Fed-batch cultivation of recombinant escherichia coli JM103 and production of the fusion protein SPA::EcoRI in a 60-L working volume airlift tower loop reactor

SPA::EcoRI fusion protein was produced by Escherichia coli JM103 carrying the multicopy expression plasmid pMTC48, the multicopy repressor plasmid pRK248, and the multicopy protection plasmid pEcoR4 in a 60-L working volume airlift tower loop reactor on M9 minimal medium with glucose. Cell mass concentration, total cell count, number of colony-forming units, specific growth rate, yield coefficient, and metabolite (acetate, pyruvate, succinate, lactate, ethanol) concentrations were monitored during the growth phase and gene expression. Gene expression was induced by temperature shift or chemically by isopropyl-thiogalactosidase in the airlift tower loop reactor (ALTR) at constant cultivation time and in a small stirred tank reactor at different cultivation times. During induction, the cultivation medium was supplemented with concentrated Luria-Bertani (LB) medium. The intracellular enzyme activity was evaluated as a function of the time after the start of the induction. It was found that the reduction of the glucose concentration and increase of the dissolved oxygen concentration reduced the acetate produced and increased the intracellular enzyme activity. (c) 1993 John Wiley & Sons, Inc.     

On-line determination of intracellular beta-galactosidase activity in recombinant Escherichia coli using flow injection analysis (FIA).

A flow injection analysis (FIA) system was developed for the determination of cytoplasmic beta-galactosidase activity in recombinant Escherichia coli. The FIA system and its application for on-line monitoring of beta-galactosidase production during cultivation of recombinant E. coli in a 60-l airlift tower loop reactor is described. The results demonstrate that an FIA assay in conjunction with a cell disintegration step can be applied successfully for on-line monitoring of intracellular protein formation.     

Cultivation of E. coli in single- and ten-stage tower-loop reactors

E. Coli was cultivated in batch and continuous operations in the presence of an antifoam agent in stirred-tank and in single- and ten-stage airlift tower reactors with an outer loop. The maximum specific growth rate, mu(m), the substrate yield coefficient, Y(x/s), the respiratory quotient, RQ, substrate conversion, U(s), the volumetric mass transfer coefficient, K(L)a, the specific interfacial area, a, and the specific power input, P/V(L), were measured and compared. If a medium is used with a concentration of complex substrates (extracts) 2.5 times higher than that of glucose, a spectrum of C sources is available and cell regulation influences reactor performance. Both mu(m) and Y(X/S), which were evaluated in batch reactors, cannot be used for continuous reactors or, when measured in stirred-tank reactors, cannot be employed for tower-loop reactors: mu(m) is higher in the stirred-tank batch than in the tower-loop batch reactor, mu(m) and Y(x/s) are higher in the continuous reactor than in the batch single-stage tower-loop reactor. The performance of the single-stage is better than that of the ten-stage reactor due to the inefficient trays employed. A reduction of the medium recirculation rate reduces OTR, U(s), Pr, and Y(X/S) and causes cell sedimentation and flocculation. The volumetric mass transfer coefficient is reduced with increasing cultivation time; the Sauter bubble diameter, d(s), remains constant and does not depend on operational conditions. An increase in the medium recirculation rate reduces k(L)a. The specific power input, P/V(L), for the single-stage tower loop is much lower with the same k(L)a value than for a stirred tank. The relationship k(L)a vs. P/V(L) evaluated for model media in stirred tanks, can also be used for cultivations in these reactors.     

Comparison of the performances of stirred tank and airlift tower loop reactors.

Following a consideration of the prerequisites for reactor comparison and the fundamental differences between stirred tank and airlift tower loop reactors, their performances are compared for the production of secondary metabolites: penicillin V by Penicillium chrysogenum, cephalosporin C by Cephalosporium acremonium, and tetracycline by Streptomyces aureofaciens. In stirred tank reactors, cell mass concentrations, volumetric productivities, and specific power inputs are higher than in airlift tower loop reactors. In the latter, efficiencies of oxygen transfer are higher, and specific productivities with regard to power input, substrate and oxygen consumptions, and yield coefficients of product formation with regard to substrate and oxygen consumptions are considerably higher than in stirred tank reactors. The prerequisites for improved performance are discussed.     

