Effect of various process parameters on morphology, rheology, and polygalacturonase production by Aspergillus sojae in a batch bioreactor

The effects of pH, agitation speed, and dissolved oxygen tension (DOT), significant in common fungal fermentations, on the production of polygalacturonase (PG) enzyme and their relation to morphology and broth rheology were investigated using Aspergillus sojae in a batch bioreactor. All three factors were effective on the response parameters under study. An uncontrolled pH increased biomass and PG activity by 27% and 38%, respectively, compared to controlled pH (pH 6) with an average pellet size of 1.69 +/- 0.48 mm. pH did not significantly affect the broth rheology but created an impact on the pellet morphology. Similarly, at constant agitation speed the maximum biomass obtained at 500 rpm and at 30 h was 3.27 and 3.67 times more than at 200 and 350 rpm, respectively, with an average pellet size of 1.08 +/- 0.42 mm. The maximum enzyme productivity of 0.149 U mL-1 h-1 was obtained at 200 rpm with an average pellet size of 0.71 +/- 0.35 mm. Non-Newtonian and pseudoplastic broth rheology was observed at 500 rpm agitation speed, broth rheology exhibited dilatant behavior at the lower agitation rate (200 rpm), and at the medium agitation speed (350 rpm) the broth was close to Newtonian. Furthermore, a DOT range of 30-50% was essential for maximum biomass formation, whereas only 10% DOT was required for maximum PG synthesis. Non-Newtonian shear thickening behavior (n > 1.0) was depicted at DOT levels of 10% and 30%, whereas non-Newtonian shear thinning behavior (n < 1.0) was dominant at 50% DOT. The overall fermentation duration (50-70 h) was considerably shorter compared to common fungal fermentations, revealing the economic feasibility of this particular process. As a result this study not only introduced a new strain with a potential of producing a highly commercially significant enzyme but also provided certain parameters significant in the design and mathematical modeling of fungal bioprocesses.     

Accumulation of organic acids in cultivations of Neisseria meningitidis C

Aiming at the industrial production of serogroup C meningococcal vaccine, different experimental protocols were tested to cultivate Neisseria meningitidis C and to investigate the related organic acid release. Correlations were established between specific rates of acetic acid and lactic acid accumulation and specific growth rate, during cultivations carried out on the Frantz medium in a 13 l bioreactor at 35°C, 0.5 atm, 400 rpm and air flowrate of 2 l min⁻¹. A first set of nine batch runs was carried out: (1) with control of dissolved oxygen (O₂) at 10% of its saturation point, (2) with control of pH at 6.5, and (3) without any control, respectively. Additional fed-batch or partial fed-batch cultivations were performed without dissolved O₂ control, varying glucose concentration from 1.0 to 3.0 g l⁻¹, nine of which without pH control and other two with pH control at 6.5. No significant organic acid level was detected with dissolved O₂ control, whereas acetic acid formation appeared to depend on biomass growth either in the absence of any pH and dissolved O₂ control or when the pH was kept at 6.5. Under these last conditions, lactic acid was released as well, but it did not seem to be associated to biomass growth. A survey of possible metabolic causes of this behavior suggested that N. meningitidis may employ different metabolic pathways for the carbon source uptake depending on the cultivation conditions.     

In situ removal and purification of biosurfactants by automated surface enrichment

Erysipelothrix rhusiopathiae, the causative agent of swine erysipelas, was cultivated in a 5-L stirred and aerated bioreactor under different dissolved oxygen tensions (0%, 5%, and 30% of saturation) for evaluation of the influence of oxygen on cell growth as well as on the production of the main antigenic component of the vaccine against erysipelas, a 64–69 kDa protein (SpaA). The microorganism presented different growth profiles for different aeration conditions. However, at the end of the batch cultivations, similar cell concentrations were obtained under the studied conditions. In order to maximize biomass titers and antigen production, the microorganism was cultivated in fed-batch operation mode under aerobic conditions. Under this condition, there was a fivefold increase in biomass production in comparison to the results attained in batch cultivations. To follow up antigen expression, samples collected during batch cultivations were concentrated and treated with choline for antigen extraction. Antigen expression was then assessed by sodium dodecyl sulfate polyacrylamide gel electrophoresis and by murine immunization tests. It was observed a direct influence of oxygen availability upon antigen expression, which is favored in the presence of oxygen. Analysis of the samples collected throughout the fed-batch process also revealed that antigen production is growth associated.     

