Fermentor Design and Bioreactor

FERMENTER DESIGN DESIGN AND AND HIGH PERFORMANCE BIOREACTER

Fermenter  Fermenter is usually cylindrical vessel it! a d"med #"r dis!ed$ dis!ed$ t"%s &"tt"m' It is made (r"m steel steel s" t!ey d" d" n"t rust' T!e t!ic)ness "( steel de%ends u%"n t!e rise "( t!e (ermenter' It is a system !ic! %r"vide c"ntr"ll c"ntr"lled ed envir"nmental c"nditi"n ("r t!e *r"t! "( micr"&es #and "r %r"duct "( s%eci(ic meta&"lites$ in li+uid culture in li+uid cultures !ilst %reventin* t!e entry and *r"t! "( c"ntaminatin* micr"&es (r"m "utside envir"nment'

Fermenter  Fermenter is usually cylindrical vessel it! a d"med #"r dis!ed$ dis!ed$ t"%s &"tt"m' It is made (r"m steel steel s" t!ey d" d" n"t rust' T!e t!ic)ness "( steel de%ends u%"n t!e rise "( t!e (ermenter' It is a system !ic! %r"vide c"ntr"ll c"ntr"lled ed envir"nmental c"nditi"n ("r t!e *r"t! "( micr"&es #and "r %r"duct "( s%eci(ic meta&"lites$ in li+uid culture in li+uid cultures !ilst %reventin* t!e entry and *r"t! "( c"ntaminatin* micr"&es (r"m "utside envir"nment'

Fermenter 

T!is main c"m%"nents "( a (ermenter and t!eir uses Base

c"m%"nent includin* drive m"t"r, !eaters, %um%s, *as c"ntr"l etc'  -essel access"ries Peri%!eral e+ui%ment suc! as rea*ent &"ttles Instrumentati"n and sens"rs

Fermentati"n "%erati"n m"de Batc!

culture (ermentati"n Fed culture (ermentati"n ( ermentati"n C"ntinu"us culture (ermentati"n

Ma."r c"m%"nent "( a (ermenter 

C"m%"nent %arts "( a ty%ical vessel i$ -essel Sin*le alled cylinder "( &"r"silicate *lass it! a (lat &"tt"m Glass .ac)eted system ty%ically r"und &"tt"m steel vessel Steel vessel ii$ T"% %late T"% %late is m"de (r"m /0123 stainless steel and is c"m%ressed "nt" t!e vessel (lan*e &y an easily released clam%in* system' iii$ Seal A seal se%arate t!e vessel *lass (r"m t!e t"% %late' iv$ P"rt (ittin*s P"rt (ittin*s "( vari"us si4es are %r"vided ("r inserti"n "( %r"&es, inlet %i%es, e5it *as c""ler, c"ld (in*ers, sam%les %i%es etc'

v$ In"culati"n %"rt A s%ecial in"culati"n %"rt ill !ave a mem&rane seal !eld in %lace it! a c"llar' vi$ Sam%lin* device Culture can &e it!dran int" a sam%lin* device "r a reserv"ir &"ttle &y a sam%le %i%e' vii$ Gas s%ar*er  A *as s%ar*er is (i5ed "n t!e t"% %late and t!is terminates in a s%ecial assem&ly !ic! insures t!at inc"min* air is dis%ersed e((iciently it!in t!e culture &y t!e (lat &laded /Rus!t"n ty%e3 im%ell"rs (i5ed t" drive s!a(t' viii$ Drive m"t"r  It %r"vides stirrin* %"er t" t!e drive s!a(t and is usually (itted in t!e drive !u& "n t!e vessel t"% %late'

i5$ E5it *as c""ler  It "r)s li)e a sim%le 6ie&i* c"ndenser t" rem"ve as muc! m"isture as %"ssi&le (r"m t!e *as leavin* t!e (ermenter t" %revent e5cessive li+uid l"sses durin* (ermentati"n and ettin* "( t!e e5it air (ilter' 5$ Pt7188 A narr" %latinum resistance tem%erature sens"r, tem%erature c"ntr"l is eit!er &y direct !eatin* usin* a !eater %ad "r &y circulatin* arm ater ar"und t!e vessel .ac)et' 5i$ C"ld (in*er  I( a direct !eatin* is used, a c"ld (in*er is used t" c""l t!e vessel c"ntents' It %asses t!e a cl"sed %i%e "r c"il !ic! %asses t!r"u*! (ermenter t"% %late and all"s c""lin* ater t" circulate, t" act as !eat e5c!an*er it! t!e culture' 5ii$ %H electr"de9 5iii$ R"tameter 

