Biochemical Engineering Journal 29 (2006) 62–68

Modeling of azadirachtin production by Azadirachta indica andits use for feed forward optimization studies

Gunjan Prakash, Ashok K. Srivastava ∗

Plant Cell Culture Laboratory, Department of Biochemical Engineering and Biotechnology, Indian Institute of Technology Delhi,Hauz Khas, New Delhi 110016, India

Received 13 October 2004; accepted 22 February 2005

Abstract

Azadirachtin, obtained from Azadirachta indica (neem) has emerged as wonderful natural biopesticide for pest control due to its efficacy,biodegradability and minimum side effects. To overcome constraints associated with its limited, variable and unreliable supply from naturaltrees, plant cell suspension culture of A. indica was developed (obtained from callus of seeds). The kinetics of cell growth/azadirachtinformation and substrate consumption of A. indica suspension culture was established in bioreactor with low shear setric impeller underoptimized culture conditions (statistically optimized independently). The culture featured a growth of 15.5 and 0.05 g/l azadirachtin in 10dfmft1f©

K

1

IAawbctskd

1d

ays of cultivation after which azadirachtin concentration declined. To prevent any inhibition caused by the excess feeding of substrate duringed-batch cultivation, inhibition of the carbon, nitrate and phosphate were separately established. Based on batch kinetics/inhibitory data aathematical model was proposed and model parameters were evaluated. The model was extrapolated to fed-batch cultivation and nutrient

eeding strategy (with respect to carbon, nitrate and phosphate) were computer simulated for a suitable fed-batch cultivation of A. indica cellso enhance growth and product formation. Fed-batch cultivation demonstrated a growth of 20.06 g/l biomass and 0.082 g/l of azadirachtin in4 days of cultivation. A 30% increase in cell growth and 83% increase in azadirachtin production were obtained when a computer simulatedeeding strategy was experimentally implemented in 3 l bioreactor.

2005 Elsevier B.V. All rights reserved.

eywords: Azadirachtin; Growth kinetics; Substrate inhibition; Bioreactor; Mathematical modeling; Fed-batch cultivation

. Introduction

Azadirachta indica commonly known as neem tree is anndian medicinal plant, which grows well in arid regions ofsian and African subcontinent. The seed kernels of neem

re source of Azadirachtin and related Limonoids (AZRL),hich has been well known since ancient times as a potentiopesticide against a variety of insects [1]. This remarkableompound shows an array of effects as antifeedent, oviposi-ion deterrent, molt inhibitor, repellent, growth retardant andterilant [2]. Currently azadirachtin is extracted from seedernels of naturally grown A. indica and its availability isependent upon a reliable supply of quality seeds. However

∗ Corresponding author. Tel.: +91 11 26591010; fax: +91 11 26582282.E-mail address: ashokks@dbeb.iitd.ernet.in (A.K. Srivastava).

in neem tree, seeds are produced once in a year and due tooperational and storage problems only a fraction of theseseeds are used for azadirachtin production. Moreover largevariations in azadirachtin content exist based on the diversegenotype and wider geographical distribution of neem trees[3]. Besides, the adulteration of neem seeds with funguspathogen creates the risk of contamination with carcino-genic compounds during processing which results in qualitycontrol problem in azadirachtin containing formulations. Pro-duction of azadirachtin from plant cell suspension culturecan overcome all these problems and therefore possibility ofazadirachtin production from plant cell culture technology isbeing viewed [4].

Plant cell culture is an useful method for the productionof valuable compounds originating from plant cells howevercommercial production of secondary metabolites are onlypossible when large-scale cultivation in bioreactor is estab-

369-703X/$ – see front matter © 2005 Elsevier B.V. All rights reserved.oi:10.1016/j.bej.2005.02.027

G. Prakash, A.K. Srivastava / Biochemical Engineering Journal 29 (2006) 62–68 63

lished. For process design and scale-up, detailed knowledgeof the kinetics of growth, nutrient uptake and product forma-tion is essential.

