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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, 20–100% (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,
0.3–2 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, 1–4% (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

1 atm. 60%. and 100%) was investigated on biomass and azadirachtin production in the hairy root culture of Azadirachta indica as model system. The gaseous composition in plant tissue culture is an important factor affecting the plant physiology [4]. Thus. the optimization of the air supply rate can prove to be more effective in the successful in vitro mass cultivation of the hairy roots. 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]. the major bottleneck in mass cultivation of hairy roots has been severe oxygen transfer limitations observed during its scale-up.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. 80%. Hairy roots are heterotrophic. 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%. Enrichment of air with pure oxygen can be an attractive alternative but this also would increase the process cost significantly. proper mass transfer of oxygen from the gaseous to the liquid phase is an essential requirement in the liquid culture (cultivation) of hairy roots. 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]. Thus. Thus. 11]. Hence. There are several literature reports of oxygen-use kinetics for root cultures. In fact. a promising approach to resolve this problem can be the use of oxygen carriers (vector) in the medium with high oxygen solubility. Mass transfer resistances near the liquid and solid boundary affect the oxygen delivery to the growing hairy roots [3]. 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. excessive aeration may also become detrimental to the root growth and secondary metabolite production [13]. respiratory organisms that rely on oxygen for energy generation and other metabolic functions. The efficient exchange of gases between the roots and their environment is one of the biggest challenges in high density culture of transformed roots. this can cause mechanical stress to the tissues and hence lead to a non-productive process. Because of the solid phase nature of the roots and the development of oxygen gradients within the root tissues. 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. High oxygen demand during the hairy root cultivation can be met by increasing the stirrer speed and/or the air supply rate. hairy roots can be oxygen limited even in shake flask cultures [2]. In a report. 21% O2 in the air). 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. Thus. Oxygen supply to the roots growing in liquid becomes more limited due to low oxygen solubility in aqueous media used for its growth [5–8]. Oxygen supply is also found to be significantly effective for secondary metabolite accumulation in plant cell cultures. it is necessary to accelerate the diffusion of oxygen into the aqueous phase to meet the demand of actively growing tissue or cells [9]. However. Restriction of nutrient oxygen delivery to the central mass of tissue in a rotary shake flask gives rise to a pocket of senescent tissues. Since oxygen is only sparingly soluble in water (0.25 mM/L at 25 °C. these substances . 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]. Oxygen vectors are defined as such hydrophobic liquids in which oxygen has higher solubility than in water. However. which suggest that the optimal cultivation conditions exist at medium dissolved oxygen (DO) levels greater than saturated ambient conditions [10.

Materials and Methods Development of the Hairy Root Culture of A. Az-122. In the present study. Az-129.I): (Final FW−Initial FW)/Initial FW) (FW: fresh weight) and metabolite accumulation (azadirachtin: in milligrams per gram) after 40 days of liquid culture. A.Appl Biochem Biotechnol stand a good chance of enhancing the oxygen availability to the growing tissue in aqueous liquid medium.75% (w/v) Agar) for 30 days. 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 Gamborg’s (B5) [16] medium. to confirm the transformed nature of the selected hairy root line. Further. indica Log-phase Agrobacterium rhizogenes cultures (108 cell ml−1) grown in yeast mannitol broth (YMB) at 220 rpm and 28 °C was used for infection. 80 and 100%) was . 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. Az-86. six relatively fast growing root lines were selected (Az-35. each root was excised and was maintained separately as one individual transformed hairy root line. 60. After initiation and growth for 20– 25 days. 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. Az-104. polymerase chain reaction (PCR) technique was used to locate T-DNA (rol A gene) incorporation in the host plant chromosomal DNA. 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 0.8. Temperature was maintained at 25 °C and the initial pH of the medium was maintained at 5. 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. 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. Az-147) and analyzed for growth (in terms of growth index (G. Subsequent subculturing of the infected explants was done under same incubation conditions on the solid medium with graded antibiotic concentration (250 to 100 mg l−1 cefotaxime) to prevent the overgrowth and subsequently no growth of bacteria until hairy roots emerged (in 15–20 days) from the site of infections. 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. Among the various hairy root lines induced. indica explants were submerged in 5 ml of the bacterial culture containing acetosyringone (100 μM) for 5 min. Establishment of Growth and Production Kinetics in Shake Flask Hairy Root Cultivation Growth and azadirachtin production kinetics in A.

