Basic Biotechnology 2nd Ed - C. Ratledge, B. Kristiansen (Cambridge, 2001) WW

BaSK Biotechnology Biotechnology 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 o~ytic add cyde: to prod!.Jtl! Intl!rmedllte~ lnd Me!X)' (ATP).

Citrie aeid cyele

GROWTH ANO METABOclSH

pan of cnvironmeo ral biotedtnology which is {'xpJaineu in detall in OJ.apter24 . To illustrate this diversity, the exa.mple of microbial degradation of fanyacids will be collsidered. The ability ofmicro.arg;misms to growon oils and fats is widespread. The difference betweeo 3D Di! and a fue is wherher one is liquid or salid at ambient temperatures: mey are both chemically the same, that is chey are fatty acyl triesters of glyceroL:

CH,OH I CHOH I CH,OH

CH,O.OC{CH,I.-CH, I

giycerol

triacylglycerol

wbere o. m and p are rypically 14 or 16: me long alkyl chain maybe saturated as indicated ormay b.aw ooeor more double bonds givingunsat· unted. ur polyun satttrated . fauy aeyl groups. TIle oils, when added to microbiaJ cull'ures, are initially hydrolysed by aUpase enzyme into ils constituent f.J.tty acids and glycerol. The latter is then metaboLised byconversioo lO gIycel'3Jde hyde 3·phosphate (ree Fig_ 2.6). The fany acids are raken iota the ceU aod immediately converted into their coenzyme A thioesters. The fatty acyl-CoA esters are degraded in a cyclic sequcnce of reactious (sce Fig. 2.13) in which the

Acetyl-CoA

($Ce A¡. 2. 10), are ( 1) isocltr.ate

¡

trasO! and (2) malatB synthue. ~

Isocitral6

~S""i"," 1

Glyoxylate

'umm"

Malate ~

¡

Oxaloacelate _ _ ATP

--J.

0

Hcw ceH ensures equal wpplies of onIoacetaU (OAA) and acetyl-CoA (AcCoA) for citric acld biosynt~esls . Tht activily of pyruvate urboxytUt (2) Is nimubttd by acetyl-CoA fOl'med by ~1"UY2[e ¿dYydrogen:ue ( 1)

The ¡Iyoxylate by-pm.. Th e acklitlonal reactloos. beyond tho~ 01 me trlarboxrl;( acl¿ cyde

Citrato

~j®

y

• Acetyl-CoA

tne

I Cf!,O.OCíCH,I,-CH,

I

~

Oxaloacetatll

Citrato

CHO.OC-{ CH2)m-C~

1Acotyl·CoA

Pyru vate

ca,

Aop .-/l - Phospnonnolpyruvate

~

_ _ ...J

~cheme ¡ llO shows oow che bypass functions lO permit rugar formatlon from acetyl-CoA. wim lhe ad~ ruetlo n (1) pOO~hoeno lpyruvat.e

arboxykl nne. folowed by nlVemd IIyc~s (d. Fi,. 2. 1'1).

27

28- 1_ RATlfOGE " ._ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __

IRCH,.CH¡.CODH)

,8-0xldaticll\ cyde of

f8try acid

"., K"~:oA "~r\.. En:r:yom.s are:

wn ZJ crotona¡e): (1) 3.hydroxyac:y\-

ATP= t m H-S-COA

G)

AOP

+

HR tnlMpO(t chain diviCc and elllctrom;wd protons cm iIow tllroulh bom me deSCTbed. The $¡teS of phospho.-yladon 01 AOP to ATP ar. onIy Imlications ~ the actual fonna!ion of ATP is carl"ied out

~u

~ATP $)'fltha~es that are driven by

Outslde

( / __ nH'

\

'- -3"··~

me physlcal mO'lt!menc of prot.lm thrgtJ&h the membrwte (see ~ho Fig. 2.161.

Membrano

Inside

Electron tran spon chain AOP+ PI

Thecouplng m.charlÍsm of ET? The elec.lrOn transportcaml!l"s (tee Fil. 2. 1S)

arll locatlld wlthln a Il1l!mbr.me. As thll redllcW1t (AHJ Is oxidited, mis seu upa fTJO"VefTIeIlt 01 protonli throllgh the. membranll. The$e protoN are then pUfT'olXjd back acro" ene membr¡¡ne driving the ATP sym~ 1! Ima cauplin,ADP with PI [O JÍ""t! ATP.

31

'1

32

RATLEDGE

MolesATP prodLJced per

mole hexose

Glycolysis (gluco$e (O pyruYare): Net yield ofATP =2 mol NADH=2molx3 PyI1N(l(C

2" 6

ro oce!yl-ú:>A: NADH= I mol x3(x2 for 2pyruvate)

6

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

Total

18 4 '2 38

r.'0lIt: • Under ~n.:u.rob;c cn X 174

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

1 1 1 1 1 1 1

lambda T4 M ycoplosmo geniwlium Borre/io burgdOt(eri

Campylobocter lelunJ Rhodobaclff sphaerOldes Bacillus sub61is Esch erichio wli

2

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

3

Socchoromyces cerevisioe

16

eul.~.." i

¡¡

t:.~!""'!!i"6~·-·'·

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

71

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 recombinants Mter 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 él. 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 speciñc 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

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

4,4,7 Nucleic aeid probes and hybridisation Nucleie 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

LeS!! hydrophobie. wilh . 19nal peptid ase re.::ogni,ion site ( ~ 8 rlsl tloosJ

lb) Socretion vector: lnéuc ibh'l promoter

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

---Ciimm;,';-i_ _llIIlHe.-

me

/

l equence.

(c)

\

Signal

Clerning

Anllbiatic

$&Quunce

!lite

."lst' nc8gl'lnll

-

Rtlpllcatiern o rigin

Secretion lIac1or wi\h insert: -Yi¡i IlIl1 WVUZUII

e

largal gene MC¡uence fus ed in·frame 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 perm¡¡nent 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 01 tl"al'lstrlptlon Inltlarlnn poinl$ u51ng

SI

noclUSI.

mRNA Í!5 hybrid~d lO

a dflMWred ONA rr'alrnent th;¡.t has been bbelled at iu 5'...end. The ONA fra¡rrterlt 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 St3rt

DNAII mRNA

~....

TIE

Gene 2

Gene 1

9

.....

'i

I!

Hybridlse extracted mRNA

transcripts 10 a deneluroo, 5'-laba lled, ONA Irll9mont

5'·labelled

1

;;... _e:

'M

polnf. Thll RNAJDNA hybrid moleal~

__o

hu sift&le-stnnded

UQnsiOlU Úlatal"O!. d~raded by

1M sln¡le-smond-:;pecific anivity of

S I nuc!e¡¡s-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 me Mu;¡¡m;aml Gilbert chernial d~'Bge

method.

,, , ,,, ,,

,,, ,,,

I

ElectrophoreS8

,. product on a denaturing

,

T polyaerylamlde gel

GG

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!eLwar 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"Ípt. 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

Sonthern blotting DNA fragments ilre separated according ro size bye1ectrophoresis. tr.msrerred to a membrane and probed with a labelle-~

S'

are B

3'

"::>-

"gB

J'

•

:::::o

Spetlfit gen!! deletlon In f~~ment~ funll. Upstreml (5 ' ) and down5tr'Um (l') reglons of a~ le¡¡5t I kb In Jlze, Immedlalfllyadjacllnt te thII chromowmal ¡en. to be deleted (gene X). Incorponl\eese two complemenW'y RNA moIecules blnd together ro form iI.

-GeneX-

IT

p

I---Genax-I

T

J} s'

3'

AMA Sensa mRNA

S'

MM3'

S'

+

~

3'

........ ··.. ··..... AAAA

Antisense mRNA 3'

---MM

Duple x RNA

S'

duplex RNA Formation of duplex RNA in the nudeus reduces me amount of m3tun- mRNA available IOr translatJon in che cytoplasm.

which is espedally useful when tbe gene in question is essential fOI" growth. is to decrease the amount ofa protein produced within cells_ TIüs uses a process known as 'antisense' in which a messenger KNA (mRNA) sequence complementary to the sense roRNA interferes with [he production afthe protein encoded by the targetgene fFig. 5.11). This. approach is becoming more commonIy used fol' the functi:onal assess· ment of daned genes inyeasts and filamentous fungi altbough it has not a1ways proved successful.

GENETIC ENGINEERING: FUNGI

Fungal transformllnta

• • • •

! Grow mycallll

1.

from p~rlfled funpi tranifol"rnllnl colonIas on agar pl.it.s

a,..

iOCKuI.ued ¡nlO gTOWth medium Wl

mlerowe plates. Mtr 16-24 Il growm. funpl mycella aJ'& v~sfl!ml!d from lile orltlnal microtitnl pbte toa n_ platll PUrifyIO..-.g Ia~_

~ lnCIc\II3Ie miGrotitre ~"1.

