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Biochemical Engineering Journal 40 (2008) 218–226

Statistical elicitor optimization studies for the enhancement of azadirachtinproduction in bioreactor Azadirachta indica cell cultivation

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 8 May 2006; received in revised form 7 December 2007; accepted 18 December 2007

bstract

Suspension culture of Azadirachta indica produces the biopesticide azadirachtin. Some elicitors (salicylic acid, chitosan, jasmonic acid, methylasmonate, yeast extract and yeast extract carbohydrate fraction) at different concentrations were added in shake flask suspension culture of A.ndica. Chitosan, salicylic acid and jasmonic acid stimulated the highest increase in azadirachtin content, which ranged from 2 to 3-fold greaterhan the control. The combined effect of these elicitor(s) on azadirachtin content was then studied by Response Surface Methodology. A synergisticffect of these elicitor(s) on azadirachtin production resulted in 5-fold higher azadirachtin production (15.9 mg/g DCW versus 3.2 mg/g in controlultures). Exposure time studies with elicitor(s) addition on 8th day revealed that highest azadirachtin accumulation reached after 48 h of combinedddition of elicitor(s) (17.4 mg/g). Cultivation of A. indica cells was also carried out with combined (statistically optimized) elicitors addition on 8th

ay in Stirred Tank Bioreactor. This led to more than 3-fold greater azadirachtin accumulation (161.1 mg/l) as opposed to control bioreactor witho elicitor addition (50 mg/l) in 10 days of cultivation period. The present study not only identifies the elicitor(s) and their respective concentrationsor enhanced azadirachtin synthesis but also establishes the role of combined elicitors to improve secondary metabolite production of plant cellultures more efficiently.

2008 Elsevier B.V. All rights reserved.

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eywords: Azadirachtin; Bioreactor; Growth kinetics; Elicitor; Statistical desig

. Introduction

The neem tree (Azadirachta indica A. Juss, family Meliaceae)s a multipurpose important tree and is being utilized in agricul-ure, medicines, environment protection, forestry and cosmetics,tc. Its potential is also being investigated to combat diseases likealaria, cancer, AIDS, and in population control [1]. The bio-

ogical activity of neem is mainly due to the presence of largeumber of secondary metabolites particularly in seed extract.zadirachtin is the principal secondary metabolite of neem seed

xtract and has been established as an efficient biopesticidegainst a wide range of insects and pests [2]. There are numerousimitations in obtaining elite varieties of neem seeds contain-ng high azadirachtin content. Seeds are usually produced only

nce in a year and cannot be stored for longer duration. Theonsiderable variation in azadirachtin content occurs depend-ng upon environmental and genetic heterogeneity in individual

∗ Corresponding author. Tel.: +91 11 26596109; fax: +91 11 26582282.E-mail address: ashokks@dbeb.iitd.ac.in (A.K. Srivastava).

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369-703X/$ – see front matter © 2008 Elsevier B.V. All rights reserved.oi:10.1016/j.bej.2007.12.017

eem trees [3]. In search of an alternative, plant cell and tissueultivation has been actively investigated for mass productionf azadirachtin. However, the yields and productivity have beenery low in the developed systems [4]. Exogenous addition ofelicitor’ molecules of biotic and abiotic origin has been reporteds one of the most promising strategies for the enhanced pro-uction of commercially important plants derived compounds5]. Effect of elicitor may vary from plant-to-plant species andhere is no universal effect of a particular elicitor on differentlants or cell culture systems [5]. Therefore, selection of rightlicitors and its appropriate dose optimization with respect tolant cell of interest is necessary to ensure the enhancement inroduct yield.

Effect of addition of certain biotic and abiotic elicitors at apecific time had been reported on A. indica suspension cul-ure for azadirachtin synthesis [6]. However, considering theomplexity of the biosynthesis of azadirachtin, there exists a pos-

ibility to synergistically improve the content by the addition ofredefined mixture of elicitors in the suspension cultures. There-ore, in the present investigation the individual and interactingffects of different elicitors (salicylic acid, chitosan, jasmonic

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G. Prakash, A.K. Srivastava / Biochemi

cid, methyl jasmonate, yeast extract, yeast extract carbohydrateraction) at different concentrations were studied to select themportant one predominantly affecting the azadirachtin biosyn-hesis in shake flask and bioreactor.

