Articles Service
Research article
Effects of Impeller Geometry on the 11α-Hydroxylation of Canrenone in Rushton Turbine-Stirred Tanks
1Department of Biological Engineering, Shanghai Institute of Technology, Shanghai 201418, P.R. China
2Department of Biological Engineering, East China University of Science and Technology, Shanghai 200237, P.R. China
J. Microbiol. Biotechnol. 2021; 31(6): 890-901
Published June 28, 2021 https://doi.org/10.4014/jmb.2104.04002
Copyright © The Korean Society for Microbiology and Biotechnology.
Abstract
Keywords
Graphical Abstract
Introduction
The steroid compound 11α-hydroxycanrenone is an important intermediate for the synthesis of the antihypertensive drug eplerenone, which can be obtained by 11α-hydroxylation of canrenone [1]. Studies have revealed that biotransformation is a common method for the synthesis of steroid hormone drug intermediates [2, 3]. For example,
It is known that aerobic fermentation is affected by the type and geometry of the impeller in stirred tanks [7-9]. The gas dispersion, flow pattern and mixing in bioreactors are all affected by these factors. The gas distribution has a great influence on the dissolved oxygen (DO) content in fermentation broth [10, 11]. Shin
In addition, the properties of the fermentation broth and microbial morphology, which greatly influence fermentation, are affected by the impeller type [19, 20]. Li
The hydrodynamics in bioreactors can be better analyzed using computational fluid dynamics (CFD) [24-26]. In the study of Amer
As mentioned above, the outcome of biotransformation is closely related to the impeller type in the bioreactor. Therefore, the geometric parameters of impellers are crucial to biotransformation. However, there are few studies on the effect of impeller design on the production of 11α-hydroxycanrenone. Contente
Materials and Methods
Bioreactor and Impellers
The experiments were performed in elliptical-bottomed cylindrical, 1,000 ml, stirred-tank bioreactors with a liquid volume of 700 ml and a liquid height of 102 mm. The experimental device is shown in Fig. 1A, in which the diameter of the tank is 100 mm, and four baffles are evenly distributed in the stirred tank that are 10 mm wide and 165 mm long. On the circular distributor with a diameter of 48 mm, twenty holes venting to the bottom of the tank are evenly distributed. Four kinds of Rushton turbine impellers were employed: a 50 mm four-blade impeller (1-1), 50 mm six-blade impeller (1-2), 60 mm four-blade impeller (2-1) and 60-mm six-blade impeller (2-2). The geometry of each impeller is shown in Fig. 1B, and the dimensions of the impellers are shown in Table 1.
-
Table 1 . Dimensions of four impellers.
Impeller Blade number Impeller diameter (mm) 1-1 4 50 1-2 6 50 2-1 4 60 2-2 6 60
-
Fig. 1. Geometry of (A) bioreactor and (B) impeller.
Materials and Reagents
Canrenone (≥ 98% purity) was purchased from Shanghai Yuanye Bio-Technology Co., Ltd., China. All chemicals and reagents used were of analytical grade or higher.
Microorganism Cultivation and Biotransformation Experiments
The strain
-
Table 2 . Operating conditions used for biotransformation experiments.
Conversion time (h) Agitation speed (rpm) Aeration rate (vvm) 0-12 350 1.5 12-24 450 2.0 24-36 500 2.5 36-60 500 2.5 avvm: air volume/culture volume/minute.
Cold Model Experiments
The hyphae were cultured for 48 h and washed with phosphate-buffered saline (PBS) three times and then collected. Equal weight hyphae were stirred at 500 rpm and 2.0 vvm for 12 h in four kinds of tanks containing 700 ml PBS, and 2.5 g/l amino acids were added to prevent premature autolysis. The contents of protein and amino acids were detected to characterize the permeability of hyphae. The hyphae were stirred for 12 h in four bioreactors and then washed with PBS three times and collected again. Equal weight hyphae were cultured at 200 rpm and 28°C for 6 h in 250 ml flasks containing 50 ml PBS, 2.5 g/l glucose and 2 g/l canrenone, and conversion of 11α-hydroxycanrenone was measured by high-performance liquid chromatography (HPLC). The conversion ratio of 11α-hydroxycanrenone with the hyphae collected in the shake flask cultured at 200 rpm and 28°C for 12 h was taken as the control to calculate the enzyme activity retention (EAR) values according to the following formula:
Analytical Methods
Analysis of the conversion ratio. HPLC (Agilent 1260, Agilent Technologies, Inc., USA) samples were extracted by ethyl acetate and filtered through a 0.22 μm filter. The column (Agilent 5 HC-C18 250 × 4.6 mm) temperature was 30°C, and the mobile phase was a mixture (v:v, 8:2) of methanol and doubly distilled water (ddH2O) with a flow rate of 0.8 ml/min at a detection wavelength of 280 nm. The concentration of canrenone in the aqueous phase was determined using the same chromatographic conditions after the fermentation broth was filtered through a 0.22 μm filter. The consumption rate of canrenone per 12 h was calculated according to the following formula:
Determination of the DO, pH and viscosity. The DO and pH of fermentation broth were measured using the bioreactor's self-equipped DO electrode and pH electrode. An SNB-1 digital viscometer (Shanghai Precision and Scientific Instrument Co., Ltd., China) was used to measure the apparent viscosity of fermentation broth.
Characterization of microbial activity. The fermentation broth was centrifuged for 5 min at 5,000 ×
Measurement of hyphal inclusions. The leakage of protein and changes in amino acids were used as evaluation indicators for hyphal permeability. Under cold-mode conditions, the supernatant was taken after the culture was centrifuged (8,000 ×
Numerical Simulation Methods
ANSYS ICEM CFD 16.0 (ANSYS Inc., USA) was used to generate the mesh of the bioreactor model. The bioreactor was divided into three parts: a tank part including a tank shell with four baffles and two stirring parts. An unstructured mesh was adopted, in which the elements of the entire model were tetrahedrons, and the quality of all the grids was larger than 0.3.
Ansys CFX 16.0 (ANSYS Inc.) was used for the simulation of the fluid dynamics in the stirred tank. The simulation conditions were set according to the fermentation conditions of 48 h. The tank part was set to the stationary domain, and the two stirring parts were set to the rotating domain at a speed of 500 rpm. The gas phase and liquid phase were both created in all domains. The gas phase was set as air at 25°C and dispersed fluid. The liquid phase was defined as a non-Newtonian fluid, and the rheological equation was μa=7.0356 ×
Results
Bioconversion of Canrenone
The effects of blade number and impeller diameter on the biotransformation when using the Rushton turbine were compared. Fig. 2A shows that the conversion ratio under agitation with 1-2 was 3.40% higher than that with 1-1. A conversation ratio of 92.65% was obtained under agitation with 2-1, which was 11.43% higher than that with 1-1. We concluded that the conversion ratio of canrenone can be increased by increasing both the blade number and impeller diameter on the basis of a 50 mm four-blade impeller. The latter was more effective than the former. However, with the agitation of a large-diameter or six-blade impeller, increases in the blade number or the diameter will lead to a decrease in the conversion ratio. Fig. 2A shows that the conversion ratio under agitation with 2-2 was 14.42% lower than that with 2-1. The lowest conversion ratio of 79.29% was obtained from the reactor stirred by the impeller with a large diameter and six blades.
-
Fig. 2. Effects of impeller geometry on (A) canrenone conversion, (B) dry biomass weight, (C) canrenone consumption and (D) canrenone concentration in aqueous phase.
According to Fig. 2B, the biomass concentration mixed with 2-2 was the highest, although the conversion ratio under this condition was the lowest. The results showed that the dry biomass weights were 9.10, 11.45, 12.88, and 14.15 g/l at 60 h when stirred by 1-1, 1-2, 2-1, and 2-2, respectively. Increasing the blade number and impeller diameter can both accelerate microbial growth. Compared to the other three kinds of impellers, 2-2 was beneficial to microbial growth. It is speculated that the hyphal concentration was not the only factor affecting the conversion ratio in the 11α-hydroxylation of canrenone.
To elucidate the above results, the canrenone consumption rate per mass biomass over a time interval of 12 h is shown in Fig. 2C. It can be seen from the figure that a low consumption rate of canrenone was obtained when the broth was stirred by 2-2 within 12-60 h, although the canrenone consumption rate was the highest within 0-12 h. The low utilization rate at late biotransformation caused a decrease in the conversion ratio. It is speculated that the decrease in the utilization rate is caused by a decrease in biological activity.
It is known that the solubility of canrenone affects the rate of 11α-hydroxylation. The concentration of canrenone in the aqueous phase shown in Fig. 2D gradually decreased with the 11α-hydroxylation of canrenone. After 36 h of biotransformation, the concentration of canrenone in the aqueous phase with 2-2 increased gradually, and the final detection concentration was 3.56 mg/l, which was 191.80% higher than that with 1-1. This result indicated that the utilization rate of canrenone was reduced in the late conversion stage when stirred by 2-2, which is consistent with the canrenone consumption rate mentioned above.
