Development of a suitable bioreactor system for azadirachtin production by hairy roots of azaidirachta indica.

DEVELOPMENT OF A SUITABLE BIOREACTOR SYSTEM FOR AZADIRACHTIN PRODUCTION BY HAIRY ROOTS OF

A2 ョ DIRA CHTA 刀 VDIC 月

by

SMITA SRI VASTAVA

DEPARTMENT OF BIOCHEMICAL ENGINEERING&BIOTECHNOLOGY

Thesis submitted

in the fulfilment of the requirements of the degree of Doctor of Philosop取 to the

Indian Institute Of Technology, Delhi

July, 2008

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CERTIFICATE

This is to certify that the thesis entitled "Development of a suitable bioreactor system

Azadirachta indica"

for azadirachtin production by hairy roots being submitted by Ms. Smita Srivastava to

of he

Indian Institute of Technology, Delhi, for the award of the degree of "Doctor of Philosophy" is a record of the bonaffide research carried out by her, which has been prepared under my supervision and guidance in conformity with rules and regulations of the "Indian Institute of Technology, Delhi". The results described in it have not been submitted in part or full to any other University or Institute for the award of any Degree/Diploma.

Dr. A. K. Srivastava Professor&Head,

Department of Biochemical Engineering&Biotechnology, indian Institute of Technology, Delhi,

New Delhi

一

i 10016

India.

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ABSTRACT

Plants are the important source of food, ffiber, color, fflagrance and medicine.

However yield and productivity fflom the natural plants are signifficantly low, making the plant production route uneconomical . Alternate in vitro production technologies like specialized plant cell/hairy root cultivation processes are being developed to mass produce the plant secondary metabolites. In vitro cultivations of hairy roots have a distinct advantage of rapid growth in hormone fflee medium. They have better genetic and biochemical stability in comparison to cell/tissue cultivations. Scientiffic communities have focused their attention toward Pits exploitation as a source of rare and valuable secondary metabolites.

Hairy root culture was developed in the present study to establish and demonstrate an alternative commercially viable production technology for Azadirachtin against extraction fflom the seeds ofAzadirachta indica.

The hairy roots of Azadirachta indica were initiated in the present study by transformation of the plant cells by Agrobacterium rhizogenes. i 75 hairy root lines were induced fflom the diLerent explants of in vitro germinated seedlings fflom the elite high Azadirachtin yielding varieties of seeds collected fflom different agroclimatic regions of the country. Further, screening and selection of a single hairy root line (Az-35) was done in liquid culture on the basis of its highest Growth Index (1 .75) and Azadirachtin accumulation (content) (3.8 mg/g). The integration of the T-DNA region of the Ri-plasmid ofA. rhizogenes in the selected A. indica hairy root line was conffirmed using the Polymerase Chain Reaction technique. The selected hairy root line was maintained on

a favorable solid medium of the following composition: MS medium major and minor salts, B5 medium vitamins and 30 g/I sucrose. The initial pH of the medium was adjusted to5.8 andte卿erature was maintained at 25 o C.

Different hairy root culture medium recipe (s), growth factors and environmental conditions were studied and their optimum concentrations yielding high Azadirachtin and biomass production were derived in the shake flask. Substrate utilization, growth and production kinetics was established in the liquid culture of hairy roots under optimized medium and environmental conditions（叩m: 80, te_叩erature: 26 o C, pH: 5.7, medium composition: (major nutrients: Sucrose 一 40 g/i; Potassium dihydrogen phosphate _一 o. i 9 g/I; Potassium nitrate一3.1 g/I; Ammonium nitrate- 1.65 g/i; Magnesium sulphate一 0.41 g/i along with MS medium minor salts and B5 medium vitamins), Inoculum size:

3 glI dry weight (DW) and age: 30 days, growth regulator: I .0 mgI! Indole-3-acetic acid (IAA) and 0.025 mg/I Gibberellic acid (GA3), permeabilizing agent: 0.5%v/v Di-n-butyl phthalate (DNBP)). A maximum biomass of 21 .3 g/i and Azadirachtin production of 78.8 1 mg/i in 25 days of the cultivation period (equivalent to an overall volume垣C productivity of 3. 1 5 mg/i d) was obtained with a residual sucrose concentration of 10.7 gIl. The disappearance of the nutrients was also reflected by a decrease in the medium conductivity fflom its initial value of 10.2 mS to 3.3 mS in 25 days of the hairy root cultivation period.

