biochemical engineering in biotechnology

8/6/2019 Biochemical Engineering in Biotechnology

1/20

Pure &Appl . Chern.,Vol. 66, No. 1, pp . 117-136, 1994Printed in Great Britain.@ 1994 IUPAC

INTERNATIONAL UNION OF PUREAND APPLIED CHEMISTRYAPPL IE D CHE MIST RY DIVISIONCOMMISSION O N B I O T E C H N O L O G Y

Interrelations of Chem istry and Biotechnology -IV?BIOCHEMICAL ENGINEERINGIN BIOTECHNOLOGY

(Technical Repo rt)

Prepared for publication byM. MOO -YOUN G and Y. CHIST I

D epa rtm ent of C hemical Engineering, University of W aterloo, W aterloo,Ontario, Canada N2L 3G1

*Membership of the Commission during the period (1991-93) in which this document wasprepare d was as follows:Chairman: J . L. Fox (USA); Vice Chairman: H. G . W . Leuenberger (Switzerland); Secretary:H. K. Kolbl (FRG ); Titular Membe rs: A . M. Boronin (Russia); Y. Yam ada (Japan ); AssociateMembers: G . W. Engels (FR G) ; J .-M . Masson (France); M. Moo-Young (Cana da); B. Nagel(FRG); S . Ramachandran (India); National Representatives: M. A . L. El-Sheikh (Ar ab Republicof E gypt); R. P. Gregson (A ustralia); M . van Montagu (Belgium); V. Moritz (Brazil); V. N .Beshkov (Bulgaria); J. K63 (Czechoslovakia); L. Kjaergaard (Den mark ); D . Kyriakidis (Greece);J. Ho116 (H ungary ); I. Go ldberg (Israel); E . Rizzarelli (Italy); M. Ariffin Hj A ton (M alaysia);G . B . Petersen (New Ze aland); M. J. T. Carron do (Portugal); D.-S. Lee (Republic of Kore a);P. Adlercreutz (Sweden); 0.Yenigun (Turkey); P. N. Campbell (UK).?The Comm ission has chosen this series with a view to intensify the interre lations of chem istryand biotechnology. By improving the knowledge of chemists in the field of biotechnology it ishoped to initiate m ore ideas for applying biological metho ds in chem istry, inspire mo re use ofchemical knowledge in the biological sciences and help scientists in bo th fields to work closertogether. In the articles in the ser ies, to be published in this journal, outstanding exp erts in theirrespective fields will give (a) an overview on topics related to the practical use of biotechno-logical metho ds in organic chemistry, (b) an outlook on upcoming research topics, their impacton existing areas and their potential fo r future developm ents.Th e Comm ission solicits com men ts as well as suggestions for future topics, and will aim to helpin providing answers to any questions in this field.Nam es of countries given after Mem bers nam es are in accordance with the ZUPAC Handbook1991-93; chang es will be ef fected in the 1994-95 editio n.Republication of this report is permitted without the need for form al IU PA C permission on condition that anacknowledgement, with full reference together with IUPAC copyright symbol (0994 IUPAC), is printed.Publication of a translation into another language is subject to the additional con dition o f prior approva l from herelevant IU PA C National Adherin g Organization.


2/20

Biochemical engineering in biotechnology(Technical Report)

Abstract. The role of biochemical engineering in biotechnology, as the technology ofindustrial exploitation of biochemical systems, is illustrated. The scientific disciplinesunderlying biotechnology are pointed out. Biochemical engineering is described as anestablished discipline, drawing upon chemical, biological and engineering sciences andconcerned with design, development, implementation and op eration of processes and processplant for handling biological m aterials and b iocatalysts. The principal aspects of bioprocessingare outlined , including processing steps, biocatalysts, bioreactors and downstream processingconsiderations, to impart a flavour of the biochemical engineering activities and theirinteraction with chemical sciences.As shown by exam ples, applications of bioprocessing andbiochemical engineering are wide ranging, covering such major facets of civilization ashealthcare, agriculture and food, resource recovery, bulk and fine chemicals, energy andenvironmental pollution abatement.

I N T R O D U C T I O NBiotechnology is considered variously as the science of genetic m anipulation, o r th e technology ofindustrial exploitation of biochemical systems, genetically modified or not. Fo r th e p urpose of thisarticle, we subscribe to the second definition which pre date s modern developments in m anipulationof genes, more popularly called genetic engineering. Th e latter, combined w ith other aspects ofmolecular biology and biochemical sciences (e.g., biomimetic chemistry, biochemistry, bioinorganicchemistry, molecular imm unology, microchemical analysis and protein engineering), will remain thepowerful fundamental scientific base1,2 of industrial biotechnology in the foreseeable future. Thismultidisciplinary nature of biotechnology is recognized in Figure 1,where it is seen to b e a subsetcomposite of biological, chemical and engineering branches of k n ~ w l e d g e . ~

Biological and biochemical systems - microorganisms, animal and plant cells, living or dea d,genetically modified, otherwise selected, or wild and material derived from these organisms (e.g.,enzymes, antibodies, cellular organelles, etc.) - are the workhorses of industrial biote~hnology.~These systems, or biocatalysts, carry out a vast array of biochemical reactions which produce usefulchemicals (e.g., foods, pharm aceu ticals, fuels, esticides, fine chemicals, fertilizers, plastics, andbiotransformati~ns)~~~~~~~~nd degrade unwanted ones (e.g., waste treatment);3p15-17 hence, th eimpact of biotechnology on all facets of human civilization. Biotechnology, relying on renewableresources, is a sustainable technology which may eventually replace many of the current non-renewable-resource-dependent production methods. Depletion of finite resources, resulting changesin production economics, environmental factors and improvements in biotechnology-basedproduction, will drive the industrial restructuring. The present, more defined niche of industrialbiotechnology - generally in areas in which a biochemical product or service cannot be obtainedby more traditional means, or when alternative technologies ar e clearly uneconomic - is bound toexpand. Moreover, in contrast to many other high technology developments which have largelyremained confined to the more advanced nations, biotechnology promises faster and relevantreturns even in th e lesser developed economies.18 This realization has s purred noticeable activity

industrial catalysts to name a ? I3 modify existing ones (e.g., numerous

118


3/20

Biochemical engineering in biotechnology 119

in the Third World countries which isalready bearing fruit.Just as the chemical industrybefore it, industrial biotechnolojgneeded people capable of developing

production processes with theb i o s c i e n t i s t s ( b i o c h e m i s t s ,microbiologists, geneticist, etc.), andtransforming these processes intoeconomically viable large-scale plantsfor products and services. Theseprocesses and process plants had to bedesigned, built, operated and controlled.Thus arose the profession ofbiochemical or bioprocess engineer.Combiningbioprocess-relevantchemicalengineering knowhow (process analysisand control, bioreaction and reactore n g i n e e r i n g , 19520 t r a n s p o r tphenomena,21y22 fluid mechanics,thermodynamics, unit operations,bioseparations,3~~~*~~rocess design and

ENGINEERINGSCIENCES

Figure 1. Multidisciplinarynature of biotechnology.

economic^^^) with the specificities of biological systems (mi~robiology,3~ i o ~ h e m i s t r y , ~ ~ ~ ~ ~b i o c a t a l y ~ i s , ~ ~ ~ ~ ~terile process engineeri~~g~~-~~),iochemical engineering is now a distinctdiscipline30 proven through the 1940s development of the antibiotics indust to the latestappreciate biochemical engineering and its interactionswith other chemical disciplines, we will lookat bioprocessing in some detail. General features of bioprocessing will be examined first, followedby bioreactors and biocatalysts; the basics of design of bioreactors will be seen, as well as somedownstream supporting operations.

