advances in biochemical engineering

0960–3085/01/$10.00+0.00# Institution of Chemical Engineers

Trans IChemE, Vol 79, Part C, March 2001

ADVANCES IN BIOCHEMICAL ENGINEERING

The following abstracts are taken from papers which were due to be presented at the Advances inBiochemical Engineering Conference in April 2001. Regretfully the conference was cancelled.

ADSORPTION KINETICS FOR THE REMOVAL OF LEAD FROM EFFLUENTBY CHITOSAN

J. C. Y. NG and G. McKAY

Department of Chemical Engineering, The Hong Kong University of Science and Technology, Hong Kong, SAR, China

Chitosan is poly-b-(1-4-linked D-glucosamine) and is being assessed as an adsorbent for theremoval of metal ions from ef� uents. Chitosan is a polymer, which can be obtained from theshells of seafood like prawns, crabs and lobsters. Chitosan has free amino groups, which canattract metal ions.

This paper is designed to study the removal of heavy metal ions using (i) equilibriumadsorption and (ii) batch kinetic techniques. The sorption of lead ions onto chitosan has beenstudied. In this research, the sorption equilibrium and the sorption kinetics of lead ions fromsolution onto chitosan have been studied including the bench scale design of the batch reactor;DSC, TGA, BET, SEM and NMR will also be used to report the pure physical states of chitosanand the chitosan-lead complex.

(i) Equilibrium Adsorption: In order to study the sorption capacity, the sorption equilibrium oflead ion onto chitosan was investigated.Equilibrium isotherms were measured for this singlecomponent system at two-� xed pH values. The experimental isotherm data were analysedusing Langmuir, Freundlich and Redlich-Peterson equations. Correlation coef� cients weredetermined for each isotherm analysis.

Error analysis selection: Due to inherent bias in using the correlation coef� cient resulting fromlinearization, alternative single component parameters have been determined by non-linearregression. Five errors functions have been used and examined, namely: Sum of the SquaresErrors; Hybrid Fractional Error Function; Marquardt’s Percent Standard Deviation; AverageRelative Error; and Sum of the Absolute Errors. The best error function method provided thebest parameters for the � ve isotherm equations in this system, and will be demonstrated forerror comparison purposes.

(ii) Batch Kinetics: In order to study the kinetics of lead ion removal, an agitated batch sorberwas designed. The experimental data were analysed and examined by kinetic equations,namely: Pseudo-� rst order, Pseudo-second order, Elovich order and Modi� ed second order.Based on the single component test results, the most appropriate reaction kinetic model canbe determined by mathematical modelling and comparing theoretically predicted data withthe experimental data points.

Keywords: Chitosan; equilibrium isotherm; order; adsorption; batch kinetic; error function.

APPLICATION OF METABOLIC ENGINEERING FOR ACTINORHODINPRODUCTION IN STREPTOMYCES COELICOLOR

F. MAVITUNA and F. NAEIMPOOR

Department of Chemical Engineering, UMIST, Manchester, UK

Antibiotics are one of the most important groups of secondary metabolites and streptomycetesproduce more than 75% of all antibiotics. Streptomyces coelicolor A3(2) produces four known

47

antibiotics; actinorhodin, undecyiprodigiosin, methylenomycin and the calcium-dependentantibiotic (CDA). Actinorhodin is a polyketide, a dimeric molecule derived from one acetyl-CoA precursor and seven malonyl-CoA extender units. It is red in acid and blue in alkali.Streptomyces coelicolor A3(2) is genetically the most-studied strain in streptomycetes alongwith Streptomyces griseus. Because of the availability of information on its genetics,metabolism, physiology and its various mutant strains, it is an excellent model microorganismfor metabolic engineering studies.Metabolic � ux analysis (MFA) is a powerful tool of metabolic engineering. The theoretical andcomputational aspects of MFA involves the determination of metabolic pathway � uxes (speci� crates of reactions) through a stoichiometric model of the cellular pathways, using mass balancesfor intracellular metabolites.

The authors applied metabolic � ux analysis to Streptomyces coelicolor using a network ofmore than 200 reactions describing the major pathways as well as the secondary metabolism forthe production of actinorhodin and excretion of certain metabolites. Two types of speci� cgrowth rates, stoichiometric and theoretical, were de� ned, maximized and compared in order toinvestigate the microbial potential. Metabolic � ux distributions in Streptomyces coelicolorduring growth under nitrogen limitation with nitrate or ammonia as the nitrogen source werealso investigated. Use of nitrate resulted in lower speci� c growth rates compared to ammonia.Excretion of some organic metabolites was observed in both limitation cases. The modelindicated that the highest actinorhodin production rate was with nitrogen limitation butnevertheless this was accompanied with the undesired excretion of certain metabolites.

The most obvious application of MFA to secondary metabolite production is to targetgenetic manipulations in order to alter the metabolism in the desired direction. From theprocess engineering point of view, it can also be used effectively to design better mediacomposition with precursor feeding and to choose the operating mode and conditions forincreased product formation.

Keywords: metabolic ¯ux analysis; antibiotics; Streptomyces coelicolor; actinorhodin; growthrates; nitrogen limitation; metabolite excretion.

AN APPROACH TO ROBUST AND FLEXIBLE MODELLING AND CONTROLOF pH IN REACTORS

M. M. MWEMBESHI1, C. A. KENT

1and S. SALHI

2

1School of Chemical Engineering, University of Birmingham, Birmingham, UK2School of Mathematics and Statistics, University of Birmingham, Birmingham, UK

pH is an important operating variable in a wide range of bioprocess operations, and may have tobe controlled within relatively tight bounds. In such cases, advanced process control offers thepotential for more accurate and robust performance. However, this may require accurate processrepresentation. One of the fundamental problems in pH control of many of these operations isthe variation in the buffering characteristics of the process stream, which cannot easily bepredicted or quanti� ed. Another is the fact that, for processes with several variable in� uentstreams (such as many wastewater neutralization reactors), the same sequence of input values(most commonly, in� uent pH and � ow-rates) may give rise to different outputs (reactorpH, . . .), depending upon the reactor composition. Such effects add a further complexity to themodelling of an already ill-de� ned and highly non-linear system.

The work reported here demonstrates the potential application of static feedforward neuralnetworks to perform the non-linear system identi� cation required to produce a global one-step-ahead predictor that captures the process stream characteristics. To assist in the training andtesting of the networks investigated, a simpli� ed, ‘global � rst principles’ (FP) model of the pHof such systems was developed. This model predicted well the pH of a variety of mixtures ofacids, and was used to simulate input-output data for both training and testing of the networks.

In order to improve network validation and design, the input information vectors wereincreased by introducing four auxiliary variables. These were derived from acid-base principlesof physical chemistry, and were estimated from transformations of input and output variables,notably pH values, � ow rates, and pK values of suspected species present.

In training and testing investigations, two training algorithms were compared using untreatedand pre-treated (standardized) data: the Levenberg-Marquardt routine, and an heuristic

48 ADVANCES IN BIOCHEMICAL ENGINEERING


gradient-descent routine with momentum and adaptive learning rate. Both schemes producednetwork designs which predicted well. However, using mean-square error as a performancecriterion, it was observed that the heuristic scheme performed consistently better on pre-treated,rather than treated data, whereas the results from the studies using the Levenberg-Marquardtscheme were less conclusive. Nevertheless, the heuristic scheme trained much faster than theLevenberg-Marquardt routine, an important consideration were such a network to be used inon-line pH estimation and control. These results suggest that further investigations into the useof auxiliary variables and alternative training algorithms in neural network-based modelling andcontrol of complex neutralization processes are justi� ed.

BIODEGRADATION OF HIGH CONCENTRATION ISOPROPANOL VAPOURIN A BIOFILTER INOCULATED WITH A SOLVENT-TOLERANT MICROBIAL

CULTURE

M. T. BUSTARD1 and P. C. WRIGHT2

1Department of Mechanical and Chemical Engineering, Heriot-Watt University, Edinburgh, UK2Vissanu Meeyoo, Centre for Advanced Materials and Environmental Research, Mahanakorn University of Technology, Bangkok, Thailand

The aerobic biodegradation of high vapour phase concentrations 2-propanol (IPA) by apreviously enriched solvent-tolerant bacterial consortium within a 1.9 l bio� lter was investi-gated. Successful bio� ltration of solvent vapour concentrations of up to 10,500ppm wasimplemented. Previously enriched solvent-tolerant bacterial cells were immobilized ontoporous glass cylinders as a means of bioprocess intensi� cation. Bio� lter start-up and acclima-tion were studied and acetone concentration tracked as an indicator of IPA utilization, as thesole carbon source within a minimal salts medium (MSM).

Removal ef� ciencies of up to 100% for both IPA and acetone were successfully demon-strated by this bio� ltration system during steady state operating conditions. IPA vapour feedconcentrations were varied between 6500 and 10,500ppmv, during bio� lter start-up andacclimation, where the lowest removal ef� ciency obtained was 47% during the initial 48 h ofoperation. It is also proposed that the metabolic lag resulting from switching from alcoholdehydrogenase to acetone carboxylase and the axial strati� cation of such enzymatic activity aremajor rate-limiting steps in the deep oxidation of IPA to acetone. The results demonstrate thepotential of a previously enriched solvent-tolerant bacterial consortium in bio� lter systems, forthe aerobic treatment of concentrated VOC gases at levels up to 2 orders of magnitude higherthan other studies.

