Biotransformation of glucose to gluconic acid by Aspergillus niger—study of mass transfer in an airlift bioreactor

A glucose–gluconic acid biotransformation system was suggested for the experimental study of oxygen transfer in bioreactors. This biosystem was used for the investigation of the effect of the flow rate and biomass concentration on the volumetric oxygen transfer coefficient k_La in a 10 dm³ internal-loop airlift bioreactor. For this purpose, the fermentation broth of the mycelial strain Aspergillus niger was employed, representing a three-phase system, where bubbles come into contact with dense rigid pellets. The results showed that the presented biotransformation system can be successfully utilised for the determination of the oxygen transfer rate in airlift bioreactors. The experiments showed a strong positive influence of the air flow rate on the rate (r_Glu), specific rate of gluconic acid production (k_Glu/X) as well as on the volumetric oxygen transfer coefficient (k_La). This confirmed an expected limitation of production rate by the oxygen transport from the gas to the liquid phase in the whole range of air flow rates applied. Moreover, consistent curves of the production rate r_Glu and k_La values vs. biomass concentration c_X (amount of enzymes) were observed. These exhibited a local maximum for c_X equal to 6.68 g dm⁻³. On the other hand, the specific production rate monotonously decreased with increasing biomass concentration. A decline of k_La values at higher c_X values was attributed to a bubble coalescence promoting effect of mycelial pellets.     

Effect of organic solvents on oxygen mass transfer in multiphase systems: Application to bioreactors in environmental protection

The absorption of oxygen in aqueous–organic solvent emulsions was studied in a laboratory-scale bubble reactor at a constant gas flow rate. The organic and the gas phases were dispersed in the continuous aqueous phase. Volumetric mass transfer coefficients (k_La) of oxygen between air and water were measured experimentally using a dynamic method. It was assumed that the gas phase contacts preferentially the water phase. It was found that addition of silicone oils hinders oxygen mass transfer compared to air–water systems whereas the addition of decane, hexadecane and perfluorocarbon PFC40 has no significant influence. By and large, the results show that, for experimental conditions (organic liquid hold-up ≤10% and solubility ratio ≤10), the k_La values of oxygen determined in binary air–water systems can be used for multiphase (gas–liquid–liquid) reactor design with applications in environmental protection (water and air treatment processes).     

Engineering characterisation of a single well from 24-well and 96-well microtitre plates

The detailed engineering characterisation of shaken microtitre-plate bioreactors will enhance our understanding of microbial and mammalian cell culture in these geometries and will provide guidance on the scale-up of microwell results to laboratory and pilot scale stirred bioreactors. In this work computational fluid dynamics (CFD) is employed to provide a detailed characterisation of fluid mixing, energy dissipation rate and mass transfer in single well bioreactors from deep square 24-well and 96-well microtitre plates. The numerical predictions are generally found to be in good agreement with experimental observation of the fluid motion and measured values of the key engineering parameters. The CFD simulations have shown that liquid mixing is more intensive in 96-well than in 24-well bioreactors due to a significant axial component to the fluid velocity. Liquid motion is strongly dependent on the orbital shaking amplitude which generally has a greater impact than the shaking frequency. Average power consumptions of 70–100 W m⁻³ and 500–1000 W m⁻³, and overall mass transfer coefficient, k_La, values of 0.005–0.028 s⁻¹ and 0.056–0.10 s⁻¹ were obtained for 24-well and 96-well bioreactors respectively at an orbital shaking amplitude of 3 mm and shaking frequencies ranging from 500 rpm to 1500 rpm. The distribution of energy dissipation rates within each bioreactor showed these to be greatest at the walls of the well for both geometries. Batch culture kinetics of E. coli DH5 showed similar maximum specific growth rates and final biomass yields in shaken 24-well and shake flask bioreactors and in stirred miniature and 20 L bioreactors at matched k_La values. The CFD simulations thus give new insights into the local and overall engineering properties of microwell bioreactor geometries and further support their use as high throughput tools for the study and optimisation of microbial and mammalian cell culture kinetics at this scale.     

