Significance of seed culture methods on mycelial morphology and production of a novel anti-cancer anthraquinone by marine mangrove endophytic fungus Halorosellinia sp. (No. 1403)

The effects of seed culture methods on the mycelial morphology and production of a novel promising anti-cancer anthraquinone 1403C by marine mangrove saprophytic fungus Halorosellinia sp. (No. 1403) was investigated. Inoculums were prepared using different seed culture methods, i.e., mycelia obtained by grinding biomass that was harvested from baffled flask culture (M1); biomass harvested from baffled flask culture (M2); biomass obtained from unbaffled flask culture with glass beads (M3); biomass attained from unbaffled flask culture (Control). The corresponding fermentations using M1, M2 and M3 enhanced 1403C production by 243.5%, 194.8% and 70.2%, respectively, as compared to that using Control (0.33 ± 0.03 g/l). Interestingly, 1403C production increased with the increase of ratio of number of clumps to pellets. Maximum 1403C production from baffled flask cultures was 4.8-fold of that from unbaffled flask culture. Increasing shaking speed from 170 rpm to 260 rpm could highly improve 1403C production by 151.8%.     

The role of volumetric power input in the growth,morphology, and production of a recombinant glycoprotein by Streptomyces lividans in shake flasks

The impact of flask geometry on Streptomyces lividans growth and morphology, production and O-mannosylation of a recombinant O-glycoprotein (APA from Mycobacterium tuberculosis) was described and associated to the evolution of the volumetric power input (P/V) in three shake flask geometries. During the exponential growth, the highest P/V was found in baffled flasks (BF) with 0.51 kW/m³, followed by coiled flasks (CF) with 0.44 kW/m³ and normal Erlenmeyer flasks (NF) with 0.20 kW/m³ (flasks volume of 250 mL, filling with 50 mL and agitated at 150 rpm). During the stationary phase, P/V decreased 20% in BF and CF, but increased two times in NF, surely due to changes in mycelial morphology and its effects on rheology. Also, NF cultures were carried out at a filling volume and agitation of 15 mL, 150 rpm (15 mL-NF), and 25 mL, 168 rpm (25 mL-NF), in order to raise P/V closely to the values obtained in CF. However, different growth, morphology and recombinant protein productivity were obtained. These data indicate that P/V is not a definitive parameter that can determine bacteria growth and morphology, not even glycoprotein production. But it can be proposed that the oxygen transfer in the center of the pellets and hydromechanical stress might be the more relevant parameters than P/V.     

Relation between pristinamycins production by Streptomyces pristinaespiralis,power dissipation and volumetric gas–liquid mass transfer coefficient,kLa

During bioreactor cultures, microorganisms are submitted to non-optimal conditions such as nutritional and hydrodynamic stresses which may lead to modifications of the physiological cell response; this is especially true for filamentous microorganisms like Streptomycetes also subjected to significant morphological changes. In the present work, growth and production of pristinamycins by Streptomyces pristinaespiralis in shaking flasks have been related to power dissipation. The filamentous bacteria were grown in different flask conditions with various total and working volumes and at two agitation rates, to test the influence of power dissipation and gas–liquid mass transfer coefficient on growth and antibiotics production. As a first step, computational fluid dynamics–volume of fluid (CFD–VOF) calculations were shown to be able to predict power dissipations for the various operating conditions in Newtonian flow conditions. Then, in non-Newtonian flow conditions (biomass concentration superior to 14 g L⁻¹), the rheological model of Sisko was implemented in CFD simulations for the calculation of the fluid viscosity and then of power dissipation. Whereas microbial growth was correlated to k_La, the antibiotics production onset was linked to the volume mean power dissipation. Once a minimal cell concentration of 15 g L⁻¹ was reached, the concentration of antibiotics was correlated to power dissipation with an optimal range of production, between 5.5 and 8.5 kW m⁻³. Higher power dissipation entailed a drop in production which could be explained by hydrodynamic cell damages.     

