Modeling solid-to-solid biocatalysis: integration of six consecutive steps

A quantitative model for the conversion of a solid-substrate salt to a solid-product salt in a batch bioreactor seeded with product crystals is presented. The overall process consists of six serial steps (with dissolution and crystallization each in themselves complex multistep processes): solid-salt dissolution, salt dissociation into an ionic substrate and a counter-ion, bioconversion accompanied by biocatalyst inactivation, complexation of the ionic product with the counter-ion, and salt crystal growth. In the model, the consecutive steps are integrated, including biocatalyst inactivation and assuming that salt dissociation and complexation of ions are at equilibrium. Model parameters were determined previously in separate independent experiments. To validate the model, either dissolved or solid Ca-maleate was converted to solid Ca-D-malate by permeabilized Pseudomonas pseudoalcaligenes in a batch bioreactor seeded with Ca-D-malate crystals. The model very well predicted the concentrations of all components in the liquid phase (Ca-maleate, Ca(2+), maleate(2-), D-malate(2-), and Ca-D-malate) and the amounts of the solid phases (Ca-maleate. H(2)O and Ca-D-malate. 3H(2)O), especially when high initial amounts of Ca-maleate. H(2)O and Ca-D-malate. 3H(2)O were present.     

Relationships between hydrodynamics and rheology of flocculating yeast suspensions in a high-cell-density airlift bioreactor

In this article a hydrodynamic and rheological analysis of a continuous airlift bioreactor with high-cell-density system is presented. A highly flocculating recombinant strain of Sacharomyces cerevisiae containing genes for lactose transport (lactose permease) and hydrolysis (beta-galactosidase) was exploited to ferment lactose from cheese whey to ethanol. The magnetic particle-tracer method was used to assess the effect of operational conditions (air-flow rate, biomass concentration) on hydrodynamic behavior of an airlift bioreactor during the fermentation process. Measurements of liquid circulation velocity showed the existence of a critical value of biomass concentration at which a dramatic deceleration of net liquid flow appeared with increasing biomass quantity. Rheological analysis revealed exponential increase of viscosity of the yeast floc suspension at the same biomass concentration of about 73 g/dm3 corresponding to 42.8% v/v of solid fraction. These facts have a particular importance for the successful processing of a high-cell-density airlift bioreactor as only a circulated flow regime will be favorable to keep the solid particles in suspension state and evenly distributed throughout the bioreactor.     

Control of the specific growth rate of Bacillus subtilis for the production of biosurfactant lipopeptides in bioreactors with foam overflow

This paper formulates a feeding law for a bioprocess dedicated to the production of an antibiotic surfactant using Bacillus subtilis. The specificity of the process relies on the use of the surface active property of the product to extract it by foaming. The control law is designed to maintain a constant specific biomass growth rate while taking into account the particularity of the process. This law can be regarded as a generalization of the conventional exponential feeding strategy and is generic enough to encompass the case of continuous processes with partial recycling. Conventional exponential feeding strategies indeed fail to account for the loss of biomass induced by the foaming. Previous experiments have provided a model of the process and values for its parameters. From this information, a feeding rate law was computed using the feeding strategy proposed in this paper and applied to an experimental culture. This experiment allows discussion of the modeling of the biomass extraction method used in this study. The results on the estimated specific growth rate highlight the complete agreement between the expected and experimental features. Further process optimization studies can now be performed on the basis of the constant specific biomass growth rate.     

Fed-batch sorbitol to sorbose fermentation by A. suboxydans

Batch kinetics for sorbitol to sorbose bioconversion was studied at 20% sorbitol concentration. The culture featured 90% conversion of sorbitol to sorbose in 20 hours. Increasing the initial substrate concentration in the bioreactor decreased the culture specific growth rate. At 40% initial sorbitol concentration no culture growth was observed. The batch kinetics and substrate inhibition studies were used to develop the Mathematical Model of the system. The model parameters were identified using the original batch kinetic data (S _o =20%). The developed mathematical model was adopted to fed-batch cultivation with the exponential nutrient feeding. The fed-batch model was simulated and implemented experimentally. No substrate inhibition was observed in the fed-batch mode and it provided an overall productivity of 12.6?g/l-h. The fed-batch model suitably described the experimentally observed results. The model is ready for further optimization studies.     

