Appl Biochem Biotechnol (2015) 177:373–388 DOI 10.1007/s12010-015-1750-8
Use of Model-Based Nutrient Feeding for Improved Production of Artemisinin by Hairy Roots of Artemisia Annua in a Modified Stirred Tank Bioreactor Nivedita Patra 1,2 & Ashok K. Srivastava 1
Received: 8 April 2015 / Accepted: 6 July 2015 / Published online: 24 July 2015 # Springer Science+Business Media New York 2015
Abstract Artemisinin has been indicated to be a potent drug for the cure of malaria. Batch growth and artemisinin production kinetics of hairy root cultures of Artemisia annua were studied under shake flask conditions which resulted in accumulation of 12.49 g/L biomass and 0.27 mg/g artemisinin. Using the kinetic data, a mathematical model was identified to understand and optimize the system behavior. The developed model was then extrapolated to design nutrient feeding strategies during fed-batch cultivation for enhanced production of artemisinin. In one of the fed-batch cultivation, sucrose (37 g/L) feeding was done at a constant feed rate of 0.1 L/day during 10– 15 days, which led to improved artemisinin accumulation of 0.77 mg/g. The second strategy of fed-batch hairy root cultivation involved maintenance of pseudo-steady state sucrose concentration (20.8 g/L) during 10–15 days which resulted in artemisinin accumulation of 0.99 mg/g. Fed-batch cultivation (with the maintenance of pseudosteady state of substrate) of Artemisia annua hairy roots was, thereafter, implemented in bioreactor cultivation, which featured artemisinin accumulation of 1.0 mg/g artemisinin in 16 days of cultivation. This is the highest reported artemisinin yield by hairy root cultivation in a bioreactor. Keywords Artemisia annua . Artemisinin . Batch cultivation . Fed-batch cultivation . Mathematical model
* Ashok K. Srivastava
[email protected];
[email protected] 1
Department of Biochemical Engineering and Biotechnology, Indian Institute of Technology Delhi, New Delhi 110016, India
2
Present address: Department of Biotechnology and Medical Engineering, National Institute of Technology Rourkela, Odisha 769008, India
374
Appl Biochem Biotechnol (2015) 177:373–388
Introduction Artemisinin and its derivatives are highly effective for the treatment of both chloroquinesensitive and chloroquine-resistant strains of Plasmodium falciparum which causes cerebral malaria. However, studies on artemisinin monotherapy indicate that there are some cases of artemisinin resistant falciparum strains [1]. Therefore, artemisinin is given in combination with other anti-malarial drugs, e.g., chloroquine. Artemisinin-based combination therapy has been indicated to be effective against both the sexual and asexual stages of malarial parasite in patients. The major source of artemisinin is mainly from the fieldgrown high-yielding cultivars of Artemisia annua plants. Some recent breakthrough production protocols have also been reported in literature using heterologous productionbased techniques wherein artemisinin has been produced from genetically modified yeast/ bacterial fermentation [2] and tobacco [3]. However, the reliability of production protocol and future market of this semi-synthetic artemisinin is still elusive [4]. These results were primarily based on the major advances in the understanding of biosynthesis and phytochemistry of artemisinin and its plant source [5] especially in this post-genomics era [6]. Total chemical synthesis had also been attempted in the early 1980s; however, the process was uneconomic with the result, and several modifications have been reported in the various steps of the process [7]. It has been invariably observed that the artemisinin content in field-grown plants (leaves, flowers, and trichomes mainly) of Artemisia annua is about 1.5 % on dry weight basis [8] and its demand exceeds meagre supply from plant sources. Therefore, there is a desperate need to find out alternative production protocols for mass production of artemisinin at competitive price. Bioreactor hairy root cultivation of A. annua could serve as one such promising production technique for economic mass scale production. This involves induction of hairy root from the plant part, optimization of medium/ environment conditions for shake flask cultivation, and assessment of batch kinetic data for growth and metabolite production. Engineering optimization protocols can then be applied to translate the observed growth and production kinetic data to mathematical model, which can, thereafter, be used to identify strategies of hairy root cultivation for enhanced artemisinin accumulation [9, 10]. Nutrient composition plays a vital role in hairy root propagation for artemisinin accumulation [11]. Sucrose concentration in the growth culture medium is of significant importance as inducer nutrient and osmoregulator [12, 13]. Besides, bioreactor cultivation of hairy roots is reasonably complex, and the problem is further aggravated by the lack of appropriate online sensing devices particularly for the assessment of limiting nutrient/intracellular secondary metabolite concentrations [14]. Fed-batch cultivation eliminates the problem of substrate inhibition and helps in process optimization by gradual feeding of substrate as and when required. However, simple fed-batch cultivations, e.g., repeated fed-batch cultivation for Artemisia annua hairy roots, do not lead to significant improvement in concentration/yield or productivity [15]. Model-based design of nutrient-feeding strategies has lot of potential for reliable process optimization. In the present study, batch growth and product formation kinetics of hairy root cultivation of Artemisia annua was established. A modified Monod’s model was, thereafter, developed, and its process parameters were estimated from the experimental data by minimizing the difference between model simulation and original experimental batch kinetic data of shake flask hairy root cultivation. The model was, thereafter, extrapolated to predict different nutrient-feeding strategies in selected fed-batch
Appl Biochem Biotechnol (2015) 177:373–388
375
cultivations. The most suitable fed-batch cultivation strategy was then experimentally implemented for mass scale hairy root cultivation in shake flasks as well as a liquidphase modified stirred tank bioreactor.
