Toward intensifying design of experiments in upstream bioprocess development: An industrial Escherichia coli feasibility study

In this study, step variations in temperature, pH, and carbon substrate feeding rate were performed within five high cell density Escherichia coli fermentations to assess whether intraexperiment step changes, can principally be used to exploit the process operation space in a design of experiment manner. A dynamic process modeling approach was adopted to determine parameter interactions. A bioreactor model was integrated with an artificial neural network that describes biomass and product formation rates as function of varied fed‐batch fermentation conditions for heterologous protein production. A model reliability measure was introduced to assess in which process region the model can be expected to predict process states accurately. It was found that the model could accurately predict process states of multiple fermentations performed at fixed conditions within the determined validity domain. The results suggest that intraexperimental variations of process conditions could be used to reduce the number of experiments by a factor, which in limit would be equivalent to the number of intraexperimental variations per experiment. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1343–1352, 2016     

Cometabolic degradation of trichloroethylene in a bubble column bioscrubber

A bubble column bioreactor was used as bioscrubber to carry out a feasibility study for the cometabolic degradation of trichloroethylene (TCE). Phenol was used as cosubstrate and inducer. The bioreactor was operated like a conventional chemostat with regard to the cosubstrate and low dilution rates were used to minimize the liquid outflow. TCE degradation measurements were carried out using superficial gas velocities between 0.47and 4.07 cm s(-1) and TCE gas phase loads between 0.07 and 0.40 mg L(-1) Depending on the superficial gas velocity used, degrees of conversion between 30% and 80% were obtained. A simplified reactor model using plug flow for the gas phase, mixed flow for the liquid phase, and pseudo first order reaction kinetics for the conversionof TCE was established. The model is able to give a reasonable approximation of the experimental data. TCE degradation at the used experimental conditions is mainly limited by reaction rate rather than by mass transfer rate. The model can be used to calculate the reactor volume and the biomass concentration for a required conversion. (c) 1995 John Wiley & Sons Inc.     

Numerical simulation of global hydro-dynamics in a pulsatile bioreactor for cardiovascular tissue engineering

Previous numerical simulations of the hydro-dynamic response in the various bioreactor designs were mostly concentrated on the local flow field analysis using computational fluid dynamics, which cannot provide the global hydro-dynamics information to assist the bioreactor design. In this research, a mathematical model is developed to simulate the global hydro-dynamic changes in a pulsatile bioreactor design by considering the flow resistance, the elasticity of the vessel and the inertial effect of the media fluid in different parts of the system. The developed model is used to study the system dynamic response in a typical pulsatile bioreactor design for the culturing of cardiovascular tissues. Simulation results reveal the detailed pressure and flow-rate changes in the different positions of the bioreactor, which are very useful for the evaluation of hydro-dynamic performance in the bioreactor designed. Typical pressure and flow-rate changes simulated agree well with the published experimental data, thus validates the mathematical model developed. The proposed mathematical model can be used for design optimization of other pulsatile bioreactors that work under different experimental conditions and have different system configurations.     

Toluene degradation kinetics for planktonic and biofilm-grown cells of Pseudomonas putida 54G

Toluene degradation kinetics by biofilm and planktonic cells of Pseudomonas putida 54G were compared in this study. Batch degradation of (14)C toluene was used to evaluate kinetic parameters for planktonic cells. The kinetic parameters determined for toluene degradation were: specific growth rate, mu(max) = 10.08 +/- 1.2/day; half-saturation constant, K(S) = 3.98 +/- 1.28 mg/L; substrate inhibition constant, K(I) = 42.78 +/- 3.87 mg/L. Biofilm cells, grown on ceramic rings in vapor phase bioreactors, were removed and suspended in batch cultures to calculate (14)C toluene degradation rates. Specific activities measured for planktonic and biofilm cells were similar based on toluene degrading cells and total biomass. Long-term toluene exposure reduced specific activities that were based on total biomass for both biofilm and planktonic cells. These results suggest that long-term toluene exposure caused a large portion of the biomass to become inactive, even though the biofilm was not substrate limited. Conversely, specific activities based on numbers of toluene-culturable cells were comparable for both biofilm and planktonically grown cultures. Planktonic cell kinetics are often used in bioreactor models to model substrate degradation and growth of bacteria in biofilms, a procedure we found to be appropriate for this organism. For superior bioreactor design, however, changes in cellular activity that occur during biofilm development should be investigated under conditions relevant to reactor operation before predictive models for bioreactor systems are developed. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 53: 535-546, 1997.     