Nucleation and growth of microbial lipase crystals from clarified concentrated fermentation broths

Bulk crystallization is emerging as a new industrial operation for protein recovery. Characterization of bulk protein crystallization is more complex than protein crystallization for structural study where single crystals are grown in flow cells. This is because both nucleation and crystal growth processes are taking place while the supersaturation falls. An algorithm is presented to characterize crystallization using the rates of the two kinetic processes, nucleation and growth. The values of these rates allow ready comparison of the crystallization process under different operating conditions. The crystallization, via adjustment to the isoelectric pH of a fungal lipase from clarified fermentation broth, is described for a batch stirred reactor. A maximum nucleation rate of five to six crystals formed per microliter of suspension per second and a high power dependency ( approximately 11) on the degree of supersaturation were found. The suspended protein crystals were found to grow at a rate of up to 15-20 nm/s and also to exhibit a high power dependency ( approximately 6) of growth rate on the degree of supersaturation.     

The tuning of a model-based estimator for the specific growth rate of Candida utilis

Control of microbial conversion processes is frequently inhibited by the infeasibility of measuring important process variables. In order to circumvent this lack of measurements, an accurate or valuable and conveniently measurable on-line hardware measurement can be combined with the balance equations describing the process to obtain estimates of less easily measurable variables. In this article the on-line estimation of the specific growth rate of Candida utilis is evaluated. The observer-based estimator requires a hardware measurement of the biomass during fermentations in conjuction with a model of the process; therefore the Biomass Monitor, giving an on-line measurement of viable biomass, is used in the bioreactor experiments described. The optimal tuning of the estimation for the experimental conditions is described and several alternative adaptations of the design of the estimator are presented. The influence of implemented time intervals for discretization of the estimator on the reliability of the estimated growth rate values is discussed. Additionally, the necessary choice of an initial value of the estimated specific growth rate has proven to be of great importance in practice.     

Kinetic analysis of bioconversion of cellulose in attrition bioreactor

The attrition bioreactor (ABR) combines wet ball milling and enzymatic hydrolysis in one process step. It was found that the ABR did not accelerate enzyme deacti-vation. Interfacial forces, not shear forces, caused the most deactivation. Elimination of the air-liquid interface by covering the reactor substantially increased enzyme stability. A simple exponential kinetic model was tested to predict the cellulose conversion in an ABR. Kinetic parameters were estimated from batch runs performed at various enzyme and substrate concentrations.     

Tropisms of Avena coleoptiles: sine law for gravitropism,exponential law for photogravitropic equilibrium

The quantitative relation between gravitropism and phototropism was analyzed for light-grown coleoptiles of Avena sativa (L.). With respect to gravitropism the coleoptiles obeyed the sine law. To study the interaction between light and gravity, coleoptiles were inclined at variable angles and irradiated for 7 h with unilateral blue light (466 nm) impinging at right angles relative to the axis of the coleoptile. The phototropic stimulus was applied from the side opposite to the direction of gravitropic bending. The fluence rate that was required to counteract the negative gravitropism increased exponentially with the sine of the inclination angle. To achieve balance, a linear increase in the gravitropic stimulus required compensation by an exponential increase in the counteracting phototropic stimulus. The establishment of photogravitropic equilibrium during continuous unilateral irradiation is thus determined by two different laws: the well-known sine law for gravitropism and a novel exponential law for phototropism described in this work.     

In Situ μGISAXS: II. Thaumatin Crystal Growth Kinetic

The formation of thaumatin crystals by Langmuir-Blodgett (LB) film nanotemplates was studied by the hanging-drop technique in a flow-through cell by synchrotron radiation micrograzing-incidence small-angle x-ray scattering. The kinetics of crystallization was measured directly on the interface of the LB film crystallization nanotemplate. The evolution of the micrograzing-incidence small-angle x-ray scattering patterns suggests that the increase in intensity in the Yoneda region is due to protein incorporation into the LB film. The intensity variation suggests several steps, which were modeled by system dynamics based on first-order differential equations. The kinetic data can be described by two processes that take place on the LB film, a first, fast, process, attributed to the crystal growth and its detachment from the LB film, and a second, slower process, attributed to an unordered association and conversion of protein on the LB film.     