Anthraquinones production in Rubia tinctorum cell suspension cultures: Down scale of shear effects

The effect of turbulence on suspended cells is one of the most complex problems in the scale-up of cell cultures. In the present paper, a direct comparison of the effects of turbulence on suspension cultures of Rubia tinctorum in a standard bioreactor and in shake flask cultures was done. A procedure derived from the well known global method proposed by Nishikawa et al. (1977) [39] was applied. Standard flasks and four-baffled shake flasks were used. The effect of turbulence and light irradiation on cell viability, biomass, and anthraquinones (AQs) production was evaluated. The biomass concentration and AQs production obtained using baffled shake flasks agitated at 360 rpm were similar to that achieved in R. tinctorum suspension cultures growing in a stirred tank bioreactor operating at 450 rpm, previously published (Busto et al., 2008 [17]). The effect of light on AQs production was found to be very significant, and a difference of up to 48% was found in cells with and without illumination after 7 days of culture. It is concluded that this down-scaled and simple flask culture system is a suitable and valid small scale instrument for the study of intracellular mechanisms of turbulence-induced AQs production in R. tinctorum suspension cultures.     

Comparison of oxygen mass transfer coefficient in simple and extractive fermentation systems

Aeration and agitation are important variables to ensure effective oxygen transfer rate during aerobic bioprocesses; therefore, the knowledge of the volumetric mass transfer coefficient (k_La) is required. In view of selecting the optimum oxygen requirements for extractive fermentation in aqueous two-phase system (ATPS), the k_La values in a typical ATPS medium were compared in this work with those in distilled water and in a simple fermentation medium, in the absence of biomass. Aeration and agitation were selected as the independent variables using a 2² full factorial design. Both variables showed statistically significant effects on k_La, and the highest values of this parameter in both media for simple fermentation (241 s⁻¹) and extractive fermentation with ATPS (70.3 s⁻¹) were observed at the highest levels of aeration (5 vvm) and agitation (1200 rpm). The k_La values were then used to establish mathematical correlations of this response as a function of the process variables. The exponents of the power number (N³D²) and superficial gas velocity (V_s) determined in distilled water (α = 0.39 and β = 0.47, respectively) were in reasonable agreement with the ones reported in the literature for several aqueous systems and close to those determined for a simple fermentation medium (α = 0.38 and β = 0.41). On the other hand, as expected by the increased viscosity in the presence of polyethylene glycol, their values were remarkably higher in a typical medium for extractive fermentation (α = 0.50 and β = 1.0). A reasonable agreement was found between the experimental data of k_La for the three selected systems and the values predicted by the theoretical models, under a wide range of operational conditions.     

Construction of recombinant Escherichia coli for enhanced bioconversion of colchicine into 3-demethylated colchicine at 70 l bioreactor level

A recombinant strain of Escherichia coli with CYP102A1 gene was developed for the demethylation of colchicine into their derivatives. The CYP102A1 gene responsible for demethylation was isolated from Bacillus megaterium ACBT03 and amplified using suitable primers. The amplified product was cloned into pET28a+ expression vector using host E. coli BL21(DE3) cells. The CYP3A4 (product of CYP102A1 gene) protein expression and other parameters like substrate toxicity, product toxicity and enzyme activity were optimized in shake flasks; and further scaled-up to 5 l bioreactor with 3 l working volume. In 5 l bioreactor, dissolved oxygen (DO) was optimized for maximum specific growth and enhanced 3-demethylated colchicine (3-DMC) production. The optimized conditions from shake flasks were scaled-up to 70 l bioreactor and resulted into ∼80% conversion of 20 mM colchicine in 48 h with a volumetric productivity of 6.62 mg l⁻¹ h⁻¹. Scale-up factors were measured as volumetric oxygen transfer coefficient (k_La) i.e., 56 h⁻¹ and impeller tip velocity (V_tip) i.e., 7.065 m s⁻¹, respectively. The kinetic parameters K_m, k_cat, and k_cat/K_m of the CYP3A4 enzyme using colchicine as the substrate were determined to be 271 ± 30 μM, 8533 ± 25 min⁻¹, and 31.49 μM min⁻¹, respectively, when IPTG induced recombinant E. coli culture was used.     

