Substrate effects in tower loop reactors

Summary Hansenula polymorpha was cultivated in a bubble column loop bioreactor employing ethanol and/or glucose as substrates. By varying the substrate concentration, the cultivations were carried out in non-limited, substrate limited and oxygen transfer limited growth ranges. The influence of the transitions from one range to another on reactor performance (OTR,k _L a, a) and cell productivity () were investigated. When employing ethanol as a substrate, the concentration considerably influences the fluid dynamics, mass transfer phenomena and cell productivity. When employing glucose as a substrate, glucose repression occurs. At high glucose concentrations no transition into the oxygen transfer limited growth is possible. The ethanol produced during the glucose repression influences the fluid dynamics, mass transfer phenomena and productivity. With decreasing glucose concentration the glucose repression can be gradually eliminated.     

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

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

New insights on O<Subscript>2</Subscript> uptake mechanisms in two-phase partitioning bioreactors

Numerous reports show that both transfer and uptake of poorly-water soluble substrates are significantly enhanced in two-phase partitioning bioreactors (TPPBs). A number of hypotheses have been put forward to explain these enhancements and among them, the occurrence of direct substrate or oxygen uptake from the vector/water interface has been suggested. The objective of this paper was to quantify the direct oxygen uptake from the vector/water interface in a culture of Pseudomonas putida, performed in a stirred tank reactor, using glucose as substrate and silicone oil as vector. Despite of a sufficient dissolved O₂ concentration in the vector phase (17 mg l⁻¹) and a significant vector surface area (4,000 m⁻¹) no significant direct O₂ uptake from the vector/water interface was observed, compared to O₂ uptake from the aqueous phase. From these results it was concluded that, direct O₂ or substrate uptake from the vector/water interface might not be significant in TPPBs.     

Structured model for cell growth and enzyme production by recombinant Escherichia coli

Summary A structured cell model has been developed to describe the cultivation of recombinant Escherichia coli K12 with multicopy plasmid under the control of a lambda P_R-promoter and a temperature-sensitive lambda cI 857 repressor. The model, based on measurements of a batch culture in a stirred tank reactor, allows statements to be made on the time variation of intracellular processes. Based on cell regulation, the substrate transfer into the cell was considered to be the rate-limiting step for substrate utilization. The model describes substrate utilization, cell growth, and product formation by means of a system of time-dependent, coupled, and partly non-linear differential equations. The solution of these equations allows calculation of the time variation of the concentrations of substrates (glucose and amino acids), dissolved oxygen and cells in the broth as a function of time and the process parameters.Offprint requests to: K. Schügerl     

Software sensors for fermentation processes

Four software sensors based on standard on-line data from fermentation processes and simple mathematical models were used to monitor a number of state variables in Escherichia coli fed-batch processes: the biomass concentration, the specific growth rate, the oxygen transfer capacity of the bioreactor, and the new R _O/S sensor which is the ratio between oxygen and energy substrate consumption. The R _O/S variable grows continuously in a fed-batch culture with constant glucose feed, which reflects the increasing maintenance demand at declining specific growth rate. The R _O/S sensor also responded to rapid pH shift-downs reflecting the increasing demand for maintenance energy. It is suggested that this sensor may be used to monitor the extent of physiological stress that demands energy for survival.     

Indirect estimation of cell mass and substrate concentration using a computer-coupled mass spectrometer

On-line estimation of cell mass and substrate concentration based on exhaust gas analysis was developed. The O₂, CO₂, H₂O, and N₂ contents at the inlet and outlet of fermentor, analyzed by a computer-coupled quadrupole mass spectrometer, were used to calculate the oxygen uptake rate and carbon dioxide evolution rate, and these rates were further used to evaluate cell mass and substrate concentration in a recombinant Escherichia coli fermentation. Cell mass, glucose concentration, specific growth rate, and specific consumption rate of glucose were well estimated by this method; the oxygen uptake rate gave more accurate estimates for these state variables than did the carbon dioxide evolution rate.     