. Materials and methods

.1. Development of callus and suspension cultures

The A. indica cell line (AG-43) used in the present studyas developed from the seed kernel of Trivendrum, India orig-

nating tree [7]. Suspension cultures were established from thisell line and cultivated in statistically optimized medium con-aining glucose: 37.5 g/l, nitrate: 5.7 g/l, phosphate: 0.094 g/l,noculum: 5 g/l (DCW basis) with minor/vitamins/iron similaro Murashige and Skoog (MS) medium [8] and 8 mg/l indoleutyric acid + 4 mg/l benzyladenine [9].

.2. Elicitor preparation

Stock solutions of salicylic acid and yeast extract were pre-ared by dissolving them in distilled water and adjusting the pHo 5.8. For the preparation of chitosan, 1 g of crab shell chitosanas dissolved in 2 ml of glacial acetic acid (drop wise addition)

t 60 ◦C for a period of 15 min. The final volume was madep to 100 ml with water and pH of the solution was adjustedo 5.8 with 1 M NaOH [10]. Yeast extract carbohydrate frac-ion was isolated from the yeast extract by ethanol precipitation

11]. These solutions were sterilized by autoclaving at 120 ◦Cnd 1 atm over 20 min and used as an elicitor. Jasmonic acidnd methyl jasmonate were dissolved in 95% ethanol and filterterilized.

S2

able 1rial experimental recipe of Central Composite Design matrix of three variables inespect to cell growth and azadirachtin

xperiment Actual and coded values

Salicylic acid (A) (mg/l) Jasmonic acid (B) (mg/l)

1 69.7 (0) 106 (0)2 69.7 (0) 0 (−2)3 1.38 (−1) 2.1 (−1)4 1.38 (−1) 2.1 (−1)5 69.7 (0) 106 (0)6 69.7 (0) 106 (0)7 138 (1) 2.1 (−1)8 138 (1) 2.1 (−1)9 1.38 (−1) 210 (1)0 69.7 (0) 106 (0)1 184.5 (2) 106 (0)2 69.7 (0) 106 (0)3 69.7 (0) 106 (0)4 1.38 (−1) 210 (1)5 138 (1) 210 (1)6 0 (−2) 106 (0)7 138 (1) 210 (1)8 69.7 (0) 106 (0)9 69.7 (0) 106 (0)0 69.7 (0) 280 (2)

gineering Journal 40 (2008) 218–226 219

.3. Screening of elicitors

Elicitation studies were carried out with salicylic acid (SA:4, 28, 70 mg/l), chitosan (CH: 50, 100, 250, 500 mg/l), jasmoniccid (JA: 10, 20, 50, 100 mg/l), methyl jasmonate (MJ: 12, 24, 60,20 mg/l), yeast extract (YE: 10, 20 50 mg/l) and yeast extractarbohydrate fraction (YECF: 0.5, 2, 3%, v/v) in duplicate. Forlicitation, 9-day-old suspension cultures were transferred to0 ml of optimized medium [9] and supplemented with desiredoncentration of elicitors. Same amount of ethanol or water wasdded to the control cultures. Control and elicited plant cellasks were harvested on 12th day of cultivation and analyzedor dry cell weight and azadirachtin content.

.4. Response Surface Methodology

The combined effect of selected elicitors (salicylic acid, chi-osan, jasmonic acid) was then elucidated by the Responseurface Methodology [12], which established the interactionf these elicitors on overall azadirachtin synthesis. A Centralomposite Design (CCD) with six axial points, eight quadrantoints and six replicates at the center points leading to a totalf 20 sets of experiments in duplicate (Table 1) was developedsing Design-Expert version 5.0.9 software (Stat-Ease Corpo-ation, USA). Cells were harvested on 12th day of cultivationnd analyzed for dry cell weight and azadirachtin content.