The above results show that the impeller geometry may influence the microbial viability, so the metabolic activity and hyphal morphology of
Variation in the Physicochemical Properties of Fermentation Broth
The DO value, reducing sugar content, pH and apparent viscosity of fermentation broth were evaluated to characterize the biological activity. The results are shown in Fig. 3.
-
Fig. 3. Effects of impeller geometry on (A) dissolved oxygen, (B) glucose concentration, (C) pH and (D) apparent viscosity of fermentation broth.
The results shown in Fig. 3A illustrated that the geometric parameters of the impeller affected the DO content of fermentation broth. The time average DO values with 1-1, 1-2, 2-1, and 2-2 were 2.14%, 3.76%, 9.26%, and 22.31%, respectively, within 60 h. As Fig. 3 indicates, the impeller with more blades and a large diameter was conducive to high DO values in fermentation broth. The DO values obtained from the agitation of 60 mm impellers (2-1 and 2-2) were significantly higher than those with 50 mm impellers (1-1 and 1-2). After 48 h of biotransformation, the DO level increased rapidly when mixing with 2-2. Although the biomass concentration was the highest when stirred by 2-2, the conversion ratio decreased due to the decrease in microbial metabolic activity in the late stage of the biotransformation.
From Fig. 3B, it was observed that increasing the blade number and impeller diameter enhanced the metabolism of reducing sugars. There was a reduced metabolic rate of reducing sugars mixed by 2-2 after 24 h, which indicated that the metabolic activity of hyphae was significantly reduced. Various organic acids are produced when reducing sugars are utilized rapidly, which decreases the pH of the fermentation broth; the variation in pH shown in Fig. 3C confirmed this occurrence. When the ratio of carbon to nitrogen in the fermentation broth decreased, nitrogen sources were metabolized rapidly, and the pH of the system began to increase. In addition to nitrogen source metabolism, the ammonia released by hyphal autolysis caused an increase in pH. When agitating with 2-1 and 2-2, the pH of the culture increased sharply at 44.5 h and 43.0 h, respectively, which indicated that impellers with large diameters accelerated hyphal autolysis.
In terms of the apparent viscosity of the cultures during biotransformation, we found that there was a great difference when various impellers were employed (Fig. 3D). The apparent viscosity of the culture gradually increased when 1-1 was used within 60 h. For agitation with 1-2, 2-1 and 2-2, the apparent viscosities all showed a downward trend after increasing. We also found that the apparent viscosity was the lowest (1.334 Pa·s) when stirred by 2-2 at 60 h, although the hyphal concentration was the highest compared with the other three kinds of impellers. The results signified that the biomass concentration was not the only factor affecting the apparent viscosity of the fermentation broth.
The results shown in Fig. 3 indicated that the geometric parameters of impellers created significant differences in the properties of fermentation broth. Rapid increases in the pH and DO content with 2-2 were observed at the end of conversion. The autolysis of filamentous fungi will cause an increase in pH, a reduction in the oxygen utilization rate and a decrease in the apparent viscosity of broth [34]. Therefore, it is reasonable to speculate that the damage to fungal activity with 2-2 resulted in the low biotransformation rate observed in the late stage of biotransformation. This indicates that the transformation activity of
During autolysis, the morphology of the hyphae changes, and intracellular substances are released at the same time. Thus, the hyphal morphology and leaked substances were investigated to confirm that the 11α-hydroxylation activity of
Hyphal Morphology
Fig. 4 shows SEM images of hyphae stirred by four impellers. The hyphae after agitation by 1-1 are smooth and thick. By contrast, the hyphal surface is rough and the thickness is not uniform when stirred by 1-2 and 2-1, in which the lack of uniformity with 2-1 is more serious than that with 2-2. The hyphae were broken most seriously, with thinner hyphae and fewer branches, when the bioreactor was equipped with 2-2. This result showed that increasing the blade number and impeller diameter can accelerate the autolysis of hyphae, where the influence of the latter was more significant than that of the former. The apparent viscosity of fermentation broth decreased sharply due to the severely broken hyphae after 36 h when using 2-2. These results were consistent with the report that the apparent viscosity is related to the concentration and morphology of microorganisms [35].
-
Fig. 4. SEM images of hyphal morphology. Hyphae was collected and observed at biotransformation of 48 h.
Leakage of Hyphal Inclusions in the Cold Model Experiment
To further investigate the effect of impeller geometry on the activity of
-
Fig. 5. Effects of impeller geometry on (A) leaky protein, (B) amino acid concentration and (C) EMR of
A. ochraceus .
According to the results of protein and amino acids concentration, we concluded that a large-diameter impeller accelerated the release of hyphal inclusions. In this case, the EAR value of filamentous fungi was calculated, as given in Fig. 5C. There were significant differences between the EAR values of different impeller stirrers, which indicated that mechanical agitation had an effect on the hydroxylation activity of fungi. The EAR values of 1-2, 2-1, and 2-2 were 12.93%, 31.75%, and 74.86% lower than that of 1-1, respectively, which indicated that the decreases in the 11α-hydroxylation activity of
The above results show that the DO content, microbial metabolism, hyphal morphology and biotransformation activity were all affected by the geometric parameters of the impellers, which in turn influenced the conversation ratio of canrenone. The variation in the properties of the broth is caused by the hydrodynamics in the stirred tank. Therefore, the hydrodynamics were investigated in bioreactors equipped with the four kinds of impellers using numerical simulation.
Numerical Simulation Results
Gas holdup. The distribution of the gas on the mid-plane of the bioreactor is shown in Fig. 6. The gas holdup in the lower region of the tank is higher than that in the upper region. This condition was created by the downward discharge flow in the lower circulation area, prolonging the residence time of the bubbles. It was obvious that the gas distribution of the six-blade impeller and 60 mm diameter impeller on the mid-plane was more uniform than that of the four-blade impeller and 50 mm diameter impeller. An impeller with a large diameter and more blades was effective for gas dispersion.
-
Fig. 6. Effects of impeller geometry on air volume fraction.
Fluid pattern. Fig. 7A shows the flow pattern at the mid-plane of the bioreactors. Under the stirring of double Rushton impellers, the fluid was discharged from the impeller tip toward the tank wall. As the fluid hit the wall, it was divided into two loops circulating at the top and the bottom of the blade, with obvious radial flow. The fluid circulations do not interact with each other when stirred by 1-1, and the proportion of the high velocity region was the smallest and only appeared near the blade. When the blade number increased to six (1-2), the fluid circulation began to contact. When the diameter of the impeller increased to 60 mm (2-1 and 2-2), the fluid circulation areas expanded to the tank wall and crossed. The flow pattern of 2-2 was more regular than that of 2-1. At the same time, the high-speed fluid region around the impeller further expanded. The mixing dead zone was decreased, especially the region between the upper and lower impellers and at the tank wall. A Rushton impeller with a large diameter and more blades was beneficial to fluid mixing.
-
Fig. 7. Effects of impeller geometry on (A) flow pattern, (B) radial velocity and (C) axial velocity of the fluid. The position of velocity generation is located on the yellow horizontal line in Fig. 7A, extending from the center of the stirring shaft to the tank wall.
The axial velocity along the radial positions of the tank near the blade is shown in Fig. 7B. This figure shows that the axial velocity of the fluid is highest when stirred by Rushton impellers with more blades and large diameters. The circulation area of the flow field mixed with 60 mm impellers is significantly wider than that of the 50 mm impellers. In this case, the width of the circulation area using 2-2 was approximately 1/2 of the tank diameter, which was the largest in this study.
Shear strain rate and stirring power. Hydrodynamic shear was produced when the broth was agitated. Fig. 8 shows the shear strain rate distribution of the fluid stirred by different impellers. The results indicated that the shear strain rate in the blade region was higher than that in the other regions in the bioreactor, and it decreased with increasing distance to the impeller. Compared with the 50 mm diameter impeller, the shear strain rate increased at the region of the liquid surface and the tank wall when equipped with a 60 mm diameter impeller in the bioreactors. When using 2-2, the low shear area only appeared at the top of the mid-plane of the bioreactor.
-
Fig. 8. Effects of impeller geometry on shear strain rate.
The average shear strain rate and specific stirring power are given in Table 3. The average shear strain rate of the fluid was 19.36 s-1, and the P/V was 1.46 kW/m3 when 1-1 was used. For 1-2, 2-1, and 2-2, the average shear strain rates were 23.53%, 120.59%, and 192.16% higher than that of 1-1, respectively; the P/V values were 29.34%, 82.39%, and 123.40% higher than that of 1-1, respectively. These results showed that increasing the blade number and impeller diameter increased the shear strain rate and the specific power of the impeller. The effect of the impeller diameter was more obvious than that of the blade number.
-
Table 3 . Effect of impeller geometry on power and average shear strain rate.
