BaSK BiotechnologyBiotechnology impinges on everyone-'s lives. It is one oftlle maJor rechnologies oftbe twenty..first century. les huge, wide-r.mging, multhlisdplinaTY activities nelude recombiIlanr ONA: techniques, doning 3ud geoetics, and the application ofmicrobiology to the production ofgoods as prosak as bread, beer, cheese and antibiotics. Jt continue$ tO revolutionise 1:realnlents ofmany diseases, and is used lo provide clea.n tedlnologie5 and todeal with environmental probJems. Basic Blo:t'chtwlogyis a textbook. thal gives aJuU accouDtofthecu.r rentstate of biotechnology, providing!he ~ader with iruight, inspiratlon and instruction. The fundamental aspects lhat Uflderpin biotechnoiogy are explained through examples fiom me pbarmaceutical, food and environmental industries. Olapters on the public pen::eption ofbiot.echnology and the business aud economics of the subject are cesses is also generated. The same cODsideratiofls. however, apply to aU substances that a.re useP,The irreversibility of the second gIycolytic enzyme. phosphofTuclO' (producing frucrose 1.6-bisphosphate). is circumvented by t he aaioD ofructose btsphosphatQ.n::i!ia5e

fructose 1.6-bisphl>sphate + El:t0 ~ fructose 6-phosphate + Piom'this point. hexose sugars can be forroed by the reversal ofglyand A:NADH= I mol x3(x2 for 2pyruvate)

TricorboxyJic add cyc/e:NADH = 3 mol )( 3 (x 2 for '2 acetyl-CoA) FADH2 = I mol x 2 (x 2 fo r 2 acetylCoA) ATP= I mol (x2foracetyl-CoA}

Total

r.'0lIt: Under ~n.:u.rob;c cn Represslon:In the absence of any Indudng molecuhl the mes$t1'lser RNA (mRNA) from the reJUlatory gelMl produces a pl'O'te lo tN.t binds te an'oper.Itor' _ funher down dl4t

J\f\J' m"NA, J\f\J' mRNA ~ JV\F" mANAl

1

Protena produced

ONA mo!ecllle. As a resultofthisbinding. tM

openuor gene ~

Inact/vated ~nd no signal is given teallow the strUcwl'lll genes (tlut

WCluld makllcdve enzymes) te be eXprased. (b) InductiO!1: in me presellCe

ot ~n Induclrlg molecule.

tlw. protein arlslllffrom me "'g"blo')' , _ 1 now no 'on&er , able ce bllld te> d'M>~r g~. Consequently, me openlotor 'switche,' Qfl the 1171.JCt:UraJ cenes and active prou:ins (enI)'mes) are

now made.

the most useful substrate ror production of energy aod provision of metabolites. The mechanisms by which catabolite repression is acl1ieved varies from organism to organismoA simple caseis with E. colt where control is ecule, cyclic AMP (cAMP). (lo cAMP (he single exerted via 3n effector moL phospho groupofAMP-see Fig. 2,4 - bridges across IToro the 3' -hydroxy group ofnoose to tbe 5'hydroxy group, tbereby forming acydicdiphosphoester.) cAMP interacts with a specific protein. catabolite activator protein(CAP;! (also known as the CRP "" catabolite receptorprotcin), and the cAMP-CAP comple>: binds to DNA causing the genes that follow afier (ordownstream of) the binding site to be tl'anscribed (see Ag. 2.22). Tbese genes may tben be used to syntbesise new proteins for uptake and metabolism ofthe next substrate (e.g. lactose if [he cells aregrowing on a glucoseflactose mixture).1his positive system ofgenetic conl:l'ol is the reverseofthe negativecoDl:I'ol systern described in Fig. 2.21. The key molerule is Lherefore cAMP. As long as gluoose or its catabolites are present, cAMP is not formed as ilS syntbesising enzyrne (adeny 1a1'e cydase) is inhibited by these catabolites and thtlS Jactase uptake and metabolism cannotoccur. The catabolites therefore repress the syn tbesis ofnewenzymes. Thc repression is removed when the catabolites disappear- i.e. a11 the gtucose has been consumcd.

2.8.6 Modification o( enzyme activieyOnce an enzyme has been syntbesised, its activity can be modulated by a variety ofmeans.

GROWTH AND HETABOlISM

41

ONA

Catabo!lI:. rep!'Msion.Th. mech;r,nism lhown s medjted by cyOlC AMP (cAMP). An operon Is strate (or eJectron donar). me electron acceptor and biomass occur. TIlis gives d = -1.857. being identical lo the full solution ofeonservatiOD eODstraims obtained before_ The other coefficients follow from appcation of (be regular conservauon constraints. i.c. the N-soutce coefficient fl"Om the N-balance. me HCO; from the C-balance etc. From me example severa! ponts mu!I: be stressed: the balance ofdegree of reduction specifies atways a linear relation between the stoichiometric coeffi.cients ofelectron donor, electron acceptorand biornass, making this relation extremclyuseful in prarnce; the bala nce ofdegree ofreduction is not a newconstraint. it bjusta l>uirable combinatio," oftheC. H. N charge conservation constraints.

Other useful applications of me conservatiotl constraims are outlined in tbe references and indude: selection ofthe yield measuremenlS wbicb provide the least errors i.n tbe calculated otber yields (dlle ro e rror propagation in the meas' uremcuts); improvementoftbe elTon in all yie1ds by measuring more than the minimal required yields (measurementredundnncy allowing data reconciliation); use ofredundant measurements to investigate l he occurrence of systematicmeasure.ment errors 01" errors i.n the system definidon (e.g. a product has been forgatteo).

3.3

Stoichiometry predictions based on Gibbs energy dissipation

A number cf methods have previously been proposed to estimate biomass yields fl'ox) from corre.1atioDS. Aparticularly sitnple but useful and recent method has been the thermodynamically based approach using Gibbs energy dissipation per unit biomass (l(YGX) in kJ perC-mol X. This is a stoichiometric quantity which can similar to che. biomass yie.ld Y on e.lectron donor as in Eqn j3.3)1be written as DX

__ = _'_ + me 1Yexy~xJ.

(3.4)

mG is the biomass specific rate of Gibbs energy dissipation fur maincenance purposes in kJ per C-mol X h and y~ is me maximal bioOlasS yield on Gibbs energy in Cmol X kJ- l. Eqn (3.4) show$ that the Gibbs eneTg)' dissipation contams a growth and a mainte.nance re.la ted termo Simple correlations have becn propased for 1{yGX' and for me (see Further reading. Section 3.5). Thcse correlations cover a wide IdIlge of microbiat growth systems and temperatures (heterotrophic. autotrophic. aerobic, anaerobic, den.itrifying growth systems on a wide rangeorCsources. gTOwth systems with and withoutre.versed electron

transport - RE'I).

3.3. 1 Correlation for maintenance Gibbs energyThe following corre1ati on has becn fouud to be valid for maintenance .

Gibbs cnergy(3.5) 298 This correlation holds for a temperature range of5 to 7S OC. for aerobic and anaerobic conditions. It does not depend an tbe C-source or electron donor OT acceptOr being applied and only shows a significaD[ ~m perature effec[. This see.ms IOglcal beca use maintenance only involves Gibbs enerID'. irrespective ofthe electron dOllorfacceptor combination which provides this Gibbs energy. Illc=4.sexp [ 690.00 8.314 T

(! -.)_ )]

STOICHIOMETRY AND KINETICS OF GROWTH

5:

3.3.2 Carrelatian far Gibbs energy needed far grawthFor the growth-rela ted Gibbs energy requiremenr lfYGf. the following correlations can be used: Por her.erotrophic Ol" autotrophic growth witbour RET:1 Yg';" = 200 + 18(6 - c)U + exp[({3.8 - 'Y.)2r l ~{3.6 + OAc)[ (3.6a)

Por autotropbic growth requiring reversed dectron transporl :1 """iiii=3500Ye and (he number of C-atoms {parameter el per mole ofesaurce. Eqn (3.6a) shows that lfYr.x ranges between about 200 and 1000 kJ af Gibbs energy requirement per Cmol biomass dependenr on the e source use X 174

Type bacteriophage bacteriophage bacteriophage bacteriophage eubacterium eubacteril.m eubacterium eubacterium eubacterium euoocterium eubactenum archaea

Number Size1 1 1 1 1 1 1

Nucleicacid Topology ssRNA ssDNA dsDNA dsDNAdsO NA dsO NA dsD NA dsDNA dsDNA dsDNA dsDNA dsDNA dsONA

lambda T4M ycoplosmo geniwlium Borre/io burgdOt(eri

Campylobocter lelunJ Rhodobaclff sphaerOldesBacillus sub61is Esch erichio wli

2

3.6 knt S.4 knt 485 kbp 174 kbp S80kbp lA Mbp 1.7 Mbp 3.0 Mbp + 0.9 Mbp 4.2 t1bp 4.6 Mbp9.45 Mbp

drcular linear linear linear circular linear circular 2 x circular circular circularND

Myxococcus xonthus Methanococrus jonnaschii An:haeog/obus fUlgidus arcnaea Schizosoccharomyces pombe eukaryoteSocchoromyces cerevisioe

1.66 Mbp1

316

eul.~.."i

t:.~!""'!!i"6-' ~

, . .",;,i. .Cyeras repealed 20-35 times leading 10 exponential doubling 01 the terget sequenee

72

I

HARWOOD AND WIPAT

4.5.2). More. recenrly ithas been discovered tbat bacteria are ableta ralee up DNA when given a high voltage pulse. In this process, called electroporation. mixtures of ce11s and exogenous DNA are subjected to a brief (typicallyofmillisecond duration) electric pulse ofup to 2500 volts. The high field strength induces pores to [onn in the ceH membrane, pennitting the entry of the negative1y charged DNA that is itself mobilised by the electncal gradient. In many cases electroporation is more effic.ient than transfonllation and sorne types of bacteria may only be transformed by this procedure.