BIOSYNTHESIS OF THE EXOPOLYSACCHARIDE GELLAN

I. GIAVASIS, B. McNEIL and L. M. HARVEY

Department of Bioscience and Biotechnology, University of Strathclyde, Glasgow, UK

Gellan gum is an exopolysaccharide produced by the bacterium SphingomonasPaucimobilis. Itis a relatively new, versatile ingredient with applications in the food industry, as well as thepharmaceutica1 and chemical industry. Although the physico-chemical properties of gellanhave been well studied, the fermentation process involved has not yet received much attention.

This presentation discusses the effect of several fermentation parameters on the physiologyand performance of Sph Paucimobolis. In particular the effect of stirring rate, oxygen supplyand transfer, and composition of the fermentation medium on the productivity and the growthrate of the microorganism are studied. In addition, the in� uence of different fermentationparameters on the molecular weight and the rheological characteristics of gellan gum areinvestigated. Possible ways for manipulating the molecular weight and thus, the properties ofthe � nal product, are proposed (for example by changing the source of nitrogen in thefermentation medium). Finally, different fermentation strategies, e.g. batch, fed-batch, bi-staged(with different temperature or pH conditions at each stage) are examined. Fed-batch experi-ments offer extended fermentation time (and more gellan production), while bi-staged


ADVANCES IN BIOCHEMICAL ENGINEERING 49

fermentations are designed for optimal, fast growth in the � rst stage and optimal productivity inthe second stage.

The fermentations were carried out in a 15 l bioreactor (B.Braun Biostat, ED ES10) undercontrolled conditions of temperature, pH, aeration and stirring rate. For the rheologicalmeasurements a RM180 Rheomat rheometer was used (Mettler-Toledo), equipped with acylinder type head. Molecular weight of gellan was determined by means of intrinsic viscositymeasurements, osometry and gel permeation chromatography (GPC).

CHARACTERIZATION OF THE HYDRODYNAMICS OF A BEERFERMENTATION WITH MIXING THROUGH CO2 EVOLUTION

L. BOON1*, J. BOYD

1, A. HIND

2and J. VARLEY

1

1Department of Chemical Engineering and Chemical Technology, Imperial College, London, UK2Brewing Research International, Nut®eld

The majority of research associated with brewing has been concentrated on the (bio)chemistryand microbiology of the process. Relatively little is known of the mixing aspects, knowledge ofwhich is indispensable when aiming, for example, to shorten the process time while maintain-ing product quality.

Investigation into the hydrodynamics of beer fermentations is complex due to its dependenceon the geometry and scale of the fermenter. Most industrial sized vessels are cylindroconicalwith liquid circulation induced by CO2 evolution through nucleation, which takes place at thesedimented yeast1–2. By applying regime analysis and available models for airlift reactors andbubble columns to cylindroconical vessels of three different scales, i.e. lab (10 l), pilot (300 l)and industrial scale (400 m3), it should be possible to improve the understanding of how scaleaffects the hydrodynamics within the fermenter.

A bench scale cylindroconical vessel (volume of 10 l with a height to diameter ratio of 4 : 1)has been designed for the purpose of studying the hydrodynamics and their in� uence on thekinetics. Results will be presented for fermentations carried out under different processconditions, e.g. different temperatures, sparging of N2-gas or recirculation of the generatedCO2. During a fermentation, the dissolved O2, pH, temperature and CO2 gas evolution aremonitored, while off line analyses include speci� c gravity (which can be converted intosugar=substrate concentration), dry weight and concentrations of ethanol and fusel oils.Furthermore, consideration will be given to the acoustic technique3 for detection of bubblenucleation sites and the bubble size of nucleated CO2 bubbles.

Keywords: beer fermentation; mixing; measurement of acoustic emissions; CO2-evolution;CO2-nucleation.

REFERENCES

1. Delente, J. and Gurley, J., 1968, Tech Q Master Brew Assoc Am, 5: 187–197.2. Pandiella, S. S., 1995, EvolucioÂn de CO2 y FluidodinaÂmica en el Proceso de FermentacioÂn de Cerveza (PhD-Thesis,

Universidad de Oviedo).3. Boyd, J. W. R. and Varley, J., 1998, AIChEJ, 44(8): 1761–1739.

THE DEVELOPMENT OF AN AGITATED CELL LYSIS REACTOR FORPLASMID DNA PRODUCTION FOR GENE THERAPY

S. CHAMSART1, A. W. NIENOW

1, A. G. HITCHCOCK

2and J. A. J. HANAK

2

1Centre for Bioprocess Engineering, School of Chemical Engineering, The University of Birmingham, Birmingham, UK2Cobra Therapeutics Ltd, The Science Park, Keele, Staffordshire, UK

For the production of gene therapy products at any scale beyond the laboratory level, abiochemical engineering approach is essential if it is to be done ef� caciously. Non-viral genetherapy appears particularly attractive using plasmid DNA (pDNA), manufactured to the



standards required by regulatory authorities for clinical products and potentially in quantitiesseveral orders of magnitude higher than previously achieved. In a viable process1, pDNA isproduced in optimized E. coli fed batch fermentations. However, the most critical of theextraction and puri� cation steps is cell lysis. Starting with a suspension, cells are lysed byconcentrated alkali and then neutralized to give a � occulated precipitate, ideally suitable for� ltration. Earlier literature however, has suggested that such a process requires very gentleagitation due to shear sensitivity of the DNA molecules and is therefore probably not scaleable.

The present work has shown that the lysate is rheologically-complex, exhibiting a viscositywhich is a complex function of shear rate, giving a maximum value of about 30–40 mPas. It isalso viscoelastic, exhibiting a Weissenberg effect when stirred. These properties are mainlydependent on the presence of chromosomal DNA and hardly change even when exposed to alaminar shear sweep at rates up to 1300 s71, suggesting that molecular degradation does notoccur2. However, such properties make the complete mixing in, of both the alkali solutionwhilst lysing and the acid when neutralizing, dif� cult3. Nevertheless, tests under controlledlaminar shear rate conditions and in small stirred reactors do not indicate signi� cantdegradation of molecular structure, i.e. < 2% chromosomal DNA in the clari� ed pDNAsolution, no pDNA fragmentation and a plasmid yield of 1 mg¡1g cells4.

Using a 5-l reactor agitated with Intermig agitators because of their ability to mix both lowand high viscosity � uids, the concentrated alkali was incorporated successfully to give a lysateof similar rheological properties to those obtained at the bench scale. This result was achievedeven though the power required to drive the agitators is signi� cantly enhanced due to theviscoelasticity. Neutralization was also accomplished successfully, mixing in the concentratedacid and producing large, easily � lterable � ocs. The � ltrate again contained < 2% chromoso-mal DNA and a plasmid yield of 1 mg¡1g cells.

These results suggest that scale up to an even larger stirred lysis bioreactor withaccompanying � ltration should be feasible and a patent has been obtained5.

REFERENCES

1. D. L. Varley et al., 1998, Bioseparation, 8: 2091.2. S. Chamsart et al., 2000, Biotech Bioeng, in press.3. S. Chamsart et al., 2000, Biotech Bioeng, in press.4. S. Chamsart et al., 2000, Biotech Bioeng, in press.5. A. W. Nienow et al., 2000, Int Pat No 00=53304, Sept.

DIELECTRIC PROPERTIES OF EMBRYOGENICAND NON-EMBRYOGENIC PLANT CELLS

C. D. FALOKUN and G. H. MARKX


Somatic embryogenesis of plants in suspension is a potentially useful system for the rapidpropagation of plant material. A suspension culture contains many cells in different develop-mental stages, but only some will actually produce embryos. Examination of suspension andcallus cultures suggests that embryogenic cells are characterized by having a denser cytoplasmthan other cells, and are also generally smaller.

The authors have initiated a research program in which they aim to characterize thedifferences in the dielectric properties of embryogenic and non-embryogenic cells, and separatethem on the basis of these differences. Such an in vitro selection program will have to be robust,selective and be capable of distinguishing subtle differences among cells from one genotype.The authors envisage that the separation method will be based on Dielectrophoretic Field-FlowFractionation techniques (DEP-FFF).

Carrot (Daucus carota) was chosen as a model system. Callus was initiated from seedlings(1% gelrite, 3% sucrose, 1 mg=ml 2,4-D, MS) and used to establish a suspension culture (samemedium as calli but without gelrite).

The dielectric properties of single plant cells were obtained through electrorotation, and theobtained data were analysed using appropriate models based on Maxwell-Wagner’s theories ofinterfacial polarization. The cells were found to have a wide spread in their internalconductivity, with dense cells having a higher internal conductivity (> 6 1072S m71) and



non-dense cells having a lower internal conductivity (< 1 l072S m71). The internal conduc-tivities obtained were lower than expected from the literature, and may indicate a loss ofviability during the experimental protocol. Modelling the DEP response on the basis of theelectrorotation data suggested that negative DEP and hence separation of cells may beachievable in the frequency range 1 MHz–10 MHz. Observation of DEP behaviour in asystem containing interdigitated electrodes showed that no negative DEP occurred in thefrequency range 1 MHz–100 MHz, suggesting that (a) the actual internal conductivity is higherthan those values obtained by electrorotation, (b) that the permittivity of the cells at lowerfrequencies is higher than that of water, and (c) that frequencies above 100 MHz are needed toachieve ef� cient separation of cells by hyperlayer DEP-FFF.