Optimization of system for higher production dependscritically on the development of mathematical models, whichcan describe its dynamics reasonably well. Although the pro-duction of azadirachtin has been studied in shake flask byseveral workers [5–8], until now there have been no reports oftheir large-scale bioreactor cultivation, not to mention of theirbioprocess modeling. For the development of large-scale cellcultivation process for efficient production of azadirachtin,batch growth kinetics of A. indica was studied in 3 l stirredtank bioreactor (STR) with low shear setric impeller. Thelimitation and inhibition due to substrates (carbon, nitrate,phosphate) on specific growth rate was studied and quanti-fied by a mathematical model. The model parameters wereidentified by using the original experimental data. The batchmodel was then extrapolated to computer simulate and designoptimum nutrient feeding strategy in fed-batch cultivationfor enhanced biomass and azadirachtin accumulation. Theselected model-based feeding strategy was experimentallyimplemented in 3 l bioreactor to increase the cell biomassand azadirachtin accumulation.

2. Materials and methods

2

kutas8w2pp

2

brmtactigtbd

log phase of culture. Specific growth rate (µ) was calculatedfrom (ln X − ln X0) versus time (t) plot where, X0 is the initialbiomass at the time of inoculation.

2.3. Batch cultivation of A. indica in bioreactor

Batch cultivation of A. indica cells was conducted in a 3 lstirred tank bioreactor with specially designed low shear, set-ric impeller (Applikon Dependable Instruments, The Nether-lands). The optimized values of the medium componentswere used for the study of growth and product formation.Bioreactor was operated at 125 rpm in the dark. The pH of theculture medium was adjusted to 5.8 before autoclaving andcontrolled by automatic addition of 0.5 M aq. NaOH/0.5 Naq. HCl during cultivation by bio-controller (ADI 1030, App-likon Dependable Instruments, The Netherlands). Tempera-ture was controlled at 27 ◦C by circulating chilled water fromchiller unit into the cooling finger of the bioreactor and evap-oration was checked by directly passing 5 ◦C water throughthe exit gas condenser. Dissolved oxygen level was main-tained above 30% saturation throughout the cultivation byadjusting aeration rate between 0.2 and 0.4 vvm. Samples(25 ml) were collected every second day and analyzed forDW, azadirachtin content, residual glucose, nitrate and phos-phate concentrations.

2

3Tstt((dsifldr

2

bdtwtEccaw

.1. Cell culture of A. indica

Suspension culture of A. indica was developed from seedernel callus (developed after vigorous screening) and grownnder statistically optimized (independently) culture condi-ions of glucose: 25.0 g/l, nitrate: 5.7 g/l, phosphate: 0.094 g/lnd inoculum: 5 g/l (DW basis) with minor/vitamins/ironimilar to Murashige and Skoog (MS) medium [9] andmg/l indole butyric acid + 4 mg/l benzyladenine. Cellsere grown in 500 ml shake flask at 125 rpm, 27 ◦C and4 h dark regime. Nitrate was supplied in the form ofotassium nitrate and Phosphate as di-hydrogen potassiumhosphate.

.2. Inhibition studies due to substrates

The effect of varying initial substrate concentrations [car-on (C), nitrate (N) and phosphate (P)] on the specific growthate (µ) of A. indica was investigated in shake flask using opti-ized media as described above. The initial pH was adjusted

o 5.8 before autoclaving. Flasks were incubated at 27 ◦Cnd at 125 rpm for 24 h under dark conditions. The carbononcentration was varied from 25 to 150 g/l, nitrate from 5.7o 21.0 g/l and phosphate from 0.094 to 0.4 g/l to study thenhibitory concentrations of these nutrients on specific cellrowth. Initially 5 g/l cells were inoculated in medium con-aining various concentrations of substrates (C, N and P) andiomass concentration (X) was estimated by measuring thery weight (DW) of the cells at an interval of 12 h during the