Optical method for the determination of the oxygen-transfer capacity of small bioreactors based on sulfite oxidation.

The growth of microorganisms may be limited by operating conditions which provide an inadequate supply of oxygen. To determine the oxygen-transfer capacities of small-scale bioreactors such as shaking flasks, test tubes, and microtiter plates, a noninvasive easy-to-use optical method based on sulfite oxidation has been developed. The model system of sodium sulfite was first optimized in shaking-flask experiments for this special application. The reaction conditions (pH, buffer, and catalyst concentration) were adjusted to obtain a constant oxygen transfer rate for the whole period of the sulfite oxidation reaction. The sharp decrease of the pH at the end of the oxidation, which is typical for this reaction, is visualized by adding a pH dye and used to measure the length of the reaction period. The oxygen-transfer capacity can then be calculated by the oxygen consumed during the complete stoichiometric transformation of sodium sulfite and the visually determined reaction time. The suitability of this optical measuring method for the determination of oxygen-transfer capacities in small-scale bioreactors was confirmed with an independent physical method applying an oxygen electrode. The correlation factor for the maximum oxygen-transfer capacity between the chemical model system and a culture of Pseudomonas putida CA-3 was determined in shaking flasks. The newly developed optical measuring method was finally used for the determination of oxygen-transfer capacities of different types of transparent small-scale bioreactors.     

Different bioreactor configurations for the decolourisation of the azo dye reactive black 5 by Geotrichum sp. CCMI 1019

Decolourisation of the azo dye Reactive Black 5 by Geotrichum sp. CCMI 1019 was studied using stirred tank reactors (STR) and two types of bubble columns (porous plate (PP) bubble column and aeration tube (AT) bubble column). For the bubble columns, the k_La increased with the gas fractional hold-up (ε_G) and the aeration rate. A linear relationship between ε_G and superficial gas velocity was obtained for all reactors. At same aeration rates, the PP bubble columns showed higher k_La and hold-up values than the AT bubble column. In the STRs, large and dense aggregates were formed which adhered to surfaces whereas bubble columns gave smaller and less compact pellets.

Manganese peroxidase and laccase were detected in the extracellular media in all reactors. However, laccase was only detected after the onset of decolourisation, suggesting that additional enzymes may be involved. Mn peroxidase activity was detected (about 46 U/ml) in both the STRs and AT bubble columns but higher values (110 U/ml) were obtained with the PP bubble columns.

Out of the three reactor systems studied, the AT bubble columns gave the most favourable results for Reactive Black 5 decolourisation. Rapid and complete colour removal was obtained throughout the visible spectrum. Bubble columns are simple in design as well as operation and may be useful for the bioremediation of textile wastewater.     

The influence of pH and dilution rate on continuous production of xylitol from sugarcane bagasse hemicellulosic hydrolysate by C. guilliermondii

Continuous fermentation of sugarcane bagasse hemicellulosic hydrolysate by the yeast Candida guilliermondii FTI 20037 was used for xylitol production from xylose. Experiments were carried out in a reactor with 1.25 l of treated hydrolysate, at 30 °C and 300 rpm. A 2² full-factorial central composite design was employed for experimental study and analysis of the results. A statistical analysis of the results showed that the effects of the pH and dilution rate (D), the interactions between these variables and the second-order effect of D on the xylitol volumetric productivity (Q_p) were significant at a 95% confidence level. The second-order effect of pH was also significant at a 90% confidence level. The k_La effect on the Q_p was not significant. A volumetric productivity of 0.68 g/l h, representing 95.8% of the predicted value (0.72 g/l h), was obtained.     