Effect of oxygen supply and dilution rate on the production of 2,3-butanediol in continuous bioreactor by Klebsiella pneumoniae

Kinetics of 2,3-butanediol production by Klebsiella pneumoniae (NRRL B199) from glucose have been studied in a continuous bioreactor. The effect of oxygen supply rate and dilution rate on the product output rate and yield of 2,3-butanediol were investigated. For a feed glucose concentration of 100 g l⁻¹, the optimum oxygen transfer rate is between 25.0–35.0 mmol l⁻¹ h⁻¹. Under these conditions, maximum product concentration obtained was 35 g l⁻¹ at a dilution rate of 0.1 h⁻¹ and the maximum product output rate obtained was 4.25 g l⁻¹ h⁻¹. The product yield based on the substrate utilized approached the theoretical value (50%) at low values of oxygen transfer rate but decreased with increasing oxygen transfer rate.     

Control of catalysis in flavin-dependent monooxygenases

Flavoprotein monooxygenases reduce flavins, speed their reaction with oxygen, and stabilize a C4a-oxygen adduct long enough to use this reactive species to transfer an oxygen atom to a substrate. The flavin-oxygen adduct can be the C4a-peroxide anion, in which case it reacts as a nucleophile. The protonated adduct - the C4a-hydroperoxide - reacts as an electrophile. The elimination of H₂O₂ competes with substrate oxygenation. This side-reaction is suppressed, preventing the waste of NAD(P)H and the production of toxic H₂O₂. Several strategies have been uncovered that prevent the deleterious side-reaction while still allowing substrate hydroxylation.     

Prostaglandin E2 production by renal inner medullary tissue slices: Effect of metabolic inhibitors

Increasing oxygen from 5 to 95% has previously been shown to increase prostaglandin (PG) production in renal inner medullary slices. The possible role of oxidative phosphorylation in this process was investigated. The oxidative phosphorylation inhibitors, dinitrophenol (DNP), oligomycin, and cyanide were evaluated for their effects on PGE₂ production and ATP levels. None of the inhibitors affected PGE₂ synthesis, although they lowered ATP levels at the concentrations tested. In contrast, incubation of inner medullary tissue slices with 0% oxygen resulted in decreases both in PGE₂ and ATP levels. This suggest that the effect of oxygen on prostaglandin synthesis may be due to substrate limiting effects rather an effect on oxidative phosphorylation.When 22 mM 2-deoxyglucose was added to the incubation medium or when glucose was ommitted, PGE₂ levels increased. Sodium fluoride, presumably acting as a glycolytic inhibitor, increased PGE₂ levels, with a maximal effect at 10mM. ATP levels were 37% of control values with 20 mM NaF. This indicates that glucose may inhibit prostaglandin synthesis.These results indicate that oxygen (substrate) availability can limit inner medullary PGE₂. In view of the low pO₂ in the inner medulla, especially during antidiuresis, oxygen can potentially regulate prostaglandin productin in this tissue.     

Closure effects on oxygen transfer and aerobic growth in shake flasks

Oxygen mass transfer in shake flasks is an important aspect limiting the culture of aerobic microorganisms. In this work, mass transfer of oxygen through a closure and headspace of shake flasks is investigated. New equations for prediction of kGa in shake flasks with closures are introduced. Using Pseudomonas putida, microbial growth on glucose (fast metabolism) and phenol (slow metabolism) in shake flasks with closures were studied, considering both substrate and oxygen restrictions. A combined model for oxygen mass transfer and microbial growth is shown to accurately predict experimental oxygen concentrations and oxygen yield factors during growth experiments more accurately than previous models.     

Oxidative metabolism of dermatophytes

A method for preparing young, actively respiring dermatophyte mycelia was obtained through the use of concentrated spore inocula and short growth periods in static culture. These hyphal elements were uniform in appearance, and vacuoles were absent. Concentrated mycelial suspensions were obtained which could be transferred easily and accurately. Glucose stimulated oxygen uptake in young mycelia which had been grown in a medium with low carbohydrate content. The level of endogenous respiration was affected by exogenous glucose only when this substrate stimulated oxygen uptake by less than 14%. Low nicotinamide adenine dinucleotide phosphate (NADP) dehydrogenase activity was noted in microconidia which have a low endogenous Q_o2 value, whereas the activity of this enzyme was greater in macroconidia and mycelia which possess higher endogenous Q_o2 values. Microsporum gypseum oxidizes 50% of exogenous glucose and assimilates the remainder. A large percentage of this substrate was assimilated into nitrogenous substances.     