.4. Fed-batch cultivation of A. indica

Fed-batch cultivation of A. indica was implemented inl stirred tank bioreactor with a working volume of 2.0 l.he medium composition and culture conditions were keptame as that of batch cultivation. The cultivation was con-inued as batch for initial 8 days. On eighth day of cul-ivation fresh feeding of nutrients containing phosphate1.0 g/l at 0.05 l/day) in MS medium and carbon as glucose500 g/l at 0.05 l/day) was started and continued till 12thay of cultivation. From 13th day feeding of glucose wastopped while nitrate (35.0 g/l) and phosphate (0.5 g/l) feed-ng in MS medium was started at a flow rate of 0.05 l/dayow rate for 2 days. Phosphate and nitrate were fed asi-hydrogen potassium phosphate and potassium nitrate,espectively.

.5. Analytical procedure

For DW estimation, cells were harvested and collectedy centrifugation at 3000 rpm for 15 min and washed withistilled water. The fresh cells were dried at 28 ± 2 ◦Co a constant dry weight. Glucose, nitrate and phosphateere measured spectophotometrically [10–12]. Extrac-

ion of azadirachtin was done as described earlier [7].xtract was thereafter analyzed by high performance liquidhromatography (Agilent technologies HP1100) on C-18olumn (Waters, USA) at a flow rate of 0.5 ml/min withcetonitrile:water (10:90) as mobile phase using 214 nmavelength.

64 G. Prakash, A.K. Srivastava / Biochemical Engineering Journal 29 (2006) 62–68

Fig. 1. Batch kinetics of A. indica in 3 l bioreactor. Symbols: � DW, �azadirachtin, � residual glucose, © residual nitrate, � residual phosphate.

2.6. Quantification of relevant growth and productionparameters

Parameters used for analyzing the efficiency of growthand azadirachtin production in suspension cultures were asfollows:

Average growth rate (QX) :

maximum cell density − initial cell density

(initial cell density)(culture time)(day−1)

Volumetric productivity (QP) :

amount of product

(culture volume)(culture time)[g/(l.d)]

3. Results and discussion

3.1. Batch kinetics of growth and azadirachtinproduction

The batch kinetic profile of DW, azadirachtin, residualglucose, nitrate and phosphate at optimal culture conditionsare shown in Fig. 1 (average values of three independentbw1it

to 4th day and then occurred at a slower rate upto 10th dayof cultivation. This is generally observed in plant cells that,phosphate is removed from the medium during the lag phaseand consumed during the growth phase. The accumulationof dry biomass was dependent on the concentration of glu-cose and phosphate in the medium. Reduction in dry biomasswas observed when culture featured complete consumptionof glucose and phosphate at 10th day of cultivation duringthe growth. Nitrate uptake occurred at a relatively slower ratein comparison to glucose and phosphate and approximately32% of nitrate remained unconsumed by the end of cultivation(14 days). A maximum of 0.05 g/l azadirachtin was obtainedon 10th day of cultivation after which the azadirachtin con-centration demonstrated a decrease (Fig. 1). The produc-tion of azadirachtin mainly followed the growth-associatedkinetics during the major portion of cultivation (4–10 day)in the bioreactor. This was in contradiction to productionof podophyllotoxin (produced by Podophyllum hexandrum)which featured combined growth and non-growth associatedkinetics in bioreactor [13]. The key parameters for growth andproduct formation of A. indica are summarized in Table 1.To the best of our knowledge this is the first report on batchcultivation of A. indica suspension culture in bioreactor forazadirachtin production.

3.2. Development of the mathematical model

3

ub

1

2

tf

µ

TcirSS

TP r azadir

M th (g/l)

BF

ioreactor experiments). A maximum biomass of 15.57 g/l,ith an average growth rate of 0.214 days−1 was obtained on0th day of cultivation. From substrate consumption profilet was observed that glucose and phosphate uptake was rela-ively fast. Phosphate uptake was particularly faster from 0th

able 1erformance of the batch and fed-batch modes of cultivation of A. indica fo

odes of cultivation Cultivation time (day−1) Grow

atch 14 15.52ed-batch 14 20.06

.2.1. Unstructured kinetic modelThe batch kinetics with optimized culture conditions was

sed for the development of unstructured model. Model wasased on the following assumptions:

. Major nutrients; carbon, nitrate and phosphate were thelimiting nutrients while the rest of the minor nutrientswere always available in excess in the culture broth.

