Appl Biochem Biotechnol

DOI 10.1007/s12010-011-9531-5

Strategies to Overcome Oxygen Transfer Limitations

During Hairy Root Cultivation of Azadiracta indica
for Enhanced Azadirachtin Production
Smita Srivastava & Ashok Kumar Srivastava

Received: 25 October 2011 / Accepted: 29 December 2011

# Springer Science+Business Media, LLC 2012

Abstract The vast untapped potential of hairy root cultures as a stable source of
biologically active chemicals has focused the attention of scientific community toward
its commercial exploitation. However, the major bottleneck remains its successful
scale-up. Due to branching, the roots form an interlocked matrix that exhibits resistance to oxygen transfer. Thus, present work was undertaken to develop cultivation
strategies like optimization of inlet gas composition (in terms of % (v/v) O2 in air),
air-flow rate and addition of oxygen vectors in the medium, to curb the oxygen
transfer limitations during hairy root cultivation of Azadirachta indica for in vitro
azadirachtin (a biopesticide) production. It was found that increasing the oxygen
fraction in the inlet air (in the range, 20100% (v/v) O2 in air) increased the
azadirachtin productivity by approximately threefold, to a maximum of 4.42 mg/L
per day (at 100% (v/v) O2 in air) with respect to 1.68 mg/L per day in control (air
with no oxygen supplementation). Similarly, increasing the air-flow rate (in the range,
0.32 vvm) also increased the azadirachtin productivity to a maximum of 1.84 mg/L
per day at 0.8 vvm of air-flow rate. On the contrary, addition of oxygen vectors (in
the range, 14% (v/v); hydrogen peroxide, toluene, Tween 80, kerosene, silicone oil,
and n-hexadecane), decreased the azadirachtin productivity with respect to control
(1.76 mg/L per day).
Keywords Hairy roots . Oxygen . Air-flow rate . Oxygen vectors . Azadirachtin . Growth .
Productivity

S. Srivastava
Department of Biotechnology, Indian Institute of Technology Madras, Chennai 600 036, India
A. K. Srivastava (*)
Department of Biochemical Engineering and Biotechnology, Indian Institute of Technology Delhi,
New Delhi 110 016, India
e-mail: ashokiitd@hotmail.com
A. K. Srivastava
e-mail: ashokks@dbeb.iitd.ac.in

