The influence of geometry on hydrodynamic and mass transfer characteristics in an external airlift reactor for the cultivation of filamentous fungi

The influences of geometric configuration, mycelial broth rheology and superficial gas velocity (U_sg) were investigated with respect to the following hydrodynamic parameters: gas holdup (), oxygen transfer coefficient (K_La) and mixing time (t_m). Increases in U_sg and height of gas separator (H_t) caused an increase in and K_La, and a decrease in t_m. Consequently, a diameter ratio (D_d/D_r) of 0.71 and H_t 0.20 m were found to be the best geometry and operation parameters to achieve high aeration and mixing efficiency for the high viscous broth system in the cultivation of filamentous fungi. An external airlift reactor (EALR) was developed and designed for the cultivation of filamentous fungi. The EALR with two spargers excels in reliability and high aeration and mass transfer coefficiency, resulting in a fast mycelial growth and high biomass productivity in the cultivation of the fungus Rhizopus oryzae.     

Interactions of cell morphology and transport processes in the lovastatin fermentation

The cholesterol lowering drug, Lovastatin (Mevacor), acts as an inhibitor of HMGCoA reductase, and is produced from an Aspergillus terreus fermentation.Pilot scale studies were carried out in 800 liter fermenters to determine the effects of cell morphology on the oxygen transport properties of this fermentation. Specifically, parallel fermentations giving (i) filamentous mycelial cells, and (ii) discrete mycelial pellets, were quantitatively characterized in terms of broth viscosity, availability of dissolved oxygen, oxygen uptake rates and the oxygen transfer coefficient under identical operating conditions.The growth phase of the fermentation, was operated using a cascade control strategy which automatically changed the agitation speed with the goal of maintaining dissolved oxygen at 50% saturation. Subsequently stepwise changes were made in agitation speed and aeration rate to evaluate the response of the mass transfer parameters (DO, OUR, and k _L a). The results of these experiments indicate considerable potential advantages to the pellet morphology from the standpoint of oxygen transport processes.List of Symbols DO % sat. Dissolved oxygen concentration - k _L a h^–1 Gas-liquid mass transfer coefficient - OUR mmol/dm³h Oxygen uptake rate - P/V KW/m³ Agitator power per unit volume - V _s m/s Superficial air velocity - _app cP Apparent viscosity     

The scale-up of plant cell culture: Engineering considerations

The enormous versatility of plants has continued to provide the impetus for the development of plant tissue culture as a commercial production strategy for secondary metabolites. Unfortunately problems with slow growth rates and low products yields, which are generally non-growth associated and intracellular, have made plant cell culture-based processes, with a few exceptions, economically unrealistic. Recent developments in reactor design and control, elicitor technology, molecular biology, and consumer demand for natural products, are fuelling a renaissance in plant cell culture as a production strategy. In this review we address the engineering consequences of the unique characteristics of plant cells on the scale-up of plant cell culture.Abbreviations a gas-liquid interfacial area per volume - C dissolved oxygen concentration - C^* liquid phase oxygen concentration in equilibrium with the partial pressure of oxygen in the bulk gas phase - K_L overall mass transfer coefficient - k_L liquid film mass transfer coefficient - m_O2 cell maintenance coefficient for oxygen - OTR oxygen transfer rate - OUR oxygen uptake rate - pO₂ partial pressure of oxygen - STR stirred-tank reactor - v.v.m. volume of gas fed per unit operating volume of reactor per minute - X biomass concentration - Y_x/O2 biomass yield coefficient for oxygen - specific growth rate     

Optimization of <Emphasis Type="Italic">Streptomyces peucetius</Emphasis> var. <Emphasis Type="Italic">caesius</Emphasis> N47 cultivation and ε-rhodomycinone production using experimental designs and response surface methods