Material and Methods Growth and Artemisinin Production Kinetics in Shake Flask Hairy Root Cultivation Hairy roots were obtained by genetic transformation of Artemisia annua according to a protocol reported in literature [16]. Molecular characterization of hairy roots was done using PCR for rol A (root locus) gene. The confirmation of transformed status for Artemisia annua hairy roots was done by a method reported earlier [17]. The growth conditions of hairy root cultivation were optimized as discussed elsewhere [18]. The statistically optimized media emerging from Plakett-Burman design and response surface methodology was of the following composition: (NO3/NH4+) ratio of 3.5, 0.5 mM KH2PO4, 37.13 g/L sucrose, and 10 μg/L GA3 in (MS/4) medium [11]. Shake flask cultivation was carried out under optimized cultivation conditions [18]. Hairy roots were maintained in 100 ml of optimized media rotating on a gyratory shaker at 70 rpm. Erlenmeyer flasks (liquid-phase culture) were incubated at 25 °C and 16/8 h light/dark conditions. The hairy root inoculum consisted of 1 g/L (dry weight basis) of hairy roots (15-day-old culture of hairy roots grown in liquid MS media (pH 5.6) in shake flasks set at an agitation speed of 70 rpm). The media pH was adjusted to 5.6 before autoclaving. The independent flasks of hairy roots were harvested every 2 days for 20 days and analyzed for biomass (in g/L, on dry weight basis), artemisinin content (in mg/L), and residual substrate (sucrose in g/L).
Development of Mathematical Model for Artemisinin Production The average values and standard deviation of process variables—biomass (x), substrate (s), and product concentrations (p) after every 2 days—were obtained experimentally from shake flask studies (as discussed in section 2.1) from three repeat experiments. It was observed that 20.8 g/L sucrose (out of the initial concentration of 37.5 g/L) was utilized while 16.7 g/L sucrose remained un-utilized after 18 days of cultivation [13] (Fig. 1). Therefore, the actual substrate (S—16.7 g/L) consumed for growth and product formation in the cultivation (calculated by difference for all data points) was used for the model development (Fig. 2). These values of process variables at different times were used to determine the key rates, specific growth rate (μ)/specific substrate consumption rate (qs)/specific product formation rate (qp). The correlations between the specific rates and process variables were then developed to determine the approximate values of the model parameters by graphical approach. The exact values of model parameters were, thereafter, optimized by minimizing the error between model simulations and experimental observations. For this, an objective function SSWR (sum of squares of weighed residues) was minimized by non-linear regression technique using the original algorithm [19] assisted by the methodology and computer program described elsewhere [20]. These optimized values of model parameters were, thereafter, used for model simulations (ymodel). The experimental error between the
376
Appl Biochem Biotechnol (2015) 177:373–388
Fig. 1 Batch growth kinetic studies for biomass production, artemisinin production, and sucrose consumption (reported earlier [13])
experimental data point and the model simulations (ymodel–yexperimental) was evaluated for different process variables at all data points. The mean deviation of any dependent variable and the variance of the residual error were then calculated to assess the statistical validity of the batch model by evaluation of degree of reliability as reported in literature [21] where in the F-test was used to calculate the λ which has the F(m, nm) distribution. The batch kinetic model was extrapolated to fed-batch cultivation by adding the dilution terms in the batch model. The detailed methodology of the model development, its extrapolation to fed-batch cultivation, and statistical validation protocol has been adequately described elsewhere [22].
Fig. 2 Comparison of model simulation (solid lines) with experimental data (points) for biomass production, artemisinin production, and sucrose consumption
Appl Biochem Biotechnol (2015) 177:373–388
377
Essentially for statistical validity test, the mean residual of each variable Δj was calculated as follows: n
Δ j ¼ 1n ∑ Δi j Where Δij is the difference between the experimental value and its correi¼1
sponding model simulation value. and
Δi j ¼ Ymodel −Yexp n ¼ no: of data points
The variance of the error of the residual (Sj) was, thereafter, calculated as follows: 2 1 X Δi j −Δi n−1 i¼1 j ¼ 3ðx; s; pÞ For data characterization, the F-test was used to calculate the λ which had the F(m, distribution: m 2 ðn−mÞn X Δ j λ¼ ðn −1Þm j¼1 S j m ¼ no: of process variables n
Sj ¼
n-m)
On comparison of value of Bλ^ from F-tables, confidence level of the model was identified.
Fed-Batch Cultivation of Artemisia annua Hairy Roots Using Constant Substrate Feed Rate To design nutrient feeding strategy for fed-batch cultivation, the developed model was extrapolated to predict maximum biomass and artemisinin production by the selection of appropriate inlet substrate concentration, its feed rate, and time of feeding. The model simulations for fedbatch cultivation were done offline using different feed rates at high initial substrate concentration. The nutrient feed rate which predicted the maximum product accumulation was selected for experimental fed-batch cultivation at constant feed rate. Since plant cells are subjected to osmotic stress at high substrate concentrations and have low substrate uptake rate, therefore, a reasonably high feed substrate concentration (37.5 g/L) was utilized in the model simulations. Addition of constant feed concentration was identified as 0.01 L/day which was initiated on the 10th day and continued till the 15th day as a single dose. This fed-batch cultivation strategy was adopted during shake flask cultivation. A. annua hairy roots (1 g/L DW basis) were aseptically transferred to 500-ml shake flask containing 100 ml optimized medium. The different shake flasks were incubated at 25±1 °C under 16/8 L/D photoperiod for 15 days on a gyratory shaker maintained at 70 rpm. The value of biomass, artemisinin, and substrate concentration was estimated after every 2 days by harvesting an independent flask till the 15th day. The fed-batch shake flask cultivation was repeated three times, and average values have been reported in this investigation.
