Airlift Fermenter Research Articles

Continuous cellobiose hydrolysis using self-immobilized β-glucosidase fromAspergillus phoenicis QM 329 in a fluidized-bed reactor

Aspergillus phoenicis QM 329 was grown in the shape of beads in shake flasks and in an air-lift fermentor. The production of β-glucosidase started when the carbon source, glucose, was consumed. The β-glucosidase activity was retained in the beads at a pH below 6.0. The influence of bead diameter on enzyme activity and the pH and temperature optima for cellobiose hydrolysis has been studied. The enzyme-containing beads were used in a fluidized-bed reactor for continuous cellobiose hydrolysis, and a productivity of 2.0 g/L-h at a substrate conversion of 76% was obtained. The self-immobilized β- glucosidase is a stable and reusable enzyme with a half-life of 700 h when operating at 50°C and pH 4.8.

Cell adsorption and local accumulation of extracellular polysaccharide in an immobilized Acinetobacter calcoaceticus system

Acinetobacter calcoaceticus can be immobilized on Celite by adsorption. The salt concentrations suitable for immobilized cell fermentation are between 10 and 50 mM phosphate concentration. Low salt concentrations cause desorption of immobilized cells while high salt concentrations inhibit the adsorption of cells on Celite. It is also found that cell adsorption is better at lower pH than at higher pH. An airlift fermentation using immobilized cells at 300 g/L Celite loading shows that about 70% of the total polymer produced is accumulated in Celite pores at a concentration (15.4 g/L) almost threefold higher than that in the bulk liquid (5.7 g/L).

The removal of pyritic sulphur from coal by Leptospirillum-like bacteria

The microbial oxidation of pyritic sulphur was studied in a 4.5-l airlift fermentor at pH 1.5 and 100 g/l pulp density. By microbial leaching with Leptospirillum-like bacteria 85% of the pyritic sulphur was removed within 40 days; 30% of the removed pyrite was oxidized to elemental sulphur, the rest being transformed to soluble sulphate. Accumulation of elemental sulphur could be avoided by using a mixed culture of Leptospirillum-like bacteria and Thiobacillus ferrooxidans. Apart from oxidation of elemental sulphur neither the pure nor the mixed culture showed a significant difference as to removal of pyrite.

Pilot scale production of a human monoclonal antibody against human immunodeficiency virus HIV-1

HUman monoclonal antibodies against the transmembrane protein gp41 of HIV-1 were isolated and purified on a pilot scale. A purification scheme was established for the production of human monoclonal antibodies on the gram scale. 50 1 of culture supernatant can be treated in one purification cycle. The hybridomas were mass cultured in an airlift fermenter. The culture broth was clarified by microfiltration and chromatographed on CM-Sepharose fast flow and protein A Superose. Scale up of the high performance affinity chromatography from 1 ml protein A Superose up to 40 ml is described. All desalting steps were performed by gel filtration on Sephadex G-25 coarse. The yield of the whole purification procedure is in the range of 50–60%. The purity is higher than 99.9%. DNA and reverse transcriptase could not be detected. The whole method is designed as a basis for scale up to industrial scale. Results from quality control assays have proven the validity of this approach.

Hydrodynamics in bubble column bioreactors with fermentation broths having a yield stress

The hydrodynamics in a bubble column bioreactor with fermentation broths having a yield stress are studied. Specifically, the liquid velocity at the reactor axis, the axial dispersion coefficient, and the gas hold-up are examined. The liquid velocity at the reactor axis and the gas hold-up are measured in a 40-1 bench-scale bubble column fermentor using carboxypolymethylene (Carbopol) aqueous solutions as simulated broths. Theoretical correlations for the liquid velocity at the reactor axis, the axial dispersion coefficient, and the gas hold-up are derived on the basis of an energy balance and the mixing length theory. The correlations are compared with the present data and a reasonable agreement is found. The theoretical predictions are also in satisfactory agreement with the re-examined data for actual fermentation broths which are Chaetomium cellulolyticum and Neurospora sitophila cultured in a 1000-1 pilot-plant scale airlift fermentor.

In situ immobilized β-glucosidase fromAspergillus phoenicis QM 329

Aspergillus phoenicis QM 329 was found to grow in the shape of beads in shake flasks and in an air-lift fermentor. Initial culture pH, pH profile during cultivation, carbon source, inoculum size and fermentor configuration influenced the retainment of the β-glucosidase activty in the mycelium. Glucose and soluble starch produced beads with the highest activity and the best stability. Glucose-derived beads were more homogeneous in size and shape than the starch-derived beads. These beads kept their integrity for 10 d at 50° C. After 48 h hydrolysis of 50 g/l cellobiose 75% of the initial enzyme activity remained. The beads could be air-dried and alcohol-sterilized with only minor loss of activity. The size of the beads could be controlled by varying the size of the conidia inoculum. In an air-lift fermentor with a working volume of 1500 ml,900 ml beads with an average diameter of 2 mm (estimated to 37–45,000 beads) were produced in less than 3 d with glucose as carbon source. The beads held a β-glucosidase activity of 0.140 IU/bead determined with the pNP assay.