Characterization of a pilot plant airlift tower loop bioreactor: II. Evaluation of global mixing properties of the gas phase during yeast cultivation

Saccharomyces cerevisiae was cultivated in a 4-m(3) pilot plant airlift tower loop reactor with a draft tube in batch and continuous operations and for comparison in a laboratory airlift tower loop reactor of 0.08 m(3) volume. The reactors were characterized during and after the cultivation by measuring the distributions of the residence times of the gas phase with pseudostochastic tracer signals and mass spectrometer and by evaluating the mixing in the liquid phase with a pulse-shaped volatile tracer signal and mass spectrometer as a detector. The mean residence times and the intensities of the axial mixing in the riser and downcomer, the circulation times of the gas phase, and the fraction of the recirculated gas phase were evaluated and compared.     

Improvement of Panax notoginseng cell culture for production of ginseng saponin and polysaccharide by high density cultivation in pneumatically agitated bioreactors

A Panax notoginseng cell culture was successfully scaled up from shake flask to 1.0-L bubble column reactor and concentric-tube airlift reactor. High-density bioreactor batch cultivation was carried out using a modified MS medium. The maximum cell density in batch cultures reached 20.1, 21.0 and 24.1 g/L in the shake flask, bubble column and airlift reactors, respectively, and their corresponding biomass productivity was 950, 1140 and 1350 mg/(L x d) for each. The productivity of ginseng saponin was 70, 96 and 99 mg/(L x d) in the flask, bubble column and airlift reactors, respectively; and the polysaccharide productivity reached 104, 119 and 151 mg/(L x d) for each. Furthermore, a fed-batch cultivation strategy was developed on the basis of specific oxygen uptake rate (SOUR), i.e., sucrose feeding before a sharp decrease of SOUR, and the highest cell density of 29.7 g/L was successfully achieved in the airlift bioreactor on day 17 with a very high biomass productivity of 1520 mg/(L x d). The concentrations of ginseng saponin and polysaccharide reached about 2.1 and 3.0 g/L, respectively, and their productivity was 106 (saponin) and 158 mg/(L x d) (polysaccharide). This work successfully demonstrated the high-density bioreactor cultivation of P. notoginseng cells in pneumatically agitated bioreactors and the reproduction of the shake flask culture results in bioreactors. The cell density, biomass productivity, production titer and productivity of both ginseng saponin and polysaccharide obtained here were the highest that have been reported on a reactor scale for all the ginseng species.     

Cultivation of Tetrahymena thermophila in a 1.5-m3 airlift bioreactor

A large-scale cultivation system for the mass cell production and extraction of the protozoon Tetrahymena thermophila has been developed on the basis of a low-cost complex nutrient medium. Cell growth and the production of extracellular proteases were investigated using a 15-l stirred-tank reactor and 13-l and 1500-l airlift reactors. Processes using defined and complex medium formulations were compared. After cell mass production by 1200 l cell suspension in the large airlift bioreactor, two different extraction methods, based on the use of an extraction decanter and a sedimentation procedure, were compared and followed by cell lyophilization. Cell sedimentation was shown to be the more efficient extraction method as it enabled cell retention/separation while preserving the cell structure. Maximum cell growth was achieved in the stirred-tank bioreactor, supporting the hypothesis that higher shear forces reduce the particle size of the medium, which is responsible for an optimized nutrient supply. The highest glucose uptake rates were found in defined medium lacking the nutrient particles that are present in complex medium formulations. The cell-specific proteolytic activity in culture supernatants of airlift bioreactors using complex medium conditions was higher than that of a culture broth with cells grown under defined medium formulations. Received: 24 September 1998 / Received revision: 23 November 1998 / Accepted: 29 November 1998     

Transport phenomena, reactor design and scale-up

This review will cover the area of impeller-mixed stirred-tank reactors. In addition, it will consider bubble columns, in which air or gas is passed up a liquid filled column through distribution plates covering the full area of the column, and also airlift reactors, in which the air is confined in a channel by means of a loop or draft tube designed to impart a certain type of overall circulatory pattern to the entire tank.There is considerable interest in the kinetics inside the solid part of various kinds of immobilized solid pellet type of enzymes and catalysts. The use of these particles in fixed bed reactors is also covered.     