. Temperature and pH were constant throughout the culti-vation.

Based on the batch kinetic profile of biomass accumula-ion, product formation and substrate utilization in bioreactor,ollowing model equation was proposed:

= µm

[S1

S1 + KS1

] [S2

S2 + KS2

] [(S3)n

(S3)n + (KS3 )n

](1)

he specific growth rate (µ) was modeled by both Monod (forarbon; S1 and nitrate; S2) and by Sigmoidal growth kinet-cs (for phosphate; S3). �m is the maximum specific growthate. KS1 , KS2 and KS3 are the saturation constant based on1, S2 and S3, respectively. ‘n’ represented the exponent inigmoidal kinetics for phosphate uptake.

achtin production

Azadirachtin (g/l) Qx (day−1) Qp (g/l.d)

0.045 0.151 0.00320.082 0.215 0.0058

G. Prakash, A.K. Srivastava / Biochemical Engineering Journal 29 (2006) 62–68 65

Fig. 2. Effect of increasing carbon concentration on specific growth rate (µ).

3.2.2. Incorporation of substrate inhibition in the kineticmodel

To account for any possible inhibition during feeding infed-batch cultivation, different concentrations of major nutri-ents (C, N and P) were tested for their inhibition level.Figs. 2–4 represents effect of increasing initial glucose,nitrate and phosphate concentrations, respectively, on theculture specific growth rate (µ). From the figures it can beconcluded that culture growth decreased with increasing S1,S2 and S3 concentration. The complete inhibition (at whichµ becomes zero) was found to be at 162.27 g, 26.35 g and0.63 g for S1, S2 and S3, respectively (from inhibition data;Figs. 2–4 and experimentally verified seperately). Based onthe inhibition studies it was observed that following modelcould adequately describe the experimental inhibition pat-tern:

µ = µm

[KI

KI + S

](2)

F

Fig. 4. Effect of increasing phosphate concentration on specific growth rate(µ).

where KI represents the inhibition constant for a particularsubstrate (C, N or phosphate). Combined inhibited growthrate can therefore be described as:

µ = µm

[KI1

KI1 + S1

] [KI2

KI2 + S2

] [KI3

KI3 + S3

](3)

where, KI1 , KI2 and KI3 are the inhibition constants for S1,S2 and S3, respectively. Incorporating kinetic and inhibitionterms in the batch kinetic model, the following equation canbe formulated for specific growth rate:

µ = µm

[S1

S1 + KS1

] [S2

S2 + KS2

] [(S3)n

(S3)n + (KS3 )n

]

×[

KI1

KI1 + S1

] [KI2

KI2 + S2

] [KI3

KI3 + S3

](4)

The specific product formation rate, qp, was considered tobe containing both growth and non-growth associated terms.Following equation was used for specific rate of azadirachtinformation:

qp = 1

X

dP

dt= K1µ + K2 (5)

where, K1, K2 represent the growth associated and non-growth associated product formation constants, respectively,for azadirachtin production.

tnnor

q

wn

ig. 3. Effect of increasing nitrate concentration on specific growth rate (µ).

The specific substrate consumption rate (qS) was assumedo be due to growth of cells, formation of product and mainte-ance coefficient, which features substrate consumption foron-growth-related activities of the plant cells, e.g. transportf nutrient to the cells, etc. The specific glucose consumptionate can be represented by the following equation:

S1 = −[

1

YX/S1

(dX

dt

)+ 1

YP/S1

(dP

dt

)+ mS1

](6)

here, YX/S1 and mS1 are the yield of biomass and mainte-ance coefficient due to S1, respectively.