Directly and/or indirectly related biosynthetic precursors of Azadirachtin were added in the medium to enhance the Azadirachtin production in the hairy roots of A.

indica・Maximum i_叩royement in the Azadirachtin production (up to 70.42 mg/i) and its equivalent volumetric productivity (up to 2.8 1 mg/I d) could be achieved fflom that

obtained in control with no precursor in the medium (44 mg/i of Azadirachtin production in 25 days and its equivalent volumetric productivity of i .76 mg/I d) when the growth medium was supplemented with 50 mg/i of Cholesterol. The Azadirachtin yield (content) in the hairy roots was found to increase fflom 3.3 mg/g in control to 5.82 mg/g on Cholesterol (50 mg/I) addition.

The possibility of Azadirachtin yield (mg/g) enhancement in the hairy roots of A.

indica was investigated by the addition of potential elicitors (biotic and abiotic) in the cultivation medium. Among ali the elicitors examined the addition of 1%(v/v) of Curvularia lunata fungal culture ffiltrate resulted in highest Azadirachtin yield enhancement (up to 7.1 mg/g with respect to control value of 3.3 mg/g with no elicitor in the medium). The addition of elicitor in the medium in the beginning of the cultivation period detrimentally affected the biomass production (g/i) as a result the overall Azadirachtin production (mg/i) and its equivalent volumetric productivity (mg/i d) got affected. Hence optimization of the time of addition of the selected elicitor in the medium was done to achieve max_面um Azadirachtin production (mg/l) and its equivalent overall volumetric productivity (mg/i d). During the optimization of the time of addition, the selected elicitor was proposed to be added along with the selected precursor in the medium under optimized cultivation conditions in order to have a synergistic effect on the overall Azadirachtin production (mg/i) and its equivalent voiumetric productivity (mg/i d). It was found that maximum Azadirachtin production ( 1 1 3 .4 mg/l, (with an Azadirachtin accumulation in the hairy roots of 5.4 mg/g and biomass production of 21 g/i)) and its equivalent overall volumetric productivity (4.53 mg/I d) was achieved when 山e co_価mned ad_山tion was done on i 5th day ofthe growth cycle.

In order to scale-up the hairy root cultivation the conventional bioreactor designs were modiffied and some new custom made bioreactors were got fabricated as per the culture characteristics and requirements (of support for growth, nutrient and oxygen transfer, liquid hold up, Experiments were conducted on these different biore actor designs to select an app ate bioreactor conffiguration (with simpler and economical reactor design) maximum biomass production (g/i) and Azadirachtin accumulation (mglg) in hairy roots could be achieved, thereby resulting in maximum Azadirachtin production (mgl!) and its equivalent overal! volumetric productivity (m朗 d). In the on different bioreactors, following overall volumetric productivities of Azadirachtin (mg/i d) were obtained, respectively (Stirred Tank Reactor (0.33 mg/i d), Bubble Column Reactor (0.43 mgl! d), Modiffied Bubble Column Reactor (with PUF) (1.14 mg/i d), Rotating Drum Reactor (O

Bioreactor (0.49 mg/i d) and Nutrient Mist Bioreactor (1

一『一 9 8 nU

mg/i d), Nutrient Spray

・

、

mg/I d). It was invariably observed that the Azadirachtin productivities obtained in the different bioreactors studied were less than that obtained in the sh水e flask (1 .76 mg/i d), presumably due to larger mass transfer limitations encountered d面ng scale-up. all the bioreactor cultivation studies done, maximum Azadirachtin overa!! volumetric productivity (1.14 mg/I d) was achieved in Modiffied Bubble Column Reactor (with Polyurethane foam (PUF) as root support).

Finally, conditions (ob

the tamlle

selected hairy root line was cultivated under optimized cultivation d fflom the shake flask studies) in the Modified Bubble Column Reactor with a minor modiffication of incorporating a Setric impeller beneath the PUF root support (for enhanced mixing of the cultivation medium and to

ensure adequate oxygen transfer). This resulted in a 5-fold increase in biomass (up to 15.2 gIl fflom an initial value of 3 g/I) and Azadirachtin production of 97.28 mg/i in 25 days of the cultivation period (with an Azadirachtin accumulation of 6.4 mg/g in the hairy roots).

The effficacy of the A. indica hairy roots as a potential biopesticide candidate was demonstrated through a bioassay carried out on the desert locust Schistocerca gregaria.

