Cultivation of recombinant E. coli and production of fusion protein in 60-1 bubble column and airlift tower loop reactors.

E. coli K12 with multicopy plasmid (lambda PR-promoter and temperature-sensitive lambda cI 857 repressor) was cultivated in 60-l bubble column and airlift tower loop reactors. The medium composition, cell concentration, and intracellulary enzyme activity were monitored on-line during batch, fed-batch, and continuous cultivations. The specific growth rates, cell mass yield coefficients, plasmid stabilities, productivities of the amount of active fusion protein (beta-galactosidase activity), concentrations and yields of acetic acid, and volumetric oxygen transfer coefficient were evaluated for different medium compositions and cultivation conditions. The enzyme activity was also monitored during the temperature induction. The results evaluated in the 60-l bubble column and airlift tower loop reactors are compared with those evaluated in a 1-1 stirred-tank reactor.     

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.     

Co-immobilized aerobic/anaerobic mixed cultures in stirred tank and gaslift loop reactors

Co-immobilized Aspergillus awamori and Zymomonas mobilis cultures were investigated in a stirred tank reactor on synthetic medium with starch as substrate at various dissolved oxygen concentrations. In a gaslift loop reactor, freely suspended and immobilized A. awamori were cultivated on synthetic medium and soluble potato starch. In the same reactor, the growth and ethanol production of freely suspended and immobilized Z. mobilis cultures were studied on synthetic medium and glucose. Co-immobilized A. awamori and Z. mobilis were cultivated in batch and continuous operations in the gaslift loop reactor on synthetic medium with starch substrate at different dissolved oxygen concentrations. The interrelations between the different process variables are discussed.     

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.     

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.     

Production of paramylon,a β‐1,3‐glucan,by heterotrophic cultivation of Euglena gracilis on potato liquor

Euglena gracilis is shown to be able to grow on potato liquor as the main medium component leading to an interesting biotechnological product represented by paramylon – a β‐1,3‐glucan – and, at the same time, revaluating an otherwise annoying waste stream of the potato‐starch industry. Paramylon mass fractions of about 75% are obtained for biomass concentrations of 15 g/L during simple batch cultivation under heterotrophic conditions. Supplementation of the growth medium with glucose and the vitamins B₁ and B₁₂ are shown to improve growth rate as well as paramylon content. E. gracilis grows best at about 27.5°C without requiring pH control.     

Production of paramylon,a β‐1,3‐glucan,by heterotrophic growth of Euglena gracilis on potato liquor in fed‐batch and repeated‐batch mode of cultivation

Recently, it had been shown that Euglena gracilis was able to grow heterotrophically not only on synthetic media, but also on media based on potato liquor. Supplementation with glucose in both cases led to the accumulation of paramylon, a β‐1,3‐glucan. Thus, such a process may yield a valuable product accompanied by the revaluation of an otherwise annoying waste stream of the potato‐starch industry. Actually, process strategies have been evaluated in order to optimise the concentration of paramylon obtained at the end of the cultivation process. Therefore, cultivation processes based on fed‐batch and in particular repeated‐batch strategies have been studied. It is shown that repeated‐batch operation maybe particularly suited for such a process since E. gracilis seems to adapt gradually to the cultivation medium so that the concentration of media components may be increased step by step. Repeated‐batch cultivation of E. gracilis leads to biomass concentrations in access of 20 g/L with a consistent paramylon mass fraction of about 75%. Cultivations have been carried out at an operating temperature of 27.5°C. As had been found earlier already, pH control is not required during cultivation. On the basis of these results it is clear that repeated‐batch cultivation represent a simple and economic way for the production of paramylon by heterotrophic cultivation of E. gracilis.     

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.     

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.     

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.     

