Oxygen transfer in a multiple air-lift loop reactor

The Multiple Air-lift Loop reactor (MAL) is a new type of bioreactor, in which a series of airlifts with internal loops is incorporated into one vessel. As such, the MAL is an approximation of an aerated plug-flow fermenter. Gas/liquid oxygen transfer was studied as a function of the gas flow rate in a MAL. The second MAL-compartment in the series was investigated in particular, and a Rectangular Air-lift Loop reactor (RAL) was used as a reference. Both a dynamic and a steady-state method were used for the determination of the overall volumetric oxygen-transfer coefficient. Both methods gave the same results. The oxygen transfer coefficient in the second MAL-compartment was low compared to that of conventional internal-loop reactors. Wall effects probably caused bubble coalescence and a reduction in the oxygen transfer. For the RAL it was found that oxygen transfer was comparable to that in a bubble column.     

Investigations of cephalosporin C production in an airlift tower loop reactor

Summary Cephalosporin C was produced by Cephalosporium acremonium in a 60 l airlift loop reactor on complex medium (with 30 kg/m³ peanut flour) in fed-batch operation. A final product concentration of 5 kg/m³ and a maximum productivity of 45 g/m³ h were attained. On-line analysis was used to determine ammonia, methionine, phosphate, reducing sugar and cephalosporin C by an autoanalyser, glucose by a flow injection analyser and cephalosporin C, penicillin N, deacetoxycephalosporin C, deacetylce-phalosporin C and methionine by HPLC. The volumetric productivity of the stirred tank reactor was higher than that of the airlift reactor because of differences in cell concentration. Specific productivities in relative to cell mass were similar in the two reactors. The substrate yield coefficient in the airlift reactor was twice that in the stirred tank reactor.Nomenclature E _o2 efficiency of oxygen transfer with regard to the specific power input - K _La volumetric mass transfer coefficient - OTR oxygen transfer rate - P power input - PR volumetric productivity of CPC - q _a volumetric aeration rate/broth volume (vvm) - SPR specific productivity with regard to RNA - V _L broth volume in reactor - z relative height of the aerated reactor     

Influence of medium composition on penicillin V production in a stirred tank reactor.

Penicillin production with a high-producing strain Penicillium chrysogenum was investigated under well-controlled conditions in a stirred tank reactor with complex media containing lard oil and lactose on the one hand, and lactose on the other hand. With lard oil, cell growth and product formation rates were higher, and the production time was shorter by 40 h than without lard oil. On account of the longer production time without lard oil, the amount of beta-lactam compounds was higher (29.93 g l-1), but the mole fraction of the decomposed products (penicilloic acid and penilloic acid) was larger (0.282) than the amount of penicillin V (23.25 g l-1) and the decomposed mole fraction (0.0747) with lard oil. The final product concentrations were about the same (20.86 g l-1 or 35,462 IU ml-1 with lard oil, and 20.43 g l-1 or 34510 IU ml-1 without lard oil). The mole fractions of the by-product (p-OH-penicillin V) were 0.0365 and 0.066. The substitution of lard oil with lactose is possible without a considerable reduction of process performance.     

The influence of acetic acid on penicillin production

Summary Fed-batch fermentations with Penicillum chrysogenum, strain S 3723, were fed with glucose as carbon source or with a mixture of glucose and acetic acid. When 20% of the carbon source was acetic acid, yields of penicillin-V were 25% higher than in fermentations where glucose was the only carbon source in the feed. The increased yield was due to higher specific productivity and/or cell mass. The effect was seen in fermentations where the carbon source was fed at a constant rate and the pH kept automatically at 6.5 by addition of inorganic acid or base, as well as in fermentations where pH controlled the addition of feed.     