Hydrodynamics and oxygen transfer in pneumatic bioreactor devices

Gas holdup and oxygen transfer studies in non-Newtonian suspensions of cellulose fibres conducted in two large (0.098 m(3) each) reactors are described. Both reactors-a bubble column and a similar internal loop airlift-were unusual in that they had rectangular cross-sections. In all cases gas holdups and k(L)a(L) declined with increasing solid concentration and, under identical conditions, the bubble column performed better than the airlift. The fluid systems used were carefully selected to represent mould fermentation broths.The behavior of true mass transfer coeffcient k(L) with changes in bubble size is discussed for these systems.     

Bakers' yeast cultivation on by-products and wastes of potato and wheat starch production on a laboratory and pilot-plant scale

Saccharomyces cerevisiae was cultivated in a 10 litre laboratory stirred tank, an 80 litre laboratory airlift tower loop and a 4 m³ pilot-plant airlift tower loop reactor using by-products and wastes of potato and wheat starch production in batch and continuous cultures. Potato protein liquor, potato liquor retentate, potato liquor residue, and wheat process water were used as nutrients and glucose from the enzymatic conversion of potato starch as energy source. Besides the performance of the cultivation (cell concentrations, specific growth rates in the first (glucose) and second (ethanol) growth phases, productivities, yield coefficients), the qualities of the effluents (concentrations of phosphate, dissolved organic carbon, dissolved organic nitrogen, and chemical oxygen demand) were also determined in the different reactors as functions of the operational parameters. The optimal conditions were evaluated with regard to cultivation performance and effluent quality. These performances do not vary with the scale of the reactors. The performance of continuous cultures is considerably better than that of batch cultures.     

Comparison of two different pneumatically mixed column photobioreactors for the cultivation of Artrospira platensis (Spirulina platensis)

Internal loop airlift and bubble column photobioreactors (PBR) were compared with respect to their performances during cultivation of Artrospira platensis (Spirulina platensis). Culture conditions were kept the same and different parameters were examined through the experiments. It was observed that a higher dry biomass weight and chlorophyll-a concentration was obtained in the airlift PBR yielding a maximum growth rate of 0.45 day(-1), while 0.33 day(-1) was reached in the bubble column PBR. Subsequently, a 17-day of production was carried out in the selected PBR to fully determine the performance of the PBR. Maximum growth rate of 0.47 day(-1) was reached during long term cultivation.     

Growth and biochemical characterization of microalgal biomass produced in bubble column and airlift photobioreactors: studies in fed-batch culture

Relatively large (0.19 m column diameter, 2 m tall, 0.06 m³ working volume) outdoor bubble column and airlift bioreactors (a split-cylinder and a draft-tube airlift device) were compared for monoseptic fed-batch culture of the microalga Phaeodactylum tricornutum. The three photobioreactors produced similar biomass versus time profiles and final biomass concentration (4 kg m⁻³). The maximum specific growth rate observed within a daily illuminated period in the exponential growth phase, had a value of 0.08 h⁻¹ on the third day of culture. Because of night-time losses of biomass, the specific growth rate averaged over the 4-days of exponential phase was 0.021 h⁻¹ for the three reactors.

The biomass in the vertical column reactors did not experience photoinhibition under conditions (photosynthetically active daily averaged irradiance value of 1150±52 μE m⁻² s⁻¹) that are known to cause photoinhibition in conventional thin-tube horizontal loop reactors. Because of good gas-liquid mass transfer, the dissolved oxygen concentration in the reactors at peak photosynthesis remained <120% of air saturation; thus, oxygen inhibition of photosynthesis and photo-oxidation of the biomass did not occur. Carbohydrate accumulation (up to 13% w/w) by the biomass was favored during light-limited linear growth. A declining light intensity caused a more than five-fold increase in cellular carotenoids but the chlorophylls increased only by about 2.5-fold during the course of the culture. In the stationary phase, up to 2% of the biomass was chlorophylls and carotenoids constituted up to 0.5% of the biomass dry weight.     

