Appl Microbiol Biotechnol (2005) 68: 425–435

DOI 10.1007/s00253-005-0003-0


F. R. Schmidt

Optimization and scale up of industrial fermentation processes

Received: 8 March 2005 / Revised: 12 April 2005 / Accepted: 15 April 2005 / Published online: 7 July 2005
# Springer-Verlag 2005

Abstract To increase product yields and to ensure con- up strategies while considering future technologies, with
sistent product quality, key issues of industrial fermen- emphasis on Escherichia coli as one of the most com-
tations, process optimization and scale up are aimed at monly fermented organisms.
maintaining optimum and homogenous reaction conditions
minimizing microbial stress exposure and enhancing met-
abolic accuracy. For each individual product, process and Introduction
facility, suitable strategies have to be elaborated by a com-
prehensive and detailed process characterization, identifi- The scale up of fermentation processes as a central prob-
cation of the most relevant process parameters influencing lem in biotechnology was first recognized and described
product yield and quality and their establishment as scale- during industrial penicillin production at the beginning
up parameters to be kept constant as far as possible. Phys- of the 1940s. At present, several aspects of this subject are
ical variables, which can only be restrictedly kept constant supported as joint research projects by European and na-
as single parameters, may be combined with other pertinent tional research foundations.
parameters to appropriate mathematical groups or dimen- Fermentation scale up is aimed at the manufacture of
sionless terms. Process characterization is preferably based larger product quantities, if at all possible, with a simul-
on real-time or near real-time data collected by in situ and taneous increase or at least consistency of specific yields
on-line measurements and may be facilitated by supportive and product quality. As will be illustrated in the next sec-
approaches and tools like neural network based chemo- tions, the changed geometric and physical conditions in
metric data analysis and modelling, clarification of the larger scales, however, lead to a less favourable mixing
mixing and stream conditions through computational fluid behaviour and to impaired physiological reaction condi-
dynamics and scale-down simulations. However, as fer- tions which in turn may lead to a decreased process con-
mentation facilities usually are not strictly designed ac- stance and reproducibility, to reduced specific yields and to
cording to scale-up criteria and the process conditions in an increase in unwanted side products and thus ultimate-
the culture vessels thus may differ significantly and since ly to a diminished batch-to-batch consistency and product
any strategy and model can only insufficiently consider and quality, key issues in industrial production processes.
reflect the highly complex interdependence and mutual
interaction of fermentation parameters, successful scale up
in most cases is not the result of a conclusive and straight- Scale up related problems
lined experimental strategy, but rather will be the outcome
of a separate process development and optimization on Reduced mixing quality and enhanced stress exposure
each scale. This article gives an overview on the problems
typically coming along with fermentation process optimi- The problem of reduced mixing quality in larger scales is
zation and scale up, and presents currently applied scale- aggravated with increasing vessel sizes: the opposite sub-
strate and oxygen gradients along the vessel height which
are formed as a result of the conventional fermenter design
F. R. Schmidt (*) according to which substrate feed usually occurs from the
Sanofi-Aventis Deutschland, Biocenter H 780, top and aeration from the bottom, are more pronounced in
Industriepark Höchst, larger reactors due to (1) longer distances to be covered
65926 Frankfurt, Germany
e-mail: leading to larger substrate and oxygen depletion zones, (2)
Tel.: +49-69-3056757 larger volumes of culture broth to be stirred and therewith
Fax: +49-69-30516371 longer mixing times and (3) stronger hydraulic pressure

(2) a deceleration of the metabolic One of the most important consequences of stress-induced speed. coli hirudin in po- 15. a simultaneous oxygen limi- tation further induces the formation of ethanol.g. 2003) only partially protects the cell against glucose oscillations favour plasmid stability and recom- detrimental stress effects. A prioritized goal of process optimization and scale up teins. curacy by minimizing microbial stress exposure. poration of amino acids into native and recombinant pro. 2000. larly in larger scales. stress-triggered proves the physiological conditions and the metabolic ac- changes in glycolysis. 2001) with numbers of up to 1. resulting in zonal overheating. the lower mixing genetic background of the host (details discussed by Friehs rates in large scales thus lead to the formation of zones with 2004. misfolding and aggregation of heat-sensitive proteins (Mogk 2003). for Escherichia coli at 30 mg/l) result in acetate overpro- duction (overflow metabolism). 