Streptomyces peucetius var. caesius is an aerobic bacterium that produces doxorubicin as a secondary metabolite. A mixture design was applied for the screening of suitable complex medium components in the cultivation of S. peucetius var. caesius N47, which is an -rhodomycinone-accumulating mutant strain. -Rhodomycinone is a non-glycosylated precursor of doxorubicin. Best growth results were obtained with soy peptone and beef extract. A central composite face-centered (CCF) experimental design was constructed for the investigation of pH, temperature and dissolved oxygen (DO) effects on the cultivation growth phase. Another CCF was applied to the production phase to investigate the effects of aeration, pH, temperature and stirring rate on -rhodomycinone production. An increase in cultivation temperature increased both cell growth and glucose consumption rate. Best -rhodomycinone productivities were obtained in temperatures around 30°C. DO control increased all growth phase responses, but aeration in the production phase coupled with pH decrease resulted in rapid -rhodomycinone decay in the medium. In non-aerated production phases a pH change resulted in better productivity than in experiments without pH change. A pH increase with a temperature decrease seemed most beneficial for productivity. This implies that dynamic control strategies in batch production of -rhodomycinone could increase the overall process productivity.     

Effect of hydromechanical forces on the production of filamentous haemagglutinin and pertussis toxin ofBordetella pertussis

Summary The production ofBordetella pertussis extracytoplasmic filamentous haemagglutinin (FHA) and pertussis toxin (PT) in a bioreactor under stirring conditions was studied in order to investigate the effect of hydromechanical forces on yields of both antigens. It was shown that FHA loses its haemagglutinin activity when the power transmitted by the agitator and the aerator per unit volume increases, whereas PT production is not affected. The loss of FHA activity can be explained by the action of shear forces on the filamentous structure of this antigen.Nomenclature C^* dissolved oxygen saturation concentration - C₁ dissolved oxygen concentration - D impeller diameter - power transmitted by the agitator and the aerator per unit of liquid volume - E_m maximum local energy dissipation rate per unit of liquid volume - K_La volumetric oxygen transfer coefficient - N impeller speed - Pg power input in aerated system - qO₂m maximum specific oxygen consumption rate - R_e Reynold number (D²N /) - VVM volume of air per volume of fermentation broth per minute - Xm maximum of biomass concentration - o Kolmogorov-microscale - fermentation broth viscosity - fermentation broth kinematic viscosity - fermentation broth density - expt experiment     

A comparative evaluation of oxygen mass transfer and broth viscosity using Cephalosporin-C production as a case strategy

The production of Cephalosporin-C (CPC) a secondary metabolite, using a mold Acremonium chrysogenum was studied in a lab scale Internal loop air lift reactor. Cephalosporin-C production process is a highly aerobic fermentation process. Volumetric gas–liquid mass transfer coefficient (k_La) and viscosity (η) were evaluated, during the growth and production phases of the microbial physiology. An attempt has been made to correlate the broth viscosity, η and volumetric oxygen transfer coefficient, k_La during the Cephalosporin-C production in an air lift reactor. The impact of biomass concentration and mycelial morphology on broth viscosity has been also evaluated. The broth exhibits a typical non-Newtonian fermentation broth. Rheology parameters like consistency index and fluidity index are also studied.     

Lysine degradation in Candida maltosa: occurrence of a novel enzyme,acetyl-CoA: L-lysine N-acetyltransferase

The yeast Candida maltosa can utilize L-lysine as sole nitrogen and sole carbon source accompanied by accumulation of -N-acetyl-L-lysine, indicating that lysine is metabolized by way of N-acetylated intermediates. A novel lysine acetyltransferase catalyzing the first step in this pathway, the N-acetylation of the -amino group of L-lysine, was found in this yeast. The enzyme, acetyl-CoA:L-lysine N-acetyltransferase, is strongly induced in cells grown on L-lysine as sole carbon source. The enzyme is specific for both L-lysine and acetyl-CoA. The K _m values are 10 mM for L-lysine and 0.33 mM for acetyl-CoA. The enzyme has a maximum activity at pH 8.1.Dedicated to Prof. Dr. F. Böttcher in occasion of his 60th birthday     

The effects of the oxygen transfer coefficient and substrate concentration on the xylose fermentation by Debaryomyces hansenii