66 G. Prakash, A.K. Srivastava / Biochemical Engineering Journal 29 (2006) 62–68

From Eqs. (5) and (6), it follows:

−dS1

dt=

(1

YX/S1

+ K1

YP/S

)dX

dt+

(K2

YP/S

+ mS1

)X (7)

Thus,

1

X

dS1

dt= −(αµ + β) (8)

Where,

α = 1

YX/S1

+ K1

YP/S

(9)

β = K2

YP/S

+ mS1 (10)

Specific substrate consumption rate equations formulatedfor S2(qS2 ) and S3(qS3 ) are represented by the following equa-tion:

qS2 = −[

1

YX/S2

µ + mS2

](11)

qS3 = −[

1

YX/S3

µ + mS3

](12)

where, YX/S2 and YX/S3 were the yield of biomass while mS2

and mS3 were the maintenance coefficient due to nitrate andp

3p

r[mcegOmmR

S

wrdwvea

wplr

Table 2Model parameters for Batch Cultivation

Parameters Units Value

µm (day−1) 0.65Ks1 (g/l) 17.66Ks2 (g/l) 1.04Ks3 (g/l) 0.14E-5n Dimensionless 0.3611KI1 (g/l) 162.27KI2 (g/l) 26.35KI3 (g/l) 0.63K1 (g/g) 0.003695K2 (g/g) 0.000107� (g/g) 2.22� (g/g/d) 0.0272YX/S2 (g/g) 0.0588YX/S3 (g/g) 0.0079mS2 (g/g/d) 0.023mS3 (g/g/d) 0.00011

formation and substrate utilization were well described by theunstructured kinetic model.

3.3. Extrapolation of batch mathematical model tofed-batch

In order to design a suitable nutrient feeding strategy,the batch model was extrapolated to fed-batch cultivationby incorporating the dilution terms D. The fed-batch modelequations are summarized below:

F = F1 + F2 + F3 (14)

dX

dt= µX −

[F

VX

](15)

dS1

dt= −[αµ + β]X +

[F1S01

V

]−

[FS1

V

](16)

F(Dp

hosphate, respectively.

.2.3. Evaluation and optimization of the modelarameters

For optimal estimation of model parameters, a non-linearegression technique [14] assisted by a computer program15,16] was used to minimize the deviation between theodel predictions and batch experimental data. For the cal-

ulation of the model predictions, the system of differentialquations of the model was solved using an integration pro-ram based on the Runge-Kutta method of fourth order.riginal method of Rosenbrock [17] was used in the opti-ization program for the direct search of the minimum of aultivariable function. The minimization criteria used in theosenbrock computer algorithm was as follows:

SWR =n∑

i=1

m∑j=1

∆2ij

W2j

(13)

here, SSWR represents sum of the square of weighedesidues, n and m represent the total number of experimentalata points and total number of variable, respectively, Wj, theeight of each variable (usually the maximum value of eachariable) and ∆ij is the difference between the model andxperimental value. The values of the optimized parametersre given in Table 2.

The model equations described above in Eqs. (1)–(12)ere fitted by computer using the optimal values of the modelarameters. A comparison between the model fitted (smoothine) and experimental data (points) is shown in Fig. 5. As theesults shows, the kinetic profiles of cell growth, azadirachtin

ig. 5. Batch kinetics of A. indica. Comparison of the experimental datapoints) and model-based simulation results (smooth lines). Symbols: �W, � azadirachtin, � residual glucose, © residual nitrate, � residualhosphate.

G. Prakash, A.K. Srivastava / Biochemical Engineering Journal 29 (2006) 62–68 67

dS2

dt= −

[µX

YX/S2

+ mS2X

]+

[F2S02

V

]−

[FS2

V

](17)

dS3

dt= −

[µX

Yx/S3

+ mS3X

]+

[F3S03

V

]−

[FS3

V

](18)

dP

dt= (K1µ + K2)X − F

VP (19)

dV

dt= F (20)

where, F is the total flow rate (l/day), V, the volume (liter), F1,the glucose flow rate, F2, the nitrate feed rate, F3, the phos-phate feed rate (all in liter/day). S01 , S02 and S03 are the initialglucose, nitrate and phosphate concentration, respectively.Although the product was intracellular in nature, dilutionterm was used for product formation also, with the assump-tion that the intracellular product will get diluted due togrowth of the cells. The fed-batch was started as a batch,which continued until eighth day during which the batchmodel equations were simulated on the computers.