hybridoma and recombinant cell culture processes and enzyme bio rea~ to r s .~ *~*~:5*31 To better

BIO PRO CESSINGA bioprocess is typically made up of three steps shown in Figure 2. The raw material which maybe of biological or non-biological origin, must be converted to a form suitable for processing. Thisis done in a pretreatment step which may involve such operations as sorting, size reduction,chemical or enzymatic hydrolysis, medium formulation and sterilization and other preparatoryoperation^.^^ The pretreatment step is followed by one or more bioreaction stages where the aimmay be the conversion of a substrate (raw material) to biomass (microbial or eucaryotic cells), orbiomass and some biochemical including enzymes. Alternatively, the conversion may use dead ornon-viable whole cells (immobilized or freely suspended) or purified enzymes as the biocatalyticagency. Bioreactors form the core of the bioreaction step.30 The material produced in bioreactorsmust usually be processed further in the downstream section of the process to convert it to a usefulform. Downstream processing consists of predominantly h sical separation operations which aresolid-liquid separation (centrifugation, filtration, sedimentation, f l ~ a t a t i o n ) : ~ ~ ~ell-disruption,precipitation, crystallization, adsorption, chromatographic separations, membrane separations(~ltrafiltration?~ i~rofiltration~~),iquid-liquid ex tra~ tion?~istillation, evaporation and drying(spray drying, freeze drying, drum and tray drying,etc.).3>z,24730938-42he purified product may haveto be in different physical forms (liquid, slurry, powder, crystalline, emulsions) and additional stepsfor product stabilization and formulation may be needed.

aimed at purification and concentration of the product.28 12 Some commonly used operations are

The design of a bioprocess and the engineering of the process equipment require a carefulconsideration of the physical and chemical properties of the material being handled and a


4/20

120 COMMISSION ON BIOTECHNOLOGY

delimitation of the maximum processingstresses (temperature, pH, shear forces,contamination, pressure, exposure todenaturing chemicals) that the materialmay safely tolerate.30 Typically, abioprocess must operate within thephysiological ranges of pH andtemperature (pH - 7.0; temperature s37 OC), the specific conditions beingvery process dependent. Sometimesduring processing exposure of materialto relatively severe environmentalconditions is unavoidable. In such cases,the duration of exposure is minimisedand other precautions (e.8, lowtemperature; addition of chemicals toreduce oxidation, efc.) are taken toreduce the impact of exposure.Generally, microorganisms and enzymesare not damaged by pressures whichwould normally be encountered ( s 2MPa) in bioreactors but carbon dioxideassociated toxicity effects due toincreased solubility of C02 at higherpressures may become important.30

Velocity gradients within fluids,or close to moving surfaces (e.g.,agitators; solid particles, liquid dropletsor gas bubbles), can potentially damagefragile biocatalyst. The sensitivity ofcatalysts to these shearing forces mayvary widely. In general, bacteria andyeasts which grow as small individualcells are quite shear tolerant.Geneticallyengineered species and wall-

PROCESS STAGES OPERAT I0NS

~~

Sort ingSievingComminutionHydrolysisMedium formulat ionS ter i l i za t ionI I I

BIOREACTION+-iomass product ionMetabolite biosynthesisImmobilized enzyme andcel l catalys isBiotransformationsI I I1(3roduct F i l t r a t i onCentr ifugat ionSedimentat onFlocculat ionCell Disrupt ionExtract ionU l t ra f i t ra t ionPrecipi o t ionCrystalizat ionChroma og raph yEvaporat ionDryingPackaging

Figure 2. Bioprocess stages and unit operations.

less mutants (aid pro top last^^^) are frequently susceptible to shear damage due to their weaker cellwall/membrane structures or modified physical feature^.^,^^,^^ Filamentous bacteria and mycelialfungi which have larger particle dimensions do show signs of mechanical damage in high shearfield^.^,^,^^ Similarly, plant and animal cells are more sensitive to shear.44 Enzymes, with theexception of multienzyme complexes and membrane-associated enzymes, are not damaged by shearin the absence of gas-liquid interface^.^^*^^ Many biofluids (e.g., some fermentation broths, blood)show shear-dependent flow b e h a v i ~ u r , ~ ~ ~ ~ut quantification of shear fields in process equipmentis not easy.&

Pretreatm ent considerationsExcept in biological treatment of wastes, most productive fermentations requiring microbial oreucaryotic cell cultivation depend on maintenance in culture of the single species with which thefermenter was initially i n o c ~ l a t e d . ~ ~ ~ ~ ~ontamination from other microorganisms and viruses isprevented by sterilization of the liquid and gaseous feeds to the fermenter,5~28~30~47terileengineering of the bioprocess plant and use of sterile processing techniques.5*6v26*28t2g

Liquids such as water and salt solutions can be inexpensively sterilized by filtration throughabsolute or properly designed depth filters which retain the contaminating microorganisms.26~2g~30This method is applicable when the liquid is free of other suspended solids. Most industrial


5/20

Biochemical engineering in b iotechnology 12 1

fermentation media contain components with suspended solids (e.g.,molasses, corn ste ep liquor,yeast extract, soya bean meal, fish meal). These components must be heat sterilized eitherseparately or with the m e d i ~ m . ~ , ~ , ~ ~hermal denaturation of one or more enzymes in thecontaminating microorganisms is used to render them non-viable. However, the temperaturesnecessary to cause effective microbial kill also cause reactions which destroy such essentialthermolabile media constituents as vitamins and growth factors. Additionally, reactions occurbetween the components of the medium, e.g., the Mailard reaction between carbohydrates andproteins, leading to formation of products which adversely affect microbial growth, lowering thefermentation yield.30 Success of industrial fermentations d epe nds o n attaining a n econom ic balancebetween insufficient sterilization with consequent frequent contamination-associated batch lossesand yield reduction due to over-cooking. The rate constant for thermal denaturation ofcontaminating microbial spores (kd) s a function of temperature

where AE is the activation energy for destruction of a particular microo rganism ,A is the Arrheniusparameter and T is the absolute temperature. Bacterial spores such as those of BaciZZwstearothennophilw and Clostridum botulinum are some of the most heat resistant, and sterilizationprocesses a re designed to be effective against them.30 Th e activation energy for th e destruction ofthese spores is 2.5-2.9 x 108 Jokmol-l and A is - 1.6 x ld6 - l . Similarly, the denaturation of thenutrients (k,) also depends on temperature-AE,IR Tk, -A, e

From equations (1) and (2) it follows tha t

The activation energy for the thermal deactivation of most nutrients ( A E , ) is much lower than A Efor microbial d e s t r u c t i ~ n . ~ ~ * ~ ~or example, AE, for thiamine hydrochloride (vitamin B6) is only 9.2x lo 7 J*kmol-l. Thus, from this simple analysis of denaturation kinetics, we see that sterilizationat high temperature for a shorter time is preferred for maximizing microbial destruction relativeto nutrient loss (equation 3 ) , i.e., higher tem perature operation would achieve a higher kd/knS3Ohisindeed is the basis of HTST (high-temperature-short-time) sterilization now being increasinglyadopted in the bioprocessing industry.29

Medium formulationCareful formulation of growth and production media is a prerequisite for successfulf e r m e n t a t i ~ n . ~ * ~ * ~ ~icrobial nutritional requirements and env ironmental needs have to be met aswell as several techno-economic constraints. Medium develop men t aims to maximize product yieldat m inimal medium cost. The choice of the medium affects downstream processing and upstream(pretreatment) activities; therefore, medium design should be carried out in an overall processcontext.30 Th e use of a purer carbon source such as glucose compa red, say, with molasses mayreduce purification problems and simplify pollution control and waste treatment.