Keywords: somatic embryogenesis; plant cell culture; dielectrophoresis; Field-FlowFractionation.

EFFECT OF CARBOHYDRATE ELICITORS ON OVERPRODUCTION OFFUNGAL CULTURES

T. KESHAVARZ and C. BUCKE

Fungal Biotechnology Group, Biotechnology Department, University of Westminster, London

Traditionally, overproduction of metabolites from animal, plant and microbial culture has beenachieved through adoption of different techniques such as medium development, inoculumimprovement, fermentation design and bioreactor design. Over the last twenty years, a newapproach has been made based on the use of ‘elicitors’ in cultures. Elicitors are compounds thatwhen added in small amounts (e.g. mg ml71) to a culture; affect some of its characteristics suchas morphology and metabolite production.

A wide range of elicitors, classi� ed into biotic or non-biotic, could improve productivity of aculture. For example, certain oligosaccharides termed oligosaccharins, have a regulatory effecton physiology and defence processes of some animal, plants and bacteria. Stimulation ofhuman monocytes activity1, promotion of germination and shoot elongation of some plants2

and increased growth rate of bi®dobacteria3 has been achieved through addition of alginate

oligosaccharides as elicitors.The authors have reported, for the � rst time, the effect of carbohydrates elicitors on fungal

cell cultures4,5. In the � rst set of studies, Penicillium chrysogenum was used as a fungal modelsystem. Shaken � ask and bioreactor studies by addition of 100 mg l71 oligosaccharins, to 48 hcultures obtained from hydrolysis of alginate, resulted in an increase of 50% to 142% in theyield and % in the volumetric productivity of penicillin G. The concentration of intermediatesof penicillin G pathway such as ACV and IPN was also affected. In addition, it was found thatoligosaccharins could affect the onset and degree of sporulation and concentration of thepigment chrysogenin in a wild type strain as well. Overproduction of glucose oxidase activityby up to 70% cultures of Penicillium variabile used as a different fungal system was alsoachieved after supplementation of the culture with 200mg l71 oligosaccharins.

The studies suggest that elicitation of fungal cultures is a complex process involving changesat different parts of the metabolic pathway. Factors affecting the outcome and the extent ofelicitation include age of the culture type, amount and size (degree of polymerization) of theelicitor, and time of its addition. While the elicitation effect on certain fungal cultures has beenestablished, the mechanism of elicitation is not fully understood. It is plausible to assume thatoverproduction of secondary metabolites in fungi is due to stimulation of its defencemechanism. In order to throw light on the mechanism of elicitation, it is essential to knowthe inter-relation between the elicitor and the cell. The authors are currently investigating someaspects of this process.

REFERENCES

1. Otteriei, M., Skjak-Braek, G., Smidsrod, O. and Espevik, T., 1991, Induction of cytokine production from humanmonocytes stimulated with alginate, J Immune Therapy, 10(4): 286–291.

2. Yonemoto, Y., Tanaka, H., Yamashita, T., Kitabatake, N., Ishida, Y., Kimura, A. and Murata, K., 1993, Promotion ofgermination and shoot elongation of some plants by alginate oligomers prepared with bacterial alginate lyase, J Fermand Bioeng, 75(1): 68–70.



3. Akiyama, H., Endo T., Nakakita, R., Murata, K., Yenemoto, Y. and Okayama, K., 1992, Effect of depolymerizedalginates on the growth of Bi®dobacteria, Biosci Biotech Biochem, 56: 355–356.

4. Ariyo, B., Bucke, C. and Keshavarz, T., 1997, Alginate oligosaccharides as enhancers of penicillin production incultures of Penicillium chrysogenum, Biotech Bioeng, 53: 17–20.

5. Petruccioli, M., Federici, F., Bucke, C. and Keshavarz, T., 1999, Enhancement of glucose oxidase production byPenicillium variable P16, Enzyme Microb Technol, 7: 397–401.

THE EFFECT OF FLOCCULATION ON DEWATERING OF YEAST IN 10 mmHYDROCYCLONES

J. J. CILLIERS1 and S. T. L. HARRISON2

1Department of Chemical Engineering, UMIST, Manchester, UK2Department of Chemical Engineering, University of Cape Town, South Africa

Filtration and centrifugation are conventionally used to separate yeast from suspensions. Minihydrocyclone (10 mm diameter) have previously been shown to yield limited dewatering ofnon-� occulating baker’s yeast. This indicated the potential of the application of hydrocyclonesto the dewatering of � occulating brewer’s yeast, where the effective particle size is larger andcan be manipulated. However, the conventionalwisdom in hydrocyclone technology is that thehigh shear forces in the free vortex will break up � occulated particles, and that the separationcannot, therefore, be enhanced in this way. This is expected to be especially so in mini (10 mmdiameter) hydrocyclones, where acceleration forces of more than 60,000g are generated.

In this study, the effect of � occulation on the dewatering of brewer’s yeast suspensions wasinvestigated. The effective � oc size and concentration were varied and the separationperformance quanti� ed. The required degree of yeast � occulation was achieved using aceticacid, to yield � oc sizes between 10 and 200mm. The feed concentration range investigated wasbetween 2 and 10 g l71.

It was found that the yeast � ocs were not broken up by passing through the hydrocyclone.This allows enhanced separation, as compared to results obtained for single cell baker’s yeast.Increasing the � oc size signi� cantly enhanced the separation, as would be expected if the � ocsare not broken up by the shear. Increasing the feed concentration decreased both the recovery ofyeast to the concentrated product, and also the concentration ratio between feed and product.This is in agreement with previous � ndings for baker’s yeast. Typical results indicate that theconcentration can be doubled and more than 70% of the yeast recovered in a single pass thoughthe system.

EFFECT OF THE 3rd DIMENSION ON DIELECTROPHORETICATTRACTION OF CELLS TO PLANAR ELECTRODES

B. ALP and G. H. MARKX


Dielectrophoresis (DEP) is the movement of particles in non-uniform electric � eld. It is causedby differences in the polarizability of the particles compared to that of the suspending medium,and depending on their relative polarizability positive dielectrophoresis towards high � eldstrength regions, or negative dielectrophoresis away from high � eld strength region is possible.Due to interfacial polarization at the surfaces of the different structures in a cell biological cellsshow a polarizability that is highly frequency dependent.

Most commonly, the dielectrophoretic behaviour of cells is studied using planar electrodesmade using photolithography. The interpretation of the results of DEP experiments using suchelectrodes often assumes that only the electric � eld in the plane of the electrodes is important.This neglects not only the fact that the electric � eld is 3-dimensional, but also that the cellstructures that are formed near the electrodes have a thickness that is more than one cell layer.

Using absorbance measurements and microscopic observations the authors have studied theeffect of the 3-dimensional nature of the electric � eld and cell aggregates on the dielectro-



phoretic behaviour of cells at different applied voltages and cell concentrations. Also, theelectric � eld pattern above the electrode was calculated. Baker’s yeast (S. cerevisiae) was usedsuspended in a medium of low conductivity, and an applied frequency of 1 MHz. Super� uouscells were washed from the electrodes using a gentle stream. Under these conditions onlypositive dielectrophoresis occurs.

When the electric � eld is applied, the cells were observed to move � rst to the edges of theelectrodes. At the lower voltages the cells attached to the electrode produce a monolayer on thesurface. When the voltage is increased, and suf� cient cells are available the cells tend to moveonto each other and the thickness of the layer of the cell increases to form a multicellular layer.Although the thickness of the layer can be increased by increasing the voltage, the thickness ofthe layer is limited by the decline in the electric � eld strength as a function of the height abovethe electrodes. The effect of the 3-D nature of DEP on the measurement of the DEP force usingabsorbance measurements will be discussed.

Keywords: dielectrophoresis; absorbance; electric ®eld pattern; yeast.

EFFICIENT PHOTOSYNTHETIC PRODUCTION OF MICROALGALBIOMASS USING A HELICAL TUBULAR PHOTOBIOREACTOR IN JAPAN IN

TERMS OF TEMPERATURE CONTROL UNDER FIELD CONDITIONS

M. MORITA, Y. WATANABE and H. SAIKI

Bio-Science Department, Abiko Research Laboratory, Central Research Institute of Electric Power Industry (CRIEPI), Japan

The authors have been investigating the microalgal CO2 � xation=conversion technology forconverting CO2 from thermal power plants into valuable microalgal biomass. When thephotobioreactor incorporating microalgae is installed for CO2 � xation=conversion under � eldconditions for one year, it is necessary to maintain the culture medium temperature in anappropriate range for effective production of microalgal biomass. The authors previouslyevaluated the heat balance in the photobioreactor to estimate the culture medium temperature.The predictions of the time-dependent changes in the culture medium temperatures weresuccessful under laboratory and � eld conditions using a heat balance model among three majorsections (photostage, degasser and helical heat exchanger) of the conical helical tubularphotobioreactor (HTP), a novel design of a photobioreactor with high photosynthetic produc-tivity. In this research, using the model previously established, the time-dependent changes inthe culture medium temperature for every season in Abiko City, Chiba Prefecture in Japan(CRIEPI location) were predicted, as an example of middle Japan. The energy required forheating or cooling was also predicted using the model where the conical HTP would be usedunder � eld conditions. Ef� cient photosynthetic production of microalgal biomass was proposedthroughout the year.

