Bioconversion of colchicine into its pharmacologically active derivative 3-demethylated colchicine (3-DMC) mediated by P450BM3 enzyme is an economic and promising strategy for the production of this inexpensive and potent anticancer drug. Continuous stirred tank reactor (CSTR) and packed-bed reactor (PBR) of 3 L and 2 L total volumes were compared for the production of 3-demethylated colchicine (3-DMC) a colchicine derivative using
Bioreactor design is of crucial importance in the development of bioprocesses. Once a microorganism is selected and the culture and/or production conditions are optimized at laboratory scale, the next issue is proceeding to larger scale so that bulk quantities of the desired product can be produced optimally in a cost-effective manner. The selection of a proper reactor type is critical to such processes and plays a major role in large-scale production. The bioreactors widely used in today’s fermentation industry are mainly those developed in the past decades purely for chemical reactions [
Colchicine is a well-documented pseudo alkaloid obtained from
In a rule of thumb, the classical method for determining optimal production conditions in fermentation process is varying in one process parameter while keeping others at a constant level. This practice is time-consuming and generates a need for large number of experiments, and the results are not so accurate and reproducible. In such practice, interaction effect between the variables is not taken into consideration. In recent times, response surface methodology (RSM) a good statistical experimental design tool has proved its worth, and it is now commonly used in optimization experiments of fermentation processes using biological system [
In the present work, we investigated the demethylation of colchicine in a continuous-flow packed-bed reactor in an effort to better understand possible adverse effect of colchicine bioconversion associated with the use of stirred tank reactors. When this experiment was carried out in a packed-bed reactor, the dissolved oxygen was required to be 60% v/v prior to entering the reactor. Because of the necessity of controlling dissolved oxygen and pH and making the process smoother and homogeneous, another fermenter was used to control conditions for the dissolved oxygen and other parameters. It is highly desirable to employ reaction conditions that minimize undesired secondary reactions of the indicated types. Tubular packed-bed reactors offer a number of advantages with higher conversion per unit mass of catalyst, recyclability, continuous operation, and minimum product inhibition when compared to stirred tank reactors.
Shake flask experiments were carried out using 100 mL and 250 mL Erlenmeyer flasks containing 15 mL and 25 mL medium having 7 g/L colchicine respectively. After inoculation, flasks were incubated over night at 28°C, 200 rpm.
The culture was incubated for 72 h in the same conditions as described above, and every 12 h, samples were taken to evaluate the growth level and the 3-DMC production by HPLC [
All the fermenter operational conditions optimization trials were carried out in 5 L fermenter (Sartorious Inc., Germany) for bioconversion experiments [
The packed-bed reactor consisted of 20 cm of tubing (2.5 cm i.d.) containing 25 g of immobilized
Prior to initiating flow of colchicine to the reactor, nitrogen was passed through the packed bed for 5 min to remove air. Each experiment was initiated by quickly flushing the reactor with a total volume of the mixture of substrates equal to at least twice the void volume of the reactor. After quasi-steady-state operating conditions were achieved, several samples of the effluent stream were manually collected over a time frame corresponding to at least three reactor space times. (The reactor space time is the ratio of the void volume of the reactor to the total volumetric flow rate of the two feed stocks.) For a set of experiments corresponding to a specified operating temperature, the experiments at different space times were conducted in random order.
The void volume (1.3 cm3/g of catalyst) was calculated using the difference between the weights of the packed-bed reactor (tubing + catalyst) when filled with a colchicine of known density and the corresponding weight of the packed-bed reactor in the absence of this fluid. Corrections were made for the regions of the tubing outside the packed bed. Reactor space times were calculated as the ratio of the void volume of the reactor to the total volumetric flow rate of the two feedstocks.
The optimization experiments were statistically designed and performed using response surface methodology (RSM) using Design-Expert from Stat-Ease, Inc. DO concentration, substrate concentration, and process time were considered as important variables in the experimental design and had been considered for optimization. The other process components were kept at constant levels throughout the experimental runs. The parameters, for example, DO, substrate conc., and process time, were simultaneously varied as depicted in Table
Design matrix generated by DoE showing response of each run.