Mass transfer studies of external‐loop airlifts and a bubble column

AbstractGas holdups (ϵGR) and liquid‐phase volumetric oxygen transfer coefficients (kLaT) were measured for a bubble column (BC) and three different external‐circulation‐loop airlift (ECL‐AL) fermentors of 50 to 60 L working volume, using viscous non‐Newtonian aqueous solutions of various carboxymethylcelluloses. Some measurements also were done with a viscous Newtonian system (51.8 wt. % sucrose solution).Discussed in this paper are correlations of ϵGR and kLaT with riser superficial gas velocity (0.02 ≦ UGR ≦ 0.26 m/s), the ratio of the downcomer and riser crosssectional areas (0[BC] ≦ Ad/Ar ≦ 0.444), and the effective viscosity of the liquid phase (0.02 ≦ ηeff ≦ 0.5 Pa · s), over the parameter ranges indicated. It is shown that both ϵGR and KLaT are highly dependent upon UGR and Ad/Ar. The effective viscosity has a significant effect on KLaT, but has only a relatively weak affect on ϵGR. The KLaT correlation developed for non‐Newtonian systems was extended to include the results obtained for the viscous Newtonian system studied by incorporating the effects of liquid‐phase molecular diffusivity, density, and interfacial tension as determined by Nakanoh and Yoshida (1980).

Characterization of an Immobilized Biocatalyst System for Production of Thermostable Amylase

AbstractAn immobilized biocatalyst system for enzyme production using a thermophilic organism is described. Bacillus stearothermophilus (ATCC 29609) immobilized on Rohm and Haas IRA–938 ion exchange resin was utilized for the production of a highly thermostable α‐amylase in a bench scale airlift fermentor. The equilibrium between suspended bacterial cells and the resin fit a Langmuir‐type sorption isotherm. Cell and α‐amylase productivity data for free cell and immobilized cell continuous culture were compared as functions of the dilution rate. Cell growth rates for free and immobilized cells were consistent with the Monod kinetic model, α‐Amylase production for free cells was described by an unstructured model associated with cell growth. The immobilized cell systems gave higher α‐amylase productivity than the free cell system but did not achieve the productivities predicted by the unstructured kinetic model.

Continuous and Batch Production of Chloroperoxidase by Mycelial Pellets of Caldariomyces fumago in an Airlift Fermentor

Batch and continuous production of the extracellular heme glycoprotein chloroperoxidase (CPO) was studied with an airlift fermentor. We induced Caldariomyces fumago CMI 89362 to form pellets by transferring a small inoculum volume in preculture prior to growth in a 1-liter fermentor. Continuous replacement of the fructose-salts medium (dilution rate, 0.008 h) supported continuous CPO formation at an average concentration of 128 +/- 10 mg of CPO liter for 8 days. Optimum CPO production rates averaged 1.2 +/- 0.1 mg of CPO h at dilution rates below 0.033 h. Varying the carbohydrate content of the feed solution or the time of starting the feed did not significantly alter the amount of CPO produced. Batch fermentation in the airlift fermentor resulted in maximum CPO concentrations of 280 +/- 80 mg of CPO liter, although on two separate occasions CPO concentrations reached 400 to 450 mg liter, which was double the amount obtained by free hyphae in shake flask culture.

Comparison of airlift and stirred reactors for fermentation with Aspergillus niger

The design and fabrication of a simple laboratory airlift fermentor are described. This reactor excels in reliability and low power requirement. Its efficiency has been proved for the production of gluconic acid by Aspergillus niger. As in the case of bubble columns, the desired morphology of A. niger, i.e pellet growth, is obtained in the laboratory airlift fermentor as well as in a pilot scale airlift fermentor of 260 l working volume. Thus, pellet formation occurred under rather low stress conditions as compared to stirred tanks where high agitation conditions are expected to be required to achieve similar results.

Liquid circulation time in concentric‐tube airlift columns with non‐newtonian fermentation broths

AbstractLiquid circulation times were measured in a 40‐dm3 concentric‐tube airlift fermenter with simulated media over a wide range of rheological properties. Circulation times decreased with increasing shear‐thinning, and the influence of draft‐tube geometry on circulation time was found to depend on the shear‐thinning of the media. A semi‐theoretical correlation for both Newtonian and non‐Newtonian fermentation media was developed.