Little energy for heating or cooling was considered to be necessary in spring and autumnbecause the culture medium temperature was predicted to be maintained within an appropriaterange of Chlorella sp. strain HA-1 (CRIEPI isolates, mesophilic strain). On the other hand, theculture medium temperature was predicted to be more than 35 C in � ne weather in summer.The application of high-temperature-tolerant Chlorella, which was isolated from a hot spring,was considered to be appropriate for the summer season. An outdoor culture experimentincorporating Chlorella sorokiniana strain HO-1 (CRIEPI isolates, a high-temperature-tolerantstrain) was carried out in summer. High photosynthetic productivity was obtained withoutcooling. As a result, ef� cient photosynthetic production of microalgal biomass was consideredto be achievable throughout the year by the combination of various strains that had differentcharacteristics relative to temperature.

Keywords: microalgae; carbon dioxide; bioreactor; heat balance; temperature change.



EVALUATION OF OPTIONS FOR BIOPROCESSING OF TRANSGENICPLANTS USING ULTRASCALE-DOWN TECHNIQUES

E. KESHAVARZ-MOORE, M. E. K. NICHOLS, T. STANISLAUS, I. E. C. MOTT,A. HUGHES and P. A. SHAMLOU

The Advanced Centre for Biochemical Engineering, Department of Biochemical Engineering, University College London, London, UK

Recent scienti� c advances have led to new challenges in bioprocessing of transgenic plantmaterials. Material availability for research and process development are limited because ofinitial low expression systems, long cultivation times coupled with seasonality of production. Inaddition, in many cases there is no precedent in processing. The authors used ultrascale-downtechniques to carry out rapid evaluation of bioprocess options in the production of non-foodmaterials from transgenic plants. Two examples are reported here. Firstly, biodegradable plasticco-polymer P(3HB-3HV) was produced from transgenic rapeseed. Recovery required removalof the oil. Ultrascale-down of pressing and solvent extraction stages to 10¡8 of the industrialscale to remove the oil was carried out with ten gram quantities of rapeseed. Results showedthat mechanical extraction led to the aggregation of biopolymer particles from 2 microns to80 microns. Oil was driven into the pores of the particles. The rate limiting process step wasidenti� ed as the solvent removal of this oil because solvent access was restricted. In a secondstudy, the processing of cowpea mosaic virus from cowpea plants was studied for potentialtherapeutic use. Using two gram quantities of plant leaves the authors showed that freezethawing followed by homogenization was an effective means of releasing the virus withminimal losses. Ammonium sulphate and PEG precipitations were investigated as possibleoptions for virus recovery.

FERMENTATION UNDER INCREASED HEADSPACE PRESSURE: EFFECTOF PRESSURE ON THE VOLUMETRIC MASS TRANSFER COEFFICIENT

B. MAIER, C. DIETRICH and J. BUÈ CHS

Department of Biochemical Engineering, Aachen University of Technology, Aachen, Germany

Bioreactor headspace pressurization represents an excellent method of enhancing the masstransfer of low soluble gaseous substrates such as oxygen into culture media. Industrialfermentation processes are often oxygen limited. Therefore, an enhancement of the oxygenmass transfer increases the productivity and the economic ef� ciency of aerobic fermentationprocesses. Despite these promising possibilities industrial fermentations are normally operatedunder ambient or only slightly increased pressure. the headspace pressure is seldom recognizedas an engineering parameter in a bioprocess development.

In this study the effect of headspace pressurization on the gas-liquid mass transfer in the rangof 0.1–1 MPa has been investigated. It is shown that the sulphite oxidation method is a reliablemethod to determine the mass transfer characteristics of a gas-liquid reactor even underpressurized conditions. However, the kinetic of the chemical reaction has to be carefullyconsidered. The often incorrect use of the sulphite oxidation method has led to inconsistentresults and therefore the reliability of the method is often questioned.

The investigations revealed that the volumetric mass transfer coef� cient kLa in a stirred tankreactor under a constant volumetric gas � ow rate inside the bioreactor is not effected by theheadspace pressure within a pressure range from 0.1 up to 1 MPa. Therefore, the mass transfercharacteristics obtained under atmospheric pressure can be used for the design of pressurizedgas-liquid reactors. Contradictory � nding of other groups are probably due to an incorrectapplication of the sulphite oxidation method.



FIXED BED STUDIES FOR THE SORPTION OF METAL IONS ONTO PEAT

D. C. K. KO, J. F. PORTER and G. MCKAY

Department of Chemical Engineering, Hong Kong University of Science and Technology, Hong Kong, SAR, China

The potential use of peat as a biosorbent for the removal of heavy metals from wastewater hasbeen reported in literature. Peat has received increasing attention as one of the most inexpensiveand readily available adsorbents compared to other carbon-based adsorbents. In the presentstudies, peat has been used as an adsorbent for the removal of heavy metals in a � xed bedsystem. Experiments have been performed to investigate the effect of � owrate and bed depth onthe peat-metal ion system. A � lm-pore diffusion model has been used to predict thebreakthrough behaviour. The three major parameters in the mathematical model are theexternal mass transfer coef� cient, k, the intraparticle mass transfer coef� cient, Deff, and thesolid-phase loading capacity, qe. One of the main problems in this type of modelling is theprediction of qe, particularly for sorbent-solute systems which take a long time to achieveequilibrium saturation. Some researchers have used the equilibrium isotherm capacity, someutilize a � xed fraction of the isotherm capacity and other workers perform the numerical orgraphical mass balances at the breakthrough curves which is time consuming and tedious. Thepresent method incorporates a novel empirical solution for qe which is correlated with theservice time. Good agreement between the predicted theoretical breakthrough curves and theexperimental results is observed.

Keywords: adsorption; peat; metal ion; ®xed bed column; mass transfer coef®cient;mathematical modelling.

THE FORMULATION OF HARD-SURFACE CLEANING PRODUCTS WITHENHANCED BIOCIDAL ACTIVITY-COMPOSITION AND MECHANISM

M. V. JONES1, K. L. RABONE1, B. A. TIMIMI2 and G. J. T. TIDDY2

1Unilever Research Port Sunlight Laboratory, Wirral, UK2Department of Chemical Engineering, UMIST, Manchester, UK

An important requirement of modern hard surface products is that they exhibit biocidal activityas well as delivering good detergency. Although non-ionic surfactants have traditionally beenregarded by microbiologists as mild and microbiologically inactive1, detergent alcoholethoxylates capable of delivering good hard surface detergency also inhibit bacterialgrowth2. Recently the authors have investigated the in� uence of co-actives such as smallamphiphilic compounds on the behaviour. Remarkably, some give strong additional biocidalactivity, being particularly active against Gram-negative bacteria. These results are describedhere.

To control the formulation it is necessary to have a good model for the biocidal activity. In arecent review of disinfectant mechanisms, Denyer and Stewart3 regards most disinfectants aschemical biocides with a relative lack of selectivity. The actual target frequently cited is thecytoplasmic membrane, which comprises a lipid bilayer with embedded proteins, lipo-polysaccharides and a range of other bio-active subtrates. In order to explain the mechanismthe authors examined the in� uence of the formulation constituents on whole cells and on lipidmembrane extracts. Using a � uorimetric technique, a result of the attack of the alcoholethoxylate in combination with a small alcohol co-active was shown to be that the cytoplasmicmaterial of the cell leaks out, leading to the death of the micro-organism. An obviousmechanism of membrane disruption is that the formulation simply solubilizes all or part ofthe lipids, thus releasing the cell contents. An alternative mechanism is that the formulationalters membrane ‘� uidity’ and=or curvature, having an in� uence on the lamellar=gel and=or thelamellar=reversed hexagonal phase transitions of the lipids. The authors have investigated thisaspect using microscopy and NMR studies of E.coli membrane lipid extracts. The resultsindicate the presence of gel-state lipids in the membranes, and that the co-actives do indeedmoderate the gel fraction.



REFERENCES

1. Russell, A. D., Hugo, W. B. and Aycliffe, G. A. J., (Editors (1992)) Principles and Practice of Disinfection

Preservation And Sterilization, (2nd Ed Blackwell Science Publications) 2: 38.2. Moore, S. L., 1997, The Mechanisms of Antibacterial Action of Some Non-Ionic Surfactants (Dr Phil Thesis,

University of Brighton).3. Denyer, S. P. and Stewart, G. S. A. B., 1998, Mechanisms of action of disinfectants, Inter Biodeterioration and

Biodegredation, 41(3–4): 261–268.