The medium must provide sufficient carbon, nitrogen, minerals and other nutrients to yieldthe required amount of cell mass and product. Minimum requirements are estimated from thesto ichiometry of g rowth and p roduct f o r m a t i ~ n . l ~ * ~ ~n general,

C-source + N-source + minerals + specific nutrients (vitamins, horm ones) + 0 2 ..* cellmass + product + C 0 2 + H20.

Most nutrients are supplied at levels well above the minimal needs.


6/20

12 2 COMMISSION ON BIOTECHNOLOGY

Al R

STIRRED BUB BLE FLUIDIZED EXTERNAL INTERNALTANK COLUMN BED LOOP LOOP

Al RLIFTIRLIFTFigure 3. Some common ferm entation bioreactors.

Fo r the production of metabolites, understandin the biochemistry of pro duc t form ation hasdirected by precursor feeding (e.g., in penicillin fermentations), limiting some nutrients andproviding some others. Even trace amoun ts of some media co mponen ts (e.g., various metal ions incitric acid production by Aspergirrus nige9) can have a major impact on the yield of the desiredproduct or its degree of contamination with other unwanted byproducts. These yield-suppressoryand stimulatory effects are usually related to inhibition or activation of enzyme action, or toenhan cem ent or suppression of production of particular enzymes by the med ia comp onents o r bysome product of metabolism of these component^.^*^^^^ Frequently, know ledge of the biosyntheticpathways of ferm enta tion products has lagged behind industrial practice of these fermentations; trialand error methods have been the basis of medium formulation and continue to play a dominantrole.

In comparison with microbial culture media, the media used in animal cell culture aregenerally better defined, containing most of the 20 amino acids, several vitamins, simple sugars ascarbon sources (e.g., glucose), salts for osmotic pressure adjustment as well as for nutrients andtrace elements. In addition, these media are usually supplemented with blood sera (e.g., bovineserum, fetal calf serum, horse serum) at 5-20 % by volume. Se rum provides essential hormones,lipids and othe r growth factor^.^,^

Natural media components such as blood sera, molasses, corn steep liquor, often showtremendous variability between batches. Good chemical and biochemical quality control of thesesubstances, relying on many of the techniques of modern analytical chemistry, is essential tomaintaining consistency in fermentation performance.

an important role in medium formulation pra~tice.~,25 The course of a fermentation can be

B I O RE A C TO R S A N D B I O R E A C T I O NIndustrial bioreactors (fermenters) for sterile operation are usually designed as pressure vessels,irrespective of whether the stirred tank, bubble column, fluidized bed or the airlift configuration isemployed.27 The se basic bioreactor configurations are shown in Figure 3.

A typical ferm enter has the features shown in Figure 4.Th e re actor vessel is provided witha vertical sight glass, and side ports for pH, temperature and dissolved oxygen sensors are a


7/20

Biochemical engineering in biotechnology 123minimum requirement. A steam-sterilizable sample valve is provided.Connections for acid and alkali (for pHcontrol), antifoam agents and inoculumare located above the liquid level in thereactor vessel. An air (or other gasmixture) sparger supplies oxygen (andsometimes C 0 2 or ammonia for pHcontrol) to the culture.A harvest nozzleis located at the lowest point on thereactor vessel. When mechanicalagitation is used, either a top or bottomentering agitation system may beemployed. The shorter agitator shaft onbottom entry design often eliminates theneed for steady bearings within thevessel, hence an easier to clean andsterilize bioreactor configuration can bea~hieved.~ ' he bottom placement ofagitator and its drive allows the top ofthe bioreactor for a spinning orvibrating cell retention mesh which issometimes used in animal cell culturebioreactors. The shaft of the high speedmechanical foam breaker (Figure 4) isprovided with a single or doublemechanical seal at the point of entry inthe reactor vessel. Steam sterilizabledouble seals with sterile waterlubrication are preferred, and may be anecessary requirement whenbiohazardous material must becontained (e.g., during production ofvaccines and other high bioactivitysubstances). Double seals are used alsoon the bottom mounted agitator shaft;however, sealless magnetically coupleddesigns are distinctly su erior whentorque limitations permit.2Y

The reactor is either providedwith a manhole, or the top isremovable. Flat headplates arecommonly used on smaller vessels, buta domed or elliptical construction of thehead is less expensive for larger units.The head plate supports a rupture disc,air exhaust ports, nozzles for media or

t

AIR-l 3 517

STEAM1

134JFigure 4. A typical fermenter: (1) reactor vessel;(2) jacket; (3) insulation; (4) shroud; (5) inoculumconnection; (6) ports for pH, temperature an ddissolved oxygen sensors; (7) agitator; (8) gas sparger;(9) mechanical seals; (10) reducing gearbox;(11) motor; (12) harvest nozzle; (13) jacketconnections; (14) samplevalve with steam connection;(15) sight glass; (16) connections for acid, alkali andantifoam chemicals; (17) air inlet; (18) removable top;(19) medium or feed nozzle; (20) air exhaust nozzle;(21) instrument ports (several); (22) foam breaker;(23) sight glass with light (not shown) and steamconnection; (24) rupture disc nozzle.

feed addition, and for sensors (e.g., foam electrode) and instruments (e.g., pressure gauge); a foambreaker may also be located on the headplate. A sight glass with light is provided and can beinternally cleaned by a jet of steam condensate. The vessels are invariably jacketed. Chloride-freefibreglass insulation, fully enclosed in a protective shroud (Figure 4), is applied to the jacket. Thejacket and the reactor are provided with overpressure protection: a relief valve on the jacket or itsassociated piping, a bursting disc on the bi~reactor.~' ot all of the items shown in Figure 4 arerequired in every application.


8/20


The vessel should be fully draining, it should have a minimum number of ports, nozzles,connections and othe r attachments consistent with the cu rrent and anticipated future n eeds of theprocess. Th e bioreactor should be free of crevices, and stagn ant areas where po ckets of liquids andsolids may a c c u m ~ l a t e . ~ ~ ~ ~ ~he vessel should have few internals; the design should take intoacco unt the clean-in-place (CIP ) Au toma ted cleaning of process machinery by CIPtechniques relies primarily on the action of cleaning chemicals on soil. Selection of cleaning andsanitizing agents with respect to soil-type, water quality and compatibility with the processequipment are important considerations. The cleaning action of chemicals is aided by heat, highvelocity flow or j et sprays.27

All materials of construction must withstand the physical-chemical conditions encou nter edduring cleaning (e.g., highly alkaline detergents), and processing. The materials which come incontac t with the pro cess fluids should be non-reactive, non-additive, non-absorptive, and durable.27All product contact surfaces must be capable of being easily cleaned using cleaning-in-placetechniques. Sterilization should be easy. For a great majority of applications, austenitic stainlesssteels are the preferred material of construction for b i o r e a ~ t o r s . ~ ~As other important considerations, the bioreactor must be designed to satisfy the oxygen

demand of the microorganisms;3~21~22~4s~4ghere must be sufficient agitation for suspension ofmixing of additives (nutrients, chemicals for pH and foam control) and mass transferbetween g a s - l i q ~ i d , ~ ~ ~ ~ ~ - ~ ~o l i d - l i q ~ i d , ~ ~r liquid-liquid (e.g., hydrocarbon dispersions in waterM)phases. M ultiphase hydrodynam ics m ust be given a t t e n t i ~ n . ~ ~ ~ ~ ~ * ~ ~he heat generated duringbioreaction must be evalua ted using thermochem ical an d kinetic data19*30*57~5snd the reactor m ustensure adequ ate heat removal for temp erature control; heat supply capability for sterilization m ustalso be c o n ~ i d e r e d . ~ ~ * ~ ~ome fermentations (e.g., animal cell cultures) can be very sensitive totemperature fluctuations.