Modeling of heat and mass transfer for solid state fermentation process in tray bioreactor

A mathematical model is developed to simulate oxygen consumption, heat generation and cell growth in solid state fermentation (SSF). The fungal growth on the solid substrate particles results in the increase of the cell film thickness around the particles. The model incorporates this increase in the biofilm size which leads to decrease in the porosity of the substrate bed and diffusivity of oxygen in the bed. The model also takes into account the effect of steric hindrance limitations in SSF. The growth of cells around single particle and resulting expansion of biofilm around the particle is analyzed for simplified zero and first order oxygen consumption kinetics. Under conditions of zero order kinetics, the model predicts upper limit on cell density. The model simulations for packed bed of solid particles in tray bioreactor show distinct limitations on growth due to simultaneous heat and mass transport phenomena accompanying solid state fermentation process. The extent of limitation due to heat and/or mass transport phenomena is analyzed during different stages of fermentation. It is expected that the model will lead to better understanding of the transport processes in SSF, and therefore, will assist in optimal design of bioreactors for SSF.     

Optimization of bioreactor using metabolic control analysis approach

A new methodology based on a metabolic control analysis (MCA) approach is developed for the optimization of continuous cascade bioreactor system. A general framework for representation of a cascade bioreactor system consisting of a large number of reactors as a single network is proposed. The kinetic and transport processes occurring in the system are represented as a reaction network with appropriate stoichiometry. Such representation of the bioreactor systems makes it amenable to the direct application of the MCA approach. The process sensitivity information is extracted using MCA methodology in the form of flux and concentration control coefficients. The process sensitivity information is shown to be a useful guide for determining the choice of decision variables for the purpose of optimization. A generalized problem of optimization of the bioreactor is formulated in which the decision variables are the operating conditions and kinetic parameters. The gradient of the objective function to be maximized with respect to all decision variables is obtained in the form of response coefficients. This gradient information can be used in any gradient-based optimization algorithm. The efficiency of the proposed technique is demonstrated with two examples taken from literature: biotransformation of crotonobetaine and alcohol fermentation in cascade bioreactor system.     

Characteristics of glucose uptake by glucose- and NH4-limited grown Penicillium ochrochloron at low, medium and high glucose concentration

Glucose uptake by Penicillium ochrochloron (formerly Penicillium simplicissimum) was studied from 0.01 to 400 mM glucose using chemostat culture and bioreactor batch culture. The characteristics of glucose uptake varied considerably with the conditions of growth, harvest and uptake assay. Glucose-limited grown mycelium showed one saturable transport system [K(S) below 0.01 mM; v(max) 1.1-1.2 mmol (g dry weight)(-1)h(-1)] plus a first order process (permeability P=1.2x10(-7)cm s(-1)). Ammonium-limited grown mycelium showed only one saturable transport system [K(S) 0.3-0.7 mM; v(max) 0.5-0.8 mmol (g dry weight)(-1)h(-1)]. During exponential growth at high glucose concentration (300-400 mM) a first order process was found with a P value of 5.6-9.3x10(-7)cm s(-1). After ammonium exhaustion a second first order phase showed a lower P value (6.1-9.3x10(-8)cm s(-1)). A similar change in permeability was also found after a re-evaluation of published data for Gibberella fujikuroi, Aspergillus niger, Aspergillus awamori and Saccharomycopsis lipolytica. For the first order processes simple diffusion was ruled out as a mechanism for glucose uptake. Glucose uptake by P. ochrochloron was controlled more strongly by metabolism than by transport and was not rate limiting for overflow metabolism.     