0000 00 000000 000000000000 000000000000 000000000000 000000000000 000000000000 000000000000 00000 00 00000

1 Cen wall digestion

Screenil:g fun,gal

UIDsrormanu by PCR. Sporu

3. Tf1ifl5I" myaIIi. ¡rIlO T....." ,,*_p/III" 4 ....lId ceII ..... - 95%) S. cen:risiae genes appear not ro require CCAAT-boxes for function althoughthis motifisknown tofunction in A.1'IiduJolls. [tshould be stressed that in the majority of fungal promotet'S mat have been ¡solated, the funcrlonal significance DI sequences identified in rhem has uot been detennined and therefore remains uuclear. With a constitutive promoter, the basal level of transcription is determined by me binding to the 'core promoter' ofa proteio cornplex which eontains RNA polymerase and tbe so. 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 normal¡sed 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 ís 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 u¡úcellular 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

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

135

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 ¡ c¡i.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 remarkabLe robustness and general validity 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 -1r i(npaller sparger-\t::::tlt--.Y

L ..

@Hi

~

A stirred tank

bloreaaor.

(o)

Sorne commonly usad imP-l ¡r¡

>

¡r¡

O

'" ;;'"

"

.....

1M'

A flvidbed bed

I biofe;JttOf".

SenUng ~"

Fluidised biocBllIlyst

Llquid

-

~,

• • • • •• • • • • • •

Rueyele

Pump

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

7.2.4 Fluidised beds F1uidised 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

TI

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 minimumfluid¡sacian 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

pad ¡"¡'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'"- 1

(8.16)

The average sbear ratecan beestimated from : y=lON

(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

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), k¡a 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 conC 300 01 3), Furthermore, liquid rransport and mixing will become very sJow compared to mass tr.msfer and reaction. and tbus rule tbe overall reac tion raleo Cooling limitations may become more significant. • The average power inputshould notexceed 5 kW m- ~.]fhigher, the mkro-organisms may be mechanicalIy damaged in areaswhere the value is locally much higher: in addition the energy costs and investmenr costs for rhe motorwill become excessively high. • !he pressure corrected superficial airvelocity should be beJow 0.10 m S- l. Compressor costs are restricting, and high gas bold-up will ¡ncrease ar the rost ofbrorhspace. • The head-space pressure has a maximum fue mechanicalreasons_ln addition CO 2 partial pressure will also ¡ncrease. and inbibit growth and production. • The gas pbdse canDor be consídered ídeally mixed. The O}partial presrure will faU as the bubbles travel up through tbe reactor and rhis reduces rhe driving force for mass transfer.

Low

o)(ygen

Air liquid now and DI tranger In a large biore:lctor. Most

ofthe 0 1 is transferTed In me region n...artlle impeller. In the circulation loop. Le. me path the brom travels Imm thft lmpeller; Out

into the body 01 th. reactor and back to the mpder. more 0lls consumid than cransferred and tite

0 1 conCf)mraúon wi!! decrease. In a ful.sale stlr~d t:ank reactor the l!quld circuladon loop can be as long as 10m. Wlth ~ IIquid 'ieloclty of r m 5- 1 che. me~n circulltion timI wiD be 10 seconds. As. a worst

cas.e Istimat. (no tr.msl.r at atI ouuide the impeller re¡ion).ltwlll Clke 10 s befOl'l'- the 0 1 wil become dep!eted In the /uo¡:>. lherefon=, theO l concent~tIon In me bottom Co:!mp;irtmellt sl'lould be JO tllgl'llhalloal OepIetlon. whlch can be 6elrlmentallorthe microbial state and produa.

fonnati..1.ures

F'erstraction

Partition

Extraction of SITIall molecules from aqueous/organic solutions

interactions between the protein surface and chargedsurfactam . and is thus dependenton pHand ionicstrength.

9.5.3 Membrane flltradon The use of me.mbrane technology for separadon of biomollX'llles aud ['anides and concentration of process fluids has expandeed stream is maintained parallel to the separation sut:fuce, with (he aim tO provide sufficient shear force clase to the membrane surface, thereby preventing particlllace matter from settling on, or within. the membrane scruc· ture. In pr.1ctice, the membranes in cross·flow filtratioo are also rubject (O fouling. but the cake thic:k:ness is Limited to a thin layer as rompared to the dead end mode. Although mast filtrarlon media are relatively merr. the formarlon of gcl layer is inevitable. The probIem of permeate flux reduction can be minimised by optimising the filter selection. operating pressure, Oow properties of feed . and frequent back·flusbing. Microfilrers and ultrafilters are avaiIable in materials sumas ceramies and steel tbat can be aggress ive:ly deaned and srerilised in place. Membranes composed ofpolymerie materials such as polyvinyldifiuoride ¡PVDF) and polyethersulphone (PES) are also used, but are more difficul t to clea n and may require chem icaLrather than steam sterilisati on. Membrane ruten are commonly pIate and frame systems. e mploying eartridge filten within which the membrane is present in a highly folded formal. This gives a large filtradon sumee area in a compact space with no dead spaces. Another fonn is lbe hoUow·fibre system, wh.ich eomprises a bundle ofhoUow capiUaries packed in a tube. TIlc liquid to be filtered is pumped through tlle central eore ofthe hoUow fibres. The permeate passing through (he capillarywalls can be drained as a pe:rmeate from one end of tbe caruidge. while tbe concentrated retentate.emerges from tbe othel' cnd. Membrane adsorbers New micro¡m.acroporous membrane matrices with ion exchange groups and affinity ligands. called membrane adsorbers . have been develaped which bind proteins from the c1arified feed pumped over them.Desorpdoo ofrhe protein is laterperformed using solutions as in chromatogl'aphy(see Section 9.6).A staekofmembranes provides a total surtace area for adsorption equivalent to chromatography gels, giviog similar high resolutioo separatioo as chromarograpic metbods. In membranes, liquid transport is by coovection as opposed to the ditTusional Oow in gels Isee Section 9.6), which increases the speed ofseparation ttemendously. Pervaporation Pcrvaporation is a membrane based process havingpotential for rey eJements of process monitoring and control are: (il measurements bywhich inforUlation aboutthecurrent process state is beillg acquired. and (B) models that dynamically ÍDte rrel.l te tbe various process variables, which are importantwith.respect to the task to be solved . Ofparticular importance are rhose variables by which the

stare afthe process ca(l be described unambiguausly.ll1ese variables. however. are not necessarily the most importam ones from [he practical point' ofview. Ofimmediate practical importance are the variabl es which describe the perfor:mance ofthe process. In arder to gel access to the performance, its relatio nships to the variables which can be measured dh'ectly and to the variables thal can be manipulated are of importance. Thus. modelling for process supetvision and control needs a quantitative definition ofme objectives afme process aud rhe particular task to be solved . For supervision and control applications in industrial environmenu. [be complexity of tbe models muS( be Jeept as low as possible to minimise me expenses ofmanpower needed te maintain them. I( only makes seose to implement complex precess conlTollers, arter it was made sure: that they will work significanlJy better than conventional simpler ones. It is the cost[beneflt-ratio thar is me final c.rirerion (or whether simpleT ormore complex contrallers are used and this must indude me cost ofprovidingthe relevan t man-

po_o lt is of advantage te formula te tbe mulo-dimensional problems of proces5 modellLng, 5upervision and control using a vector representation. TIüs nor only helps to keep tbings dear, buthelps to translate them into modern software mols which are mainly matrix based. The matrix notation \Ised in rhis artide was adapted to software products available on the maTket such as MATLAB OI SOLAB where tbe variable. x, i5 generilly assumed to be a matrix. Vectors and tbe Ul>1JaJ scalar Quantities are.considered matrices ofspecia l dimensions.

10.2

I Structure of process models

Process identification is tbe procedure of de\'cloping a process model

from prior knowledge and experimental data. The dassical approach to process modclling is the development ofa mamematical model in the forro of adynamic differe.ntial equation sysrem deriverl from rnechanistic considerations. The prior knowledge usually leads to the structure of a parameterised model. leaving me parameters assooatcd with tbis model structure to be estima ted from process data. However, suirable model structures for sorne parts of the process may not always be known. Then, 'blackbox' ideutification methods that make onJy minimal a.ssumptions about the strucn¡re of these sub-processes aTe al ternatives. The state of a biopTocess is mainly determined by tbe amount, n , (measured in mol) of irs key components. !be vectoT, n , may be composed oftbe amollnts ofthe substr:ate. bromass. productetc_ The basis of a bioproce5s model is a baJance eqllation (bar can describe tbe changes of n as a fllnction of time. Please uotc that bold cbaracters or abbrcviations such as n indicare mal che corresponding quantity is a ve U cril

(10.28)

Finally, the specific product developmenl rate, "fr. must descrWed. It mostoften primarily depends 00 the sped6c biomass growth rate. lL. in sorne more or less complex relationship, The desired produce may be a recombinant protein. Then. a highly non-linear relationship. sucb as e.g.: w

(10.29)

l+ (~r

may be u sed in the simuJation. The sigmoidal form ofthis rate expression reflects the experience thar lhe cells need sorne growth to start protein produrnon and that there is a final specific production rate limit. 1l"uw' that is asymptotically approached at higner specific growth mtes. IL. The parameters of this expression must be deterntined from experimental data, in particular from p.(t) and 1T{t) estimations discllssed latero With these specific rate expressions, the absolutevolumetMe rate vector. R, can be detennined as:

R=Xlp.. -

fT,

a. '!TI

(10.30)

With R. the entire dynamic system is determined. Hence, when the required parameters, kinetic constants and yields can be supplied . we only need tO salve Eqn (10.5). This, once again, i5 very ea5)' when a modern software toollike MATLAB is used. Pigure 10.1 sbows a typjcal result oi a process simulation where

Il'1daC"'Ute. a~ eompaN!d.,.,.;th musur.G claa. clepi cce cl by tlt. symbolJ.ln me Iower p;irt the carre~ncllng feedrat.e (gluc:ose saludon) In.:! cultUN! _ight prom ...! are shown.