Bioreactor for cultivation of red beet hairy roots and in situ recovery of primary and secondary metabolites

To arrive at an appropriate bioreactor design and in situ recovery of the products, red beet hairy roots were used as a model system where the levels of betalain pigments (betacyanins and betaxanthins) were followed as secondary metabolite and the peroxidase enzyme as primary metabolite. Medium volume and other kinetic parameters were found to play significant roles by way of directly affecting the biomass yield rather than a specific metabolite. The hydrodynamic stress created on the roots by large culture volume could be minimized by pulse‐feeding of medium in shake‐flasks; and by separating the biomass chamber from the aerated medium reservoir in circulatory fed‐batch bioreactor. Accordingly the bioreactor was modified to provide anchorage and air‐enrichment chamber which resulted in higher formation of both the metabolites than in shake‐flasks. Various down‐stream processing aspects such as in situ release of pigments by non‐destructive methods, followed by adsorption through a column and recovery by desorption were optimized for betalains. A strategy for simultaneous recovery of pigment and peroxidase was worked out using aqueous two phase extraction (ATPE).     

Heat transfer simulation in solid substrate fermentation

A mathematical model was developed and tested to simulate the generation and transfer of heat in solid substrate fermentation (SSF). The experimental studies were realized in a 1-L static bioreactor packed with cassava wet meal and inoculated with Aspergillus niger. A simplified pseudohomogeneous monodimensional dynamic model was used for the energy balance. Kinetic equations taking into account biomass formation (logistic), sugar consumption (with maintenance), and carbon dioxide formation were used. Model verification was achieved by comparison of calculated and experimental temperatures. Heat transfer was evaluated by the estimation of Biot and Peclet heat dimensionless numbers 5-10 and 2550-2750, respectively. It was shown that conduction through the fermentation fixed bed was the main heat transfer resistance. This model intends to reach a better understanding of transport phenomena in SSF, a fact which could be used to evaluate various alternatives for temperature control of SSF, i.e., changing air flow rates and increasing water content. Dimensionless numbers could be used as scale-up criteria of large fermentors, since in those ratios are described the operating conditions, geometry, and size of the bioreactor. It could lead to improved solid reactor systems. The model can be used as a basis for automatic control of SSF for the production of valuable metabolites in static fermentors.     

Microbial production of polyhydroxybutyrate with tailor-made properties: an integrated modelling approach and experimental validation

The microbial production of polyhydroxybutyrate (PHB) is a complex process in which the final quantity and quality of the PHB depend on a large number of process operating variables. Consequently, the design and optimal dynamic operation of a microbial process for the efficient production of PHB with tailor-made molecular properties is an extremely interesting problem. The present study investigates how key process operating variables (i.e., nutritional and aeration conditions) affect the biomass production rate and the PHB accumulation in the cells and its associated molecular weight distribution. A combined metabolic/polymerization/macroscopic modelling approach, relating the process performance and product quality with the process variables, was developed and validated using an extensive series of experiments and measurements. The model predicts the dynamic evolution of the biomass growth, the polymer accumulation, the consumption of carbon and nitrogen sources and the average molecular weights of the PHB in a bioreactor, under batch and fed-batch operating conditions. The proposed integrated model was used for the model-based optimization of the production of PHB with tailor-made molecular properties in Azohydromonas lata bacteria. The process optimization led to a high intracellular PHB accumulation (up to 95% g of PHB per g of DCW) and the production of different grades (i.e., different molecular weight distributions) of PHB.     