Aerobic batch cultivation in micro bioreactor with integrated electrochemical sensor array

Aerobic batch cultivations of Candida utilis were carried out in two micro bioreactors with a working volume of 100 μL operated in parallel. The dimensions of the micro bioreactors were similar as the wells in a 96‐well microtiter plate, to preserve compatibility with the current high‐throughput cultivation systems. Each micro bioreactor was equipped with an electrochemical sensor array for the online measurement of temperature, pH, dissolved oxygen, and viable biomass concentration. Furthermore, the CO₂ production rate was obtained from the online measurement of cumulative CO₂ production during the cultivation. The online data obtained by the sensor array and the CO₂ production measurements appeared to be very reproducible for all batch cultivations performed and were highly comparable to measurement results obtained during a similar aerobic batch cultivation carried out in a conventional 4L bench‐scale bioreactor. Although the sensor chip certainly needs further improvement on some points, this work clearly shows the applicability of electrochemical sensor arrays for the monitoring of parallel micro‐scale fermentations, e.g. using the 96‐well microtiterplate format. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Factors influencing the production of a novel compound, 7,10-dihydroxy-8(E)-octadecenoic acid, by Pseudomonas aeruginosa PR3 (NRRL B-18602) in batch cultures

Pseudomonas aeruginosa PR3 (NRRL B-18602) converts oleic acid to a novel compound, 7,10-dihydroxy-8(E)-octadecenoic acid (DOD). Parameters that included medium volume, cell growth time, gyration speed, pH, substrate concentration, and dissolved oxygen concentration were evaluated for a scale-up production of DOD in batch cultures using Fernbach flasks and a bench-top bioreactor. Maximum production of about 2 g DOD (38% yield) was attained in Fernbach flasks containing 500 ml medium when cells were grown at 28 degrees C and 300 rpm for 16-20 h and the culture was adjusted to pH 7 prior to substrate addition. Increases of medium volume and substrate concentration failed to enhance yield. When batch cultures were initially conducted in a reactor, excessive foaming occurred that made the bioconversion process inoperable. This was overcome by a new aeration mechanism that provided adequate dissolved oxygen to the fermentation culture. Under the optimal conditions of 650 rpm, 28 degrees C, and 40-60% dissolved oxygen concentration, DOD production reached about 40 g (40% yield) in 4.5 L culture medium using a 7-L reactor vessel. This is the first report on a successful scale-up production of DOD.     

Dependence of morphology on agitation intensity in fed-batch cultures of Aspergillus oryzae and its implications for recombinant protein production

We previously reported that, although agitation conditions strongly affected mycelial morphology, such changes did not lead to different levels of recombinant protein production in chemostat cultures of Aspergillus oryzae (Amanullah et al., 1999). To extend this finding to another set of operating conditions, fed-batch fermentations of A. oryzae were conducted at biomass concentrations up to 34 g dry cell weight/L and three agitation speeds (525, 675, and 825 rpm) to give specific power inputs between 1 and 5 kWm(-3). Gas blending was used to control the dissolved oxygen level at 50% of air saturation except at the lowest speed where it fell below 40% after 60-65 h. The effects of agitation intensity on growth, mycelial morphology, hyphal tip activity, and recombinant protein (amyloglucosidase) production in fed-batch cultures were investigated. In the batch phase of the fermentations, biomass concentration, and AMG secretion increased with increasing agitation intensity. If in a run, dissolved oxygen fell below approximately 40% because of inadequate oxygen transfer associated with enhanced viscosity, AMG production ceased. As with the chemostat cultures, even though mycelial morphology was significantly affected by changes in agitation intensity, enzyme titers (AGU/L) under conditions of substrate limited growth and controlled dissolved oxygen of >50% did not follow these changes. Although the measurement of active tips within mycelial clumps was not considered, a dependency of the specific AMG productivity (AGU/g biomass/h) on the percentage of extending tips was found, suggesting that protein secretion may be a bottle-neck in this strain during fed-batch fermentations.     