Addition of the Oxygen Vectors The effect of the addition of different oxygen vectors (n-hexadecane. Some of these compounds (like n-hexadecane. The experiments were done in 1-L reagent bottles rotating on a gyratory shaker at 60 rpm containing 150 mL of liquid MM2 medium.8.8. 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. supplementing better biomass and secondary metabolite production. Selection of these compounds was done on the basis of literature which suggests the use of these compounds as oxygen vectors. indica. Temperature was maintained at 25 °C and initial pH of the medium was set at 5. Effect of Air-Flow Rate The effect of air-flow rate (0. etc. . and toluene) at the concentration levels of 1%. 1.3 to 2.3. 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. Experiments were conducted under 16/8 h L/D illumination conditions. Inoculum was prepared by growing the roots on solidified MM2 medium (with 0.Appl Biochem Biotechnol investigated on biomass and azadirachtin production in the hairy root culture of A. indica was investigated.0 vvm) on the growth and azadirachtin accumulation in the hairy root culture of A.8.0 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.8. 2.75% (w/v) Agar). toluene. indica hairy roots. The experiments were done in 1 L reagent bottles rotating on a gyratory shaker at 60 rpm containing 150 mL of liquid MM2 medium.75% (w/v) Agar). Temperature was maintained at 25 °C and initial pH of the medium was set at 5. The inoculum size used was 3 g/L (DW) with a subculture age of 30 days.0. 0.6 volume of air per working volume per minute (vvm) and its effect on biomass (in grams per liter. 2%. 0. The inoculum size used was 3 g/L (DW) with a subculture age of 30 days. Temperature was maintained at 25 °C and initial pH of the medium was set at 5. resulting in better mass transfer of substrates across the cell membrane. Tween 80. The inoculum size used was 3 g/L (DW) with a subculture age of 30 days. silicone oil.6. and 4% (v/v) was studied on hairy root growth (biomass) and azadirachtin production in liquid culture of the A. DW) and azadirachtin accumulation in hairy roots (in milligrams per gram).) can also act as cell permeability enhancers. The mixture of pure oxygen and air was fed inside the liquid medium (in the reagent bottle) at a flow rate of 0. Inoculum was prepared by growing the roots on solidified MM2 medium (with 0.75% (w/v) Agar). 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. 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. kerosene. Inoculum was prepared by growing the roots on solidified MM2 medium (with 0. DW) and azadirachtin accumulation (in milligrams per gram). Tween 80. Flasks were harvested after 25 days for the estimation of biomass (in grams per liter. The air was bubbled inside the medium at different flow rates (0. Experiments were conducted under 16/8 h L/D illumination conditions. hydrogen peroxide. Experiments were conducted under 16/8 h L/D illumination conditions.

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.5 mL/min. the dried mass of roots was extracted (twice) with methanol for 30 minutes (1 g in 10 mL). This aqueous methanolic layer was partitioned twice against dichloromethane (with volume equivalent to the methanolic layer). USA HP1100). USA. Tween 20). Extraction Protocol for Azadirachtin from the Hairy Roots of A. The sample was incubated for 20 hours at 30 °C in dark.D490) vs. A-7430). The dried crude extract of azadiractin from the hairy roots was redissolved in methanol prior to analysis by HPLC (Agilent Technologies. Sigma. The azadirachtin content of the sample was obtained from a previously prepared standard graph (Azadirachtin 95%. The viability of the test samples was expressed . Azadirachtin eluted at a retention time of ∼2.06 M Na2HPO4–KH2PO4 and 0.1 minutes was detected at 214 nm by using a diode array detector. USA) as per the protocol described elsewhere [17]. Separation of azadirachtin was achieved by using a Novapack C-18 column (250×4. Agilent Technologies. To the methanolic layer. 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]. In order to avoid the degradation or chemical conversion of azadirachtin in the sample to some other analogue. Catalogue No. Elution was performed using acetonitrile/water (10: 90 (v/v)) as the mobile phase at 0.6 mm. 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. 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. water was added in the ratio of 60:40 (v/v).6% w/v. the samples were stored at −20 °C.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. concentration (in grams per liter). 3. Residual Sucrose Estimation Residual sucrose in the medium was estimated by the phenol–sulfuric acid method [18].05% (v/v). 45 °C). This biochemical assay depends on the activity of the mitochondrial respiratory chain in living tissues which reduces tetrazolium salts to formazan products. Quantification of Azadirachtin The amount of azadirachtin present in the sample was estimated by HPLC (HP1100. Dichloromethane fractions were pooled and evaporated under vacuum below 40 °C. Finally. 2. 5 triphenyltetrazoliumchloride in 0. indica For extraction of intracellular azadirachtin. 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. which was accounted as the DW of the roots.