Production of a microbial lipase by Staphylococcus carnosus (pLipMut2) in a bubble column reactor and a centrifugal field bioreactor

Extracellular lipase production by the recombinant strain Staphylococcus carnosus (pLipMut2) has been studied. First substrate optimization was carried out in shaken cultures. As a result, the best substrate yield of 20 units/g (peptone + yeast extract) and maximum lipase activity in the culture supernatant of 1.7 units/cm³ could be obtained by a nutrient rich complex medium consisting of 75 kg/m³ yeast extract, 15 kg/m³ tryptone, 5 kg/m³ glucose and 0.5 kg/m³ K₂HPO₄. Higher initial substrate concentration caused inhibition of growth. Antifoam agent at higher levels than 1 cm³/ dm³ resulted in a negative influence on lipase yield. Comparative fermentation studies have been carried out in a bubble column reactor and in a centrifugal field bioreactor. Direct proportionality between growth, lipase production and oxygen consumption was observed. In the bubble column reactor usual superficial air velocities (4 cm/s) caused intensive foam generation, thus fermentation was only possible after installation of a broader column head to allow coalescence. In the centrifugal field bioreactor higher productivities were obtained without foam problems at superficial gas velocities which were one order of magnitude lower than in the bubble column. Fermentations have been performed batchwise and without holding pH constant. Neither pH control nor glucose feeding could improve the substrate yield further. Compared to former fermentation studies with the strain S. carnosus (pLipPS1) lipase yield (lipase activity/cell density) could be improved by 300% and substrate yield (lipase activity/substrate concentration) by 600%.     

Metabolic Interactions Between Glucose, Glycerol, Alanine and Acetate in Leishmania braziliensis panamensis Promastigotes

¹³C-nuciear magnetic resonance (NMR) spectroscopy was used to investigate the products of glycerol and acetate metabolism released by Leishmania braziliensis panamensis promastigotes and also to examine the interaction of each of these substrates with glucose or alanine. The NMR data were supplemented by measurements of the rates of oxygen consumption and substrate utilization, and of ¹⁴CO₂ production from ¹⁴C-labeIed substrate. Cells incubated with [2-¹³C]glycerol released acetate, succinate and D-lactate in addition to CO₂. Cells incubated with acetate released only CO₂. More succinate C-2/C-3 than C-l/C-4 was released from both [2-¹³C]glycerol and [2-¹³C]glucose, indicating that succinate was formed predominantly by CO₂ fixation followed by reverse flux through part of the Krebs cycle. Some redistribution of the position of labeling was also seen in alanine and pyruvate, suggesting cycling through pyruvate/oxaloacetate/phosphoenolpyruvate. Cells incubated with combinations of 2 substrates consumed oxygen at the same rate as cells incubated with 1 or no substrate, even though the total substrate utilization had increased. When promastigotes were incubated with both glycerol and glucose, the rate of glucose consumption was unchanged but glycerol consumption decreased about 50%, and the rate of ¹⁴CO₂ production from [l,(3)-¹⁴C]glycerol decreased about 60%. Alanine did not affect the rates of consumption of glucose or glycerol, but decreased ¹⁴CO₂ production from these substrates by increasing flow of label into alanine. Although glucose decreased alanine consumption by 70%, it increased the rate of ¹⁴CO₂ production from [U-¹⁴C]- and [l-¹⁴C]alanine by about 20%. This is consistent with rapid equilibration of alanine with pyruvate derived from glucose and yet little decrease in the specific activity of the large alanine pool.     