Potential of enzymatic kinetic resolution using solid substrates suspension: improved yield, productivity, substrate concentration, and recovery

In the literature the enzymatic kinetic resolution of a suspension of a solid substrate has largely been treated as a conventional kinetic resolution of a fully dissolved substrate. In this paper it is shown that this type of kinetic resolution is different in several important aspects. Quantitative models are developed for two types of such suspension processes. These models are used to compare the merits of these processes with the conventional kinetic resolution process where fully dissolved substrate is used. In the suspension processes the liquid phase concentration of substrate enantiomer that should be converted can be kept close to the maximum value, i.e., the solubility, when process conditions are properly chosen, whereas in a conventional process this concentration gradually decreases. Calculations show that this leads to a productivity that is about 6-fold higher in the suspension processes. Also, for enzymes with a low enantioselectivity, a severalfold increase in yield of remaining enantiopure substrate is predicted compared to the conventional kinetic resolution of dissolved enantiomers. Other potential advantages of using suspension reactions are that the initial substrate concentration may be higher (up to 25% (w/w)) and that the desired remaining substrate may be recovered by simply filtering off the solid crystals. Experimental evidence that these merits can be exploited is only partly given, using the few available examples from the literature.     

An analysis of a trickle-bed bioreactor: carbon disulfide removal

An analysis of the local processes occurring in a trickle-bed bioreactor (TBB) with a first-order bioreaction shows that the identification of the TBB operating regime requires knowledge of the substrate concentration in the liquid phase. If the substrate liquid concentration is close to 0, the rate-controlling step is mass transfer at the gas-liquid interface; when it is close to the value in equilibrium with the gas phase, the controlling step is the phenomena occurring in the biofilm. CS2 removal rate data obtained in a TBB with a Thiobacilii consortia biofilm are analyzed to obtain the mass transfer and kinetic parameters, and to show that the bioreactor operates in a regime mainly controlled by mass transfer. A TBB model with two experimentally determined parameters is developed and used to show how the bioreactor size depends on the rate-limiting step, the absorption factor, the substrate fractional conversion, and on the gas and liquid contact pattern. Under certain conditions, the TBB size is independent of the flowing phases' contact pattern. The model effectively describes substrate gas and liquid concentration data for mass transfer and biodegradation rate controlled processes.     

Growth, metabolic, and antibody production kinetics of hybridoma cell culture: 1. Analysis of data from controlled batch reactors.

A mouse-mouse hybridoma cell line (167.4G5.3) was cultivated in a 1.5-L stirred-tank bioreactor under constant pH and dissolved oxygen concentration. The transient kinetics of cell growth, metabolism, and antibody production were followed by biochemical and flow cytometric methods. The cell-specific kinetic parameters (growth and metabolic rates) as well as cell size were constant throughout the exponential phase. Intracellular protein and RNA content followed a similar trend. Cell growth stopped when the glutamine in the medium was depleted. Glucose could not substitute for glutamine, as glucose consumption ceased after glutamine depletion. Ammonia and lactate production followed closely glutamine and glucose consumption, respectively. Alanine, glutamate, serine, and glycine were produced but other amino acids were consumed. The cells are estimated to obtain about 45% of the total energy from glycolysis, with the balance of the metabolic energy provided by oxidative phosphorylation. The antibody was produced at a constant rate in both the exponential and decline phases of growth. The intracellular antibody content of the cells remained relatively constant during the exponential phase of growth and decreased slightly afterwards.     

Cell growth and differentiation on feeder layers is predicted to be influenced by bioreactor geometry