2000. citrate cycle and of pathways in. sitions 29 and 59. following subsections. growth rates and substrate stress genes (a survey on bacterial stress proteins is given by concentrations. whereas those at the bottom are exposed to glucose thus a cause for an increase in by-products at the expense starvation. Plasmid stability is influenced by the plasmid and surplus heat generated by agitation and metabolic properties. 14. the resulting shifts in the To identify process-specific stress factors and to understand amino acid pool in turn lead. Eq. Plasmid stability formiate. Table 1) (discussed in more detail by Larsson et al. through reduction of temperature.g. Excess glucose concentrations (threshold value of the desired main product yields in larger scales. 1997). 1999. The subsequent activation of parameters like temperature. stead of isoleucin into recombinant E. For instance. simultaneously a more precise replica- tion and amino acid incorporation. and Kiick et al. 1999). including size and nucleotide sequence. analysed in detail. Amino acid misincorporations are tations. Enfors et al. by the processes. which ultimately reduce growth to the separation of their replication and expression from and productivity and increase by-product formation. (2002) report the incorporation of β-methylnorleucin in- . A concept to render plasmid upon heat exposure (Umakoshi et al. more difficult to control and less easy to location of proteins and membranes have been observed be maintained in larger scales. Further possible mea- sures to enhance the accuracy of metabolic processes are (1) Metabolic shifts a reduction the generation time and generation numbers throughout the process. to amino acid various physical and physiological parameters have to be misincorporations in freshly synthesized proteins. Combined with a decreased transportation tion stage.426 gradients influencing the oxygen transfer rate (OTR. Even though translation processes Process characterization are considered to be generally performed quite precisely (Weickert and Apostol 1998). (1997) concentrations and simultaneously suffer from oxygen limi. despite activation of binant protein production rate. An essential prerequisite for high product yields particu- The produced acids can become reassimilated in oxygen.000 in the expression phase and. Enfors et al. atively influenced. Castan and Enfors 2001). 1998). Plasmid stability and plasmid numbers are thus neg- et al. A misincorporation of norvalin instead 1996. conditions. 1998. development more stress-resistant microbial strains. Xu et al. mids. in which cultures pass a higher num- rich zones. due constant metabolic shifts. Thiry and Cingolani 2002) as well as by process enhanced stress conditions. but in any case first lead to a temporary acid. of leucin and methionine into human recombinant haemo- Cells at the fermenter top are exposed to excess glucose globin in E. hydrogen. detrimental metabolites ter cells. lactate and succinate (mixed acid fermentation. An overview on analytical methods ularly in phases of high protein production rates which are currently applied and yet in development is given in the characterized by amino acid shortages. “wobbling” of tRNA). the physiological responses to the vessel specific physical racy of the translation machinery (unspecificity of amino. Bylund et al. and (3) the metabolic shifts affecting product quality is the misincor. coli dnaK and clpB. 2001). ber of generations due to larger culture broth volumes and ification of the microenvironment. Bylund et al. the replication of which can be induced separately tinuous activation and shut down of the corresponding from growth in the desired fermentation phase (e. 1999. is the stable propagation of plasmids to daugh- and elimination of carbon dioxide. The evidently stability and expression rates more independent from such unavoidable detrimental effects of the repeated and cyclic physiological influences is the application of runaway plas- passing of different stress zones and the subsequent con. due to the inherent inaccu. enabling copy physiology (Schweder et al. the reasons for which are complex and not yet fully thus consists of an appropriate process design which im- clarified: according to current knowledge.coli has been published by Apostol et al. (2001). 1999. Lin and Neubauer (2000) show that rapid Kwint et al. partic. whereas high glucose con- heat stress genes like E. volved in anaerobic and mixed acid fermentation lead to shifts in the coupled amino acid biosynthetic pathways (Fenton et al. Nordstrom and Uhlin 1992). Muramatsu et al. the mutual influences and interactions of the acyl-tRNA synthases. later eventually also of longer inoculation chains from the cell bank to the produc- larger regions. e. the growth phase. reducing centrations diminish plasmid stability (Neubauer et al. metabolic changes and damages such as trans. Ansorge stress genes are believed to lead to a completely altered and Kula 2000.