Strategies for dephenolization of raw olive mill wastewater by means of <Emphasis Type="Italic">Pleurotus ostreatus</Emphasis>

The reduction of polyphenols content in olive mill wastewater (OMW) is a major issue in olive oil manufacturing. Although researchers have pointed out the potential of white-rot fungus in dephenolizing OMW, the results available in the literature mainly concern pretreated (sterilized) OMW. This paper deals with the reduction of polyphenols content in untreated OMW by means of a white-rot fungus, Pleurotus ostreatus. Dephenolization was performed both in an airlift bioreactor and in aerated flasks. The process was carried out under controlled non-sterile conditions, with different operating configurations (batch, continuous, biomass recycling) representative of potential industrial operations. Total organic carbon, polyphenols concentration, phenol oxidase activity, dissolved oxygen concentration, oxygen consumption rate, and pH were measured during every run. Tests were carried out with or without added nutrients (potato starch and potato dextrose) and laccases inducers (i.e., CuSO₄). OMW endogenous microorganisms were competing with P. ostreatus for oxygen during simultaneous fermentation. Dephenolization of raw OMW by P. ostreatus under single batch was as large as 70%. Dephenolization was still extensive even when biomass was recycled up to six times. OMW pre-aeration had to be provided under continuous operation to avoid oxygen consumption by endogenous microorganisms that might spoil the process. The role of laccases in the dephenolization process has been discussed. Dephenolization under batch conditions with biomass recycling and added nutrients proved to be the most effective configuration for OMW polyphenols reduction in industrial plants (42–68% for five cycles).     

Use of respiratory quotient as a control parameter for optimum oxygen supply and scale-up of 2,3-butanediol production under microaerobic conditions

The respiratory quotient (RQ) was found to be a suitable control parameter for optimum oxygen supply for the production of 2,3-butanediol + acetoin under microaerobic conditions. In laboratory scale continuous cultures optimum production of 2,3-butanediol + acetoin was obtained at an RQ value between 4.0 to 4.5. This agreed well with optimum RQ value (4.0) stoichiometrically derived from the bioreactions involved. In fed-batch cultures product concentrations as high as 102.9 g/L (96.0 g/L butanediol + 6.9 g/L acetoin) can be achieved within 32 h cultivation with an RQ control algorithm for oxygen supply. Under similar conditions only 85.7 g/L product (77.6 g/L butanediol + 8.1 g/L acetoin) was obtained with control of constant oxygen supply rate throughout the cultivation.In pilot scale batch cultures under identical oxygen supply rate the achievable RQ value was found to be strongly influenced by the reactor type and scale. The initial oxygen supply rate influenced the achievable RQ as well. However, in all the reactors studied the specific product formation rate of cells in the exponential growth phase was only a function of RQ. The same optimum RQ value as found in continuous cultures was obtained. It was thus concluded that RQ can be used as a control parameter for optimum production of 2,3-butanediol + acetoin in both laboratory and pilot plant scale reactors. (c) 1994 John Wiley & Sons, Inc.     

Effects of Cultivation Parameters on the Morphology of Rhizopus arrhizus and the Lactic Acid Production in a Bubble Column Reactor

The use of filamentous Rhizopus for lactic acid production is facing a challenge due to its low yield mainly caused by the difficulty to control its morphology in submerged fermentation processes. This study was aimed at investigating the impacts of cultivation parameters on the morphology of Rhizopus arrhizus DAR 36017 and lactic acid production using waste potato starch in a laboratory scale bubble column reactor (BCR). The fungal morphology was significantly influenced by carbon sources, process pH, starch concentrations, sparger designs and aeration rates. The favorable morphology for lactic acid production was a freely dispersed small pellet, which was achieved under operation conditions at pH 5.0–6.0, starch concentrations of 60–120 g/L and aeration rates of 0.2–0.8 vvm using a sintered stainless steel disc sparger. Optimal cultivation conditions at pH 6.0 and an aeration rate of 0.4 vvm resulted in the formation of freely dispersed small pellets and 103.8 g/L lactic acid with a yield of 87 % from 120 g/L liquefied potato starch in 48 h. The overall results in terms of lactic acid yield and productivity are comparable to those reported in previous studies using immobilized Rhizopus cells in batch fermentations.     

Efficient In Vitro Micropropagation and Regeneration of Humulus Lupulus on Low Sugar, Starch-Gelrite Media

Several Czech and foreign hop mericlones were tested in vitro for efficiency of green callus formation and plant regeneration from internodal or nodal explants. Modified MS media gelled either with agar, starch or a mixture of potato starch and Gelrite, supplemented with different concentrations of either glucose or maltose, were investigated. Two mericlones of Czech hop (Osvald 72 no. 5216 and Sládek no. 6908) were studied in more details because of their different regeneration capacities. The HPLC analysis of medium sugar concentrations after the explant cultivation has revealed slow uptake of sugar from the medium. Presence of glucose at concentration of 45 g dm^–3 in agar medium resulted in a decreased number of nodes compared to the control with 30 g dm^–3 of glucose. The use of a mixture of potato starch plus Gelrite instead of routinely used agar and decreasing the medium glucose concentration to 15 g dm^–3 proved to be most efficient for multiplication rate. The use of this medium results in lower cost of micropropagation of healthy hop cultures without exhibition of vitrification.     