Appl Biochem Biotechnol

Introduction
The vast untapped potential of hairy root cultures as a stable source of biologically active
chemicals has focused the attention of the scientific community for its exploitation. However,
the major bottleneck in mass cultivation of hairy roots has been severe oxygen transfer
limitations observed during its scale-up. Hairy roots are heterotrophic, respiratory organisms
that rely on oxygen for energy generation and other metabolic functions. Substantial progress
has been made in understanding the challenging mechanisms of oxygen limitation and transfer
for large-scale growth of the hairy root cultures [1]. Because of the solid phase nature of the
roots and the development of oxygen gradients within the root tissues, relatively small
reductions in the dissolved oxygen concentration in the medium can lead to a significant
decrease in the growth rate of the culture and may also affect the synthesis of certain secondary
metabolites. In fact, hairy roots can be oxygen limited even in shake flask cultures [2].
Restriction of nutrient oxygen delivery to the central mass of tissue in a rotary shake flask
gives rise to a pocket of senescent tissues. Mass transfer resistances near the liquid and solid
boundary affect the oxygen delivery to the growing hairy roots [3]. The efficient exchange of
gases between the roots and their environment is one of the biggest challenges in high density
culture of transformed roots. The gaseous composition in plant tissue culture is an important
factor affecting the plant physiology [4]. Oxygen supply to the roots growing in liquid becomes
more limited due to low oxygen solubility in aqueous media used for its growth [58]. Since
oxygen is only sparingly soluble in water (0.25 mM/L at 25 C. 1 atm, 21% O2 in the air), it is
necessary to accelerate the diffusion of oxygen into the aqueous phase to meet the demand of
actively growing tissue or cells [9]. Thus, proper mass transfer of oxygen from the gaseous to
the liquid phase is an essential requirement in the liquid culture (cultivation) of hairy roots.
There are several literature reports of oxygen-use kinetics for root cultures, which suggest that
the optimal cultivation conditions exist at medium dissolved oxygen (DO) levels greater than
saturated ambient conditions [10, 11]. Oxygen supply is also found to be significantly effective
for secondary metabolite accumulation in plant cell cultures. In a report, partial pressure of
oxygen was also found to have an impact on cell growth and metabolite production by Panax
notoginseng cells in 1 L airlift bioreactors [12].
One of the elegant ways by which the oxygen transfer in the medium can be enhanced is by
modifying the composition of the oxygen in the gaseous phase. Thus, the present study was
undertaken in which the effect of a mixture of pure oxygen and air (where the % (v/v) of pure
oxygen in the mixture varied as 40%, 60%, 80%, and 100%) was investigated on biomass and
azadirachtin production in the hairy root culture of Azadirachta indica as model system.
Aeration is required in the liquid cultures of hairy roots to meet the demand of essential
gases (O2 and CO2) for carrying out various metabolic functions in the cells of the tissue.
However, excessive aeration may also become detrimental to the root growth and secondary
metabolite production [13]. Hence, the optimization of the air supply rate can prove to be
more effective in the successful in vitro mass cultivation of the hairy roots. High oxygen
demand during the hairy root cultivation can be met by increasing the stirrer speed and/or the
air supply rate. However, this can cause mechanical stress to the tissues and hence lead to a
non-productive process. Enrichment of air with pure oxygen can be an attractive alternative
but this also would increase the process cost significantly. Thus, a promising approach to
resolve this problem can be the use of oxygen carriers (vector) in the medium with high
oxygen solubility. Oxygen vectors are defined as such hydrophobic liquids in which oxygen
has higher solubility than in water. It has been observed that the addition of a non-aqueous
organic phase may induce a significant increase of the oxygen transfer rate from air to cells
without requiring a supplementary intensification of mixing [14]. Thus, these substances

Appl Biochem Biotechnol

stand a good chance of enhancing the oxygen availability to the growing tissue in aqueous
liquid medium. In the present study, the effect of the addition of different oxygen vectors in
the concentration range of 1 to 4% (v/v) was hence investigated on the growth (biomass) and
azadirachtin production in the liquid culture of the A. indica hairy roots as model systems.

Materials and Methods

Development of the Hairy Root Culture of A. indica
Log-phase Agrobacterium rhizogenes cultures (108 cell ml1) grown in yeast mannitol broth
(YMB) at 220 rpm and 28 C was used for infection. A. indica explants were submerged in
5 ml of the bacterial culture containing acetosyringone (100 M) for 5 min. The treated
explants were incubated for co-cultivation at 25 C in 16/8 hL/D photoperiod for 48 h on
different combinations of Murashige and Skoog (MS) [15] and/or Gamborgs (B5) [16]
medium. Subsequent subculturing of the infected explants was done under same incubation
conditions on the solid medium with graded antibiotic concentration (250 to 100 mg l1
cefotaxime) to prevent the overgrowth and subsequently no growth of bacteria until hairy
roots emerged (in 1520 days) from the site of infections. After initiation and growth for 20
25 days, each root was excised and was maintained separately as one individual transformed
hairy root line. Among the various hairy root lines induced, six relatively fast growing root
lines were selected (Az-35, Az-122, Az-104, Az-129, Az-86, Az-147) and analyzed for
growth (in terms of growth index (G.I): (Final FWInitial FW)/Initial FW) (FW: fresh
weight) and metabolite accumulation (azadirachtin: in milligrams per gram) after 40 days of
liquid culture. Further, to confirm the transformed nature of the selected hairy root line,
polymerase chain reaction (PCR) technique was used to locate T-DNA (rol A gene)
incorporation in the host plant chromosomal DNA.
Establishment of Growth and Production Kinetics in Shake Flask Hairy Root Cultivation
Growth and azadirachtin production kinetics in A. indica hairy root culture in liquid medium
was established in 250-mL Erlenmeyer flasks rotating on a gyratory shaker at 60 rpm
containing 38 mL of the liquid MM2 medium of the following composition: MS medium
major salts+MS medium minor salts+B5 medium vitamins+30 g/L sucrose. The hairy root
inoculum (3 g/L dry weight (DW)) used in the study was prepared by growing the hairy
roots on solidified MM2 medium (with 0.75% (w/v) Agar) for 30 days. Temperature was
maintained at 25 C and the initial pH of the medium was maintained at 5.8. The experiment
was conducted under 16/8 h L/D illumination conditions and the flasks (containing roots)
were harvested at an interval of 5 days up to 40 days for generating the profiles for biomass
production (in grams per liter, on DW basis) and azadirachtin accumulation (content) in
hairy roots (in milligrams per gram) along with its overall production (in milligrams per liter)
with substrate (residual sucrose: in grams per liter) utilization.
Strategies to Overcome the Mass Transfer Limitations in the Hairy Root Cultivation
Effect of Oxygen (O2)
The present study was undertaken in which the effect of a mixture of pure oxygen and air
(where the% (v/v) of pure oxygen in the mixture varied as 40, 60, 80 and 100%) was