Ethanol production from glucose by free and agar-entrapped batch cultures ofSaccharomyces cerevisiae at different oxygenation levels

Summary The influence of the oxygenation level on glucose alcoholic fermentation by free- and immobilized-cell batch cultures ofSaccharomyces cerevisiae (CBS 1200) was investigated. Ethanol production by suspended cultures was stimulated by moderate oxygen flow rates (OFRs) but inhibited by higher OFRs (1.25 mol O₂ h^–1 dm^–3). On the contrary, agar-entrapped yeasts displayed the best fermentation performance under strict anaerobiosis and were less affected than free cultures by high OFRs. These results are dicussed by referring to the substrate mass transfer limitations inherent in immobilized-cell systems.     

Relationship between substrate concentration,growth rate,and respiration rate of Escherichia coli in continuous culture

Summary Using a continuous flow technique the relationship between growth rate and substrate concentration was investigated with glucose as the limiting factor of a culture of Escherichia coli. Graphical and numerical analysis of the experimental data demonstrated that the application of the Michaelis-Menten equation produced erroneous results, whereas, the constants obtained from the Teissier equation were in agreement with the experimental data. On this basis, new equations defining the steady state cell and substrate concentration in continuous flow cultures were developed and tested against experimental data.Comparison of the specific growth rates, substrate uptake rates and oxygen consumption rates demonstrated that all were directly proportional to each other and could be related to each other by mathematical equations. Specifically it was shown that as the growth rate increased from 0.06 to k _m =0.76 the substrate uptake rate increased from 134 to 1420 mg glucose per gram cell weight per hour and the oxygen consumption rate increased from 48.6 to 505 mg O₂ per gram cell weight per hour. Independent of the growth rate 37% of the carbohydrate consumed were oxidized. The yield factor varied from 0.44 at low growth rates to 0.54 at high growth rates. Analysis of the growth rate-substrate uptake rate relationship indicated that a minimum substrate uptake rate of 55 mg glucose per gram cell weight per hour existed below which cell reproduction would cease. This was supported by the fact that steady state conditions could not be maintained in the culture at D values below 0.02 when the substrate supply rate decreased below 45 mg glucose per gram cell weight per hour.Material contained in this paper was submitted as a thesis in partial fulfillment of the requirements for the Ph. D. degree of Dr. R. S. Lipe.     

Investigations of cephalosporin C production in an airlift tower loop reactor

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

Comparison of single- and three-stage tower loop reactors

Summary Hansenula polymorpha was cultured for long periods in 254 cm high single and three-stage countercurrent tower loop reactors 20 cm in diameter using ethanol as a substrate in the absence and presence of antifoam agents (Desmophen 3600 and/or soy oil). In the absence of antifoam agents in the three-stage column, much higher volumetric mass transfer coefficients were attained than in the corresponding single-stage column. The cell productivity in the former, however, was only slightly higher than in the single-stage column due to considerable enrichment of the cells in the foam and nonuniform cell concentration distribution in the three-stage column. In the presence of antifoam agents the three-stage column has a higher cell productivity, OTR, k_L a and a lower specific energy requirement with regard to the absorbed oxygen and/or produced cell mass than the single stage column. The reactor performance is especially high if the bubbling layer height is reduced to 20 cm. Soy oil has considerably less foam eliminating property than Desmophen. Since the soy oil is metabolized by the yeast, large amounts are needed to operate these reactors.     

Mass transfer and liquid mixing in an airlift reactor with a net draft tube

Mass transfer and liquid mixing in an airlift reactor with a net draft tube were experimentally investigated. Four different column diameters were considered. The mass transfer was measured using the volumetric gas-liquid mass transfer coefficient which was determined by the dynamic method. The mass transfer coefficients in the airlift reactors with different column diameters were not always higher than those in the bubble columns. The liquid mixing was measured using mixing time which was determined by a pulse technique. Under the same superficial gas velocity, the mixing times of the airlift reactors with a net draft tube were always less than those of the bubble columns.List of Symbols C mol·dm^–3 bulk concentration of dissolved oxygen - C ₀ mol·dm^–3 initial concentration of dissolved oxygen - C _e mol·dm^–3 saturated concentration of dissolved oxygen - ¯C dimensionless dissolved oxygen concentration - D _c cm diameter of column - D _N cm diameter of the nozzle hole - D _T cm diameter of the net draft tube - H _L cm static liquid height - H _T cm height of the net draft tube - k _L a hr^–1 volumetric mass transfer coefficient - L _T cm length of the net draft tube - t _M sec mixing time of the liquid phase - t ₀ sec mixing time of the liquid phase in a bubble column - V _L dm³ volume of the liquid phase - U _g cm/s superficial air velocity     