2003) are cur- 2004. 1999). cell several parameters simultaneously have been established viability. 1994. 2003. 2002). as shown for the The on-line connection of mass spectrometric. 1999. Multiparameter flow cytometry permits. or (3) measure- quotient (RQ. further discussion and refer. NIR. 427 Analysis of physiological parameters tation at 350 nm and the fluorescence measurements at 450 nm (more details and literature in Vaidyanathan 1999). Matz and Lennemann 1996). 2004. mass spectroscopy (Buchholz of capacitance measuring electrodes (Aber Instruments et al. van der Merbel 1997) ed number of reactants is fixed in smallest space or are and flow injection analysis (FIA. Solle et al. (2) near-infrared spectroscopy (NIR. Hall et al. Established analytical ments in infrared (Pollard et al. tained signals and data through chemometric modelling out prior sampling and filtration has been presented by (Boehl et al. increasing important role (Ferreira et al. by refractional 2001). (2003). e. membrane potential. Unscrambler 9. 2002). cell viability. 1993. yield and quality and to change the according set points and pounds (Vaidyanathan et al. Hammond and and the composition of the exhaust gas. Schügerl et 2. besides the pH as discussed. For (e. 1998) and electronic tongues (Turner et al. giving information Brooks 1992) for the measurement of nutrient and product about respiratory states as indicated. biomass. Kell et al. 1990). ences given by Vaidyanathan et al. Rocha and Ferreira 2002. biochemical and physiological pro- by gel-embedded biocatalysts. hybridizing techniques. the activity of which can cess parameters and to design the according process mod- be measured by means of pH changes or by fluorophores els.0. also the employment of various analytical procedures monitoring analysis of the cellular DNA. maceutical natural product screening. Broader applicable are physical op- todes enabling the simultaneous measurement and quanti- fication of several parameters and metabolites through Process optimization and scale up continuous scans or excitations and subsequent absorption and scattering measurements within defined wavelength Process control ranges like (1) 2D fluorescence spectroscopy (Boehl et al. Norway) seem to gain an their robustness. In general. 2003. Trelea et al. Table 1). wavelength spectra (Vaidyanathan et al. as cepts practically applicable for industrial purposes could illustrated in the next section. An on-line FIA For analysis. 1991. To gain a picture 2001. cell development stages (Hewitt and Nebe-von Caron 2001. Neils et al. of suitable strategies for process optimization scale up. 2001). chemically as by ion ex. see Turner 1994). Hooked up to expert systems. Despite eters are preferably performed by optical probes (optodes). 2004) as comprehensive as possible by enhancing the amount of or flow cytometry. working either on a physical base. membranes in a generated electric field. Wolfbeis 2002.500-nm scan in the antibiotic partial oxygen and carbon dioxide pressures in culture broth fermentation at Pfizer (Hammond 1992. as in combination with robust automated sampling and sam. onto which the need- 1999. elec- determination of NAD(P)H concentrations through exci. 2001). 1995. intracellular pH. brought together in microchannels and the analytes are Tothill et al. biochips (Enfors et al. The application of the hitherto employed enzymes ing physiological drifts and their influences on product has been restricted mostly to the analysis of sugar com. Farinas et al. 2001. Collins et al. multi-channel arrays with parallel in addition to the analysis of usual metabolites. RNA and protein content. this concept will be facilitated by a further miniaturization Ex situ methods can be designed as real-time procedures of the sensors and chips employed (Moser et al. van de Merbel et separated in microcapillars (lab-on-a-chip technology. by the respiratory as well as side product concentrations. 1999). Huang et al. 2002) to avoid falsified or gappy the human olphactory sensoric system. 