Power consumption in shaking flasks on rotary shaking machines: II. Nondimensional description of specific power consumption and flow regimes in unbaffled flasks at elevated liquid viscosity

This article is the second part of a series presenting and modeling the hydrodynamics and specific power consumption in shaking flasks on rotary (orbital) shaking machines. In part I, a new method was introduced that enables the accurate determination of the specific power consumption in shaking flasks. The method was first applied to investigate unbaffled flasks with a nominal volume of < or =1 L at low viscosity. In part II, the results for the specific power consumption of unbaffled shaking flasks at elevated viscosities are investigated after varying shaking frequency, flask size, filling volume, and shaking diameter. The theory introduced in part I is extended to liquids of elevated viscosities using nondimensional equations. With these results, the specific power consumption in unbaffled shaking flasks can now be fully described. For the first time, the phenomenon of the liquid being "out of phase" is observed and described. This occurs at certain operating conditions and is characterized by an increasing amount of liquid not following the movement of the shaking table, thus reducing the specific power consumption. This, of course, has much relevance for practical work with microbial cultures. The phenomenon of being "out-of-phase" is described in the form of a newly defined nondimensional phase number (Ph) in analogy to a partially filled, rotating horizontal drum. The Ph can be used to determine reasonable operating conditions for shaking flask experiments when using viscous media, avoiding unfavorable "out-of-phase" operation.     

Optimization of the aeration and agitation speed of Aeribacillus palidus 418 exopolysaccharide production and the emulsifying properties of the product

The specific properties of exopolysaccharides (EPS) from thermophilic microorganisms have attracted interest in their optimized production. In this study, the ability of Aeribacillus pallidus 418 to grow and produce polysaccharide in a 5-l stirred tank bioreactor was investigated. Agitation rates of 100, 200, 600, 900, and 1100 revolutions per minute (rpm), at an air flow rate of 0.5 gas volumes per unit medium volume per minute (vvm), and aeration rates of 0.25, 0.5, 1.0, and 1.5 vvm, at an agitation rate of 900 rpm, were examined. A maximum EPS yield of 170 μg/ml has been registered in a single impeller bioreactor equipped with an original Narcissus impeller at agitation speed of 900 rpm, with an aeration rate of 0.5 vvm. The bioprocess oxygen uptake rate (OUR) and oxygen mass transfer coefficient (K_La) were evaluated. The emulsifying properties of the specific EPS produced by A. pallidus 418 were determined. Stable oil-in-water emulsions, a low level of separated water phase and high dispersion stability were found, which together demonstrate the prospects for the industrial exploration of EPS production. Enhanced synergism between the A. pallidus 418 synthesized EPS and various commercially used hydrocolloids was observed; superior synergy was achieved in combination with xanthan gum.     

Effect of the aeration rate and agitation speed on β-carotene production and morphology of Blakeslea trispora in a stirred tank reactor: mathematical modeling

The effect of aeration rate and agitation speed on β-carotene production and morphology of Blakeslea trispora in a stirred tank reactor was investigated. B. trispora formed hyphae, zygophores and zygospores during the fermentation. The zygospores were the morphological form responsible for β-carotene production. Both aeration and agitation significantly affected β-carotene concentration, productivity, biomass and the volumetric mass transfer coefficient (K_La). The highest β-carotene concentration (1.5 kg m⁻³) and the highest productivity (0.08 kg m⁻³ per day) were obtained at low impeller speed (150 rpm) and high aeration rate (1.5 vvm). Also, maximum productivity (0.08 kg m⁻³ per day) and biomass dry weight (26.4 kg m⁻³) were achieved at high agitation speed (500 rpm) and moderate aeration rate (1.0 vvm). Conversely, the highest value of K_La (0.33 s⁻¹) was observed at high agitation speed (500 rpm) and high aeration rate (1.5 vvm). The experiments were arranged according to a central composite statistical design. Response surface methodology was used to describe the effect of impeller speed and aeration rate on the most important fermentation parameters. In all cases, the fit of the model was found to be good. All fermentation parameters (except biomass concentration) were strongly affected by the interactions among the operation variables. β-Carotene concentration and productivity were significantly influenced by the aeration, agitation, and by the positive or negative quadratic effect of the aeration rate. Biomass concentration was principally related to the aeration rate, agitation speed, and the positive or negative quadratic effect of the impeller speed and aeration rate, respectively. Finally, the volumetric mass transfer coefficient was characterized by the significant effect of the agitation speed, while the aeration rate had a small effect on K_La.     