4.4.6 Selection and screening of recombinantsMter most doning procedures it is necessary to scteen the resulring clones to solate tbose carrying the required gene er fragmentofDNA. At the simplest leve! this may be done by selecting bacterial transformants tbar contain a copy ofthe vector. This is achieved by incorporating an antibiotic-resistance marker gene into tbe vector so that onIy transformed bacteria which have received a copy of the vector are able to grow on medi a containing the a ppropriate antibiotic. More advanced systems have been developed to allow the discrimination of t[ansiormants containing a vector with ar without a cloned inserto These systems indude the use of gene disrllption methods which result in the loss of a particular trait uponinsertion offoreign DNA (Section 4.5.]). Cones containing a specific gene or fragment can be identified directly by selection techniques 01" indirectly by restriction e.ndonuclease rnapping, PCR or hybridisation techniques. If the target gene is expressed. irs presence may be se.lected by compleme.ntation of a defeet in the cloning host (e.g. restoration ofthe ability to utilise a particular substra.te or to growin the absence of ill1 otherwise esse.ntiaJ nutrient). ln the case of restricrion mapping, plasmid DNA extracted from l. number of representative dones is digested with specific restriction endonucleases. Only dones containing the required gene ar DNA fragmentwill generate the correet pa ttern ofbands after agarose gel electro phoresis. Restriction mapping is onIy feasible if the target clones are likely to occur at a high freqllency amongst the poplIlation to be screened. Diagnostic PCR, using oligonuc1eotide pLimers specic to the targetDNA sequence. may also be used ro identifYclones containing tbe required gene or DNA fragmento Since PCR may be used directly on unprocessed samples of colorues, it is feasible to test many more clones. Ifthe target DNAis likely to accur at a low frequencyin a population of clones, as would be the case with a genomic library (Section 4.5), a large number of clones need to be screened. In tbis case the methoq of choice is hybridisation of the bacteria colony that grows from a single cel! (ar in the case ofphage vectors. the. viral plaque). Colany ar plaque hybridisation makes use of1abelled nuc1eic add probes (DNA or RNA) that are able to detect the presence ofspecific DNA sequences within individual colonies oL.plaques. Biomass from individual transformant colorues or plaques is transfereed to a membrane ontowhichdenatured DNA, released by breaking the cells apen, will bind. The membrane is then e.xposed to a lahelled peohe (Section 4.4...7) which binds specifically

GENETIC ENGINEERING: PROKARYOTES

73

to the immobilised target DNA, revealing me idenlity of (alonies orplaques containingthe appropriate doned DNA.

4,4,7 Nucleic aeid probes and hybridisationNucleie acid probes are osed to detect specific target DNA molecules. The soluble probe binds (Le. hybridises) to the targetDNA thatis immobilised anta a nylon or nitrocellu1ose membrane. Hybridisation is used for a variety ofbiotechnological applications induding lhe detection of cloned DNA (Section 4 .4.6), analysis of gelletic org-.misation and the diagnosis ofgenetic diseases. Althougb nucleicacid hybl'idisarion techo niques are used in a wide variety ofcontexts, the same basic principIes apply. Nudeic add hybridisatioD exploits the ability of single-stranded probe nudeic acid (DNA or RNAI to anneal lO complementary single sll'anded target sequences lDNA or RNA) within a population of non complementary nudeic aod molerules. The original technique, reterred to as Soutbern blotting afrer its inventor, Ed Southern, involved the size separation ofl'eSO'iction endonuclease digested fragments of DNA by gel electrophoresis. and their transfer by blotting onto nitroceUulose membranes. 111e probe nudeic acidjs applied as an aqueous solution ando under appropriate hybridisa tion conditions. binds ro immobilised target ONA. lbe loeation of bOlmd probe nucleic acid on the membrane is indieated by tbe presence of a readily and sensitively detecte8 residuul

slgl\alloquence. (e} Secreti(1Il vector ..... ith m. ONA leqUef'oCe of

lb) Socretion vector:lnuc ibh'l promoter

che tilrgetproteio1 fu~ed ....fram. dowNlream el ,lgnal

me

---Ciimm;,';-i_ _llIIlHe./Signal$&Quunce

l equence.

Anllbiatic

."lst' nc8gl'lnll

Rtlpllcatiern o rigin

(c)

Secretion lIac1or wi\h insert:-Yii IlIl1 WVUZUII

-

e

largal gene MCuence fus ed inframe with thlt s ign ai sequence

Secretion Veclors Currently most systems for the production of TeCombinant protcins lead to the in tracdluLar acrumulation ofthe productoHoweyec, mm cellular accumulation can ead to lower production levels, protein aggregation, proteoJy5is and permnent 105s or biologicaJ activity (Section 4.9.41 T.his can sometimes be oven:ome by 5ecrcting the target . protein diIeclly into the cuJture medium since secretelng 01tl"al'lstrlptlon Inltlarlnn poinl$ u51ng

PromoterDNAII mRNA

SI

noclUSI.

mRNA !5 hybrid~d lO

a dflMWred ONA rr'alrnent th;.thas been bbelled at iu 5'...end. The ONA frarrterlt is cho~en 50 mat la S' .,.d b Intel'nal to the t:lrxet mRNA. whUfI th,l' -eod extenm beyond da putativa mRNA St3rtpolnf. Thll RNAJDNA hybridmoleal~

'i

~....

Gene 1

Gene 2

TIE9

.....Hybridlse extracted mRNA

I!

5'labelled

1

transcripts 10 a deneluroo, 5'-laba lled, ONA Irll9mont;;... _e:'M

hu sift&le-stnnded

__o

UQnsiOlU latal"O!. d~raded by

1M slnle-smond-:;pecific anivity of

S I nuc!es-e. TheJ'-endofthe DNA l~lIlnt is ootennlned by r unning II o n .. denaturing gel pinst a DNA sequencll8 of me ongml fngmem geoer.J.ted by meMu;m;aml Gilbert cherniald~'Bge

method.

, , , , , , , ,

,GG

,. product on a denaturingT polyaerylamlde gel

I

ElectrophoreS8

ee

1

, , , , , ,

-j

nylon membranes by blotting and theu covalentlycross-linked. Specific mRNA species are detected by hybridisation (Section 4.4.7), using labelled oligonucleotide. DNA or RNA pro bes. The use ofmarkers wilh different mo!eL war sizes allows the sizes af specific transcripts to be estimated whkh provides ciLles as ro the organisation ofthe transcripriona! unit from which rhe transcriprwas syntbesisro. Sl-nudease and primer extension analyses facilitate the identificarion ofthe 5' -prime eods ofmRNA transcripts or the processed products of primary transcripts, In rhe case of Sl-mapping (Fig. 4.14), mRNA is hybridised [O a speciflc spedes of ssDNA that overlaps tbe start afthe targct !r.lnscl"pt. Tbe resu!ting RNAfDNA hybrid malecule has an overll'lpofDNAat theJ'-end thatisdigested bythe singl~strand specific Sl-nuc1ease. The size ofilie processed ssDNA molecule, which is labelled al u unmodified 5'-end, is determined by denaturing polyacrylamide gel elecrropboresis using a ONA sequence ladder as molecular size markcr. ID the case of primer extension analysis (Fig. 4.15). a 5' -end la belled oligonudeotide, hybridising about 60-100 nucleatides downstream ol' the predicted tr.:mscription initiation rite. is used to prime the synth esis of a ONA copy of the mRNA tr.mscript, using the enzyme reve rse tl"atlscriprase. Syntbesis ofthe complementary DNA strand terminales at me 5'-end of the transcriPl, ro genera te a prodUL1: of defined length. Again t.b.is can be sized using a ONA sequence ladder. geneI 2). Polymerase chaiu reaction{pcR) A procedore for =pont!lltlal ampllficadon of DN .... fragmenn (see Fig. 4.7). P.romoler TIle region ofDNA upstream (Le. S') ofa gene which rontains signa!s fOf initiating and regulating tr.Illsoiptiou afthe gene. Protoplasts CeUs frOID which the cell wall has becn removed by the action of carbohydl'llse enzymes. These ceUs are bounded by me plasma membrane and are osmoticaJly fragile. Recombination The exchan;e ofDN.... beN."eeD two DNAmolecules a r the incorpol'lldon of one DNA molecuJe inm anotber. e.g. be~1l [he ch romosome and introduced DNA. Rl!sllictlon enzyme Enzyme which deaves ar or oear a specific, sbort ONA seguence (restrictiol1 site/_ Shuttle ftCtOr A vector tbatcan replicate iodependently in mo re than one type of organism. e.g. can 'shutlle' betweeo a bac[eflum and a ~ast.

