, for the production of bcarotene. A central composite rotatable experimental design (CCRD) for three independent variables was used to obtain the combination of values that optimizes the response within the region of three dimensional observation spaces, which allows one to design a minimal number of experiments. The experiments were designed using the software, Design Expert Version 6.0.10 trial version (State Ease, Minneapolis, MN). The medium components selected for the optimization were sucrose (carbon source), potassium nitrate and ammonium sulphate (nitrogen source) and sodium di-hydrogen phosphate (phosphate source). The concentration of minor nutrients, vitamins and plant growth hormones were kept constant. pH was adjusted to 5.8 0.1. The different liquid media combinations were autoclaved and used for inoculation. Callus maintained on solid B5 1 (B5 + 0.1 2,4-D + 0.1 BA) media was aseptically transferred to liquid B5 1(B5 + 0.1 2,4D + 0.1 BA). The fully grown suspension culture was harvested for b-carotene extraction after 20 days. Regression analysis was performed on the data obtained from the design experiments. Decoding of the variables was done according to the Eq. 1. xi Xi Xcp DXi i 1; 2; 3; . . .; k 1

where: xi, dimensionless value of an independent variable; Xi, real value of an independent variable; Xcp, real value of an independent variable at the center point; and DXi, step change of real value of the variable i corresponding to a variation of a unit for the dimensionless value of the variable i. The experiments were carried out at least in duplicate, which was necessary to estimate the variability of measurements, i.e. the repeatability of the phenomenon. Replicates at the center of the domain in three blocks permit the checking of the absence of bias between several sets of experiments. The relationship of the independent variables and the response was calculated by the second order polynomial equation: k k X X Y b0 bi Xi bii Xi Xj i1 i1 X X i jbij Xi Xj 2
i\j

Y is the predicted response; b0 a constant; bi the linear

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coefcient; bii the squared coefcient; and bij the cross-product coefcient, k is number of factors. The second order polynomial coefcients 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 signicance 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 NaH2PO4H2O 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 asks 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 specic 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 l 3 x dt td 0:693 l 4

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 lter paper disks (diameter-70 mm) were placed in the oven (60C) at least 24 h before use. The dried lter paper (W1) disk was weighed directly after removal from the oven (1 disk/sample aliquot). The disk was placed on Buchner lter (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 lter (W2) disk was reweighed immediately. The disk on the lter bed was replaced and positioned on the lter top centrally. Known volume (20 ml) of properly mixed culture was ltered. 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 containing 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 suspension 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 60C 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 tartarate in 100 ml water). The tubes were then placed in a boiling water bath for 10 min. after which they were cooled and 10 ml D/W was added to make up the volume to 12 ml. The red colored complex

The overall maximum biomass, maximum b-carotene production, % increase in biomass and

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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 inefciency 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

the mathematical model relating the production of b-carotene with the independent process variables, Xi and the second order polynomial coefcient 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 signicant. The Model F-value of 27.14 implies that the model is signicant. 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 signies that the model is signicant. The P values were used as a tool to check the signicance of each of the coefcients, 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 coefcient estimate, are given in Table 3. The smaller the magnitude of the P, the more signicant is the corresponding coefcient. Values of P less than 0.0500 indicate model terms are signicant. The coefcient estimates and the corresponding P values suggests that, among the test variables used in the study, X1 (sucrose), X2 (nitrogen), X3 (phosphate), X1 9 X3 (sucrose 9 phosphate) and X2 9 X3 (nitrogen 9 phosphate) are signicant 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 insignicant. The corresponding second-order response model for Eq. 2 that was found after analysis for the regression was

b-carotene lg=g DW 13:502 0:814 Sucrose 0:728 Nitrogen 1:523 Sucrose2 3:351 Nitrogen2 2:353 Phosphate2 1:176 Sucrose Phosphate 1:335 Nitrogen Phosphate 5

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128 Table 2 Central composite rotatable design (CCRD) matrix of independent variables and their corresponding experimental and predicted values of b-carotene Media componentsa Sucrose (%) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
a