Membrane sparger in bubble column,airlift, and combined membrane–ring sparger bioreactors

The bubble column and the two internal loop airlift reactors (riser/downcomer area ratios of 0.11 and 0.58) characterized in this study were equipped with a rubber membrane sparger, which produced small bubbles, giving high mass transfer coefficients. The low mixing intensity in the bubble column was increased by an order of magnitude in the airlift reactors. We designed a novel aeration and mixing system by adding a ring sparger to the membrane sparger in the bubble column and maintained the advantages of both airlift configuration (good mixing properties) and bubble column configuration (efficient aeration, without any internal constructions). The combined membrane–ring sparger system has unique features with respect to the efficiency of utilization of substrate gasses and energy. Model experiments showed that the small bubbles from the membrane sparger do not coalesce with the large bubbles from the ring sparger. If different gases were added through the two spargers it was possible to transfer a hazardous or expensive gas quantitatively to the liquid through the membrane sparger (dual sparging mode). In the combined membrane–ring sparger system the energy input for mixing and mass transfer is divided. Therefore, the energy consumption can be minimized if the flow distribution of air through the membrane and ring sparger is controlled by the oxygen demand and the inhomogeneity of the culture, respectively (split sparging mode). The dual sparging mode was used for mass production of the alga Rhodomonas sp. as the first step in aquatic food chains. Avoiding mechanical parts removes an important risk of malfunction, and a continuous culture could be maintained for more than 8 months. © 1999 John Wiley & Sons, Inc. Biotechnol Bioeng 64: 452–458, 1999.     

Process optimization of a continuous airlift tower-loop reactor

Based on the experimental investigations with H. polymorpha and Methylomonas M 15 in bench-scale airlift tower-loop reactors, a general distributed parameter model was developed and used to simulate to cultivation process in a 40-m-high production reactor. This general model was simplified with regard to the gas phase and loop balances and was employed to optimize cell productivity and/or profit in a 20-m-high pilot-plant airlift tower-loop reactor. Maximum cell productivity always occurs in the oxygen-transfer-limited growth range. In case of a high "penalty factor" for nonconsumed substrate, maximum profit is attained at the boundary between substrate and oxygen-transfer-limited growth. Oxygen-transfer limitation exists in the lower half of the tower, whereas in the upper half, substrate limitation prevails. The longitudinal dissolved oxygen concentration passes a minimum in this case as has been determined experimentally in the bench-scale column. The simulation results agree fairly well with the data measured in the pilot plant.     

Generation of small bubbles and small bubble-liquid mass transfer in airlift reactors containing highly viscous liquids

The time-dependent gas hold-up is investigated during the aeration of the Saccharomyces cerevisiae suspension, the aqueous saccharose solutions and the glycerol solutions in the external loop airlift reactor. Due to the time-dependent bubble size distribution the fraction of the small bubble hold-up in the total gas hold-up decreases with an increase of the gas flow rate and with a decrease of the viscosity. The course of the accumulation process of the small bubbles is described by the first-order kinetic equation. The small bubble accumulation rate is investigated in the airlift reactor and the bubble column. It is showed that the small bubbles form and disappear exclusively in the riser of the airlift reactor. It is found that the small bubble-liquid mass transfer coefficient is several times larger than the overall oxygen transfer coefficient.     

Enhanced productivity of Chaetoceros calcitrans in airlift photobioreactors

The various modes of cultivation of Chaetoceros calcitrans in airlift photobioreactors (ALPBRs) were examined. The batch system illustrated that the airlift configuration was superior to the bubble column as the airlift supported the circulation of the cell within the system, leading to a better light utilization. The cultivations in both semi-continuous and continuous systems resulted in a high cell productivity, although the steady state cell concentrations in both systems were lower than that obtained from the batch system. The behavior of the large-scale airlift system was not significantly different from the conventional bubble column where the diatom could only be produced at low cell density. Despite this, among all of the systems investigated in this work, the large-scale system gave the highest productivity. The main limiting factor for the large-scale airlift culture was the availability of light. Based on economical analysis, the continuous cultivation in the 2.8L ALPBR with a medium feed rate of 3 mL min(-1) was most attractive where the operation cost could be maintained at a minimum of approx. 7.95 x 10(-4)THBL(-1)h(-1). However, this continuous small-scale system still suffered from relatively low cell productivity (8.10 x 10(4)cellss(-1)).     