Appl Biochem Biotechnol

investigated on biomass and azadirachtin production in the hairy root culture of A. indica.
The experiments were done in 1-L reagent bottles rotating on a gyratory shaker at 60 rpm
containing 150 mL of liquid MM2 medium. Temperature was maintained at 25 C and initial
pH of the medium was set at 5.8. Inoculum was prepared by growing the roots on solidified
MM2 medium (with 0.75% (w/v) Agar). The inoculum size used was 3 g/L (DW) with a
subculture age of 30 days. Experiments were conducted under 16/8 h L/D illumination
conditions. The mixture of pure oxygen and air was fed inside the liquid medium (in the
reagent bottle) at a flow rate of 0.6 volume of air per working volume per minute (vvm) and
its effect on biomass (in grams per liter, DW) and azadirachtin accumulation in hairy roots
(in milligrams per gram) was analyzed to achieve maximum azadirachtin production (in
milligrams per liter) and its equivalent volumetric productivity (in milligrams per liter per
day) after 25 days of cultivation period.
Effect of Air-Flow Rate
The effect of air-flow rate (0.3 to 2.0 vvm) on the growth and azadirachtin accumulation in the hairy root culture of A. indica was investigated. The experiments were done
in 1 L reagent bottles rotating on a gyratory shaker at 60 rpm containing 150 mL of
liquid MM2 medium. Temperature was maintained at 25 C and initial pH of the
medium was set at 5.8. Inoculum was prepared by growing the roots on solidified
MM2 medium (with 0.75% (w/v) Agar). The inoculum size used was 3 g/L (DW) with
a subculture age of 30 days. Experiments were conducted under 16/8 h L/D illumination conditions. The air was bubbled inside the medium at different flow rates (0.3, 0.6,
0.8, 1.0, 2.0 vvm) and roots were harvested after 25 days of cultivation period at each
air-flow rate for the estimation of biomass (in grams per liter, DW) and azadirachtin
accumulation (in milligrams per gram). The results obtained were analyzed for achieving maximum azadirachtin production (in milligrams per liter) and its equivalent
volumetric productivity (in milligrams per liter per day) in 25 days.
Addition of the Oxygen Vectors
The effect of the addition of different oxygen vectors (n-hexadecane, Tween 80, silicone
oil, kerosene, hydrogen peroxide, and toluene) at the concentration levels of 1%, 2%,
and 4% (v/v) was studied on hairy root growth (biomass) and azadirachtin production in
liquid culture of the A. indica hairy roots. Selection of these compounds was done on
the basis of literature which suggests the use of these compounds as oxygen vectors.
Some of these compounds (like n-hexadecane, Tween 80, toluene, etc.) can also act as
cell permeability enhancers, resulting in better mass transfer of substrates across the cell
membrane, supplementing better biomass and secondary metabolite production. Experiments were done in 250-mL Erlenmeyer flasks rotating on a gyratory shaker at 60 rpm
containing 38 mL of MM2 medium with a known concentration of an oxygen vector.
Temperature was maintained at 25 C and initial pH of the medium was set at 5.8.
Inoculum was prepared by growing the roots on solidified MM2 medium (with 0.75%
(w/v) Agar). The inoculum size used was 3 g/L (DW) with a subculture age of 30 days.
Experiments were conducted under 16/8 h L/D illumination conditions. Flasks were
harvested after 25 days for the estimation of biomass (in grams per liter, DW) and
azadirachtin accumulation in hairy roots (in milligrams per gram). The results obtained
were analyzed for achieving maximum azadirachtin production (in milligrams per liter)
and its equivalent volumetric productivity (in milligrams per liter per day) in 25 days.