Bioprocess parameters and oxygen transfer characteristics in beta-lactamase production by Bacillus species

After screening potential beta-lactamase producers in a medium containing penicillin G, an inducible (Bacillus subtilis NRS 1125) and a constitutive (Bacillus licheniformis 749/C ATCC 25972) beta-lactamase producer were selected. As the highest enzyme activity was obtained with B. licheniformis 749/C, the effects of the concentration of carbon sources, i.e., glucose, fructose, sucrose, citric acid, and glycerol, and nitrogen sources, i.e., (NH(4))(2)HPO(4), NH(4)Cl, yeast extract, casamino acids and peptone, pH, and temperature on beta-lactamase production were investigated with B. licheniformis 749/C in laboratory scale bioreactors. Among the investigated media, the highest volumetric activity was obtained as 270 U cm(-)(3) in the medium containing 10.0 kg m(-)(3) glucose, 1.18 kg m(-)(3) (NH(4))(2)HPO(4), 8.0 kg m(-)(3) yeast extract, and the salt solution at 32 degrees C and pH(0) = 6.0. By using the designed medium, fermentation and oxygen transfer characteristics of the bioprocess were investigated at V = 3.0 dm(3) bioreactor systems with a V(R) = 1.65 dm(3) working volume at Q(O)/V(R) = 0.5 vvm and N = 500 min(-1). At the beginning of the process the Damk?hler number was <1, indicating that the process was at biochemical reaction limited condition; at t = 2-5 h both mass-transfer and biochemical reaction resistances were effective; and at t = 6-10 h (Da >1) the bioprocess was at mass transfer limited condition. Overall oxygen transfer coefficients (K(L)a) varied between 0.01 and 0.03 s(-)(1), enhancement factor (K(L)a/K(L)a(O)) varied between 1.2 and 2.3, and volumetric oxygen uptake rate varied between 0.001 and 0.003 mol m(-)(3) s(-)(1) throughout the bioprocess. The specific oxygen uptake and the specific substrate consumption rates were the highest at t = 2 h and then decreased with the cultivation. The maximum yield of cells on substrate and the maximum yield of cells on oxygen values were obtained, respectively, as Y(X/S) = 0.34 and Y(X/O) = 1.40, at t = 5 h, whereas the highest yield of substrate on oxygen was obtained as Y(S/O) = 6.94 at t = 3.5 h. The rate of oxygen consumption for maintenance and the rate of substrate consumption for maintenance values were found, respectively, as m(O) = 0.13 kg kg(-)(1) h(-)(1) and m(S) = 3.02 kg kg(-)(1) h(-)(1).     

Oxygen transfer rate,respiration and yields in batch and chemostat cultures ofKlebsiella aerogenes

The effect was studied of oxygen supply on the changes in total and specific rate of oxygen consumption by the cells, oxygen transfer rate, saturation concentrations of dissolved oxygen and the yields of batch and continuous cultivations. Experiments were done on the microorganismKlebsiella aerogenes CCM 2318 growing on synthetic glucose medium. Continuous cultivations were carried out at dilution rates of 0.96 and 0.178 h⁻¹. The rate of oxygen transfer was determined by the sulphite method and the coefficient K_La was assessed using the dynamic method with a correction for changes in the saturations of dissolved oxygen. A lowered oxygen supply in batch cultivations caused deformations in the course of cell respiration. Comparison of results of batch and continuous cultivations showed that the highest yields Y_x/s and Y_x/o are attained at low dilution rates without oxygen limitation. Batch cultivations, on the other hand, exhibit the lowest yields and the highest cell respiration levels. In both types of cultivations, a respiration peak was ascertained under the conditions of growth limitation by oxygen.