Kinetics of methanogenic degradation of phenol by activated-carbon-supported and granular biomass

A continuous-feed recycle bioreactor was used to study the kinetics of methanogenic degradation of phenol at 35 degrees C by bacteria supported on a bed of granular activated carbon (GAC). At dilution rates well above the growth rate of the culture, the cells not only populated the GAC, but also formed a layer of granular biomass. This layer was stabilized by the presence of the GAC, and accounted for over half of the phenol-degrading activity in the bioreactor. The specific phenol degradation rates for GAC-attached biomass, suspended biomass, and granular biomass were all in the range 0.15 to 0.22 mg phenol/mg volatile solids per day as measured under pseudo-steady-state conditions. (c) 1992 John Wiley & Sons, Inc.     

Mathematical modeling of a horizontal tubular loop bioreactor for biomass production from natural gas

In this study, mathematical modeling of a horizontal tubular loop bioreactor (HTLB) was considered for biomass production from natural gas. Gas inlet segments, static mixers, gas–liquid separator, and liquid pump of the HTLB were mathematically modeled according to the ideal stirred reactors, and the horizontal parts, riser, and down-comer sections were modeled in line with the dispersed plug-flow reactors as well. The set of ordinary and partial differential equations were coupled to calculate the oxygen and methane concentrations in the liquid through the length of bioreactor and time. Moreover, the tuned kinetic and hydrodynamic parameters of SCP process in the HTLB were determined based on the mathematical model at various operational conditions. The model was validated by considering experimental dissolved oxygen, methane, and biomass concentrations in liquid at different ratios of air to methane and liquid flow rates. The results showed satisfactory agreement between the developed model and the experimental data.     

Optimisation of a cost-effective culture medium for the large-scale production of Bacillus subtilis UTB96

Fermentation optimisation to achieve high biomass and high efficiency of a biocontrol product is millstone in biocontrol sciences. Here, a Placket–Burman design used for comparison and screening of some environmental factors that are effective on bacterial biomass of Bacillus subtilis UTB96. A response surface methodology used to determine the optimal points for three factors including pH, temperature and C/N ratio where the biomass is high. The results of response surface methodology application showed that the optimum conditions for maximum production of biomass in the medium occurs at pH 7, temperature 30?°C and C/N ratio of 23:1. Bacteria derived from optimised conditions either from laboratory or semi-industrial bioreactors, showed a considerable increase in biomass and also, their antagonistic activity against Phytophthora drechsleri in a plate assay. However, optimisation of culture medium in a laboratory bioreactor decreased the antagonistic activity against Aspergillus flavus. Application of the optimised culture medium in both semi-industrial and laboratory bioreactors reduced the length of the lag phase of bacterial growth.     

Production of somatic embryos in a helical ribbon impeller bioreactor

Embryogenic cultures of a transformed Eschscholtzia californica cell line were carried out in a 11-L helical ribbon impeller bioreactor operated under various conditions to evaluate the performance of this equipment for somatic embryo (SE) production. All bioreactor cultures produced SE suspensions with maximum concentrations at least comparable to those obtained from flask control cultures ( approximately 8-13 SE . mL(-;1)). However, an increase of the mixingspeed, from 60 to 100 rpm, and low sparging rate ( approximately 0.05 VVM, k(L) a approximately 6.1 h(-;1)) for dissolved oxygen concentration (DO) control yielded poorer quality embryogenic cultures. The negative effects on SE production were attributed mainly to the low but excessive shear experienced by the embryogenic cells and/or embryoforming aggregates. High DO ( approximately 60% of air saturation) conditions favored undifferentrated biomass production and high nutrient uptake rates at the expense of the slower SE differentiation process in both flask and bioreactor cultures. Too low DO (-5-10%) inhibited biomass and SE production. The best production of SE ( approximately 44 SE . mL(-1) or approximately 757 SE . g dw(-1) . d(-1)) was achieved by operating the bioreactor at 60 rpm while controlling DO at approximately 20%by surface oxygenation only (0.05 VVM, k(L) a approximately 1.4 h(-;1)). This production was found to be a biomass production/growth-associated process and was mainly limited by the availability of extracellular phosphate, magnesium, nitrogen salts, and carbohydrates. (c) 1994 John Wiley & Sons, Inc.     