Impeller Average shear strain rate (s-1) P/V (kW/m3) 1-1 19.36 1.46 1-2 25.04 1.80 2-1 35.31 3.21 2-2 43.25 4.26
Discussion
The bioconversion ratio of canrenone is determined by various factors including the DO content, the metabolism of
The biochemical reaction rate of
Oxygen needs to be dispersed and dissolved in the aqueous phase to be used by microbial cells in the process of oxygen consumption biotransformation [40, 41]. Therefore, it is essential to maintain the supply and delivery of oxygen for transformation. According to the gas holdup distribution in the bioreactor, increasing the blade number and impeller diameter can improve both the uniformity of gas in the fluid and increase the DO content. The results showed that 332.71% and 493.35% increases in the time average DO content were achieved by increasing the impeller diameter by 20% when mixing with the four-blade impeller and six-blade impeller, respectively. High power input is beneficial for air dispersion [42]. The simulation results showed that the stirring power of the Rushton impeller was higher with more blades or large diameters. It is easy for air to reach the state of complete dispersion in high-speed mixing fluid, which enables gas recycling [43]. At the same time, the apparent viscosity of fermentation broth decreases with increasing shear strain rate in the high-speed mixing area [44]. All these conditions are effective for increasing the gas-liquid contact area, reducing the gas transfer resistance and increasing the DO content in liquid phase [45]. Thus, the fermentation broth mixed with 2-1 or 2-2 had a high DO content. On the one hand, the high DO content accelerates the aerobic metabolism of reducing sugar, which provides the sufficient energy and carbon skeleton for the growth of microorganisms [46]. A high DO content is conducive to the enzyme expression during hydroxylation [47-49]. Under the condition of vigorous growth of
Compared with the other three kinds of impellers, we found that the dry biomass concentration from mixing by 2-2 was the highest at 60 h, which was 55.49% higher than that mixed by 1-1. However, the conversion ratio of 2-2 with the highest biomass was 79.29%, which was the lowest in this experiment. Therefore, the detailed influence of impeller geometry on microbial metabolism was explored further.
According to the physicochemical properties of the broth and SEM results regarding hyphal morphology, we concluded that increasing the blade number and impeller diameter of the Rushton turbine accelerated hyphal autolysis. The sharp increase of DO was related to the decrease of microbial activity. Within 36-60 h of biotransformation, the 11α-hydroxylation rates of canrenone under the agitation of the 50 mm impellers were significantly higher than that of the 60 mm impellers, even though the DO and biomass concentration of the former were lower than that of the latter. The yield of fermentation is affected by the hyphal morphology, which is greatly influenced by the shear in the tank [50, 51]. Filamentous fungal fermentation broth with differentiated and poor flexibility hyphae is more susceptible to shearing force because it is usually a non-Newtonian fluid [52]. The hyphae were damaged and autolysis was accelerated under the condition of continuous high shear stress, which was more obvious after the stable growth period. Autolysis was the most serious when agitation was provided by 2-2, resulting in severe damage to the hyphae. The average shear strain rate of 43.25 s-1 was beyond the tolerance range of the hyphae in the late stage of biotransformation under this condition, resulting in a significant decrease in the metabolic activity of
In addition to the reduction in metabolic activity, it was speculated that the decreased hydroxylation capacity of
In this study, the effects of the blade number and diameter of a Rushton impeller on the 11α-hydroxylation of canrenone were compared. We concluded that fluid flow and shear both impact the biological parameters and play an important role in the 11α-hydroxylation of canrenone. Increasing the blade number and the impeller diameter improved the fluid mixing in the stirred tank. The DO content of fermentation broth can be increased, and it had the positive effects on the microbial growth and 11α-hydroxylation of canrenone. At the same time, it was essential to apply an appropriate shear strain rate because high shear stress reduced the biotransformation activity. According to the characteristics of the 11α-hydroxylation of canrenone, it can be inferred that the increased conversion ratio of canrenone by the alteration of impellers was mainly because of the high DO content in the early stage and the good hyphal morphology in the late stage of bioconversion. In addition, both of the above conditions are beneficial for the activation of metabolism in
In summary, the fluid flow and shear in mixing fermentation broth must be controlled at the same time to ensure that the 11α-hydroxylation of canrenone can be carried out efficiently. Since reports about the influence of the impeller geometry on the biosynthesis of 11α-hydroxycanrenone have rarely been published, this research provides basic data for the industrial production of this compound.
Acknowledgments
This work was supported by the Science and Technology Commission of Shanghai Municipality (Grant no. 17441905400).
Conflict of Interest
The authors have no financial conflicts of interest to declare.
References
- Huang DM, Zhang TZ, Cui FJ, Sun WJ, Zhao LM, Yang MY,
et al . 2011. Simultaneous identification and quantification of canrenone and 11-α-hydroxy-canrenone by LC-MS and HPLC-UVD.J. Biomed. Biotechnol. 2011 : 917232. - Al-Aboudi A, Kana'an BM, Zarga MA, Bano S, Atia tul W, Javed K,
et al . 2017. Fungal biotransformation of diuretic and antihypertensive drug spironolactone with Gibberella fujikuroi, Curvularia lunata, Fusarium lini, andAspergillus alliaceus.Steroids 128 : 15-22. - Donova MV. 2017. Steroid bioconversions.
Methods Mol. Biol. 1645 : 1-13. - Petrič Š, Hakki T, Bernhardt R, Žigon D, Črešnar B. 2010. Discovery of a steroid 11α-hydroxylase from
Rhizopus oryzae and its biotechnological application.J. Biotechnol. 150 : 428-437. - Mao S, Hua B, Wang N, HU X, Ge Z, Li Y,
et al . 2013. 11α hydroxylation of 16α, 17-epoxyprogesterone in biphasic ionic liquid/water system byAspergillus ochraceus.J. Chem. Technol. Biotechnol. 88 : 287-292. - Hannemann F, Bichet A, Ewen KM, Bernhardt R. 2007. Cytochrome P450 systems-biological variations of electron transport chains.
Biochim. Biophys. Acta 1770 : 330-344. - Amanullah A, Tuttiett B, Nienow AW. 1998. Agitator speed and dissolved oxygen effects in Xanthan fermentations.
Biotechnol. Bioeng. 57 : 198-210. - Grein TA, Loewe D, Dieken H, Weidner T, Salzig D, Czermak P. 2019. Aeration and shear stress are critical process parameters for the production of oncolytic Measles virus.
Front. Bioeng. Biotechnol. 7 : 78. - Amanullah A, Serrano-Carreon L, Castro B, Galindo E, Nienow AW. 1998. The influence of impeller type in pilot scale Xanthan fermentations.
Biotechnol. Bioeng. 57 : 95-108. - Hudcova W, Machon W, Nienow AW. 1989. Gas-liquid dispersion with dual Rushton impellers.
Biotechnol. Bioeng. 34 : 617-628. - Kracík T, Moucha T, Petříček R. 2020. Gas-liquid contactors' aeration capacities when agitated by Rushton turbines of various diameters.
ACS Omega 5 : 5072-5077. - Albaek MO, Gernaey KV, Hansen MS, Stocks SM. 2011. Modeling enzyme production with
Aspergillus oryzae in pilot scale vessels with different agitation, aeration, and agitator types.Biotechnol. Bioeng. 108 : 1828-1840. - Shin W-S, Lee D, Kim S, Jeong Y-S, Chun G-T. 2013. Application of scale-up criterion of constant oxygen mass transfer coefficient (
kL a) for production of itaconic acid in a 50 L pilot-scale fermentor by fungal cells ofAspergillus terreus .J. Microbiol. Biotechnol. 23 : 1445-1453. - Jayus, McDougall BM, Seviour RJ. 2005. The effect of dissolved oxygen concentrations on (1→3)- and (1→6)-β-glucanase production by
Acremonium sp. IMI 383068 in batch culture.Enzyme Microb. Technol. 36 : 176-181. - Revstedt J, Fuchs L, Kovács T, Trägårdh C. 2000. Influence of impeller type on the flow structure in a stirred reactor.
AIChE J. 46 : 2373-2382. - Govardhan M, Venkateswarlu G. 2003. Effect of impeller geometry and tongue shape on the flow field of cross flow fans.
J. Therm. Sci. 12 : 118-125. - Li ZJ, Shukla V, Wenger KS, Fordyce AP, Pedersen AG, Marten MR. 2002. Effects of increased impeller power in a production-scale
Aspergillus oryzae fermentation.Biotechnol. Prog. 18 : 437-444. - Wang Z, Xue J, Sun H, Zhao M, Wang Y, Chu J,
et al . 2020. Evaluation of mixing effect and shear stress of different impeller combinations on nemadectin fermentation.Process Biochem. 92 : 120-129. - López JLC, Pérez JAS, Sevilla JMF, Porcel EMR, Chisti Y. 2005. Pellet morphology, culture rheology and lovastatin production in cultures of
Aspergillus terreus .J. Biotechnol. 116 : 61-77. - Buffo MM, Esperança MN, Farinas CS, Badino AC. 2020. Relation between pellet fragmentation kinetics and cellulolytic enzymes production by
Aspergillus niger in conventional bioreactor with different impellers.Enzyme Microb. Technol. 139 : 109587. - Li ZJ, Shukla V, Wenger K, Fordyce A, Pedersen AG, Marten M. 2002. Estimation of hyphal tensile strength in production-scale
Aspergillus oryzae fungal fermentations.Biotechnol. Bioeng. 77 : 601-613. - Ghobadi N, Ogino C, Ogawa T, Ohmura N. 2016. Using a flexible shaft agitator to enhance the rheology of a complex fungal fermentation culture.