2003. The realization of data due to time differences between sampling and analysis. Nishi et al. metabolites and products. Solle et al. trochemical and multi-array gas sensors by the neuronal net . neural network tools Arndt and Hitzmann (2004) and Kleist et al. whereby the most fre. hidden relations between a multitude change reactions (Spichiger-Keller 1997) or enzymatically of various physical. process profiles. Among the physiologically most relevant parameters are.g. interpretation and correlation of the ob- glucose analysis method of the native culture broth with. However. chromatog. Matz and Lennemann1996. In this way. By means raphy (van de Merbel 1997). Hewitt et al. it has been achieved with biochips developed for the phar- ple preparation methods like ultrafiltration (Schaefer et al. even non-obvious.g. 2000. optimize process control and to facilitate the identification 1997).g. 2001). inherent limitations (Chen and Rollins 2000. 1991). the Vaccari et al. despite intensive research. cell size and and new concepts like artificial noses (Dickinson et al. they allow immediate (chemo. highly complex and sor principles. 1999). if possible. or visible methods and tools are colorimetric procedures. viable cell counts can be performed by seizing electrophoretic methods (Klyushnichenko 2004. which has been concentrations of substrates.. neural network tools help to quent applications are reported for glucose (Tothill et al. analysable substances. al. data are. only a few con. Griffiths and Hall and short-term reactions to process deviations by anticipat- 1993). ultraviolet. 2003). Macaloney et al.and biosensors.g. Camo. Gabig-Ciminska et al. 18. recognizing and revealing non-linear. in situ measurements of biological param. 1994). Stark et al. 1997. Schweder et al. adequately trained neural networks are capable of or spectroscopic measurements (for further physical sen. 1996. 2004. preferably captured as real-time values allowing the analysis of a large number of analytes by by in situ on-line measurements as occurring for physical cross-talking semispecific sensors which act in analogy to parameters (Harms et al. UK). enzymatic and Ltd. the employed as a 700. have been developed. the electric capacitance and conductivity of cells with intact 1989. Eq. the rently tried to be integrated into fermentation technology. e. e.

FG=volumetric gas flow rate. n=stirrer speed. V=volume. VR=fermenter reaction volume volume per minute. Tmn=dimensionless mixing time. pO2=partial pressure of oxygen. Pg=power input in aerated vessel. tm=mixing time. η=dynamic viscosity   Aeration rate (volume per ð9Þ AR ¼ FG =VR m3 =m3 min AR=aeration rate.dI . volumetric ð1Þ P¼ 2nM ¼NPo n3 dI5 kgm2 s2 ¼ W P=power input. implicated in mixing.Table 1 Fermentation parameters. CL=measured oxygen saturation concentration in the liquid phase. CER=carbon dioxide emission rate. Fractional gas hold up (ɛ) ð12Þ " ¼ VG =VR ɛ=fractional gas hold up.h i Modified dimensionless ð8Þ T0m n ¼ nTm ðdF =dI Þ2 Re ¼ Tm =dF 2  dF=inside vessel diameter. P=power input. as dimensionless groups as described and discussed. Dimensionless power ð2Þ NPo ¼ P=n3 dI5 dI=impeller diameter number (NPo) Impeller tip speed (vtip) ð3Þ vtip ¼ 2  n dI ½m=sec n=stirrer speed. n=stirrer speed.Re. a.S. dI=impeller diameter. A=fermenter cross section Gas hold up (τ) ð11Þ  ¼ VR =FG τ=gas hold up.8 ðj=dI Þ0. aeration. M=momentum. pi=vessel back pressure [bar]. dI=impeller diameter Respiratory quotient (RQ) ð18Þ RQ ¼ CER=OUR ¼pCO2E  pCO2i =pO2i  pO2E ½mol CO2 =mol O2  RQ=respiratory quotient. g=aerated. FG=volumetric gas flow rate. n=stirrer speed. Wang–Cooney j=buffle width. qg=gas throughput. dI=impeller diameter. preferably but not necessarily. n=stirrer speed. CG=oxygen saturation concentration in the gas phase. n=stirrer speed. a=specific interfacial surface area. g =0 ¼qg =ndI3 NQg=gassing number. VG=dispersed gas volume     Gassing number (NQg) ð13Þ NQg ¼ f Fr. g =0 . b and c as specific fluid constants to be coefficient (kLa) determined experimentally  a kLa Scale-dependent ð17Þ kL a ¼ k 0 Pg =VR ðvs Þb ðB=6Þ0. pO2G=partial pressure of oxygen in the gas phase. S=fluid constant.g. 526pi =36 þ T ½mg=1 coefficient. by Hubbard (1987) and Wang and Cooney (1997) Parameter/coefficient Mathematical characterisation Symbol explanation   Power input (P). 