Fermentation, growth and product formationA sterilized batch fe rme nter containing a properly developed medium, sufficient aeration (oxygensupply; C02 emoval), supplies of p H control chemicals and antifoams must be inoculated with thedesired microbial specia to initiate the fermentation. The inoculum consists of a microbialsuspension in rapid exponential growth added a t a concentration of 5-10 % of working volume ofthe f e r m e ~ ~ t e r . ~ ~nimal and plant cell cultures, being slower growing, require larger inocula (upto 25 %) to avoid having long fermentation times (costs) in the production vessels. Becauseindustrial fermenters tend to be quite large (up to 10 m3 in animal cell culture; 100-300 m3 inmicrobial fermentations), inoculum prepara tion from seed bank often requires several fermentationsteps in series: shake flasks (T-flasks and roller bottles in animal cell culture), seed fermenter andsecondary (or tertiary) seed f e r m e n t a t i ~ n . ~ ~ ~ ~ ~ ~ ~ ~or spore forming microorganisms, quantities ofspores produced for inoculation in a seed stage may be blown into the production ferm enter withthe ingoing sterile air.

Unlike most microorganisms which are capable of growing in suspension unattached to asolid surface, certain animal cells are anchorage dependent.6 For these, growth surface in large scaleculture is provided most comm only by suspending solid (e.g., glass, polystyrene) microspheres (150-300 pm diam eter) in the culture medium.6 When required, as during inoculation of the next largerferm enter in the produ ction train, the cells can be dislodged from the microcarriers by ad dition oftrypsin, a proteolytic enzym e. Following inoculation the growth of the microorganisms follows thetypical pattern shown in Figure 5. An initial eriod of no cell growth, the lag period, is soongrowth h is to ry of the inoculum, the inoculum size and the composition of the m e d i ~ m . ~ ~ * ~ ~hecomposition in the seed and the production fermenters should be identical to avoid excessive lag.Additionally, as pointed out earlier, rapidly growing cells (late exponential growth phase) shouldbe used for inoculation and the volume of the inoculum (typically 510% of final working volumeas mentioned earlier) should be such that possible osmotic shock effects on dilution in the largervessel are minimal.30 Also, the environmental conditions (p H , tem perature, dissolved oxygen level)in the s eed and produ ction stages should be identical during inoculation. So me animal cell cultures

fo llo wed by th e ex po nentia l gro wth p h a ~ e . ~ * ~ ~ ,: y60 The length of the lag phase is related to the


9/20

Biochemical engineering in biotechnology 12 5

Figure 5. Progression of cell growth.

may not grow if the cell concentrationat inoculation is less than 1-3 x 105cells/ml Consistency through theinoculum building stages is essential forreproducible behaviour of theproduction fermentation.61Although the lag phase ispreparatory to rapid exponential growthphase, it ties up the fermentation plantunproductively and fermentationoptimization should attem t to reduce

continuous culture methods). Duringexponential growth, cell mass (ornumber) increases exponentially. For acell mass concentration X, at thebeginning of exponential growth (X,usually equals the inoculumconcentration in the fermenter) theconcentration X at any time t can bedescribed by the kinetic equation

or eliminate the la$ f (e.g., by

- I Lag phaseII i

1 I I I0 FERMENTATION TIME

where p and kd are the specific cell growth and death rates, respectively. During exponential growthkd is usually much smaller than p and a biomass doubling (td) can be shown to be

In general, the doubling times (time to double cell mass) for various classes of microbial andeucaryotic cells increase in the order: bacteria, yeasts, moulds, protozoa, mammalian and insectcells, plant cells.30 For any given species, the doubling time is dependent on the growth mediumand environmental factors. In the limit, microbial growth is a reflection of biochemical reactionswithin the cell and like chemical reactions, growth is speeded by increase in temperature until thepoint where thermal denaturation of the enzyme catalysts causes precipitous decline in growthrate.ll In nature, extremophilic microorganisms thrive at temperatures exceeding 100 OC (e.g., involcanic vents, hot springs).2 With improved understanding of the molecular and other featureswhich permit extremophilic enzyme action at such high temperatures, industrially useful enzymesmay potentially be synthesized for enhanced rate bioreactors.

B l O R E M E D l A T l O N A N D W A S T E T R E A T M E N TBiological treatment of liquid, solid and gaseous effluents for environmental pollution control orabatement differs from most other industrial bioprocesses in two main ways: the scale of operationis generally very large and mixed microbial populations under non-sterile conditions are thebiocatalytic entities responsible for degradation of waste.19 Treatment of domestic and municipalwastewater by biological means (e.g., activated sludge process, trickle bed biofilters, etc.) is a wellestablished, predominant technology of treatment of these Many industrialwastewaterscontaminated with relatively recalcitrant organics are also amenable to biological treatment usingmicrobial PO ulations which have adapted (natural selection) to degrading these wastes forsoils polluted with wastes of petrochemical Both in situ treatment and treatment in~ u r v i v a I . ~ ~ * ~ ~ ~Bioremediation techniques are increasingly being applied to decontamination of


10/20


bioreactors such as the airlift devices are being implemented.22*5s*67@n important considerationin these processes is minimization of costs.Gaseous streams polluted with low loadings of organics and odorous substances can betreated in biofilters now being deve10ped.l~ norganic pollutants such as heavy metals, even in verydilute waste streams, can be adsorbed or otherwise accumulated by certain microbes (e.g., some

algae) thereby affording a method of concentration and treatment. Although biological wastetreatment has a long and proven record of application, much greater contribution can yet beexpected from bioprocessing in environmental quality improvement. Many traditional chemicalindust processes as well as newer ones will depend on biotechnology for their waste managementneeds?*65 Elucidation of the degradative mechanisms and kinetics for the pollutants and designof reactors for their treatment would require innovative approaches from chemists, biochemists,microbiologists and biochemical engineers.

BIO CATALY S ISNearly all biochemical reactions taking place in plants, animals and microorganisms are catalysedby enzymes. Because of the vast array of reactions and the diverse life forms, an equally largenumber of enzymes exist in nature. Produced inside cells, the enzymes may be retained within (Le.,intracellular enzymes) the cell or secreted (Le., extracellular enzymes) to the outside. Biochemicalreactors or bioreactors use the enzyme systems of microbial and other cells to carry out therequired biosyntheses, biodegradation or b i o t r a n s f o r m a t i ~ n s . ~ * ~ ~ ~ ~ ~ @part from cell growth andaccompanying metabolite production in fermenters, non-viable or non-growing live cells and cell-free enzymes, either immobilized or freely suspended in their native state, are important forms ofindustrial b i o c a t a l y s t ~ . ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~Cell-free enzyme sSoluble en es in their native state are used in industrial reactors as well as being commodityUsually, inexpensive extracellular microbial enzymes are used in soluble form. These are mostlyhydrolytic enzymes such as amylases (starch hydrolysis), cellulases (cellulose hydrolysis), pectinasesand proteolytic enzymes.31

As commodity products, cellulases and proteinases are used in detergent^:^ pectinases inenhanced recovery of juices from fruits, proteinases in meat processing and cheese making,amylases in brewing, to cite but a few examples. In most applications the activity and the pH andtemperature optima of the enzyme are dependent on its source and enzyme selection is oftentailored to the process needs. For example, alkaline bacterial proteinases, such as subtilisin, areactive over the pH range 7-113' and are used in detergents. Neutral bacterial proteinases aremetaloproteins active over a pH range 6-9.31Similarly, bacterial amylases suitable for 70-85 "C or90-105 O C temperature optima are available.31

chemicak2T ,1 69 A soluble enzyme is not easily recovered after use and is lost with the product.