GENETIC ENGINEERING: FUNGI

97

Sonthern blotting DNA fragments ilre separated according ro size bye1ectrophoresis. tr.msrerred to a membrane and probed with a labelleI'llwlamyd n (G 418)-resistance gene (Kan) under the control of a runla! prornotet (P) and short !link;"g sequences ofonly about 40 bp (O) whlch corrtipClnd 10 !he ends oftMl:ene lO be deleted. A doubl. o:roJ~r by homologou1retOlTlblnation between tht..SOl endsand me chrorTlOSOIl"OfI ~d:s 10 lhe

deletlon of d-..gene. At th e tronOYer 11tU, cJvumo$OlNl ONA molKule$al""ll brokenenymlc.1l/y nd DNA $tr;lnd, ei'!d\arJ&ed with mat ofthe ncomin cassette ONA viii DNA repair mechani$m whlch re-joins me DNA molecules_ BecaU5e many genes I I""Il essentiaJ ror surviYlll, gene deleooo is flrst arded OUt in dlploid ceh wheNl on/yone of lile [WO copies o/ th.gene is deleted. Y~diploid tr.ansformoonu COI"Iulni ng Ihe dele'".ed

gene ue seletttd on moolum contailllnll the ;votiblotic G418, which is more effective l'"

he~u5prole;n

G ~~C.

(e) Endopeptld... el....eg. In tIMo I.t. PC tory pathll\lIlY

LLI O

(001,,0 .nd .'!aa.. o,ca,rla. p.otl! n and /"utterolollO'ls prou!n 10

O...

O

lQ

cK~T C?OOH~ FoIdlng and proCeJ$ln&

of ffcre10ry l'ullon pIO{ell$InflIunenlOU$ '~ (a)

Entry of

JUs(ent polypepdde Imo the tumen oIthe e~opWm!c reticulum lE"R).

n. sInal s.qllel"lCt whkh d lrecaerTll'y of!h. polypepdde is r.mo-v.d by slgnal pcpcld.ase so th:.Ic ma emarln poIypepudc wlthln malllmen IlW me slpl SllqUiIn". SIP Is m ~bI.r1d~l1t chaperone wlchil me IUll1en whtch 11 nroclated wich nrly prot.eln fQldtn, e-.n:nt$ , Other chaperones

an d foldues (lee tElxt) are also preient. (b) Fokl ing of!he fuI! length fu$ion proce~, within the ER.(e) The rus Ion proceln isclea~.d

withln til . Golgl body by a lpeclfl(

peptldan (KEX2I n S.cereviJioe) to release me hetw-otogous prote;n tO m e ceUextMlo r fOIIowl"g tnnsport of me prot eln by membra.r.eboun d ycsJ.t:1e5 (el).

126

ARCHER. MACKENZIEANO JEENES

a1ready c1ear tb3t. as wirh ycast expression, severa} factors can conspire to present a bottle-neck and l llat their relative importance depends on the heterologous protein. Foreign genes which use CodOllS Ilotcommon in fungi , the presence of secuences which destabilise mRNA, differences in the protein folding/secretOlY pathway and the abuodance of protcases aO contribttte ro me observed bottlenecks. lo addition, although hyperglycosylation of heterologous proteins Is not such a problem with1i.1amentous fungi as it is with S. cerevisiae, it can still be a difficulty. In addioon. [he patterns of gIycosylation diffcr from those seen in mammalian ceJls wh ich could be importa nt for therapeutic protein production. TIte glycan su-uctures in fungaJ glycoproteillS are being analysed aod the genes tbat enrode enzytDes respons ible fur gIyc3n assembly are being doncd, providing the possibilil)' in che fuorre of manipuladng glycan synthesis. The essential details ofthe secretory pathway in filamentous fun gi appearto be qualjtativelyverysimilar ro those in theyeastsystem whch has becn studied more exrensively. Sorne of the genes that encode chaperones aud foldases have -been d oncd, as have genes that encocle proteins lnvolved in vesicular traosport.Although successful manipulation o( the protein sec.retory pathwayusrng these genes has notyetbeen reported, the nccessary rools to do so are becoming available.

5.7 I Further readingAtuubel. F. M_ Brent. R., Kingston. R. E.. Mool'e, D. D.. Seidman.J. e . Smith,J_A.and Sttuhl. K. (1995). CllrrmtE'roI(l(.l,'S in Molmslnr f:liology. John W iJey, New

York,Broda, P" Oliver, S, G. aud Sinu, P_ F. G. (1.993). The cuknl)'tlllC Cfflcttw:OrganisutionHIt! Rlgulatioll. Cambridge University Press. Cambridge. Gel lissen, e, and HoUcubcrg, C. P. (1997). Application of ycasts in gene expresslcn studies: a comparison ofS[J[charomycts cercvisiac, HanuIJu la polymorpha and f(!uyveromym: lactis - a review. Gm~ 190. 87- 97. Gow, N.A. R. and Gadd. G.M. (eds.) (1995). The Growing Funsus. Olapman and Hall, London . Kinghorn,j.R. and Tumer, G. (eds.) (1992). J\pplied Molf'CU larCenalCJ 01 FilatlU'ntollsFungi. Blackie Academic!lr Profusional. GlilsgtJW. I.uban.J. 31ld GoEr, S.I'. (1995). The.ycasl lwo-bybrid s~te m for studying proteinprorein inter.1roolls. Cun: apill. Biotl'dlnol. 6, 59- 64. Oliver, R. P. and Scb weizer, M. (eds.). (1999). MoIcculur FlIngtll Diolug)'. Cambridge

Universiry Press. Cambridge.Wolf. 1. Often it is convellient lO noI'malise che rates witb respect lO the amount ofbiomass presento since the rates hereby easily can be compared between fermentation experiments. even when the amountofbiamass changes. Such normalsed rates are referred to as specific tates, and t hese are often represented as r. where che subscript indicates whether it is a substrate (s) oI' a metabolic product (P). The specificgrowth rateofthe total biomass s also a very important variable. and it is generatly designated /L. The specific growt.h l'ate is relaree! ro the doubling time ttl (b) oftbe biomass through:,~

In 2

,

"

(6.1)

The doubling time td is equal (O the generation time rar a cell . .e. [he lengthofa cell cyde for ucellular organisms. which is frequentlyused by life scientists to quantify the rate ofccll growt.h. The speciftcrates. ar the flow in and out ofthe cell. are very impor tant design paramcters since they are related to the productivity of the celL Thus , the specific productivity of a given met.abolite directIy indicates the capacity of tile cells ro synthesise uds metabalite. FuI'thennore. iftbesped.fic raCe is multiplied by lhe biomass concentra tion in tbe bioreactor one obtains the volu.meni.c productivity. or the capacity of tbe biomass papulation per reactor volume. In simple IUnetic models the speci1ic rates are specified as runctions oC the vari-

MICROBIAL PROCESS KINET1CS

133

ables in tbe system, e.g. the 5ubstrate coneentratioo!!. In more complex models where tbe rates ofthe intracellular reactiODs are spedfied as functions ofthe variables in the ~ys(em, me substrate uptake rates aad productformation rates are given as funcnons ofthe intraeeUular reaetion mtes. Another dass oC very important design parameren; are the yield coefficients, which qu:mtify the amount of substr.lte re( "

e, ~2K

Logistic law

Contais ldnetics, an influence of the biomass eoncentration, x, is induded. i,e. at high biomass concenttations there i.s an inhibition of cell growth.1t is unlikely that the biomass concentration as such inhibits cell growth but there may well be 3n indireeteffect, e.g. the tormation of an inhibitory compound by the biomass or high biamass concentI-ations may glve a very viscous medium that results in mass trarufer problems. Similarly the Logistic Law expresses a negative inf1uenee ofthe biomass concentration on the specificgrowth rateo These different expressions clear1y demonstrate tbe empirical nature of these kinetic models. and it is therefore futile to discuss which model is to be preferred. sinee. they are 3U simply data fitters. and Qne shauld simply choose the madel that gives the best deseriptian afthe system beiug studied. All tbe kinetic eXQressians presented in Table 6.2 assume that there is anly ane limiting substrate. but afien more than one subsrrate

136

NIELSEN

concentratioh lnOueuces rhe specific growth rateo In these situations, complex interactioos can occur which are difficulc to model wirh unstructured models unless many adjustable parameters are included. SeveraJ different multiparameter. unstructured models fOf growth on multiple substrates have been proposed where it is often difficulr te dis ringuish between whether a second substrate is growth enhancing 01' limiting growth. A general metic expression thar accounes for both types ofsubstrates is: =

(1 + L.: ~ -~~j,~-;-) rr JA.u,.x,J ' .1 ci.e + K~j

! c. j

+ K..j

(6.6)

The presence of growth-enhancing substrates increases tbe specific growth rate whel'eas the essential substrates are necessaryfor growth to take place. A special case ofEqn (6.6) is the growth in tbe presence oftwo essential substrates. el,! and e.2:

.-

J.LUI:ax.I IJ-IllU. :.l C.,lCs.:.!