.5. Effect of exposure time

Optimum concentrations of elicitors obtained from Responseurface Methodology (salicylic acid: 137.3 mg/l, jasmonic acid:.9 mg/l and chitosan: 16.5 mg/l) were added on 8th day of cul-

actual and coded concentration units along with the study of responses with

Responses

Chitosan (C) (mg/l) DCW (D) (g/l) Azadirachtin (E) (mg/g)

0 (−2) 12.2 8.5505 (0) 15.5 4.8

1000 (1) 15.0 3.210 (−1) 13.5 7.3

505 (0) 10.4 5.4505 (0) 10.2 4.8

1000 (1) 10.0 17.610 (−1) 10.7 18.710 (−1) 12.5 5.2

505 (0) 10.3 5.3505 (0) 15.5 13.2505 (0) 14.7 5.4505 (0) 10.3 5.2

1000 (1) 12.5 9.31000 (1) 7.7 1.6505 (0) 12.7 4.3

10 (−1) 16.5 5.4505 (0) 10.3 5.5

1337 (2) 13.2 8.5505 (0) 13.6 9.6

2 cal Engineering Journal 40 (2008) 218–226

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Table 2The least square fit and the parameter estimates (significance of regressioncoefficients)

Term Regression analysis for azadirachtin production

Coefficient estimate t-Value Significant level

Intercept 5.30A, salicylic acid 2.06 13.04 <0.0001***

B, jasmonic acid −5.66 −35.82 <0.0001***

C, chitosan −0.96 −6.08 0.0009**

A2 1.27 18.14 <0.0001***

B2 0.73 10.46 <0.0001***

C2 1.19 16.91 <0.0001***

AB −4.16 −44.21 <0.0001***

AC −0.61 −6.45 0.0007**

BC 0.69 7.32 0.0003**

A3 0.21 2.66 0.0377B3 2.51 31.30 <0.0001***

C3 0.34 4.21 0.0056*

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20 G. Prakash, A.K. Srivastava / Biochemi

ivation and cells were harvested after 24, 48, 72 and 96 h tonalyze for azadirachtin accumulation to identify the optimalxposure time.

.6. Bioreactor cultivation with elicitor

After optimizing the elicitor(s) concentration in shake flask,he plant cell cultivation was performed in 3 l Stirred Tank Biore-ctor to study the influence of elicitor addition on azadirachtinroduction in a bioreactor cultivation system. A. indica cellsere cultivated in 3 l Stirred Tank Bioreactor (Applicon,ependable Instruments, The Netherlands) (2.4 l working vol-me) at 0.2 vvm of initial airflow rate and 27 ◦C in darkness.he statistically optimized values of the medium componentsere used for the study of growth and product formation in theatch cultivation [13]. Low shear setric impeller at an agitationate of 125 rpm was used for mixing. After 8 days, cultures werereated with the optimized combination of elicitors; salicylic acid137.3 mg/l), jasmonic acid (2.9 mg/l) and chitosan (16.5 mg/l).he cultivation was further continued for additional 4 days. Thearvested cells were used for dry cell weight and azadirachtinnalysis.

.7. Dry cell weight and azadirachtin estimation

Dry cell weight (DCW) and azadirachtin were quantified astated earlier [9]. For DCW estimation, cells were harvested andollected by centrifugation at 3000 rpm for 15 min and washedith distilled water. The fresh cells were dried at 28 ± 2 ◦C to a

onstant dry weight. Extraction of azadirachtin was done withethanol [9]. Extract was thereafter analyzed by high perfor-ance liquid chromatography (Agilent technologies HP1100)

n C-18 column (Waters, USA) at a flow rate of 0.5 ml/minith acetonitrile:water (10:90) as mobile phase using 214 nmavelength.

.8. Statistical analysis

Variations within the elicitors were analyzed using theesign-expert software version 5.0.9. Treatments were com-ared to controls with ANOVA statistical analysis using thene factor design of design expert (P ≤ 0.001) and reported aseans ± standard error (S.E.). ANOVA values between the elic-

tors were calculated and null hypothesis (F-test for confidenceevel 0.10) was applied to analyze the significance betweenlicitors on azadirachtin synthesis. Data were also tested for nor-al distribution and found to be normally distributed for entire

ange.

. Results and discussions

.1. Screening of elicitors

Over accumulation of secondary metabolites in plant cell cul-ivation in response to the elicitor addition has been well reporteds a result of activation of defense response of plants [5]. Effectsf addition of different elicitors (individually, 21 sets of experi-

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Magnitude of significance.

ent, Table 2) on DCW and azadirachtin content are describedn detail.