Bioprocess Biosyst. Eng. 39 : 1793-1801. - Jüsten P, Paul GC, Nienow AW, Thomas CR. 1998. Dependence of
Penicillium chrysogenum growth, morphology, vacuolation, and productivity in fed-batch fermentations on impeller type and agitation intensity.Biotechnol. Bioeng. 59 : 762-775. - Gu D, Liu Z, Tao C, Li J, Wang Y. 2019. Numerical simulation of gas-liquid dispersion in a stirred tank agitated by punched rigidflexible impeller.
Int. J. Chem. React. Eng. 17 : 588-597. - Chen P, Sanyal J, Duduković MP. 2005. Numerical simulation of bubble columns flows: effect of different breakup and coalescence closures.
Chem. Eng. Sci. 60 : 1085-1101. - Gelves R, Dietrich A, Takors R. 2014. Modeling of gas-liquid mass transfer in a stirred tank bioreactor agitated by a Rushton turbine or a new pitched blade impeller.
Bioprocess Biosyst. Eng. 37 : 365-375. - Amer M, Feng Y, Ramsey JD. 2019. Using CFD simulations and statistical analysis to correlate oxygen mass transfer coefficient to both geometrical parameters and operating conditions in a stirred-tank bioreactor.
Biotechnol. Prog. 35 : e2785. - Duan S, Yuan G, Zhao Y, Ni W, Luo H, Shi Z,
et al . 2013. Simulation of computational fluid dynamics and comparison of cephalosporin C fermentation performance with different impeller combinations.Korean J. Chem. Eng. 30 : 1097-1104. - Xia J-Y, Wang Y-H, Zhang S-L, Chen N, Yin P, Zhuang Y-P,
et al . 2009. Fluid dynamics investigation of variant impeller combinations by simulation and fermentation experiment.Biochem. Eng. J. 43 : 252-260. - Contente ML, Guidi B, Serra I, De Vitis V, Romano D, Pinto A,
et al . 2016. Development of a high-yielding bioprocess for 11-α hydroxylation of canrenone under conditions of oxygen-enriched air supply.Steroids 116 : 1-4. - Miller GL. 1959. Use of dinitrosalicylic acid reagent for determination of reducing sugar.
Anal. Chem. 31 : 426-428. - Snyder JC, Desborough SL. 1978. Rapid estimation of potato tuber total protein content with coomassie brilliant blue G-250.
Theor. Appl. Genet. 52 : 135-139. - Moore S. 1968. Amino acid analysis: aqueous dimethyl sulfoxide as solvent for the ninhydrin reaction.
J. Biol. Chem. 243 : 6281-6283. - Harvey LM, McNeil B, Berry DR, White S. 1998. Autolysis in batch cultures of
Penicillium chrysogenum at varying agitation rates.Enzyme Microb. Technol. 22 : 446-458. - Riley GL, Tucker KG, Paul GC, Thomas CR. 2000. Effect of biomass concentration and mycelial morphology on fermentation broth rheology.
Biotechnol. Bioeng. 68 : 160-172. - Tokura Y, Uddin MA, Kato Y. 2019. Effect of suspension pattern of sedimentary particles on solid/liquid mass transfer in a mechanically stirred vessel.
Ind. Eng. Chem. Res. 58 : 10172-10178. - Lin Y, Zhang Z, Thibault J. 2011. New impeller for viscous fermentation: power input and mass transfer coefficient correlations.
Ind. Eng. Chem. Res. 50 : 3510-3516. - Tang W, Pan A, Lu H, Xia J, Zhuang Y, Zhang S,
et al . 2015. Improvement of glucoamylase production using axial impellers with low power consumption and homogeneous mass transfer.Biochem. Eng. J. 99 : 167-176. - Dohi N, Takahashi T, Minekawa K, Kawase Y. 2004. Power consumption and solid suspension performance of large-scale impellers in gas-liquid-solid three-phase stirred tank reactors.
Chem. Eng. J. 97 : 103-114. - Rao DVK, Ramu CT, Rao JV, Narasu ML, Rao AKSB. 2008. Impact of dissolved oxygen concentration on some key parameters and production of rhG-CSF in batch fermentation.
J. Ind. Microbiol. Biotechnol. 35 : 991-1000. - Tang YJ, Li HM, Hamel JFP. 2009. Effects of dissolved oxygen tension and agitation rate on the production of heat-shock protein glycoprotein 96 by MethA tumor cell suspension culture in stirred-tank bioreactors.
Bioprocess Biosyst. Eng. 32 : 475-484. - Fujasová M, Linek V, Moucha T. 2007. Mass transfer correlations for multiple-impeller gas-liquid contactors. Analysis of the effect of axial dispersion in gas and liquid phases on "local"
kL a values measured by the dynamic pressure method in individual stages of the vessel.Chem. Eng. Sci. 62 : 1650-1669. - Bao Y, Wang B, Lin M, Gao Z, Yang J. 2015. Influence of impeller diameter on overall gas dispersion properties in a sparged multiimpeller stirred tank.
Chin. J. Chem. Eng. 23 : 890-896. - Kilonzo PM, Margaritis A. 2004. The effects of non-Newtonian fermentation broth viscosity and small bubble segregation on oxygen mass transfer in gas-lift bioreactors: a critical review.
Biochem. Eng. J. 17 : 27-40. - Najafpour GD. 2015. Gas and liquid system (aeration and agitation), pp. 51-102.
In: Najafpour GD (ed),Biochemical Engineering and Biotechnology , 2th Ed. Elsevier, Amsterdam, Netherlands. - Clark TA, Maddox IS, Chong R. 1983. The effect of glucose on 11β- and 19-hydroxylation of Reichstein's Substance S by
Pellicularia filamentosa .Appl. Microbiol. Biotechnol. 17 : 211-215. - Chen K-C, Wey H-C. 1990. 11β-Hydroxylation of coxtexolone by
Curvularia lunata .Enzyme Microb. Technol. 12 : 305-308. - Gbewonyo K, Buckland BC, Lilly MD. 1990. Development of a large-scale continuous substrate feed process for the biotransformation of simvastatin by
Nocardia sp.Biotechnol. Bioeng. 37 : 1101-1107. - Clark TA, Chong R, Maddox IS. 1982. The effect of dissolved oxygen tension on 11β- and 19-hydroxylation of Reichstein's Substance S by Pellicularia filamentosa.
Appl. Microbiol. Biotechnol. 14 : 131-135. - El-Enshasy H, Hellmuth K, Rinas U. 1999. Fungal morphology in submerged cultures and its relation to glucose oxidase excretion by recombinant
Aspergillus niger.Appl. Biochem. Biotechnol. 81 : 1-11. - Chen X, Zhou J, Ding Q, Luo Q, Liu L. 2019. Morphology engineering of
Aspergillus oryzae for L-malate production.Biotechnol. Bioeng. 116 : 2662-2673. - Ghobadi N, Ogino C, Yamabe K, Ohmura N. 2017. Characterizations of the submerged fermentation of
Aspergillus oryzae using a Fullzone impeller in a stirred tank bioreactor.J. Biosci. Bioeng. 123 : 101-108. - Žnidaršič P, Komel R, Pavko A. 1998. Studies of a pelleted growth form of
Rhizopus nigricans as a biocatalyst for progesterone 11 α-hydroxylation.J. Biotechnol. 60 : 207-216. - Shen Y, Wang L, Liang J, Tang R, Wang M. 2016. Effects of two kinds of imidazolium-based ionic liquids on the characteristics of steroid-transformation
Arthrobacter simplex .Microb. Cell Fact. 15 : 118-127.
Related articles in JMB

Article
Research article
J. Microbiol. Biotechnol. 2021; 31(6): 890-901
Published online June 28, 2021 https://doi.org/10.4014/jmb.2104.04002
Copyright © The Korean Society for Microbiology and Biotechnology.
Effects of Impeller Geometry on the 11α-Hydroxylation of Canrenone in Rushton Turbine-Stirred Tanks
Shaofeng Rong1, Xiaoqing Tang1, Shimin Guan1*, Botao Zhang2, Qianqian Li1, Baoguo Cai1, and Juan Huang1*
1Department of Biological Engineering, Shanghai Institute of Technology, Shanghai 201418, P.R. China
2Department of Biological Engineering, East China University of Science and Technology, Shanghai 200237, P.R. China
Correspondence to:Shimin Guan, guanshimin0000@163.com
Juan Huang, hjhuangjuan@126.com
Abstract
The 11α-hydroxylation of canrenone can be catalyzed by Aspergillus ochraceus in bioreactors, where the geometry of the impeller greatly influences the biotransformation. In this study, the effects of the blade number and impeller diameter of a Rushton turbine on the 11α-hydroxylation of canrenone were considered. The results of fermentation experiments using a 50 mm four-blade impeller showed that 3.40% and 11.43% increases in the conversion ratio were achieved by increasing the blade number and impeller diameter, respectively. However, with an impeller diameter of 60 mm, the conversion ratio with a six-blade impeller was 14.42% lower than that with a four-blade impeller. Data from cold model experiments with a large-diameter six-blade impeller indicated that the serious leakage of inclusions and a 22.08% enzyme activity retention led to a low conversion ratio. Numerical simulations suggested that there was good gas distribution and high fluid flow velocity when the fluid was stirred by large-diameter impellers, resulting in a high dissolved oxygen content and good bulk circulation, which positively affected hyphal growth and metabolism. However, a large-diameter six-blade impeller created overly high shear compared to a large-diameter four-blade impeller, thereby decreasing the conversion ratio. The average shear rates of the former and latter cases were 43.25 s-1 and 35.31 s-1, respectively. We therefore concluded that appropriate shear should be applied in the 11α-hydroxylation of canrenone. Overall, this study provides basic data for the scaled-up production of 11α-hydroxycanrenone.