Fed-Batch Cultivation Involving Pseudo-Steady State with Respect to Substrate in Shake Flask The model simulations indicated that for high growth of biomass and product formation, it was necessary to ensure non-limiting, non-inhibitory substrate concentration during the cultivation
378
Appl Biochem Biotechnol (2015) 177:373–388
of the hairy roots. This was ensured by feeding the model-simulated substrate feed rate (F) which featured dS/dt=0 (and therefore constant substrate concentration) during 10–15 days of cultivation. Improved biomass and product accumulation was observed by simulation of this cultivation strategy; therefore, it was decided to implement it experimentally (in both shake flask and bioreactor cultivation). It was observed that the optimal concentration of sucrose was 37.5 g/L in the production medium (as identified by statistical media optimization protocol) out of which 16.7 g/L was remaining in the broth. This indicated that the net concentration of sucrose utilized was 20.8 g/L (Fig. 1). It was therefore considered appropriate to maintain a constant (non-limiting non-inhibitory) substrate concentration of 20.8 g/L from the 10th to 15th day (major hairy root growth period) in the shake flask cultivation by addition of medium (substrate concentration S0 =37 g/L and other nutrients) at variable flow rates under sterile conditions. A. annua hairy roots (1 g/L DW basis) were aseptically transferred to 500-ml shake flask containing 100 ml statistically optimized production medium. The different shake flasks were incubated at 25±1 °C under 16/8 L/D photoperiod for 15 days on a gyratory shaker maintained at 70 rpm. The value of biomass, artemisinin, and substrate concentration was estimated after every 2 days by harvesting an independent flask till the 15th day. The fed-batch experiments were repeated three times, and average values have been reported in this investigation.
Batch Cultivation in Modified Stirred Tank Bioreactor (STR) Stirred tank bioreactor utilized in this investigation was a liquid-phase bioreactor with a unique design wherein a low shear setric impeller was used for maintenance of minimum shearing to the growing hairy roots in an otherwise segregated and agitated area. Essentially perforated Teflon mesh was used in the stirred tank bioreactor primarily to separate the growing shear sensitive hairy roots from the highly agitated zone of agitator. A sintered sparger was used in the bioreactor for aeration. The detailed design and salient features of modified stirred tank reactor has been discussed in detail elsewhere [13].
Fed-Batch Cultivation Involving Pseudo-Steady State with Respect to Substrate in Modified Stirred Tank Bioreactor The fed-batch simulation studies in shake flask indicated that maintenance of pseudo-steady state of substrate was the best strategy for enhanced artemisinin production. This strategy was, thereafter, adopted in a 3-L modified stirred tank reactor also (Applikon Dependable Instruments, The Netherlands). The roots were first grown in the batch mode till the 10th day, and thereafter, during 10–15 days, the reactor was maintained at a pseudo-steady state concentration of 20.8 g/L sucrose by programmed feeding of sucrose (37 g/L) at variable flow rates as established by the simulation of the mathematical model to ensure dS/dt=0 (and S=constant) as adequately explained in Section 2.4.
Analytical Methods Medium from the bioreactor was collected under sterile conditions after every 2 days for the analysis of sucrose, nitrate, and phosphate. Hairy root biomass was estimated (on dry cell weight basis) only at the end of cultivation whereas nitrate was estimated by colorimetric method [23], and viability estimation of hairy roots was done using
Appl Biochem Biotechnol (2015) 177:373–388
379
tetrazoliumtrichloride (TTC) dye assay [24]. The phosphate and sucrose were analyzed spectrophotometrically [25, 26]. Extraction protocol and quantification of artemisinin from hairy root culture was as follows. Hairy roots were separated at the end of cultivation and used for estimation of total artemisinin content. Extraction of artemisinin for hairy roots was performed as per the protocol described in the literature [27]. The separation of extracted and derivatized artemisinin samples was done using Agilent 1200 HPLC system, C18 column, and the mobile phase as 0.01 M sodium phosphate buffer and methanol (in 50:50 ratio). The retention time for artemisinin was in the range of 3.5–4.5 min and was the same as that of commercially available artemisinin standard (Sigma, USA).
Results and Discussion Growth and Artemisinin Production Kinetics in Shake Flask Hairy Root Cultivation Figure 1 describes the typical batch growth and product formation kinetics during shake flask cultivation. Artemisinin formation was found to be growth-associated. After 10 days of cultivation, log phase ends and substrate limitation sets in. Thereafter, a decline in rate of biomass formation and artemisinin accumulation was observed. Relatively less sucrose consumption rate was also observed during the stationary phase of growth (after 10 days) leading to accumulation of lower biomass in shake flask. This short growth phase maybe due to uncontrolled pH, accumulation of byproducts due to rapid growth of biomass and inadequate aeration as well as substrate limitation typically observed during batch shake flask cultivation. The final biomass obtained during shake flask cultivation was 12.49 g/L, and artemisinin accumulation was 3.38 mg/L.
Development of Batch Kinetic Model The batch kinetic studies with respect to biomass, artemisinin accumulation, and residual sucrose are shown in Fig. 1 which formed the basis of the development of unstructured mathematical model. The model had the following assumptions: 1. Carbon source (sucrose) was assumed to be the major limiting nutrient while the rest of the medium components were assumed to be available in excess. 2. The temperature and pH was maintained constant throughout the hairy root cultivation. In order to ensure adequate nutrient feeding, it was essential to study the effect of increasing feed of major substrate (carbon) on the growth of hairy roots. This also helped to develop a correlation between increasing substrate feed and specific growth rate (μ vs. So). The complete inhibition (at which μ=0) by the major limiting nutrient (sucrose) was observed to be 116.9 g/L of sucrose. It was also observed that increased initial phosphate concentration did not show any inhibitory effect on specific growth rate (μ). In literature, also increasing the initial phosphate concentration in MS media exhibited no significant effect on artemisinin content [28]. Similarly, nitrate concentration remains non-limiting till stationary phase for Artemisia annua hairy root cultures [10].