Appl Biochem Biotechnol

Analytical Methods
Fresh Weight and Dry Weight Estimation of Hairy Root Biomass
The harvested roots were blotted on a filter paper to remove excess water and weighed for
FW estimation and then dried at 35 C until a constant weight was obtained, which was
accounted as the DW of the roots.
Extraction Protocol for Azadirachtin from the Hairy Roots of A. indica
For extraction of intracellular azadirachtin, the dried mass of roots was extracted (twice) with
methanol for 30 minutes (1 g in 10 mL). To the methanolic layer, water was added in the
ratio of 60:40 (v/v). This aqueous methanolic layer was partitioned twice against dichloromethane (with volume equivalent to the methanolic layer). Dichloromethane fractions were
pooled and evaporated under vacuum below 40 C. The dried crude extract of azadiractin
from the hairy roots was redissolved in methanol prior to analysis by HPLC (Agilent
Technologies, USA HP1100). In order to avoid the degradation or chemical conversion of
azadirachtin in the sample to some other analogue, the samples were stored at 20 C.
Quantification of Azadirachtin
The amount of azadirachtin present in the sample was estimated by HPLC (HP1100, Agilent
Technologies, USA) as per the protocol described elsewhere [17]. Separation of azadirachtin
was achieved by using a Novapack C-18 column (2504.6 mm, 45 C). Elution was
performed using acetonitrile/water (10: 90 (v/v)) as the mobile phase at 0.5 mL/min.
Azadirachtin eluted at a retention time of 2.1 minutes was detected at 214 nm by using a
diode array detector. The azadirachtin content of the sample was obtained from a previously
prepared standard graph (Azadirachtin 95%, Sigma, USA; Catalogue No. A-7430).
Residual Sucrose Estimation
Residual sucrose in the medium was estimated by the phenolsulfuric acid method [18]. The
protocol involved the treatment of 1 mL sample with 1 mL 5% phenol and 5 mL concentrated sulfuric acid for 10 min before being placed in a water bath at 30 C for 20 min.
Finally, the absorbance of the treated sample was taken at 490 nm for the total sugar
concentration estimation in the sample using a standard plot of optical density at 490 nm
(O.D490) vs. concentration (in grams per liter).
Viability Estimation of the Hairy Root Tissue Using Tri-phenyl tetrazolium Chloride Assay
Tetrazoliumtrichloride (TTC) dye is reduced to a pink triphenylformazan complex by live
root tissues with a peak absorbance at 520 nm [19]. This biochemical assay depends on the
activity of the mitochondrial respiratory chain in living tissues which reduces tetrazolium
salts to formazan products. Two hundred milligrams of fresh fine hairy roots were cut into 1
to 2 mm long fragments and were put into 10-ml vials containing 6 mL TTC-solution (0.6%
w/v, 2, 3, 5 triphenyltetrazoliumchloride in 0.06 M Na2HPO4KH2PO4 and 0.05% (v/v),
Tween 20). The sample was incubated for 20 hours at 30 C in dark. The formazan product
was extracted for 15 min in 95% (v/v) ethanol (up to 60 C) and the light absorption was
measured at 520 nm on spectrophotometer. The viability of the test samples was expressed

Appl Biochem Biotechnol

as relative viability (as percentage with respect to the control samples) assuming control
samples as 100% viable.
The experiments reported were done in duplicate and average values were reported for
the different parameters analyzed. The variation in the experimental data collected has been
reported in the form of error bars (which depicts absolute amount of variation from the
average data shown on the graph) in the respective figures and e (e: error, i.e. absolute
amount of variation from the average value of the reported parameter) in the respective
tables. Missing error bars depict insufficient data collected.