Death rate in a small air-lift loop reactor of vero cells grown on solid microcarriers and in macroporous microcarriers

The death rate of Vero cells grown on Cytodex-3 microcarrierswas studied as a function of the gas flow rate in a smallair-lift loop reactor. The death rate may be described byfirst-order death-rate kinetics. The first-order death-rateconstant as calculated from the decrease in viable cells, theincrease in dead cells and the increase in LDH activity islinear proportional to the gas flow rate, with a specifichypothetical killing volume in which all cells are killed ofabout 2.10(-3)m(3) liquid per m(3) of air bubbles.In addition, an experiment was conducted in the sameair-lift reactor with Vero cells grown inside porous Asahimicrocarriers. The specific hypothetical killing volumecalculated from this experiment has a value of 3.10(-4)m(3) liquid per m(3) of air bubbles, which shows thatthe porous microcarriers were at least in part able to protectthe cells against the detrimental hydrodynamic forcesgenerated by the bubbles.     

Death rate in a small air-lift loop reactor of vero cells grown on solid microcarriers and in macroporous microcarriers

The death rate of Vero cells grown on Cytodex-3 microcarriers was studied as a function of the gas flow rate in a small air-lift loop reactor. The death rate may be described by first-order death-rate kinetics. The first-order death-rate constant as calculated from the decrease in viable cells, the increase in dead cells and the increase in LDH activity is linear proportional to the gas flow rate, with a specific hypothetical killing volume in which all cells are killed of about 2·10^–3 m³ liquid per m³ of air bubbles. In addition, an experiment was conducted in the same air-lift reactor with Vero cells grown inside porous Asahi microcarriers. The specific hypothetical killing volume calculated from this experiment has a value of 3·10^–4 m³ liquid per m³ of air bubbles, which shows that the porous microcarriers were at least in part able to protect the cells against the detrimental hydrodynamic forces generated by the bubbles.     

Coenzyme Q10 production in a 150-l reactor by a mutant strain of Rhodobacter sphaeroides

For the commercial production of CoQ₁₀, batch-type fermentations were attempted in a 150-l fermenter using a mutant strain of R. sphaeroides. Optimum temperature and initial aeration rate were found to be 30°C and 2 vvm, respectively. Under optimum fermentation conditions, the maximum value of specific CoQ₁₀ content was achieved reproducibly as 6.34 mg/g DCW after 24 h, with 3.02 g/l of DCW. During the fermentation, aeration shift (from the adequate aeration at the early growth phase to the limited aeration in active cellular metabolism) was a key factor in CoQ₁₀ production for scale-up. A higher value of the specific CoQ₁₀ content (8.12 mg/g DCW) was achieved in fed-batch fermentation and comparable to those produced by the pilot-scale fed-batch fermentations of A. tumefaciens, which indicated that the mutant strain of R. sphaeroides used in this study was a potential high CoQ₁₀ producer. This is the first detailed study to demonstrate a pilot-scale production of CoQ₁₀ using a mutant strain of R. sphaeroides.     

Identification of mass-transfer parameters and process simulation of SCP production process in airlift tower reactors with an external loop

A distributed parameter model for simulation of SCP-production processes in tower reactors with an outer loop was developed by considering substrate, cell, and CO(2) balances in the liquid phase, and O(2) and CO(3) balances in the ges phase and taking into account variations of dissolved oxygen concentration, pressure, and k(L)a along the column, as well as double substrate Monod kinetics. This model was used to describe the cultivation of Hansenula polymorpha in a tower-loop reactor (height 275 cm, diameter 15 cm). Parameter identification and process simulation were carried out by a hybrid computer. The variation of identified mass transfer parameters with fermentation time and operation mode is considered employing ethanol and glucose substrate, respectively. Relationships among k(L)a, substrate concentration, and superficial gas velocity were developed to facilitate the layout and simulation of pilot-plant reactors.     

Enzymatic splitting of penicillin V for the production of 6-APA using immobilized penicillin V acylase

Penicillin V acylase from Fusarium sp. SKF 235 was immobilized on several cation-exchange resins, of which Amberlite CG-50 was preferred. Maximum activity of the immobilized penicillin V acylase was 250 to 280 IU/g dry beads. The pH and temperature optima of the enzyme shifted from 6.5 to 6.8 and 55°C to 60°C, respectively, as a result of immobilization. However, the K _m for penicillin V remained at 10mm. Parameters for producing 6-aminopenicillanic acid were investigated and the immobilized penicillin V acylase was used for 68 cycles in a stirred tank reactor.     