Keywords: 11α-Hydroxycanrenone, impeller geometry, fungal metabolism, CFD, fluid velocity, shear strain rate
Introduction
The steroid compound 11α-hydroxycanrenone is an important intermediate for the synthesis of the antihypertensive drug eplerenone, which can be obtained by 11α-hydroxylation of canrenone [1]. Studies have revealed that biotransformation is a common method for the synthesis of steroid hormone drug intermediates [2, 3]. For example,
It is known that aerobic fermentation is affected by the type and geometry of the impeller in stirred tanks [7-9]. The gas dispersion, flow pattern and mixing in bioreactors are all affected by these factors. The gas distribution has a great influence on the dissolved oxygen (DO) content in fermentation broth [10, 11]. Shin
In addition, the properties of the fermentation broth and microbial morphology, which greatly influence fermentation, are affected by the impeller type [19, 20]. Li
The hydrodynamics in bioreactors can be better analyzed using computational fluid dynamics (CFD) [24-26]. In the study of Amer
As mentioned above, the outcome of biotransformation is closely related to the impeller type in the bioreactor. Therefore, the geometric parameters of impellers are crucial to biotransformation. However, there are few studies on the effect of impeller design on the production of 11α-hydroxycanrenone. Contente
Materials and Methods
Bioreactor and Impellers
The experiments were performed in elliptical-bottomed cylindrical, 1,000 ml, stirred-tank bioreactors with a liquid volume of 700 ml and a liquid height of 102 mm. The experimental device is shown in Fig. 1A, in which the diameter of the tank is 100 mm, and four baffles are evenly distributed in the stirred tank that are 10 mm wide and 165 mm long. On the circular distributor with a diameter of 48 mm, twenty holes venting to the bottom of the tank are evenly distributed. Four kinds of Rushton turbine impellers were employed: a 50 mm four-blade impeller (1-1), 50 mm six-blade impeller (1-2), 60 mm four-blade impeller (2-1) and 60-mm six-blade impeller (2-2). The geometry of each impeller is shown in Fig. 1B, and the dimensions of the impellers are shown in Table 1.
-
Table 1 . Dimensions of four impellers..
Impeller Blade number Impeller diameter (mm) 1-1 4 50 1-2 6 50 2-1 4 60 2-2 6 60
-
Figure 1. Geometry of (A) bioreactor and (B) impeller.
Materials and Reagents
Canrenone (≥ 98% purity) was purchased from Shanghai Yuanye Bio-Technology Co., Ltd., China. All chemicals and reagents used were of analytical grade or higher.
Microorganism Cultivation and Biotransformation Experiments
The strain
-
Table 2 . Operating conditions used for biotransformation experiments..
Conversion time (h) Agitation speed (rpm) Aeration rate (vvm) 0-12 350 1.5 12-24 450 2.0 24-36 500 2.5 36-60 500 2.5 avvm: air volume/culture volume/minute..
Cold Model Experiments
The hyphae were cultured for 48 h and washed with phosphate-buffered saline (PBS) three times and then collected. Equal weight hyphae were stirred at 500 rpm and 2.0 vvm for 12 h in four kinds of tanks containing 700 ml PBS, and 2.5 g/l amino acids were added to prevent premature autolysis. The contents of protein and amino acids were detected to characterize the permeability of hyphae. The hyphae were stirred for 12 h in four bioreactors and then washed with PBS three times and collected again. Equal weight hyphae were cultured at 200 rpm and 28°C for 6 h in 250 ml flasks containing 50 ml PBS, 2.5 g/l glucose and 2 g/l canrenone, and conversion of 11α-hydroxycanrenone was measured by high-performance liquid chromatography (HPLC). The conversion ratio of 11α-hydroxycanrenone with the hyphae collected in the shake flask cultured at 200 rpm and 28°C for 12 h was taken as the control to calculate the enzyme activity retention (EAR) values according to the following formula:
Analytical Methods
Analysis of the conversion ratio. HPLC (Agilent 1260, Agilent Technologies, Inc., USA) samples were extracted by ethyl acetate and filtered through a 0.22 μm filter. The column (Agilent 5 HC-C18 250 × 4.6 mm) temperature was 30°C, and the mobile phase was a mixture (v:v, 8:2) of methanol and doubly distilled water (ddH2O) with a flow rate of 0.8 ml/min at a detection wavelength of 280 nm. The concentration of canrenone in the aqueous phase was determined using the same chromatographic conditions after the fermentation broth was filtered through a 0.22 μm filter. The consumption rate of canrenone per 12 h was calculated according to the following formula:
Determination of the DO, pH and viscosity. The DO and pH of fermentation broth were measured using the bioreactor's self-equipped DO electrode and pH electrode. An SNB-1 digital viscometer (Shanghai Precision and Scientific Instrument Co., Ltd., China) was used to measure the apparent viscosity of fermentation broth.
Characterization of microbial activity. The fermentation broth was centrifuged for 5 min at 5,000 ×
Measurement of hyphal inclusions. The leakage of protein and changes in amino acids were used as evaluation indicators for hyphal permeability. Under cold-mode conditions, the supernatant was taken after the culture was centrifuged (8,000 ×
Numerical Simulation Methods
ANSYS ICEM CFD 16.0 (ANSYS Inc., USA) was used to generate the mesh of the bioreactor model. The bioreactor was divided into three parts: a tank part including a tank shell with four baffles and two stirring parts. An unstructured mesh was adopted, in which the elements of the entire model were tetrahedrons, and the quality of all the grids was larger than 0.3.
Ansys CFX 16.0 (ANSYS Inc.) was used for the simulation of the fluid dynamics in the stirred tank. The simulation conditions were set according to the fermentation conditions of 48 h. The tank part was set to the stationary domain, and the two stirring parts were set to the rotating domain at a speed of 500 rpm. The gas phase and liquid phase were both created in all domains. The gas phase was set as air at 25°C and dispersed fluid. The liquid phase was defined as a non-Newtonian fluid, and the rheological equation was μa=7.0356 ×
Results
Bioconversion of Canrenone
The effects of blade number and impeller diameter on the biotransformation when using the Rushton turbine were compared. Fig. 2A shows that the conversion ratio under agitation with 1-2 was 3.40% higher than that with 1-1. A conversation ratio of 92.65% was obtained under agitation with 2-1, which was 11.43% higher than that with 1-1. We concluded that the conversion ratio of canrenone can be increased by increasing both the blade number and impeller diameter on the basis of a 50 mm four-blade impeller. The latter was more effective than the former. However, with the agitation of a large-diameter or six-blade impeller, increases in the blade number or the diameter will lead to a decrease in the conversion ratio. Fig. 2A shows that the conversion ratio under agitation with 2-2 was 14.42% lower than that with 2-1. The lowest conversion ratio of 79.29% was obtained from the reactor stirred by the impeller with a large diameter and six blades.
-
Figure 2. Effects of impeller geometry on (A) canrenone conversion, (B) dry biomass weight, (C) canrenone consumption and (D) canrenone concentration in aqueous phase.
According to Fig. 2B, the biomass concentration mixed with 2-2 was the highest, although the conversion ratio under this condition was the lowest. The results showed that the dry biomass weights were 9.10, 11.45, 12.88, and 14.15 g/l at 60 h when stirred by 1-1, 1-2, 2-1, and 2-2, respectively. Increasing the blade number and impeller diameter can both accelerate microbial growth. Compared to the other three kinds of impellers, 2-2 was beneficial to microbial growth. It is speculated that the hyphal concentration was not the only factor affecting the conversion ratio in the 11α-hydroxylation of canrenone.
To elucidate the above results, the canrenone consumption rate per mass biomass over a time interval of 12 h is shown in Fig. 2C. It can be seen from the figure that a low consumption rate of canrenone was obtained when the broth was stirred by 2-2 within 12-60 h, although the canrenone consumption rate was the highest within 0-12 h. The low utilization rate at late biotransformation caused a decrease in the conversion ratio. It is speculated that the decrease in the utilization rate is caused by a decrease in biological activity.