Result and Discussion

Development and Establishment of the Hairy Root Culture of A. indica
One hundred seventy-five transformed root lines of A. indica could be successfully induced
from different explants of A. indica, when infected by different strains of A. rhizogenes. Out
of the six relatively fast growing hairy root lines, Az-35 root line was finally selected for
further studies because of maximum growth (G.I (1.75)) and azadirachtin content
(3.8 mg g1) in liquid culture (as shown in Table 1). PCR amplification of the rol A gene,
present in the selected hairy root line Az-35 and the Agrobacterium strain (A. rhizogenes
LBA 920) responsible for its induction, was carried out using left and right primers, 5ggatggaactagccggaataa-3 and 5-cgtaggtttgtttagaactgc-3, respectively. The DNA of the
hairy root line Az-35 and the plasmid DNA of A. rhizogenes LBA 920 showed the presence
of rol A gene on PCR amplification, which was otherwise found absent in the untransformed
root line DNA (Fig. 1). This led to the establishment of the transformed nature of the hairy
root line Az-35. The size of the amplified product was found to be between 200 to 300 bp
(equal to the size of the aligned rol A gene sequence found conserved in different strains of
A. rhizogenes during Clustal W analysis) by running it along with a DNA ladder (100 bp)
during agarose gel electrophoresis (Fig. 1).
Growth and Azadirachtin Production Kinetics in Shake Flask Hairy Root Cultivation
In order to study the growth kinetics of the hairy root culture at submerged conditions of
cultivation, shake flask study was carried out with culture conditions as described in the
Materials and Methods section. The specific growth rate of the culture was calculated by
linear regression of the plot of natural logarithms of the cell dry weight versus time. The average
specific growth rate of the hairy root culture was found to be 0.07 day1. The profiles of hairy
root growth and product formation are shown in Fig. 2. The hairy root biomass (in grams per

Table 1 Selection of a fast growing and high azadirachtin

yielding hairy root line

No.

Root line

Growth index

Azadirachtin content (mg/g)

1
2

Az-35

1.75

3.8

Az-122

0.08

3.7

Az-104

0.03

3.3

4
5

Az-129
Az-86

0.05
0.03

1.7
3.2

Az-147

0.08

2.3

Appl Biochem Biotechnol

Fig. 1 Agarose gel analysis of
PCR amplicons to confirm the
transformed nature of the hairy
root line Az-35 (Lane 1 100 bp
DNA ladder; Lane 2 untransformed root DNA of A. indica as
negative control, Lane 3 hairy
root line (Az-35) DNA of
A. indica; Lane 4: plasmid DNA
of A. rhizogenes LBA 920 as
positive control)

300 bp

liter, DW) increased exponentially over a period of 25 days. The azadirachtin concentration (in
milligrams per liter) obtained during the liquid culture of the hairy roots was dependent on both
the biomass production (in grams per liter) and the azadirachtin yield (content; in milligrams per
gram) as per the mathematical relationship given below:
Azadirachtin productionmg=l Biomassg=l  Azadirachtin accumulationmg=g
The azadirachtin production increased from approximately 5.7 to 44 mg/L in 25 days
before decreasing rapidly to 23.3 mg/L after 40 days (Fig. 2). This decrease was related to
the fact that, highest yield of azadirachtin (3.3 mg/g) was obtained after 25 days of growth,
which subsequently decreased rapidly to 1.4 mg/g in 35 days. Also, there was no significant

Fig. 2 Growth kinetics of the hairy root culture in the liquid medium (biomass (in grams per liter) filled
diamond; azadirachtin (in milligrams per liter) filled square; sucrose (in grams per liter) filled circle;
azadirachtin (in milligrams per liter) filled upright triangle)