0=not aerated. a′. Q=volumetric flow rate. Nfl=pumping number Dimensionless mixing time ð6Þ Tm n¼f ðRe. e. η=dynamic viscosity. i=air feed. Re=Reynolds number. dI=impeller diameter. E=exhaust air .Þ¼V =Q¼V = Nfl n dI3 ½sec Tm=mixing time. ρ=density. P=power input. Fr=Froude number. n=stirrer speed. b=vessel specific coefficients. OUR=oxygen uptake rate. NPo Þ Tm=mixing time. according to V=volume of culture broth. η=dynamic viscosity Mixing time (Tm) ð5Þ Tm ¼ f ðn. n=stirrer mixing time speed. kL=mass transfer ð15Þ CG ¼ 0. Vs=superficial gas velocity. pO2L=partial pressure of oxygen in the liquid phase. ρ=density of the medium. Re=Rey- (Tmn) nolds number NPo=dimensionless power number Modified dimensionless 0 NṔo=modified dimensionless power number T´mn=modified dimensionless ð7Þ NPo ¼ NPo Re3 dF =dI ¼ PdF 2 =3 power number mixing time. dI=impeller diameter Reynolds number (Re) ð4Þ Re ¼ ndI2 = Re=Reynolds number. ν=kinematic viscosity. vvm) Superficial gas velocity (vs) ð10Þ vs ¼ FG =A ½m=sec vs=superficial gas velocity. NPo=dimensionless power number. ρ=fluid density. coefficients and terms. VR=fermenter reaction volume. dI=impeller diameter   Oxygen transfer rate (OTR) ð14Þ OTR ¼kL aðCG  CL Þ¼kL a LO2 ðpO2G  pO2L Þ kg O2 =m3 h with OTR=oxygen transfer rate from gas to liquid phase. oxygen and heat transfer. LO2=oxygen solubility in the liquid phase. ρ=density of the medium. VR=fermenter reaction transfer volume. B=number of stirrers. suitable as scale-up variables to be kept constant alone or combined with 428 each other or other process relevant variables. T=temperature [°C] Volumetric oxygen mass ð16Þ kL a¼a0 ðP=VR Þb vcs ½s1  kLa=oxygen transfer coefficient. . Re=Reynolds number.3 ½s1  k′. FG=volumetric gas flow rate. NPo=dimensionless power power input (P/V) number (impeller specific). vs=superficial gas velocity. pCO2=partial pressure of carbon dioxide.

Qhxch=energy losses through cooling. A step ahead in this regard would be a further enhancement of the mixing quality by a reactor and process design permitting a multilevel injection of both. Table 1 (continued) (1996) and Bylund et al. (2001) established an rate (mol O2/s) integrative kinetic model combining these parameters by aid of the simulation program SCILAB (Inria. Yho=- adaptive control as well as for the development of appro- priate process models and its connection to a real-time ex- pert system based on the Gensym G2 system has been described by Cimander et al. see text bility spectrometry. Yim et al. Kishimoto and co- workers. and acetate concentrations as well as of the specific growth rate on the basis of the carbon dioxide emission rate (CER. air and substrates into high turbulence zones. who developed a model for on-line measurements of cell. A=surface area. (2003) identified five growth phases of Saccharomyces cerevisiae by neu- rocomputation of exhaust gas data measured by ion mo- working volume and aeration according to the Wang–Cooney equation (Wong et al. Further strategies aiming at the avoidance of oxygen limitation and glucose overflow metabolism consist of the employment of alternative carbon sources like glycerol (Lee 1996) or galactose (Kim et al. The observed pattern of glucose os- Qt ¼ Qmet þ Qag þ Qaer  Qevap  Qhxch ½kWh ¼ kJ with cillation can even serve as a key parameter condition in later scale-up stages (Lin and Neubauer 2000). (2003). Qag=energy approximate metabolic constant (460 kJ/mol O2). (1998) suggested a glucose feed at the dynamic zones of the fermenter bottom together with the injected air. To achieve a more uni- Heat transfer coefficient Energy balance (Qges) Parameter/coefficient form glucose distribution in large-scale tanks. Wong et al. For details and further explanations. ammonium consumption. 