221

222

LÜBBERT ANO SIMlJT1S

t hese model components were used .The kineric bortle neck mechanism is of general use llot only for [he accompanying example of E. col! growth, bu( alsofor most other sysrems ofpractical interest.

10.4 I Advanced modelling considerations 10.4.1 Methods of lean modelling When a rnodcl is (O be used in process optimisation or for on-line process measurcmeut orcontrol , ir must be salvable quickly. ¡ inee [he model evaluation must be frcque ndy repeared. This requi res that the m ode! must be strictly resrricted (O rhose aSpccts thatdirectly influence tbe process performan ce, Rcstrictioo to these mostimportant variables i~ nor the only possibility to reduce the computing time, An important further meaSUl:C is ro identifYvariables that are importanr, but change with much smaller timeconstants in comparison with the keyvariables mbstrate, biomass oc produet concentration, These variables can be assumed [Q be atany time in an equilibrium state with the keyva riables. Hencc. their dyna mic changes nero Ilor be considered sepa.rately. In otherwords. they can be statically related ro cheother stare varjables. ln chis way che numbe.r of diffcrcntial equatioDs can be decreascd and thus the compudug time fo r the simulanoo, All important example is che dissolved 0 2l:onccnrradon . pOr on e of the most importanr variables in aerobic production processcs. Since p02 is irnmediateIy adapting ro rhe biomass gl'owth rate , its rate of change can be neglecred. The specific O) consumpnoo rateo f(I!$,ls not lble te folow m e ;t(tuuinllsiz:nal immediat.lly; Innl1ad it riSfU in a del3)'ld w ay to a leYe~ A. The oolTt!SpOnding lime con sum, T, can be esllmated lrum theaJn,dna "",Iue$ of me Inlb"secu ofthe tangetll thm ugtl turnl~ por ... t of the I'1!lf>Clnu ell~ iU'od ab!ICis!.l and tu p;! raIle l al t lle leve! finally approac:hod by thc rv$pOnse curve_

me

-

- P-

- -·¡U...set

- -¡< -- -- ·- ·J.Lset

O.,

0.10~.L-'!---'--:4_L-!6:-L-;8~.L-!.'0 Tim e (h)

Mi el

o

,

4

6

8

lO

Tim e (h)

RelllJ lt o bralned wlth 3 stan dard PIO controlle r forthe specifK: growth rate oJL. at an E. co/i cu ltivarlo n on glUI;ose , wnere m e contfolle r parameters were determlned In diffef'{!nt phases of the process. The liMt.part crI me figure w.u. obr.ained wim p3.r1metl1rs determined d urillg m e In!tia! pha.se of Dne ofthe prmollS runs of the pmten. and the right w\th panmeters determined ooring me end1lhul1 o f olle or!he pnlYious rons o fme proc;ess. AJ, can be 1een partlc:ubrly in tlle right p.!.f'1: where ,,"is strongly osdllatiog. thtI controller Is IlDlmle 10 perronn me expecled task.l\3mely te keep the specific¡rowth rate dose to me set-point pmlile.

..

--/L

Typia t U'sult of 1 PID conrrolllr tor che 5p«1fk: growth

....... /L_se! 0.8

... I&, p.. ..,¡th p.1rnllleler adaptaticn.

if

0.7

O.•

me

05

"c

"-

The improvtmtnt In (om~rison wilh Itle usual PlO controller. depicted in Ag. 10.5, bccome1 oo...ious. OllCfI apio. the dl1hed c;u ...... 1s 1M sec polnl protile, wh lte [he full !ine deplcu (antroUe we are going to work on is che production of fabulase . an imaginary inuacelJular enzyme used in the fragrance i.ndustry. The goal is lo produce 10 lOnnes per year,

11.5.1 Process details The process details are given in rabie 11.'1. 111e figures are obtained in laboratory and pilot-plant tests . and ir is assumed tbat the technology can be scaled up successfully to prod uction scale. Please note tilar tbere are nodefined VQlume resrrictions for a pilotplant.lt merely refers to a reacto("volume. wh.ich is trnditionally one oTdcrofmagnitude less than your production vessel, wbatever the volume lhis may be. A pilO[ plant is rhe reactor stage where yOl! tl'y out your laboratory results under (semi) produ ction conditions. If you succeed here. yeu can assume that you can achico."e rhe sarne (and sometimes better) cerolt in tbe production·scaJe reactoes.

PROCESS ECONOM ICS

Ge nér'allaycut of a

R.w matenaJS

-g--g--~-

production plan! wi(h unit proce.'J~e.'S and utir.ties. 8iotechoo!ogn>lanu at"f! in general vuy simi lar, (he main difference

being me n:lUlre of!he utalyst,

whether it ~ 3n enzyme or microorganism, Compared ro gel'H!l""3 l chemical planu, biotechnology

Mo:dium

Stor~e t4nJ,:

Product isolation

---->

"'"'

1---'

• ¡.--

G lucose

Soy-flour, potassium and magnesium salts 120h Bh 61

4Skg m-3 2 kg m- ) 0.36 28 oc:

Not ..: • An inlr.l.cellula.r enzymt' •

• The genes tor fabll la$ewerr found in ~ IMcrel'ium. bllt the l'omp.lnyhas expl'ess~-rhe gelle'! inA.---' Exil gas Air filter

Other !utrients

$Gluco,a

Wat"

Holding

Blending tank

Air

co'""p-,Le'.s-o-,--------~~

0----- ------'

A,,,Hte,

¡:f .

Holding tank

,

Ammonium sulphate

Salid waste

Jo

Precipitation

Centrifuga

Homogeniser

L ----'~D~;:~~-'----~,~~,~~~~~ LlquJ wasta

U

Freeze drier

Centrifuge Di",... rn 01 da planl Ior produaion offabulase (dl'1lWn with permlsslon !mm IntellIgen Ine. New }torsey. USA).

fermenter

Fabulase

PROCESS ECO NOMICS

It""

Number

Co5t(K€)

Holding tanks Blending tanks Conoouous steriliser Production reactor Seed and inoculum readors Ho mogeniser P~ c i pitation tank Centrifuges

2

70 100 lL5 2324 240 185 700 335 1100 145 39 1300

Comp~ssors

Freeze dryer Air fitters Auxillary processing equipment

2 1 1 2 1 2 2 1

2

Total pun:hase costs

6663

It,m

e"" (K€)

Equipmenl InstaJlation cost (250% of equipment cost - indudes lnstn.Jmentation) Total di~ct costs Construction expenses (70% of direct costs) Total direct and indirect costs Contingency ( 10% of directand indirect costs)

6663 16658

Total capital costs

2332 1 16325 39646 3964 436 10

Utilities Assumed lO be IO%oftb e production casts. see aboYe. Waste treatment The assu med costs for treatment of Iiquid and salid wastes are 0.001 €Jkg and 0.01 €fkg. respectively. Venting ofgas srreams wHl in dtis case nol involve any cosrs.

Labour costs Te is estimate:d tbat t he 24 operators (three shifts of su and one st.md-by shift) will be required [O run the plantoworking a 37.5 hourweek witb-4 weeks holiday a year.Additionallabour costs will be supervision (1 0%of operator costs).laboratory (15% ofoperator coses) and mainten ance and social costs (50% oftotallabour costs).

249

250

KRISTIANSEN

CO>I(K€)

Raw materials Utilities Waste treatmerrt Labour (@20€ per hour) Administration and overheads (40% of Iabour) Depreciation (10% of opital costs) Contingency ( 2% • " ) Insurance (1 % • ) Taxes (2.5% " )

762

1173 11 2 160 864 436 1 872

436 1090

Total production cosís

11729

Item

CO>I (K€J

Capital costs Start up ( 0 $ (5% of capital costs) Total investment Income rromsales @1700€kg-1

43610 2181

4579 (

Taxes (@ 40%) Netprofrt.

17 192 11 729 5463 2 185 3278

Expected retum on investment

16.2%

Production costs

Gross profrt

Other COSt5 of sal es. R&D expenses. patent and royaJties (osts mil nOl be included. Thcse items are very product specifit with typical figures of 10%. 5% and 5%ofproduction costs respectiveJy. lhe production cost (or the production of 10 tonnes fabula se per yearisgiven in TablelL7.