Run | A Dissolved oxygen (%) | B Colchicine (g/L) | C Process time (h) | Predicted 3-DMC (g/L) |
---|---|---|---|---|
1 | 50.00 | 5.00 | 48.00 | 4.32 |
2 | 32.50 | 7.50 | 39.00 | 4.60 |
3 | 32.50 | 7.50 | 39.00 | 4.60 |
4 | 15.00 | 5.00 | 48.00 | 1.45 |
5 | 32.50 | 7.50 | 39.00 | 4.60 |
6 | 32.50 | 7.50 | 39.00 | 4.60 |
7 | 32.50 | 11.70 | 39.00 | 3.18 |
8 | 32.50 | 7.50 | 39.00 | 4.60 |
9 | 32.50 | 7.50 | 39.00 | 4.60 |
10 | 50.00 | 10.00 | 30.00 | 4.60 |
11 | 32.50 | 7.50 | 54.14 | 5.10 |
12 | 15.00 | 10.00 | 48.00 | 2.74 |
13 | 50.00 | 10.00 | 48.00 | 7.90 |
14 | 32.50 | 3.30 | 39.00 | 2.43 |
15 | 61.93 | 7.50 | 39.00 | 5.95 |
16 | 15.00 | 10.00 | 30.00 | 0.91 |
17 | 15.00 | 5.00 | 30.00 | 1.12 |
18 | 3.07 | 7.50 | 39.00 | 0.67 |
19 | 32.50 | 7.50 | 23.86 | 2.04 |
20 | 50.00 | 5.00 | 30.00 | 3.10 |
The obtained data in real time was added in each set corresponding to the individual experiments. Design expert uses statistical designs and tools for predicting the significance and accuracy of the model as well as the predicted trends, in the attempted design space. The results were fitted into various RSM models out of which central composite design (CCD) predicted curves with better regression coefficients and nonsignificant lack of fit. Table
Concentration ranges of independent process variables (dissolved oxygen, colchicine concentration, and process time) used in RSM.
Factor | Name | Units | Type | Min. | Max. | −1 actual | +1 actual | Mean | Std. Dev. |
---|---|---|---|---|---|---|---|---|---|
A | DO | (%) | Numeric | 3.07 | 61.93 | 15 | 50 | 32.5 | 14.46 |
B | Colchicine | (g/L) | Numeric | 3.3 | 11.7 | 5 | 10 | 7.5 | 2.07 |
C | Process Time | (s) | Numeric | 31.91 | 52.09 | 36 | 48 | 42 | 4.96 |
The present study, performed for the possible application of PBR system in biotransformed products. Previous researchers have published various research articles on the use of PBR system for biotransformation [
Our previous reports on bioconversion of colchicine,
Before starting the bioconversion experiments on PBR (Figure
Schematic diagram showing biocatalysis process for conversion of colchicine to 3-demethylated colchicine through packed-bed reactor (PBR).
The optimization of bioconversion parameter was analyzed by response surface methodology. Table
ANOVA for response surface quadratic model to verify whether developed model is significant or nonsignificant.
Source | Sum of squares | Degrees of freedom | Mean square |
|
|
---|---|---|---|---|---|
Model | 66.63 | 6 | 11.1 | 176.23 | <0.0001* |
A-DO | 45.96 | 1 | 45.96 | 729.41 | <0.0001 |
B-Colchicine | 13.71 | 1 | 13.71 | 217.54 | <0.0001 |
C-Process time | 3.37 | 1 | 3.37 | 53.51 | <0.0001 |
AB | 2.82 | 1 | 2.82 | 44.76 | <0.0001 |
AC | 0.56 | 1 | 0.56 | 8.83 | 0.0108 |
BC | 0.21 | 1 | 0.21 | 3.3 | 0.0923 |
Residual | 0.82 | 13 | 0.063 | ||
Lack of fit** | 0.82 | 8 | 0.1 |
Response surface methodology showing bioconversion of colchicine to 3DMC with response to dissolved oxygen (DO).
Response surface methodology showing bioconversion of 3DMC with response to dissolved oxygen (DO) and process time.
Comparison of CSTR as well as PBR was performed by taking significant parameters for the study, namely, intial cell conc. (
Performance comparison of STR and PBR of lab scale for the production of 3-DMC at an equal DO level.
Parameters | CSTR | PBR |
---|---|---|
|
1.2 | 25 |
|
180 | 25 |
|
35 | 35 |
Productivity (g/L/h) | 4.78 [ |
6.58 |
Bioconversion time (h) | 72 | 32 |
(CSTR) continuous stirred tank reactor; (PBR) packed-bed reactor;
On the other hand, a set of triplicate experiments were performed in which a proposed PBR system worked out for bioconversion efficiency for continuous six batch experiments in which 35 mM colchicine concentration used initially. Batch-wise experiments were performed (Figure
Showing batch-wise reuse of PBR system having constant flow of colchicine, that is, 35 mM converted into 3-DMC.
The results here presented comprise a new approach to the bioconversion of colchicine into their respective derivative, that is, 3-demethylated colchicine (3-DMC). Furthermore, experimental design has provided an influential tool not only to study but also in optimization of bioconversion conditions that allows a significant enhancement of decisive feature of this process. Scanty reports are available on the PBR as an alternative form for CSTR mainly for the cultivation of the microorganisms which are sensitive to shear stress. In the present study, experimental design has been employed to evaluate the performance of continuous PBR system designed for the attainment of high bioconversion rate. Hence proposed continuous PBR reactor illustrates highly selective 3-DMC production achieved, which is considerably higher (Table
Kashyap Kumar Dubey sincerely acknowledges UIET, M.D. University, for providing the facilities for research work.