Relevant production of xylitol by Debaryomyces hansenii requires semiaerobic conditions since in aerobic conditions the accumulated reduced adenine dinucleotide coenzyme is fully reoxidized leading to the conversion of xylitol into xylulose. For oxygen transfer coefficient values from 0.24 to 1.88 min^-1, in shake flasks experiments, biomass formation increased proportionally to the aeration rate as shown in the oxygen transfer coefficient and xylose concentration isoresponse contours. The metabolic products under study, xylitol and ethanol were mainly growth associated. However, for oxygen transfer coefficient above 0.5 min^-1 higher initial xylose concentration stimulated the rate of production of xylitol. This fact was less evident for ethanol production. The direct relationship between increased biomass and products formation rate, indicated that the experimental domain in respect to the aeration rate was below the threshold level before the decreasing in metabolic production rates reported in literature for xylose-fermenting yeasts. The fact that ethanol was produced, albeit in low levels, throughout the experimental design indicated that the semiaerobic conditions were always attained. Debaryomyces hansenii showed to be an important xylitol producer exhibiting a xylitol/ethanol ratio above four and a carbon conversion of 54% for xylitol.Abbreviations K_La oxygen transfer coefficient - DO(T) dissolved oxygen (tension) - OUR oxygen uptake rate - NAD(H) oxidised (reduced) nicotinamide adenine dinucleotide - NADP(H) oxidised (reduced) nicotinamide adenine dinucleotide phosphate - CRC catabolic reduction charge - C oxygen concentration in the culture medium - C^* oxygen concentration at saturation conditions - Y_i response from experiment i - parameters of the polynomial model - x experimental factor level (coded units) - R² coefficient of multiple determination - t time     

Oxygen transfer in an airlift reactor with multiple net draft tubes

A pilot scale airlift reactor with multiple net draft tubes was developed to improve oxygen transfer in the reactor. The reactor was 0.29 m in diameter and 2 m height. A steadystate sulfite oxidation method was applied to determine an overall volumetric mass transfer coefficient. Oxygen transfer of the proposed airlift reactor can be 60–100% higher than that of bubble columns under the same operating conditions.List of Symbols C ^* mol·dm^–3 saturated concentration of dissolved oxygen - C _L mol·dm^–3 bulk concentration of dissolved oxygen - G mol/min nitrogen flow rate - k _L a hr^–1 the volumetric gas-liquid mass transfer coefficient - Mo ₂ g/mol molecular weight of oxygen - OTR g/min the oxygen transfer rate - U _g cm/s superficial air velocity - V _L dm³ volume of the liquid phase - _in oxygen mole ratio in the inlet gas - _out oxygen mole ratio in the outlet gas     

Oxygen mass transfer in a bubble column bioreactor containing lysed yeast suspensions

Summary Liquid-phase volumetric oxygen transfer coefficients were evaluated in a bubble column containing yeast suspensions, using the instationary oxygen absorption method and a polarographic oxygen electrode. The electrode time lag was found to be independent of both the system studied and the operating conditions. The volumetric oxygen mass transfer coefficients k _L a could be reasonably predicted by calculating k _L from the equation derived by Bhavaraju et al. or the empirical equation of Calderbank and Moo-Young and a from the experimental gas hold-up values.Nomenclature a Exponent in Eq.6 or specific gas-liquid interfacial area based on reactor volume m - b Exponent in Eq. 6 - C Constant in Eq 6 or oxygen concentration in the liquid phase g/ml - C ^* Equilibrium oxygen concentration g/ml - C ₀ Oxygen concentration in the liquid phase at t=0 g/ml - C _E Oxygen concentration as determined by the polarographic electrode g/ml - D _B Bubble equivalent diameter mm - D _l Oxygen diffusivity in the liquid phase m²/s - g Acceleration of gravity m/s² - K Consistency index Pasⁿ - K _L Liquid-phase mass transfer coefficient m/s - n Power law exponent - Pe _sw Peclet number based on bubble swarm velocity - S _C Schmidt number - Sh Sherwood number - i Time s - U _B Bubble rise velocity in infinite medium m/s - U _g Superficial air velocity based on column cross-sectional area m/s - U _sw Bubble swarm velocity defined by Eq.15 m/s - Y _MSW Mass transfer coeficient correction factor for mobile interfaces in pseudo-plastic fluids Eq. 7 - Y _MSW Mass transfer coefficient correction factor for immobile interface in pseudo-plastic fluids Eq. 8 Greek letters _l Density of liquid g/ml - _sus Density of unaerated suspension g/ml - _{wet cell} Density of yeast wet cells g/ml - _l Viscosity of the liquid Pas - _app Apparent viscosity of power law fluid Pas - _E Electrode time lag s - _l Time lag due to resistance of the gas-liquid interface s - _g Gas hold-up, volume fraction occupied by the gas phase - _l Liquid hold-up - _c Wet cell volume fraction     