(C,.I + KI~c.2 + K,)

(6.7)

Jfbotb substrates are ar concenrrations where tbe specific growth rate for eaeh substrate reaches 90% ofits maximum value. Le. ' ..,=0 .9 K,. then tbe total rate ofgrowth is limited to 81%ofthe maximum possible vaJue. TItis is hal'dly praespite its simple structure che linear GIre equation (6.12) o[Pict is found to hold for many difIerent species, and Thble 6 .3 compiles true yield coeftidents and maintenance coefficients for various microbial spooes. !he empicically derived, linear coITelations are veryuseful to correlate growth data, especially in steady smb" continuolls cultures where linear correlations similar to Eqn (6_U) are found for mos! of the important specific rates. The remarkabL robustness and general valide ity of the Linear oorrelations indicares tbat they have a fundamemal basis and t his basis is likely to be the continuous supply and consumption of ATP, sincc these two pTOCCSSCS are tightly coupled in all ce.lls. Thus. m e role oC the energy producing substrate is to provide ATP lo drive both the biosyntbetic and polymerisation react:ions of tbe cell and the different mamtenance processes accO[ding ro the linear re1ationship:(6.14)

which is a formal a nalogue to the linear correlation ofPict. and states [bal (he ATP being produced is balanced by its oonsumption for growth and ror maintcnancc. lftheATP yield on theenergy-prodllcing sllbstrate is coostant, Le, r...TI' is proportional to r" ir is quite obvious thatEqn (6.14) can be used lo derive rhe litlCar correlation Eqn (6.12), y .....TP used in Eqn (6.14) is -l r

f4 f-< O

'" '" ;;>

;

"

I biofe;JttOf".

1M'

A flvidbed bedSenUng~"

-

Llquid~,

Fluidised biocBllIlyst

Pump

Rueyele

th;;m compensate for any additional resistance to flow due to the separator.

7.2.4 Fluidised bedsF1uidised bed bioreactors are suited to reactions involving: a fluidsuspended particulate biocataLyst such as me hnmobilised enzyme and ceU partides or microbial fiocs.An up-Oowing stream oftiquid is used to suspend or 'f1uidise' che solids as in Fig. 7.5. Geometrically, che reactor is similar [o a bubble column except that the top section is expandec1 to reduce che superficial velocity of the Ouidising liquid ro a level below titat nceded [O keep tbe solids in suspe.nsioll. COllsequently, the solids sedimentin the expanded zone and drop back into (he narrower reactor column beJow; hence. the solids are retained in the reactor whereas the liquid flows out. A liquid fluidised hed may be spa:rged with air orsome O[her gas to produce a gas-liquid-solid Huid bed. lftbe solid partides are too light. tbey may have to be artificially weighted. for example by embedding stainless steel balls in aD otherwise Iig ht solid matrix. A high density of solids improves solid- liquid mass tt"ansfer by increasing the re[ative velocity belWCen the phases. Denser solids are also easier [O sediment

BlOREACTOR DESIGN

157

but me densi(}' should not be too high relative to that ofme Iiquid . or fluidisation wilJ be difficu)t. Liquid Ouidised beds tend ro be fuirly quiescem but introduction of a gas subsrantially e nhances turbuJence and agitanan. Even withrclatively Iight partides. the superfidal liquid velocity needE'd to suspend mE' soJids may be so high that the liquid lE'avE's tbE' reactor much too quickly, i.E'. thesoLid- liquid contact time is insufficient for tbe reaction. ln mis case. me Iiquid may have to be recycLed to eruure a suffidently longcumularive contact time with the biocatalyst. The minimumfluidsacian velocity - t.e. the superficial liquid velocity needed to just suspend the solids from a settled state - depends on severa! factors. induding the density difference between (he phases. the dia.meter of the particles. and theviscosity ofthc liquido

TI

padulent enough ro aid periodk movement of cells fram the deeper peorly lit intenorto the regiolls-ncarcrthewalh_ GeneraUy. a minimum Reynolds number value of 104 is recommended. While turbulence is needed (O improve radiallllXing. too much turbuJence can be harmfuJ. Tbe veJoc ity everywhere shouJd be sufficienI (O prE'Vt'm sedlmentation of cells. Typicallinear veJocities through receiver tubes [end ro be 0.3-0.5 m S- l. Bccause of the need tu m ainta.in adequate sun light penetratian. a tubular solar receiver canno[ be scaled up bysimply increasing [he tube diametel~ The diameter should not exceed 6 cm. although this constraim may be relaxed somewba t bydeploying specially designed sratic mixers rhatimprove radial mixinginside the tube. Without themixers, reducing !he tulle di and D. G. Springham, 005.). pp. 177- 222. Harwood Acaderuic Publ i1; tl(~rs. NC'W York. Doran, p, M. (1995). Bioprows Engillct'rlttg Prllldples. Academk Press. London. Grima, E. M., Fernndez, F. G. A.. Camacho. F. G. ;lnd Chis[i. Y. (1999). Photobioretctors: ligbt rr:gimt>, mas! trans(er, and sCilleup.]. Bioti!chnoL 70,231 -247. L yderseTl. B. K.. D' Elia. N. A. and fllelsoll. K. L (ed s.)(l~). Hiopl'Oce.u Engineering:SyslrnlJ. F.quipmenr tina J-(cllitiu. John Wiley. Ncw York.

Van't Riel. K.and Tramper. j. (1991). Bane Bioro'actorlkstgn . Mareel Dekkff. New York.

Chapter8

Mass transferHenkJ. NoormanNo mendature Introducton Tite mass tra nsfer steps Mass transfer equiltions Determining tbe vo]u metdc mass tr.1Rsfer coefficienls The effect ofscall' on mass ttansfer Furthe'r reading

- 1NomenclatureRom= a inrertacial arca per unit quid volume a' interfacial area per unit total rcardan valumee e,(gas plus liquid) conte.ntration in liquid phase concenlr.ltion al ,quid side ofinterface sarur:l.tion ('" equi.l ibrium) concentratiol1 in liquldpbast' (= p/ffJ

e

CI d D D H

biomass concentrarion liqllid film lhicknes$ dfusion coeffici entor etTectivt' diffilSiviry impellerdiameter HC!nrycoeffldenl

moLm kgm'm m1 s 1 m

1

barm' mol ~ 1

H v liquJd hl1:lght

m

)

molar mass flux

J,

molar lllilSS fiux across gaJi film JI molar ma5S flux ilcroSS liquid film k mass transfer coeffident k, gas film mass transfer coefficient. "1 liquid film mass transfur coeffident kla volllmetricmass transfetcoefficient K OYel'all nl.:lSS transfu coeffidentK11

mol m- 2,s mol m ~J ,s mol m- ~ s ms- I ms- I ms- L1S~1

ms- l

COllsistcncy index

power law index N impeUer rotational speed OTR OJ transfer r "'o. where 1-1-0:= O.05Pa s. Ifthc broth behaves like a pseudopIastic fluid (viscosity isd ecreased at higher shear rates) or dilatant fluid (viscosityis incre3sed with higher shear rates), an average viscosity can be taken over the reactor:

.,.= K '5'"- 1The average sbear ratecan beestimated from :y=lON

(8.16)

(8. 17 )

The parameters K and n in Che rheology model depend on the biomass concentratioo. Typically K is proportional [O C;. with the value of el' r.mging from 1.5 to4. Por pseudoplastic broths n < 1, fordilatant liquids n> 1. whereas for Newmruan media 11 =-1.