Proof-of-concept of a novel micro-bioreactor for fast development of industrial bioprocesses

The experimental performance of a novel micro-bioreactor envisaged for parallel screening and development of industrial bioprocesses has been tested in this work. The micro-bioreactor with an internal volume of 4.5 mL is operated under oscillatory flow mixing (OFM), where a controllable mixing and mass transfer rates are achieved under batch or continuous laminar flow conditions. Several batch fermentations with a flocculent Saccharomyces cerevisiae strain were carried out at initial glucose concentrations (S(0)) range of approximately 5-20 g/L and compared to yeast growth kinetics in a stirred tank (ST) bioreactor. Aerobic fermentations were monitored ex situ in terms of pH, DO, glucose consumption, and biomass and ethanol production (wherever applicable). An average biomass production increase of 83% was obtained in the micro-bioreactor when compared with the ST, with less 93.6% air requirements. It also corresponded to a 214% increase on biomass production when compared with growth in a shaken flask (SF) at S(0) = 20 g/L. Further anaerobic fermentations at the same initial glucose concentration ranges gave the opportunity to use state-of-the-art fiber optics technology for on-line and real-time monitoring of this bioprocess. Time profiles of biomass concentration (measured as optical density (OD)) were very similar in the ST bioreactor and in the micro-bioreactor, with a highly reproducible yeast growth in these two scale-down platforms.     

A mathematical description of recombinant yeast

A model was formulated to examine specific experimental data of growth and heterologous product formation with recombinant Saccharomyces cerevisiae while incorporating available literature. The model simulated dry cell weight, glucose, ethanol, dissolved oxygen, human Epidermal Growth Factor (hEGF) production, fraction of recombinant cells, oxygen uptake rate, and carbon dioxide production rate for batch, fed batch, and hollow fiber bioreactor configurations. Nineteen differential equations, 24 analytical equations, and 48 parameters were required. Due to the lack of detailed studies needed for the ADH-II and the TCA enzyme pool, 8 of the 48 parameters were adjustable. Simulation results are presented for verification of the model which successfully described the observed phenomena for the fermentations of S. cerevisiae strain AB103. 1 pYalphaEF-25. Also presented is a statistical analysis of the model's fit and model parameter sensitivity.     

Factors Influencing the Production of a Novel Compound, 7,10-Dihydroxy-8(<Emphasis Type="Italic">E</Emphasis>)-octadecenoic Acid,by <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> PR3 (NRRL B-18602) in Batch Cultures

Pseudomonas aeruginosa PR3 (NRRL B-18602) converts oleic acid to a novel compound, 7,10-dihydroxy-8(E)-octadecenoic acid (DOD). Parameters that included medium volume, cell growth time, gyration speed, pH, substrate concentration, and dissolved oxygen concentration were evaluated for a scale-up production of DOD in batch cultures using Fernbach flasks and a bench-top bioreactor. Maximum production of about 2 g DOD (38% yield) was attained in Fernbach flasks containing 500 ml medium when cells were grown at 28°C and 300 rpm for 16–20 h and the culture was adjusted to pH 7 prior to substrate addition. Increases of medium volume and substrate concentration failed to enhance yield. When batch cultures were initially conducted in a reactor, excessive foaming occurred that made the bioconversion process inoperable. This was overcome by a new aeration mechanism that provided adequate dissolved oxygen to the fermentation culture. Under the optimal conditions of 650 rpm, 28°C, and 40–60% dissolved oxygen concentration, DOD production reached about 40 g (40% yield) in 4.5 L culture medium using a 7-L reactor vessel. This is the first report on a successful scale-up production of DOD. Received: 26 September 2002 / Accepted: 24 October 2002     