Enhancement of citric acid production by Aspergillus niger using n-dodecane as an oxygen-vector

Due to the significant oxygen requirement during citric acid production and the relatively low solubility of oxygen in water, aeration is critical. The potential use of n-dodecane as an oxygen-vector for improvement of citric acid production by Aspergillus niger was studied. The volumetric fraction of oxygen-vector has a great influence on the volumetric oxygen transfer coefficient k_La. With the addition of an oxygen-vector to the fermentation medium with a final concentration of 5%, the k_La value reached a maximum value (130 h⁻¹), which is twice that of the control experiment. The addition of 5% (v/v) n-dodecane enhanced citric acid accumulation, reduced residual sugar concentration and stimulated mycelial growth. Adding n-dodecane had no adverse effects on the cells of A. niger. The results of enzyme assays indicated that no significant differences were observed between the activity of citrate synthase of two kinds of mycelial cell-free extracts.     

Influence of sparger on energy dissipation, shear rate, and mass transfer to sea water in a concentric-tube airlift bioreactor

Data on volumetric mass-transfer coefficient, K_La_L, in a 12 × 10⁻³ m³ airlift bioreactor are reported. Measurements were made in sea water. The superficial gas velocity ranged up to 0.21 m/s. Four cylindrical spargers (60–1000 μm pore size) were tested. In bubbly flow, the sparger pore size strongly influenced the K_La_L; the highest K_La_L values were obtained with the smallest pore size. In contrast, in the transition and heterogeneous flow regimes, the pore size had little influence on K_La_L. The best correlation of the mass transfer data was obtained when both gas holdup and liquid superficial velocity were taken as independent variables. Shear rates were estimated in the different zones of the reactor. The highest values were found in the bottom zone of the reactor and in the gas-liquid separator. The penetration and isotropic turbulence models were used to develop a semi-theoretical equation relating the volumetric mass-transfer coefficient to shear rate; hence providing a better understanding of how the operational variables may be manipulated to attain a moderate shear rate and an appropriate level of mass transfer, two extremely important parameters for the growth of sensible microorganisms as those used in marine biotechnology.     

Critical assessment of the steady-state Na2SO3 feeding method for kla measurement in fermentors

Important aspects of k(l)a measurement in agitated aerated vessels are briefly characterized from the standpoint of reliability of the measured data. It seems that most of the k(l)a data, based on a number of variants of the steady-state and dynamic methods in noncoalescent liquids, do not have a clear physical meaning, because they are affected by the differences between the actual driving force and the driving force assumed by the model used for its evaluation. A reliability test is given for the Na(2)SO(3) feeding steady-state method (FSM), by comparing the results of air and pure oxygen absorption in a noncoalescent liquid (0.5M Na(2)SO(4) solution) with the results obtained by the independent pressure step dynamic method (RDM). The RDM is one of a few variants of the dynamic method which gives correct k(l)a data unaffected by nonideal mixing of the gas phase in the reactor. It was found that the FSM yields correct k(l)a values only when pure oxygen is used for absorption. When air is absorbed, the FSM gives k(l)a values in the region of k(l)a > 0.1 s(-1) substantially (to 55%) lower than those for pure oxygen absorption.     

A simple Na(2)SO(3) feeding method for K(L)a measurement in large-scale fermentors

A simple and convenient method for measuring K(L)a in large-scale fermentors was proposed. This method was based on the measurement of the dissolved oxygen concentration under steady state conditions established by an equivalency of the sulfite ion feed and chemical oxidation rates. This method had the following advantages: It was a steady state method, and so it was not necessary to consider the response lag of a dissolved oxygen probe and the response lag due to gas phase mixing in fermentors. The oxygen content of the effluent gas in this measuring system was nearly the same as that of the sparged air. Therefore, it was possible to use the oxygen partial pressure of the sparged air for the calculation of the driving force of oxygen transfer. The detailed information on the kinetics of sulfite oxidation was not necessary, because the dissolved oxygen concentration in steady state was not influenced by sulfite oxidation rates. The K(L)a measurement was finished in as short a period as 150 s, even in a fermentor with a volume of 10 m(3). Since the amount of Na(2)SO(4) accumulation in the test fermentors was very small because of the quick measurement, the K(L)a values obtained by this method were applicable to the electrolyte-free system. Furthermore, we could discharge the used liquid from the fermentors into a drain without any pretreatment due to the low salt concentration.     