Production of carbonyl reductase by Metschnikowia koreensis

A new strain of the yeast Metschnikowia koreensis was grown in shake flasks and a stirred bioreactor for the production of carbonyl reductase. The optimal conditions in the bioreactor for maximizing the biomass specific activity of the enzyme were found to be: a medium composed of glucose (20 g/L), peptone (5 g/L), yeast extract (5 g/L) and zinc sulfate (0.3g/L); the pH controlled at 7; the temperature controlled at 25 °C; an agitation speed of 500 rpm; and an aeration rate of 0.25 vvm. In the bioreactor, a biomass specific enzyme activity of 115.6 U/gDCW was obtained and the maximum biomass concentration was 15.3 gDCW/L. The biomass specific enzyme activity obtained in the optimized bioreactor culture was 11-fold higher than the best result achieved in shake flasks. The bioreactor culture afforded a 2.7-fold higher biomass concentration than could be attained in shake flasks.     

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.     

Biodegradation of benzene,toluene, ethylbenzene,and o-xylene by a coculture of Pseudomonas putida and Pseudomonas fluorescens immobilized in a fibrous-bed bioreactor

A fibrous-bed bioreactor containing the coculture of Pseudomonas putida and P. fluorescens immobilized in a fibrous matrix was developed to degrade benzene (B), toluene (T), ethylbenzene (E), and o-xylene (X) in synthetic waste streams. The kinetics of BTEX biodegradation by immobilized cells adapted in the fibrous-bed bioreactor and free cells grown in serum bottles were studied. In general, the BTEX biodegradation rate increased with increasing substrate concentration and then decreased after reaching a maximum, showing substrate-inhibition kinetics. However, for immobilized cells, the degradation rate was much higher than that of free cells. Compared to free cells, immobilized cells in the bioreactor tolerated higher concentrations (>1000 mg l⁻¹) of benzene and toluene, and gave at least 16-fold higher degradation rates for benzene, ethylbenzene, and o-xylene, and a 9-fold higher degradation rate for toluene. Complete and simultaneous degradation of BTEX mixture was achieved in the bioreactor under hypoxic conditions. Cells in the bioreactor were relatively insensitive to benzene toxicity; this insensitivity was attributed to adaptation of the cells in the bioreactor. Compared to the original seeding culture, the adapted cells from the fibrous-bed bioreactor had higher specific growth rate, benzene degradation rate, and cell yield when the benzene concentration was higher than 100 mg l⁻¹. Cells in the fibrous bed had a long, slim morphology, which is different from the normal short-rod shape found for suspended cells in solution.     

Unstructured models for suspension cultures of Taxus media cells in a bioreactor under substrate-sufficient conditions

For a better understanding of the simulation, optimization, and process control in cell cultures, good kinetic models are necessary for large scale plant cell culture. In this paper, the systematic kinetics of taxol production by Taxus media cell suspension cultures in a stirred 15-L bioreactor under substrate-sufficient conditions and the absence of inducer intervention were studied. A kinetic model of cell growth was established by logistic equation, and kinetic unstructured models of substrate consumption, product synthesis and rheological behavior were constituted, which incorporated energy spilling. These models were verified by comparing the simulation results with those obtained experimentally. These results showed that energy spilling was a key factor that must be considered in constructing unstructured kinetic models of Taxus media cell suspension cultures in a stirred bioreactor under substrate-sufficient conditions. Besides, an optimized operation measure of decreasing energy spilling was proposed. An increase of 17.64% in cell biomass and 14.88% in taxol concentration were obtained when the strategy of limiting added carbon several times was experimentally implemented in a 15-L bioreactor. Results demonstrated that these established models should be helpful in the process prediction and operation optimization to guide the production and amplification of Taxus media cell suspension cultures in a bioreactor.     