3.4. Nutrient feeding strategy in fed-batch cultivation

Nutrient feeding strategy was designed to eliminate sub-strate limitations at different times in the vigorously growingpptomdaitcD8di0pcgldbbtrtwtbwt

Fig. 6. Fed-batch cultivation with glucose, phosphate and nitrate feeding.Comparison of experimental data (points) and model predicted (smoothlines) values. Symbols: � DW, � azadirachtin, � residual glucose, © resid-ual nitrate, � residual phosphate.

increasing the volume of reactor. From computer simulation,it was observed that this fed-batch cultivation resulted in highbiomass (22.25 g/l) and azadirachtin concentration (0.09 g/l)on 14th day, which was significantly higher than batch culti-vation.

3.5. Model validation

As mentioned above, an unstructured kinetic model wasdeveloped and tested to describe the kinetics of suspensionculture of A. indica in a 3 l stirred tank bioreactor. To fur-ther establish the validity of model simulations the batchmodel was extrapolated to fed-batch cultivation, which wasexperimentally used for design of nutrient feeding strate-gies for C, N and P as described above. Fig. 6 representsthe experimental and model-simulated profiles of growth,azadirachtin formation and substrate(s) consumption duringthe fed-batch cultivation of A. indica. The observed exper-imental fed-batch kinetic profile exhibited similar trendsthroughout the cultivation process with minor variations ascompared to model. In batch cultivation, azadirachtin accu-mulation was decreased after 10 days while in fed-batchcultivation the azadirachtin increase continued even after 10days presumably due to availability of limiting nutrients.Biomass concentration of 20.06 g/l as compared to modelprediction of 22.25 g/l and 0.082 g/l azadirachtin as opposedtboaaf

an

lant cell population in bioreactor. The normal trial and errorrocedure (usually followed) were thus eliminated by usinghe feed forward model simulation under different fed-batchperating conditions. Several offline computer simulations ofathematical model for fed-batch cultivation were done to

evelop nutrient feeding strategies for limiting nutrients (C, Nnd P). From the offline simulation and batch kinetics (Fig. 1),t was observed that till 10th day nitrate was still present inhe medium but all available glucose and phosphate wereonsumed which probably led to no further enhancement inW and a decline in azadirachtin formation. Therefore tillth day bioreactor was operated in the batch mode. From 8thay onwards fresh feeding of nutrients containing phosphaten MS medium (1.0 g/l at 0.05 l/day) and glucose (500 g/l at.05 l/day) was started to increase the available glucose andhosphate levels for the growing plant cells. Feeding wasontinued till 12th day, which featured large accumulation oflucose and phosphate. Due to the availability of these twoimiting nutrients, significant increase in biomass was pre-icted by the model (up to 19 g/l), however this increase iniomass led to the consumption of available nitrate, whichecame limiting on the 12th day of cultivation. To overcomehis limitation, nitrate feeding (35.0 g/l) at 0.05 l/day flowate was started with continued phosphate feeding (0.5 g/l) athe same flow rate as earlier from 13th day. Glucose feedingas discontinued during this period so that all the glucose

hat may have accumulated during the initial feeding coulde consumed. Substrate concentrations (in the inlet feed)ere particularly taken higher so that the substrate concentra-

ion in the bioreactor can be enhanced without significantly

o model-simulated 0.09 g/l was obatined in 14 days of fed-atch cultivation. Performances of batch and fed-batch modesf cultivation in terms of growth and production parametersre shown in Table 1. Cell growth was enhanced by 30%nd production of azadirachtin was increased by 83% in theed-batch cultivation.