380
Appl Biochem Biotechnol (2015) 177:373–388
On the basis of above kinetic and inhibitory data, a model structure for specific growth rate (μ), specific substrate consumption rate (qs), and specific artemisinin production rate (qp) was proposed as follows: 1 dx S K1 ¼ μm μ¼ ð1Þ K1 þ S x dt S þ Ks
qs ¼
1 ds 1 ¼− μþm x dt Y
ð2Þ
1 dp ¼ k1μ þ k2 x dt
ð3Þ
qp ¼
Here, μm refers to maximum specific growth rate (d−1); Ks and KI are saturation and inhibition constants for substrate utilization and product formation, respectively. Sucrose was mainly consumed for growth-associated product formation. The value of specific substrate consumption rate (qs) has been represented by Eq. 2. The negative sign in the equation indicates decrease in substrate from initial values while biomass concentration increases [29]. Yield coefficient (Y) was termed as biomass formed per unit sucrose consumed. Maintenance coefficient (m) was the substrate consumed per unit biomass per unit time for maintenance-related activities of the hairy roots. The specific product formation rate (qp) was expressed as the sum of growth-associated (k1) as well as non-growth-associated (k2) product formation activities of the hairy roots. Table 1 summarizes the values of model parameters which were obtained by non-linear regression using the algorithm and computer program as described in section 2.2. The optimized model parameters were used for the model simulation. Figure 2 describes the comparison of model simulated values of different process variables (X, S, and P) with solid lines against corresponding experimental observations (points). A closer look of Fig. 2 indicates that model was highly successful in the description of experimentally observed hairy root growth and production kinetics, thereby, establishing the validity of the mathematical model. The λ-value was calculated to be 1.99 which was less than the F(3, 7) for 99 % confidence level from the F-tables thereby confirming the validity of the proposed model. In Table 1 Optimized parameters of batch kinetic model
Parameter Definition
Values
μm
Specific growth rate
0.44/day
Ks
Saturation constant for Sucrose
10.3 g/l
KI Y
Inhibition constant for Sucrose Yield of biomass with respect to sucrose
116.9 g/l 0.85 g/g
m
Maintenance coefficient for Sucrose
0.16 g/ g.d
k1
Growth-associated product formation constant 0.93 mg/ g
k2
Non-growth-associated product formation constant
0.0 g/g.d
Appl Biochem Biotechnol (2015) 177:373–388
381
literature, also it has been observed that the model for Artemisia annua features typical Monod’s kinetics behavior [30] as well as typical inhibition of specific growth rate due to high substrate concentration [14]. Similar substrate inhibition phenomena have also been observed for Azadirachta indica plant cell culture systems as well [31].
Fed-Batch Cultivation Involving Pseudo-Steady State with Respect to Substrate Normally, in batch cultivation process, major nutrient limitation was observed on the 10th day. This results in slow growth of hairy root and reduced artemisinin accumulation. Therefore, it necessitated the need of nutrient supplementation. It would be rather interesting to see the consequences of nutrient feeding at a rate which features maintenance of non-limiting and noninhibitory nutrients (particularly pseudo-steady state of substrate). The fed-batch model equations were as follows: F V dx F ¼ μx − x dt V D¼
ð4Þ ð5Þ
" # ds μx F ðs −s0 Þ þ mx − ¼− dt Y x =s V
ð6Þ
dp F ¼ ðk 1 μ þ k 2 Þx − p dt V
ð7Þ
dV ¼F dt
ð8Þ
where BF^ is the substrate (sucrose) flow rate (L/d), BV^ is the total reactor volume (L), Bs0^ is the initial sucrose concentration. Although the product formation was intracellular in nature, the dilution term was used for product formation also, with the assumption that the intracellular product will get diluted due to growth of the hairy roots. Maintenance of constant substrate concentration (20.8 g/L sucrose) during 10–15 days of hairy root cultivation resulted in 16.54 g/L biomass and 16.51 mg/L artemisinin in shake flask which was significantly higher than the corresponding values of batch cultivation. This cultivation strategy was, therefore, experimentally implemented in bioreactor cultivation as well. Figure 3 summarizes the model predicted values of comparison between biomass (X), substrate (S), and product concentration (P) with corresponding experimentally obtained values of X, S, and P. It clearly indicated that the model adequately describes the observed experimental trends of the process variables (X, S, and P) during the entire cultivation period, although the exact match of model simulation with the corresponding experimental data was not observed. It was not expected also primarily due to unstructured nature of the model which assumed that activity of the cell was described by biomass which consisted of both live and dead mass of hairy root cultures. This hypothesis may not be true during the entire cultivation period; however, it was observed that the product (artemisinin) concentration obtained in this cultivation strategy was much better than the corresponding model-predicted values of artemisinin. This deviation from the model-predicted product concentration could be possibly
382
Appl Biochem Biotechnol (2015) 177:373–388
Fig. 3 Comparison of model simulations (solid lines) with experimental data points in fed-batch cultivation using pseudo-steady state concentration for substrate
due to the twin role of sucrose which may be present not only as a nutrient but also as a sensing molecule and osmolite at higher concentration in the growth medium [11]. An overall accumulation of 16.54±0.43 g/L biomass and 0.99 mg/g (16.51 mg/L) artemisinin was observed as compared to accumulation of biomass of 12.49 g/L and 3.38 mg/L artemisinin in batch cultures (control) during the same time period (15 days) which indicated a significant improvement.