HASS TRANSFER

183

llxaOlple A pseudoplastie broth in a bioreactorhas the foUowing properties: e" "" 30 g 1- 1, K= 1, ti =0.4. The stir.rer speed is 3 revolutioru pers. Using Eqn (8.16), che apparent viseosity i5 0 .13 fu s. According to Eqn (8.15), ka is reduced to 51 %ofthe value in a lowviscosity broth. Whatwould happen fthe biomass concentration o r [be impeUerspeed doublcd? (AnsWers: with d ouble biomass conCNeen 50Iids and broth (kg m- ' )

Cost of recoveryHighest

O.2xO.2

Ix27x lO 40x40

100xl00IxI Ox(marted) IOOx l OO

0-120" 70 90 70 50 10

< < < <

'~HH~ - -u~ .....OH

- >4)ct-L-Gulofonlc aC1d'(1 ->I

Alglnat'" , compo$ed

of m~nnuron1c acid;md guluronkacid. Tht. propordom and 'equenceof mese monomers depmd on di", $OUn::e al di", polyme;r.

338

ANDERSON ANO WYNN

B losymhesls oflW1man In XOnlhOI7l0'lOJ r;am~wif. GlA, a1:~I- CoA: Pyr, P'fl"UYa~ : PEP, p h01phocnol pynlVll~e: Lpld, Ilpi!!a rrler (J Re- IS.22).

dlb.us

~J .probe

,~

1 ",+_

1 CcoUng

Lave! proba

...,--,

Alltiloam

..

_

..~ ~

$am !!le AnR!n.!1

pH

SIl9BtI

DO

""""""Sulph8lePIOdUets

Ph04Qtlate:P~U/SOI5

ClIM1INtlO'1

I SugafIOiI

,...,

==

Dissolved 0 1 DO Jevels ;ue critiCal AlI aqueous reactJons. neutral pH and am bient temperatures e Good control and monitoring of reaction through pH measlw ment and adjustment Q uid< removill of o;oluble reaction products from immobilised cataJyst Re-use of immobilised enzyrne catalyst Easy recovery of side chain for re-use Pleasant working environment for aJl personnel Improved product quaJity, less impurities Improved yields and manufacturing capacity Decreased (ost o f manufacture

16.9.4 Production of 6-aminopenicillanic acidOver the last 10 yeaTS the industry has switched from chemical hydrolysis of penicillins to enzym e hydrolysis to decrease cost and anOlio enviraumentaJ benefirs ('rabie 16.7). SpedfLc. immobiLised penicillin amidases bave bccn developed fol' penicillin e and peniciJlin V.bydrolysis. lmmobilised enzyme can be made in-hotlse o. putthased trom third parties. From the. thermadynamic equilibrium o f&APA and the side chain. hydrolysis is sornewhat greater for penidllin V than penid llin G. However peniciln G is a. m ore versatilc prod uct due to its application in rillg expansion owhich partially e xplains its fc rmen tation vol ume d ominance over penicillin V.

ANTIBloncs

367

Ihe final choice between either proces5 is afien directed by lhe company's own rustoricaJ deveJopmcnt success . In conventional Splitcing rechnology. the penicillin salr is used at 12- 15% (wfv) for enzymjc hydrolysis by tbe appropriate irnmobilised penicillin amidase system.This yields mixtures af&APA and the precur sOr acid. During the hydTolysis me pH is mai ntJilled between 7- 8 by the addition ofbase. either caustic or ammonium hydroxide. Tbe product 6-APA can be rt'Cowrcd by precipitation al pH 4 in the presence of a water immiscible-solvent rhe convenient removal ofthe precursor acid . lnoperations that have both penicilLin re rmentatian and splining processes. the reCVe.red pre nutrients to keep them at conceotrations wbich will pe.rmt optimaJ metabolic activity in tbe cultivated micro-orgarnsms. The uptake and energy metabolism ofthe main sugars utilised by baker'S yeast i5 shown in Fig.17.1. Baker's yeast is composed ofliving cells of aerobicaUy grown S, cerevtsjae.The commerdal producers use valious strain.s ofthis species. They differ from the strains ofS. cerevislae used fol' beer production mainly in rheir panero of utilisation of medium components. The product is cimer deUvered as a dried powder(dryyeast) with about 95% dry weight or as a cake with about 25-29% dryweigh. containing onlywashed cells and residual water. The yeastis used to r.se Che dough in the baking process and to give special texture and taste to the bTead. Oough raising is caused by the production of CO 2 during alcoholic ferrnentation of sugaTS available io the dough. These sugars are mainly maltose and glucose. produeed from the floor starch by the a-amylase aetivity in the Rour, or sucrose if added by the baker. lbe majn reactioo ofthe dough raising can be considered as anaerobic fermentation ofhexose to C0 2 and ethanol: (17.1) The carbon dioxide i5 entrapped in the dough and causes its exp:m sion. Tbe erhanol, even though it evapora tes in the oven , concributes (O formation ofesters. However, there are rnany other, le5S well characterised, properties of the yeast thar are important for the bread Quality, as evident from tbe difference between yeast fennented bread and bread

]80

I

ENFORS

produced-with bakingpowder. that aIso evolves COl"Thus, baker's yeast should be considered as a package ofenzymes.ratber thanjustbomass. The: composition af lhis e nzyme package is subject ro opti.mJsation by slra m developmenr and control ofthe fermentatioR process.

17.2 I Medium for baker's ye.se produceionThe stoichiometlyfor production ofbaker's yeast can be summarised as200 g glucose+ 10 gNHl + 100 g Oz + 7.5 g salts~

100gbiomass+ 140gC02 +70g HPTIlis results in me following approximateyie1d coefficienls:

117.2)

Yxs "" O.5kgkg-t y ro = 1.0 kg kg-I YJ(N= O.lkgkg- I , 111e production is an aerobic fedbatch process on a medium af motas ses. aromana orammoniumsalts. phosphates, vitamins and antifoam. Which specific vitamins and additional sal ts have to be ineluded in rhe medium depends on the strain, (he quality ofthe rugar source (moJasses) and the quality ofthe water. S. cerevislae has a rlemand formany como poncnts. as evident from the complexity of a detined med.ium for its growth (seeTable 17.1). For cornmerdal production. howeve.r, rhe mol..lsses and the process water fur.nish mos[ ofthese components, Molasse5 ofboth sugar cane and sugar beet can be used rol' baker's yeaS( production. 1b.e sugar content of the commen:ial moJasses is 45-50%. A major difference becween che two types of molasses is th3t 5Ugar bf:.et molasses contains mainly sucrose and Htt1e hiolin, whlle in sugar come rnolasses the sucrose to a large extent has been hydrolysed to glucose plus fructose, aud ir is also richer in biotin. Furthermore, motasses contaius other fermentable sugars and amino acids that are udlised by the cells. A problem with the beet molasses is that 0.5 te 3% oftbe sugar is r.tffinose, a trisaccharide (fructose-glucose-gaIactose) tbat is only partiallyhyd.rolysed by baker's yeast that does not baw a-galactosidase activity. This results in a substantial emuent ofme.libiose (glu..gal). Brewer's yeas!, on the other band, often.has a-galactosidase activity. a nd doning the gene coding for this enzyme into bake.r's yeast is tberefore aD obvious possibiliry to improve the yield and deOlul.Km DOe of Ihe enantiomerk alco hol! reacts fasterthan !:he omer te fonn /In Utass of an e enantiomer ef the esten (ldeaUy lInantiopu rt!) The sllCcess of!:he resoludon is expressed by the enantiome rk ratio E, which depend$ on he difl"erence in rree energy of activation o( me twO dianernomeric transitlon states formed whlch In wm is related tO the twQ tetrahedral intermediates.

proportional ro rhe.rate constant (1:) and the conrentrations ofthe r eartants. It is the free elle.rgy of artivatioll, 6,Gt, that decides magnitude of tbe rate constantofa reaction.lfa reaction indudes several steps, it is the stepwith the largest;lQt thatis [he rate determining step ofthe rea.ction. An enzyme-catalysed reaction follows a different mechanism from !hat of a reaction caralysed in a non-enzymic manner. TIte difference in rate ofcatalysed and uncatalysed reaction depends on their difference infree energy of activation (a6,Gl) . A relativelywell understood reamon is hydrolysis of either;, peptide or carboxylic ester bond catalysed by a seriue hydrolase, mch as trypsin, chymotrypsin or lipase Bfrom Gandida antarctica. as shown in Fig. 19.2.

19.2

1

Hydrolytic enzymes

Hydrolytic enzymes (Oass 3) are tbe m ostcornmonlyused enzymes in organic chemistry. There are several reasons for this. Firstly, they are easy to use because they do not need co-factors like the oxidoreductases. SecondJy, there is a large number of hydrolytic enzymes available because of their industrial interest. Detergent enzyrnes com-

SYNTHESIS OF CHEMICAlS USING ENZYMES

41 J

Hydrolase

(.)