The hairy root sample (its crude solvent extract) demonstrated a high level of antifeedant activity (Antifeedant Index (A.I): 83 .5 at 0.5 mg/ml concentration of the hairy root extract in Ethanol). The Electron Spray Ionization- Mass Spectroscopy (ESI-MS) analysis of the hairy root sample (its crude solvent extract) revealed the presence of Azadirachtin (-A, -B, -11H), Salannin, Nimbin, Isonimbinolide and Salannol acetate in the hairy roots of A. indica. A study was also undertaken to reduce the photodegradation of Azadirachtin present in the hairy root sample (its crude solvent extract) by the addition of selected chemicals _印hotostabilizers). Maximum improvement in the Azadirachtin degradation time (in terms of the Disappearance Time, DT50) was achieved when Terbutyihydroquinone was used as a photostabilizer in the sample (where the Disappearance Time (DT50) increased fflom 3.3 days in control (sample without stabilizer) to 4.5 days in the presence ofthe stabilizer).

CONTENTS

Title Page

List of Figures List of Tables

List of Abbreviations List of Symbols

O. v N i-

v.-vill

CHAPTER 1. INTRODUCTION AND OBJECTIVES i . i Introduction

i .2 Objectives

CHAPTER 2. LITERATURE REVIEW 2. 1 Hairy root culture

2.1 . I Discovery 2. 1 .2 Mechanism 2. 1 .3 Confirmation 2.1 .4 Characteristics

2.2 Applications ofhairy root technology

2.2. 1 Production ofvaluable plant metabolites

2.2.2 Secondary metabolite production ifiom hairy roots

12-67 つム つ」 ^ーーユ

11 つー うつ

噌1上 『11 くJ \G 11 一11 「ノ On 11 11

2.2.3 Production of compounds (metabolites) not found in 21 untransformed roots

2.2.4 Hairy root metabolic engineering 21

2.2.5 Phytoremediation 23

2.2.6 Regeneration ofwhole plants 24 2.3 Large scale production of valuable plant metabolites丘om 24

hairy roots

2.3.lLiquid-phase reactors 26

2.3.1.i Stirred Tank Reactor 26

2.3. 1 .2 Airliifi Reactors 27

2.3.1.3 Bubble ColumnReactor 28

2.3. 1 .4 Convective Flow Reactor 29

2.3.1.5 Turbine Blade Reactor 30

2.3. 1 .6 Rotating Drum Bioreactor 31

2.3.2 Limitations 32

2.3.3 Gas phase reactors 33

2.3.3.1 Trickle Bed Reactor 34

2.3.3.2 Radial Flow Reactor 35

2.3.3.3 Nutrient Mist Bioreactor 36

2.3 .4 Hybrid Reactor 38

2.4 Measurement of growth during hairy root cultivation 38 2.5 Mass transfer requirements during scale-up ofhairy roots 40 2.6 Mathematical modeling in hairy roots 42

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46

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2.7 Azadirachtin: a valuable plant secondary metabolite 2.7.1 A biopesticide

2.7.2 Chemistry of Azadirachtin 2.7.3 Mode ofaction of Azadirachtin

2.7.4 Biosynthetic pathway for Azadirachtin 2.7.5 Availability of Azadirachtin

2.7.6 Stability ofAzadirachtin 55

2.8 Plant cell/tissue culture: an alternative to in vitro 55 Azadirachtin production

2.9 Strategies for secondary metabolite productivity 58 enhancement in plant cell/tissue culture

2.9. 1 Strain improvement and selection

2.9.2 Media compositions and culture conditions

2.9.3 Application of elicitors precursors and permeabilizing agent

2. 9 .4 Genetic engineering approach

2.9.5 Somatic ryogenesis and regeneration 2.9.6 Two-phase (stage) systems

2.9.7 Immobilization

2 . 1 0 Future prospects for Azadirachtin production technology

CHAPTER 3. MATERIALS AND METHODS 68-132

3.1 Major equipments and chemicals 68

3 .2 Experimental methods 71

3.2.1 Development of the hairy roots of Azadirachta 71 indica

3.2.1.1 Explant preparation 71

3.2.1.2 Development of active A_即りbacterium 72 rhizogenes culture for infection

3.2. 1 .3 Method ofmaking wound 74 3.2. 1 .4 Addition of Acetosyringone 74

3.2. 1 .5 Exposure medium 74

3.2.1.6 Exposure time 75

3.2. 1 .7 Co-cultivation and initiation medium 75 3.2. 1 .8 Co-cultivation time and temperature 75 3 .2. 1 .9 Illumination conditions 75 3.2.3. Selection ofthe hairy root line 77 3.2.4 Confirmation of the transformed nature of the 78

selected hairy root line

3.2.4.1 Designing ofprimers 79

3.2.4.2 Total DNA and plasmid DNA isolation of the 79 hairy root line and the Agrobacterium rhizogenes strain LBA 920 respectively