Appl Biochem Biotechnol

increase in the biomass observed after 25 days (Fig. 2). This synchronized with the high
depletion of sucrose (up to 0.42 g/L) in 25 days from an initial value of 30 g/L. As a result,
due to cumulative effect of no significant increase in the biomass concentration and decrease
in azadirachtin yield after 25 days of growth, maximum azadirachtin concentration (44 mg/L)
could be obtained in 25 days (Fig. 2) which decreased rapidly thereafter. Hence, the growth
cycle period for the hairy root culture could be reduced from 40 to 25 days in order to achieve
maximum azadirachtin production from the in vitro cultivation of A. indica liquid hairy root
culture.
Strategies to Overcome the Mass Transfer Limitations in the Hairy Root Cultivation
Effect of Oxygen (O2)
Oxygen is one of the most important nutrients for cells, being a major factor in all aerobic
metabolic cycles. However, it is often the limiting nutrient for successful in vitro tissue
growth [20]. In the present study, supplementation of pure oxygen in the ambient air
(control) bubbled inside the medium significantly affected the biomass and azadirachtin
production in the hairy root culture of A. indica. As shown in Fig. 3, the total biomass
increased to a maximum of 15.3 g/L at 60% (v/v) of pure oxygen addition in the air after
which it subsequently reduced at higher levels of oxygen addition (the biomass reduced to a
minimum of 12 g/L at 100% (v/v) fraction of pure oxygen in a mixture with ambient air).
This decrease in biomass was presumably due to cytotoxicity. Cytotoxicity has been reported
due to oxidative stress caused by high oxygen concentrations in the medium [21], which
could have led to the reduction in the total biomass obtained with the increase in the oxygen
concentration in air (above 60% (v/v) of pure oxygen mixture with air). However, the
azadirachtin content in hairy roots (in milligrams per gram) was found to increase with the
increase in oxygen concentration in air (from 3.2 mg/g azadirachtin in control (with no
supplementation of oxygen in the ambient air supplied) to 5.9 mg/g in pure oxygen (100%
(v/v) oxygen in the inlet gas); Fig. 3). Similar to the results obtained in the present study,

Fig. 3 Effect of oxygen on biomass production and azadirachtin accumulation in hairy root culture of
A. indica

Appl Biochem Biotechnol

increase in the artimisinin production has been reported by Kim et al. [22] in oxygen-rich
cultures of Artemisia annua hairy roots. The increase has been related to artimisinin being a
highly oxygenated molecule and also that the genes of the terpenoid biosynthetic pathway
might get affected in oxygen-rich cultures [23]. This hypothesis might also hold true for
azadirachtin, for it being a highly oxygenated triterpenoid. A decrease in the lag phase of the
culture was also observed with supplementation of pure oxygen in the ambient air supplied.
Moreover, the increase in the average growth rate of the hairy roots was also evident from
the reduced growth cycle period observed (on complete utilization of the nutrient medium;
Table 2). The increase in the average growth rate of the culture with the increased oxygen
concentration in air was related to the increased mass transfer of oxygen to the liquid
medium which reduces the oxygen transfer limitations in liquid culture of the hairy roots
(as suggested by Curtis, [24]). As can be observed in Table 2, maximum azadirachtin
production (70.8 mg/L) and volumetric azadirachtin productivity (4.43 mg/L per day) was
achieved when pure oxygen (100% (v/v) of oxygen in the inlet gas) was fed in the medium.
Effect of Air-Flow Rate
As shown in Fig. 4 increasing the air-flow rate from 0.3 to 0.8 vvm resulted in the increase of
the biomass production to a maximum of 13.3 g/L at 0.8 vvm. Above 0.8 vvm of air-flow
rate, the growth of the culture was detrimentally affected which was evident from the
decreasing values of the biomass obtained after 25 days of the cultivation period (to a
minimum of 8.5 g/L) with the increasing air-flow rate up to 2 vvm. However, between 0.6 to
0.8 vvm, no significant effect of air was observed on the growth. This was evident from the
fact that there was no significant difference found in the biomass production obtained
(13.1 g/L) at 0.6 vvm and (13.3 g/L) at 0.8 vvm after 25 days of the cultivation period.
Similarly, the azadirachtin content in the hairy roots was also found to increase with the
increasing air-flow rate from 0.3 to 0.8 vvm (with a maximum of 3.46 mg/g at 0.8 vvm) after
which it subsequently reduced at higher air-flow rates (to a minimum of 1.39 mg/g at
2 vvm). As can be observed in Fig. 4, maximum azadirachtin production of 46.02 mg/L
in 25 days of the cultivation period equivalent to an overall volumetric productivity of
1.84 mg/L per day was thus obtained at 0.8 vvm. The volumetric gas flow rate is a
particularly important parameter affecting oxygen transfer rates, degree of turbulence, heat
transfer, mixing and broth recirculation rates [25]. As a result inadequate air supply can
hinder the growth and secondary metabolite production in the hairy root culture by detrimentally affecting these factors. Moreover, the decrease observed at lower flow rates of air
could also be presumably due to the demand of the essential gases being more than supply to
the actively growing culture. The decrease in the biomass and azadirachtin production
observed at high air-flow rates was related to the shear stress associated, which was evident
Table 2 Effect of oxygen on the
growth cycle period and on
azadirachtin volumetric
productivity in the hairy root
culture of A. indica