It is known that the solubility of canrenone affects the rate of 11α-hydroxylation. The concentration of canrenone in the aqueous phase shown in Fig. 2D gradually decreased with the 11α-hydroxylation of canrenone. After 36 h of biotransformation, the concentration of canrenone in the aqueous phase with 2-2 increased gradually, and the final detection concentration was 3.56 mg/l, which was 191.80% higher than that with 1-1. This result indicated that the utilization rate of canrenone was reduced in the late conversion stage when stirred by 2-2, which is consistent with the canrenone consumption rate mentioned above.
The above results show that the impeller geometry may influence the microbial viability, so the metabolic activity and hyphal morphology of
Variation in the Physicochemical Properties of Fermentation Broth
The DO value, reducing sugar content, pH and apparent viscosity of fermentation broth were evaluated to characterize the biological activity. The results are shown in Fig. 3.
-
Figure 3. Effects of impeller geometry on (A) dissolved oxygen, (B) glucose concentration, (C) pH and (D) apparent viscosity of fermentation broth.
The results shown in Fig. 3A illustrated that the geometric parameters of the impeller affected the DO content of fermentation broth. The time average DO values with 1-1, 1-2, 2-1, and 2-2 were 2.14%, 3.76%, 9.26%, and 22.31%, respectively, within 60 h. As Fig. 3 indicates, the impeller with more blades and a large diameter was conducive to high DO values in fermentation broth. The DO values obtained from the agitation of 60 mm impellers (2-1 and 2-2) were significantly higher than those with 50 mm impellers (1-1 and 1-2). After 48 h of biotransformation, the DO level increased rapidly when mixing with 2-2. Although the biomass concentration was the highest when stirred by 2-2, the conversion ratio decreased due to the decrease in microbial metabolic activity in the late stage of the biotransformation.
From Fig. 3B, it was observed that increasing the blade number and impeller diameter enhanced the metabolism of reducing sugars. There was a reduced metabolic rate of reducing sugars mixed by 2-2 after 24 h, which indicated that the metabolic activity of hyphae was significantly reduced. Various organic acids are produced when reducing sugars are utilized rapidly, which decreases the pH of the fermentation broth; the variation in pH shown in Fig. 3C confirmed this occurrence. When the ratio of carbon to nitrogen in the fermentation broth decreased, nitrogen sources were metabolized rapidly, and the pH of the system began to increase. In addition to nitrogen source metabolism, the ammonia released by hyphal autolysis caused an increase in pH. When agitating with 2-1 and 2-2, the pH of the culture increased sharply at 44.5 h and 43.0 h, respectively, which indicated that impellers with large diameters accelerated hyphal autolysis.
In terms of the apparent viscosity of the cultures during biotransformation, we found that there was a great difference when various impellers were employed (Fig. 3D). The apparent viscosity of the culture gradually increased when 1-1 was used within 60 h. For agitation with 1-2, 2-1 and 2-2, the apparent viscosities all showed a downward trend after increasing. We also found that the apparent viscosity was the lowest (1.334 Pa·s) when stirred by 2-2 at 60 h, although the hyphal concentration was the highest compared with the other three kinds of impellers. The results signified that the biomass concentration was not the only factor affecting the apparent viscosity of the fermentation broth.
The results shown in Fig. 3 indicated that the geometric parameters of impellers created significant differences in the properties of fermentation broth. Rapid increases in the pH and DO content with 2-2 were observed at the end of conversion. The autolysis of filamentous fungi will cause an increase in pH, a reduction in the oxygen utilization rate and a decrease in the apparent viscosity of broth [34]. Therefore, it is reasonable to speculate that the damage to fungal activity with 2-2 resulted in the low biotransformation rate observed in the late stage of biotransformation. This indicates that the transformation activity of
During autolysis, the morphology of the hyphae changes, and intracellular substances are released at the same time. Thus, the hyphal morphology and leaked substances were investigated to confirm that the 11α-hydroxylation activity of
Hyphal Morphology
Fig. 4 shows SEM images of hyphae stirred by four impellers. The hyphae after agitation by 1-1 are smooth and thick. By contrast, the hyphal surface is rough and the thickness is not uniform when stirred by 1-2 and 2-1, in which the lack of uniformity with 2-1 is more serious than that with 2-2. The hyphae were broken most seriously, with thinner hyphae and fewer branches, when the bioreactor was equipped with 2-2. This result showed that increasing the blade number and impeller diameter can accelerate the autolysis of hyphae, where the influence of the latter was more significant than that of the former. The apparent viscosity of fermentation broth decreased sharply due to the severely broken hyphae after 36 h when using 2-2. These results were consistent with the report that the apparent viscosity is related to the concentration and morphology of microorganisms [35].
-
Figure 4. SEM images of hyphal morphology. Hyphae was collected and observed at biotransformation of 48 h.
Leakage of Hyphal Inclusions in the Cold Model Experiment
To further investigate the effect of impeller geometry on the activity of
-
Figure 5. Effects of impeller geometry on (A) leaky protein, (B) amino acid concentration and (C) EMR of
A. ochraceus .
According to the results of protein and amino acids concentration, we concluded that a large-diameter impeller accelerated the release of hyphal inclusions. In this case, the EAR value of filamentous fungi was calculated, as given in Fig. 5C. There were significant differences between the EAR values of different impeller stirrers, which indicated that mechanical agitation had an effect on the hydroxylation activity of fungi. The EAR values of 1-2, 2-1, and 2-2 were 12.93%, 31.75%, and 74.86% lower than that of 1-1, respectively, which indicated that the decreases in the 11α-hydroxylation activity of
The above results show that the DO content, microbial metabolism, hyphal morphology and biotransformation activity were all affected by the geometric parameters of the impellers, which in turn influenced the conversation ratio of canrenone. The variation in the properties of the broth is caused by the hydrodynamics in the stirred tank. Therefore, the hydrodynamics were investigated in bioreactors equipped with the four kinds of impellers using numerical simulation.
Numerical Simulation Results
Gas holdup. The distribution of the gas on the mid-plane of the bioreactor is shown in Fig. 6. The gas holdup in the lower region of the tank is higher than that in the upper region. This condition was created by the downward discharge flow in the lower circulation area, prolonging the residence time of the bubbles. It was obvious that the gas distribution of the six-blade impeller and 60 mm diameter impeller on the mid-plane was more uniform than that of the four-blade impeller and 50 mm diameter impeller. An impeller with a large diameter and more blades was effective for gas dispersion.
-
Figure 6. Effects of impeller geometry on air volume fraction.
Fluid pattern. Fig. 7A shows the flow pattern at the mid-plane of the bioreactors. Under the stirring of double Rushton impellers, the fluid was discharged from the impeller tip toward the tank wall. As the fluid hit the wall, it was divided into two loops circulating at the top and the bottom of the blade, with obvious radial flow. The fluid circulations do not interact with each other when stirred by 1-1, and the proportion of the high velocity region was the smallest and only appeared near the blade. When the blade number increased to six (1-2), the fluid circulation began to contact. When the diameter of the impeller increased to 60 mm (2-1 and 2-2), the fluid circulation areas expanded to the tank wall and crossed. The flow pattern of 2-2 was more regular than that of 2-1. At the same time, the high-speed fluid region around the impeller further expanded. The mixing dead zone was decreased, especially the region between the upper and lower impellers and at the tank wall. A Rushton impeller with a large diameter and more blades was beneficial to fluid mixing.
-
Figure 7. Effects of impeller geometry on (A) flow pattern, (B) radial velocity and (C) axial velocity of the fluid. The position of velocity generation is located on the yellow horizontal line in Fig. 7A, extending from the center of the stirring shaft to the tank wall.
The axial velocity along the radial positions of the tank near the blade is shown in Fig. 7B. This figure shows that the axial velocity of the fluid is highest when stirred by Rushton impellers with more blades and large diameters. The circulation area of the flow field mixed with 60 mm impellers is significantly wider than that of the 50 mm impellers. In this case, the width of the circulation area using 2-2 was approximately 1/2 of the tank diameter, which was the largest in this study.
Shear strain rate and stirring power. Hydrodynamic shear was produced when the broth was agitated. Fig. 8 shows the shear strain rate distribution of the fluid stirred by different impellers. The results indicated that the shear strain rate in the blade region was higher than that in the other regions in the bioreactor, and it decreased with increasing distance to the impeller. Compared with the 50 mm diameter impeller, the shear strain rate increased at the region of the liquid surface and the tank wall when equipped with a 60 mm diameter impeller in the bioreactors. When using 2-2, the low shear area only appeared at the top of the mid-plane of the bioreactor.
-
Figure 8. Effects of impeller geometry on shear strain rate.
The average shear strain rate and specific stirring power are given in Table 3. The average shear strain rate of the fluid was 19.36 s-1, and the P/V was 1.46 kW/m3 when 1-1 was used. For 1-2, 2-1, and 2-2, the average shear strain rates were 23.53%, 120.59%, and 192.16% higher than that of 1-1, respectively; the P/V values were 29.34%, 82.39%, and 123.40% higher than that of 1-1, respectively. These results showed that increasing the blade number and impeller diameter increased the shear strain rate and the specific power of the impeller. The effect of the impeller diameter was more obvious than that of the blade number.