Fed-Batch Cultivation of Artemisia annua Hairy Roots Using Constant Substrate Feed Rate For better understanding of behavior of hairy root cultivation, the developed model was simulated on computer at different initial substrate concentrations. These offline computer simulations and independent studies of effect of major limiting nutrient (carbon and nitrogen) clearly indicated that hairy root growth and metabolite accumulation was severely inhibited by higher initial concentrations of major substrate (sucrose). Appropriate nutrient feeding strategy was thereafter identified by computer simulations to eliminate this inhibition. Improvement in biomass and artemisinin accumulation was indicated when sucrose (along with other nutrients in the optimized concentration) was fed at a concentration of 37.5 g/l and at constant rate of 0.1 L/day during 10–15 days. The model simulations featured the production of 20.99 g/L biomass and 16.45 mg/L artemisinin accumulation by this strategy; however, the experimental implementation of this fed-batch cultivation strategy resulted in production of 22.66 g/L biomass and 17.67 mg/L artemisinin. A detailed comparison of model predicted (X, S, and P) with the corresponding experimental values in fed-batch cultivation experiments in shake flask is described in Fig. 4 which indicated that the model successfully described the trends observed for different process variables (X, S, and P). Four repeat experiments were performed along with a parallel batch cultivation control experiment in order to find out the magnitude of improvement between batch and fed-batch cultivation under identical experimental conditions. It was observed that this fed-batch cultivation strategy exhibited 1.8-fold higher biomass and 6.3-fold improvement in artemisinin production as compared to batch cultivation.
Appl Biochem Biotechnol (2015) 177:373–388
383
Fig. 4 Comparison of experimental data (points) and model simulations (solid lines) in fed-batch cultivation using constant feed rate
It was interesting to observe that fed-batch cultivation demonstrated a biomass growth of 22.66 g/L and 17.67 mg/L artemisinin accumulation after 16 days as compared to batch cultivation wherein the hairy root culture featured biomass concentration of 12.49 g/L and 3.38 mg/L artemisinin accumulation during the same time period (16 days). This clearly demonstrates significant improvement both in terms of biomass and artemisinin accumulation in fed-batch cultivation.
Batch Cultivation in Stirred Tank Bioreactor A modified 3-L stirred tank reactor (1.5-L working volume) was used for the mass production of hairy root cultures of Artemisia annua under optimum cultivation conditions. This reactor featured segregation of roots from the agitator of reactor by a perforated Teflon disc. This arrangement ensured adequate nutrient availability to growing hairy roots without any mechanical shearing. High biomass production (dry weight of 18.52±2.01 g/L) along with high artemisinin content (4.63±0.03 mg/L) in the bioreactor was obtained after 28 days of hairy root propagation (Fig. 5). The roots were harvested on 28 days as after 28 days of batch growth stationary phase sets in on the basis of substrate utilization kinetics. The improvement in biomass and artemisinin concentrations may be due to proper mixing and less shearing of hairy roots due to separation from rotating agitator in the bioreactor. The salient features of the batch hairy root cultivation in modified stirred tank reactor have been reported in detail in our recent report [13].
Fed-Batch Cultivation Involving Pseudo-Steady State with Respect to Substrate in Modified Stirred Tank Bioreactor On the basis of fed-batch model prediction as well as batch cultivation studies implemented experimentally in shake flask cultivation and, thereafter, in modified stirred tank bioreactor, it was observed that fed-batch cultivation using pseudo-steady state with respect to substrate
384
Appl Biochem Biotechnol (2015) 177:373–388
Fig. 5 Batch cultivation of hairy root culture in a 3-liter modified stirred tank reactor. a bioreactor setup; b hairy roots obtained after 28 days of growth
concentration was the better (0.99 mg/g artemisinin) cultivation strategy as opposed to constant feed rate (0.78 mg/g) feeding for high substrate consumption and biomass production. This strategy was, therefore, implemented in modified stirred tank bioreactor (and repeated four times) as described in section 2.6. The detailed protocol and experimental setup for fedbatch bioreactor cultivation is shown in Fig. 6. Model-based fed-batch cultivation (pseudosteady state feeding strategy for maintenance of constant substrate) resulted in hairy root growth of 13.68±1.26 g/L and artemisinin content of 1.0 mg/g at the end (16 days) of cultivation in a 3-L modified stirred tank bioreactor. Table 2 summarizes a comparison of bioreactor and shake flask cultivation results of this investigation. It can be concluded from the table that the cultivation of Artemisia annua hairy roots in modified STR using fed-batch cultivation resulted in the maximum artemisinin content of 1.0 mg/g DW. This was comparable to the artemisinin content obtained in fed-batch cultivation in shake flasks of 0.99 mg/g DW and was much higher than the artemisinin content obtained in batch cultivation in shake flask (0.27 mg/g DW) and modified stirred tank batch bioreactor cultivation (0.26 mg/g DW). The average growth rate was calculated as residual biomass (final–initial) per unit initial biomass per day. The average growth rate was also much higher in case of fed-batch cultivation in STR (0.86 days−1) and fed-batch cultivation in shake flask (0.93 days−1) as compared to batch cultivation in STR (0.62 days−1) and shake flask cultures (0.76 days−1) which is the highest reported in literature for large-scale hairy root cultivation. Very few fed-batch cultivation in bioreactor have been reported for hairy roots in bioreactor for the enhancement of secondary metabolite production. In batch and fed-batch cultivation of hairy roots of Beta vulgaris, the betalaine content was increased by 11 % in fed-batch cultivation mode where nutrient medium was fed once or on five different time intervals. It was reported in above studies that 13.3 g/L hairy root biomass was obtained in fed-batch mode in a 3-L bubble column reactor [32]. Recently, fed-batch cultivation based on kinetic model in closed and open loop for hairy roots of Datura innoxia was also reported in shake flask and thereafter validated in bioreactor [33]. The present investigation demonstrates that with the help of mathematical models, better regulation of biological processes at different stages of development can be done [34]. There have been very few reports on mathematical model to describe plant bioprocesses [35–37]. An integrated approach of combining various yield enhancement strategies with mathematical model-based nutrientfeeding strategy for continuous cultivation of cell suspension culture of A. indica was utilized earlier to achieve significantly higher azadirachtin productivity [38]. Measurement and use of on-line sensors for assessment of biomass in plant cell cultivation helped in process optimization [39, 40]. A mathematical model has also been shown to be beneficial for managing the gas
Appl Biochem Biotechnol (2015) 177:373–388
385
Fig. 6 Fed-batch cultivation in modified STR: a Bioreactor setup, b Bioreactor cultivation initiated in the batch mode till the 10th day and, thereafter, in fed-batch mode from day 10–15, and c Hairy root mass harvested from modified STR on the 16th day
phase composition in bioreactor cultivation for better growth and product formation as reported in literature using plant tissue/hairy root culture of A. indica [41]. To enhance the hairy root growth in a mist bioreactor system, an aerosol deposition model had also been proposed [42]. Mathematical modelling predicted that roots in a mist reactor are often too sparsely packed to capture mist particles efficiently and cannot, therefore, meet the nutrient requirements for maintenance of high growth rates. The aerosol deposition model, however, correctly predicted optimization protocol for enhanced hairy root growth in the mist reactor. Table 2 Summary of bioreactor and shake flask cultivation results Type of cultivation
Biomass (g/l)
Artemisinin (mg/g)
Time (day)
Average growth rate (d−1)QX
Volumetric productivity (mg/(l.d))QP
Batch in modified STR
18.52±2.01
0.26
28
0.62
0.16
Fed-batch in modified STR
13.68±1.26
1.0
16
0.86
0.57
Fed-batch shake flask (pseudo-steady state)
16.54±0.43
0.99
16
0.93
1.02
Fed-batch shake flask (constant feed rate)
22.66±0.67
0.77
16
1.35
1.10
Batch (shake flask)
12.49±0.09
0.27
15
0.76
0.22
386
Appl Biochem Biotechnol (2015) 177:373–388
Mathematical models for describing hairy root cultivation are highly complex, and yet a lot of potential exists for the identification of more reliable mathematical models. It is important to mention that the high-yielding hairy root line reported in this investigation can be easily used to supplement the supply of the existing artemisinin production method of natural field cultivation. Using the bioreactor hairy root cultivation technology, it was possible to produce equivalent amount of artemisinin which is normally present in the native plant species. The present investigation reports artemisinin content (13.68 mg/L artemisinin in 16 days of cultivation) which is the highest reported so far with cell/hairy root cultivation in a large-scale bioreactor. A summary of artemisinin content obtained using different reactors has been reported elsewhere by the authors recently wherein the highest artemisinin yield using batch hairy root culture was 10.33 mg/L in 28 days [13]. It is also important to mention here that bioreactor hairy root cultivation production protocol, once optimized, can be implemented throughout the year and can be easily scaled up as well. To the best of our knowledge, this is the first report of development and experimental validation of a model for growth and product formation for mass scale artemisinin production. The model was extrapolated and implemented for cultivation of hairy roots of Artemisia annua to optimize artemisinin accumulation in fed-batch mode of bioreactor cultivation. The investigations reported here not only provides a new approach for enhanced production of artemisinin at reasonably high productivity by model-based fed-batch cultivation but also successfully demonstrates highest average growth rate of hairy roots in large-scale bioreactor cultivation. It is also important to mention here that recently reported semi-synthetic artemisinin production technology [43] by recombinant E. coli (or S. cerevisiae) is for intermediate artemisinic acid as compared to pure artemisinin obtained in hairy root cultivation process reported here. This intermediate has to be converted to artemisinin; therefore, the synthetic production protocol will still take a lot of time for validation of technology before it can replace the existing method of artemisinin production obtained from field-grown seasonal plants.
Conclusion Hairy root cultivation was considered as an alternative production protocol for artemisinin production in this investigation. Batch shake flask cultivation of hairy root featured an accumulation of 12.49 g/L biomass and 0.27 mg/g artemisinin. Mathematical model was then proposed and identified for study of system behavior and process optimization. Computer simulation of the developed model was, thereafter, done to design the time of feeding, concentration, and rate of limiting nutrient during shake flask fed-batch cultivation for enhanced production of artemisinin. Artemisinin accumulation of 0.77 mg/g was experimentally observed in one such model-based fed-batch cultivation strategy which involved feeding of sucrose (37.1 g/L) at a constant feed rate of 0.1 L/D during 10–15 days. While still higher accumulation of artemisinin was observed by maintenance of pseudo-steady state of substrate (sucrose) concentration (20.8 g/L) during 10–15 days of shake flask fed-batch cultivation. Maintenance of the pseudo-steady state of substrate in fed-batch cultivation was used for growth of A. annua hairy roots in bioreactor cultivation, which yielded the highest reported artemisinin accumulation of 1.0 mg/g artemisinin in 16 days of cultivation. Acknowledgments The authors thankfully acknowledge the supply of elite seed material of A. annua from CIMAP Lucknow. The financial support by Ministry of Human Resource Development, New Delhi (India), for the execution of the above project is gratefully acknowledged by one of the authors (Nivedita Patra).