-

,:!:>(CO,RA CO:t-t

lb'

OCOR7(XOCOR

O(H OCOR (1S,2R)+

~ (X0H

~ (XOCORO H

yX~OR+

OH

le)

X~OR+H

I1 OCOR

Hydrolese

X~OR

OCOR

-

HO H

X~ORB"'!.I/"H

H ceOA

(d)

A

-->=0

B,

Baker's yeast

~

~\ A ~

+

A

..::> kJ and me lower monoeuer (1R.2S) wlR be eonsumed fasl"!. Hcnce both st~ wllI k;.ld w an Increase ofme u ppe1' enantlomerat Ihe monoenlr nage. lr the reOt.her meso-gres$ cUrYlO$look lil! the examples of Flg. 19. 5. For reaCl.lo"s wlth sma ller K., val~ a dr.lrnatic effect i$ observed for ee,. The curve reaches a rnlXlmum. a1 the n!2.ction progr'eS5es furcher. tt, is reduud and ~he curve Ilt.'ver reaches 100%as le alw~yl does in cm. Irre .... rslbte cue. The e!fea of reverslblUty If noc U dr;matlc 011 fil~. The CUr-il ips down al an urlier oogree of conve rt lon wnen Koq Islo_red. An obvloU1 way lO proued is 10 pum lhe (euuon towards the pl"Odutnoo n of urlc "del in a

JaIf1>Io!. the inlti~ 1 absorbance al291 nm is measun:d. lhen the enzyme.lJI"ic;ff (urate oxidaul).;$ addo!eI. Oxid,don ofurlc acid (with 0 1;U the oxlcbnt) oc:nrs unti l aH cM wbstr.lte has befll C OIIVf!rtet.,ru ~nd co llun~rUal ly ..v:ail:a"J...

"32

I

KRESSE

HO-~CH' OOHOH

HaOHGlucose Glucose 6-phosphate

-O-vF~H

G6P-DH

Hbf-f1OH

HONADP+ NADPH

~OH

CH"'OH COOH

OH

Glucose 6-phosphate

6-Phosphogl LlColiate

An example of a coupled enzymatIc assay system using an indicatar enzyme: glurose assay wim hexoklnase and glucose-6-phosphate dchydrogcnase. The determinario n al glucOM! In blood or load materlals comprlses the pho~horyation 01 glucose catalysed b~ yeaCesses). and is used tberapeutieaU mainly in renal an;)emia, but y aiso in other indications. e.g. in tUIllOur anaernia. Granulocyte-colony stimulating factot(G-CSp) G-CSF belongs, as EPO, to me dass ofbaematopoietic growth fueton. GCSF stimulates proliferation and differentiation ofneutrophil precursor cells to mature granulocytes. It is therefore used as ao adjunct in chemotherapy ofcaneer to treat neutropenia caused by tbe dcstruction ofwhite blood eell5 by the cytotoxic agent. Furthermore, G-CSF is also lIsed in the rreatmenrof myelosuppt"ession afier bone marrow transplantation. chronic neutropenia, acute leukaemia. aplastic anaemia. as well aS to mobilise haematopoietic precursor cells frolD peripheral blood. G-C5F is aglycoprotein containing 174 amino acid residues, Pl'oduets bave been launched which eontam either the glyco5ylated mol&lI le produced fl"om rceombinant CHO cells (Lenograstim) or altematively an ungtycosylated, but the.rapeuticallyequallyeffcctive, form producel shown aboye, the production potential ofrnanmlalian cens is not the lirniting factor but ratheritis the attainable biomass concentration. To meet this demand, fed-barch culture.s andhollow fibre reactors have been used to obtain high eell deruity cultures of hybridoma and rnyeloma cells. Glucose and gl utamine Iimitaton has been combined wi(h feeding of amino acids and serum. resulting in a total eell concentration of approximately 5 X 107 eells ml-! (ofwhichless thanhalfwasviable)over 550 b. and a:final antibody concentration of2A g 1- 1 , i.e. giving a vol umetric productivity ofO.l g 1-1day. Commercial production of monodonal antibodies in hollow:fibre reactors can yield about 700 g product per month at abont 2 g 1- 1 Each ron Jasts foe abont (hree months but tbe first [un 15 nor productive sinee t:h.is time is required for building U]) tbe biornass in the extra capi1lary space. TIIe productivity in this system 15 0.3 g l-l.day during the harvest periodo

21.8

I Genetic engineering of mammalian cells

Geneticmodificationofmammaliancclls can be used to introduce the genetic information needed for production of a specific protein or to improve the characteristics of a production ceU lineoThere are many methods thatcan be used to introduce fore.ign DNA.into a mammalian cell. amongstothers are: electroporation, lipofeetion in whlch the DNA 15 introduced via liposomes. micro-injection of the ONA directly into the ceno fusion of the manunalian cell with a bacterial protoplast containing the DNA or viral vector systems. A transfected ceH tine will express tbeintroduced DNA stablyonly ifitis integrated in the genome. In contrast to micro-organisms. lilce S. cerevisiae and E.coH. the integra-

MAMMAlIAN CELL CULTURE

46'

tion ofthe introduced DNA is ltlostly nonbomologous. The.gene eneod ing a protein product m ay the.refore be integrated into regions of the genome thar are not favourable for efficient expression of the gene. Selection forthe best producing tr.msrenants is therefore always necessary. SevffaI selectablt ruarkers for marnmalian eel1lines are available. Dominan( markel'S thatean be used irrespective ofthe host ce11 line are mostly concemed with drug resisrance. Recessive markers, tbar are used in combinarion with a specmc hosr ceO genetic background, can involve enzyme5 ofme salvage patbways afthe purioe md pyrimidioe metabolismodrug resistance or amina acid merabolism. The two mos( successful systems are tbe glutamine synthetase (GS) system and tbe dibydrofulate: reductase (dhfr) system. The enzyme glutamine synthetase catalyses the furmation ofglura mine from glutamare and ammonium iom. The GS gene can be used as a selectable marker in hybridoma and myeloma cells and other cells thatdo not possess GS. Stable transfected cells will express the GS gene and are therefure able ro growin gluramine-free media.As with the dhfr system (see below) the GS system can be used ta amplifY the product gene, a procedure aiso leading to amplification of the es gene. The me tabolicconsequence ofthis situation would be thar tbe ce.ll aculaUy overproduces glutamine. Thedhfr syS(e.m is mostlyused in combinanoo with a clhfr- CHO cell line, A dhfr- cellline is unable to synthesise tetrabydrofotate wbkh is an essential cornctor in the one-carbon metabolismoDhfr- cell Lines are only abie to grow inmedia containing thymidine. glycine and bypoxantbine, precurson and building blocks necessary to ovcrcome chis defi. ciency. Srable, trao.sfected cells that t'..'l:press tbe dhfrgene are capable of growth in unsupplemented medium. MethotTexate (MTX) can be used 10 ampli1Y rhe dhfr gene. Ibis folate anaJogue inhibits the dhfr gene producto By selecting for cells capable of growth in a medium witb increasing concentr.ttions ofMIX, cells with an increased numher of gene copies. and th~eby with enhanced expression of (he dhfr gene product. are obtained. An enhanced expression ofthe produce protein is obtained at tbe same time. A disadvantage ofilie dhfr system is rhar MTX resistance can develop that is ndependent o dhfr expression. TIte introduction of foreign genes into marnmalian eells 1S quite common, while the deletion of specific genes is notoAs mammalian cells sbow heterologous recombination, the opporrunities for sitespecific insertions and deletions are lacking as is possible in yeasts and E.. coli. Mutations ro prewnr expression of genes can be made by less specific dassical methods, like UV treatment of ceUs , combined with selection for tbe desired phenotype as has been done for the generation ofglycosylation muta.nts. A more recentapproach togene 'knock-out' is che use o antisense oligo nudeotides that hybridise with a specific mKNA. thereby preventing its transtation Lnto roa,t un: protein. Genetic modification ofmammalian cells fal" cellline improvement is notyetwide-sprea.d but is increasing in importance. Ateas ofintetest are the prolongationofproductive ceH life,growth in serumfree media.,

o

VRlEZEN, VAN DIJKEN ANO HAGGSTRM

the decrease ofby-product formarion and glycosylarion characteristics.Apoptosis , that oecurs in most rnammalian c:ell cultures, can be influenced by introducing the bd2 gene. an anti-apoprotic gene. TIls prolongs cell life, and thereby tbe productive phase of a process. An example of decreasing by-produ ct formiltion is the introrluction ofrhe GS gene. Cells with GS produce less ammonia/ammonium as they can be cultivated in media without glutamlne. As a resultof tbis . the production ofMAbsinhybridoma celis i5 increased . CHO ceHUnes with gly-

cosylanon mutations have beeo developed with the aim of generating a less heterogeneous glycosylation ofthe product formed by tbese ceUs .

21.9

I

Fu rther reading

Butler, M. (ed.) (1991). Mn!llnwlfan Cdl BiotrdmoJogy. A Practical ApprtXlrh, Oxford Univeni.ty Press. NewYork. Spier. R. E. (ed.) (2000). Tht F.m:ydopedia ofC.ell TrdInDlo.;y. John Wiley. New York.