Oxygen transfer during batch cultivation in an airlift tower fermentor

Oxygen transfer was studied in an airlift tower fermentor with motionless mixers (Kenics type). The intensity of oxygen transfer was characterized by its volumetric mass transfer coefficient, kLa, which was determined by the balance method. Experimental data of kLa were described by correlation equation and compared with results obtained for the airlift fermentor without motionless mixers with respect to energetic consumption. In addition, growth characteristics of the yeast culture Turulopsis ethanolitolerans cultivated on ethanol were also investigated.

Growth and nicotinate biotransformation in batch cultured and airlift fermenter grown soybean cell suspension cultures

Fermentation of a heterotrophic cell suspension culture of soybean (Glycine max) in a 15 l airlift fermenter for 10 to 12 days yielded appr. 4.67 kg fr. wt. (233.5 g dr. wt.) of biomass starting with an inoculum of 300 g fr. wt. Growth limiting factors were the conditions of preculturing the cells and the available amounts of phosphate and oxygen in the fermenter culture medium. Comparison of the metabolic activity of cells grown in 200 ml Erlenmeyer flasks and in the fermenter was performed by measuring the enzyme activities of nicotinamide-amidohydrolase and SAM: nicotinic acid-N-methyltransferase both in the presence and the absence of exogenous nicotinic acid during the biotransformation of nicotinate to trigonelline. The applied nicotinic acid was quantitatively taken up by the cells and an enhanced production of trigonelline could be observed. The activities of the aforementioned soybean enzymes were the same both in the batch cultured and in the airlift fermenter grown cells indicating good physiological conditions of the cells from the fermenter process.

Zum Lichteinfluß auf Fermentorkulturen von Nigella damascena L

AbstractThe growth behaviour of a Nigella damascena cell line cultivated in airlift fermenters in darkness as well as in ligth was investigated. Considerable variations were observed with regard to the morphology of the cells, the consumption of substrate and the content of yellow pigments. The UV absorption spectra of the yellow pigments show some similarities to the spectra known for thymoquinone.

A Synthetic Medium for the Production of Submerged Cultures of Lentinus edodes

A new synthetic medium was developed for the production of submerged mycelial cultures of Lentinus edodes. Glucose and ammonium chloride were the best carbon and nitrogen sources, respectively. The remaining ingredients in the medium were calcium chloride, aspartic acid, thiamine, magnesium sulphate and trace elements (Mn, Zn and Fe). The threshold level in the dry weight of mycelium was reached at an inoculum concentration of 9.9 mg/ml and was maintained through 16.5 mg/ml. The size of mycelial pellet was inversely related to the level of inoculum. Similar relationships were obtained between mycelial growth and mycelial pellet and shaking frequencies from 50–200 rpm. Optimum growth occurred at pH 4.3-4.8 and at temperatures 20–25 C. Mycelial growth in the new synthetic medium was increased five-fold by using an airlift fermentor instead of shaking. The number of days to the appearance of the first flush of sporophores was reduced by 45 da by using liquid spawn. An important difference between the ultrastructure of cells of the agar-grown filamentous hyphae and those of mycelial pellets in submerged culture is that paramural bodies were prominent in the former.

A Synthetic Medium for the Production of Submerged Cultures ofLentinus Edodes

A new synthetic medium was developed for the production of submerged mycelial cultures of Lentinus edodes. Glucose and ammonium chloride were the best carbon and nitrogen sources, respectively. The remaining ingredients in the medium were calcium chloride, aspartic acid, thiamine, magnesium sulphate and trace elements (Mn, Zn and Fe). The threshold level in the dry weight of mycelium was reached at an inoculum concentration of 9.9 mg/ml and was maintained through 16.5 mg/ml. The size of mycelial pellet was inversely related to the level of inoculum. Similar relationships were obtained between mycelial growth and mycelial pellet and shaking frequencies from 50-200 rpm. Optimum growth occurred at pH 4.3-4.8 and at temperatures 20-25 C. Mycelial growth in the new synthetic medium was increased five-fold by using an airlift fermentor instead of shaking. The number of days to the appearance of the first flush of sporophores was reduced by 45 da by using liquid spawn. An important difference between the ultrastructure of cells of the agar-grown filamentous hyphae and those of mycelial pellets in submerged culture is that paramural bodies were prominent in the former.