A novel multi-stage preculture strategy of Rhizopus oryzae ME-F12 for fumaric acid production in a stirred-tank reactor

This study has developed a novel multi-stage preculture strategy to control the morphology of filamentous Rhizopus oryzae in a 3.3-l stirred-tank reactor (STR), consequently enhancing the fumaric acid production. Using multi-stage precultures as inoculum, the morphology of R. oryzae was maintained as large clumps, small fluffy pellets, and entangled filaments. The highest fumaric acid production of 42.5 g/l with a yield of 55.6% was obtained in association with the small fluffy pellets which were formed in the third preculture at pH 3.0. The results indicated that the use of the multi-stage precultures as inoculum in the STR is a promising approach for commercial production of fumaric acid.     

The influence of mechanical forces on the morphology and penicillin production of Penicillium chrysogenum

The influence of mechanical forces resulting from the rotation of (multiple) turbine impellers on the morphology and penicillin production of Penicillium chrysogenum Panlabs P-1 was investigated in batch fermentations using semi-defined media. Experiments were carried out at three different scales of fermentation, 5 dm³,100 dm³ and 1000 dm³ working volume, with the impeller tip speed ranging from 2.5 to 6.3 m/s. Throughout all fermentations, the dissolved oxygen concentration never fell below the critical value for maximum penicillin production. Morphological measurements using image analysis showed that the mean main hyphal length and mean hyphal growth unit increased during the rapid growth period and then decreased to a relatively constant value dependent on the agitation intensity. The specific rate of penicillin production (q _pen)and the average main hyphal length during the linear penicillin production phase were lower at high agitation speed, which promoted more rapid mycelial fragmentation and a higher branching frequency. Comparison of the results from the three scales showed that impeller tip speed is a poor scale up parameter whereas a term based on mycelial circulation through the zone of high energy dissipation fitted the data well.List of Symbols C.E.R. mmol/(dm³h) Carbon dioxide evolution rate - D m Impeller diameter - D.O.T. % air saturation Dissolved oxygen tension - L _e m Mean effective length or main hyphal length - O.U.R. mmol/(dm³h) Oxygen uptake rate - P W Total power dissipation - q _pen units/(mg dry cell weight h) rate Specific penicillin production - R.Q. Respiratory quotient - 1/t _cs^–1 Circulation frequency     

The production of 6-aminopenicillanic acid by a multistage tubular reactor packed with immobilized penicillin amidase

A theoretical model equation was derived to find the correlation between the conversion and the amount of immobilized penicillin amidase in column. The theoretical values of the conversion were predicted form this correlation and compared with experimental results. It was observed in a column reactor that the pH drop along the column path was linear versus the enzyme loading and that the enzyme activity was also linearly dependent on pH up to 8.0. In order to diminish the effect of pH drop, a continuous two-stage plug-flow reactor (PFR) with pH adjustment between the two columns was used was used in the experiments, and two- and three-stage PFRs were simulated by computer. In the case of the two-stage PFR, the maximum productivity was demonstrated experimentally and theoretically as well. when an equal amount of the immobilized enzyme was packed in both columns. It was also predicted in the tree-stage PFR system that the optimal distributions of enzyme loading in three columns were found to be 1:1:1. It was demonstrated that the increased number of reactors in series could enhance the level of the maximum productivity with a given amount of enzyme loading.     

Performance of an external loop air-lift bioreactor for the production of monoclonal antibodies by immobilized hybridoma cells

Summary The performance of an external loop air-lift bioreactor was investigated by assessing the inter-relationships between various hydrodynamic properties and mass transfer. The feasibility of using this bioreactor for the production of monoclonal antibodies by mouse hybridoma cells immobilized in calcium alginate gel beads and alginate/poly-l-lysine microcapsules was also examined. When the superficial gas velocity, V _g , in the 300 ml reactor was varied from 2 to 36 cm/min, the average liquid velocity increased from 3 to 14 cm/sec, the gas hold-up rose from 0.2 to 3.0%, and the oxygen mass transfer coefficient, k _L a, increased from 2.5 to 18.1 h^-1. A minimum liquid velocity of 4 cm/s was required to maintain alginate gel beads (1000 m diameter, occupying 3% of reactor volume) in suspension. Batch culture of hybridoma cells immobilized in alginate beads followed logarithmic growth, reaching a concentration of 4×10⁷ cells/ml beads after 11 days. Significant antibody production did not occur until day 9 into the culture, reaching a value of 100 g/ml of medium at day 11. On the other hand, bioreactor studies with encapsulated hybridoma cells gave monoclonal antibody concentrations of up to 800 g/ml capsules (the antibody being retained within the semipermeable capsule) and maximum cell densities of 2×10⁸ cells/ml capsule at day 11. The volumetric productivities of the alginate gel immobilized cell system and the encapsulated cell system were 9 and 3 g antibody per ml of reactor volume per day, respectively. The main advantage of the bioreactor system is its simple design, since no mechanical input is required to vary the hydrodynamic properties.     