Chapter 22

BiotransformationsJoaquim M. S. CabralIntroduction Biocatalyst selection BiocataIyst irnmobllisaon aud perfurmallcelmmobilised enzyme reactors BiocataIysis in non-conventional media Conducling remarks

Furtherreading

22.1

I Introduction

Biotransformation deals with fue USe ofbiological cataIysts to convert a ~ubstrate iTIto a product in a limited llumber of enzymatic steps. The establishment of an efficieflt biotransfonnation process requires the extensive examinaran offactors affecting tbe development of oprimal biocata1ysts. reaction media and bioreactors (Fig. 22.1). There are many opportunities for industrial us~of biological cata1ysts for biotransformations. These nelude not ooly the tl'aditional bydrolytk (e.g. starch and protein hydrolysisj and isomerisation (e.g. glucose conversioll to fructose) reactions bur, more recently, synthesis of chiral compounds. reversal of hydrolytic reactions. complex synthetic reactiolls suenas aromatic hydroxylations and enzymatic grOllp protection chemistry and degradation of toxic and environmentally hannful compotmds. Biological cata1ysts when compared with chemical catalysts have the advantages of their n ..gioselectivi ty and srereospecifid ty wh ich lead to single enantiomeric products witb regulatory requisites for phannaceutical, fuod and agricultural use. They are also energy effective catalysts working armoderare temperarures. presslIres and pHvalues. Hiotransfonnations have been perforrned by a variety of biological catalysts, such as isolated enzymes. cells. irnmobilised enzymes and celIs. lhe dcvelopments of recombinant DNA technology have led to improvements in the enzyme production in different host org;ulisffi.S giving the bioprocess en.g:ineer a greater choice ofbiociltalyst option. The optimal biocatalyst must be selective. active and stable under

172

CABRAL

Biotransrormatlon

Substratas

Productconcentration

Ovarall volumatric

productivity

Ouality Purity Scale

Product

operational conditions in the bioreactor, which may nor be necessarily conveotional in terms of composition, concentratioo, pressure arrd temperature. lo particular ir is necessary ro evaluare rhe biocatalyst performance in non-conventional media (e.g. organic solvents arrd supercritical fluids) . A l- - .

r--l---.} '---.

.. ..

Membrana reactor

Continuous membrana

reaClor

Anorher altemative is ro d1311gC Che flow pattero. using a plug f]ow r:ype of reactor: the total recycle reactor ar batch rerurulation reactor, which may be a packed bed or Ouidised bed reactor, or even a coated tubular reactor. This type of reactor may be useful where a single pass gives inadc.quate conversioas. However, ie has found greatest applica tion in me laboratory for the acquisition of kinetic data, when tbe recyde rate is adjusted so thar tbe con version in me reactor is low and it can be considered as a differential reactor. One advantageofthis cype ofreactor is that the externa! mass transfec.effects can be reduced bythe operational high fluid velocitics.

22.4.3 Continuous reactorsThe continuous operation of immobilised cnzymes has sorne advan

rages when compared witb batch processes, such rl res ultingfrom ~ T..:e11dependern B-eeU respon,alrwolyes ancErn r6 21 kJ onlywhen pH~ < 10- u atIn, while the mi.nimum pHl value allowing the production of one mol melhane to generate - 21 kJ is also in the range 1O-~ atIn. Thus only when pR,t lies around 10- 5 atlll is the sequential conversion ofpropionate to acerate and acetare ro ntethRDe possble. Similar conc1usions can be draWIl fur tbe conversion ofbutyrate to methane and also wjth formate instead of H2 as intermediate. The understanding ofthe nature ofthe 'symbiosis' amang synrrophk o rganisms is a challenging task and essential 10 the optimisation of anaerobic biotedmology. Mast anaerobic reactors treatingwastewaten are upflow anaerobic sludge blanket, or UASB, reac[Ors (Pig. 24.5). Tbe waste water eoters the reactor at the bottom via a specia1ly designed influent distribution system and subsequently flows through a sludge hed cansisting of anaerobic bacteria growing In the fonn of granuLes which set'tle very well (50-SO m h - I ). The mixture ofsludge, biogas and water is separated in the three phase separatorsituated in the top ofthe reactor. The major advantages of anerobic waste water lreatIOeot over aerobic.: treatment are tbe sma lJ sludge production (0.1 kg per kg BODJ, tbe low energyconsumptioJl sin ce 00 aeration is required and tb e small f1oorarea, typicallyO.Ol mI perinJlabitant compared ro 0.05 rol for acdvated sludge planls (Table 24.1). Moreover.. energy is reoovered in the fonn ofbiogas (0.35 1IT1ethane per g BODs)' The rateofBOD removal in Ce o ra toxic oompound in the wau.t waler sampJe added in B. Passlble remedial actioos are (1) me additicn cf tcXlcanl-ntutralbing additlves In lhe nuln flcwlo me pblnt, e,g, powder activate'

mixed reactor

Wastewater

Organic slurry50-100 2- 5 20-40 20-40

Solid WdStes

Solid concentration in reactor(g 1- 1) Loading me (kg organics m - J'day)Hydrau1ic retention time (days) Salid retention time (days)NJt20

50-100 2-5 20-40 20-40

Solid wastes 200-400

20-40 1 0-2010-20

The rompletcly mcro reactors tteating organic slurries are operarro. at low volumctric loading rates. i.c. 2 ro S kg organics m-l' day because me particulate organics musr be solubilised befure they can be subjecced to anaerobic conversions [rabie 24.2). The rare ofsolubilisation of particulate organics may be rather slow as in the case of waste actlvated sludge which takes 1S days ro reach 90% hydrolysis. As a consequence, retention times ofat (east 20 days and up ro 60 days or (onger are usecl. Several new developmentsincrease the performallceofanaerobic digesters. For example, rhe hydraulic rercnDon time in che reactor can be uncoupled from tbe solid retention time by filtenng the treated effluent and reinjecting the solids in [he reactor until the hydrolysis products pass througb the membrane, This reactor design removes a greater proportion of sotids due to tbe Jonger solid retention time and achieves this in a srnaller (cheapel') reactor due to the smaller hydraulc Tetentioll time. Improved performance can al50 be obtained by runoing the digestion at higher temperatures since the rate ofhydrolysis ofparticulate matter increases with temperarure. Ncw insigbts in thermophilic digestion rcsulted in the construction in Dcnmark of several large-scale therrnopbilic digesrors to treat farm manure. Being rnn at higher temperatures. these reactors yield a pathogen-free efftuent, unlike the mesophilic digesters which ofien fail to meet che reguJations in terms of faeca l pathogens (Fg. 24.8). Several drawbacks llave, in the past, kept thermophilic digestion from becoming popular, for example the difficulty o( start-up and rhe sensitivity to certain stress factors such as NH 3 and H1S. 8entonite ciay can be used to rernO'ft NH 3 illhibiriOll. H1S, on the other band , can be descroyed by inj ecting electron acceptors, e.g. oxygen or nitrare in the reactor. Perhaps the major problem, at least-for sewage 5ludge digesters, is to minimise the massof N and Pbeing recycled to the main planrflowvia tbe so-called 'sJudge water'. Indeed. more tban 50% of the sludge N is hyd rolysed during digestion aud che resul ting recyde load con tains typically about 1 g NH: 1- 1 and mayrontribute 20% ofthe infiuentN load.

- -

---------- ~iltlmesof

VANDEVNERE ANO VERSTRAETE

;";;;;P"ho,,,' ,;~M:ani!ms lIpan contillUOusuposure to dUl'erellt temptr.1turel. l'1esaphnic reactol"l. trutiflg ."Ima/ rNnllf'eS or ~ i/udg u 20-30 C ....ith retentlon of ont mOl'lm do na{

2

...~

tme:s

. Iimlnare me SalonM~completely. TIt, tlItrmophilic

,.

E

40

reacton, ron at 5S C, ,uccud n IIIIng 111 pathozens after 1 ft:wdays' re~non lime.