Characterization of heat transfer of large orbitally shaken cylindrical bioreactors

Disposable shaking bioreactors are a promising alternative to other disposable bioreactors owing to their ease of operation, flexibility, defined hydrodynamics and characterization. Shaken bioreactors of sizes 20 L and 50 L are characterized in terms of heat transfer characteristics in this research work. Water and an 80% glycerol–water system were used as fluid. Results indicated large heat generation due to shake mixing which was observed by temperature difference between the fluid inside the vessel and the surrounding air outside the vessel. Maximum temperature difference of ca. 30 K was encountered for a 50 L vessel, at 300 rpm and 20 L filling volume. Outside heat transfer rate was governing the overall heat transfer process. Lateral air flow did increase heat transfer rates to large extent. An empirical correlation of overall heat transfer coefficient was obtained in terms of filling volume, rotational speed and lateral air flow rate. However, as the vessel thickness increased, the overall heat transfer process was limited by vessel wall resistance.     

Changes in alginate molecular mass distributions, broth viscosity and morphology of Azotobacter vinelandii cultured in shake flasks

The effect of different aeration conditions during the culture of Azotobacter vinelandii on the production and molecular mass of alginate was evaluated in shake flasks. In baffled flasks, the bacteria grew faster and produced less alginate (1.5 g/l) than in conventional (unbaffled) flasks (4.5 g/l). The viscosity of the culture broth was also influenced by the type of flask. Higher final viscosities were attained in unbaffled flasks [520 cP (520 mPa s)] as compared to baffled flasks (30 cP). This latter phenomenon was closely related to the changes in the molecular mass distribution. In either cases, the mean molecular mass increased with culture age; however, at the end of the fermentation, the mean molecular mass of the alginate obtained in unbaffled flasks was fivefold higher than that obtained in baffled flasks. As the culture proceeded, the cells of Azotobacter grown in unbaffled flasks increased in diameter, whereas those cultured in baffled flasks decreased in size. Received: 13 December 1996 / Received revision: 10 April 1997 / Accepted: 27 April 1997     

Power consumption in shaking flasks on rotary shaking machines: I. Power consumption measurement in unbaffled flasks at low liquid viscosity

In this first article of a series a new method is introduced that enables the accurate determination of the power consumption in a shaking flask. The method is based on torque measurements in the drive and appropriate compensation of the friction losses. The results for unbaffled shaking flasks at low viscosities are presented after varying shaking frequency, flask size, filling volume, shaking diameter, and surface quality (hydrophilic and hydrophobic) of the inner flask walls. The order of magnitude of the values of power consumption in shaking flasks is equal to, or even higher than, the values typical for agitated tank bioreactors. A physically based model equation for shaking flasks is derived that introduces a modified power number and a resulting constant as the only fitting parameter. With this equation, the measured results are correlated with sufficient accuracy. For the first time, comprehensive data for the power consumption in unbaffled shaking flasks at low viscosity is available, giving a detailed picture of the influences of the different variables.     

The physical characterisation of a microscale parallel bioreactor platform with an industrial CHO cell line expressing an IgG4

There is a growing body of evidence that the ambr™ workstation from TAP Biosystems performs well in terms of helping to select appropriate clones for scale-up studies. Here we have investigated the physical characteristics of this microscale bioreactor system and found that these are quite different from those that exist in larger scale stirred bioreactors. For example, the flow regime in the ambr™ vessel is transitional rather than turbulent and the sparged air/oxygen superficial gas velocity is relatively very low whilst the specific power input is much higher (~400 W/m³) when compared to that used at larger scales (typically ~20 W/m³). This specific power input is necessary in order to achieve k_La values sufficiently high to satisfy the oxygen demand of the cells and control of dO₂. In line with other studies, we find that the culture of CHO cells in a 15 mL ambr™ bioreactor gave similar cell growth and productivity to that achieved in a 5 L stirred bioreactor whilst the results from shake flasks were significantly different. Given the differences in physical characteristics between the ambr™ and larger stirred bioreactors, we suggest that this similarity in biological performance is due to their similar control capabilities and the ‘equivalence of the stress parameters’ across the scales when compared with shake flasks.     