I 1.8

I

The costs case - to build or not to build

To obtain information on which a dedsioll ro build. the p lallt can be based. we w iH carry out a profitability analysis. The results afsuch an analysis based on a plant lifeof15 years is given in TabJe 11.8. ln some texts, lhe tenn 'operating profif is used. This is uscd to describe the proflt generated frem plantoperations and is the same as gross prafit in rabIe 11.8 and does na t ioclude taxes, cose of capital . depreciatíon etc.

PROCESS ECONOHICS

.S E .2

Result 01' a COSl

...u. !i}-

-,

.~]

~

&!

I... Salesprice

1 0_ _--"'2_ \Il

\

4p

I ... Operatlng cosls ' ..... Investmentco~

% change in cost

Tbe expected rate ofreturn on the capital invested helps to decide whether ro invest in the process. For existillg processes. tbe generated cash flow may be a better indication of the. health or tbe company. The cash t10w is obcained by adding money spent o n che depreciabon ofthe plantta the netprofit. Forourplant. the cash tlow will be {in kiloEuros): K€ 3278 Ne.tprofit Depreciarion K€ 4134 Cash flow KE 7412 An expcct'ed retum on investment of16.2%, equivalentto apay-back period of 6.2 yea rs. is relatively low fol' the biotechnological industry and ir is unlikely that our factory will be built. However, the returo is suffidently high nOI lO be discarded irnmediately and is accordingly subjected to a cost sensitivity analysis. Hete, the efTect of changes in important cost parameters . suth as sale plice. invcstment and operating cost, are nudied. The resultof such an analysis is given in Fig. UA.

The figure shows tbat the process is sensitivc to changes in all rnl'ee parameters, although it is more sensitive to changes in rhe sale price and investment(o.ca pital)tosts as the slopes ofthese curves are approx· imateJy me same and sreeper than the slope ofthe operating costs. It may be difficult for liS to influence the sale price as this is set by a numher of external factors over wltich we have ¡ittle control and will depe.nd on such things as the number ofplayen; in the mal'ket. the age ofthe market, competing products, etc. Howevcr. the figure shows that a decrease in the in~tment costs will also lead to a higll er fOlCe of retorn, and we will therefore go back ro ourdesign to see ifwe can cut the cosrs without afTecting plaut performance. Thus we must find o ut: • Are all the processing steps required? • Can we alter the capacity ofche units? • Can we reducedowntimes (a cornmOD fault for a first dcsign effOrt is to overestimate the requirect downtime)? • Can we lIse cheaper materials of co nstruction? • Can weuse multi-purpose units? Whilstdoing this we musr I'emembe. that the new plant must give the same performance as before.lfwe can cut the capital costby10%, we wiU get a .etllrn of around 20%.1bc proeess is now beginning to look ralhe. aUractive and will warrant furth er srudy. Ir is at this point that the reader takes overo Good luck!

senmMty an¡lys!s, in whidl the ,ffect of change1 in !mPQrtant con p:trameters ane studled .

251

252

KRISTtANSEN

11.9 I Further reading AsprnPtus.AqJen Techno[ogy lne. MaJlsachusetu. ISlmulatlon $(lftware.1 Petas, M. S. and TImmerh3us, K. D. (1991). P1anWrdgn and Ecorwmlcsfor Chemiml EnginCC1'S. McCraw·Hllllnt~rnatlonal Editions. Reili Olan. H.. B. (1988).Econarnlo:Anll/y5is ofFurnentatiOl1 PrOCt$ses. CRe Press. Boca Ralon. Rorida. Seide r, W. D.. Seadtt. J. D. and Lewin, D. R. ( l993~ Process Drngn Principles.John Wiley, New 'l'ork..

Superf'roDesi'gntr, lntd.ligen lne. Nl!w Jl!rsey. New York. ISimulat ion software.1 'IUrlon. R.• Baille . R. C. Whiting. W. 8. and ShaeJwitt. j. (1998).Mnlysis, Syn thesis and lk~gn ofChcmicall'n:lassa. p ren tice Halllnternalional Series in th.e Physical and Cbemk al Engi neering Seri es.

Part 11 Practical applicatíons

Chapter 12

The business of biotechnology William Bai ns and Ch r is Evans Introduct ion What is biotedmologyused for?

Biotechnologycompan.ies. thcir care and nurturing Investmenf in biotechnology Who needs management? Patenls and biotechnology Conclusion:jumping rhe fence .Further reading

12. 1 Introduction 1

Biotechnology is the application ofbiOloglcal processes. 'New' biorech· Ilology is when mis 15 driven by systematic knowledge ofbio logical processes. In this cbapter we will di scuss tbe 'new' biotecbnoJogy industry'~ mast specracu1ar commercial maoifestation - rhe 'biotech start-up company' - and what factors contribute to the success and fa.ilure ofthe entreprelleuriaJ application ofme science describe $U¡~bI.

iIM:IMn¡ undrr.c:tl ~ mutagef1esis.

Substrate uptake Since the cost of the sugar soun;:e has a decisive influence on !he price ofrhe amino acid produceticated device to handle a reactive and diffu· $ible intermediare within me ceU.

13.8.1 Produccjon from precursors The process ofL-tryptophan production with this enzyme is based onE. ceH celli which have a high tryprophan synthase activity. Thc er, and f3 subunits encodinggenes trpA and trpB, respectively, are locared 00 the crpEDCBA operon which is regulated by repression and attenuation. In theE. coli mutantused, the repressorofthatopcron has been deleted as is partofthe attenuator region togetherwith (he first structural genes oflhe operoo.lnthe resulting strain, about 10% ofrhe total protein is tryptophan synthase with an excess ofthe{J subunit.Although indole i5 Dot the true substrate of the enzyme (see Fig, 13.20). with a sufficiently high eoncentration the enzyntc will reactwith ir. lndole is available from tbe petroch.emical industry as a romparably cheap educt. whereas tbe second educt. L-senne. is recovered from molasses during sugar reftnement using ion exdusion chromatogl'aphy, aud further

TIle ~ryp~ophan

~

~nth:l5e l1Se5m vivo Indole

).¡lycerol pholphate plus l-scrine. and In the productlon proce$$ indole plus l·5ertne.

299

300

EGGEUNG. PFEFffRLE ANO SAHM

ProducdOl"l plal'lt te fractionlte molnsH by Ion· exclusiO'l'l chromawgraptlr. wldl i$oIulon ofl·,erine. AA E. coli mUQnt overexprenln¡ tryptOphan synthue 1$ pre¡rown. Ind subsequently mixed wlth l-ser1r.e plus Indole to convert the-,e $ubnra u~s tO ,.trypwphal'l.

Sugar-beet molas.ses

Eeotí mutant

L '/íypI~

$r"fhltse I BiA ..,iIÚ"oaIoo

I Dea:Io!is;¡Iion, IIIrIlbl

ancI aystaIisaIiOn

I DIyIng. s1eYilg ami tilg 001

¡ j

-lTrpfopMil

purification steps (Fig.13.21). The rcsulting L-5erine is fed to thc previously cultivated E. ooli cells. and indole is added continuously at a con· cenfration adjusted to 10 1llM. which is controlled on-line. Tbis type of process ensures an almost quantitative conversion of indole to yield L-tryptophall with a space-time yield of abaut 75 g pet litre and day. Fu.rther-processing afthe L-tryptophansolution can be taken from Fig. 13.21 leading ro a pyrogen-free phannaceutical product oftbc highest quality.

13.9 I L·Aspartate

'OOC

~eoo' +

NH,

!f +

H3N

Aspartase

H y--.. 1

coa'

coa' Fumar~1.I!

ilnC

ammonlum serve as subsl:rU9$ fOl' the UP¡rtasll.

L-AspactiC acid is widely usengiu' 6. 455-502. ti. K.. Mikola. M. R.. Dl'lIths, K. M" WOl'den. R. M. and Prest,j . W,(l999). JUl. batch fermenter synthesis of3.Jehydroshikimic acid using F.s~rldtja coH. Biouclmol. Blorng. 64. 61- 73. Pcters-Wendisch, P.. Kreutzer. C.. K,alinowski.J .. Pátek. M.. Sahm, H, and Eikmann5, B.j. {'1998).l'yruvale carboxylase from Cmynebacterium gltltamiC'l.lm: Chancterization. expression and inactjvation o fthe ~ gene, MicrulriOlogy. UK 134, 915- 927. Schilling. B. M.. Pfefferle. W.. Bachmann. B., leuchtenberger, W. a nd Dedcwer. W. D.(1999).A spec:ial rea ctol'desigo fo r invt'Stigario l\5 of mixing t ime effe 15 mM and a pH below3.5 (tbe latter also prevenU the formation of2-oxogluconate), aud gluconic aod is therefore accumulated when these conditions are applied . Fungal gluconic add formation is catalysed by the enzyme glucose oxidase. Th e enzyme is extraceUular, ¡,€'. partiaUy cell-wall bound in Penlc1llium spp., but secrete 90% 00 a molarbasis) are usuaUy completed in less lhan 24 h. Sodium glueonate has been used as a superior alternative to the ealcium gluconare precess. as it enables the fermentation of even higher glucose concentrations (up to 350 g 1-1).ln this process. [be pH is mainrained close to pH 6.5 by the addition ofNaOH. In other respects, the process is similar to thecalcium glucol1ateprocess. This process has been employed for the developmem of conrinuous fermentations in Japan, whichclaimed the conversion of35% (wJvl glucose solutionswith 95%yicld. Severa! differem bacterial gluconic acid fermentation processes bave been described but only few ofthem are actually performed on an industrial scale_ As alre.ady mentioned, a high glucose concentration (> 15%. wlv) a ud apH below 3.5 are necessary for high yields. Severa! workers havealso shown the possibility to use immobilised eells fo[ g lucoDie acid production.