-
Table 3 . Effect of impeller geometry on power and average shear strain rate..
Impeller Average shear strain rate (s-1) P/V (kW/m3) 1-1 19.36 1.46 1-2 25.04 1.80 2-1 35.31 3.21 2-2 43.25 4.26
Discussion
The bioconversion ratio of canrenone is determined by various factors including the DO content, the metabolism of
The biochemical reaction rate of
Oxygen needs to be dispersed and dissolved in the aqueous phase to be used by microbial cells in the process of oxygen consumption biotransformation [40, 41]. Therefore, it is essential to maintain the supply and delivery of oxygen for transformation. According to the gas holdup distribution in the bioreactor, increasing the blade number and impeller diameter can improve both the uniformity of gas in the fluid and increase the DO content. The results showed that 332.71% and 493.35% increases in the time average DO content were achieved by increasing the impeller diameter by 20% when mixing with the four-blade impeller and six-blade impeller, respectively. High power input is beneficial for air dispersion [42]. The simulation results showed that the stirring power of the Rushton impeller was higher with more blades or large diameters. It is easy for air to reach the state of complete dispersion in high-speed mixing fluid, which enables gas recycling [43]. At the same time, the apparent viscosity of fermentation broth decreases with increasing shear strain rate in the high-speed mixing area [44]. All these conditions are effective for increasing the gas-liquid contact area, reducing the gas transfer resistance and increasing the DO content in liquid phase [45]. Thus, the fermentation broth mixed with 2-1 or 2-2 had a high DO content. On the one hand, the high DO content accelerates the aerobic metabolism of reducing sugar, which provides the sufficient energy and carbon skeleton for the growth of microorganisms [46]. A high DO content is conducive to the enzyme expression during hydroxylation [47-49]. Under the condition of vigorous growth of
Compared with the other three kinds of impellers, we found that the dry biomass concentration from mixing by 2-2 was the highest at 60 h, which was 55.49% higher than that mixed by 1-1. However, the conversion ratio of 2-2 with the highest biomass was 79.29%, which was the lowest in this experiment. Therefore, the detailed influence of impeller geometry on microbial metabolism was explored further.
According to the physicochemical properties of the broth and SEM results regarding hyphal morphology, we concluded that increasing the blade number and impeller diameter of the Rushton turbine accelerated hyphal autolysis. The sharp increase of DO was related to the decrease of microbial activity. Within 36-60 h of biotransformation, the 11α-hydroxylation rates of canrenone under the agitation of the 50 mm impellers were significantly higher than that of the 60 mm impellers, even though the DO and biomass concentration of the former were lower than that of the latter. The yield of fermentation is affected by the hyphal morphology, which is greatly influenced by the shear in the tank [50, 51]. Filamentous fungal fermentation broth with differentiated and poor flexibility hyphae is more susceptible to shearing force because it is usually a non-Newtonian fluid [52]. The hyphae were damaged and autolysis was accelerated under the condition of continuous high shear stress, which was more obvious after the stable growth period. Autolysis was the most serious when agitation was provided by 2-2, resulting in severe damage to the hyphae. The average shear strain rate of 43.25 s-1 was beyond the tolerance range of the hyphae in the late stage of biotransformation under this condition, resulting in a significant decrease in the metabolic activity of
In addition to the reduction in metabolic activity, it was speculated that the decreased hydroxylation capacity of
In this study, the effects of the blade number and diameter of a Rushton impeller on the 11α-hydroxylation of canrenone were compared. We concluded that fluid flow and shear both impact the biological parameters and play an important role in the 11α-hydroxylation of canrenone. Increasing the blade number and the impeller diameter improved the fluid mixing in the stirred tank. The DO content of fermentation broth can be increased, and it had the positive effects on the microbial growth and 11α-hydroxylation of canrenone. At the same time, it was essential to apply an appropriate shear strain rate because high shear stress reduced the biotransformation activity. According to the characteristics of the 11α-hydroxylation of canrenone, it can be inferred that the increased conversion ratio of canrenone by the alteration of impellers was mainly because of the high DO content in the early stage and the good hyphal morphology in the late stage of bioconversion. In addition, both of the above conditions are beneficial for the activation of metabolism in
In summary, the fluid flow and shear in mixing fermentation broth must be controlled at the same time to ensure that the 11α-hydroxylation of canrenone can be carried out efficiently. Since reports about the influence of the impeller geometry on the biosynthesis of 11α-hydroxycanrenone have rarely been published, this research provides basic data for the industrial production of this compound.
Acknowledgments
This work was supported by the Science and Technology Commission of Shanghai Municipality (Grant no. 17441905400).
Conflict of Interest
The authors have no financial conflicts of interest to declare.
Fig 1.

Fig 2.

Fig 3.

Fig 4.

Fig 5.

Fig 6.

Fig 7.

Fig 8.

-
Table 1 . Dimensions of four impellers..
Impeller Blade number Impeller diameter (mm) 1-1 4 50 1-2 6 50 2-1 4 60 2-2 6 60
-
Table 2 . Operating conditions used for biotransformation experiments..
Conversion time (h) Agitation speed (rpm) Aeration rate (vvm) 0-12 350 1.5 12-24 450 2.0 24-36 500 2.5 36-60 500 2.5 avvm: air volume/culture volume/minute..
-
Table 3 . Effect of impeller geometry on power and average shear strain rate..
Impeller Average shear strain rate (s-1) P/V (kW/m3) 1-1 19.36 1.46 1-2 25.04 1.80 2-1 35.31 3.21 2-2 43.25 4.26
References
- Huang DM, Zhang TZ, Cui FJ, Sun WJ, Zhao LM, Yang MY,
et al . 2011. Simultaneous identification and quantification of canrenone and 11-α-hydroxy-canrenone by LC-MS and HPLC-UVD.J. Biomed. Biotechnol. 2011 : 917232. - Al-Aboudi A, Kana'an BM, Zarga MA, Bano S, Atia tul W, Javed K,
et al . 2017. Fungal biotransformation of diuretic and antihypertensive drug spironolactone with Gibberella fujikuroi, Curvularia lunata, Fusarium lini, andAspergillus alliaceus.Steroids 128 : 15-22. - Donova MV. 2017. Steroid bioconversions.
Methods Mol. Biol. 1645 : 1-13. - Petrič Š, Hakki T, Bernhardt R, Žigon D, Črešnar B. 2010. Discovery of a steroid 11α-hydroxylase from
Rhizopus oryzae and its biotechnological application.J. Biotechnol. 150 : 428-437. - Mao S, Hua B, Wang N, HU X, Ge Z, Li Y,
et al . 2013. 11α hydroxylation of 16α, 17-epoxyprogesterone in biphasic ionic liquid/water system byAspergillus ochraceus.J. Chem. Technol. Biotechnol. 88 : 287-292. - Hannemann F, Bichet A, Ewen KM, Bernhardt R. 2007. Cytochrome P450 systems-biological variations of electron transport chains.
Biochim. Biophys. Acta 1770 : 330-344. - Amanullah A, Tuttiett B, Nienow AW. 1998. Agitator speed and dissolved oxygen effects in Xanthan fermentations.
Biotechnol. Bioeng. 57 : 198-210. - Grein TA, Loewe D, Dieken H, Weidner T, Salzig D, Czermak P. 2019. Aeration and shear stress are critical process parameters for the production of oncolytic Measles virus.
Front. Bioeng. Biotechnol. 7 : 78. - Amanullah A, Serrano-Carreon L, Castro B, Galindo E, Nienow AW. 1998. The influence of impeller type in pilot scale Xanthan fermentations.
Biotechnol. Bioeng. 57 : 95-108. - Hudcova W, Machon W, Nienow AW. 1989. Gas-liquid dispersion with dual Rushton impellers.
Biotechnol. Bioeng. 34 : 617-628. - Kracík T, Moucha T, Petříček R. 2020. Gas-liquid contactors' aeration capacities when agitated by Rushton turbines of various diameters.
ACS Omega 5 : 5072-5077. - Albaek MO, Gernaey KV, Hansen MS, Stocks SM. 2011. Modeling enzyme production with
Aspergillus oryzae in pilot scale vessels with different agitation, aeration, and agitator types.Biotechnol. Bioeng. 108 : 1828-1840. - Shin W-S, Lee D, Kim S, Jeong Y-S, Chun G-T. 2013. Application of scale-up criterion of constant oxygen mass transfer coefficient (
kL a) for production of itaconic acid in a 50 L pilot-scale fermentor by fungal cells ofAspergillus terreus .J. Microbiol. Biotechnol. 23 : 1445-1453. - Jayus, McDougall BM, Seviour RJ. 2005. The effect of dissolved oxygen concentrations on (1→3)- and (1→6)-β-glucanase production by
Acremonium sp. IMI 383068 in batch culture.Enzyme Microb. Technol. 36 : 176-181. - Revstedt J, Fuchs L, Kovács T, Trägårdh C. 2000. Influence of impeller type on the flow structure in a stirred reactor.
AIChE J. 46 : 2373-2382. - Govardhan M, Venkateswarlu G. 2003. Effect of impeller geometry and tongue shape on the flow field of cross flow fans.