.1.1. Effect of jasmonatesDCW was increased to 20.1 ± 0.30 g/l with the supplemen-

ation of highest concentration of jasmonic acid (100 mg/l) in. indica suspension culture. In contrast, methyl jasmonate led

o reduced cell growth at all the concentrations studied as com-ared to control cultures (15.5 ± 0.05 g/l). Highest increase inzadirachtin content (7.7 ± 0.06 mg/g) was obtained with lowestoncentration of JA (10 mg/l) (F = 4539.91, d.f. = 4, P ≤ 0.001)hile MJ exhibited a marginal increase in azadirachtin con-

ent (4.0 mg/g versus 3.2 mg/g in control) (F = 3.83, d.f. = 4,= 0.0865) only at highest concentration (120 mg/l) (Table 3).NOVA comparison also indicated the significant differenceetween these two elicitors (Table 4). JA is known to be aey signaling compound in plants activated upon insect feed-ng [14]. Involvement of JA as the signal molecule responsibleor increased synthesis of nicotine and hypericin (plant insecti-ides) is well documented [15,16]. In the present investigation,A induced the azadirachtin synthesis however MJ failed to doo. A variety of jasmonates had been applied in cell cultureedium as elicitors like JA, MJ and dihydro-methyl jasmonate

HMJA) and variability in their effects have been well reported17–20]. HMJA was found to be less effective than MJ on gin-enoside synthesis in Panax notoginseng cell culture [21] whileA improves ginsenoside accumulation in adventitious root cul-ure of Panax ginseng [22] indicating the specificity of chemicaltructure of elicitor for secondary metabolite accumulation.

.1.2. Effect of salicylic acidAddition of salicylic acid at lowest concentration (14 mg/l)

ad an inhibitory effect on DCW, however at highest con-

entrations (70 mg/l) it showed a marginal increase in DCW16.8 ± 0.25 g/l). Azadirachtin synthesis was induced at allhe concentrations studied and 2.7-fold higher azadirachtinontent (8.2 ± 0.05 mg/g as opposed to 3.2 ± 0.02 mg/g in con-

G. Prakash, A.K. Srivastava / Biochemical En

Table 3Effect of different elicitors on DCW and azadirachtin accumulation (each valuerepresents the mean ± S.E. of two samples)

Elicitor DCW (g/l) Azadirachtin (mg/g) Azadirachtin (mg/l)

Control 15.5 ± 0.04 3.2 ± 0.02 49.7 ± 1.60

Salicylic acid (mg/l)SA-14 10.3 ± 0.17 4.5 ± 0.04 47.2 ± 0.09SA-28 15.4 ± 0.03 5.5 ± 0.06 84.5 ± 1.06SA-70 16.8 ± 0.25 8.2 ± 0.05 134.5 ± 4.14

Chitosan (mg/l)CH-50 15.5 ± 0.15 8.9 ± 0.04 139.6 ± 2.01CH-100 14.8 ± 0.04 7.5 ± 0.06 112.6 ± 0.62CH-200 14.2 ± 0.08 6.9 ± 0.06 99.3 ± 0.44CH-500 14.0 ± 0.05 3.1 ± 0.06 44.3 ± 0.74

Jasmonic acid (mg/l)JA-10 16.4 ± 0.05 7.7 ± 0.06 127.9 ± 1.54JA-20 15.5 ± 0.08 6.4 ± 0.04 99.1 ± 0.11JA-50 18.5 ± 0.12 6.0 ± 0.04 111.0 ± 0.57JA-100 20.1 ± 0.30 4.6 ± 0.05 90.8 ± 0.35

Methyl jasmonate (mg/l)MJ-12 14.4 ± 0.07 2.9 ± 0.20 46.5 ± 0.37MJ-24 14.5 ± 0.09 3.4 ± 0.09 41.8 ± 2.67MJ-60 14.5 ± 0.04 3.6 ± 0.13 49.3 ± 1.84MJ-120 14.2 ± 0.10 4.0 ± 0.04 53.0 ± 1.8

Yeast extract (mg/l)YE-10 14.6 ± 0.08 3.6 ± 0.04 46.5 ± 0.37YE-50 16.6 ± 0.10 5.6 ± 0.08 53.0 ± 0.99YE-100 14.1 ± 0.09 6.5 ± 0.05 92.9 ± 1.89