ORGANIC ACIDS

Methods for product reeovery are similar for both fungal and bacte· rial fermentations but depend on the type of carbon source used and the metbod ofbroth neutralisation. Calcium gluconate is precipitated frOIn hypersaturated solutions in the cold and is subsequently released by adding stoichiometric amounts of solphuric add. By repetition of tbis step, the dear liquid is concentrated to a 50% (wfv) solution ofgluconie add. Sodium gluconate is precipitated by concentration to a 45% (w/V) solution and raising the pH to 7.5. Today. sodillm gluconate is the maio manufactured fOI"lll ofgluconie acid, and hence free gluconic actd and &glueonolactone are prepared from itby ionexchange.As gluconic aod and its lactone are in a pH· and temperature-dependellt equilibrium, eitheror both can beprepared by appropriate adj1l5tment ofthese two conditioDS.

14.3.3 Commercial applications of gluconic acid Gluconic ada is characterisea by an extremely low toxicity, low corrosivity and the ability to form water·soluble complexes with a variety of di· and trivalent metal ions. Gluconic arid is thus exceptionalIy well· suited:foruse in removing calcareous and rust deposits from metals or other surfaces, including milk or beer seale on galvanised iconor stain· less m'el. "Because ofits physiological properties it is used as an additi~ in the food, beverage and pharmaceutical industries , where it is the pee-ferred carner used in caldum and iron therapy. In severa] food-directed applications , gluconie. add 1.5·Iaetone is advantageous over gluconic arid or gluconate because it enables acidic conditions to be reached gradually over a longer period, e.g. in the preparation of pick1ed goods, curing fresh sausages or leavening during baking. Mixtures of gelatin and sodiuro gluconate are used as sizing agents in the papee industry. Textilemanufacturers employ gluconate fordesizing polyester or poIy· amide fubrics. Concrete manufacturers use 0.02-0.2 wt% ofsodium gluconate ro produce concrete highly resistant to frost and cracking. According to recent estimates, its annual worldwide production is > 60 000 toones.

14.4 I L.ctic .cid Lactic acid (Fig. 14.8) was first isolated fromsour milk in 1798, and subsequently shown to occur in two isomeric forms, Le. L(+) and D(-) isomers, and as a ra.cemic mixture ofthese. The capitalletterprefixed to the names indicate configuration in relation to isomers ofglyceralde-hyde, and the (+) and (-) syrnbols indicate the direction ofrotation ofa plane ofpolarised light. The mixture of ¡somas is called m·lactic ando

14.4.1 Production organisms and biochemical pathways Lactic acid was the first organic acid to be manufactured industrially by5fermentation(arollnd 1880 inMassacllusetts, USA). The biology and biochemistry of lactic acid bacteria have been extensively reviewed. Tradition.al1y, tbey are functionaUy classmed into hetera- and

coaH

eCOH

I H-C-QH I

I

HO-G·H I

eH,

eH,

D (.)

l (+)

m'" ackls.

) and L( +) lactic

D(

I

317

318

KUBICEK

homofermentative bacteria, each ol which in tuco can be divided accordingto t beir coccoid or Tod-shaped form .Appli:catiou of molecular genctic tedmiques ro determine che relaredncss of food-associated lactic acid bacteria has resulted in signifkant changes in their ~xo­ nomic dassification. The lactie acid bacteria assciated with foods now indude species of che genera CarllObacterium, fntcrOeocrus, Lactobaallus. Úlctococrus, I.euronostoc, OCIIOCO«US, Pidiococrus, Srreptococcvs, TdTClgnwcucrus, Vagococcus and WdseHa. The genus Lcctoootillus remams heterogeneous with over 60 spcdes. of which one-third are heterafermentative_ Heterofennentative ¡actic acid bacteria are. invol~ in mostofthe lYPieal ferwentations Icading ro food Oi feed preservatioll and tr.msforrnation. whereas the homofermentative bacteria are used for buIk lactic acid production. Generally, strains opcrating ata higher remperature (45--62 oC) are prcferred. to the laltet, as mis reduces the powcr requirements needed fur medium sterilisation, Lactt)b!J.cilltls spp. (e.g.L delbrueddi) are used with glucose as the carbon SOllrce, whereas1. delbrueckll spp. bulgaricus and 1. hdveti! are ltsed with lacrose-containing media (whe.y). L ddbrueckii spp. Jactis call fenucnt mal tose.. whereas L IlmylopJliJlIs Cóln even fennentstarch. Most lactic acid-producing micro-organisms produce only one ¡somer of lactic acid; however, sorne bacteria, whi ch unfortunatelycan occur as infectíorn during lactic acid fermenr:ation s, are known ro contain racemate.s and are thus able ro convertonc isomcri 45 °Cwitb gentle stirring (lactic acid bacteria are anaerobicorganisms and the introduetion ofOz therefore has to he avoided). The pH is maintained between 5.5 and 6.0 by tbe addition of sterile ealcium carbonate. A5 an alterna tive to neutrallsation with caldurn carbonate , ammoniacan be used. which al so aids in tbe l'ecovery oflaetic acid by esterification (see below). but this resulls in a more expensive process. Due to the COITosive properties of lactie acid, wood oI' concrete were useu s transports cis-acooirate, rather than citrate. lO exchange with malate out oC the mitochondria (Fig. 14.11). During fennentation, itaconic acid formation is also accompanied by varying arnounU oC succinic:. citramalic and itatartaric ¡¡cid. Data rurrently avaiJable s ugge~¡[ that t hese are Doe degradation prod· Ucts ofitaconic acid butratherare.formed byocher pathways.

Slmpllfied meabo\ic scheme ofil.aGO!'lIc Kid bIosym:hesls. Side t'UMns i nd IntermedJ;ltM!'IOE,~v..,t to luconlc ¡cid biosyn~515 hive bee n omiu ed.

32 1

322

KUB1CEK

The fennentation production of itaconic acid is large1y similar ro that of citric acid. ie it requires an excess of an easily metabolisable carbon S"ou.rce (glucose syrup. erude starch hydrolysates. molassesJ. and a limitarlon in metal ions by the aid of complexation and/or precipitation with hexacyanote rr.l.t or addition of copper (see Secrion 14.2.2). However, [he effect ofpH is different: several workers reported that the pH has ro be maintained betw'c.'.en 2.8 and 3.1. and lower pH values favollr the formation ofitatartanc acid.Yields of85% (wfw) ofthe tbeoretical maximum have been reported to be obtained witbin 5 days ofcultivation at rather higb temperatures (39-4rC). Recovery is usually performed by evaporation. active camon treatment and erystallisationjrecrystallisation. ltacorue acid is sold in two grades: reflned, whic.h is a pale tan to white crystalline salid, and the industrial grade which is darker in colour. The main potential of utilisatiOIl of itaconic acid is rhe manufacture of styrene butadiene co-polymers. and for lattices and paint e-mulsiOlls.

14.5.2 l·Ascorbic ,cid (vilamin C) Ascorbic acid 1s the- officia llUPAC designation forvitamin C.lr was discav~ in 1928 by Szent-Gyorgi. IUl mast significant characteristic is its reversibleox:idation lodehydro-L·ascorbicacid(Fig.14.12). with whi

"l

326

ANDERSON ANO WYNN

PHAsran ulu In Ra/no.n.iG f llllCptlo. .

c.t1s1

environment, in the same way that pla.ntand animal waste isdegraded. Their biodcgrarlability and tbe fact Ihat tbey can be produceof l-hydr-oxybutyrate &lid l_ hydroxyvaJlrate mooomers, and is

therefore desCTbed H a raMom copo1ymer. HaS'!: PHA conta!n two or more different monomers in me

CH,

I

CH

O

I '

11

O- CH-CH2- C

iH,

O - CH-CH2-

~ C

polymer chain.

glucose plus propionic acid, PHBfV (Fíg. 15,6) is a copolymer of3HB and 3HV monomers. aud its composition can be controlJed by varying the conceutrations ofglucose and propionic acid in the medium during [he polymer accumularíon pltase. PHB is hard and brirde. but rhe incorporarion ofa smaJl propartion of3-hydroxyvaJerate (3HV) monomers inro the polymerchain results in a stronger and more Oexible plastic_Tbis is exploited in the commercia l productionofPHBfV(Secdon 15.2,8), In some cases. bacteria can produce PHA monomers thar are Ilor relared ro the Structure of the carbon sources provided. For example, fluorescenl pseudomonads produce PHA containing 3-hydroxydecanoate from rnany carbon sources and sorne Rhodoroccus and Nocontia species produce PHB{V (Fig. 15.6) containing a bigh proportion of3HV mODomers, again frero 3 varietyot'carbon sources.