Large-scale growth and taxane production in cell cultures of Taxus cuspidata (Japanese yew) using a novel bioreactor

 A novel type of bioreactor was successfully developed for the production of taxol and its precursors by culturing cells of Taxus cuspidata (Japanese yew) on a pilot-scale. Rapidly growing cell lines were selected from callus cultures derived from immature embryos of yew. The cells were inoculated in 20-l capacity bioreactors of different types to test the growth performance. The models of small-scale bioreactors incorporated in this study included a balloon-type bubble bioreactor (BTBB), a bubble-column bioreactor (BCB), a BCB with a split-plate internal loop, a BCB with a concentric draught-tube internal loop, a BCB with a fluidized bed bioreactor, and two different models of stirred tank reactors. Among the reactors, BTBB appeared to be the most efficient in promoting cell growth. The doubling time of cell growth in BTBB was 12 days with a 30% inoculation cell density. The optimum time for medium replacement or feeding was 12–15 days after inoculation as determined by monitoring both the levels of sugars and medium conductivity. When yew tree cells were grown in different sizes (100–500-l) of BTBBs, more than 70% cell viability was recorded at the time of harvest. The growth pattern of the cells in the pilot-scale BTBB appeared to be the same as that of cells in the 20-l bioreactors. Approximately 3 mg/l of taxol and 74 mg/l total taxanes were obtained after 27 days of culture. Received: 6 April 1999 / Revision received: 23 August 1999 / Accepted: 31 August 1999     

Production of dextransucrase by Leuconostoc mesenteroides immobilized in calcium-alginate beads: I. Batch and fed-batch fermentations

In batch fermentation Leuconostoc mesenteroides immobilized in calcium alginate beads produced a total dextransucrase activity equal to about 93% of that by free, suspended bacterial cells under comparable conditions in a bubble column reactor. Continuous sucrose feeding (5 g/L h) to the immobilized-cell culture in the airlift bioreactor increased production of enzymatic activity by about 107% compared with ordinary batch operation of this reactor. About 14% of the enzymatic activity produced by the immobilized cells appears as soluble activity in the cell-free broth compared with about 40% in case of free cells. In an airlift bioreactor, both the soluble and the intact (sorbed and entrapped) enzymatic activity produced by the immobilized bacterial cells was about 34% greater under automatic pH control, compared to that produced in a bubble column reactor with only manual pH control. During formation of dextran by intact enzyme within cells and beads, declines are observed in apparent enzymatic activity.     

12 beta-Hydroxylation of digitoxin by suspension-cultured Digitalis lanata cells: production of digoxin in 20-litre and 300-litre air-lift bioreactors.

A biotransformation process for the production of digoxin was developed using Digitalis lanata cell suspension cultures. Digitoxin was used as the substrate for biotransformation. Digoxin production was carried out in a variety of vessels, including 1-l exsiccators, 20-l glass reactors and a 300-l air-lift bioreactor. A culture volume of 200 l was established after 28 d and the cells were then cultured semi-continuously in a 300-l bioreactor employing the draw-fill cultivation method. Maximal digoxin production was achieved in an 8% glucose medium with a production optimum after 40-60 h of incubation in the presence of 0.65-0.8 mmol digitoxin per l. Levels of 0.52, 0.53 and 0.60 mmol digoxin per l suspension were achieved in 1-l, 20-l and 300-l vessels, respectively. About 80% of the digoxin produced was found in the bathing medium.     

Comparative analysis of ethanolic fermentation in two continuous flocculation bioreactors and effect of flocculation additive

Two types of bioreactor using a flocculating strain of Saccharomyces cerevisiae and continuous ethanolic fermentation as model were compared in terms of start-up evolution, overall performance and power costs. Also, the effect of adding to the medium a polymer — Magna Floc LT25 — that increases floc porosity was studied. The main difference between the reactors lies on the system that is used to recycle the flocculated cells — one presents an external loop with mechanically forced recycling and the other has an airlift configuration with an internal loop. During start-up of both bioreactors, no significant differences between the fermentation kinetics were established, either with or without Magna Floc. In the airlift bioreactor no positive effect of the dilution rate on substrate uptake was observed. Concerning ethanol productivity, both systems behave in a similar way. The best ethanol productivity, 12.9 kg/kg/h, was obtained for the airlift system. This value is 7 times higher than in conventional systems and justifies the interest devoted to flocculation bioreactors. The results also indicate that the activity of the cells that are kept inside the airlift bioreactor is higher and compensates its lower cell retention capacity at higher dilution rates. The addition of Magna Floc to the medium causes a reduction on the ethanol yield on glucose for the external loop system, but allows for an increase in the maximal dilution rate for total glucose consumption. Such a behavior is not observed for the airlift system. The analysis of the power cost associated with the operation of the two bioreactors indicates that the differences between them are only relevant at laboratory and pilot scales. However, from an industrial scale point of view the airlift bioreactor is advantageous because no mechanical parts are involved in recycling.