Bench-scale fermentation of Trichoderma viride on wastewater sludge: Rheology, lytic enzymes and biocontrol activity

Conidiation and lytic enzyme production by Trichoderma viride at different solids concentration of pre-treated municipal wastewater sludge was examined in a 15-L fermenter. The maximum conidia concentration (5.94 × 10⁷ CFU mL⁻¹ at 96 h) was obtained at 30 g L⁻¹ suspended solids. The maximum lytic enzyme activities were achieved around 12–30 h of fermentation. Bioassay against a fungal phytopathogen, Fusarium sp. showed maximum activity in the sample drawn around 96 h of fermentation at 30 g L⁻¹ suspended solids concentration. Entomotoxicity against spruce budworm larvae showed maximum value ≈17290 SBU μL⁻¹ at 30 g L⁻¹ suspended solids concentration at the end of fermentation (96 h). Plant bioassay showed dual action of T. viride, i.e., disease prevention and growth promotion. The rheological analyses of fermentation sludges showed the pseudoplastic behaviour. In order to maintain required dissolved oxygen concentration ≥30%, the agitation and aeration requirements significantly increased at 35 g L⁻¹ compared to 30 and 25 g L⁻¹. The oxygen uptake rate and volumetric oxygen mass transfer coefficient, k_La at 35 g L⁻¹ did not increase in comparison to 30 g L⁻¹ due to rheological complexity of the broth during fermentation. Thus, the successful fermentation operation of the biocontrol fungus T. viride is a rational indication of its potential for mass-scale production for agriculture and forest sector as a biocontrol agent.     

Oxygen transfer phenomena in 48-well microtiter plates: determination by optical monitoring of sulfite oxidation and verification by real-time measurement during microbial growth

Oxygen limitation is one of the most frequent problems associated with the application of shaking bioreactors. The gas-liquid oxygen transfer properties of shaken 48-well microtiter plates (MTPs) were analyzed at different filling volumes, shaking diameters, and shaking frequencies. On the one hand, an optical method based on sulfite oxidation was used as a chemical model system to determine the maximum oxygen transfer capacity (OTR(max)). On the other hand, the Respiration Activity Monitoring System (RAMOS) was applied for online measurement of the oxygen transfer rate (OTR) during growth of the methylotropic yeast Hansenula polymorpha. A proportionality constant between the OTR(max) of the biological system and the OTR(max) of the chemical system were indicated from these data, offering the possibility to transform the whole set of chemical data to biologically relevant conditions. The results exposed "out of phase" shaking conditions at a shaking diameter of 1 mm, which were confirmed by theoretical consideration with the phase number (Ph). At larger shaking diameters (2-50 mm) the oxygen transfer rate in MTPs shaken at high frequencies reached values of up to 0.28 mol/L/h, corresponding to a volumetric mass transfer coefficient (k(L)a) of 1,600 1/h. The specific mass transfer area (a) increases exponentially with the shaking frequency up to values of 2,400 1/m. On the contrary, the mass transfer coefficient (k(L)) is constant at a level of about 0.15 m/h over a wide range of shaking frequencies and shaking diameters. However, at high shaking frequencies, when the complete liquid volume forms a thin film on the cylindric wall of the well, the mass transfer coefficient (k(L)) increases linearly to values of up to 0.76 m/h. Essentially, the present investigation demonstrates that the 48-well plate outperforms the 96-well MTP and shake flasks at widely used operating conditions with respect to oxygen supply. The 48-well plates emerge, therefore, as an excellent alternative for microbial cultivation and expression studies combining the advantages of both the high-throughput 96-well MTP and the classical shaken Erlenmeyer flask.     