Plant Cell Tiss Organ Cult (2008) 93:123132

Run

b-carotene (lg/g DW) Phosphorus (mM) 0.5 (-1) 0.5 (-1) 0.5 (-1) 0.5 (-1) 1.5 (1) 1.5 (1) 1.5 (1) 1.5 (1) 1 (0) 1 (0) 1 (0) 1 (0) 0.16 (-1.68) 1.84 (1.68) 1 (0) 1 (0) 1 (0) 1 (0) 1 (0) 1 (0) Experimental 4.76 4.00 10.12 9.48 4.08 7.77 3.85 8.17 7.48 10.17 3.97 3.34 6.79 6.16 12.41 13.64 13.74 13.88 13.81 13.63 Predicted 5.06 4.16 9.01 8.47 4.57 8.37 3.17 7.34 7.82 10.56 2.79 5.24 7.52 6.16 13.50 13.50 13.50 13.50 13.50 13.50

Nitrogen (mM) 25 (-1) 25 (-1) 75 (1) 75 (1) 25 (-1) 25 (-1) 75 (1) 75 (1) 50 (0) 50 (0) 8 (-1.68) 92 (1.68) 50 (0) 50 (0) 50 (0) 50 (0) 50 (0) 50 (0) 50 (0) 50 (0)

2 (-1) 4 (1) 2 (-1) 4 (1) 2 (-1) 4 (1) 2 (-1) 4 (1) 1.32 (-1.68) 4.68 (1.68) 3 (0) 3 (0) 3 (0) 3 (0) 3 (0) 3 (0) 3 (0) 3 (0) 3 (0) 3 (0)

Values in parenthesis are coded values of variables

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Table 3 Analysis of variance (ANOVA) for the experimental results of the central-composite design (Quadratic Model)

Factora

Coefcient estimate 13.50 0.81 0.73 -0.41 -1.52 -3.35 -2.35 0.092 1.18 -1.34

Sum of squares 280.23 9.05 7.26 2.25 33.47 161.92 79.80 0.067 11.08 14.27

Standard error 0.44 0.29 0.29 0.29 0.28 0.28 0.28 0.38 0.38 0.38

d.f.b F value Pc \0.0001* 0.0185* 0.0306* 0.1912 0.0003* \0.0001* \0.0001* 0.8137 0.0111* 0.0055*

Intercept or model X1 X2 X3 X12 X22

9 1 1 1 1 1 1 1 1 1

27.14 7.89 6.33 1.97 29.17 141.14 69.56 0.059 9.66 12.44

X1, sucrose; X2, nitrogen; X3, phosphate

b c

X32 X1 9 X2 X1 9 X3 X2 9 X3

Degree of freedom

* P \ 0.05 are signicant, R2 = 0.96

The t of the model was also expressed by the coefcient 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 concentration levels of the factors into the regression equation, the maximum predictable response for b-carotene production was calculated and was experimentally veried. 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.

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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 identied. The optimal concentrations for the three components 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 prole of carrot cells for 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. 1 Predicted Vs experimental values of b-carotene production by D. carota cell suspension

Accordingly, three-dimensional graphs were generated for the pair-wise combination of the three factors, while keeping the other two at their center

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

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Table 4 Optimized medium composition for b-carotene production by D. carota cell suspension No Media component concentration Sucrose (g/100 ml) 1a 2b 3c 4d
a b c d e

b-Carotene (lg/g DW) Nitrogen (mM) 25 50 53 53 Phosphate (mM) 1.094 1 0.97 0.97 9.631 0.25 13.501 0.48 13.614 0.37 13.660 0.54

2 3 3.25 3.25

The values before optimization The composition of center points in Table 2 The optimized values derived from RSM regression and b-carotene production in this study The predicted optimum values and predicted maximum b-carotene production derived from RSM regression in this study Results are mean SD of three determinations

12

inoculum size 8 (g DW/L)

30

12

inoculum size 20 (g DW/L)

DW (g) -carotene (g/g)

8 6 4 2 0 0 10 20 30 40 50

20 15 10 5 0

DW (g) -carotene (g/g)

TS(g/L) Glucose(g/L) Sucrose(g/L)

10

25

10 8 6 4 2 0 0 10 20 30 40 50

Time (days)
DW (g) TS (g/L) Sucrose (g/L) -carotene (g/g) Glucose (g/L)

Time (days)
DW (g) -carotene (g/g)

Fig. 3 Production prole of b-carotene by cell suspension culture of D. carota (TS is total sugars and DW indicates dry weight of cells)

Fig. 4 Production prole of b-carotene by cell suspension culture for inoculum size of 20 g DW/l (DW indicates dry weight of cells)

DW (g) -carotene (g/g)