Modeling the growth curve for Spirulina (Arthrospira) maxima, a versatile microalga for producing uniformly labelled compounds with stable isotopes

The paper presents a five-phase model to describe batch culture of Spirulina maxima under limitations of light and nutrients nitrogen andsulfur. The general equation for the exponential, linear, decelerating andstationary phases of the growth curve took into account that (i) the specificgrowth rate was proportional to the local light intensity in thephotobioreactor; (ii) light attenuation was due to cell's absorption andreflection and observed the Lambert-Beer's law with a total absorptioncoefficient (_T) that was the sum of absorption coefficient(_a) and reflection coefficient (_r); (iii) thecomposition of the alga and its absorption _a changed withtime and (iv) the specific growth rate was influenced by nutrients ofnitrogen and sulfur in the culture medium according to a Monod's law.Two successive equations describing the death phase were proposed basedon the hypothesis that the death rate accelerated with the %PSC until itreached its maximum. From that point the %PSC decreased and the deathrate reduced. Our model fitted very well the cultures grown in the photobioreactor developed in our laboratory.     

Red fluorescence spectra as a new means of determining the rate of damage to photosynthesis apparatus in plants caused by stress

A new method is presented for determining the rate of damage to photosynthesis apparatus efficiency caused by stress using the red fluorescence spectra of plant leaves. A direct connection was found between the position of the red fluorescence maximum _max and the photosynthesis apparatus efficiency . The method was tested on several examples and good results were obtained.     

A method for estimating oxygen availability of stationary plant cell cultures in liquid media

A method for estimating the oxygen availability in plant cell cultures grown in stationary liquid media (e.g. many protoplast cultures) was developed. The method is based on short-term measurements of respiration rate versus oxygen concentration on a sample of cells, suspended in liquid media. From such data it is possible to estimate the oxygen concentration at the bottom of a stagnant liquid culture, by calculating the amount of oxygen reaching the cells by diffusion. As an example, rape (Brassica napus L. cv. Omega) hypocotyl protoplasts were grown with different oxygen concentrations at the site of the cells, obtained by varying the cell density, the height of the liquid layer and the oxygen content of the gas phase. The number of surviving calli was positively correlated with the estimated oxygen availability in the range between 60 and 350 M O₂, below 60 M all cells died. This indicates that oxygen availability can be a limiting factor in the range usually encountered in protoplast cultures, and that the method can be useful when designing optimal growth conditions for stationary cultures of plant cells.Abbreviations C₁ bulk oxygen concentration in agitated medium - C_o oxygen concentration in medium at the gas-liquid interface, in equilibrium with the gas - C_x oxygen concentration at cell level - D diffusion constant of oxygen in water - K_La oxygen transfer rate - l height of liquid above cells - n number of cells per ml - R_x respiration rate per cell     

Dissolved oxygen as principal parameter for conidia production of biocontrol fungi Trichoderma viride in non-Newtonian wastewater

Dissolved oxygen (DO) concentration was selected as a principal parameter for translating results of shake flask fermentation of Trichoderma viride (biocontrol fungi) to a fermenter scale. All fermentations were carried out in a 7.5 l automated fermenter with a working volume of 4 l. Fermentation performance parameters such as volumetric oxygen transfer coefficient (k _L a), oxygen uptake rate (OUR), rheology, conidia concentration, glucose consumption, soluble chemical oxygen demand, entomotoxicity and inhibition index were measured. The conidia concentration, entomotoxicity and inhibition index were either stable or improved at lower DO concentration (30%). Variation of OUR aided in assessing the oxygen supply capacity of the fermenter and biomass growth. Meanwhile, rheological profiles demonstrated the variability of wastewater during fermentation due to mycelial growth and conidiation. In order to estimate power consumption, the agitation and the aeration requirements were quantified in terms of area under the curves, agitation vs. time (rpm h), and aeration vs. time (lpm h). This simple and novel strategy of fermenter operation proved to be highly successful which can be adopted to other biocontrol fungi.     