Oscillatory behavior of Saccharomyces cerevisiae in continuous culture: I. Effects of pH and nitrogen levels

The appearance of sustained oscillations in bioreactor variables (biomass and nutrient concentrations) in continuous cultures of Saccharomyces cerevisiae indicates the complex nature of microbial systems, the inadequacy of current growth kinetic models, and the difficulties which may arise in bioprocess control and optimization. In this study we investigate continuous bioreactor behavior over a range of operating conditions (dilution rate, feed glucose concentration, feed ammonium concentration, dissolved oxygen, and pH) to determine the process requirements which lead to oscillatory behavior. We present new results which indicate that high feed ammonium concentrations may eliminate oscillations and that under oscillatory conditions ammonium levels are generally low and oscillatory as well. The effects of pH are complex and oscillations were only observed at pH values 5.5 and 6.5; no oscillations were observed at a pH of 4.5. Under our nominal operating conditions (feed glucose concentration 10 g/L, dilution rate 0.145 h(-1), feed ammonium concentration 0.0303M, dissolved oxygen level 50%, pH 5.5, and T = 30 degrees C) we found two possible final bioreactor states depending on the transient used to reach the nominal operating conditions. One of the states was oscillatory and characteristic of oxidative metabolism and the other was nonoscillatory and fermentative.     

Effect of deletion of chitin synthase genes on mycelial morphology and culture viscosity in Aspergillus oryzae

The objective of this study was to quantify the effect of disrupting two chitin synthases, chsB and csmA, on the morphology and rheology during batch cultivation of Aspergillus oryzae. The rheological properties were characterized in batch cultivations at different biomass concentrations (from 3.4-22.5 g kg(-1) biomass) and the power-law model adequately described the rheological properties. In the cultivations there were pellets, clumps, and freely dispersed hyphal elements. The different morphological fractions were quantified using image analysis. The apparent viscosity of the fermentation broth was significantly affected by the biomass concentration, the morphology, and also by pH. The chsB disruption strain had lower consistency index K values for all biomass concentrations investigated, which is a desirable trait for industrial Aspergillus fermentations.     

A fully online sensor-equipped,disposable multiphase microbioreactor as a screening platform for biotechnological applications

A new disposable, multiphase, microbioreactor (MBR; with a working volume of 550 μl) equipped with online sensors is presented for biotechnological screening research purposes owing to its high-throughput potential. Its design and fabrication, online sensor integration, and operation are described. During aerobic cultivation, sufficient oxygen supply is the most important factor that influences growth and product formation. The MBR is a microbubble column bioreactor (μBC), and the oxygen supply was realized by active pneumatic bubble aeration, ensuring sufficient volumetric liquid-phase mass transfer (k _L a) and proper homogenization of the cultivation broth. The μBC was equipped with miniaturized sensors for the pH, dissolved oxygen, optical density and glucose concentration that allowed real-time online monitoring of these process variables during cultivation. The challenge addressed here was the integration of sensors in the limited available space. The MBR was shown to be a suitable screening platform for the cultivation of biological systems. Batch cultivations of Saccharomyces cerevisiae were performed to observe the variation in the process variables over time and to show the robustness and operability of all the online sensors in the MBR.     