15.2.4 Siosynthosis 01PHS Of all the PHA. the biosynthesis of PHB has been studied in grea1;esc detail. In most bacteria. PHB is synrbesisIW from acetyl-CoA in three sreps (Fig. 15.7). 3-Ketothiolase (encoded bygene phM) catalyses the condensation of two molecules of acetyl.coA to produce acetoacetyl-CoA, wbich 15 then reduced by an NADPH-dependenr aceroacyl-CoA reductase (PhbB) te yield R·3·hydroxybutryl-CoA, Addition of 3-hydroxybutyrare (3HB) to rhe growing PHB chain involves PHAsynthase (phbC), an

PHA. POl YSACCHARIOES ANO UPlOS

enzyme associated with me memhrane SUJTDunding PHB granules. lo Ralstonia futTopna, the genes for these enzymes are organised in an operon: phbCAB. The genes have been clonedana expressed in other bactelia and aIso in p lants (see below).

CH3 ·COSCoA Acetyl.coA

CoA

~

15.2.5 Regulation of PHB metabolism The enzymes for PHB biosynthesis are constitutive - tbey are prese.nt even during umestricted growth. This allows irnmediate PHB synthesis as soon as growtJ¡ becomes restricted by the avajlability ofan essential nutnent.ln natUI

Pyr I I 46

'" r -

Y ~-o-Man- ( 1-+4) -~- D-G IcA-( 1-+2 )-a-o-Man-6-0-Ac I

I

Divalent cations can scronggel.

c..TOSS-link

The strUCWre 01 XlIIlthao. The extellt Qf ;H;11[)'b.tion ofthe Il'QnllO$e \l nlt ;J.djacent ro die

Oackbone i5 cornmonly

~

bol:

an be sl&nlfia.ntly lo'HI1I" or Ngher.

polysaccharide chains to produce a

IS.J .J Xanthan Xanthan is produced by the Gram·negative bacterium. XtinrhamaflÜs cnmpesbis. It is the best.-studied and most widely used exopolysaccha· ride.. Xanthan is a largc polyrner. having an M r in excess of ]1)6 daltons. lt ís a branched polymer with a p.(1-+4) linked gluca.n (Le. polyrner of glucose) backbonc with a trisaccharid e sidechain on altemate glucose residues (Fig. 15.14). The pyruvate and acetare content depend on the bacterial strnín. cultm"e conditions and processing of tbe polymer. These substituents do not have a great inflt1ence on the properties ofthe polymer. Xanthan is a polyelectrolyte due to the glucuronic acid l'esidues in the side chains. Despite bcing an acidic polysaccharide. the viscosity of xanthan i5 relative ly independentofthe saltconcentration. xanthan is the m ost ünportant commercia! microbial polysaccha· cide. aud eurre ne production is around 20000 tonnes each ycal'. Kelco, nowpartofMonsalHo, is the principal manufucturer. Xanthan was fiNt used in 1967 and approved forfoad usem tbe United Statcs in 1969. (t is widcly used ful' stabilisation . suspensioD, gelling and viscosity control in the foad industry. These propenies are a lso exploited for W3)-f}-O-G Ic-( ,->

.hU StruCtu~

of curdlan.

Curd lan (Fig. 15.18) is a l --t3-tJ-glllcan produced as an exopolysaccharide by A!ct:llIgenes fuecalis varo myxogenes. Similar polysaccbarides are p~ duced by Agrobacterlum radiobacter aod ;'grobactmum rhizogenl's. and RI.izobium mfolif.

PHA. POLYSACCHARlOES ANO UPlDS

,->

- >5)-0:-D-G le-( ' ...4 )-a-o-Glc-( , ...)-a-D-Gle-(

M"U

StrllCllRofpullulan,

Unlike sclerogluC3n, rurdJan is insoluble in water and forros 3. stronggel on heating ahove SS oC and this gel furmation is irrevenible. Cnrdlan can be used.as a gellingagent in cooked foods and as a support for immobilised enzymes. The properties of enrolan resemble those of the 1~3 -J3-g1ucan, laminarin, which is fouad in many brown algae.

15_3.8 Pullulan PuUu]an (Fig. 15.19) is an ~Iucan with a trisaccharide repearing unit. It is pl'odueed commercialiy using the fungus Aureo/1a.sidellm pllUuluns. The ferm entation is relatively slow (5 days) compared witb the production ofbacterial exopolysaccharides but70%ofthe substrate (glucose) is converted to polysacc.baride. PuDu lan forros strong, resilient H1ms and libres, and can be moulded. The ti lms have a lower permeabiliry to 0 l than cellophane or polypropylene ando being a natural product, the plllluJan i.s biodegradable . Similar polyrners are produced by sorne bacteria.

15.3.9 Alginate Alginate is linear polymer composed of mannuronic ¡nd guluronic acids (Fig. 15.20). lt is produeed by the Gram-negative bacteria A2otobacter vlnelandU and Pseudvmonas species. The bacterial exopolysaC'Ch:ll;de is similar ro algal (seaweed ) alginate, except thatsome ofthe mannuronic acid residues areD-aeetylated. l'he relatiw abundance of mannuronic and guluronic adds and tbe degree of acetylation depends on the organism and growth conditions . Polymers conraining a high mannuronic acid contenr are elastic gels, whereas those wilh a high guluronic acid content adopt a different conformation and are strong. brittle gels. Alginates are not random co-polymers ofmannuronic and guluronic acids, and regions containing a single monomer (Le. -M-M-M-M-M-M- and -Gdlb.us

~J •.

,~

probe

1",+_

1 CcoUng

Lave! proba

Alltiloam

...,--,

• • •

,...,

DO

SIl9BtI

..

""""""

Ph04Qtlate:

Sulph8le

PIOdUets

_

..

I SugafIOiI

$am !!le AnR!n.!1

pH

P~U/SOI5

C«lIM1INtlO'1 ~

~

==

Dissolved 0 1 DO Jevels ;ue critiCal AlI aqueous reactJons. neutral pH and am bient temperatures Good control and monitoring of reaction through pH measlwe 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 acid Over 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 penicilün 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

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 reCúVe.red preigned at the completion of each stage and the record s checked by management and rNained as the batch record . These recoros are available to any inspections by Government regulatory bodies. Strict adherence to (hese polides will satisiY thc regulatory authorities, and will ensure confidence in the general public that their medicines are safe.

16. 16

1

Further reading

Elander, R. P. (1989). Bioprocess technology in..industlial fungi.ln FcrmCTJ!Qtllm l'roo:ss Ot'wlopll'lcnt eifIndu5trial Orgemisms. U. O. Neway, ed.). pp. 169-219. Maree! Delckl!r, Ni! w York. Hersbach, G.J. M.. Van Dl!r Beek. C. P. and Van Dick. P. W. M. (19M). TIle ¡x'nicil· Uns: properties. biosynthesis . and fi'1:mentation.ln HiotechnoloKY oflndustrlal Anfiblo'fa(E.j. Vandamme, ed.). pp. 45-140. Maree! Dekker, NewYol'k. Lowe, O.A. (1986). Manufacture ofpeniciJlim. In Bcta·LactamAnt'lhioNcs for Qinfral U~(S. F. Queener, j.A WebberandS. W. Queener, eds.), pp. 117-161 . Mareel Dekker. New York. Paradkar. A. S.. Jensen. S. .E. and Mosher. R. H. (1997). Comparative genctics and molecular biologyorbeta"lactam biw.ynthesis.ln 1Ilou,hnology of AntChiollcs, 2nd l!ditum (W. R. SlrOhI. ro .). pp. 241-277. Mared Dekker. New York. Queellt'r. S. a nd SchwarlZ. R. W. (1979). Penicillin.s: biosynthetic and se mi· sytl(hctic. [n Eronomic M¡crobiorogy. Vol. 3 (A. H. Rose. 00 ,), pp. 35-122. Academic Prcss. l.ondon. Smith. A. (1985). Ccphalosporins.ln Compreht.'J1sü -e 8iout:hno.logy. VoL 3 (M. MoaYOUllg, oo.), pp. 163-I3S. Pcl'gamon Press, N~ York. Strohl. W. R. ('1997). Lnd ustrial antibiotics: roday ólnd the future. ln f!wteochnology ofAll1ibiotia, 2nd Edition (W. R. Strobl, ed.). pp. 1-47. M;¡rce1Dekker. New York. Vandamme. E.j. (1984). Mtibiouc searcb and production: an overvi.ew. ln IllouchnologoflndustrlaJ Antlbiotict (E.J . V;mdilrnme, ed .), pp. 3-3 1. Marce l

Dekker, New York.

