A comparative study of immobilized amyloglucosidase in a packed bed reactor and a continuous feed stirred tank reactor

The catalytic activity of amyloglucosidase covalently attached to DEAE-cellulose was studied in a packed bed reactor and a continuous feed stirred tank reactor (CSTR) for the reaction maltose → glucose. At low flow rates mass-transfer limitations in the bed reactor lead to lower conversions for this reactor compared to the CSTR. Simple theoretical expressions for these reactors were compared with the experimental results. There are significant differences between the kinetic parameters and pH profile of the immobilized and free enzyme. The immobilized enzyme also showed greater stability at 50°C than did free amyloglucosidase. The temperature dependence of the reaction rate was the same for immobilized and free enzyme.     

Transient behavior of a continuous stirred tank biological reactor utilizing phenol as an inhibitory substrate

Transient experiments were conducted on a Pseudomomas utilizing phenol in a continuous culture by disturbing the influent substrate concentration and dilution rate. Two stable steady states existed for some ranges of the parameters. Highly damped oscillations were observed in approaching a new high conversion steady state or in returning to a new high conversion steady state following a small disturbance. When a large disturbance was applied there was a smooth (overdamped) approach to a new low conversion steady state. The observed oscillatory behavior for small disturbances was predicted by a modified Powell-Ierusalemskii bottleneck model, but could not be predicted by a Monod-Haldane model; neither model was accurate for predicting the effect of large disturbances. A constant wall growth factor was used to account for microbial film activity, and the existence of two stable states was directly due to the presence of the film.     

Prediction of gas-liquid mass transfer coefficient in sparged stirred tank bioreactors

Oxygen mass transfer in sparged stirred tank bioreactors has been studied. The rate of oxygen mass transfer into a culture in a bioreactor is affected by operational conditions and geometrical parameters as well as the physicochemical properties of the medium (nutrients, substances excreted by the micro-organism, and surface active agents that are often added to the medium) and the presence of the micro-organism. Thus, oxygen mass transfer coefficient values in fermentation broths often differ substantially from values estimated for simple aqueous solutions. The influence of liquid phase physicochemical properties on kLa must be divided into the influence on k(L) and a, because they are affected in different ways. The presence of micro-organisms (cells, bacteria, or yeasts) can affect the mass transfer rate, and thus kLa values, due to the consumption of oxygen for both cell growth and metabolite production. In this work, theoretical equations for kLa prediction, developed for sparged and stirred tanks, taking into account the possible oxygen mass transfer enhancement due to the consumption by biochemical reactions, are proposed. The estimation of kLa is carried out taking into account a strong increase of viscosity broth, changes in surface tension and different oxygen uptake rates (OURs), and the biological enhancement factor, E, is also estimated. These different operational conditions and changes in several variables are performed using different systems and cultures (xanthan aqueous solutions, xanthan production cultures by Xanthomonas campestris, sophorolipids production by Candida bombicola, etc.). Experimental and theoretical results are presented and compared, with very good results.     

Photosynthetically oxygenated salicylate biodegradation in a continuous stirred tank photobioreactor

A consortium consisting of a Chlorella sorokiniana strain and a Ralstonia basilensis strain was able to carry out sodium salicylate biodegradation in a continuous stirred tank reactor (CSTR) using exclusively photosynthetic oxygenation. Salicylate biodegradation depended on algal activity, which itself was a function of microalgal concentration, light intensity, and temperature. Biomass recirculation improved the photobioreactor performance by up to 44% but the results showed the existence of an optimal biomass concentration above which dark respiration started to occur and the process efficiency started to decline. The salicylate removal efficiency increased by a factor of 3 when illumination was increased from 50-300 microE/m2.s. In addition, the removal rate of sodium salicylate was shown to be temperature-dependent, increasing from 14 to 27 mg/l.h when the temperature was raised from 26.5 to 31.5 degrees C. Under optimized conditions (300 microE/m2.s, 30 degrees C, 1 g sodium salicylate/l in the feed and biomass recirculation) sodium salicylate was removed at a maximum constant rate of 87 mg/l.h, corresponding to an estimated oxygenation capacity of 77 mg O2/l.h (based on a BOD value of 0.88 g O2/g sodium salicylate for the tested bacterium), which is in the range of the oxygen transfer capacity of large-scale mechanical surface aerators. Thus, although higher degradation rates were attained in the control reactor, the photobioreactor is a cost-efficient process which reduces the cost of aeration and prevents volatilization problems associated with the degradation of toxic volatile organic compounds under aerobic conditions.     