Immo bilized biocatalyst reactorsImmobilized enzyme (and immobilized cells,70 ro top last^,^^ organelles) catalysts can be employedin a variety of reactor c o n f i g ~ r a t i o n s . ~ ~ ~ ~ ~ ~ ~ ~atalyst particles may be used in suspension as instirred tank and fluidized bed reactors (Figure 6) or they may be held in place in fixed or packedbed devices. Hollow fibre reactors containing catalyst immobilized either throughout the thicknessof the fibre wall or confined to one side of it (e.g., perfusion systems) are p o s ~ i b l e . ~ ? ~ ~lat polymermembranes containing immobilized catalyst have been used in spirally wound configurations.Immobilized particulate biocatalyst can, of course, also be used in airlift and bubble column reactorsso long as the solids loading and density are not A packed-bed-airliftcombination reactor, as shown in Figure 7, is another p~ss ib il ity.~~n such reactors a compressed


11/20

Biochemical engineering in biotechnology 12 7

Catalystparticles

a ) St i rred tank

Feedc ) Packed bed

b) Fluidized bed

Feed-+Y

Product

IFibre wall(permeable)

support i igcatalystsupport i igcatalystFeed

d ) Hollow fibre ( e ) Spiral wound membranesystem module

Figure 6. Deployment of immobilized enzymes and cells in bioreactors: Free suspension instirred tank (a) or fluidized bed (b); fied catalyst in packed bed (c), hollow fibre (d), or spiralwound polymer membrane module (e).

gas generates the necessary agitation and circulation of the fluid. In fact, airlift reactors usingmicrobial films immobilized on sand particles have been described as the future of wastewatertreatment where their low power requirements are an important ad~antage.~*,~l

Relative to soluble forms, immobilized biocatalysts are easily retained in reactors and canbe reused. Often, immobili~ation~~mproves the stability of the catalyst and can be used toadvantageously alter the apparent pH optima relative to the native form. However, immobilizationrepresents an additional cost. Nevertheless, certain expensive intracellular enzymes (e.g., glucoseisomerase) could commercially be employed only because immobilization improved the overallprocess economics by permitting repeated long term use.31Bioreactor efficiency is measured by the quantity of substrate (reactant) transformed per unittime per unit volume of the r e a c t ~ r . ~ ~ ? ~ ~bviously, the efficiency is dependent on the activity perunit mass of the immobilized catalyst. For specified initial concentration of substrate (So) and its

desired conversion x = (So - S)/S,, the conversion characteristics of different reactor configurationscan be calculated from a knowledge of the kinetics of reaction. Thus, for a reaction which obeysMichaelis-Menten kinetics


12/20


where e is enzyme concentration,S is substrateconcentration at time t ,K, is Michaelis constantand k, is the rate constant, we can developequations for predicting the conversion asexplained below for a batch stirred tankreactor.Because the change in the quantity ofthe substrate in a batch reactor equalssubstrate consumption by the reaction, we canwrite

(7 )S krES-vLa7 K,+swhere V, is the volume of the reactor and E isthe total amount of enzyme in it. Equation (7)may be integrated for S = So at t = 0 and S =S at t = t , to

(8),E t -So-S-K,ln ST- T

RISER

AlR

PACKEDB E D

Figure 7. An external-loop airlift reactor withpacked catalyst bed in downcomer.

which can be written in terms of the conversion x as

Hence, the batch time t for any degree of conversion may be calculated. We see from this exam leof course, the exact nature of the rate equations may differ.that the principles of analysis of biochemical reactors are the same as for chemical reactors;21

P R O D U C T I O N OF 6- AM INO P E NIC ILLANIC ACIDHere we illustrate, in the context of a current industrial bioprocess, some of the biochemicalengineering considerations and their relationship to process chemistry. The production of6-aminopenicillanic acid is used as an example.30

Penicillins are among the most widely used antibiotics. Benzylpenicillin (Penicillin G)obtained by fermentation with Penicillium chrysogenum is of limited use and most of it is convertedto 6-aminopenicillanic acid (6-APA) which is a precursor for the manufacture of semi-syntheticpenicillins. The chemical conversion of Penicillin G (PEN-G) to 6-APA, shown in Figure 8,has nowbeen superseded by the one-step biochemical route (Figure 8).The chemical process required lowtemperatures (hence expensive low temperature steels for processing equipment) and strictlyanhydrous conditions during parts of the process; moreover, several processing steps were needed.As a result, the process proved to be uncompetitive with the biochemical conversion. The enzymeused for the bioconversion is penicillin acylase (EC 3.5.1.11) produced by the bacterium Escherichiucoli, in addition to other microorganisms. Several process schemes may be used: (1) E. coli cellsgrown separately and killed may be contacted with a solution of PEN-G in a batch stirred reactor.After one use, however, the cells lose most of their enzyme activity due to lysis and have to bereplaced. (2) Live, immobilized E. coli cells may be brought in contact with a continuous flow ofPEN-G solution. Either packed bed or suspension reactor modes may be used. Side reactions andcontamination by metabolites and substances required to sustain the cells are possible problems.(3) The enzyme penicillin acylase may be extracted from E. coli, immobilized and used incontinuous reactors. Scheme (1)has been used commercially, but scheme (3) seems to be thepreferred industrial route. This route is examined further.


13/20


0

PE N I C I L L I N - G 6-APA(CH3)2SiClZ

+ P a ,(-4O'C)

V

Figure 8. Conversion of Penicillin G to 6-aminopenicillanic acid (&MA):Chemical vs.enzymatic route.The enzyme is produced by growing a high yielding strain of E. coli in a batch fermenter.The cells are harvested by centrifugation, resuspended in a smaller volume of buffer and disruptedby high pressure homogenization. A two stage salt precipitation follows, precipitated nucleic acidsand cell debris are removed first (centrifugation) and then the enzyme. The enzyme precipitate issolubilized, further purified to a relatively pure form and immobilized on polymer beads. Theimmobilized catalyst, which can be purchased ready to use, has a half-life of the order of severalmonths under the normal reactor conditions of 37 "C and pH 6-8, long enough for industrial

purposes.The kinetics of the deacylation reaction are such that it is strongly inhibited by 6-APA andis also susceptible to some substrate (PEN-G) inhibition. Based on detailed calculations, a packedbed catalytic reactor would be best for the conversion. However, as the deacylation reactionproceeds, the pH of the reaction medium drops and good pH control is essential to avoidingdamage to the product, substrate and the enzyme catalyst. Control of pH is difficult in packedcolumns and in this reaction is achieved by addition of alkali to a buffered reaction medium, Rapidand uniform mixing is necessary to avoid high local pH values which lower the half-life of theenzyme. To satisfy the operational requirements of good reactor mixing and at the same time toapproximate the reaction system to a plug-flow type, a battery of up to four continuous stirred tankreactors (CSTRs) in series is used for the reaction. Four CSTRs in series are about optimal,

allowing a PEN-G conversion of more than 95 %. Addition of a fifth reaction stage would increasethe conversion only slightly as the catalyst would be subject to severe product inhibition by 6-APAwhich would be at its maximum concentration in this last stage. A fifth stage would, of course, addto capital and operational costs.After the reaction, 6-APA is precipitated from the product-containingsolution by adjustmentof pH to its isoelectric point; the precipitate is filtered, washed and dried. The product obtainedis better than 98 % pure.Although the 6-APA production process is relatively straightforward, the large scaleproduction plant can be quite complex as revealed in the very basic piping and instrumentation

diagram shown in Figure 9 which does not include the downstream plant. The need for continuoussupply of PEN-G solution, automatic catalyst replacement, sequencing of flow through anycombination of the four reactors (to allow for taking spent reactors off-line and replacing with onecontaining fresh catalyst), complicate the flow scheme. The requirements for cleaning-in-place andcontrol (e.g., temperature control) further add to the complexity, leading to extensive pipework,valves, pumps and other ancillaries, most of which must be built to hygienic standards.