Yeast extract carbohydrate fraction (v/v)YECF-0.5 15.7 ± 0.10 2.8 ± 0.13 44.9 ± 1.85

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rol) (F = 763.36, d.f. = 3, P ≤ 0.001) was obtained at highest

oncentration (70 mg/l) (Table 3). Increase in azadirachtin con-entration with respect to SA addition could be attributed tohe fact that SA act as powerful inducing signals for secondary

able 4NOVA values between elicitors

Fcal FTab

A vs. JA 67.05 57.2*

A vs. CH 98.09 57.2*

A vs. MJ 77.79 57.2*

A vs. YE 58.31 55.8*

A vs. YECF 64.0 55.8*

A vs. CH 100.92 58.2*

A vs. MJ 91.84 58.2*

A vs. YECF 66.05 53.8*

A vs. YE 58.43 57.2*

H vs. MJ 129.39 58.2*

H vs. YE 98.96 57.2*

H vs. YECF 105.95 57.2*

J vs. YE 61.52 57.2*

J vs. YECF 53.89 57.2E vs. YECF 50.92 55.8

Fcal > FTab invokes the rejection of null hypothesis that states that the two vari-nces in comparison are from the same population (i.e., they are not statisticallyifferent).

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gineering Journal 40 (2008) 218–226 221

etabolite synthesis, and plays an essential role in many plantefense reactions [23]. It is known to regulate the expression ofarious defense related genes [24,25] some of which could be thenes linked to the azadirachtin pathway. SA is also regarded as aignal molecule playing an important role in Systemic Acquiredesistance (SAR) and inhibitor of ethylene biosynthesis [26].herefore, the possibilities exist that SA could be acting through

he inhibition of ethylene biosynthesis, a phytohormone whichs active in plant defense mechanism [27]. Azadirachtin beinghe defense chemical of plants against insects could be regulatedith the presence of this signal molecule.

.1.3. Effect of chitosanAddition of chitosan at lower concentration (50 mg/l) had

o inhibitory effect on DCW while at higher concentra-ions (100–500 mg/l) biomass decreased slightly (Table 3).

aximum increase in azadirachtin accumulation (among alllicitors studied individually) was obtained with the addition of0 mg/l chitosan (8.9 ± 0.04 mg/g; ∼2.8 times higher than con-rol; 3.2 ± 0.02 mg/g) (F = 2313.73, d.f. = 4, P ≤ 0.001). Above0 mg/l chitosan concentrations, azadirachtin content decreasedontinuously (Table 3). Chitosan (�-1,4-linked glucosamine)as also been proved to enhance the production of secondaryetabolites in various suspension cultures [11,28].

.1.4. Effect of yeast extract and yeast extract carbohydrateraction

A reduction in cell growth was observed by the addition ofeast extract (except at 50 mg/l concentration) and yeast extractarbohydrate fraction at higher concentrations (0.5–2.0, v/v)Table 3). Addition of YE and YECF failed to over induce thezadirachtin synthesis as compared to control at lower concen-rations (P = 0.0014 for YE10 mg/l; P = 0.071 for YECF0.5% and= 0.001 for YECF2.0%) and exhibited their influence at higher

evels only. Azadirachtin content increased to 6.5 ± 0.05 mg/gF = 1852.66, d.f. = 3, P ≤ 0.001) and 5.8 ± 0.06 mg/g (than con-rol; 3.2 ± 0.02 mg/g) (F = 234.5, d.f. = 3, P ≤ 0.001) with theddition of highest concentration of YE (100 mg/l) and YECF3%, v/v), respectively (Table 3). In the present study, carbohy-rate fraction isolated from yeast extract at higher concentrations>0.5%) was able to induce the azadirachtin biosynthesis whilet lower concentrations it could not enhance the productionTable 3). Therefore, it can be concluded that carbohydrate frac-ion of yeast extract could be acting as an elicitor and a minimumoncentration of this carbohydrate fraction is required for thelicitation.