β - glucosidase production by Aspergillus wentii in stirred tank bioreactors

Summary Batch cultivation of Aspergillus wentii, Wehmer, Pt 2804 was carried out on Mandels and Reese (1957) medium containing 3% cellulose and 0.3% peptone at 30°C in 14 and 30 liter Bioreactors. Use of a 48 hours old inoculum (10% v/v), 0.5 vvm aeration at 400–800 rpm and maintenance of dissolved oxygen and pH above 50% saturation level and between 5.2 to 4.0 respectively, produced 10.58 g cells/lit and 61.9 IU/1/hr cellobiase productivity.     

A pilot-scale study of struvite precipitation in a stirred tank reactor: conditions influencing the process

Currently, the two most developed techniques for recovering phosphorus from wastewater consist of the formation of calcium phosphates and struvite (MgNH(4)PO(4).6H(2)O). In this work the influence of the operational conditions on the struvite precipitation process (pH in the reactor, hydraulic retention time, and magnesium:phosphorus, nitrogen:phosphorus, and calcium:magnesium molar ratios) have been studied. Twenty-three experiments with artificial wastewater were performed in a stirred reactor. In order to obtain the pH value maintenance during the crystallization process, a fuzzy logic control has been developed. High phosphorus removal efficiencies were reliably achieved precipitating the struvite as easily dried crystals or as pellets made up of agglomerated crystals.     

Study of microencapsulated urease in a continuous feed,stirred tank reactor

Urease, (urea amidohydrolase, EC 3.5.1.5) co-encapsulated with haemoglobin in cellulose nitrate membranes was found to exhibit apparent Michaelis-Menten kinetics; however, a steadily increasing apparent Michaelis-Menten constant over the lifetime of the preparation was observed. The activity of the enzyme in a continuous feed stirred tank reactor (CSTR) was investigated and correlated with a mathematical model derived from basic Michaelis-Menten kinetics. Plots relating substrate conversion to feed substrate concentration and tank reactor capacity were constructed and found to be accurate to less than 15% error under the experimental conditions studied.     

Optimization and control of a continuous stirred tank fermenter using learning system

A variable structure learning automaton is used as an optimization and control of a continuous stirred tank fermenter. The algorithm requires no modelling of the process. The use of appropriate learning rules enables to locate the optimum dilution rate in order to maximize an objective cost function. It is shown that a hierarchical structure of automata can adapt to environmental changes and can also modify efficiently the domain of variation of the control variable in order to encompass the optimum value.List of Symbols f Random number - F Dimensionless flow rate (F/V ₀) - F m³/h Flow rate - F ₀ m³/h Inlet flow rate - J Objective function - K _i Dimensionless constant in Eq. (3) (k _i/s₀) - k _i · kg/m³ Substrate inhibition constant in Haldane model - K _m Dimensionless constant in equation (3) (k _s/s₀) - k _m kg/m³ Substrate inhibition constant in Haldane model - L Number of levels of the hierarchical system of automata - N Number of possible control actions - p Probability - S Dimensionless substrate concentration (s/s ₀) - s kg/m³ Substrate concentration - T Dimensionless sampling period - t h Time - v Dimensionless volume (V/V ₀) - V m³ Liquid volume in fermenter - W Input to the stochastic automaton - X Dimensionless biomass concentration - x kg/m³ Biomass concentration - Y Biomass/substrate yield coefficient - Weighting factor in Eq. (4) - Dimensionless specific growth rate (/ ^*) - ^* h^–1 Maximum specific growth rate - h^–1 Specific growth rate - Dimensionless time ( t)     

kLa of stirred tank bioreactors revisited

By means of improved feedback control k_La measurements become possible at a precision and reproducibility that now allow a closer look at the influences of power input and aeration rate on the oxygen mass transfer. These measurements are performed online during running fermentations without a notable impact on the biochemical conversion processes. A closer inspection of the mass transfer during cultivations showed that at least the number of impellers influences mass transfer and mixing: On the laboratory scale, two hollow blade impellers clearly showed a larger k_La than the usually employed three impeller versions when operated at the same agitation power and aeration rate. Hollow blade impellers are preferable under most operational conditions because of their perfect gas handling capacity. Mixing time studies showed that these two impeller systems are also preferable with respect to mixing. Furthermore the widths of the baffle bars depict a significant influence on the k_La. All this clearly supports the fact that it is not only the integral power density that finally determines k_La.     