14/20

C

N

D

AOOPG

t

t

t

t

A

MNR

W

VSM>

I

CmlNGO

RSTO

F

R

T

T

T0CF

S3

l

r

P

WA

HNO

EGSO

O

3COM0-m

1P

O

OO.MO75

PCLNG

S

O

IN

w

Tm

A

p75ws

mM

S

ZM

PWlTE

C

TOSMG

O TSWEWD

SOUMww

RSUWVZH

ODT

E

IEUPMEI

RI5IE

I

E

IT

IVlVZ

I

T

IV

IV

I

V

I

V

IP5

I

K

D

PON

F

ON

CNSR

T

C

Rm

WGNA

H

IXNT

I

S SLO

TS

WPP

LDDF

S

OT

ONRE

P

AWSAS

TMN

p4mSW

S

IE

T

T

WEGN4

MXNMN

-I-

WMPI4

L

-

S

O

IPPAL

IPE

&c

I

S IWlA1%W

mrc

E

-

P

C

1D

...

FOR

.nm"u

'LO"LI~

L

NOA

L

NC

C

PCT

C

WGlNC

R

C E

G

LwLE

I

I

1

I

I

I

1

I

1

I

I

I

I

Fg9Asmpedppnanrumenaodamohbo

osao6aminpncacadmaunp

2W 0 00z v cn0z 0z m 0 0Izs ; E t -


15/20


D O W N S T R E A M P R O C E S S I NGAs explained earlier in this article, downstream processing is essential to obtaining a saleableproduct from the raw product of the bioreaction step. This discussion is limited to factors whichmust be considered in developing any economically viable product purification and concentrationscheme based on a few of the m any downstream processing operations that ar e available. Individual

In general, a biological product is either secreted into the extracellular environment, or itis retained intracellularly. In com parison with the total am oun t of biochemicals produced by the cell,very little material is usually secreted to the outside; however, this selective secretion is itself apurification s tep which simplifies the task of the biochem ical engineer. Extracellular p roducts, beingin a less complex mixture, are relatively easy to recover. On the other hand, because a greaterquantity and variety of biochemicals are retain ed w ithin cells, intracellular substances are boun d toeventually become a major source of bioproducts. Am ong som e of the newer intracellular productsar e recom binant p roteins produ ced as dense inclusion bodies in bacteria and yeasts. Recovery ofintracellular pro ducts is more expensive as it requires such additional processing as cell disruptionP8l ~ s i s ~ ~r p e r m e a b i l i ~ a t i o n . ~ ~n principle, selective release of the desired intracellular products ispossible, but in practice it is neither easily achieved nor sufficiently selective. Hence, the desiredprodu ct m ust be purified from a relatively complex mixture, complicating processing and adding tothe cost. Nevertheless, a n increasing num ber of intracellular products a re in production. Economicsof production may be impro ved by recovering several products (intracellular an d extracellular) fromthe same fermentation batch.

Product recovery schemes commonly employ solid-liquid separations such as filtrations,centrifugation, sedimentation and floatation. Filtration and centrifugation are by far the morecommon. Solid-liquid separation is a means of concentration of cell mass prior to disruption, or amethod for total removal of cells as product o r waste. Some other co ncentration steps, applicableto products in solution, are precipitation, adsorption, chromatography, evaporation andultrafiltration. So me of these opera tions a re equally capab le purification steps (e.g.,chromatographicseparation s). Typically, volume red uction of the ferm entation broth should be o ne of the earlydownstream processing steps to reduce the capacity requirements (cost) of other processingequipment. Certain steps (e.g., some chromatographic separations; membrane separations) mayrequire a relatively clean process strea m, free of debris, lipids or m icelles which may caus e foulingof the equ ipment. Such steps are often used downstream of steps which can handle cru der material.As far as possible, the requisite purification and concentration should be achieved with thefewest processing steps; generally, no more than 6-7 steps are used, a situation quite different fromthat in chemistry and biochemistry laboratories, where the number of individual steps is often nota major consideration, purity of the product is usually more important than overall yield or costs.A train of only five steps, each w ith 90 % step yield, would redu ce the overall recovery to less than60 T o minimize reduction of the overall yield, a m ulti-step processing scheme should atte mptto use steps with higher step yield earlier in the processing sequence than the lower-yieldingoperations.Th e specifications on product purity an d concentration should b e carefully considered indeveloping a production protocol. Concentration or purification to levels beyond those dictated byneeds is wasteful. When more than one processing options are technically feasible, evaluations ofthe economics of use in terms of capital expenditure on equipment and its operating costs(processing time, yields, labour, cleaning, ma intenance, analytical supp ort) is necessary for optimal

process selection. Economic evaluations should be performed over the expected lifetime of theequipment. For example, for separation of solids from fermentation broth, centrifugation andmicrofiltration may be two competing alternatives3* In still other applications, for example when


16/20


very fragile cells are to beseparated from suspendingliquid, centrifugation may not atall be an option.For many biological

p r o d u c t s , p a r t i c u l a r l ypharmaceuticals, seeminglyminor alterations in downstreamprocessing can have importantimplications on the performanceof the product. For example,penicillins may be recovered byliquid-liquid extraction of eitherthe whole fermentation broth orsolids-free broth. The latterscheme requires an additionalsolid-liquid separation than thewhole broth process. However,the whole broth extractedproduct has been known to causemore frequent cases of allergenicreactions in comparison with the

* UROKINASEFACTORXUI

n0,Y2 10W

RENNIN *CENTAMYCIN

ETHANOL\I U 10-7 1 0 - 5 10-3 l o - ' 1 0 ' l o 3

CONCENTRATION (kg m-3 )Figure 10.Cost of some bioproducts vs. concentration in theraw fermentationbroth.

other processing alternative. In fact, some pharmaceutical companies now demand of contractsuppliers that, in addition to meeting product specifications in terms of measurable contamination,the product they supply must conform to a certain production method, in this case extraction afterremoval of fungal solids. When raw penicillin is for bulk conversion to semi-synthetic penicillins,whole broth extraction may be acceptable in view of the security afforded by the additional stepsinvolved in making and purifymg 6-aminopenicillanic acid (6-MA) from raw penicillin.

Downstream processing typically represents 6040% of the cost of production of fermentationproducts. Thus, superficially t may appear that process improvement should focus on downstream.This is not so. Even small improvements in the yield or purity of the product in the bioreaction stepcan have a significant effect on downstream recovery costs. This is clearly shown in Figure 10,wherethe cost of production (reflected in selling price) of several biochemicals is plotted as a function ofthe product concentration in the fermentation broth or the starting material. The potential for yieldimprovement at the bioreaction stage is usually high. Major yield enhancements have been fairlycommonly achieved by strain selection, medium development and environmental control. Processimprovement or intensification should emphasize a global approach. Ascertaining the impact ofprocess changes and monitoring the separation and purification steps require extensive chemicaland biochemical analytical support.