Effect of preculture on the slime production inChlamydomonas humicola Lucksch fo

Summary Batch-culturedCblamydomonas bumicola Lucksch fo. showed seasonal oscillations of slime production even in constant conditions. These alterations were found to be correlated to the natural day length during the preculture of the inocula. After preculture in short day conditions production and excretion of mucopolysaccharides started earlier than after preculture in long day conditions. Dry weight production and carbohydrate content were not correlated to preculture.     

Continuous ethanol fermentation in a tower reactor with flocculating yeast recycle: scale-up effects on process performance, kinetic parameters and model predictions

An unsegregated and unstructured model developed for a small-scale process of ethanol production in a tower reactor with cell recycle was applied to describe the experimental data obtained in a large-scale process. The model was developed considering the following points: reactor hydrodynamic behavior analogous to that of ideal CSTR, substrate limitation, inhibition phenomena linked both to ethanol and to biomass, absence of fermentation in the settler, and no loss of cell viability. The scale-up criterion consisted in maintaining an identical relation settler volume/fermentor volume on the two scales. All large-scale experiments were carried out using a flocculating yeast strain IR-2, isolated from fermented food, and identified as Saccharomyces cerevisiae. Sugarcane juice was used as the substrate source with sugar concentrations of 150?g/l. Different values of dilution rate and recycle ratio were employed (D?=?0.11–0.33?h^?1, α?=?5.4–18.0) and the temperature was of 32?°C. The kinetic parameters were similar on both scales and the model predictions agreed well with the large-scale experimental data.     

A novel process for the production of a veterinary rabies vaccine in BHK-21 cells grown on microcarriers in a 20-l bioreactor

We studied BHK-21 cells growth in a 2-l bioreactor and investigated the effects of microcarrier concentration, type of growth medium, culture mode and serum concentration. The highest cell density reached was equal to 4x10(6) cells/ml and was achieved in minimum essential medium supplemented with Hanks' salts, non-essential amino acids and 5% fetal calf serum, using a perfusion culture mode and a microcarrier concentration of 4 g Cytodex 3/l. We studied rabies virus production (PV/BHK-21 strain) by BHK-21 cells grown at the optimal conditions determined previously. We analyzed the effects of multiplicity of infection (MOI) and type of medium used for virus multiplication in spinner-flasks and showed that the highest virus titer reached (when the cells were infected at a MOI of 0.3) in M199 medium supplemented with 0.2% of bovine serum albumin was equal to 8.2x10(7) Fluorescent Focus Units (FFU)/ml. When we grew the cells in a 2-l perfused bioreactor, we obtained a maximal virus titer of 3x10(8) FFU/ml. In addition, we scaled-up to a 20-l bioreactor and obtained similar results for cell density and virus titer. The experimental vaccine we developed meets WHO requirements for vaccine potency. Each run yielded about 40,000 doses of potent vaccine.     

Medium optimization for the production of penicillin V acylase fromBacillus sphaericus

Summary Bacillus sphaericus (NCIM 2478) produced high levels of penicillin V acylase (100 U / g dry cells) when grown on cornsteep liquor — minerals medium at 25°C for 20 h. Supplementation of the medium either with 1 % (w/v) whole wheat bran or its aqueous extract brought about more than 70 % increase in the total enzyme activity. Moreover, deletion of Na₂HPO₄, MgSO₄, CaCl₂, FeSO₄, CuSO₄ and KCl from the medium affected neither growth nor enzyme production.NCL Communication No; 4964.