Gasometric measurement of sulfite oxidation

In the commonly used sulfite method the consumption of sulfite is determined by iodometry. Since however, the addition of organic substances may interfere with iodometry (e.g. due to chemical reactions with iodine) the gasometric measurement of sulfite oxidation has been developed for analysis of how different culture media may influence the oxygen transfer rate. The striking decrease of sulfite oxidation rate due to addition of culture media to the sulfite solution suggests that adsorption of orgnic components in the gas liquid interface may account for an additional diffusion barrier and thus for a decrease of the oxygen transfer coefficient which in addition gives an explanation for differences between values found by the sulfite method and by aerobic cultivations. Consequently identical values of oxygen transfer rate have been obtained for both systems whenever the sulfite system has been properly adjusted to the aerobic cultivation conditions. In so far, the gasometric sulfite method proved to be a unique tool for rapid determination of factors influencing oxygen transfer rate in fermentation processes which may give rise to a reappraisal as to the relevance of the sulfite method for oxygen transfer optimization.     

Evaluation of oxygen transfer rates in stirred-tank bioreactors for clinical manufacturing

Several methods are available for determining the volumetric oxygen transfer coefficient in bioreactors, though their application in industrial bioprocess has been limited. To be practically useful, mass transfer measurements made in nonfermenting systems must be consistent with observed microbial respiration rates. This report details a procedure for quantifying the relationship between agitation frequency and oxygen transfer rate that was applied in stirred-tank bioreactors used for clinical biologics manufacturing. The intrinsic delay in dissolved oxygen (DO) measurement was evaluated by shifting the bioreactor pressure and fitting a first-order mathematical model to the DO response. The dynamic method was coupled with the DO lag results to determine the oxygen transfer rate in Water for Injection (WFI) and a complete culture medium. A range of agitation frequencies was investigated at a fixed air sparge flow rate, replicating operating conditions used in Pichia pastoris fermentation. Oxygen transfer rates determined by this method were in excellent agreement with off-gas calculations from cultivation of the organism (P = 0.1). Fermentation of Escherichia coli at different operating parameters also produced respiration rates that agreed with the corresponding dynamic method results in WFI (P = 0.02). The consistency of the dynamic method results with the off-gas data suggests that compensation for the delay in DO measurement can be combined with dynamic gassing to provide a practical, viable model of bioreactor oxygen transfer under conditions of microbial fermentation.     

Influence of Diesel Soot Particles and Sulfite on Functions of Polymorphonuclear Leukocytes

Aqueous suspensions of diesel soot particles in combination with sulfite influence certain functions of human polymorphonuclear neutrophils in vitro. Chemiluminescence, generated after activation by opsonized zymosan as well as oxygen uptake were decreased, whereas phagocytosis was increased. An enhancement of degranulation could not be observed. The single substances show little or no effects on the above properties. The results indicate that combinations of air pollutants such as diesel soot and sulfite may modulate vital functions of activated leukocytes in vivo.     

Measurement of k(L)a by dynamic pressure method in pilot-plant fermentor

The dynamic pressure method (DPM) is used for measurement of k(L)a in a 1-m(3) pilot scale fermentor in coalescing (distilled water) and noncoalescing (0.3 M Na(2)SO(4) aqueous solution) batches. The method consists in recording oxygen concentration in a batch after a small pressure change (20 kPa) in the fermentor. The upward pressure change is brought about by temporary closing and subsequent throttling of outlet gas stream and the downward change by full reopening of the gas outlet. Absorption of pure oxygen yields the same k(L)a values as absorption of air. In noncoalescing batch, the downward k(L)a values are always higher than the upward values owing to spontaneous nucleation of bubbles. The experiments performed in a stirred cell confirm this behavior. Thus, only upward pressure change should be used for measurement. The correlation of k(L)a data measured in small (18-L) and large (1000-L) vessels based on power dissipated and superficial gas velocity are in a good agreement. Unlike the DPM, the classical dynamic methods yield, under the same conditions, excessively low values of k(L)a (the dynamic startup method) or fail to produce data at all (the dynamic method with interchange of air for N(2)). (c) 1994 John Wiley & Sons, Inc.     