A GENERAL APPROACH TO THE VALIDATION OF STERILIZINGFILTRATION USED IN ASEPTIC PROCESSING

J. M. CAPPIA

Millipore France SA, France

Sterile � lters as used in aseptic processing require speci� c quali� cation, production controls,quality assurance systems and validation programs to ensure that they perform in a reliable,predictable and reproducible way.

A number of regulatory guidelines and guidance documents such as the PDA TechnicalReport no 26 on Sterile Filtration of Liquids provide a systematic approach to validating the� lter performance. Validation studies are aimed at showing that a particular combination ofproduct, process conditions and � lter device will meet the requirements of sterilizing grade� ltration.

Complete documentation packages consisting of pre-approved protocols and � nal testreports are now currently established for bacterial retention, extractables and physical integritytesting. Critical process steps such as � lter steam sterilization must also be considered at theearly stage of the process development to establish easy validation procedures. Following SIP,pre-use integrity testing is regarded as good manufacturing practice, to ensure that the � ltrationstarts with an integral � lter.

This paper proposes a general validation master plan for sterilizing � ltration as used inaseptic processing, and describes the different types of testing and controls needed to meetcurrent US Food and Drug Administration and European Union (EU) regulatory requirements.

GRAPHICAL REPRESENTATION OF THE PROCESS TRADE OFF BETWEENPURITY AND RECOVERY FOR CHROMATOGRAPHIC SEPARATION

S. H. NGIAM, Y. H. ZHOU, M. K. TURNER and N. J. TITCHENER-HOOKER


Chromatography is a widely used separation technique in the manufacture of pharmaceuticals.Chromatograms are often the � nal output of modelling predictions but they are not very usefulas a means of quantifying the consequences for performance and the sensitivity of thechromatographic separation quality to changes in operating conditions. In response to this asimple engineering framework that enables the rapid visualization of the performance of liquidchromatographic separations has been provided in this paper. The framework comprises theconstruction of a fractionation diagram and its associated maximum puri� cation factor versus aproduct yield diagram, which may be derived from chromatographic data. The former showsthe relative change in the cumulative fractional mass of product eluted with the correspondingfractional total mass eluted while the latter re� ects the degree of trade-off between the levels ofrecovery and purity achieved in the chromatographic step. An algorithm was written inMATLAB for the optimization of puri� cation factor versus yield using a search technique.These diagrams are more straightforward and easily interpretable compared to the basicconventional chromatograms and enable the investigation of the degree of trade-off betweenpurity and recovery for any set of operating condition to be made.

Data from two different chromatographic systems were employed to demonstrate theapproach. In the � rst, a set of simulation results from a veri� ed size exclusion model separatingthree globular proteins (i.e. g-globulin, ovalbumin and Ribonuclease A) was adopted. In the



second a set of experimental results from a hydrophobic interaction STREAMLINETM Phenylexpanded bed separation of a labile enzyme alcohol dehydrogenase (ADH) from bakers yeasthomogenate has been used. With the aid of such an approach, the optimal operating conditions,in terms of acceptable yield and desired purity, can be selected easily and the product fractionsneeded to achieve this value of puri� cation factor or yield identi� ed.

Keywords: chromatography; fractionation diagram; yield; puri®cation factor;process trade-off.

IMMOBILIZED LIPASE HYDROLYSIS OF TRIGLYCERIDES;FUNDAMENTAL BIOREACTOR DESIGN

J. C. BURT and B. AL-DURI

University of Birmingham, Birmingham, UK

The aim of the project is to discern the effect of pertinent system characteristics on the successof the lipase mediated hydrolysis of Olive and Hemp oils. Olive oil was selected as it is acommonly used and well characterized substrate, whereas hemp oil is high in particular longchain polyunsaturated fatty acids (o3 and 6 ‘essential’ fatty acids) currently in demand in anumber of industries.

The system characteristics can be split into two main groups; the microscopic (and sub-microscopic) considerations and the macroscopic parameters. The microscopic characteristicsinclude such considerations as the conformational shape of the lipase upon immobilization andin response to environmental factors (pH, temperature etc.), as well as the behaviour of lipasemolecules with respect to interfacial activation and steric hinderance. Macroscopic considera-tions include the mass transfer to and from the active lipase sites and the creation of a suf� cientinterfacial area via suitable agitation.

The ultimate aim is that the effect of these factors upon the rate and degree of hydrolysis ofthe chosen substrates can be expressed within a suitable mathematical framework.

Main Findings

Lipases from Pseudomonas ¯. are more suitable for immobilization onto the selected supports(Accurel EP100 and MP4000) via adsorption, than lipases from Candida rugosa.All lipases studied (both in free and immobilized states) hydrolyse hemp oil slightly morereadily than olive oil, but the effect of system parameters seems to be very similar.The degree of hydrolysis achieved with immobilized lipases is still far short of that seen withthe free lipases especially those sourced from Candida rugosa.Increasing the rate of agitation with immobilized lipases for both substrates has little effect onconversion. This demonstrates that external mass transfer (� lm transfer) has no signi� canteffect on reaction rate.The immobilized lipase AK (Pseudomonas ¯.) had an optimum operating temperature ofapproximate1y 10 C higher than the optimum for free lipase AK, demonstrating thatimmobilization can confer an increase thermal stability. This effect was not, however,observed for the immobilized LO34P (Candida rugosa).Aqueous-organic phase ratio is an important parameter for the reaction (both for free andimmobilized lipase systems). The smaller the oil phase the higher the degree of conversionhowever the activity (expressed as the number of micromoles of fatty acid produced per unittime per unit biocatalyst) was observed to increase with increasing oil phase.

Keywords: lipase; hydrolysis; immobilized lipase.



IMPROVED MEASUREMENT AND CONTROL OF FERMENTATION FOAMS

A. K. BROWN1, C. ISBELL2, S. GALLAGHER3, P. W. DODD4 and J. VARLEY1

1Imperial College of Science Technology and Medicine, Department of Chemical Engineering and Chemical Technology, London, UK2Neusciences, Totton, UK

3Charis Technology, Maidstone, UK4Avecia Ltd., Billingham, UK

Many commercially important biological products are mass-produced using fermentationprocesses inside closed bioreactors with microbial organisms. Due to the presence ofsurface-active materials in the culture medium and in combination with intense agitation=aera-tion that is required for acceptable oxygen transfer for microbial growth, foams are easilygenerated in aerobic microbial fermentations. The presence of foams in bioreactors is notdesirable for the following reasons: (i) loss of cells and substrate into the foam can reduceprocess productivity; (ii) foam production can lead to microbial containment issues and loss ofprocess sterility; (iii) fouling of probes causes poor process control. There are various strategiesused to control fermentation foams (i.e. chemical and physical methods). Chemical antifoamingagents are very ef� cient at reducing foam. However, these chemicals can result in depletedoxygen transfer, extra downstream processing and product formation inhibition. Also, antifoamadditions become expensive for large-scale operations. Physical methods for foam-breakingsubject foam to shear stress but this can lead to cell damage; extra power requirements and arenot suitable under conditions of excessive foaming.

In commercial fermentation processes, the limitations of existing antifoam methods requirebioreactors to operate at below maximum volume capacity, resulting in reduced productivity.Currently, chemical antifoam methods rely on a single-point conductivity measurement of alocal region of foam inside the vessel headspace. This simple on-off control system merelyindicates that foam is present inside the vessel, there is no information pertaining to the physicalcondition or dynamics of the foam, thus, it cannot be predicted if foam will continue to rise orwill passively collapse without addition of antifoam. Moreover, it may be possible to controlfoam by changing only certain process conditions without affecting process performance ornecessitating chemical antifoam additions.

The research described here aims to address limitations of current technology by developingan improved measurement and control system for fermentation foams. A new foam probe hasbeen developed by Charis Technology that enables on-line foam conductivity to be measured atvarious points within the vessel headspace. These conductivity values relate to the liquid hold-up in the foam. Information from the foam probe will be used to develop neural networkmodels of the foaming process for a particular fermentation process. It is envisaged that a fullydeveloped neural network integrated with the new foam probe and existing antifoamtechnology will give superior control over current foam control methods. By ‘training’ theneural network with a range of operating conditions to obtain foams with various characteristics(i.e. different liquid hold-up=bubble size=stability), predictions of the foam properties and‘foam-out’ possibility will be determined. The neural network will then decide if antifoam isneeded or a change in operating conditions can prevent ‘foam-out’. Preliminary neural networkmodel development based on the foam probe data has shown promise for prediction of a targetresponse related to foam height at 1 or 5 min ahead of actual process time. In addition it isaimed to develop neural networks which use both foam probe data and on-line process data forpredictions of foam behaviour.