THE PRO DUCT IO N FACILITYBiochemical engineers play an important role in the design of buildings which house bioprocessingplant^.^^,^^ Plant layout, containment and treatment of biohazardous material^,^^ controlled anddirected movement of plant personnel, separation of processing areas according to cleanliness andother requirements, control of air flow and air pressure differentials in buildings, production, supplyand distribution of utilities, and matters relating to finishings for ease of cleaning and sanitizationare some of the aspects which must be considered.


17/20


C O N C L U S I O N SBiochemical engineering is a flourishing discipline which mak es industrial p ractice of biotechnologypossible. Looking through the anatomy of bioprocessing, we saw how the biochemical engineerdraws upon chemical sciences to research, design, develop and operate biochemical processes.Multidisciplinary skills in biological, chemical and engineering sciences are essential features ofbiochemical engineering. Advances in molecular genetics, biochemistry, protein chemistry,microchemical analyses, immunology and oth er biophysical sciences continue to p ose new challengesand provide new tools to the biochemical engineer, hence ensuring new products and betterproduction methods for pharmaceuticals, foods, fuels, bulk chemicals, fine chemicals, andenvironmental pollution control and resource recovery services.

N O M E N C L A T U R EAAnEAEAEneeK,kdknkr

s oRSTttdXPX6-APACIPCSTRHTST

VLx o

Arrhenius parameterArrhenius parameter for nutrient denaturationTotal amount of enzyme in reactorActivation energyActivation energy for nutrient denaturationEnzyme concentrationConstant (= 2.7183)Michaelis constantSpecific death rateSpecific nutrient denaturation rateRate constantGas constantSubstrate concentrationInitial substrate concentrationAbsolute temperatureTimeMean doubling timeLiquid volume in reactorBiomass concentration (dry)Initial biomass concentration (dry)Specific growth rateConversion6-Aminopenicillanic acidCleaning-in-placeContinuous stirred tank reactorHigh temperature short time sterilization

(as appropriatej(J*K-* kmol-')( k g ~ m - ~ )( k g ~ m - ~ )

(K)(s)(s)

(kg*m 3)( k g ~ m - ~ )(s-9(-)

PEN-G Penicillin G

REFERENCES1.2.3.4.5.

Dunnill, P. (1987), Chem. Eng. Res. Des., 65, 211-217. Biochemical engineering and biotechnology.Stiefel, E. I. (1987), Chem. Eng. Prog., 83(10), 21-34. The technological promise of the biologicalsciences.Moo-Young, M., editor (1985), Comprehensive Bwtechnology,vol. 1-4, Pergamon Press (Oxford).Neway, J. O., editor (1989),Fermentation Process Development of Industrial Organisms,Marcel Dekker(New York).Crueger,W. nd Crueger, A. (1990), Bwtechnology: A Textbook of Industrial Microbiology,2nd Edition,Science Tech Publishers (Madison).


18/20


6.7.8.

9.10.11.12.13.14.15.16.17.18.19.20.21.22.23.24.25.26.27.28.29.30.

31 .32.33.34.35 .36.37.38.

Lubiniecke, A. S.,editor (1990),Large-Scale Mammalian Cell C ulture Technology,Marcel Dekker (NewYork).Moo-Young, M., Chisti, Y. and Vlach, D. (1993), Bwtechnol. Ad v., 11(3), in press. Fermentation ofcellulosic residues t o m ycoprotein foods.Bowen, R. and Pugh, S. (1985), Chem. Ind. (Lond.),20 May, 323-326. Redox enzymes in industrial finechemicals synthesis.Elder, A. L, ditor (1970), Chem. Eng. Prog. S Ser., 66(100), 1-97. The history of penicillinproduction.Payne, C. C. (1989), Chem. I d Lond.),20 March, 182-186. Microbial control of insect pests: currentand potential uses.Moo-Young, M., Chist i,Y. and Vlach, D . (1992), Bwtechnol. Lett., 14,863-868.Fermentative conversionof cellulosic substrates to microbial protein by Neurospora sitophila.Lisansky,S. (1989), Chem. Ind. (Lond.),7 August, 478-482. Biopesticides: Th e next revolution?McNaughton, K. J. (1989), Chemical Engineer (L ond.) ,4 6 6 , 4 4 4 . Twenty years of single cell proteinventures.Banerjee, U. C., Saxena, B. and Ch isti, Y. (1992), Biotechnol. Adv ., 10,577-595. Biotransformations ofrifamycins: Process possibilities.Bohn, H. (1992), Chem. Eng. Prog., 88(4), 34-40. Consider biofiltration for decontam inating gases.Thomas, D. R., Carswell, K. S., Georgiou, G. (1992),Biotechnol. Bioeng.,40, 1395-1402. Mineralizationof biphenyl and PCBs by the white rot fungus Phanerochaete chrysosporium.Allsop, P. J., Chisti, Y., Moo-Young, M. and Sullivan, G. R. (1993), Biotechnol. Bbeng., 41, 572-580.Dynamics of phenol degradation by Pseudomonas putida.Chisti, Y. (1984),Dairy Times (Nigeria),Wednesday, 22 February, 12-13. Biotechnology: Potential androle in Africa.Bailey, J. E. and Ollis, D. F. (1986),Biochemical Engineering Fundamentals,2nd edition, McGraw-Hill(New York).Moser, A. (1988), Bwprocess Technology: Kinetics and Reactors , Springer-Verlag (New York).Moo-Young, M. and Blanch, H. W. (1981), Adv. Biochem. Eng., 19, 1-69. Design of biochemicalreactors: Mass transfer criteria for simple and complex systems.Chisti, Y. (1989), Ai d@ Bioreactors,Elsevier Applied Science (London).Belter, P. A., Cussler, E. L. and Hu, W.-S. (1988), Bwsepara tions: Downstream Processing forBiotechnology, Joh n Wiley (New York).Wheelright, S. M. (1991),Protein Purification:Design and Scale up of Downstream Processing, HanserPublishers (New York).Wang, D. I. C., Cooney, C. L., Demain, A. L., Dunnill, P., Humphrey, A. E., Lilly, M. D. (1979),Fermentation and Enzyme Technology, Joh n Wiley (New York).Chisti, Y. (1992), Chem. Eng. Prog., 88(9) ,80-85 (1992). Assure biorea ctor sterility.Chisti, Y, (1992), Chem. Eng. Prog., 88(1), 55-58. Build b ette r industrial bioreactors.Aiba, S., Humphrey, A. E. and Millis, N. F. (1973), Biochemical Engineering, 2nd e dition, Universityof Tokyo Press (Tokyo).Crueger, W. (1990), In: Finn, R. K. and Prave, P., editors, Biotechnology Focus 2, Ha nser Publishers(New York), pp. 391 -422. Sterile techniques in biotechnology.Chisti, Y. and Moo-Young, M. (1991), In: Moses, V. and Cape, R. E., editors, Bwtechnology: TheScience and the Business, Harwood Academic Publishers (New York), pp. 167-209. Fermentationtechnology, bioprocessing, scale-up and manufacture.Gerhartz, W., editor (1990), Enzymes in Industry, VC H Publishers (New York).Mackay, D. and Salusbury, T. (1988), Chemical Engineer (Lond.), 447, 45-50. Choosing betweencentrifugation and cross flow microfiltration.Bowden, C. (1985), Chemical Engineer (Lond.), 415, 50-54. Recovery of micro-organisms fromfermented broth.Macdonald, D. (1985), Chemical Engineer (L on d.) ,412, 15-17. Evaluation of sepa ration problems fordisc bowl centrifuges.Cooper, A. R. (1984), Chemktry in Britain, 20(9), 814-818. Ultrafiltration.Butcher, C. (1990), Chemical Engineer (Lo nd .),469, 50-53. Microfiltration.Abbott, N. L. and Hatton, T. A. (1988), Chem. Eng. Prog., 84(8),31-41. Liquid-liquid extraction forprotein separations.Chisti, Y. and M oo-Young, M. (1986),Em. icrobial Techno l.,8,194-204. Disruption of microbial cellsfor intracellular products.