Optimal conditions for enhanced β-mannanase production by recombinant Aspergillus sojae

Optimization of the growth conditions for maximum β-mannanase production in shake flasks by using recombinant Aspergillus sojae ATCC11906 (AsT1) was carried out by Box–Behnken design of response surface methodology. The highest β-mannanase activity on the fourth day of cultivation at 30 °C was obtained as 363 U/ml in the optimized medium consisting of 7% sugar beet molasses, 0.43% NH₄NO₃, 0.1% K₂HPO₄ and 0.05% MgSO₄ (by weight per volume) at 207 rpm. On the sixth day of cultivation under the optimized conditions, the highest β-mannanase activity was achieved as 482 U/ml which is 1.4-fold of 352 U/ml activity found on glucose medium previously.     

Oxygen mass transfer and scale-up studies in baffled roller bioreactors

Oxygen mass transfer was studied in conventional, bead mill and baffled roller bioreactors. Using central composite rotational design, impacts of size, rotation speed and working volume on the oxygen mass transfer were evaluated. Baffled roller bioreactor outperformed its conventional and bead mill counterparts, with the highest k _L a obtained in these configurations being 0.58, 0.19, 0.41 min^?1, respectively. Performances of the bead mill and baffled roller bioreactor were only comparable when a high bead loading (40 %) was applied. Regardless of configuration increase in rotation speed and decrease in working volume improved the oxygen mass transfer rate. Increase in size led to enhanced mass transfer and higher k _L a in baffled roller bioreactor (0.49 min^?1 for 2.2 L and 1.31 min^?1 for 55 L bioreactors). Finally, the experimentally determined k _L a in the baffled roller bioreactors of different sizes fit reasonably well to an empirical correlation describing the k _L a in terms of dimensionless numbers.     

Surfactant-assisted two phase partitioning bioreactors for laccase-catalyzed degradation of anthracene

The degradation of anthracene by laccase from Trametes versicolor in enzymatic reactors was evaluated. The use of a surfactant (Triton X-100) at concentration above critical micelle concentration (CMC) enhanced anthracene solubility and facilitated its degradation. Moreover, Triton exerted a beneficial effect on the laccase stability and protected it from the oxidative action of the mediator 1-hydroxybenzotriazole (HBT). In a further stage, the combined configuration of a two phase partitioning bioreactor (TPPB) operating with silicone oil as an immiscible solvent and the surfactant achieved the degradation of anthracene at higher conversion rate: 16 μmol/L_Rh. Furthermore, a model for anthracene degradation by laccase-mediator system was developed. The first order kinetic constant (k) and the overall mass transfer coefficient (K_La) were estimated by using the method of least squares. The increased K_La value obtained, 788.1 h^?1, proved that Triton also improved mass transfer. Anthracene concentration in aqueous phase was close to that corresponding to equilibrium state suggesting that mass transfer mechanism did not limit the global process. The kinetic constant, which is expected to depend on the initial concentration of enzyme, resulted in 52.2 h^?1. Enzyme inactivation occurred in two stages and could be modeled by using a three parameter biexponential model. The possibility of reusing silicone oil to dissolve more anthracene was proven in three consequent cycles with high percentages of anthracene removal.     