IMPROVED PRESENTATION FOR SHEAR RATE OF HELICAL RIBBONIMPELLER IN THE TRANSITION REGION BASED ON LDA STUDIES

M. JAHANGIRIA1, M. R. GOLKAR-NARENJIA2, N. MONTAZERINB2 and S. SAVARMANDA2

1Department of Chemical Engineering, Amirkabir University of Technology, Iran2Department of Chemical and Mechanical Engineering, Amirkabir University of Technology, Iran

It is important in bioengineering development involving polymeric viscoelastic materials toestimate accurately the prevailing shear rates in bioreactors because they are generally sensitiveto shear. The signi� cant method of Metzner and Otto (AIChE J. 3, 3, 1957) for calculation of



effective shear rate is limited to the laminar region and is no longer valid in the transitionregion. In this article, the local shear rate and velocity pro� les and Metzner and Otto conceptfor helical ribbon impeller have been studied using laser Doppler anemometry (LDA) forviscoelastic liquids. Mean and � uctuating tangential and radial velocities were measured alongthe axial centre line of the helical ribbon impeller. New correlations are suggested fordimensionless mean tangential velocity pro� les. Experimental results show that the magnitudesof dimensionless radial velocities are much less than dimensionless mean tangential velocitiessuch that no considerable radial � ow could be detected. It is also shown that the variation oflocal shear rate against the impeller speed is better correlated by the power equation, i.e. {(shearrate) ˆ ks.N b0}, (b0 is power of N) in the transition region, i.e. Reynolds number between 210and 6700. In addition, a correlation between the improved coef� cient, and the elasticity numberof a viscoelastic liquid is given, that can be helpful in designing of the mixing of bothviscoelastic and inelastic non-Newtonian � uids through relating rheological properties tokinematical and dynamical parameters of the mixing process.

Keywords: LDA; helical ribbon impeller; viscoelastic liquids; velocity pro®le; mixing.

A MAXWELL-STEFAN MODEL OF MULTI-COMPONENTULTRAFILTRATION WITH TOTAL REJECTION OF CHARGED

MACROMOLECULES

S. S. VASAN and Z. F. CUI

University of Oxford, Department of Engineering Science, Oxford, UK

Ultra� ltration is widely applied to purify proteins and macromolecules under mild operatingconditions. In the practical application, the ultra� ltration media usually contains more than oneprotein, but most experimental research and theoretical analyses are conducted with wellcharacterized protein solutions (one or two). A study of ultra� ltration performance for multiplecomponent media is of great industrial and theoretical interests. A non-parameterized model basedon Maxwell-Stefan equations has been developed for a multi-component system with considera-tions such as non-ideality, charge interactions, Maxwell-Stefan diffusivities, etc. so that theapproach is more general in nature. The model has been de-linked from hydrodynamic equationsand focuses on the pro� le and interactions in the mass transfer boundary layer. There are 4n ‡ 4variables and as many equations, for a system of n71 solutes in water (species 1). A simpli� edversion of the multi-componentmodel has been numerically solved for a system of one, two, andmany proteins that are totally rejected by the membrane, while water and co=counter ions areallowed to pass through. Some of the important predictions are presented below.

The model predicts that the concentration of protein in the boundary layer increases withTMP, so the counter ion concentration also shoots up to maintain electroneutrality. Thepermeate salt concentration also increases with TMP, while the water � ux increases andsaturates, due to increase in osmotic pressure that opposes the applied pressure. A higherprotein concentration in the bulk results in a higher concentration in the � lm, and thus, a higherosmotic pressure, and lower water � ux.

An increase in ionic strength increases the protein concentration in the � lm due to betterelectrostatic shielding, and decreases the water � ux at very high TMPs. The model also predictsthat the proteins with lesser surface charge and lower diffusivity values accumulate better in theboundary layer, and tend to exclude those with higher diffusivities or charge or both. Theeffects are quite dramatic when the protein is very close to the isoelectric point (typically abovea charge of 75), and the minimum in permeate � ux occurs at pI of the protein. A lower surfacecharge on the macromolecules implies less electrostatic repulsion, so the � lm concentration ofthe protein goes up, and this trend is true across TMPs. Presence of a second protein with lessercharge decreases the permeate salt concentration, while a second protein with lower diffusivitydecreases the permeate � ux slightly (due to increase in osmotic pressure).

The model predictions are consistent with the received wisdom and experimental observa-tions reported in the literature, and are encouraging to solve more complicated situations.



MODELLING OF THE BAEYER-VILLIGER MONOOXYGENASECATALYZED SYNTHESIS OF OPTICALLY PURE LACTONES

B. H. CHEN, S. D. DOIG, G. J. LYE and J. M. WOODLEY


While considerable progress has been made on the implementation of biocatalytic hydrolyses,there are still a number of engineering issues to address regarding the implementation ofbiocatalytic redox and carbon-carbon bond forming reactions. The use of process models linkedto graphical tools (process maps) to describe the parameter boundaries for effective reactoroperation have found particular use for these classes. Such process maps help to describe theproperties of the biocatalyst and also the properties of the reactants and products which arefrequently labile in the reactor environment. Data to form and verify such models can be rapidlycollected at the microwell-scale. The models themselves can either be mechanistic or, as usedhere, based on arti� cial neural networks. The output of such models can then be incorporated ingraphical process maps and used as a predictive tool to carry out ‘what if’ studies. These maybe, for example, on possible changes to the biocatalyst (via directed evolution) or theimplementation of innovative process solutions (such as in-situ product removal). In thisway, alternative process � ow sheets and operating strategies can be evaluated more rapidly. Inthis paper the utility of these numerical and graphical techniques is illustrated using the Baeyer-Villiger oxidation of bicyclo[3.2.0]hept-2-en-6-one by a whole cell biocatalyst expressingcyclohexanone monooxygenase.

Keywords: ANN modelling; Bayer-Villiger oxidation; process maps; windows of operation;in-situ product removal.

OPTIMIZED METABOLIC FLUX DISTRIBUTIONS FOR L-GLUTAMATEPRODUCTION IN B. FLAVUM

F. MAVITUNA1, S. TAKAC2 and G. CALIK2

1Chemical Engineering Dept., UMIST, Manchester, UK2Chemical Engineering Dept., Ankara University, Ankara, Turkey

L-Glutamate is used widely throughout the world as a seasoning as well as a starting materialfor the synthesis of various chemicals. It is produced industrially by fermentation usingBrevibacterium and Corynebacterium strains. Since the wild strains produce only smallamounts of glutamic acid extracellularly, mutant strains with altered metabolism have beenused for increased product yields. Application of metabolic engineering tools such as themetabolic � ux analysis, can provide information about the bottlenecks in the metabolism for theoverproduction of the desired metabolite.

The authors constructed a comprehensive metabolic network consisting of 110 reactions torepresent the metabolism of glutamic acid bacteria and used this in a stoichiometrically based� ux balance model for L-glutamate production. The theoretical results were compared with theexperimental results obtained from the batch fermentations of Brevibacterium ¯avum DSM-20411. The fermentation was carried out in the medium consisting 60 g l71 glucose; 6 g l71

urea; 1 g l71 KH2PO4; 0.4 g l71 MgSO4 7H2O; 0.01 g l71 FeSO4 7H2O; 80 1076g l71

thiamine; and 1076g l71 biotin at 30 C for 38 h in a 3.5 l bioreactor. Penicillin was added tothe medium at t ˆ 22 h maintaining the penicillin ratio of 2000U=g DW. The agitation andaeration rates were 250 min71 and 1 vvm, respectively. Amino acids and organic acids wereanalysed by HPLC using the PicoTag method and by electrophoresis. Glucose consumptionwas followed by the DNS method.

The stoichiometrically balanced reactions involved major pathways of catabolism, anabolismincluding energy and redox balances for glutamic acid bacteria. An optimization programmewas used to solve the pseudo-state steady metabolic � ux balance equation for differentobjective functions and several experimental conditions. The computational results werecompared with experimentally measured parameters, such as speci� c substrate uptake andproduct formation rates. The � ux distribution maps showed that the cells utilized the TCA cyclein part, whereas the glyoxylate bypass was active throughout the fermentation. The results also



indicated that the pentose phosphate shunt played an important role in glutamate fermentation.Comparison of the theoretical metabolic � ux distributions with experimental results indicatedsome targets for genetic engineering as well as physiological engineering by changing thephysico-chemical environment of the cultures for improved glutamate production.

Keywords: metabolic engineering; glutamate; Brevibacterium; metabolic ¯ux; optimization.

PRE-USE POST-STERILIZATION INTEGRITY TESTINGOF STERILIZING-GRADE FILTERS

J. M. CAPPIA

Millipore France SA, France

Integrity testing is taking on much greater signi� cance in the pharmaceutical industry. Asautoclaving and steam-in-place are the most common sources of damaging a � lter, it isrecommended testing the integrity of sterilizing-grade � lters after sterilization, before the� ltration process. Post sterilization integrity testing involves wetting of the product � lter withthe standard wetting medium or the product and is a possible source of breaching sterility of thesterilized system. From the sterile � lter to the sterile vent � lter on the sterile tank is a sealed,closed and sterile system. Water used to wet the � lter before integrity testing cannot be draineddownstream of the closed � lter system, and will remain in the sterile product tank.

This paper describes the different procedures used to test the integrity of product � lters, insitu, after sterilization, without compromising the integrity of the sterile � ltration system.

PROTEIN FRACTIONATION USING CARRIER PHASE ULTRAFILTRATION

R. GHOSH

Department of Engineering Science, University of Oxford, Oxford, UK

Protein concentration, desalting and fractionation are increasingly being carried out usingultra� ltration (UF). Concentration and desalting processes are not very technologicallydemanding. Macromolecular fractionation on the other hand is a technological challenge andhas only very recently being investigated in detail. Protein fractionation using UF is stronglyin� uenced by operating and physicochemical parameters and, hence these processes need to bevery precisely ‘� ne-tuned’ to achieve satisfactory levels of fractionation. The � ne-tuningexercise includes optimization of pH, salt concentration, permeate � ux and system hydro-dynamics. Recently, the author reported a novel technique for rapid optimization of physico-chemical and operating parameters for protein fractionation.