Hypoxic fed-batch cultivation of Pichia pastoris increases specific and volumetric productivity of recombinant proteins

High cell density cultivation of Pichia pastoris has to cope with several technical limitations, most importantly the transfer of oxygen. By applying hypoxic conditions to chemostat cultivations of P. pastoris expressing an antibody Fab fragment under the GAP promoter, a 2.5-fold increase of the specific productivity q(P) at low oxygen supply was observed. At the same time the biomass decreased and ethanol was produced, indicating a shift from oxidative to oxidofermentative conditions. Based on these results we designed a feedback control for enhanced productivity in fed batch processes, where the concentration of ethanol in the culture was kept constant at approximately 1.0% (vv(-1)) by a regulated addition of feed medium. This strategy was tested successfully with three different protein producing strains, leading to a three- to sixfold increase of the q(P) and threefold reduced fed batch times. Taken together the volumetric productivity Q(P) increased 2.3-fold.     

Effect of specific oxygen uptake rate on Enterobacter aerogenes energetics: carbon and reduction degree balances in batch cultivations

The effect of oxygen availability on the metabolism of Enterobacter aerogenes NCIMB 10102 was studied through batch fermentations of glucose performed increasing the specific oxygen uptake rate up to 72.7 mmol(O2) C-mol(DW) (-1) x h(-1). The final concentrations of fermentation products of this biosystem (2,3-butanediol, hydrogen, acetoin, formate, acetate, carbon dioxide, ethanol, lactate, succinate, and biomass) were utilized to check the use of simple carbon mass and reduction degree balances for the study of microbial energetics even in batch cultivations.     

Intracellular pH-based controlled cultivation of yeast cells: II. cultivation methodology

Intracellular pH (pH(i)) was measured on-line in a bioreactor using a fluorescent pH(i) indicator, 9-aminoacridine, and controlled fed-batch cultivations of yeast cells based on pH(i) (FB-pH(i)) were performed. In FB-pH(i) cultivations, automated glucose additions were made to the culture in response to culture pH(i). The average ethanol (an-aerobic product) yield was significantly lower [0.12 g g(-1) glucose in fed-batch pH(i) cultivations with 100 ppm glucose additions (FB-pH(i)-100 cultivation) vs. 0.48 g g(-1) glucose in batch] and cell yield was higher (0.54 g g(-1) glucose in FB-pH(i)-100 cultivation vs. 0.3 g g(-1) glucose in batch) compared to batch cultivation. An expression has been derived to calculate changes in pH(i) from measured fluorescence values when the cell concentration increases during growth. Cultivations based on pH(i), performed with different magnitudes of glucose addition (100, 50, and 10 ppm additions), showed that lower magnitudes of glucose addition resulted in lower ethanol yields while cell yield remained unaffected. The ratio of specific oxygen uptake rate to specific glucose uptake rate (OUR/GUR) increased with decreased in magnitude of glucose additions in FB-pH(i) cultivations, suggesting that the culture aerobic state was higher when the magnitude of glucose addition was lower. The average cell productivity in FB-pH(i) cultivations was 29% higher than in batch cultivation. Cells were also cultivated at high OUR conditions, and the results are compared with other cultivations. (c) 1993 John Wiley & Sons, Inc.     

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.     

Bacteriocin production by strain <Emphasis Type="Italic">Lactobacillus delbruecki</Emphasis>i ssp. <Emphasis Type="Italic">bulgaricus</Emphasis> BB18 during continuous prefermentation of yogurt starter culture and subsequent batch coagulation of milk