3.2.4.3 Polymerase Chain Reaction 79

3.2.4.4 Product analysis 80 3.4.5 Growth and maintenance of the selected root line on 80

a favorable solid medium

3.4.6 Development of the A. indica hairy root culture in 81 liquid medium (in shake flask)

3.4.6.1 Optimization ofmedium to flask volume ratio 81 3.4.6.2 Growth kinetics ofthe hairy root culture in the 82

liquid medium

3.4.7 Optimization of the culture conditions for the 83 maximum biomass and Azadirachtin production in the liquid culture ofA. indica hairy roots

3.4.7.1 Selection ofthe carbon source 83 3.4.7.2 Optimization ofillumination conditions 84 3.4.7.3 Optimization ofrotational speed (rpm) 85 3.4.7.4 Optimization of inoculum concentration (size) 85

and inoculum age

3.4.7.5 Optimization of medium components using 87 statistical design protocol

3.4.7.5.1 Selection of signifficant nutrients 87 affecting growth and Azadirachtin production using Plackett-Burman design

3.4.7.5.2 Determination of the optimum 89 concentrations of the selected variables out of the six efたctors in Plackett- Burman design for maximum growth and Azadirachtin production in A.

indica hairy roots using Response Surface Methodology (RSM).

3.4.7.6 Optimization of pH and temperature using 91 statistical design protocol

3.4.8 Exogenous additions in the medium to enhance the 93 Azadirachtin volumetric productivity in the liquid culture ofA. indica hairy roots.

3.4.8.1 Addition ofgrowth regulators 93 3.4.8.2 Addition ofpermeabilizing agents 95

3.4.8.3 Addition ofprecursors 95

3.4.8.4 Addition ofelicitors (biotic and abiotic) 97 3.4.8.1 Biotic and abiotic elicitor preparation 98 3.4.9 Establishment ofthe hairy root culture kinetics under 99

optimized cultivation conditions

3 .4. 1 0 Optimization of the day of addition of the selected i 00 elicitor and precursor in the optimized culture medium ofthe A. indica hairy roots

i 03

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3.4.11 Effect of Ethylene addition on the volumetric 101 productivity of Azadirachtin in the hairy root culture ofA. indica

3.4. 12 Scale-up of the production technology of Azadirachtin fflom the hairy root culture ofA. indica

3.4.12.1 Batch cultivation of A. indica hairy roots on different bioreactor conffigurations

3.4.12.1 . i Stirred Tank Reactor 3.4.12.1.2 Bubble Column Reactor

3.4.12.1.3 Modiffied Bubble Column Reactor 3.4. 1 2. 1 .4 Rotating Drum Reactor

3.4.12.1 .5 Nutrient Spray Bioreactor 3.4.12.1.6 Nutrient Mist Bioreactor

3.4.12.2 Scale-up of the hairy roots in the selected bioreactor conffiguration under optimized set of cultivation conditions

3 .4. 1 3 Strategies to overcome the mass transfer limitations in the h root cultivation

3 .4. 1 3 . 1 Optimization of the gas phase composition 3.4.13.1.1 Effect of oxygen

3.4. 1 3. 1 .2 Effect of carbon-dioxide 3.4.13.2 Optimization ofthe air flow rate 3.4.13.3 Addition ofthe oxygen vectors

3 .4 . 1 4 Stability enhancement of Azadirachtin against i 24 atmospheric degradation

3.4.15 Biopesticidal effficacy in the hairyroots ofA. indica 126 3 .4. 1 6 Qualitative characterization of the Azadirachtin i 28

Related Limonoids (AZRL) present in the hairy roots ofA. indica

3.4.17 Analytical methods 129

3.4. 1 7. 1 Fresh weight (FW) and Dry weight (DW) i 29 estimation

3.4.17.2 Extraction protocol for Azadirachtin Related 129 Limonoids (AZRL) ifiom the hairy roots of A.

indica

3.4.1 7.3 Quantiffication ofAzadirachtin 130 3.4. 1 7.4 Residual Sucrose estimation i 30 3.4.17.5 Reducing sugar (Glucose and Fructose) 130

estimation

3 .4. 1 7.6 Residual Nitrate estimation I 31 3 .4. 1 7.7 Estimation of the Phenolic release in the i 31

medium

3.4. 1 7.8 Viability estimation of the hairy root tissue 132 using Tri-phenyl tetrazolium chloride (TTC) assay