The results of � ne-tuning for protein fractionation are dif� cult to translate into practice whenusing conventional modes of UF (i.e. dead-end and cross-� ow � ltration). The main drawbacksof conventional modes of UF include:

(1) dif� culty in adjusting pH and salt concentration of feed solutions=suspensions;(2) change in permeate � ux during process (due to concentration polarization and fouling)

leading to change in the sieving coef� cients of the membranes;(3) non-uniform concentration polarization along the length of the membrane;(4) change in pressure drop along the length of the membrane;(5) increase in concentration of the feed along the length of the membrane.

These can be overcomeby using a novelmode of operation,which can be termed Carrier PhaseUltra� ltration (CPUF). This is based on a modi� cation of dead-end UF. In the present work thepuri� cation of lysozyme from egg white proteins using CPUF is investigated. Two membranemodules in series � tted with two different membranes are used in the puri� cation process.

Keywords: ultra®ltration; carrier phase; lysozyme; puri®cation; membrane.



PROTEIN RELEASE AT HIGH CELL DENSITY CULTURE OF E. COLIBY CHEMICAL EXTRACTION

W. S. CHOE and A. P. J. MIDDELBERG

Department of Chemical Engineering, University of Cambridge, UK

The chemical extraction method developed by Falconer et al.1,2 achieved a high release ofintracellular protein from E. coli by permeabilizing the cell envelope through the combinationof 6 M urea and 3 mM EDTA. The extraction ef� ciency proved to be competitive with that ofmechanical disruption at low cell density (OD600ˆ 4). However, to be considered as a usefulprocess alternative, this method must be proved at high cell density.

In this study, the release of protein and DNA from non-recombinant E. coli JM 101 andrecombinant E. coli HMS174(DE3) expressing L1 (the major viral coat of protein of humanpapillomavirus type 16 as an inclusion abs was investigated at high cell density (OD600ˆ 160).

Non-recombinant strain showed a rapid decrease in extraction ef� ciency as cell massincreased. This decrease was explained in terms of speci� c urea concentration available per unitcell mass. Recombinant strain exhibited a completely different extraction pro� le. Virtuallycomplete release of the total protein and the target protein L1 was achieved despite the cellmass increase. DNA release also mirrored these pro� les. This suggested that the recombinantstrain has a weaker resistance to the extraction chemicals, which was con� rmed frommechanical disruption studies conducted in parallel with chemical extraction. The target protein(L1), expressed as insoluble inclusion abs, was extracted at high ef� ciency without the use ofreducing agent. This is expected to enable the direct coupling of chemical extraction with adownstream process such as expanded bed adsorption (EBA) employing metal af� nity resin. At67.2 g l71DCW (OD600ˆ 160), the complex viscosity3 measured at 10% strain and a frequencyof 10 rad s71 was 186 mPa.s, which is expected to cause processing dif� culty. Methods arecurrently being developed to reduce this viscosity in a controlled manner.

Keywords: chemical extraction; high cell density; papillomavirus; viral coat protein; expandedbed adsorption.

REFERENCES

1. Falconer, R. J., O’Neill, B. K. and Middelberg, A. P. J ., 1998, Biotechnol Bioeng, 57: 38l–396.2. Falconer, R. J., O’Neill, B. K. and Middelberg, A. P. J., 1999, Biotechnol Bioeng, 62: 455–460.3. Mackley M. R., Marshall R. T. J., Smeulders, J. B. A. F. and Zhao F. D., 1994, Chem Eng Sci, 49: 2551–2565.

A PROTOTYPE REACTOR FOR ALKALINE LYSIS DURING THE RECOVERYOF PLASMID DNA FOR GENE THERAPY AND DNA VACCINES

L. A. S. CICCOLINI, L. KIM LEE, P. A. SHAMLOU, P. DUNNILL and N. TITCHENER-HOOKER


Gene therapy is concerned with the provision of functioning genes to cells and tissues to correctgenetic disorders and acquired diseases. Initial trials involved inherited single gene diseases butas scientists and engineers gain experience in dealing with the complex materials involved ingene therapy and DNA vaccines, the move will be towards developing cures for multi-genediseases such as diabetes. The present investigation is concerned with plasmid DNA, which islikely to be a safe base for the delivery of functional, heterologous genes to somatic cells.Widespread application of this technique however, requires the ability to produce substantialquantities of pure plasmid DNA. Plasmid DNA is a large macromolecule and the authors haveshown that it is susceptible to shear damage during preparation and puri� cation. Preparation ofpharmaceutical grade plasmid DNA also involves its separation from chromosomal DNA insolution. Chromosomal DNA is signi� cantly larger, and hence more prone to shear damagecompared to supercoiled plasmid DNA. Its degradation during separation can producefragments with molecular weights comparable to those of the plasmid DNA. The presence



of such fragments in solution containing the plasmid DNA poses considerable puri� cationproblems.

In the present investigation the authors are concerned with the design of a protoype mixing-reactor for the primary recovery of plasmid DNA from the host bacterial cells and its separationfrom chromosomal DNA. Experimental engineering data will be presented together with amethodology leading to the design of a mixing-reactor for the alkaline lysis operation. The lysisstage and the neutralization stage were considered separately, and a method of mixing devisedfor each. At lysis, mixing of the cell suspension with the lysis reagent by impinging-jets, theresultant mixture was discharged into a reaction column where no further agitation to the celllysate was applied. Neutralization using potassium acetate was initiated 300 seconds later by itsaxial-jet injection from the base of the reaction column. Feasibility studies using the builtprototype reactor involved suspensions of Escherichia coli DH5a carrying the small (6.9 kbp)high copy number plasmid pSVb. Results showed that shear sensitivity of chromosomal DNAis more pronounced when the latter molecules have precipitated out of solution to form a gel-matrix upon neutralization, compared to them being denatured in the alkaline cell lysate.Separation of the gel-matrix and crude lysate was by � otation and the crude lysate wasrecovered by drainage. The reactor con� guration gave a plasmid yield of 90% and a plasmidpurity of 56%. Contamination by suspended solids was 25 mg ss=mg SC plasmid, contaminationby soluble proteins was 18 mg sp=mg SC plasmid, and the volumetric recovery was 92%.Compared to the conventional small-scale operation, using the prototype gave an improvedplasmid yield, purity and a lower contamination by suspended solids. Volumetric recoverieswere similar but contamination by soluble proteins was higher. The results con� rm theapplicability of the proposed methodology for process design and scale-up.

REFOLDING AND AGGREGATION OF RECOMBINANT TRYPSINOGEN

A. M. BUSWELL1, M. EBTINGER2, A. A. VERTEÁ S2 and A. P. J. MIDDELBERG1

1Department of Chemical Engineering, University of Cambridge, Cambridge, UK2Lilly France Centre de Production de Fegersheim, France

The inclusion abs process route is attractive for protein manufacture both in terms of achievableexpression levels and the ease of initial puri� cation. Overall process cost is often determined byrefolding yield and the refolding operation must therefore be carefully optimized. Dilutionrefolding is the simplest and most extensively used refolding operation. Signi� cant yield lossesoften occur during dilution refolding due mainly to aggregation. The degree of aggregation andhence renaturation yield is signi� cantly affected by reactor operating variables, yet a systematicstudy has not been reported.

This study investigates the effect of operating variables on the dilution refolding ofsolubilized r-trypsinogen inclusion bodies in a pulse-fed stirred reactor. Variables investigatedwere inclusion abs washing, stirring speed, feed rate, concentration of solubilized r-trypsinogenand the concentration of urea during inclusion abs solubilization. The effect of baf� es in thereactor was also investigated. The yield of renatured r-trypsinogen varied between 12 and 20%as the operating variables were varied.

The variation of yield with operating variables clearly demonstrates the need to carefullyoptimize physical refolding conditions. It is not suf� cient to merely optimize the chemicalenvironment. Interestingly, increased mixing intensity adversely affected yield. These resultsindicate that yield is determined not only by the ef� ciency of dispersion, but also by the localchemical environment of the protein as it folds, and the rate of change of this environment. Therelationship between physical refolding variables and chemical environment is complex andrequires further characterization.

Keywords: protein refolding; protein aggregation; trypsinogen; mixing.



STRATEGIES FOR PILOCARPINE PRODUCTION BY PILOCARPUSPENNATIFOLIUS CELL CULTURES

F. MAVITUNA and C.-W. TANG

Chemical Engineering Department, UMIST, Manchester, UK

Pilocarpus pennatifolius is a tree that grows wild in South America accumulating imidazolealkaloid pilocarpine in its leaves. This chemical is used widely in glaucoma treatment. Since thechemical synthesis of this compound has proved very dif� cult and uneconomical, it is currentlyobtained by extraction from the leaves. Plant tissue culture techniques can provide analternative for the in vitro production. It is dif� cult however, to produce cell cultures from awoody plant species.