3~

Chapter 17

8aker's yeast Sven-Olof Enfors Nomenclature Introduction

MC!dium fur ba).:er's yeasr production .Aerobic ethanol formation and consumption ll1e fed-batch techniquE' used to control e:thanol production Industrial process control Process outline

Funhcr reading

1

C, C,

e, OOT

oor F 11

K," K. ,~

'" ""'" q.

'.

,~

''''''

'... '.... S I

V X

Nomenclature EtbaDol carbon concentration Sugar carboo concentratlon CeU carban concentratlon Dissolved oxygcn t.e.nsion DOT inequilibrium with gas Substrate fiow rate Convenioo COllstant Oxygen transrer coefficienl Saturadon constant Spedfic rate of ethanol coruumption Spedfic rate o r ethanol production Maintenance coefficient Specific rate of oxygen consumption Specific rate croxygen consumptioll fur sug;rroxidation Spedfic rate ohugar comumplion Specifk mte of sugar ro anaboHsm Specific rnteofethanol con.rumplion wnen over:fIow metabolism seu in Specific rateofsugar 10 aerobic ellergy metabolism Maximum nutrients to keep them at conceotrations wbich will pe.rmít 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% dryweighí. containing onlywashed cells and residual water. The yeastis used to r.üse 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

]79

]80

I

ENFORS

produced-with bakingpowder. that aIso evolves COl"Thus, baker's yeast should be considered as a package ofenzymes.ratber thanjustb¡omass. 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 produceion The stoichiometlyfor production ofbaker's yeast can be summarised as 200 g glucose+ 10 gNHl + 100 g Oz + 7.5 g salts~

100gbiomass+ 140gC02 +70g HP

117.2)

TIlis results in me following approximateyie1d coefficienls: Yxs "" O.5kgkg-t y ro = 1.0 kg kg-I YJ(N= O.lkgkg- I , 111e production is an aerobic fed·batch process on a medium af motas· ses. aromanía 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

-

,:!:>(CO,R

Hydrolase

(.)

OCOR7

lb'

(XOCOR

A

O(H OCOR (1S,2R)

CO:t-t

~ (X0H

+

OH

y

~ (XOCOR OH

le)

I1 OCOR

X~OR

OCOR

X~OR

(d)

-

Hydrolese

+ H

HOH

X~OR

B,

-->=0

A

Baker's yeast

~

+

H ceOA

X~OR B

B"'!.I/"H

~\ A ~

+

OH

..::> 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"OduIo!. the inlti~ 1 absorbance al

lo

291 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 COIIVf!rtet.,¡ru ~nd co llun~rUal ly ..v:ail:a"J...

"32

I

KRESSE

HO-~CH' OOH OH

Ha OH Glucose 6-phosphate

Glucose

CH"'OH ®

®-O-vF°~H

Hbf-f1 OH

G6P-DH

~ OH

COOH

HO NADP+

Glucose 6-phosphate

NADPH

OH

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~hory¡ation 01 glucose catalysed b~ yeaCesses). and is used tberapeutieaUy mainly in renal an;)emia, but 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 produce teios hut do so differently from marnmalian cells (see Chapter 5). The

MAMMAUAN CEU CULTURE

NeuAc(a2-6)Gal(~ 1-4)GIcNAc(Pl-4) ' " NeuAc(a2-6)Gal(pl-4)GlcNAc(Pl-2)

-

NeuAc( a2-6)Gal(p 1-4)G IcNAc(Pl -6) NeuAc(a2-6)Gal(Jl1-4)G lcNAc(Jl1-4) NeuAc(a2-3)Gal(p1-4)GIcNAc(Pl -2)

presence and conformation ofthe sugar moiel)' ofa glycoprotein ¡s, in many cases, essential for a functional product as this functionaHty proJongs the half-life in the bloodstream. and diverts the protein te its spedfic location in th~ cell. If this is 50, tbe production organism of choice is ofien a marnmaliancellline. Pbannaceutical proteins that are nOl glycosylated or need not be glycosylated for proper functi OI1. likc ¡nsulin or human growth hor:mone, human serum albumin and hae· moglobin.. may be produced more cosr.effectively with bacteria. yeasts OT filamentous fungi (see Cbapter 20).

21.4

I Protein glycosylation

Whereas protein syntbesis is guided by DNA and RNA templa tes. the addition of sugar to a protein is a process without a template, Therefore a large variation in tbe oligosaccharidc structures of glycoproteins can be round. Glycoprote-ins with the same amino acid sequence, but difIero e.nt ol-igosaccharide strucCUfes ¡ue Gllled glycoforms. The oligosaccharidestructure5 are covalently bound lO the protein either at a nitrogen (N-glycosylation)or atan oxygen (Q-glycosylation) atom. These two fonns of gl}'COsylation differ no( ooly in [he position where sugars are artache

2' .5

"'

I Media for the cultivation of mammalian cells

Mammalian cells in thebodyofan organism receive nutrients from lbe blood circulation. (en culture media for the in vlrro propagarloa of marnmalian cells must thercfore supply nutrients similar to those present in the blood strealll.lnitial attempts to grow marnmalian ceUs in vHro ¡nvolved media derived from tomple:< natural sources sllch as c1úckembryos. bIood serum ordots and lymph fluids.Since about 1950, partly defined media. consistingofa great number ofromponents. have been developed (Table 21.2). The basis for ceU culture media is a baI· anced salt solution. Thcse salt solutioos were originally used to create a physiological pH and osmolarity, required formaintainingcell viabi li ty ill \litro. To create conditions promoting proliferation. glucose, amina acids and vitamins were added ro tbe salt solut'ion, according to the requiremurs of the specific tell lineo This developmcnt resulted in various of media formulations. each des:igned for a limite tbe tricarboxylic acid cyde (TCA cyc1e). A smal) fraeríon (4-8%) of consumed glucose goes via tbe pentose phosphate pathway (PPP) which supplies ribose-5-phospbate for tbe synthesis of nudeotides. as well as reducing equivalellts (NADPH) forbí osyntbesis. ppp ¡s rhe most important partofrhe sugar metabolism for marnmalian eeJls as shown by the fo Uowingexample. Ifglucose ¡s exchanged for [ructoscovery littl e sugar is consume

bifulKliQl1al

nonle

umíll. polyphen ..'¡-pwl.- - -

.

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 reactors The continuous operation of immobilised cnzymes has sorne advan·

rages when compared witb batch processes, such ody)or. alternatively, through activatioo of the complement cascade and the binding to complement recepcors_ComplemeJlt is another family ofproteins found in the blood and whjch ate involved in immllll..e reactions. The components of complemenc are mainIy specific proteolytie enzy¡ues whose Sllbstrates are lhemselves otba complement components which are activated by proteolysis. Tbis gives rise to a classical biochemical amplification of an inidal small activation step. Once activated sorne ofthese compleroenl components also rapidly fonu covalent chemica! bonds witb antigen, thus marking tbem rOl' c1earance by complement receptors of the immune system. whilst ochers are able. ro creare pores in ceU or viral memb[anes ofinfectiou s organisms and thus kili the (elis orviruses. Each of!he different immunoglobulin (antibody) classes and also sub-elasscs exhibit a different pattern of effector functions sorne af

F, The bask; IgG invnunoglobtln smKture of 'MO heavy chalM (bla2- 3 g l-I).

24.2.2 Anaerobic treatment of waste water Un til recendy. anaerobicdigestion was only appl iedfor the stabilisatiOD of concentcated organic slllrries such as animal manures and waste sewage sludge. The consensus was that anaerobic Wolh rsoctiOlls yiHkl > 21 I 1 1 kJ mol- ' tr.mslormed. preuure

00

lr¡nU8nl

Sdlematic diagram 01 me upnow anaerobie sJudxe bI~nkl,ll re.ct.Or ext_ i~ely for

(UASB) Ulld

the trutn'Hlnt of tontentnted ....ute watlml in lempente regions and also Ior the IreU!TM!nt 01 Ul ..... (dilUUl ....aste wUflr) In trOpical ..-t¡lOIIl.