Optimizing control of a continuous stirred tank fermenter using a neural network

Feedforward neural networks are a general class of nonlinear models that can be used advantageously to model dynamic processes. In this investigation, a neural network was used to model the dynamic behaviour of a continuous stirred tank fermenter in view of using this model for predictive control. In this system, the control setpoint is not known explicitly but it is calculated in such a way to optimize an objective criterion. The results presented show that neural networks can model very accurately the dynamics of a continuous stirred tank fermenter and, the neural model, when used recursively, can predict the state variables over a long prediction horizon with sufficient accuracy. In addition, neural networks can adapt rapidly to changes in fermentation dynamics.List of Symbols F Dimensionless flow rate (F/ V₀) - F m³/h Flow rate - F ₀ m³/h Inlet flow rate - J Objective cost function - K _i Dimensionless constant in Eq. (3) (k _i /s₀) - k _i kg/m³ Substrate inhibition constant in Haldane model - k _m Dimensionless constant in Eq. (3) (k _s /s₀) - k _m kg/m³ Substrate inhibition constant in Haldane model - n prediction horizon - S Dimensionless substrate concentration (s/s₀) - s kg/m³ Substrate concentration - t h Time - v Dimensionless volume (V/V₀) - V m³ Liquid volume in fermenter - W _ij , W _jk Weight matrices in neural network - X Dimensionless biomass concentration - x kg/m³ Biomass concentration - Y Biomass/substrate yield coefficient - Weighting factor in Eq. (4) - Dimensionless specific growth rate (/ ) - 1/h Maximum specific growth rate - 1/h Specific growth rate - Dimensionless time ( t)     

Shear conditions in clavulanic acid production by Streptomyces clavuligerus in stirred tank and airlift bioreactors

In biochemical processes involving filamentous microorganisms, the high shear rate may damage suspended cells leading to viability loss and cell disruption. In this work, the influence of the shear conditions in clavulanic acid (CA) production by Streptomyces clavuligerus was evaluated in a 4-dm(3) conventional stirred tank (STB) and in 6-dm(3) concentric-tube airlift (ALB) bioreactors. Batch cultivations were performed in a STB at 600 and 800 rpm and 0.5 vvm (cultivations B1 and B2) and in ALB at 3.0 and 4.1 vvm (cultivations A1 and A2) to define two initial oxygen transfer conditions in both bioreactors. The average shear rate ([Formula: see text]) of the cultivations was estimated using correlations of recent literature based on experimental data of rheological properties of the broth (consistency index, K, and flow index, n) and operating conditions, impeller speed (N) for STB and superficial gas velocity in the riser (UGR) for ALB. In the same oxygen transfer condition, the [Formula: see text] values for ALB were higher than those obtained in STB. The maximum [Formula: see text] presented a strong correlation with a maximum consistency index (K (max)) of the broth. Close values of maximum CA production were obtained in cultivations A1 and A2 (454 and 442 mg L(-1)) with similar maximum [Formula: see text] values of 4,247 and 4,225 s(-1). In cultivations B1 and B2, the maximum CA production of 269 and 402 mg L(-1) were reached with a maximum [Formula: see text] of 904 and 1,786 s(-1). The results show that high values of average shear rate increase the CA production regardless of the oxygen transfer condition and bioreactor model.     

Biodegradation of phenol at high initial concentrations in two-phase partitioning batch and fed-batch bioreactors

A two-phase organic-aqueous system was used to degrade phenol in both batch and fed-batch culture. The solvent, which contained the phenol and partitioned it into the aqueous phase, was systematically selected based on volatility, solubility in the aqueous phase, partition coefficient for phenol, biocompatibility, and cost. The two-phase partitioning bioreactor used 500 mL of 2-undecanone loaded with high concentrations of phenol to deliver the xenobiotic to Pseudomonas putida ATCC 11172 in the 1-L aqueous phase, at subinhibitory levels. The initial concentrations of phenol selected for the aqueous phase were predicted using the experimentally determined partition coefficient for this ternary system of 47.6. This system was initially observed to degrade 4 g of phenol in just over 48 h in batch culture. Further loading of the organic phase in subsequent experiments demonstrated that the system was capable of degrading 10 g of phenol to completion in approximately 72 h. The higher levels of phenol in the system caused a modest increase in the duration of the lag phase, but did not lead to complete inhibition or cell death. The use of a fed-batch approach allowed the system to ultimately consume 28 g of phenol in approximately 165 h, without experiencing substrate toxicity. In this system, phenol delivery to the aqueous phase is demand based, and is directly related to the metabolic activity of the cells. This system permits high loading of phenol without the corresponding substrate inhibition commonly seen in conventional bioreactors. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 155-162, 1997.     