In batch cultivation, limiting nutrients are consumedfter certain period of time; fresh nutrient supply is usuallyecessary for maintenance of high cell growth and product

68 G. Prakash, A.K. Srivastava / Biochemical Engineering Journal 29 (2006) 62–68

formation rates. The fresh nutrient feeding (fed-batch plantcell cultivation) has been attempted (although not model-based) for other plant cell suspension cultures [18–20].Model-based limiting nutrient feeding in fed-batch cul-tivation is more logical and it minimizes the number ofexperiments to achieve more biomass and product forma-tion. This methodology is particularly useful in plant cellcultivations, which are invariably very time consuming. A36% increase in volumetric productivity of podophyllotoxin,an anti-cancer drug was also obtained by model-based fed-batch cultivation in Podophyllum hexandrum cell suspensionculture in bioreactor in the author’s laboratory [21].

4. Conclusions

Submerged cultivation of A. indica was carried out in 3 lstirred tank bioreactor. It featured 15.5 g/l of biomass and0.05 g/l of azadirachtin. A simple unstructured mathematicalmodel was developed on the basis of batch kinetic/inhibitorydata. The model could predict cell growth, azadirachtin for-mation and substrate(s) consumption in suspension cultureof A. indica. The model was extrapolated for fed-batch cul-tivation and it was extremely helpful for the developmentof suitable nutrient feeding strategy to overcome the limita-tion of nutrients, which occurred during the batch cultivation.F2tisdc

A

tP

R

[2] A.J. Mordue, A. Blackwell, Azadirachtin: an update, J Insect Physiol.39 (1993) 903–924.

[3] O.P. Sidhu, H.M. Behl, Seasonal variation in azadirachtin in seedsof Azadirachta indica, Curr. Sci. 70 (1996) 1084–1085.

[4] G. Prakash, S.S. Bhojwani, A.K. Srivastava, Production ofazadirachtin from plant tissue culture-state of the art and futureprospects, Biotechnol. Bioprocess. Eng. 7 (2002) 185–193.

[5] E.J. Allan, A.A. Easwara, A. Wood, The production of azadirachtinby in vitro tissue culture of neem Azadirachta indica, Pestic. Sci.42 (1994) 147–152.

[6] T. Kuruvilla, P. Komaraiah, S.V. Ramakrishna, Enhanced secretionof azadirachtin by permeabilized margosa (Azadirachta indica) cells,Ind. J. Exp. Bot. 37 (1999) 89–91.

[7] K.N. Raval, S. Hellwing, G. Prakash, R.A. Plasencis, A.K. Srivas-tava, J. Buchs, Necessity of a two stage process for the production ofazadirachtin-related limonoids in suspension culture of Azadirachtaindica, J. Biosci. Bioeng. 96 (2003) 16–22.

[8] K. Balaji, C. Veeresham, K. Srisilam, C.K. Kokate, Azadirachtin, anovel biopesticide from cell cultures of Azadirachta indica, J. PlantBiotechnol. 5 (2003) 121–129.

[9] T. Murashige, F. Skoog, A revised medium for rapid growth andbioassays with tobacco tissue culture, Physiol. Plant 15 (1962)473–497.

[10] G.M. Miller, Use of dinitrosalicylic acid reagent for determinationof reducing sugar, Anal. Chem. 31 (1959) 426–428.

[11] D.A. Cataldo, M. Haroon, L.E. Schrader, V.L. Youngs, Rapid col-orimetric determination of nitrate in plant tissues by nitration ofsalicylic acid, Commun.Soil Sci. Plant Anal. 6 (1975) 71–80.

[12] J. Murphy, J.P. Riley, A modified single solution method for determi-nation of phosphate in natural systems, Anal. Chim. Acta 27 (1962)31–36.

[

[

[

[

[

[

[

[

[

ed-batch cultivation of A. indica resulted in production of0.06 g/l biomass and 0.082 g/l of azadirachtin as comparedo 15.5 g/l biomass and 0.045 g/l azadirachtin, respectively,n batch after 14 days. The model identified in the presenttudy is system independent and may be used for processevelopment and optimization of other plant cell suspensionultures as well.

cknowledgement

Fellowship provided by Council of Scientific and Indus-rial Research (CSIR), India to one of the authors (Ms. Gunjanrakash) is gratefully acknowledged.

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