Enhanced oxygen transfer rates in fermentation using soybean oil-in-water dispersions

Summary The effect of soybean oil on the volumetric oxygen transfer coefficient during the cultivation ofAerobacter aerogenes cells is presented. For our aeration-agitation conditions (0.278 vvm and 500 rpm), it has been demonstrated that the use 19% (v/v) of soybean oil enabled a 1.85-fold increase of thek _l a coefficient (calculated on a per liter aqueous phase basis). For smaller volumetric oil fractions,k _L a increased linearly with the oil loading. Because of the oxygen-vector properties of soybean oil, this oil is able to significantly increase thek _L a of a bioreactor.Nomenclature C^*, C saturation and actual dissolved oxygen concentrations respectively (g/m³) - K_La volumetric oxygen transfer coefficient (h^–1) - K_La_initial k _La measured before the oil addition (h^–1) - M_O2 molar mass of oxygen (dalton) - N oxygen transfer rate (g/m³. h) - P_O2. P_N2 partial pressures ofO ₂ andN ₂ in the gas (atm) - P_H2OT partial pressure of water in air at the temperatureT (atm) - P_T total pressure (atm) - Q₀ volumetric flow rate of outlet air before seeding (m³/h) - Sp spreading coefficient (dynes/cm) - T absolute temperature of outlet gas (K) - V_i volume of the liquidi in the fermentor (m³) - V_M molar volume at 273 K and 1 atm (m³/mole) - _ij interfacial tension betweeni andj componants (dynes/cm) - _v volumetric fraction of the oil (v/v) - G gas - O oil - W water - i inlet - o outlet     

Pullulan fermentation in a reciprocating plate bioreactor

Reciprocating plate bioreactors are particularly well suited for conducting fermentations which give rise to highly viscous broth. To evaluate their performance for polysaccharide fermentations, a series of pullulan fermentations were performed with a particular emphasis placed on the influence of aeration on both the quantity and quality of the product. Two experiments were conducted at constant aeration rates and two others with constant dissolved oxygen concentrations. For the latter two experiments, the dissolved oxygen concentration was controlled by manipulating either the aeration flow rate or the reciprocating frequency of the perforated plates.It was found that, in general, a higher dissolved oxygen concentration leads to a higher productivity but the quality of the product, expressed in terms of the viscosity of the fermentation broth, was nevertheless reduced. It appears that the optimum yield, in terms of both quantity and quality, would be achieved at an intermediate dissolved oxygen concentration.List of Symbols DO mg/l Dissolved oxygen concentration - f Hz Agitation frequency - K _L a s^–1 Volumetric mass transfer coefficient - P g/l Pullulan concentration - Q vvm, l/min Volumetric gas flow rate - X g/l Biomass concentration Greek Letters s^–1 Shear rate - Pa.s Apparent viscosity We wish to acknowledge the financial contribution of l'Association des femmes diplômées des Universités (AFDU) and the National Science and Engineering Research Council of Canada (NSERC).     

Oxygen transfer on β- D-galactosidase production by Kluyveromyces marxianus using sugar cane molasses as carbon source

The optimal initial volumetric oxygen transfer coefficient (K_La) was 60 h^–1 for the production of -d-galactosidase from Kluyveromyces marxianus CDB 002, using sugar cane molasses as carbon source. At this K_La applying an agitation/aeration relationship of 700 rpm/0.66 vvm resulted in 812 U l^–1 h^–1 for -d-galactosidase production. This was about 50% better than a relationship of 500 rpm/2 vvm at the same K_La.     

IgE heavy chain constant region genes in atopic dermatitis and senile erythroderma patients

By Southern hybridization using a genomic DNA fragment carrying a human IgE heavy chain constant region gene (C ) as a probe, we analyzed the organization of human C genes and their flanking regions in 23 atopic dermatitis and 6 senile erythroderma patients with elevated serum IgE levels, and 6 atopic dermatitis patients with normal IgE levels. On Barn HI, Hind III, and Eco RI digestions, we detected three hybridizable fragments containing three human C genes, C 1, C 2, and C 3, respectively, in all leukocyte DNAs. These fragments were almost identical in size among patients and healthy donors. Pst I digestion generated a genetic polymorphism. We, however, could find no correlation between this polymorphism and the disorders. It was concluded that among the patients and healthy donors, there was no marked difference in the organization of the functional C gene and its flanking region containing a class switch region. Our conclusion cannot rule out the presence of genetic abnormalities of this region in some atopic dermatitis patients which are not resolvable by our method. In the course of this study, we found a novel C -like gene in placenta DNA which differs from the three C genes commonly present in normal human DNA.     