All elicitors exhibited the different trends in elicitingzadirachtin synthesis and the attained production ranged from.4 ± 0.09 to 8.9 ± 0.04 mg/g DCW. This was almost thrice themount of control cultures (3.2 ± 0.02 mg/g DCW). Individualddition of elicitors demonstrated that SA, CH and JA were theost effective ones to enhance azadirachtin synthesis in seed

ernel derived suspension culture of A. indica. The analysis of

ariance (ANOVA) for different elicitors showed that signifi-ant difference exits between elicitors except for YE and YECFnd MJ and YECF (Table 4). It was also observed that highestncrease in azadirachtin was obtained with lowest concentra-

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22 G. Prakash, A.K. Srivastava / Biochemi

ions of CH (50 mg/l) and JA (10 mg/l). This signifies that higherzadirachtin content could have been obtained if concentrationsf CH and JA would have been decreased further. Similarlyncreasing the SA concentration (>70 mg/l) could have furthernhanced the azadirachtin content. Based on these observationsnd literature reports describing the effect of combined elicitorsn alkaloid accumulation in Catharanthus roseus cell cultures29], in the suspension cultures of Taxus chinensis [30,31],hytoalexin production in rice cell cultures [32,33] and in adven-

itious root culture of Scopolia paraviflora [34] it was assumedhat addition of these three elicitors together at appropriate con-entrations could further enhance the azadirachtin content higherhan their individual additions. Therefore, SA, JA and CH werehosen for further investigation of their interacting effects andoncentrations optimization for enhanced azadirachtin produc-ion by Response Surface Methodology.

.2. Response Surface Methodology

The combined effects of SA, JA and CH were studiedy Response Surface Methodology using Central Compositeesign. Concentration-Component recipe was generated by

tatistical software (Design Expert, 5.0.9; State Ease Corpora-ion, USA). The specific interacting combinations of differentlicitors along with the measured response values (DCW andzadirachtin) are summarized in Table 1.

The summary of the analysis of variance representing theesults of the Cubic Response Surface model is listed in Table 5.he models ‘Prob > F’ value was extremely low: <0.0001, indi-ating the highly significant third-order model equation forzadirachtin accumulation. The squared regression statistic (R2),he determination coefficient, a measure of the goodness of fitf the model was highly significant at the level of 99.8% mean-ng that model was unable to explain only 0.2% of the totalariations. The adjusted (R2) value (0.996) also indicated theignificance of the model. Table 2 represents the student t distri-ution and the corresponding P values along with the parametersstimated. The P value establishes the significance of each of theoefficients, which reflects the magnitude of effect of differentariables and their interactions. Table 2 revealed the significantndividual effect of jasmonic acid (B) and salicylic acid (A),

uadratic effect of A2, B2, C2 and cubic effect of B3 and ABCP < 0.0001) on azadirachtin synthesis.

The application of RSM on the basis of parameter estimatesesulted in an empirical relationship between the azadirachtin

meii

able 5nalysis of variance (ANOVA) for the cubic model of azadirachtin production

ource of variation Sum of squares Degree of freedom

odel 380.31 13esidual 0.43 6ack of fit 0.15 1ure error 0.27 5orrected

otal 380.73 19

ome important statistics: root mean square error = 0.27; coefficient of variation = 3.5

ngineering Journal 40 (2008) 218–226

roduction and the process variables (elicitors). The followingegression equation indicated the relative azadirachtin produc-ion as a function of the test variables (A, B, C).

zadirachtin = +5.30 + 2.06 × A − 5.66 × B − 0.96 × C

+ 1.27 × A2 + 0.73 × B2 + 1.19 × C2

− 4.16 × A × B − 0.61 × A × C

+ 0.69 × B × C + 0.21 × A3 + 2.51 × B3

+ 0.34 × C3 − 1.38 × A × B × C (1)

here A = salicylic acid concentration, B = jasmonic acid con-entration, and C = chitosan concentration.

The above model equation representing azadirachtin accu-ulation was solved for different sets of elicitor concentrations

o develop contour plots and study the response of different elic-tor interactions. A critical analysis of the response reveals thatonsiderable interactions occur between SA, JA and CH. SAas always beneficial for azadirachtin production in higher con-

entrations except when it was added with JA and CH at theiraximum concentrations (Table 1). Combination of all three

licitors in their maximum concentration resulted in decreasedzadirachtin production suggesting some inhibitory effect oniosynthetic machinery of A. indica cells.