2003). (1991) describe a biomass controlling feed depending on ethanol forma- tion rate and RQ for S. F) enabling the determination of the maximum glucose and oxygen uptake rates through determination of the time which passes between glucose pulses and subsequent changes of partial oxygen pressure (pO2) and biomass concentration.5 to 30 l by limiting glucose feed and keeping the growth rate at the minimum (μ=0. Ro=molar oxygen uptake modul NeurOn-line capable of processing 1. Qt=total energy. the kLa value—as the most frequently applied physical scale-up variable—has been combined with (1) the vessel backpressure p by maintaining the product of kLa·p constant through variation of pressure. Dantigny et al. or (2) the aeration rate vvm under variation of power input. OTR or the growth pa- rameters can be applied as reference parameter for glucose ð19Þ ð20Þ ð21Þ feed in a closed loop design. β-galacto- sidase.116 h−1). Qevap=energy α=heat transfer coefficient. 1997).5 and 1 g/l of a recombinant K99 an- tigen. 2003) and of a drastic glucose re- striction. 2002). the various different physiological or directly measurable physical process parameters like pH. α-amylase. Besides the above-mentioned parameters such as the max- imum uptake capacities. Qmet=energy generated by metabolic activities. Lin et al. and vitamin B12 fermentation based on algorithms describing and steering fuzzy states and relations (Horiuchi and Kishimoto 1998). see Eq. Kolehmainen et al. The equilibration of carbohydrate and oxygen supply thus constitutes a key component of process control and optimization. Qaer=energy generated by aeration. As the maximum glucose and oxygen uptake rates are not constant but depend on growth phases Symbol explanation perature difference and rates and get reduced by induction of product synthe- sis (Neubauer et al. present a process control strategy for glutamic acid. 18.800 signal generated by agitation. glucose.4 g/l Mathematical characterisation of human granulocyte colony stimulating factor during  ¼ ðdQ=dtÞ=AdT scale up from 2. agitation and aeration for scale up of Bacillus thuringiensis fermentations (Flores et al. Table 1). Larsson et al. pO2. (2001) achieved constant yields of 4. cerevisiae. 429 For instance. (2002) report that minimal concen- trations of glucose and yeast extract yielded the highest Qmet ¼ Yho Ro ½kWh ¼ kJ concentrations of 0. dQ/dt=heat transfer. . dT=tem- simultaneously for the calculation of the process state and losses through evaporation. Such a model thus allows the alignment of the glucose feed meet- ing the maximum uptake capacity and therefore avoiding overflow metabolism.

the reaction volume is discretized zones and the zonal residence times. properties of the cul- Amanullah (2001). coli fermentations in dif. thus will facilitate the identification of key parameters influencing product yield and quality the most and there- with of suitable scale-up parameters and strategies. 2001) and bles and mass transports enables the determination of zon- physiological parameters evidently are not straight-lined al pO2 values and oxygen transfer rates. the conditions gov. 5. ferent scales with similar and identical courses of biomass. (2001). through direct numeri- action conditions and to reduce both. Table 1). Suitable for employment as physical scale-up parameters ic or radio pills) is pursued and detected optically or by are all known process parameters and coefficients exerting means of antenna or induction coils wound around the ves. 3. which permit the prediction of physiological ef- mone were 80% higher and the concentration of pro. (2000) found that the yields of human growth hor. it will by Enfors et al. the size of stress cal simulation (DNS)]. known physiological effects. are described by solving the corresponding Navier–Stokes lated and depicted through high-performance computing equations under consideration of the conservation laws of [computational fluid dynamics (CFD). magnet. ological data. (2001) for investigation and description most likely contribute at long terms to a realistic model- of gradients and oscillations of substrate concentrations ling of the interplay between physics and physiology and through computational fluid dynamics (CFD). heat transportation and mixing. like unstirred bypass loops to simulate and shear stress (Bezzo et al. 10.g. momentum and energy. glucose and oxygen concentrations. by the green as the avoidance of shearing stress and oxygen limita- fluorescing protein (GFP signaling) and the detection by tions even in critical reactor zones for the scale up of car- appropriate probes (Reischer et al. as performed thus further research (Schmalzriedt et al. and meshed into as many zones and cells as possible and acterization of the large-scale hydrodynamic and reaction subsequently. 