J. Therm. Sci. 12 : 118-125. - Li ZJ, Shukla V, Wenger KS, Fordyce AP, Pedersen AG, Marten MR. 2002. Effects of increased impeller power in a production-scale
Aspergillus oryzae fermentation.Biotechnol. Prog. 18 : 437-444. - Wang Z, Xue J, Sun H, Zhao M, Wang Y, Chu J,
et al . 2020. Evaluation of mixing effect and shear stress of different impeller combinations on nemadectin fermentation.Process Biochem. 92 : 120-129. - López JLC, Pérez JAS, Sevilla JMF, Porcel EMR, Chisti Y. 2005. Pellet morphology, culture rheology and lovastatin production in cultures of
Aspergillus terreus .J. Biotechnol. 116 : 61-77. - Buffo MM, Esperança MN, Farinas CS, Badino AC. 2020. Relation between pellet fragmentation kinetics and cellulolytic enzymes production by
Aspergillus niger in conventional bioreactor with different impellers.Enzyme Microb. Technol. 139 : 109587. - Li ZJ, Shukla V, Wenger K, Fordyce A, Pedersen AG, Marten M. 2002. Estimation of hyphal tensile strength in production-scale
Aspergillus oryzae fungal fermentations.Biotechnol. Bioeng. 77 : 601-613. - Ghobadi N, Ogino C, Ogawa T, Ohmura N. 2016. Using a flexible shaft agitator to enhance the rheology of a complex fungal fermentation culture.
Bioprocess Biosyst. Eng. 39 : 1793-1801. - Jüsten P, Paul GC, Nienow AW, Thomas CR. 1998. Dependence of
Penicillium chrysogenum growth, morphology, vacuolation, and productivity in fed-batch fermentations on impeller type and agitation intensity.Biotechnol. Bioeng. 59 : 762-775. - Gu D, Liu Z, Tao C, Li J, Wang Y. 2019. Numerical simulation of gas-liquid dispersion in a stirred tank agitated by punched rigidflexible impeller.
Int. J. Chem. React. Eng. 17 : 588-597. - Chen P, Sanyal J, Duduković MP. 2005. Numerical simulation of bubble columns flows: effect of different breakup and coalescence closures.
Chem. Eng. Sci. 60 : 1085-1101. - Gelves R, Dietrich A, Takors R. 2014. Modeling of gas-liquid mass transfer in a stirred tank bioreactor agitated by a Rushton turbine or a new pitched blade impeller.
Bioprocess Biosyst. Eng. 37 : 365-375. - Amer M, Feng Y, Ramsey JD. 2019. Using CFD simulations and statistical analysis to correlate oxygen mass transfer coefficient to both geometrical parameters and operating conditions in a stirred-tank bioreactor.
Biotechnol. Prog. 35 : e2785. - Duan S, Yuan G, Zhao Y, Ni W, Luo H, Shi Z,
et al . 2013. Simulation of computational fluid dynamics and comparison of cephalosporin C fermentation performance with different impeller combinations.Korean J. Chem. Eng. 30 : 1097-1104. - Xia J-Y, Wang Y-H, Zhang S-L, Chen N, Yin P, Zhuang Y-P,
et al . 2009. Fluid dynamics investigation of variant impeller combinations by simulation and fermentation experiment.Biochem. Eng. J. 43 : 252-260. - Contente ML, Guidi B, Serra I, De Vitis V, Romano D, Pinto A,
et al . 2016. Development of a high-yielding bioprocess for 11-α hydroxylation of canrenone under conditions of oxygen-enriched air supply.Steroids 116 : 1-4. - Miller GL. 1959. Use of dinitrosalicylic acid reagent for determination of reducing sugar.
Anal. Chem. 31 : 426-428. - Snyder JC, Desborough SL. 1978. Rapid estimation of potato tuber total protein content with coomassie brilliant blue G-250.
Theor. Appl. Genet. 52 : 135-139. - Moore S. 1968. Amino acid analysis: aqueous dimethyl sulfoxide as solvent for the ninhydrin reaction.
J. Biol. Chem. 243 : 6281-6283. - Harvey LM, McNeil B, Berry DR, White S. 1998. Autolysis in batch cultures of
Penicillium chrysogenum at varying agitation rates.Enzyme Microb. Technol. 22 : 446-458. - Riley GL, Tucker KG, Paul GC, Thomas CR. 2000. Effect of biomass concentration and mycelial morphology on fermentation broth rheology.
Biotechnol. Bioeng. 68 : 160-172. - Tokura Y, Uddin MA, Kato Y. 2019. Effect of suspension pattern of sedimentary particles on solid/liquid mass transfer in a mechanically stirred vessel.
Ind. Eng. Chem. Res. 58 : 10172-10178. - Lin Y, Zhang Z, Thibault J. 2011. New impeller for viscous fermentation: power input and mass transfer coefficient correlations.
Ind. Eng. Chem. Res. 50 : 3510-3516. - Tang W, Pan A, Lu H, Xia J, Zhuang Y, Zhang S,
et al . 2015. Improvement of glucoamylase production using axial impellers with low power consumption and homogeneous mass transfer.Biochem. Eng. J. 99 : 167-176. - Dohi N, Takahashi T, Minekawa K, Kawase Y. 2004. Power consumption and solid suspension performance of large-scale impellers in gas-liquid-solid three-phase stirred tank reactors.
Chem. Eng. J. 97 : 103-114. - Rao DVK, Ramu CT, Rao JV, Narasu ML, Rao AKSB. 2008. Impact of dissolved oxygen concentration on some key parameters and production of rhG-CSF in batch fermentation.
J. Ind. Microbiol. Biotechnol. 35 : 991-1000. - Tang YJ, Li HM, Hamel JFP. 2009. Effects of dissolved oxygen tension and agitation rate on the production of heat-shock protein glycoprotein 96 by MethA tumor cell suspension culture in stirred-tank bioreactors.
Bioprocess Biosyst. Eng. 32 : 475-484. - Fujasová M, Linek V, Moucha T. 2007. Mass transfer correlations for multiple-impeller gas-liquid contactors. Analysis of the effect of axial dispersion in gas and liquid phases on "local"
kL a values measured by the dynamic pressure method in individual stages of the vessel.Chem. Eng. Sci. 62 : 1650-1669. - Bao Y, Wang B, Lin M, Gao Z, Yang J. 2015. Influence of impeller diameter on overall gas dispersion properties in a sparged multiimpeller stirred tank.
Chin. J. Chem. Eng. 23 : 890-896. - Kilonzo PM, Margaritis A. 2004. The effects of non-Newtonian fermentation broth viscosity and small bubble segregation on oxygen mass transfer in gas-lift bioreactors: a critical review.
Biochem. Eng. J. 17 : 27-40. - Najafpour GD. 2015. Gas and liquid system (aeration and agitation), pp. 51-102.
In: Najafpour GD (ed),Biochemical Engineering and Biotechnology , 2th Ed. Elsevier, Amsterdam, Netherlands. - Clark TA, Maddox IS, Chong R. 1983. The effect of glucose on 11β- and 19-hydroxylation of Reichstein's Substance S by
Pellicularia filamentosa .Appl. Microbiol. Biotechnol. 17 : 211-215. - Chen K-C, Wey H-C. 1990. 11β-Hydroxylation of coxtexolone by
Curvularia lunata .Enzyme Microb. Technol. 12 : 305-308. - Gbewonyo K, Buckland BC, Lilly MD. 1990. Development of a large-scale continuous substrate feed process for the biotransformation of simvastatin by
Nocardia sp.Biotechnol. Bioeng. 37 : 1101-1107. - Clark TA, Chong R, Maddox IS. 1982. The effect of dissolved oxygen tension on 11β- and 19-hydroxylation of Reichstein's Substance S by Pellicularia filamentosa.
Appl. Microbiol. Biotechnol. 14 : 131-135. - El-Enshasy H, Hellmuth K, Rinas U. 1999. Fungal morphology in submerged cultures and its relation to glucose oxidase excretion by recombinant
Aspergillus niger.Appl. Biochem. Biotechnol. 81 : 1-11. - Chen X, Zhou J, Ding Q, Luo Q, Liu L. 2019. Morphology engineering of
Aspergillus oryzae for L-malate production.Biotechnol. Bioeng. 116 : 2662-2673. - Ghobadi N, Ogino C, Yamabe K, Ohmura N. 2017. Characterizations of the submerged fermentation of
Aspergillus oryzae using a Fullzone impeller in a stirred tank bioreactor.J. Biosci. Bioeng. 123 : 101-108. - Žnidaršič P, Komel R, Pavko A. 1998. Studies of a pelleted growth form of
Rhizopus nigricans as a biocatalyst for progesterone 11 α-hydroxylation.J. Biotechnol. 60 : 207-216. - Shen Y, Wang L, Liang J, Tang R, Wang M. 2016. Effects of two kinds of imidazolium-based ionic liquids on the characteristics of steroid-transformation
Arthrobacter simplex .Microb. Cell Fact. 15 : 118-127.