Appl Biochem Biotechnol (2015) 177:373–388
387
References 1. Starzengruber, P., Swoboda, P., Fuehrer, H. P., Khan, W. A., Hofecker, V., Siedl, A., Fally, M., Graf, O., TejaIsavadharm, P., Haque, R., Ringwald, P., & Noedl, H. (2012). Current status of artemisinin-resistant falciparum malaria in South Asia: a randomized controlled artesunate monotherapy trial in Bangladesh. PloS One, 7, 1–7. 2. Arsenault, P. R., Wobbe, K. K., & Weathers, P. J. (2008). Recent advances in artemisinin production through heterologous expression. Current Medicinal Chemistry, 15, 2886–2896. 3. Zhang, Y., Nowak, G., Reed, D. W., & Covello, P. S. (2011). The production of artemisinin precursors in tobacco. Plant Biotechnology Journal, 9, 445–454. 4. Peplow, M. Malaria drug made in yeast causes market ferment, Nature, 2013 Feb 2014;2494(7436):2160– 2011. 5. Brown, G. D. (2010). The biosynthesis of artemisinin (Qinghaosu) and the phytochemistry of Artemisia annua L. (Qinghao). Molecules, 15, 7603–7698. 6. Graham, I. A., Besser, K., Blumer, S., Branigan, C. A., Czechowski, T., Elias, L., Guterman, I., Harvey, D., Isaac, P. G., Khan, A. M., Larson, T. R., Li, Y., Pawson, T., Penfield, T., Rae, A. M., Rathbone, D. A., Reid, S., Ross, J., Smallwood, M. F., Segura, V., Townsend, T., Vyas, D., Winzer, T., & Bowles, D. (2010). The genetic map of Artemisia annua L. identifies loci affecting yield of the antimalarial drug artemisinin. Science, 327, 328–331. 7. Chen, H. J., Han, W. B., Hao, H. D., & Wu, Y. K. (2013). A facile and scalable synthesis of qinghaosu (artemisinin). Tetrahedron, 69, 1112–1114. 8. Kumar, S., Gupta, S. K., Singh, P., Bajpai, P., Gupta, M. M., Singh, D., Gupta, A. K., Ram, G., Shasany, A. K., & Sharma, S. (2004). High yields of artemisinin by multi-harvest of Artemisia annua crops. Industrial Crops and Products, 19, 77–90. 9. Wyslouzil, B. E., Whipple, M., Chatterjee, C., Walcerz, D. B., Weathers, P. J., & Hart, D. P. (1997). Mist deposition onto hairy root cultures: Aerosol modeling and experiments. Biotechnology Progress, 13, 185– 194. 10. Kim, Y., Wyslouzil, B. E., & Weathers, P. J. (2001). A comparative study of mist and bubble column reactors in the in vitro production of artemisinin. Plant Cell Reports, 20, 451–455. 11. Patra, N., Sharma, S., and Srivastava, A. K. (2011) Statistical media optimization for enhanced biomass and artemisinin production in Artemisia annua hairy roots, In Chemistry of Phytopotentials: Health Energy and Environment Perspectives (Srivastava, M. M., Khemani, L. D., and Srivastava, S., Eds.), pp 173–176, Springer-Verlag Berlin Heidelberg 12. Arsenault, P. R., Vail, D. R., Wobbe, K. K., & Weathers, P. J. (2010). Effect of sugars on artemisinin production in Artemisia annua L.: transcription and metabolite measurements. Molecules, 15, 2302–2318. 13. Patra, N., & Srivastava, A. K. (2014). Enhanced production of artemisinin by hairy root cultivation of Artemisia annua in a modified stirred tank reactor. Applied Biochemistry and Biotechnology, 174, 2209– 2222. 14. Lee, J., Lee, S. Y., Park, S., & Middelberg, A. P. (1999). Control of fed-batch fermentations. Biotechnology Advances, 17, 29–48. 15. Kim, Y. J., Weathers, P. J., & Wyslouzil, B. E. (2002). Growth of Artemisia annua hairy roots in liquid- and gas-phase reactors. Biotechnology and Bioengineering, 80, 454–464. 16. Putalun, W., Luealon, W., De-Eknamkul, W., Tanaka, H., & Shoyama, Y. (2007). Improvement of artemisinin production by chitosan in hairy root cultures of Artemisia annua L. Biotechnology Letters, 29, 1143–1146. 17. Srivastava, S., & Srivastava, A. K. (2012). In vitro azadirachtin production by hairy root cultivation of Azadirachta indica in nutrient mist bioreactor. Applied Biochemistry and Biotechnology, 166, 365–378. 18. Patra, N., & Srivastava, A. K. (2014). Mass scale artemisinin production in a stirred tank bioreactor using hairy roots of Artemisia annua. International Journal of Bioscience, Biochemistry and Bioinformatics, 4, 467–474. 19. Rosenbrock, H. H. (1960). An automatic method of finding the greatest or the least value of a function. The Computer Journal, 3, 175–184. 20. Volesky, B., & Votruba, J. (1992). Modelling and optimization of fermentation processes. Amsterdam: Elsevier. 21. Bard, Y. (1974). Nonlinear parameter estimation. New York; London: Academic. 22. Kaur, G., Srivastava, A. K., & Chand, S. (2012). Mathematical modelling approach for concentration and productivity enhancement of 1,3-propanediol using Clostridium diolis. Biochemical Engineering Journal, 68, 34–41. 23. Cataldo, D. A., Haroon, M., Schrader, L. E., & Youngs, V. L. (1975). Rapid colorimetric determination of nitrate in plant-tissue by nitration of salicylic-acid. Communations in Soil Science and Plant, 6, 71–80.