Vbrio cho/ent8SalmonflJl8

1 dav

1 week 1 monlh

This extra nutrient load may cause problem s in view ofthe ncw, more

stringent. standards concerning the nutrient content of discharged efflu t!lln. This may also be the case for P as sorne investigators have found tbar up to 60% of the sludge-bou nd P may be rcleased during anaerobic digestion. Various treatments llave in the past been opurnise-d lO precipitare P chemica.l1y. The cost of mese treatmellts ha\le. however. prevenled lhem from bcing used in practice. 111e pH-controlled precipitation with lime sceros attrdctive bccause the tllgh pH may a lso serve tO remov'. me ammoruum by sttipping. TIle con auociatcd witb mE' lime addition can be greatly reduced by pre-aetatiog the effluene in ordE'r to fE'move the buffering capacity assodaled with [he alkalinty. Th.is method can al50 be combined with dlE'additiODof Al or Fe salrs. preferably from a cheap souece suc.b as AlfFe-rich sludge ITom drinldng water production p lants. Still olnother methoed to evapor.lte tha waste water generated during the mechanical dewatering ofthe digested p3.!>"te. Dewnered paste (500IlS0\ids 1- 1 i:; subjected toa short( 1-2 weeks) aerobk pon-treaonent ) ylel dlng a humus-like material The varlous points where odours are produced. e.g. the aerobic post_componing. are ventilated and the wane air Is treated In a biofilter where vol~tile organic co mpounds are renK.>ved.

to waste a lOlor energy and creare secondary pollution. Pollutant concentrations in industrial emissions, for example. are. of the order of 100 ml m-O. To burn these gases in an incinerator. at least 50 litres methane need to be added per m l in order to ensure complete destruction. A bioreattor mayo in most tases. achieve the same oxidation provided thc VOCs are brougbt in close contact witb degradative microbes. 0l' lizO and nutrients. Biodegradation. rates vary with me pollutant being degraded: quicldybiodegraded: aleaboIs. ketones. aldehydes. organic acids. organo-N: slowlybiodegTdded: phenols. hydrocarbons. soIvents (e.g. chIoroethene): very sIowlybiodegraded: poly-halogenated and poly-aromatic hydrocarbons.

ENVIRONMENTALAPPUCAnONS

54i

Biofilter

r--- Wate~

Supportmaterial

Wasteair -

- jU Humldlflor Biofilter Nutrients. pH control l

BioscrubberClesn sir

IJ-U

I

Activated s tudge

Waste al rSpray chamber

t t{acrubber)Compact wast e wate r treatment

Air

"""Despite [he broad spectrum ofairpolluta.nts amenable to biofiJter [reatment, the introduction ofthis new technology is slow. perhaps because its low cost does not ensure h.igh profit margins and because m e physico-chemical air pollution conu"Ol indu stry is well entrenchcd. Various types ofreactor designs are used to treat air biologically (Fig. 24.11).ln biofilters, contaminated air flows slowly tbrougb a "Wetporous medium - compost, peat. orwood chips - which support Jo degl, Th. s~u'mtlalneps al"fl solubilisatOll In a

scrubber, N ren'l0V31 ina tlloreactor. w lphite reducrlon to

sulphide in a UASB reilctor.sulptllde partial ox1datlon 10eI~en~1

S'ln a submerged oltic

atLlched biofllm reacto r and Il'lcavery of solid sulphur, Thel;quld phne ts contll'llJOYSly~",,",.

2 Fell{ED'TA) (NO) + electron donar -. 2 Fell(EDTA) + Nz + COl + H ~O Anelectron donar. e.g. methanol oretbanol. needs to be added inomer to sustain the reaction. In tbe two following Steps. fl:SOJ is sequentially

ENVlRONMENTALAf'PUCATIONS

504

reduced biologically to ~S and finally partially re-oxidized to solid ejemental sulpbur:~SO,+3~ --+ ~S+

H2S+3 ~O

YI O~ --+ So + HzO

The rrouction ofH~SOJ takes phlce in a UASB reactor (Fig. 24.5) seeded with sulphale-reducing bacteria. Rocculant polymers are added, logether with fue necessary nuttients a nd reducing equivalenu (ethanol or 1\) to adj ust tbe (BOD HZSOJ) molar ratio at a value of one. lo the third bioreactor, erobie bacteria oxidisesulphide back tosolid SO (end-product). TIte further oxldation of S ~ lo A!S03 and H]S04 is preventeandtreat sites cvaluate-d by a committce under the auspicesof the US Nationa! Researcb Council (NRq in 1992. only eight had reportedly reached the cleanup goals, which in aH cases were the maximum contaminantlevels for constituents regulared under the Safe Drinking Water Act. Of the eight successful sites. six were polluted with petro/eum hydrocarbons which would also have becn elinlinated via naturnJ attenuation. 'Ole NRe CommiUee concluded tbat pump-andtrear me thods Wtle quire Jimited in theil' abilityto remove contaminallt mass fiom rbe subsurface because of sub-surface heterogeneities, prescnce offractu res, lowpermeability !ayers. stTangly adsorbed compounds, and slow mass transfer in the sub-surl'ace. Even with the best extraction 'metbocls, very afien only a small fraction of soilbound contami.nants can be mobi1Euro~

ENVIRONMENTAL APPLlCATIO NS

SSS

Pump-a nd-treat

'M,,,strip colurnn

Th Il'puI"p-and-

treat' r"eml. 95. 97 artiftdaJ. 120 bacterial.6O.61 replicatian fork. 62 bacto:!rial artillela!. 78 fungal.61 vira!. 61 Cbylllooln, 11, 125 Cit:ricacid. 306-1:S Clollil\g(DNA) libarles,76-81.H17- 15 Qoningveolism and e nel.'gyyields.460 pharm;IC1!utic.a1 pro teiru, 452 prorcW glymsytlllion. 453-5 Malllmalilln ce lls. 437 MaS51ransfer coefftclenl.!, I83-5 equations. 177-83 Fickequation. t77 gas tliJn tr.msport. 178 liquld film transport, 178 gas-liquid. 180 Iiquid-solid. 183 axygen trarufer measurement. 184 srne down. 186 s(al e up a nd mass transfer 1116 rramfcr across lile ccll envelopc. 176--'7 Il'ilnsfer berween pbaJies, 176 traJlsfer insidea single phase. 176 rwo.fllm theOll', 178 volu mdric mas~ tr:m sfer coefficie ntS. 183-" Measure ment ,llld control adaptlvc conool. 235 advanced modelJing consideratiollS.222 arti.tlclal nl'm:al ne t:works. 223 ITO$$validation Itthni que. 227 fu~zy expert sys tems. 225 fu~1.y rule system s. 22ol. 225 model parnmeters. 225 model validaton. 2.27 off-limomeasul'li!ment. 226 olJoline measuremenu, 226.:.12 8-9

lactlc acid. 34. 317-19 I..a.c!oroum wsei. 87 Leurononoc: rr.esenttft)jde3". 315 Linearr4te equadollS. 137 ,.Linohmic acid. 340 Linkage g roupa. 98 Up.ascs. 4'13. 473L~,in{'.283.289-93

mass bala.lCC' equatioD., 2"16 l model pred i ctj~ control. 235. 237 time hori'.!:on. 236 prOCe5s iden tificadon. 215 proce5s node~. 215-16 process $upervisi on. 228 rtac:tion equatiom , 216 spcci.fic by-prod u ct COIl$Umptioll rate.220 specific by-productgeneratioD rene.sJX'Cific growth rJte, 220 spo;.or~

culture syuems,

466 microc;l rriacterial. 318 biochenlkil1 P;tllWa:yS. 317- 19 production o rg:mis1ns. 317-19USe! ,321

synth\$c.343unU lO ra ted, 341-2 lipid a t'C"U blu latioil.. 342 m a.liC"en :yme-.344

m RNA.4o-l . S)- S, 112Mntanll"OmpleruentalioD. 109 Mu tan l isoladon. 108 M,vroplusmllgnital!um,61 Mywrort11S ....arrtll ll5. 6"1 Nested prim e!' PCR, U2 Neurusparu musc, 100. 108

m iCJ"O-()rga nisnu as oil f./cturks. J.45 n u trion al im por tance uf polyunsalul"ated fatly actds.3llna lllide aden ine din uc1eotide(liAD). 20 rt'clu red NAD. 20 Northcrn blotting, 73. 8.J Nudcic acid p l"obe ~. 73- 4 NudoosoOle.~ . 99N~t a tin . 352

sucdnk acid. 324 tartarie acid . 3 24 3-Oxid.ation cyd~. 23 Ol'idative phosphorylatiOI1 , 30 Orldo-reduClilses. 419 Oxygen. stt "IInd~r Mass m nsfe.r

.!liOllOl. 330 biosynthe.'i is ofPHru. 3J0 bjosy n r hesis of PHB. 328 biosyn dll~!'iis ofPHUJV. 330 t"Ompvsir ion ofPHi\, 328 l"Onrinuom nutrientlim.ltation, 327 medica! appliCl lions ofPHIl, 3J1 nUl rient Ii mi tatiun. 326 PHA 1"l5eI"VI!" maler ials, 326 PHB gr.u m les. 332 physical pmperties ufPFlA. 3:.17 p la n ts a5 pros~di\IC so u rce$ of PHA.323 )lotyhydroxybutyf""te.326 productioo ofl'HA by rcco\nbin allt bacteria. 332 regula lion ofl'HB miltaooli slIl, 329 Mic :obia l polys.. tcharld cs 1 a lg illate, 337-8 biosynlhesis.338 curdlan. JJG dextran, 335-6 exopolysa