% (v/v) pure oxygen Growth cycle Azadirachtin Azadirachtin

period (days) (avg mg/L) (mg/L per day)
in a mixture with
ambient air
40

22

51.2

2.32

60

20

62.7

3.14

80
100

18
16

51.6
70.8

2.87
4.42

control

25

42.0

1.68

Appl Biochem Biotechnol

Fig. 4 Effect of air-flow rate on biomass and azadirachtin accumulation in the hairy root culture of A. indica

from the significant loss in the viability of the tissue (65% viability observed at 2 vvm in
comparison to that obtained at 0.8 vvm). Moreover, the stripping of key volatiles and gases
like CO2 and ethylene from the culture medium at high air-flow rates [13] could also be
responsible for the inhibition in the growth and secondary metabolite production observed at
high air-flow rates.
Addition of the Oxygen Vectors
As can be observed in Table 3, in the concentration range of oxygen vectors studied (1% (v/v) to
4% (v/v)), the growth response observed, in general, varied from low biomass production (in
n-hexadecane, Tween 80, kerosene) to zero growth (in hydrogen peroxide and toluene) after
25 days of the cultivation period with respect to control (13.3 g/L in 25 days with no oxygen
vector present in the medium). Similarly, the secondary metabolite (azadirachtin) accumulation
(content, in milligrams per gram) was also in general detrimentally affected with respect to
control (3.3 mg/g) by the addition of these compounds (n-hexadecane, Tween 80, silicone oil,
hydrogen peroxide and toluene) in the medium (Table 3). The fact that some of these
compounds (like n-hexadecane, Tween 80, hydrogen peroxide, etc.) are also known to act as
cell permeability enhancers could be related to the decrease in biomass and azadirachtin yield
obtained in comparison to control. The increase in cell permeability by the addition of these
compounds to an extent which is detrimental/ irreversible can lead to cell death/viability,
thereby resulting in less biomass and secondary metabolite production. However, it was
observed in the present investigation (as shown in Table 3) that silicone oil increased the
biomass production (to 14.3 g/L) with respect to control (13.3 g/L) at lowest concentration
studied (1% (v/v)). On the contrary, its addition decreased the azadirachtin accumulation
(content) in the hairy roots (from 2.8 to 1.9 mg/g) with respect to control (3.3 mg/g) in the
entire concentration range studied (from 1% (v/v) to 4% (v/v), respectively). Hence, despite the
increase in the biomass production to 14.3 g/L at 1% (v/v) silicon oil addition with respect to the
control (13.3 g/L), the overall azadirachtin production (40.04 mg/L) achieved in 25 days of the
cultivation period (equivalent to an overall volumetric productivity of 1.6 mg/L per day) could
not be increased from that obtained in control (with 44 mg/L of azadirachtin production in
25 days equivalent to an overall volumetric productivity of 1.76 mg/L per day). Similarly, as

Appl Biochem Biotechnol

Table 3 Effect of the oxygen vectors on the biomass and azadirachtin production in the hairy root culture of
A. indica
Oxygen vectors added

n-Hexadecane

Concentration
(% v/v)

Biomass
(g/L)

Azadirachtin
(mg/g) (avg mg/L)

Azadirachtin productivity
(mg/L per day)

9.00.2

1.040.23

9.36

0.37

7.3

0.85

6.20

0.24

6.01.1

0.640.01

3.84

0.15

Tween 80

1
2

6.00.9
5.81.2

1.510.34
1.090.16

9.06
6.32

0.36
0.25

4.00.4

0.60.3

2.40

0.09

Silicone oil

14.30.9

2.80.5

40.04

1.60

13.80.8

2.10.2

28.98

1.15

8.51.2

1.90.1

16.15

0.64

7.3

3.9

28.47

1.13

5.00.6

2.90.3

14.50

0.58

Kerosene

Hydrogen
peroxide

4
1

3.81.0
1.70.4
6.46
0.25
No growth observed (death of the tissue)