For the determination of the optimum operating concentra-ion and analysis of the interaction of elicitors on azadirachtinormation, the Response Surfaces were generated and studiedn detail for all the possible elicitor combinations by keepingne elicitor’s concentration constant at a time. Some of theseesponse Surfaces are shown in Fig. 1(a–c) with one variableept at central level (SA, JA and CH, respectively) and the otherwo varying within the experimental range (JA and CH; CH andA and JA and SA, respectively). Based on the different responseurves generated, optimum concentration of all the elicitorsere determined as follows: SA = 137.3 mg/l; JA = 2.9 mg/l andH = 16.5 mg/l. An azadirachtin production of 18.4 mg/g wasredicted by the Eq. (1) corresponding to these optimum con-entrations of the elicitor(s). In order to confirm the predictedesults of the model, experiments using the optimum concentra-ions of the elicitors were performed and 15.9 mg/g azadirachtinas obtained which represents the 87% validity of the predicted

odel for azadirachtin production. The combined elicitors syn-

rgistically promoted greater azadirachtin production than eachndividual elicitor addition. It was 1.8-fold higher than maximumncrease obtained by individual elicitor addition (8.9 mg/g with

Mean square F-value Probability P > (F)

29.25 411.99 <0.00010.0710.15 2.76 0.15780.055

6; R2 = 0.99; Adj R2 = 0.99.

G. Prakash, A.K. Srivastava / Biochemical Engineering Journal 40 (2008) 218–226 223

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ig. 1. Response Surface graph of azadirachtin production between (a) jasmonicasmonic acid; (c) salicylic acid and jasmonic acid at constant chitosan.

0 mg/l CH). The reasons why these combined elicitors gave aiverse of effects on azadirachtin production are still not knownet. The mechanism of action of each elicitor in the present studys different and it could be complicated for every combined treat-ent since it depends on the interactions between physiological

ffects caused by different treatments. A synergistic effect ofultiple elicitors on production of secondary metabolites isost often seen when a plant-derived (endogenous) elicitor (like

alicylic acid) and a microbe-derived (exogenous) elicitor likechitin or chitosan) are applied together [35]. Addition of methyl

asmonate with chitosan derived oligosaccharides resulted in fur-her enhancement of taxol in the Taxus canadensis cultures [36].imilarly, in T. chinensis cultures the synergistic effects of addi-

ion of more than one elicitor together had been investigated

dbic

and chitosan at constant salicylic acid; (b) chitosan and salicylic acid at constant

30,31]. A synergistic effect of more than one elicitor (fungalreparations and chemicals) on alkaloid accumulation was alsobserved in C. roseus cell cultures [29].

Different elicitor affects the cell physiology and activation ofefense compound in different ways, which could antagonize orarmonize with each other, leading to negative or additive inter-ctions, respectively [35]. However, One Variable At a TimeOVAT) optimization procedure is laborious/time consumingnd interaction of various elicitors could not be studied togetherith this method. The application of statistical experimental

esign is a very useful method to study the different interactionsetween all process variables. Response Surface Methodologys one such technique, which is widely used in different plant cellultivation processes to study the interaction of various param-

224 G. Prakash, A.K. Srivastava / Biochemical Engineering Journal 40 (2008) 218–226

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ters for process optimization [37,38]. Application of statisticalechniques is also gaining importance to study the interaction oflicitor’s action in plant cell cultivation systems for optimizationf secondary metabolites production [31,36].

.3. Effect of incubation time of elicitors on biosynthesis ofzadirachtin

Optimization of the incubation time of the elicitors is impor-ant in order to maximize the final metabolite concentration in theultivation [38]. Growth kinetic study showed that azadirachtins growth associated and therefore, the elicitors were addedn 8th day (when cells were in log phase) in shake flask.he relationship of the incubation time of A. indica cultures

or the mixture of elicitors (SA, JA and CH at their optimumoncentrations) on azadirachtin concentration is presented inig. 2. Azadirachtin concentration increased from 2.2 ± 0.13 to.6 ± 0.35 mg/g after 24 h and then to 17.4 ± 0.01 mg/g after8 h of addition of elicitors. After 48 h the levels of azadirachtinecreased with additional incubation time and remained con-tant thereafter. The DCW decreased slightly in the elicitedultures and could reach to 14 ± 0.2 g/l only on the 12th dayf cultivation as opposed to 15 ± 0.23 g/l in the non-elicitedultures. The result shows that the amounts of azadirachtin pro-uced varied with duration of the incubation period of elicitors.n immediate increase followed by a decrease in secondaryetabolite production with addition of elicitor molecule is gen-