S37

obtained. Becauseadapration ofthe association is probably a1so based on the proliferatioo of tbe right plasmids. tbere is clearly a nee;d for bctter insight in genetic evolution. plasmid transfer and species interaction in anaerohic communities dealing with xellobiotics, Another potential benefit associated with the largc-scale availabiüty of~pecial­ ised microbialconsortia i5 'biochemical re-routing'. Le. the inc\uction of desicable biorne.mical pathways. as fur example the dcgl'adadon ofmal· odol"Ous primary amines. anaerobic arnmonium oxidation or horno· acetogenesis. Deve10pment oC perfonnance-enhancing additives Biomass rNention through adequate granulation is of utmost importance in UASB [echnology. first in moer [O obtain a good eftIuenr quality andsecond. in order to ensure a mínimal ceH residence time of7 to 12 days wh.ich is required to avaid the wash-out of [he slov.¡est-growing anaerobic bacteria . Dile way to foster granular growtb is lO add palymees. cLay oc surfactants whkh llave aphysico-tabUise the populations of other very valuable wicro-organisms. 5Uth as bactivorous protozoa which are esscntial to obtain good qualityeffluents, orto show rile development oE detrimental micro-organisms, such as the filamcntaus bacteria which cause sludge bulking,

elean procoss water Thls

process flcw

diagram iUl,.I$lt1.tU tht state-of-me-

art technology cmplcytd In the textile industry lO c.onven la rge vol~m es 01 wastt W,ter Inte higl!-

qlJ;lWty proce.» wnerus,d for w,15l!ill&- ¡cuuri.", blnching. dyelng and prillt~. Biologkal tre.1tmenlS are cOfl,bined with physlcCKhemital treaunet1lf In orde r te achleve tlle requlnu:! purlty. Th e final biof¡ltnticm step 011

actiV;!.ted arbon. tombinlng

, physical sorptlon with in S1W

biodegracbtion.ls necen;¡,ry to remove texic compoonds produc.ed durlng the ozoni$ation uep.

539

Slope measures aClivity

e

x mg acetate

Time jmin)

me.

Respirogr.lm obalned wlth a biosensor useO to mearure on·n~e BOO and potentilll to,oclty ofWUle water befort II tllu,r$ lo treatrnent pJ.nl. TIle addltlor. of ac6elte to an aenteCe 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'

Effiuerrt treated

Wastewater

Organic slurry

Solid WdStes

Solid concentration in reactor(g 1- 1) Loading me (kg organics m - J'day)

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

Biofilter

r--- Wate~

Support material

Wasteair -

- jU Humldlflor

Biofilter

Bioscrubber Clesn sir

Nutrients. pH control l

IJ-U

I

Activated s tudge

t t

Waste al r Spray chamber

{acrubber)

Air

Compact wast e wate r treatment

""" 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'mtlal

from flue gases

neps al"fl solubilisatÍOll In a scrubber, N ren'l0V31 ina tlloreactor. w lphite reducrlon to

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

S'ln a submerged oltic

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

Ni trogen oxides (NO.el and sulphur d ioxide fS02) are majar aiT poll utants tbrmed during tbecombustion ofroal and oil and released in flue gases. 1'here is considerable mterest in tbe development of an ef:ficienl and low-cost biotechnologyfor the simultaneous reruqval ofthese ah pollutanu. since convcntional physico-chemical technologies areeitbervery expensive or inefficie nt. Anew system is currently being propase 99%)via sulphide precipitation.

24.6

I Soil remediation

Oneofthe major problems facing tbe indusmalised world roday is !he contaminalion of solls, groundwater and sediments. The total world hazardous wasre remediation market i5 approximarcly US $16 billion pe.r year. Thcre are al least 350000 concaminated sites in Westeru Europe a lone and ir may cost as much as US $400 billion to dean jUSt rhe riskiest ofthese sjtes O\'er rhe next 20--25 years . The mast COffimon contaminants are chlorinated solvents, hydrocarbons. polychlorobiphenyls and metals. BioremediatioD, i.c. the use ofmicrO-Ol'ganisms ro degrade or detoxifY poUurants. is becoming incrcasingly lLsed mostly in cases ofhydrocarbons pollurions. However, bioremediatiOll is nor yet universaUy undcl'stood 01' trustcd by those who must approve ofits use and its suocess is stil1 3n intensively debated issue. Qne reason is the 1ack of predictability of bioremediation, due to insufficient information on: • bioavallability. Le. how ro obtain good (ontact between contaminanf molec::uJes .:md micrOand·treat 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-and·trear me thods Wtle quire Jimited in theil' abilityto remove contaminallt mass fiom rbe subsurface because of sub-surface heterogeneities, prescnce offractu res, low·permeability !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 soil·bound contami.nants can be mobi1· Euro~

ENVIRONMENTAL APPLlCATIO NS

'M,,,

Pump-a nd-treat

SSS

Th Il'puI"p-and-

treat' r"em a w

surface

~

InJection well

z

::>

waler la~

-

direction offlow

olean groundwater

I polluted groundwater I ¡sed. leaving a large residual fraetion in the seU. As a result of this failure. remediarlon policy and technical developments are shifting towards íncreased lIse oflTlsltl/ containment practices. e.g. biofencing (Fig. 24.15), ratber than filU treatment scenarios. In cases where full treatment is necessary. less stringent cleanup goals are seto based on risk assessment taking ioto account typeofland use. Aside frem the much-studied genericcompounds discussed a bove in mis cbapter. there is a hoS{ oftoxic compOunds usually present ar U"3ce level and whose fate remains poorly studied. One example a re !he polych10rinated dioxins and finans which are fOroled as by-products of chemical syntbesis processes. ThE1 are alsoproduced by combustion of

"-

p.

VANDEVIVERE AND VEPSTRAETE

garbage. waste oils, soils pollmed wüb oils, chemical wastes containing PCBs, and by vmous oUler high temperatl1re prcx:esses. Because of the high toxicity ofsome diorins andfurans, thesecompounds are ofmajor eco-toxicological concern. Ongo¡ng research and development has attempted to minimise theirfonnationin inr.:tner.nors and emission via tiy ashes. Yet, the biological breakdown ofUlese compounds in theenvi· ronment is of considerable importance. tndeed. they are often present in wastcs which are extreme1y difficult to t:reat properly by incineration (e.g. pollured soils and riversediments). Theyarealso ptesent in Oyashes ofincinerators which are depositcd in landfills and, notwithstanding aB precautions, can contaminare landfillleachates.

24.7.2 Natural attenuation and monitoring Several factors have recently generaroo a lot ofinterest in rrew monüor· ing techniques. Ofie such factor is the ract that remediatían tecbnolo-gies are ofien insuffident te meet stringent c1eanup targets. This limitation is making legislators reassess che target pollutant levels and making them consider the use ofrisk-based end-points in place of abso· luteend·pointvalues. 111e newconcept ofrisk·based end·points requires the development ofnew a.n..a.Iytical tools which assess the bioavailable rath~ th3.l1 the total poUulant concentration. These new rools typical1y relyon bioassays beca use the traditional analyticalmethods c;:muot dis· tinguish polJutants that are :'lva.i lable to biological systems from those thar ex:ist in ¡nerl. or complexed, unavailable ronns. Subjecting a poi· luted soil to a perlod ofinrensive microbial activity can reduce the toxicityby a factor of5 to 10. ThisecotOJCicological informatian can be easily deduced by runn.ing a simple bioassay with soil leachatcs. One type of bioassay is based on me inhibitian ofthe natural bioluminescence of tbe marine organism Phorobacreriurn pllOS"pl!orezmz, which is med , for example. in the Microtox, Lurnistox and Biotox tests . Thcse assays are, howevec, not specifk since light inhibition will occur upon exposure to any toxicant. This li.m.itation is circumvented in a new class ofbacterial biosensors which are spec:i..fI.c ro certain types oftaxicants. Forexample. biosensors able to detect bioava.ilable mecals, were conSITl1cted by pladng lu¡¡- genes ofVibrlo fischeri as reporter genes under rhe control of genes ¡nvolved in the regulation ofheavy metal resistance in the bacte-rium Alall1gC'"l'S eurrophus. Theo recombinant strains. upon mixing with metal·poIJuted soi ls orwater. emit Iight in proportion [ O the COUceD.IT"d· tion ofspecific bioavailable nletals. Light emission is easily measures radial fIow. :t04 sizc-exclusion,205 ~'onccncration ofproducu, 195 evaporation, 195 cl'ntrlfugal forceO la tion oE soluble el12ymes. 403 Iiltration, oI04 Ul trafil tratiou. 4()4 la rge-scalt' production. 396 Jegisl:lI:ive/sMt'ty, 401- 8 m anUÍilctU n:! rs of, 392

microbiaJ enzytnl'S rl'placing p lanl enzym e~. 398 micrubial soul'ccs. 395 prim'lI'ys tru cturC . 410 prod uced by Aspagíl1us"'S..... 396 p urific ~tion, 404-6 precipita tion, 104 separation by cltromatogr.lphy, 405.524 rt'COYely. 402-3 t'-" tracellular. 402 intrllcellu lar.40'2 N ductiOIlS and o xida tions. 419-:12 monlHJJo/gen ases. 4.20, 421 n ·ge ner.ltio n n t' co-lilL1urs, 4'2 \ $t'condary structure. 410 $lllLdirected mutag(!]l~ S, 4{1J sourccs of ...amylasc, 396 sou n:es of glucose homer~ e, 396 'pt!ci~lity. 393 Ll'rtiary structurc, 0110 tOlal turnO'o'ern umber (fCNI, 421 F.rythrupoieti n, 437

F,smtrlchiu ro!! c hro mosomes.59- 62 ft' rmeutltion , .ICCI/n _leTEnzymf'l¡ growt:b yield, aerobic/ana.erobic, 43 Ethanol.3