Modeling and optimization of hairy root growth in fed‐batch process

This article proposes a feeding strategy based on a kinetic model to enhance hairy roots growth. A new approach for modeling hairy root growth is used, considering that there is no nutrient limitation thanks to an appropriate feeding, and the intracellular pools are supposed to be always saturated. Thus, the model describes the specific growth rate from extracellular concentration of the major nutrients and nutrient uptakes depend on biomass growth. An optimized feeding strategy was determined thanks to the model to maintain the major nutrient levels at their optimum assuming optimal initial concentrations. The optimal feed rate is computed in open loop using kinetic model prediction or in closed loop using conductivity measurements to estimate biomass growth. Datura innoxia was chosen as the model culture system. Shake flask cultures were used to calibrate the model. Finally, cultures in bioreactor were performed to validate the model and the control laws. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Using CFD simulations and statistical analysis to correlate oxygen mass transfer coefficient to both geometrical parameters and operating conditions in a stirred-tank bioreactor

Optimization of a bioreactor design can be an especially challenging process. For instance, testing different bioreactor vessel geometries and different impeller and sparger types, locations, and dimensions can lead to an exceedingly large number of configurations and necessary experiments. Computational fluid dynamics (CFD), therefore, has been widely used to model multiphase flow in stirred-tank bioreactors to minimize the number of optimization experiments. In this study, a multiphase CFD model with population balance equations are used to model gas–liquid mixing, as well as gas bubble distribution, in a 50 L single-use bioreactor vessel. The vessel is the larger chamber in an early prototype of a multichamber bioreactor for mammalian cell culture. The model results are validated with oxygen mass transfer coefficient (k_La) measurements within the prototype. The validated model is projected to predict the effect of using ring or pipe spargers of different sizes and the effect of varying the impeller diameter on k_La. The simulations show that ring spargers result in a superior k_La compared to pipe spargers, with an optimum sparger-to-impeller diameter ratio of 0.8. In addition, larger impellers are shown to improve k_La. A correlation of k_La is presented as a function of both the reactor geometry (i.e., sparger-to-impeller diameter ratio and impeller-to-vessel diameter ratio) and operating conditions (i.e., Reynolds number and gas flow rate). The resulting correlation can be used to predict k_La in a bioreactor and to optimize its design, geometry, and operating conditions.     

Optimal parametric sensitivity control for the estimation of kinetic parameters in bioreactors

In this paper the well-known problem of optimal input design is considered. In particular, the focus is on input design for the estimation of kinetic parameters in bioreactors. The problem is formulated as follows: given the model structure (f,g), which is assumed to be affine in the input, and the specific parameter of interest theta;(k) find a feedback law that maximizes the sensitivity of the model output to the parameter under different flow conditions in the bioreactor and, possibly, minimize the input or state costs. Analytical solutions to these problems are presented. As an example a bioreactor with a biomass that grows according to the well-known Monod kinetics is considered.     

Neural network modeling of the light profile in a novel photobioreactor

An artificial neural network (ANN) was implemented to model the light profile pattern inside a photobioreactor (PBR) that uses a toroidal light arrangement. The PBR uses Tequila vinasses as culture medium and purple non-sulfur bacteria Rhodopseudomonas palustris as biocatalyzer. The performance of the ANN was tested for a number of conditions and compared to those obtained by using deterministic models. Both ANN and deterministic models were validated experimentally. In all cases, at low biomass concentration, model predictions yielded determination coefficients greater than 0.9. Nevertheless, ANN yielded the more accurate predictions of the light pattern, at both low and high biomass concentration, when the bioreactor radius, the depth, the rotational speed of the stirrer and the biomass concentration were incorporated in the ANN structure. In comparison, most of the deterministic models failed to correlate the empirical data at high biomass concentration. These results show the usefulness of ANNs in the modeling of the light profile pattern in photobioreactors.