19/20


39.40.41.42.43.

44.45.46.47.48.49.50.51.52.53.

54.55.56.57.58.59.60.61.62.63.64 .65.66.

67.68.69.70.

Hochuli, E. (1992),Pure &Appl. Chem.,64(1), 69-184. Purification techniq ues for biological products.Fleming, H. L. 1992), Chem. Eng. Bog., 88(7), 46-52. Consider membrane pervaporation.Redman, J. (1990), Chemicul Engineer (Lond.), 69, 6-49. Pervaporation h eading for new h orizons.Chisti, Y. and Moo-Young, M. (1990), Biotechnol. Ad v., 8 , 699-708. Large scale protein separations:Engineering aspects of chromatography.Crueger, A. (1990), In: F inn, R. K. and P rave, P., e ditors, Biotechnology Focus 2, Hanser Publishers(New York ), pp. 365-390. Microbial protoplasts - their applications in biotech nology and genetics.Chisti, Y. and Moo-Young, M. (1993), ChimicuOm*Chemhtty T du y) ,ll(3-4),25-27. Effec tively usefragile biocatalysts in biore actors.Moo-Young, M. and Chisti, Y. (1988), Biotechnology,6(1l), 1291 12%. Conside rations for designingbioreactors for shea r-sensitive culture.Chisti, Y. and Moo-Young, M. (1989),Biotechnol. Bioeng.,34, 1391-1392. On th e calcu lation of shearrate and apparent viscosity in airlift and bubble column bioreactors.Shuler, M. L.and Kargi, F. (1992), Bioprocess Engineering: Basic Concepts, Prentice Hall (EnglewoodCliffs).Chisti, Y. (1989), Chemica l Engineer (Lond.), 457, 1-43. Airlift reactors - design and diversity.Chisti, Y. and Moo-Young, M. (1988), Biotechnol. Bioeng., 31, 87-494. Hydrodynamics and oxygentransfer in pn eum atic bioreactor devices.Chisti, Y., Kasper, M. and Moo-Young, M. (1990), Canad. J . Chem. Eng., 68, 45-50. Mass transfer inexternal-loop airlift bioreactors using static mixers.Chisti, Y. and Moo-Young, M. (1988), J . Chem. Technol. Biotechnol.,42,211-219. Prediction of liquidcirculation velocity in airlift reactors with biological media.Chisti, Y., Halard, B. and Moo-Young, M. (1988), Chem. Eng. Sci., 43, 451-457. Liquid circulatio n inairlift reactors.Mao, H. H., Chisti, Y. and Moo-Young, M. (1992), Chem. Eng. Commun., 113, 1-13. Multiphasehydrodynamics and solid-liquid mass transport in an external-loop airlift reactor - A comparativestudy.Miura, Y. (1978), Adv. Biochem. Eng., 9, 31-56. Mechanism of liquid hydrocarbon uptake bymicroorganisms and growth kinetics.Chisti, Y. and Moo-Young, M. (1988), Chem. Eng. J., 39, B31-B36. Gas holdup behaviour infermentation broths and other non-newtonian fluids in pneumatically agitated reactors.Chisti, Y. and Moo-Young, M. (1988), Chem. Eng. J., 38, 49-152. Gas h oldup in pneum atic reactors.Ouyoung, P. K.,Chisti, Y. and Moo-Young, M. (1989), Chem. Eng. Res. Des., 67,451-456. Heattransfer in airlift reactors.Chisti, Y. and Moo-Young, M. (1987), Chem. Eng. Commun., 60, 195-242. Airlift reactors:Charac teristics, applications and design considerations.Chisti, Y. (1993),Bwproc. Eng., 9, 91-1%.Animal cell culture in stirred bio reactors: Ob servations onscale-up.Lee, J. M. (1992), Biochemical Engineering, Prentice Hall (Englewood Cliffs).Webb, C. and Atkinson, B. (1992), Chem . Eng. J., 50, B9-Bl6. The role of chemical engineering inbiotechnology.Eckenfelder, W. W., Goodman, B. L. and Englande,A. J. (1972),Ad v. Biochem. Eng.,2,145-180. Scale-up of biological wastewater treatment reactors.Hill, G. A,, Tomusiak, M. E., Quail, B. and Van Cleave, K. M. (1991), Environ. Bog.,0(2), 147-153.Bioreactor design effects on biodegradation capabilities of VOCs in wastewater.Choi, Y.-B., Lee, J.-Y., Kim, H A . (1992),Biotechnol. Bioeng., 40,1403-1411.A novel bioreactor forthe d egradation of inhibitory solvents: Experim ental results and mathe matical analysis.Eckenfelder,W.W., Argaman, Y. and Miller, E. (1989), E n v h n . Bog., 8(1), 40-45. Process selectioncriteria for th e biological treatm ent of industrial wastewaters.McDennott, J. B., Unterman, R., Brennan, M. J., Brooks, R.E., Mobley, D. P., Schwartz, C. C. andDietrich, D. K. (1989), Environ. hog., 8(1), 46-51. Two strategies for PCB soil remediation:biodegradation and surfactant extraction.Halden, K. (1991), Trans. Inst. Chem. Eng., 69(B), 173-179. Meth anotroph ic b acteria for the in-siturenovation of polluted aquifers.Redman, J. (1987), Chemica l Engineer (Lond.), 41,12-13. Deep shaft treatment for sewage.Malmos, H. 1990), Chem. Ind. (Lond), 19 M arch, 183-186. Enzymes for detergents.Kennedy, J. F, Melo, E. H. M. and Jumel, K. (1990), Chem . Eng. Bog.,6(7), 81-89. Immobilizedenzymes and cells.


20/20

136 COMMISSION ON BIOTECHNOLOGY71.72.

73.74.75.76.77.78.

Varey, P. (1992), Chemical Engineer (Lond.), 29, s37. Air lift for purity.Moo-Young, M. and Chisti, Y. (1992), In: Ladisch, M. R. and Bose, A, editors, HarnessingBwtechnologyfm he 21st Century,Am erican Chemical Society (Washington), pp. 174-177. Transportphenomena in novel bioreactors: Design of airlift-based devices.Dabora, R. L.and Cooney, C. L. (1990), Adv. Biochem. EnglBwtechnol., 43, 11-30. Intrace llular lyticenzyme systems and their use for disruption of Escherichia coli.Domenburg, H. and Knorr, D. (1992), Process Biochem.,27, 161-166. Relea se of intracellularlystoredanthraquinones by enzym atic permeabilization of viable plan t cells.Fish, N. M. and Lilly, M. D. (1984), Biotechnologv,2,623-627. Th e interactions between ferm entationand protein recovety.Nelson, K. L. (1991), In: Prokop, A, Bajpai, R. K. and Ho, C. S., editors, Recombinant DNATechnologyand Applications, McGraw-Hill (New York), pp . 509-565. Biopharm aceutical plant design.Hill, D. and Beatrice, M. (1989), BwPhann, October, 20-26. Biotechnology facility requirements, PartI. Facility and systems.Lee, R. and Waltz, J. (1990), Chem. Eng. Prog., 86(12), 44-48. Consider continuo us sterilization ofbiopro cess wastes.

biochemical engineering in biotechnology

Documents