CHAPTER 4. RESULTS AND DISCUSSION 133-231 4.1 Development of the hairy root culture of Azadirachta 133

indica

4.3 Conffirmation of the transformed nature of the selected 139 hairy root line (Az-35)

4.2.1 Primers designed 140

4.2.2 DNA isolation of the bacterial strain i 40 4.2.3 DNA isolation of the selected hairy root line and 140

normal root line for PCR analysis

4.2.4. The conffirmation in the PCR reaction 141 4.3 Growth and maintenance of the selected root line on a 143

favorable solid medium

4.4 Development of the hairy root culture in the liquid medium 143 4.5 Optimization of the culture conditions for maximum 146

Biomass and Azadirachtin production in the liquid culture ofA. indica hairy roots

4.5.1 Selection ofthe carbon source 146 4.5.2 Optimization ofthe illumination conditions 149 4.5.3 Optimization ofthe rotational speed (rpm) 151 4.5.4 Optimization of the inoculum concentration and 152 inoculum age

4.5.5 Optimization of the medium components using 155 statistical design protocol

4.5.5.1 Selection of signifficant nutrients affecting 155 growth and Azadirachtin production using Plackett-Burman design

4.5.51 Determination of the optimum concentrations 158 of the selected variables out of the six medium components used in Plackett-Burman design for maximum growth and Azadirachtin production in A. indica hairy roots using Response Surface Methodology (RSM).

4.5.6 Optimization of the pH and temperature using a 162 statistical design protocol

4.6 Exogenous additions in the medium for enhanced 166 Azadirachtin volumetric productivity in the liquid culture ofA. indica hairy roots

4.6. 1 Addition of growth regulators 166 4.6.1 . i Addition ofGibberellic acid (GA3) 166 4.6.1.2 Addition ofAuxins and Abscisic acid 169 4.6.2 Addition ofpermeabilizing agents 172

4.6.3 Addition ofprecursors 176

4.6.4 Addition ofelicitors (biotic and abiotic) 179 4.6.4. 1 Effect of signal molecules i 79 4.6.4.2 Effect ofabiotic elicitors 182 4.6.4.3 Effect ofbiotic elicitors 183

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4.7 Establishment of the hairy root culture kinetics under I 89 optimized cultivation conditions

4.8 Addition of the selected elicitor and precursor under 191 optimized cultivation conditions in the hairy root culture ofA. indica

4.9 Effect of Ethylene addition on the volumetric productivity i 93 ofAzadirachtin in hairy root culture ofA. indica

4.10 Scale-up of the production technology of Azadirachtin 196 fflom the hairy roots ofA. indica

4.10.1 Batch cultivation of A. indica hairy roots on different bioreactor conffigurations for selection of an appropriate bioreactor conffiguration

4.10.1 .i Stined Tank Reactor (STR) 4.10. 1 .2 Bubble Column Reactor (BCR)

4.10.1.3 Modified Bubble Column Reactor (MBCR) (with Polypropylene mesh support)

4. 1 0. 1 .4 Rotating Drum Bioreactor 4.10.1.5 Nutrient Spray Bioreactor 4.10. 1 .6 Nutrient Mist Bioreactor

4.10. 2 Scale-up of the hairy roots in the selected 207 bioreactor conffiguration under optimized cultivation conditions

4. 1 1 Strategies to overcome the mass transfer limitations in the 210 hairy root cultivation

4.11 . i Optimization of the gas phase composition 210

4.11.1.1 Effect of oxygen 210

4.11 .I .2 Effect ofcarbon dioxide 212 4.11.2 Optimization ofthe air flow rate 214 4. 1 1 . 3 Addition of the oxygen vectors 216 4. 1 2 Stability enhancement of Azadirachtin昭ainst 219

atmospheric degradation

4. 1 3 Biopesticidal effficacy in the hairy roots ofA. indica 222 4. 1 4 Qualitative characterization of the Azadirachtin Related 225

Limonoids (AZRL) present in the hairy roots ofA. indica

CHAPTER 5. SUMMARY AND CONCLUSIONS 232-255

5.1 Summary 232

5.2 Conclusions 242

CHAPTER 6. RELATED SCIENTIFIC CONTRIBUTIONS MADE 256-257 DURING THE RESEARCH PERIOD (January, 2005 to

July, 2008)

REFERENCES 258-293

APPENDIX 294-297

RESUME OF THE AUTHOR