388
Appl Biochem Biotechnol (2015) 177:373–388
24. Brunner, I., Brodbeck, S., & Walthert, L. (2002). Fine root chemistry, starch concentration, and ‘vitality’ of subalpine conifer forests in relation to soil pH. Forest Ecology Management, 165, 75–84. 25. Miller, G. H. (1959). Use of dinitrosalicylic acid reagent for determination of reducing sugar. Analytical Chemistry, 31, 426–428. 26. Murphy, J., & Riley, J. P. (1962). A modified single solution method for determination of phosphate in natural systems. Analytica Chemica Acta, 27, 31–36. 27. Smith, T. C., Weathers, P. J., & Cheetham, R. O. (1997). Effects of gibberellic acid on hairy root cultures of Artemisia annua: growth and artemisinin production. In Vitro Cell Develpment in Plants, 33, 75–79. 28. Paniego, N. B., & Giulietti, A. M. (1996). Artemisinin production by Artemisia annua L.-transformed organ cultures. Enzyme and Microbiol Technology, 18, 526–530. 29. Shuler, M. L., & Kargi, F. (2002). Bioprocess engineering-basic concepts (2nd ed.). New Jersey, USA: Pearson Prentice Hall. 30. Monod, J. (1942). Recherches sur la Croissance des Cultures Bacte’riennes. Paris: Herman. 31. Prakash, G., & Srivastava, A. K. (2006). Modeling of azadirachtin production by Azadirachta indica and its use for feed forward optimization studies. Biochemical Engineering Journal, 29, 62–68. 32. Pavlov, A., Georgiev, M., & Bley, T. (2007). Batch and fed-batch production of betalains by red beet (Beta vulgaris) hairy roots in a bubble column reactor, Zeitschrift fur Naturforschung. C, Journal of biosciences, 62, 439–446. 33. Mairet, F., Villon, P., Boitel-Conti, M., & Shakourzadeh, K. (2010). Modeling and optimization of hairy root growth in fed-batch process. Biotechnology Progress, 26, 847–856. 34. De Vos, D., Dzhurakhalov, A., Draelants, D., Bogaerts, I., Kalve, S., Prinsen, E., Vissenberg, K., Vanroose, W., Broeckhove, J., & Beemster, G. T. (2012). Towards mechanistic models of plant organ growth. Journal of Experimental Botany, 63, 3325–3337. 35. Maschke, R. W., Geipel, K., & Bley, T. (2015). Modeling of plant in vitro cultures—overview and estimation of biotechnological processes. Biotechnology and Bioengineering, 112(1), 1–12. 36. Choi, Y.-E., Kim, Y.-S., and Paek, K.-Y. (2006) Types and designs of bioreactors for hairy root culture, In Plan Tissue Culture Engineering (Gupta, S. D., and Ibaraki, Y., Eds.), pp 161–172, Springer Netherlands. 37. Choi, J. W., Cho, G. H., Byun, S. Y., & Kim, D. I. (2001). Integrated bioprocessing for plant cell cultures. Advances in Biochemical Engineering/Biotechnology, 72, 63–102. 38. Prakash, G., & Srivastava, A. K. (2011). Integrated yield and productivity enhancement strategy for biotechnological production of azadirachtin by suspension culture of Azadirachta indica. Asia-Pacific Journal of Chemical Engineering, 6, 129–137. 39. Chattopadhyay, S., Bisaria, V. S., Scheper, T., & Srivastava, A. K. (2002). Non-invasive methods for determination of cellular growth in Podophyllum hexandrum suspension cultures. Biotechnology and Bioprocess Engineering, 7, 331–334. 40. Srivastava, S., Harsh, S., & Srivastava, A. K. (2008). Use of NADH fluorescence measurement for on-line biomass estimation and characterization of metabolic status in bioreactor cultivation of plant cells for azadirachtin (a biopesticide) production. Process Biochemistry, 43, 1121–1123. 41. Palavalli, R. R., Srivastava, S., & Srivastava, A. K. (2012). Development of a mathematical model for growth and oxygen transfer in in vitro plant hairy root cultivations. Applied Biochemistry and Biotechnology, 167, 1831–1844. 42. Wyslouzil, B. E., Whipple, M., Chatterjee, C., Walcerz, D. B., Weathers, P. J., & Hart, D. P. (1997). Mist deposition onto hairy root cultures: aerosol modeling and experiments. Biotechnology Progress, 13, 185– 194. 43. Paddon, C. J., Westfall, P. J., Pitera, D. J., Benjamin, K., Fisher, K., McPhee, D., Leavell, M. D., Tai, A., Main, A., Eng, D., Polichuk, D. R., Teoh, K. H., Reed, D. W., Treynor, T., Lenihan, J., Fleck, M., Bajad, S., Dang, G., Dengrove, D., Diola, D., Dorin, G., Ellens, K. W., Fickes, S., Galazzo, J., Gaucher, S. P., Geistlinger, T., Henry, R., Hepp, M., Horning, T., Iqbal, T., Jiang, H., Kizer, L., Lieu, B., Melis, D., Moss, N., Regentin, R., Secrest, S., Tsuruta, H., Vazquez, R., Westblade, L. F., Xu, L., Yu, M., Zhang, Y., Zhao, L., Lievense, J., Covello, P. S., Keasling, J. D., Reiling, K. K., Renninger, N. S., & Newman, J. D. (2013). Highlevel semi-synthetic production of the potent antimalarial artemisinin. Nature, 496, 528–532.