2 6 Az-147 0.75 3.3 4 5 Az-129 Az-86 0.8 Az-122 0.08 2.7 3. indica could be successfully induced from different explants of A.3 . rhizogenes LBA 920) responsible for its induction.I (1. 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. shake flask study was carried out with culture conditions as described in the “Materials and Methods” section.08 3. rhizogenes LBA 920 showed the presence of rol A gene on PCR amplification. The experiments reported were done in duplicate and average values were reported for the different parameters analyzed. Az-35 root line was finally selected for further studies because of maximum growth (G. 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.75)) and azadirachtin content (3. respectively. This led to the establishment of the transformed nature of the hairy root line Az-35.Appl Biochem Biotechnol as relative viability (as percentage with respect to the control samples) assuming control samples as 100% viable. 2. Out of the six relatively fast growing hairy root lines. when infected by different strains of A. which was otherwise found absent in the untransformed root line DNA (Fig. 1).e.7 3 Az-104 0.8 mg g−1) in liquid culture (as shown in Table 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.03 3. The hairy root biomass (in grams per Table 1 Selection of a fast growing and high azadirachtin yielding hairy root line No. was carried out using left and right primers. The DNA of the hairy root line Az-35 and the plasmid DNA of A. The profiles of hairy root growth and product formation are shown in Fig. Root line Growth index Azadirachtin content (mg/g) 1 2 Az-35 1.03 1.05 0. indica. 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. 5′ggatggaactagccggaataa-3′ and 5′-cgtaggtttgtttagaactgc-3′.07 day−1. rhizogenes during Clustal W analysis) by running it along with a DNA ladder (100 bp) during agarose gel electrophoresis (Fig. PCR amplification of the rol A gene. Missing error bars depict insufficient data collected. absolute amount of variation from the average value of the reported parameter) in the respective tables. Result and Discussion Development and Establishment of the Hairy Root Culture of A. rhizogenes. The average specific growth rate of the hairy root culture was found to be 0. present in the selected hairy root line Az-35 and the Agrobacterium strain (A. 1). indica One hundred seventy-five transformed root lines of A.

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.4 mg/g in 35 days. 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. indica as negative control. in milligrams per gram) as per the mathematical relationship given below: Azadirachtin productionðmg=lÞ ¼ Biomassðg=lÞ  Azadirachtin accumulationðmg=gÞ The azadirachtin production increased from approximately 5. azadirachtin (in milligrams per liter) filled upright triangle) .3 mg/L after 40 days (Fig.7 to 44 mg/L in 25 days before decreasing rapidly to 23. rhizogenes LBA 920 as positive control) 1 2 3 4 300 bp liter. 2). 2 Growth kinetics of the hairy root culture in the liquid medium (biomass (in grams per liter) filled diamond.Appl Biochem Biotechnol Fig. which subsequently decreased rapidly to 1. Lane 4: plasmid DNA of A. sucrose (in grams per liter) filled circle. there was no significant Fig. indica. Lane 2 untransformed root DNA of A. DW) increased exponentially over a period of 25 days.3 mg/g) was obtained after 25 days of growth. Lane 3 hairy root line (Az-35) DNA of A. highest yield of azadirachtin (3. azadirachtin (in milligrams per liter) filled square. Also. This decrease was related to the fact that.