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 and reaches to a maximum. This content of cells decreases when they enter the stationary phase. This may be due to unstability of b-carotene. Table 5 shows amount of sucrose consumed for b-carotene production and growth of cells. This is represented in the form of yield factors (YX/S and YP/S).
Table 5 Sucrose consumption, b-carotene production and yield factors for inoculum size of 8 g DW/l Sucrose (g/l) Sucrose utilization (%) 96.25 Max. DCW (g/l) 153.24 Max. bcarotene (mg/l) 1.566 Yield factor YX/S 0.358 Yield factor YP/S 0.355

16 12 8 4 0 0

inoculum size 40 (g DW/L)

10

20

30

40

50

Time (days)
DW (g) -carotene (g/g)

20

Fig. 5 Production prole of b-carotene by cell suspension culture for inoculum size of 40 g DW/l (DW indicates dry weight of cells)

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20

inoculum size 80 (g DW/L)

16 12 8 4 0 0 10 20 30 40 50 60

Time (days)
DW (g) -carotene (g/g)

Fig. 6 Production prole of b-carotene by cell suspension culture for inoculum size of 80 g DW/l (DW indicates dry weight of cells)

The growth pattern and b-carotene production for inoculum size 1 g DW/50 ml media (20 g DW/l), 2 g DW/50 ml media (40 g DW/l) and 4 g DW/

18 15

50 ml media (80 g DW/l) is as shown in Figs. 4, 5, 6 respectively. In all the four cases, b-carotene reaches to a maximum of approximately 10 lg/g DW during exponential phase and ten declines. This was in accordance with the reports by Shimizu et al. (1979), who reported that carotenoids were synthesized in the early logarithmic phase and synthesis rate sharply delined as the culture aged. The Fig. 7 shows comparison of growth prole for the different inoculum size. The exponential phase is steeper and between shorter time period in the case of 20 g DW/l inoculum size when compared to the other inoculum sizes. The exponential phase for 80 g DW/l is very long. The lag period increased with increasing inoculum size. Kinetic parameters like specic growth rate (l) and doubling time (td) were estimated and comparison is shown in Table 6. The l and td were found to be higher for 20 g DW/l inoculum size. The td increased rapidly from 40 g DW/l inoculum size. An inoculum size beyond the optimum value led to competence between the cells for intake of nutrients. Table 6 shows yield factor, YP/X for the four different inoculum sizes.

DW (g)

DW (g) -carotene (g/g)

12 9 6 3 0

Conclusion
0 10 20 30 40 50 60

Time (days)
8 g DW/L 40 g DW/L 20 g DW/L 80 g DW/L

Fig. 7 Comparison of growth proles of different inoculum size (DW indicates dry weight of cells)

Response surface methodology could be used for the optimization of media components for the maximum production of b-carotene. RSM resulted in the maximum production of b-carotene as 13.61 lg/g DW compared to 9.63 lg/g DW before optimization. Specic growth rate and doubling time were found to be higher for 20 g DW/l inoculum size.

Table 6 Kinetic parameters for the different inoculum size Inoculum (g/l) 8 20 40 80 lmax (day-1) 0.259 0.277 0.123 0.074 td (h) Max. cell DW % Increase in Max. b-carotene conc. b-carotene content of cells (g/l) DW (mg/l) (lg/g DW) 18.155 9.54 6.105 3.1035 1.566 (24 days) 2.136 (26 days) 2.340 (26 days) 2.321 (26 days) 10.371 10.831 9.873 9.83 Yield factors, Yp/x 0.817 0.841 0.452 0.811

64 153.24 60 210.8 135 284.2 224 328.28

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Plant Cell Tiss Organ Cult (2008) 93:123132 Rao JK, Chul-Ho K, Sang-Ki R (2000) Statistical optimization of medium for the production of recombinant hirudin from Saccharomyces cerevisiae using response surface methodology. Process Biochem 35:639647 Shimizu K, Kikuchi T, Sugano N Nishi A (1979) Carotenoid and steroid synthesis by carrot cells in suspension culture. Physiol Plant 46:127132 Stafford A, Morris P, Fowler MW (1986) Plant cell biotechnologya perspectiv. Enzyme Microb Technol 8:578587 Tsapatsaris S, Kotzekidou P (2004) Application of central composite design and response surface methodology to the fermentation of olive juice by Lactobacillus plantarum and Debaryomyces hansenii. Int J Food Microbiol 95:157168 Xu CP, Kim SW, Hwang HJ, Choi JW, Yun JW (2003) Optimization of submerged culture conditions for mycelial growth and exobiopolymer production by Paecilomyces tenuipes C240. Process Biochem 38:10251030

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