Consecutive immobilized enzymatic reactions in continuous stirred tank reactor systems

The performance characteristics of two-enzyme reaction in a continuous stirred tank reactor (CSTR) are analytical investigated in this work. A model is formulated to describe the substrate concentration variations by taking into account the external and internal diffusion resistances. It is found that the reaction system exhibits the characteristics of reaction control or diffusion control depending on the operating conditions. The single CSTR model is also extended to describe the multiple CSTR system. The latter model enables the prediction of the number of CSTRs in series required to achieve a prescribed substrate conversion.     

Biodiesel synthesis in a solvent-free system by recombinant Rhizopus oryzae: comparative study between a stirred tank and a packed-bed batch reactor

A simultaneous synthesis of biodiesel, as fatty acid methyl esters, and monoacylglycerols catalysed by the recombinant Rhizopus oryzae lipase immobilized by adsorption on Relizyme OD/403M is presented. The use of this 1(3)-positional specific lipase prevents the formation of glycerol as a by-product, thus avoiding its drawbacks. The synthesis was carried out in a solvent-free system and it has been studied in two different reactor systems: stirred tank and packed-bed reactor. Stirred tank reactor presented a high-initial reaction rate and achieved a 33.6% yield, which corresponds to a value of 50.4% of the maximum yield that can be achieved with a 1(3)-positional specific lipase. In packed-bed reactor there was a smaller initial reaction rate, but it was achieved a 49.1% yield, which corresponds to a 73.6% of the maximum yield. When a second batch is performed, the yield decreased only 4% when packed-bed reactor is employed whereas a drastic decrease is observed in a stirred tank operation. Therefore, packed-bed reactor showed a best performance and minor damage to the biocatalyst.     

Operational stability of immobilized d-glucose isomerase in a continuous feed stirred tank reactor

d-Glucose isomerization has been studied using immobilized cells of Streptomyces phaeochromogenes in a continuous feed stirred tank reactor (CSTR) where the external film diffusion resistance was negligible. Experiments conducted with various sizes of enzyme particles indicated that a strong internal diffusion resistance improved the apparent stability of these particles. The performance equations of the CSTR were constructed by associating the material balances for the inside porous support matrix with the bulk liquid phase, and enzyme deactivation was also taken into consideration. An iterative method together with the orthogonal collocation method is proposed for the evaluation of effectiveness factor and the substrate concentration profile within the enzyme particles. The numerical results offer an alternative analytical proof for the observation that under strong internal diffusion control the apparent operational stability of immobilized enzyme is improved.     

Comparison of a production process in a membrane-aerated stirred tank and up to 1000-L airlift bioreactors using BHK-21 cells and chemically defined protein-free medium

The applicability of a protein-free medium for the production of recombinant human interleukin-2 with baby hamster kidney cells in airlift bioreactors was investigated. For this purpose, a BHK-21 cell line, adapted to grow and produce in protein-free SMIF7 medium without forming spheroids in membrane-aerated bubble-free bioreactors, was used as the producer cell line. First, cultivation of the cells was established at a 20-L scale using an internal loop airlift bioreactor system. During the culturing process the medium formulation was optimized according to the specific requirements associated with cultivation of mammalian cells under protein-free conditions in a bubble-aerated system. The effects of the addition of an antifoam agent on growth, viability, productivity, metabolic rates, and release of lactate dehydrogenase were investigated. Although it was possible to establish cultivation and production at a 20-L scale without the use of antifoaming substances, the addition of 0.002% silicon-oil-based antifoaming reagent improved the cultivation system by completely preventing foam formation. This reduced the release of lactate dehydrogenase activity to the level found in bubble-free aerated stirred tank membrane bioreactors and led to a reduction in generation doubling times by about 5 h (17%). Using the optimized medium formulation, cells were cultivated at a 1000-L scale, resulting in a culture performance comparable to the 20-L airlift bioreactor. For comparison, cultivations with protein-containing SMIF7 medium were carried out at 20- and 1000-L scales. The application of protein supplements did not lead to a significant improvement in the cultivation conditions. The results were also compared with experiments performed in a bubble-free aerated stirred tank membrane bioreactor to evaluate the influence of bubbles on the investigated culture parameters. The data implied a higher metabolic activity of the cells in airlift bioreactors with a 150% higher glucose consumption rate. The results of this study clearly demonstrate the applicability of a protein-free chemically defined medium for the production of recombinant proteins with BHK cells in airlift bioreactors.