9 mg/g in pure oxygen (100% (v/v) oxygen in the inlet gas). indica liquid hairy root culture. As shown in Fig. However. 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. This decrease in biomass was presumably due to cytotoxicity. In the present study. maximum azadirachtin concentration (∼44 mg/L) could be obtained in 25 days (Fig. 3. 3). the azadirachtin content in hairy roots (in milligrams per gram) was found to increase with the increase in oxygen concentration in air (from 3. 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. 2) which decreased rapidly thereafter. Fig. 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. being a major factor in all aerobic metabolic cycles. Similar to the results obtained in the present study. However. indica . Hence.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). Cytotoxicity has been reported due to oxidative stress caused by high oxygen concentrations in the medium [21]. Fig.42 g/L) in 25 days from an initial value of 30 g/L. due to cumulative effect of no significant increase in the biomass concentration and decrease in azadirachtin yield after 25 days of growth. indica. This synchronized with the high depletion of sucrose (up to 0. it is often the limiting nutrient for successful in vitro tissue growth [20]. 3 Effect of oxygen on biomass production and azadirachtin accumulation in hairy root culture of A. As a result.2 mg/g azadirachtin in control (with no supplementation of oxygen in the ambient air supplied) to 5. 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). the total biomass increased to a maximum of 15.Appl Biochem Biotechnol increase in the biomass observed after 25 days (Fig. 2).

8 vvm of air-flow rate.32 60 20 62. This was evident from the fact that there was no significant difference found in the biomass production obtained (13. 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. 4.8 vvm resulted in the increase of the biomass production to a maximum of 13. 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.42 control 25 42.6 vvm and (13. As can be observed in Table 2. 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]. Effect of Air-Flow Rate As shown in Fig. 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. between 0. Above 0. The volumetric gas flow rate is a particularly important parameter affecting oxygen transfer rates.0 1. no significant effect of air was observed on the growth.6 70.8 vvm after 25 days of the cultivation period. 4 increasing the air-flow rate from 0.Appl Biochem Biotechnol increase in the artimisinin production has been reported by Kim et al. This hypothesis might also hold true for azadirachtin.46 mg/g at 0.1 g/L) at 0.8 2. As can be observed in Fig.87 4.3 g/L) at 0.6 to 0. [24]). [22] in oxygen-rich cultures of Artemisia annua hairy roots. degree of turbulence. maximum azadirachtin production (70.8 vvm. 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. As a result inadequate air supply can hinder the growth and secondary metabolite production in the hairy root culture by detrimentally affecting these factors.2 2.68 .8 vvm (with a maximum of 3. mixing and broth recirculation rates [25].02 mg/L in 25 days of the cultivation period equivalent to an overall volumetric productivity of 1. Similarly.8 vvm) after which it subsequently reduced at higher air-flow rates (to a minimum of 1.8 mg/L) and volumetric azadirachtin productivity (4.3 to 0. 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. However.14 80 100 18 16 51.8 vvm. 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 8.43 mg/L per day) was achieved when pure oxygen (100% (v/v) of oxygen in the inlet gas) was fed in the medium.3 to 0. Moreover. Moreover.84 mg/L per day was thus obtained at 0.7 3.3 g/L at 0. heat transfer.5 g/L) with the increasing air-flow rate up to 2 vvm. maximum azadirachtin production of 46. the azadirachtin content in the hairy roots was also found to increase with the increasing air-flow rate from 0. The decrease in the biomass and azadirachtin production observed at high air-flow rates was related to the shear stress associated. Table 2).39 mg/g at 2 vvm).8 vvm. 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.