Oxygen transfer to mycelial fermentation broths in an airlift fermentor

Oxygen transfer rates and gas holdups were measured in mycelial fermentation broths of Chaetomium cellulolyticum and Neurospora sitophila, each cultured in a 1300-L pilot-plant-scale airlift fermentor. These cultures exhibited highly non-Newtonian flow behavior coupled with a substantial decrease in oxygen transfer rates. The volumetric mass transfer coefficients in these cultures were found to be 65-70% lower than those in water. The data were compared with the available correlations obtained for simulated fermentation broths. In general, the data for C. cellulolyticum are in satisfactory agreement with the correlations for the model media but the data for N. sitophila are higher than that predicted by the correlations. Model media based correlations are found to be applicable to the fermentation processes if the culture medium does not possess a high yield stress.

Growth and Respiratory Characteristics of Batch and Continuous Cell Suspension Cultures Derived from Fertile and Male Sterile Petunia hybrida

Growth rate of Petunia hybrida cells, cultivated in batch suspension was the same for cells derived from fertile (RMF) and male sterile (RMS) Petunia hybrida cv. Rosy Morn. The oxygen uptake by the RMS cells was twice that of the RMF cells. Analysis of the oxygen uptake data showed, that the capacity of both the mitochondrial cytochrome and alternative pathway was larger in the RMS cells; the non-mitochondrial residual respiration was about the same for both cell types (at least in the logarithmic and early stationary growth phase). The engagement of the alternative pathway in uninhibited respiration generally was low. When the dry weight production per mole of respiratory ATP was calculated, the RMS-cells had a much lower «growth efficiency« than the RMF cells, the latter forming about twice as much dry matter per mole of ATP. When these Petunia cells are cultivated in a Kurz airlift fermentor, at various glucose concentrations and at various dilution rates, the conversion of glucose in cellular dry weight appeared to be less efficient in RMS-cells (0.2 g dry weight produced per g of glucose) than in RMF-cells (0.4–0.5 g dry weight per g of glucose). Also for these continuously cultured cells, the respiration of the RMS-cells was higher and the dry weight production per mole of respiratory ATP was lower than for the RMF-cells, again indicating a more «efficient» growth for these RMF-cells. However, the absolute value of these parameters showed big differences for cells grown in batch and in continuous culture. The possible relation between the inefficient growth and respiration of the RMS-cells and the fact that the plants from which these cells were derived are cytoplasmic male sterile is discussed.

A comparison of some methods of estimating volumetric mass‐transfer coefficients in an external‐loop airlift fermenter

Volumetric mass-transfer coefficients were measured in an 11-L external-loop airlift fermenter with deionized water, a fermentation medium, and during a fermentation. Both a Mackareth oxygen electrode and a novel rapid-response probe were used. When the conventional step-change dynamic method was used for water, the long, nonlinear response time of the Mackareth electrode made correction of its readings difficult; this problem did not occur when the rapid-response probe was used. A comparison was made with a method of mass-transfer coefficient determination which does not involve any assumptions about the gas residence time distribution. However, this method requires that the liquid phase is well-mixed and this requirement was not met in the airlift fermenter. Comparison of the present results with other K(L) a determinations for airlift fermenters showed that K(L) a in water depends on the active gas holdup, the value of K(L) a/epsilon at 20 degrees C being ca. 0.37 s(-1). Although higher gas holdups were obtained with the fermentation medium than for water, the values of K(L) a/epsilon were lower, ca. 0.22 s(-1) at 20 degrees C.

Production of phenylalanine and organic acids by phosphoenolpyruvate carboxylase-deficient mutants ofEscherichia coli

Isogenic strains ofEscherichia coli were grown aerobically in minimal medium in a 2-liter airlift fermentor to determine whether appc (phosphoenolpyruvate carboxylase) mutation had the effect of directing glucose carbon into phenylalanine synthesis. Two host strains, YMC9 (ppc+) and KB285 (ppc−) were used, either with (Phec) or without (Phe0) a plasmid which determines constitutive phenylalanine production. Carbon consumption and metabolic products were monitored. Phenylalanine production occurred only in strains carrying the Phec plasmid.ppc− strains produced less cell mass and more acetate, pyruvate, and phenylalanine (in the Phec strains) than did isogenicppc+ strains. Lactate and ethanol production were not detected in any of the strains. Phec strains produced less acetate and pyruvate than their Phe0 homologs. Importantly,ppc−/Phec produced at least six times as much phenylalanine (0.32 g phenylalanine/g dry weight cells) asppc+/Phec. Even in this case, however, phenylalanine was produced at ten-fold lower levels than acetate. Thus, although theppc− mutation stimulates phenylalanine production, it also stimulates the production of unwanted by-products such as acetate and pyruvate.