Effects of cultivation techniques and media on yields and morphology of the basidiomycete Armillaria mellea

Summary The yield of cell mass and the morphology of Armillaria mellea, strain ATCC 11114, was studied using a variety of cultivation methods: solid media, standing liquid culture, shake flasks, tower reactors and impeller-stirred reactors. Two different media, malt extract broth and a glucose/asparagine/peptone-medium, and the corresponding agar media, were used. Yields were higher in the malt extract media than in the glucose media. Generally the highest yields were obtained on solid media while agitated cultures gave the lowest yields. Morphological characteristics such as pellet formation, adhesion to surfaces and pigment production were significantly affected by culture conditions.     

Dynamic modeling and simulation of continuous airlift bioreactors

For dynamic behaviors of continuous airlift bioreactors, a mathematical model based on a tanks-in-series model with backflow has been developed. The equations describing the dynamics of airlift bioreactors are material balances for micro-organism, substrate, dissolved oxygen and oxygen in gas-phase and heat balances. Non-ideal mixing of liquid and gas phases is taken into account using a tanks-in-series model with backflow. The batch operation, startup operation and the consequence of plant failure were simulated and the effects of design and operating parameters for an airlift bioreactor on its dynamic behaviors were discussed. The concentration profiles of micro-organism, substrate, dissolved oxygen and oxygen in gas-phase and the temperature profile in an airlift bioreactors and their dynamics were obtained. The computational results indicate that the transients of a chemostat in the case of bubble column bioreactor are slower compared with those in the case of airlift bioreactor. The proposed simulator is more precise as compared with models published previously in the literature and therefore provides more reliable and rational examination of continuous airlift bioreactor performance.     

Investigation of the use of agricultural byproducts for fungal protein production

Cellulose and nutrient salts as well as potato pulp and potato protein liquor (PPL), were used as substrates for the cultivation of Chaetomium cellulolyticum in batch and repeated-batch operations. Using cellulose as the substrate a linear relationship existed between the rates of cell mass formation and acid production. The repeated-batch process was controlled by NaOH consumption using a simple computer model. When the production of cell mass and acid stopped because of a lack of substrate cellulose was fed into the reactor. This occurred within 10 min at which point no NaOH-feed was needed to maintain a constant pH. Repeated-batch operations yielded higher cell concentrations and productivities than batch operations. The relationship between the NaOH and H₂SO₄ consumed, and the fungal mass concentration was complex in cultivation media containing potato pulp and PPL, because various substrates were consumed by the fungus simultaneously and successively. Therefore, for repeated-batch cultivation a constant time interval was used. Repeated-batch cultivation of the fungus on potato pulp and PPL did not yield higher cell concentrations and productivities than did batch cultivation. With the optimal pulp-to-PPL ratio a maximum specific growth rate of 0.61 h¹ was obtained. These investigations indicate, that potato pulp and PPL are well suited to fungal protein production by Chaetomium cellulolyticum for fodder supplement.     

Influence of total and oxygen partial pressure on growth and metabolism of Methylomonas clara

The effect of total and oxygen partial pressure on metabolic activities of Methylomonas clara has been investigated in batch and continuous cultivation experiments using a pressurizable airlift loop reactor. At Oxygen partial pressures of more than 1000 mbar growth of M. Clara is retarded and completely inhibited at 1200 mbr. However, after several hours of incubation at elevated oxygen partial pressures, biomass formation is nearly doubled in subsquent continuous operation, Cells pretreated with oxygen are characterized by a change of cytochrome content; they excret carboxylic acids into the medium. The results indicate that, by sparging an aerobic culture intermittently wich pure oxygen, formation of biomass might be enbanced. (c) 1992 John Wiley & Sons, Inc.     

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)).