The authors used the leaves and stems of a specimen tree grown from seed in theirlaboratories to initiate callus cultures on different solid media using different photo regimes:16 h light=8 h dark or complete darkness. There was no signi� cant difference between the darkand cycled light photo regime for callus initiation however, white callus was induced in the darkand yellow in cycled light photo regime. With the four different initiation media tested,production of callus was best on Woody Plant medium (W3) supplemented with Murashige andSkoog vitamins, 1 mg l71 NAA (naphthaleneacetic acid), 0.5 mg l71dimethylallyladenine,0.5 mg l71 zeatine and 20 g l71 sucrose. Leaf was the best explant for callus initiation, over90% of the explants produced callus in 35 days.

The suspension cultures normally consisted of very large aggregates. The authors obtained� ne cultures using a homogenization method. After homogenizing in a Waring 8010s blender,the suspension culture took about 2 to 4 weeks to recover. The � ne suspension cultures were alsoimmobilized in reticulate polyurethane foam particles. Modi� ed Berlin’s production mediumwith 80 g l71 sucrose was used to induce pilocarpine production. Growth, viability, sugar uptakeand pilocarpine production were monitored in batch cultures in shake � asks and a 2 l bioreactor.Pilocarpine, sucrose, glucose and fructose were analysed by HPLC. Cultures had faster growthrates in complete darkness and reached stationary phase after 20 days. Pilocarpine productionstarted on day 9 in suspension culture and reached the maximum concentration, 124 mg l71 onday 15. In immobilized cultures, pilocarpine production started on day 18 and reached themaximum concentration, 96 mg l71 on day 21. No pilocarpine was detected after day 21 in thesuspension culture. Immobilized culture continued to produce pilocarpine after day 24.

Keywords: pilocarpine; Pilocarpus pennatifolius; plant cell cultures; immobilized cells.

A 3-D NETWORKS-OF-ZONES ANALYSIS OF GAS-LIQUID MIXING,OXYGEN MASS TRANSFER AND NUTRIENT PARTIAL SEGREGATION IN A

STIRRED BIOREACTOR

H. HRISTOV1, R. MANN

1, V. LOSSEV

2, S. D. VLAEV

3and P. SEICHTER

4

1Department of Chemical Engineering, UMIST, Manchester, UK2Research Institute for Antibiotics, Razgrad, Bulgaria

3Institute of Chemical Engineering, Bulgarian Academy of Sciences, So®a, Bulgaria4Techmix, Brno, Czech Republic

Gas-liquid stirred vessel two-phase mixing accompanied by bioreaction has been analysed by a3-D networks-of-zones in which non-axisymmetric elements can be incorporated. Particularlythe effect of the feed of nutrient from a single dip-pipe can be included so that previous 2-Dlimitations of axisymmetry are avoided (Vlaev et al.1, 2000). The turbulent swirl � ow createdby the impeller uses clockwise and anti-clockwise swirl coef� cients, which can be estimatedusing image-reconstruction 3-D visual modelling (Rahimi et al.8, 2000).

The simulations can provide detailed predictions of the local gas hold-up distribution, thelocal mass transfer area, the partial segregation of both the oxygen and the nutrient and theextent of oxygen absorbed by bubbles. The overall gas hold-up and mass transfer area areobviously summations of the local values and the local and overall reaction rates can bepredicted as well as the local and overall oxygen absorption � uxes.



Simulations are presented for a 3 2 (10 10) 60 networks-of-zones for a 3 m3 triple-impeller industrial pilot-plant bioreactor. The theoretical predictions are demonstrated usingcolour-augmented 3D contour maps and solid-abs isosurface images created by AVS graphics.

Severe non-uniformity of gas hold-up distribution and consequently spatially uneven oxygenmass transfer create signi� cant partial segregation of both oxygen and nutrient. The simulatedbioreactor is predicted to be far from perfectly mixed, so that Tylosin producing micro-organisms will experience large variations in dissolved oxygen and nutrient concentrations asthey circulate around the stirred fermenter.

Keywords: gas-liquid mixing; stirred bioreactor; oxygen mass transfer; networks-of-zones;partial segregation.

USE OF YATES’S ALGORITHM TO DETERMINE THE EFFECTS OF THEOPERATING PARAMETERS ON THE PRODUCTION OF

S. CEREVISIAE

N. BURSALI, B. AKAY, S. ERTUNC, H. HAPOGÆ LU and M. ALPBAZ

Ankara University, Department of Chemical Engineering, Ankara, Turkey

It is generally proposed and necessary to determine the effect of the operating parameters on theprocess whether chemical or biochemical. In order to determine the effects of the operatingparameters, experimental studies are necessary. It is possible to use two different experimentalpolicies; one of them is the traditional method which is called One-factor at a time, the other isthe Factorial Experimental Design Technique. There are a lot of advantages in using FactorialExperimental Design Technique over the One-factor at a time method. Recently there has beena lot of research related to the use of Factorial Experimental Design Technique. It is importantto emphasize the application of this technique together with the use of the Yates’s Algorithm.This is a quicker method for calculating effects of the operating parameters on the process.

In this study it was proposed to determine the effects of four operating parameters whichwere temperature, pH, agitation rate and air � ow rate on the S. cerevisiae production underaerobic conditions in a batch operated jacketed bioreactor. Two-level Factorial ExperimentalDesign Technique was used in order to construct the experimental design matrix. Thistechnique estimates not only the main effects but also the interactions between the factors.The experimental design matrix was constructed according to this technique which consist of24ˆ 16 experiments. Each of the experiments was realized for 8 h because at the end of thistime a stationary phase was observed. Due to the fact that replication increases the economicload and work load, each experiment was only carried out once.

The experimental results of the design matrix were analysed to determine the effects of eachfactor and interaction factors according to the Yates’s Algorithm. It is possible to judge thesigni� cance of effects from an estimate of variance obtained by replication when available,from higher order interactions (assumed due to the noise), or by plotting effects on normalprobability paper. Above all, in this study it was proposed to use Yates’s Algorithm which givesrapid calculation of the effects of the operating parameters.

Finally by examining the results of the Yates’s Algorithm, it was concluded that main effectof the temperature and maybe the effect of interactions between the four factors aredistinguishable from the noise. A rise in temperature from 27 C to the 32 C causes an increasein yield by about 0.83%.

Keywords: S. cerevisiae production; process optimization; Yate’s Algorithm; operatingparameters; Factorial Experimental Design; statistical optimization.



USING A GRAPHIC USER INTERFACE FOR VISUALIZING DATA FOR RAPIDBIOPROCESS EVALUATION

P. GRIFFITHS, Y. ZHOU and N. TITCHENER-HOOKER

Department of Biochemical Engineering, University College London, London, UK

Recent developments in biological science have enabled the discovery of advanced therapeuticdrugs. The pace of development in the delivery of these candidate drugs now needs to bematched by advances in the methods employed for large-scale production. Unlike in othersectors, computers have not been used extensively for the design and evaluation of biopro-cesses. Instead traditional design methods, which are largely empirical, relying heavily onexpensive pilot plant trials, have been used. A direct consequence is that in order to get theproduct to market rapidly often bioprocess designs are sub-optimal. The use of simulationtechniques is one important way of speeding up the design process and of evaluating rapidlydifferent process options. This paper presents results of work at UCL conducted to examine therapid process evaluation of multi-step sequences. It proposes the use of a Graphic UserInterface (GUI) to assist in this and illustrates the approach by application to real bioprocessdesign problems.

Bioprocess unit operations variables can be divided into two types, control variables andperformance of output variables. Control variables (i.e. fermenter growth rates, disruptionpressures and centrifuge � owrates) are controlled by the operator whilst output variables (i.e.yield and purity) are determined by where the control variables are set. This paper presents amethodology that enables the rapid evaluation of any feasible set. Such a set corresponds to theunique combinations of control variables which result in the output variables reaching pre-setprocess performance criteria. The feasible set is visualized in the form of a three-dimensionalspace whose axes are de� ned by the control variables. Insight generated through such plots canbe used to help choose the most appropriate operating conditions needed to realize a speci� edlevel of performance.

In the present investigation this techniquewas applied to experimental data from a pilot scaleprocess for the production of a labile intercellular enzyme, alcohol dehydrogenase (ADH)expressed in Sacchromyces cerevisae. The GUI was used to enable the user to evaluate andcompare different processing options. For the ADH process the feasible region, under theconditions examined, was largest for a growth rate of around 0.19 h-1. smaller growth ratesresulted in the production of smaller cells and therefore smaller cell debris, which requiredmore stringent centrifugal operating conditions so as to reduce the levels of debris contamina-tion to pre-speci� ed levels. At higher growth rates the transition from oxidative to oxio-reductive growth reduced the yields of alcohol dehydrogenase but at the same time the cellwalls weaken resulting in smaller debris. The combined effect leads to smaller operatingregions at higher growth rates. Examples of the GUI being applied to identify conditions inwhich the feasible space becomes discontinuous will also be presented. Such events areimportant since operation in such regions is not possible. The GUI provides a rapid means ofidentifying these events and hence the fast evaluation of the corresponding bioprocessconditions needed to stay within the feasible space.



advances in biochemical engineering

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