Tissue function is comprised of a complex interplay between biological and physicochemical rate processes. The design of bioreactors for tissue engineering must account for these processes simultaneously in order to obtain a bioreactor that provides a uniform environment for tissue growth and development. In the present study we consider the effects of fluid flow and mass transfer on the growth of a tissue in a parallel-plate bioreactor configuration. The parenchymal cells grow on a preformed stromal (feeder) layer that secretes a growth factor that stimulates parenchymal stem cell replication and differentiation. The biological dynamics are described by a unilineage model that describes the replication and differentiation of the tissue stem cell. The physicochemical rates are described by the Navier-Stokes and convective-diffusion equations. The model equations are solved by a finite element method. Two dimensionless groups govern the behavior of the solution. One is the Graetz number (Gz) that describes the relative rates of convection and diffusion, and the other a new dimensionless ratio (designated by P) that describes the interplay of the growth factor production, diffusion, and stimulation. Four geometries (slab, gondola, diamond, and radial shapes) for the parallel-plate bioreactor are analyzed. The uniformity of cell growth is measured by a two-dimensional coefficient of variance. The concentration distribution of the stroma-derived growth factor was computed first based on fluid flow and bioreactor geometry. Then the concomitant cell density distribution was obtained by integrating the calculated growth factor concentration with the parenchymal cell growth and unilineage differentiation process. The spatiotemporal cell growth patterns in four different bioreactor configurations were investigated under a variety of combinations of Gz (10(-1), 10(0), and 10(1)) and P(10(-2), 10(-1), 10(0), 10(1), and 10(2)). The results indicate high cell density and uniformity can be achieved for parameter values of P = 0.01, ..., 0.1 and Gz = 0.1, ..., 1.0. Among the four geometries investigated the radial-flow-type bioreactor provides the most uniform environment in which parenchymal cells can grow and differentiate ex vivo due to the absence of walls that are parallel to the flow paths creating slow flowing regions. (c) 1996 John Wiley & Sons, Inc.     

Continuous enantioselective esterification of trans-2-phenyl-1-cyclohexanol using a new Candida rugosa lipase in a packed bed bioreactor

Enantioselective resolution of trans-2-phenyl-1-cyclohexanol (TPCH) by a Candida rugosa lipase, obtained by fermentation in the laboratory, and immobilised on EP100 polypropylene powder has been carried out using isooctane as solvent and propionic acid as esterifying agent. The study have included the utilisation of this biocatalyst in a batch process and the optimisation of the esterification conditions by means of a Box-Hunter-based experimental design. The main variables controlling the process, concentration of acid and alcohol, have been numerically optimised using initial esterification rate as objective function. The optimal concentrations for the batch process were 50 mM for the alcohol and 71 mM for the acid. This esterification reaction kinetics corresponded to a reversible Michaelis-Menten kinetic law for the optimal conditions, which has permitted to select a plug-flow packed bed bioreactor as the most appropriate configuration to minimise the residence time and to avoid shear stress effect on the biocatalyst. The behaviour of the continuous packed bed bioreactor at two different residence times (302 and 582 min) was in accordance with predictions from batch experiments, with slightly deviations (less than 10%). Continuous experiments maintained high values of enantioselectivity (enantiomeric factor was practically 1) and conversion near equilibrium value (35%) when long-time operation was carried out. Besides, long-time stability of biocatalyst has permitted to scale-up the production of enantioenriched (1R,2S)-TPCH propionate to yield gram quantities.     

Intrinsic autotrophic biomass yield and productivity in algae: modeling spectral and mixing-rate dependence

For non-inhibitory irradiances, the rate of algal biomass synthesis was modeled as the product of the algal autotrophic yield Φ(DW) and the flux of photons absorbed by the culture, as described using Beer-Lambert law. As a contrast to earlier attempts, the use of scatter-corrected extinction coefficients enabled the validation of such approach, which bypasses determination of photosynthesis-irradiance (PI) kinetic parameters. The broad misconception that PI curves, or the equivalent use of specific growth rate expressions independent of the biomass concentration, can be extended to adequately model biomass production under light-limitation is addressed. For inhibitory irradiances, a proposed mechanistic model, based on the photosynthetic units (PSU) concept, allows one to estimate a target speed νT across the photic zone in order to limit the flux of photons per cell to levels averting significant reductions in Φ(DW) . These modeled target speeds, on the order of 5-20 m s(-1) for high outdoor irradiances, call for fundamental changes in reactor design to optimize biomass productivity. The presented analysis enables a straightforward bioreactor parameterization, which, in-turn, guides the establishment of conditions ensuring maximum productivity and complete nutrients consumption. Additionally, solar and fluorescent lighting spectra were used to calculate energy to photon-counts conversion factors.