By screening for bacteriocin-producing lactic acid bacteria of 1,428 strains isolated from authentic Bulgarian dairy products, Lb. bulgaricus BB18 strain obtained from kefir grain was selected. Out of 11 yogurt starters containing Lb. bulgaricus BB18 and S. thermophilus strains resistant to bacteriocin secreted by Lb. bulgaricus BB18 a yogurt culture (S. thermophilus 11A+Lb. bulgaricus BB18) with high growth and bacteriocinogenic activity in milk was selected. Continuous (pH-stat 5.7) prefermentation processes were carried out in milk at 37 degrees C in a 2l MBR bioreactor (MBR AG, Zurich, Switzerland) with an IMCS controller for agitation speed, temperature, dissolved oxygen, CO2 and pH. Prefermented milk with pH 5.7 coagulated in a thermostat at 37 degrees C until pH 4.8-4.9. S. thermophilus 11A and Lb. bulgaricus BB18 grew independently in a continuous mode at similar and sufficiently high-dilution rates (D=1.83 h(-1)-S. thermophilus 11A; D=1.80 h(-1)-Lb. bulgaricus BB18). The yogurt cultures developed in a stream at a high-dilution rate (D=2.03-2.28 h(-1)). The progress of both processes (growth and bacteriocin production) depended on the initial ratio between the two microorganisms. The continuous prefermentation process promoted conditions for efficient fermentation and bacteriocinogenesis of the starter culture during the batch process: strong reduction of the times for bacteriocin production and coagulation of milk (to 4.5-5.0 h); high cell productivity (lactobacilli-4x10(12) CFU ml(-1), streptococci-6x10(12) CFU ml(-1)); high productivity of bacteriocins (4,500 BU ml(-1))-1.7 times higher than the bacteriocinogenic activity of the batch starter culture.     

Reaction engineering studies of acetone-butanol-ethanol fermentation with Clostridium acetobutylicum

Acetone-butanol-ethanol (ABE) fermentation by Clostridium acetobutylicum has been extensively studied in recent years because the organism is recognized as an excellent butanol producer. A parallel bioreactor system with 48 stirred-tank bioreactors on a 12 mL scale was evaluated for batch cultivations of the strictly anaerobic, butanol-producing C. acetobutylicum ATCC 824. Continuous gassing with nitrogen gas was applied to control anaerobic conditions. Process performances of ABE batch fermentations on a milliliter scale were identical to the liter-scale stirred-tank reactor if reaction conditions were identical on the different scales (e.g., initial medium, pH, temperature, specific evaporation rates, specific power input by the stirrers). The effects of varying initial ammonia concentrations (0.1-4.4 g L(-1) ) were studied in parallel with respect to glucose consumption and butanol production of C. acetobutylicum ATCC 824 as a first application example. The highest butanol yield of 33% (mol mol(-1) ) was observed at initial ammonia concentrations of 0.5 and 1.1 g L(-1) . This is the first report on the successful application of a 48 parallel stirred-tank bioreactor system for reaction engineering studies of strictly anaerobic microorganisms at the milliliter scale.     

Pellet morphology, culture rheology and lovastatin production in cultures of Aspergillus terreus

Pellet growth of Aspergillus terreus ATCC 20542 in submerged batch fermentations in stirred bioreactors was used to examine the effects of agitation (impeller tip speed u(t) of 1.01-2.71 ms(-1)) and aeration regimens (air or an oxygen-enriched mixture containing 80% oxygen and 20% nitrogen by volume) on the fungal pellet morphology, broth rheology and lovastatin production. The agitation speed and aeration methods used did not affect the biomass production profiles, but significantly influenced pellet morphology, broth rheology and the lovastatin titers. Pellets of approximately 1200 microm initial diameter were reduced to a final stable size of approximately 900 microm when the agitation intensity was >/=600 rpm (u(t)>/=2.03 ms(-1)). A stable pellet diameter of approximately 2500 microm could be attained in less intensely agitated cultures. These large fluffy pellets produced high lovastatin titers when aerated with oxygen-enriched gas but not with air. Much smaller pellets obtained under highly agitated conditions did not attain high lovastatin productivity even in an oxygen-enriched atmosphere. This suggests that both an upper limit on agitation intensity and a high level of dissolved oxygen are essential for attaining high titers of lovastatin. Pellet size in the bioreactor correlated equally well with the specific energy dissipation rate and the energy dissipation circulation function. The latter took into account the frequency of passage of the pellets through the high shear regions of the impellers. Pellets that gave high lovastatin titers produced highly shear thinning cultivation broths.     

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