A Novel Amplified Enzymatic Assay Method for Hypoxanthine

A sensitive and rapid enzymatic assay for hypoxanthine, using a Clark oxygen electrode as sensor, is proposed. In the presence of sodium sulfite, oxidation of hypoxanthine by milk xanthine oxidase caused very rapid oxygen consumption in excess of the stoichiometric requirement for hypoxanthine oxidation. Hypoxanthine from 0.5 to 10 μM can be assayed within a few minutes by addition of 25 mM sodium sulfite to the reaction mixture. This assay proved to be over 10-times more sensitive and much more rapid than the control method without sulfite.     

A Novel Amplified Enzymatic Assay Method for Hypoxanthine

A sensitive and rapid enzymatic assay for hypoxanthine, using a Clark oxygen electrode as sensor, is proposed. In the presence of sodium sulfite, oxidation of hypoxanthine by milk xanthine oxidase caused very rapid oxygen consumption in excess of the stoichiometric requirement for hypoxanthine oxidation. Hypoxanthine from 0.5 to 10 μM can be assayed within a few minutes by addition of 25 mM sodium sulfite to the reaction mixture. This assay proved to be over 10-times more sensitive and much more rapid than the control method without sulfite.     

Air pressure effects on biomass yield of two different Kluyveromyces strains

The use of air pressure as a way of improving oxygen transfer in aerobic bioreactors was investigated. To compare the air pressure effects with traditional air bubbled cultures, experiments using a pressure reactor and a stirred flask, with the same oxygen transfer rate, were made. Kluyveromyces marxianus is an important industrial yeast and some of it show a “Kluyver effect” for lactose: even under oxygen limited growth conditions, certain disaccharides that support aerobic, respiratory growth, are not fermented. This study deals with the effect of increased pressure on the physiological behavior of two Kluyveromyces strains: K. marxianus ATCC10022 is a lactose-fermenting strain, whereas K. marxianus CBS 7894 has a Kluyver-effect for lactose. For K. marxianus ATCC10022 an air pressure increase of 2 bar led to a 3-fold increase in biomass yield. When air pressure increased an enhancement of ethanol oxidation of cell yeasts was also observed. Batch cultures of K. marxianus CBS 7894 exhibited different growth behaviour. Its metabolism was always oxidative and ethanol was never produced. With the increase in air pressure, it was possible to increase the productivity in biomass of K. marxianus CBS 7894. As a response to high oxygen concentrations, due to the increase in oxygen partial pressure, oxidative stress in the cells was also studied. Antioxidant defences, such as superoxide dismutase, catalase, and glutathione reductase, were at high activity levels, suggesting that these yeast strains could tolerate the increased pressures applied.     

Artificial inhibitory effects of sulfite on photosystem II activity measured by oxygen evolution in chloroplasts

In this report we demonstrate sulfite interaction with oxygen and PSII electron acceptors (ferricyanide and para-benzoquinone) during measurement of oxygen evolution in chloroplasts. Redox potentials of oxygen, ferricyanide and para-benzoquinone allow them to compete for sulfite. Without taking this into account, sulfite inhibition of oxygen evolution can be overestimated, since sulfite consumes oxygen and reduces ferricyanide or para-benzoquinone during the measurement. In order to correctly measure the rate of oxygen evolution in chloroplasts, it is necessary to avoid presence of sulfite during the measurement. After overcoming the artifact, mentioned above, we confirm the sulfite inhibition of oxygen evolution in chloroplasts but at a lesser extent than earlier reported. This, however, is a pretreatment effect.Abbreviations Chl Chlorophyll - EDTA Ethylenediamine Tetraacetic Acid - FeCN Potassium Ferricyanide - Hepes N-2-Hydroxyethylpiperazine-N¹-2-ethanesulfonic acid - pBQ Para-benzoquinone - PSII photosystem II