2
4

Toluene

No growth observed (death of the tissue)

2
4
Control (no oxygen
vector)

13.3

3.3

43.89

1.76

shown in Table 2, kerosene addition at the lowest concentration studied (1% (v/v)) did increase
the azadirachtin content in the hairy roots (to 3.9 mg/g) with respect to control (3.3 mg/g) but at
higher concentrations (2% (v/v) to 4% (v/v)) it subsequently reduced the azadirachtin accumulation in the hairy roots (to a minimum of 1.7 mg/g at 4% (v/v) concentration) as compared to
control (3.3 mg/g). The biomass production was detrimentally affected (from 7.3 g/L to 3.8 g/L)
with respect to control (13.3 g/L) by the addition of kerosene oil (at 1% (v/v) to 4% (v/v)
concentration, respectively) in the medium. Hence, despite the increase in the azadirachtin
content of the hairy roots (to 3.9 mg/g) with respect to control (3.3 mg/g) at 1% (v/v) kerosene
oil, the overall azadirachtin production (28.47 mg/L) obtained in 25 days (equivalent to an
overall volumetric productivity of 1.13 mg/L per day) could not be increased as opposed to that
in control (with 44 mg/L of azadirachtin production in 25 days equivalent to an overall
volumetric productivity of 1.76 mg/L per day). The inhibition in the growth and secondary
metabolite production observed, in general, by the addition of these agents could be related to
their ability to cause toxicity to plant cells above a certain concentration level [26, 27]. The other
presumable reason could be the decrease of the gasliquid mass transfer coefficient by the
addition of these agents (as suggested by Dumont et al. [28], in one of the related studies).

Conclusion
Oxygen supplementation (40% to 100% (v/v)) in the ambient air sparged inside the liquid
medium was optimized to obtain maximum azadirachtin volumetric productivity (in

Appl Biochem Biotechnol

milligrams per liter per day) in the hairy root culture of A. indica. It was found that
increasing the oxygen fraction in air increased the azadirachtin productivity to a maximum
of 4.42 mg/L/day (at 100% (v/v) oxygen in air) with respect to 1.68 mg/L per day in control
(air with no oxygen supplementation). The increase in the average growth rate of the culture
with the increased oxygen concentration in inlet air was based on the increased oxygen
transport rate (OTR) obtained due to higher oxygen concentration gradient created between
the gas and liquid phase. Increased OTR facilitated increased availability of dissolved
oxygen to the culture suspended in liquid medium. However, use of pure oxygen in the
strategy can lead to significant enhancement in the production cost at large scale. Thus, in
order to increase the dissolved oxygen content in the medium without sparging pure oxygen
gas, chemicals acting as oxygen vectors (auxiliary liquids immiscible in the aqueous phase
in which oxygen is highly soluble, like hydrogen peroxide, toluene, Tween 80, kerosene,
silicone oil and n-hexadecane) were added in the medium. However, no appreciable increase
either in the biomass production or in the azadirachtin content in the hairy roots as opposed
to that in control (with no oxygen vector added) could be observed by their addition. The
results demonstrate that further concentration optimization is required to be done to exploit the
benefit of oxygen vectors in hairy root cultivations. Similarly, in the range (0.3 to 2 vvm) of airflow rate studied, maximum production of biomass (13.3 g/l) and azadirachtin (46 mg/l in
25 days) could be obtained at an air-flow rate of 0.8 vvm. Increasing the air-flow rate further,
reduced the growth of the hairy root culture presumably due to increasing shear stress and
stripping of essential volatiles like CO2 and ethylene from the medium.
From the studies done, it can also be postulated that an integrated strategy involving
oxygen vector addition (at optimum concentration level) with supplementation of pure
oxygen at low concentration in inlet air sent at an optimum flow rate can result in high
azadirachtin productivity in A. indica hairy root culture with relatively lower increase in the
production cost.

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