ral phenomena observed for various plant cell culture systems.n a similar manner, Addition of autoclaved and non-autoclavedlicitor (prepared from Sebacina vermifera, a mycorrhiza likeungi) added during the early log phase of same culture of A.ndica resulted in higher azadirachtin production after 3 and 6ays, respectively, indicating the effect of incubation time of

licitor on cell culture [39]. The present results are also con-istent with the observed maximum increase in anthraquinoneontent after 48 h of incubation and decline thereafter for addi-ional incubation time with elicitors [40] and observed decrease

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ig. 3. Growth and azadirachtin production in control and elicited bioreactor.rrow indicates the time of addition of elicitors.

n tropane alkaloid level and thiarubrine-A yield after 12 and2 h of exposure of elicitors, respectively [41]. Catabolism oronversion to other unknown compounds could be responsibleor this phenomenon.

.4. Bioreactor process with combined addition of elicitorsor azadirachtin production

To scale up plant cell cultivation upto commercial level,nvestigations in the bioreactors are necessary [42]. Therefore,he effect of combined addition of elicitors on cell cultures of A.ndica was examined in a bioreactor (with setric impeller). Sincehe azadirachtin production was intracellular and growth associ-ted, elicitors were added in the late exponential stage (8th day)f cultivation in the bioreactor (i.e., when sufficient biomass hadccumulated). The growth and azadirachtin production profilen bioreactor with and without elicitor addition is representedn Fig. 3. Biomass in combined elicitor-treated bioreactor wasess (14.0 ± 0.2 g/l) than that of the control one (15.5 ± 0.22 g/l)ecause of the possible inhibition due to elicitors. Contrary toiomass, more than 3-fold increase in azadirachtin production161.1 ± 2.05 mg/l; 11.5 mg/g) was obtained by A. indica sus-ension cultures treated with SA (137.3 mg/l), JA (2.9 mg/l) andH (16.5 mg/l) in bioreactor as compared to the control cultures

50 ± 1.09 mg/l; 3.2 ± 0.02 mg/g). The azadirachtin production161.1 mg/l) and volumetric productivity (16.1 mg/l d) was sig-ificantly higher in elicited bioreactor cultivation than theontrol bioreactor (5 mg/l and 5 mg/l d, respectively). Such anmprovement effect obtained from shake flask to bioreactoruggests reproducible elicitation effect of the combined elici-ors upon scale-up. Bioreactor cultivations with elicitor additionre increasingly being investigated to improve the secondaryetabolite production and to reduce the process cost in several

lant cell/hairy root cultivation systems. About 2-fold higherotal alkaloid production than the control cultures was observed

ith a combined elicitation of malate (40 mg/l) and sodium

lginate (1.5%, w/v) for 3 days in bioreactor [29]. A 2-foldnhancement in taxoid production has also been demonstratedy repeated elicitation of methyl jasmonate in bioreactor as com-

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G. Prakash, A.K. Srivastava / Biochemi

ared to single elicitor addition in cell cultures of T. chinensis30]. A strategy of elicitation in bioreactor at late exponentialhase has been reported for improvement of betalain produc-ivity (47% higher) in hairy root cultures of Beta vulgaris inubble Column Bioreactor [43]. The elicitors identified in theresent study are very cost effective (at their final concentrationss very less amount is required for addition in the medium) andherefore would not significantly increase the overall processost. Moreover, azadirachtin is a high value product and resultshowed that substantial increase in its concentration could bechieved with the addition of very less amount of elicitorsecreasing the overall production cost. Hence, use of combinedlicitors in bioreactor may be a practical way for large-scalezadirachtin production. A low-cost production process is ofreat importance for feasible industrial application. Applicationf high cell density cultivation with combined elicitor treatmentan lead to significantly higher azadirachtin production by A.ndica suspension cultures. The present study suggests an effec-ive strategy for the enhancement of azadirachtin production inioreactor.

cknowledgements

One of the authors (Ms. Gunjan Prakash) is grateful to theouncil of Scientific and Industrial Research (CSIR), Govt. of

ndia, New Delhi, for providing the fellowship.

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