Similarly. 4 Effect of air-flow rate on biomass and azadirachtin accumulation in the hairy root culture of A.3 mg/g) by the addition of these compounds (n-hexadecane. Tween 80.3 g/L).3 mg/g) in the entire concentration range studied (from 1% (v/v) to 4% (v/v).3 g/L in 25 days with no oxygen vector present in the medium). despite the increase in the biomass production to 14. the secondary metabolite (azadirachtin) accumulation (content.76 mg/L per day).8 vvm).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. its addition decreased the azadirachtin accumulation (content) in the hairy roots (from 2. as .) 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. hydrogen peroxide and toluene) in the medium (Table 3).Appl Biochem Biotechnol Fig. etc. Addition of the Oxygen Vectors As can be observed in Table 3. kerosene) to zero growth (in hydrogen peroxide and toluene) after 25 days of the cultivation period with respect to control (13.04 mg/L) achieved in 25 days of the cultivation period (equivalent to an overall volumetric productivity of 1. hydrogen peroxide. Similarly. respectively). Hence. Tween 80. the growth response observed. thereby resulting in less biomass and secondary metabolite production.8 to 1. Tween 80.3 g/L) with respect to control (13. it was observed in the present investigation (as shown in Table 3) that silicone oil increased the biomass production (to 14. in general.9 mg/g) with respect to control (3.3 g/L) at lowest concentration studied (1% (v/v)). 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. indica from the significant loss in the viability of the tissue (65% viability observed at 2 vvm in comparison to that obtained at 0. The increase in cell permeability by the addition of these compounds to an extent which is detrimental/ irreversible can lead to cell death/viability.3 g/L at 1% (v/v) silicon oil addition with respect to the control (13. On the contrary. silicone oil. The fact that some of these compounds (like n-hexadecane. in the concentration range of oxygen vectors studied (1% (v/v) to 4% (v/v)). in milligrams per gram) was also in general detrimentally affected with respect to control (3. However. the overall azadirachtin production (40. varied from low biomass production (in n-hexadecane.

60 2 13.1 0.0±0.0±0.15 4 8.6±0.4 6.8±0. kerosene addition at the lowest concentration studied (1% (v/v)) did increase the azadirachtin content in the hairy roots (to 3.1±0.76 mg/L per day).3 3.0±1.3 43.58 Kerosene Hydrogen peroxide 4 1 3.9±0.9±0.3 14.04±0.3 g/L) by the addition of kerosene oil (at 1% (v/v) to 4% (v/v) concentration.25 No growth observed (death of the tissue) 2 4 Toluene 1 No growth observed (death of the tissue) 2 4 Control (no oxygen vector) 13.36 0.8 g/L) with respect to control (13.8±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. The biomass production was detrimentally affected (from 7.36 0.9 mg/g) with respect to control (3.24 4 6.7 mg/g at 4% (v/v) concentration) as compared to control (3.8±1.04 1. The other presumable reason could be the decrease of the gas–liquid mass transfer coefficient by the addition of these agents (as suggested by Dumont et al.23 9.8±0.46 0.3 2.5±1.9 28.25 4 4.2 1.06 6.16 9.09 Silicone oil 1 14. Hence.0 1.3±0.15 Tween 80 1 2 6. respectively) in the medium.37 2 7.Appl Biochem Biotechnol Table 3 Effect of the oxygen vectors on the biomass and azadirachtin production in the hairy root culture of A. in one of the related studies).09±0.3 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 1.9 mg/g) with respect to control (3.3 0.85 6.4 0.5 40. the overall azadirachtin production (28.2 1. 27].01 3. indica Oxygen vectors added n-Hexadecane Concentration (% v/v) Biomass (g/L) Azadirachtin (mg/g) (avg mg/L) Azadirachtin productivity (mg/L per day) 1 9.50 0.64 1 7.2 1.1 16.15 0.6 2.34 1.7±0. by the addition of these agents could be related to their ability to cause toxicity to plant cells above a certain concentration level [26.2 28.47 1.47 mg/L) obtained in 25 days (equivalent to an overall volumetric productivity of 1.98 1.13 2 5.20 0.3 mg/g) at 1% (v/v) kerosene oil. 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 .64±0.0±0.51±0.3 mg/g).76 shown in Table 2.40 0.32 0.84 0. in general.3 g/L to 3.0±0.89 1. The inhibition in the growth and secondary metabolite production observed.9 2.8 2.3 3. [28]. despite the increase in the azadirachtin content of the hairy roots (to 3.9 5.

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