Optical sensor enabled rocking T-flasks as novel upstream bioprocessing tools

During the past decade, novel disposable cell culture vessels (generally referred to as Process Scouting Devices or PSDs) have become increasingly popular for laboratory scale studies and seed culture generation. However, the lack of engineering characterization and online monitoring tools for PSDs makes it difficult to elucidate their oxygen transfer capabilities. In this study, a mass transfer characterization (k_La) of sensor enabled static and rocking T‐flasks is presented and compared with other non‐instrumented PSDs such as CultiFlask 50®, spinner flasks, and SuperSpinner D 1000®. We have also developed a mass transfer empirical correlation that accounts for the contribution of convection and diffusion to the volumetric mass transfer coefficient (k_La) in rocking T‐flasks. We also carried out a scale‐down study at matched k_La between a rocking T75‐flask and a 10 L (2 L filling volume) wave bioreactor (Cultibag®) and we observed similar DO and pH profiles as well as maximum cell density and protein titer. However, in this scale‐down study, we also observed a negative correlation between cell growth and protein productivity between the rocking T‐flask and the wave bioreactor. We hypothesize that this negative correlation can be due to hydrodynamic stress difference between the rocking T‐flask and the Cultibag. As both cell culture devices share key similarities such as type of agitation (i.e., rocking), oxygen transfer capabilities (i.e., k_La) and disposability, we argue that rocking T‐flasks can be readily integrated with wave bioreactors, making the transition from research‐scale to manufacturing‐scale a seamless process. Biotechnol. Bioeng. 2012;109: 2295–2305. © 2012 Wiley Periodicals, Inc.     

Bioprocess development of the production of the mutant P-219-L human d-amino acid oxidase for high soluble fraction expression in recombinant Escherichia coli

In order to examine the structure–activity relationship and the substrate specificity of human d-amino acid oxidase (h.DAO), a single amino acid mutation had been established as proline-219-luecine (P-219-L). The gene encoding mutant h.DAO has been cloned and expressed in Escherichia coli BL21 (DE3). It was observed that the host cell was negatively affected by the expressed mutant h.DAO, resulting in a remarkable decrease in the cell growth and consequently the amount of the produced enzyme. To overcome this problem, we investigated several factors that may affect the cell growth rate and the mutant h.DAO production such as optimization of the glucose concentration as a main carbon source and the yeast extract concentration as a main nitrogen source, optimization of dissolved oxygen (DO%) concentration and the addition of benzyl alcohol (BA, which can artificially induce a strong heat shock response at low temperature), to enhance the production of natively folded soluble fraction of the recombinant protein. These parameters were tested on both shake flask level and fed-batch bioreactor level. The Western blot analysis and the enzyme activity assay indicated the higher level of the mutant expression towards enhancement of the conditions by using our designed approach.The specific activity (which was used as an indicator for the level of the desired protein produced = U/mg protein) and the OD_600 nm of the host cells (which was used as an indicator for the cell growth), reached to be 0.061 U/mg protein and 3.44, respectively upon using fed-batch culture system containing the optimized medium composition (15 g/l glucose and 5 g/l yeast extract). While upon using the shake flask level, these values were 0.032 and 1.1, respectively. Enhancement of the cell growth and the enzyme production was noticed after DO% optimization upon using 500 rpm agitation speed and 1.8 v.v.m. (volume volume minute) aeration. The specific activity for the mutant enzyme and the OD_600 nm of the host cells reached to be 0.14 U/mg protein and 7.1, respectively. Finally upon using the optimized culture composition (15 g/l glucose and 5 g/l yeast extract), optimized DO% (using 500 rpm agitation speed and 1.8 v.v.m.) and 0.1 mM BA at the fed-batch bioreactor level, the specific activity and the OD_600 nm of the host cells increased significantly to be 0.21 U/mg protein and 11.3, respectively at 24 h culture. These results indicate the importance of our approaches to overproducing mutant h.DAO in soluble form in E. coli.     

Characterisation of the gas-liquid mass transfer in shaking bioreactors

The maximum gas-liquid mass transfer capacity of 250ml shaking flasks on orbital shaking machines has been experimentally investigated using the sulphite oxidation method under variation of the shaking frequency, shaking diameter, filling volume and viscosity of the medium. The distribution of the liquid within the flask has been modelled by the intersection between the rotational hyperboloid of the liquid and the inner wall of the shaking flask. This model allows for the calculation of the specific exchange area (a), the mass transfer coefficient (k(L)) and the maximum oxygen transfer capacity (OTR(max)) for given operating conditions and requires no fitting parameters. The model agrees well with the experimental results. It was furthermore shown that the liquid film on the flask wall contributes significantly to the specific mass transfer area (a) and to the oxygen transfer rate (OTR).