9. As supportive approaches. aeration and agitation rate to glass vessels. tools of Fluent Inc. In this way. Broadly applied in the field of amount of parameters which can be simulated in small numerical flow simulations are the flow modelling software scales. the physical conditions governing these cells erning in large vessels and their relevant zones are simu. The generation and distri- through transfer into small reactors (scale down). (USA) (Fluent 4. Scale down approach mixing time distributions. the application of which however is restricted power input (Eq. during which the trajectory and velocity of tracer particles (fluorescence dyes. overflow dynamics and metabolic fluxes (structured metabolic mod- metabolism. Bylund els) leads to integrative models [integrated fluid dynamics et al. mixing rates and profiles. which de. ready existing or still to be developed reactor and stirrer cose concentrations or pH fluctuations. are predictable. duction triggered by microenvironmental fluctuations and acterization of the hydrodynamic conditions. with parameters like substrate concentrations and gradients. bution of the resulting field variables enable the vectorial representation of physical conditions at any point and therewith detailed information with respect to trajectories. such as procedure. particularly those affecting sel (flow follower techniques). fects and reactions. Physical scale-up parameters ments (Davidson et al. When comparing E. Generally. hy- drodynamic conditions. Evidently.g. Prob- Computational fluid dynamics lems related to the scale up of physical parameters are illustrated in the next section. Enfors et al. the viability of cells is impaired in simulation of the trajectories and distributions of gas bub- smaller scales (Hewitt et al. mixed acid fermentation. (2003). 2003). For the mathematical calculation and mixing quality in larger scales to ensure homogenous re. knowledge about the conditions to be requires a deeper understanding of the dynamics of meta- transferred is not yet sufficiently comprehensive. is the laser Doppler method. (IFD)]. mass transports Scale down designs. hydrodynamic model development can be per- formed mathematically abstract as well as data driven on the basis of experimental flow and circulation measure. also scale-up influences governing stress conditions by tracking of the correspond.g. the residence times in the respective zones. The characterization of the ed process parameters. 6 and MixSim). population respiration. Even though it is emphasized that the teolytically digested side products 30% lower in the small integration of CFD and structured biokinetics at present scale. A 2000). mixing time . (2002) report a standard- ing stress gene expression (see above) can be performed ization of hydrodynamic and mixing conditions as well on-line by means of bioluminescence. requires the char. 1. 2003) in dependence of al- and investigate effects of oxygen limitations. 2000. Cant 2002] and mass. Williams et al.430 Process design tects the scattered and reflected light pattern (Doppler shift) generated by floating particles passing a dual laser wave The realization of concepts aimed at the improvement of interference field. flow behaviour. The sort and tilage cell fermentations. e. e. A further optical detection oxygen supply. Enfors et al. appear to be restricted (see Bylund et al. excess glu. (Eq. 2003). Current bolic and regulatory networks and cascades of signal trans- efforts thus try to complete the picture by a detailed char. 2004). Alves and Vasconcelos 1996). The combination transferable. Hewitt et al. however. are illustrated by design (Kelly and Humphrey 1998). On one side. on the basis of given geometric and rhe- conditions. cell lysis. description of turbulent flows [e. (2000) ture broth like density and viscosity as well as the adjust- and Onyeaka et al.