Interrelation between penicillin productivity and growth rate

Summary Fermentations were carried out in an 801 tower-loop reactor with pellets of Penicillium chrysogenum. The development of the inner structure of the pellets with regard to various fermentation conditions was observed by means of histological preparations of the pellets. Under conditions of energy-source-limitation mycelial tip growth and lysis of other mycelial parts exist simultaneously. Thus the net growth rate (formation rate of cell mass) is higher than the gross growth rate (multiplication rate of cell mass). Under conditions of nitrogen limitation, gross growth rate and net growth rate are identical. A very strict correlation between gross growth rate and penicillin production rate was found as long as sufficient oxygen supply could be maintained and carbon catabolite repression was avoided. The energy source requirement of the biomass can be described with the sum of three terms that correspond to gross growth, lysis compensation growth and maintenance.Symbols a Constant 1/l h - b Constant - K Decay rate constant for product 1/h - K ₁ Substrate inhibition constant g/l - K _op Controls saturation constant for oxygen g/l - K _p Saturation constant for substrate g/l - m Maintenance coefficient 1/h - ms Apparent maintenance coefficient 1/h - O Dissolved oxygen concentration g/l - P Product concentration g/l - p Exponent of O - q Specific productivity 1/h - S Substrate concentration g/l - t Time h - t ₁ Beginning of production phase h - t ₂ Time of pellet dissolution h - V Liquid volume of fermentation broth l - X Dry cell mass concentration g/l - Y Yield of dry cell mass from energy substrate - g Specific gross growth rate of biomass 1/h - l Specific lysis rate of cell mass 1/h - n Specific net growth rate of cell mass 1/h - p Maximum specific rate of product formation 1/h     

Analysis of ligand-binding to the kringle 4 fragment from human plasminogen

The interaction of the isolated human plasminogen kringle 4 with the four -amino acid ligands -aminocaproic acid (ACA), N-acetyl-l-lysine (AcLys), trans-aminomethyl(cyclohexane)carboxylic acid (AMCHA) and p-benzylaminesulfonic acid (BASA) has been further characterized by ¹H-NMR spectroscopy at 300 and 600 MHz. Pronounced high-field shifts, reaching 3 ppm, are observed for AMCHA resonances upon binding to kringle 4, which underscores the relevance of ligand lipophilic interactions with aromatic side chains at the binding site. Ligand titration curves for the nine His and Trp singlets found in the kringle 4 aromatic spectrum reveal a striking uniformity in the kringle response to the various ligands. The average binding curves exhibit a clear Langmuir absorption isotherm saturation profile and the data were analyzed under the assumption of one (high affinity) binding site per kringle. Equilibrium association constants (K _a ) and first order dissociation rate constants (k _off) were derived from linearized expressions of the Langmuir isotherm and of the spectral line-shapes, respectively. The results for the four ligands, at 295 K, pH^* 7.2, indicate that: (a) AMCHA exhibits the strongest binding (K _a =159 mM ^-1) and ACA the weakest (K _a =21 mM ^–1) with AcLys and BASA falling in between; (b) ACA dissociates readily (k _off = 5.3 × 10³ s^–1) and AMCHA associates the fastest (k _off = 2.0 × 10⁸ M ^–1 s^–1) while the kinetics for BASA exchange is relatively slow (k _off = 0.8 × 10³ s^–1, k _on = 0.6 × 10⁸ M ^–1s^–1); (c) the ligand-binding kinetics is close to diffussion-controlled.Abbreviations ACA -aminocaproic acid - AcLys N-acetyl-l-lysine - AMCHA t-aminomethyl(cyclohexane)carboxylic acid - BASA p-benzylaminesulfonic acid - K4 kringle 4 - NOE nuclear Overhauser effect - ppm parts-per-million - pH^* glass electrode pH reading uncorrected for deuterium isotope effects - K _a ligand-kringle 4 equilibrium association constant - k _off ligand-kringle 4 dissociation rate constant - k _on ligand-kringle 4 association rate constant