For each individual product.) The performance of sive and detailed process characterization to identify key further various types and designs of stirrers is presented and stress factors and key parameters influencing product yield discussed by Junker et al. 7) and the modified di. oxygen mass transfer coefficient (kLa. agitation and aeration for scale up of point to keep physical parameters constant throughout the Bacillus thuringiensis fermentations. Wang and Cooney 1997). the calculations require a precise measure. mixing performance with a minimum of power input and A wholistic scale-up strategy consists of a comprehen- shear stress without foam generation. and of an appropriate process control efficients and curves are thus crucial scale-up aids. p by maintaining the product of kLa·p constant through not or only restrictedly possible from the physical view. Oniscu et al. but is limited fer rate as a product of kLa and mass transfer potential. gen supply like agitation (via energy input) and aeration as surements were found to be in definite correlation with superficial gas velocity (Eq. Classical examples are (1) the mixing time. like recom. short tubes instead of blades and is reported to exhibit best a suitable scale-up strategy has to be elaborated. 1995). limitedly compensated and kept constant by increasing working volume and aeration according to the Wang– stirrer speeds and energy inputs and (2) the volumetric Cooney equation (Eq. 2003). (1998). it is. A constant volumetric power input reflects these parameters in dependence of the fermenta- has indeed been successfully applied as scale-up parameter tion scale. Reynolds number for interferon α E. In shear stress. . 15. which energy input P/V itself. 4). mathematically driven approaches are (Eq. 12).8 kW per 1 m3) and in fermentations process-relevant parameter. variation of pressure. generally applicable strategy es- to be the Visco-Jet of Inotec. among many others. coli fermentations which inevitably increases in larger vessels due to the from 5 to 200 l by keeping constant the product of kLa larger volumes to be stirred and which cannot be un. 17. Russell This relates also to the kLa value (Eq. Diaz and Acevedo 1999. (2002) successfully scaled up E. 16). for each specific mensionless power number (Eq. 6). as a component metabolic activities and with OTR and are employable as of dimensionless terms. Wang and Cooney 1997). however. which possesses cone formed tablished. 2001) and dimensionless mixing time groups and characteristic curves (examples given. (1997) integrated the backpressure most cases. is not the most gallon. Diaz and Acevedo (1999) argue that the oxygen for the early industrial penicillin fermentations (1 hp per transfer capacity. the employ a stirrer speed and glucose feed steered pO2 of 20% dimensionless power number (Eq. 10) and which. by (Eq. Constance of even a single specific parameter growth was reduced and the biomass production and spor- mostly leads to an uncontrolled and unpredictable change ulation efficiency remained constant. 5.g. 19. 431 (Eq. and process design ensuring optimum mixing and reaction In addition. While the specific scale up. 13) and the modified di. To avoid shear stress and to keep constant by an appropriate choice and adjustment of the the energy input (P) at the lowest possible level. et al. Wong realizable. A common simple and robust method is the maintenance ing dimensionless coefficients (e. scale. 1). gassing number (Eq. OTR (Eq. which is et al. nology and the comparably large body of literature. 2000) as well as computational tools (Fig. 14. by the Buckingham of a constant pO2 in the culture broth by variation of stirrer Pi method as cited by Hubbard 1987). depending on the scale-up factor. and aeration rate (vvm) under variation of power input. currently the mostly applied physical scale-up variable 17). appropriate variables and coefficients and there- mensionless mixing time (Eq. equivalent to 1. reactor scale up (see Yawalkar 2002). Berkholz et al. Riesenberg et al. driven models (Berkholz and Guthke 2001. however.and 450-l (Eq. liquid phase and suggest an OTR based scale-up strategy For this reason. These examples demonstrate that. 21). pO2. process-specific parameter correlations. (1990) Well-established examples are. 20. e. Anderson et al. process. and biocalorimetric variables like heat fluxes and heat since it includes the relevant parameters influencing oxy- transfer coefficients (Eq. coefficients. is steered at the minimum limit. 14. Stirrer performance co. 8). Eq. process and facility. any of these parameters and coefficients can conditions. i. Flores et al. coli cultures. in fermentations requiring high energy inputs. OTR and tolerable inputs and of the temperature distribution in the vessel. the pO2 relevant influencing process and fermenter parameters. 2).e.g. energy up in the form of a limited power input. supported by appropriate knowledge and data be combined with other variables to set up and create new. the difference between the pO2 values in the gas and the binant E. coli fermentations in a 30. which are kept speed and aeration rate. indicated by the kLa value. 2002). terms. 11. latter two coefficients and of the corresponding curves enable the identification of appropriate types of stirrers capable of exerting the desired mixing performance at a Conclusion given stirrer speed with a minimum of energy consump- tion and therewith of compensating the above-mentioned Despite the central role of the scale-up issue in biotech- limitations of volumetric energy inputs at larger scales. is also frequently employed for growth phase indicators (Türker 2004. but the effective oxygen trans- with low energy inputs (see Kim et al. there (A quantum leap in stirrer technology in this regard seems seems to be no common. 15). The comparison of the with scale-up strategies have to be identified individually. Taking into account the limitation of a kLa-oriented scale ment and knowledge of heat fluxes and sources. gas hold up and fractional gas hold up (Eq. 16. and quality the most. Biocalorimetric mea. fermentation time of other variables into dimensions that are technically not could be shortened and toxin yields were increased. pursued both for process and reactor scale up by form.

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