Peri%!eral %art and access"ries9 a$ Rea*ent %um%s Pum%s

are n"rmally %art "( instrumentati"n system ("r measurin*' %H Anti("am C"ntr"l

Peristaltic

%um%s are used and t!e (l" rate is (i5ed it! a time :s!"t and delay; (eed system "( c"ntr"l'  Fl"

rate de%ends "n t!e &"re "( t!e tu&in* used< %eristaltic tu&in* lin)s t!e reserv"ir &"ttles it! t!e vessel multiay inlet' T!e

tu&in* is clam%ed s!ut durin* aut"clavin*, already c"nnected t" vessel and "%ened ("r active additi"n "( rea*ent'

&$ Medium (eed %um%s and reserv"ir &"ttles Medium

(eed %um%s are "(ten varia&le s%eed t" *ive t!e ma5imum %"ssi&le ran*e "( (eed rates' 

T!e

s%eed "( t!e "%erati"n "( t!e %um% can &e set manually "r !"le system %ut under t!e c"m%uter c"ntr"l' 

T!e

reserv"ir &"ttles are usually lar*er =7>8 litre'

 Reserv"ir

&"ttle may !ave c!an*ed several times durin* a l"n* c"ntinu"us culture e5%eriment' 

E((luent

%um% is als" used ("r t" rem"ve culture (luid (r"m t!e (ermenter vessel int" a st"ra*e reserv"ir &"ttle' c$ R"tameter?*as su%%ly R"tameter

is used t" c"ntr"l t!e air (l" rate int" a (ermenter vessel' A %ressure re*ulat"r valve &e("re t!e r"tameter ensures sa(e "%erati"n' 

A

sterile (ilter #usually 8'>> @m$ is used as a &rid*e &eteen tu&in* (r"m t!e r"tameter t!at c"nnected t" t!e air s%ar*er "( t!e (ermenter' 

A

sec"nd (ilter "n t!e e5it *as c""ler st"%s micr"&es &ein* released int" atm"s%!ere'

d$ Sam%lin* device T!is

all" culture (luid is rem"ved ase%tically durin* (ermentati"n at t!e intervals decreased &y users acc"rdin* t" t!e need "( t!e e5%eriment'

Alternative -essel Desi*ns Alternative

vessel desi*n tried !en standard vessel c"n(i*urati"n d"es n"t all" ade+uate *r"t! "( t!e "r*anism' a$ Air li(t T!is

vessel desi*n eliminate t!e need "( a stirrer system' A rin* s%ar*er is n"rmally used t" %r"duce a c"lumn "( air &u&&les !ic! rises u%' T!is is called air li(t system' T!is %r"duce a d"n (l" "( a medium !ic! is c!anneled &y a drau*!t tu&e (i5ed in t!e centre "( t!e (ermenter'

&$ Fluidi4ed &eds T!e

micr"&es? cell are tra%%ed in a %!ysical medium and !eld in t!e vessel &y mes!' T!e medium is reticulated via a %um% and t!is can &e easily ada%ted t" *ive a c"ntinuum? semi c"ntinu"us (l" t" all" t!e tra%%ed cells t" a((ect t!e c!emical c!an*es "n t!e c"nt"rti"ns "( t!e medium it!"ut &ein* as!ed "ut al"n* it! s%ent medium' c$ H"ll" (i&re T!e

cells are em&edded in (i&re c"ntained in cartrid*e !ic! is &at!ed in circulatin* culture medium' e'*' used ("r t"5icity "r interactive studies and mammalian cell culture' d$ In situ sterili4a&le (ermenters T!e

use "( aut"clava&le vessels "( *reater t!an =718 litre "r)in* v"lume are n"t sa(e and vessels a&"ve t!is si4e an dually m"de "( stainless still and are desi*ned t" &e sterili4ed in situ usin* !"use steam "r an electrical steam *enerat"r  Heatin* is %r"vided via a d"u&le ac)et '

Di((erent ty%e "( instrumentati"n Fermenter instrumentati"n can ran*e (r"m sim%le anal"*ue c"ntr"l m"dules arran*ed as /stac)3 t" %"er(ul, em&edded micr" %r"cess"r !ic! "%erate as "n &"ard c"m%uters t" directly "%erates t!e !eaters, %um%s, valves and "t!er c"ntr"l actuat"rs "( a (ermenter'

Anal"*ue c"ntr"l less7 rac) system T!e

individual m"dules ("r t!e measurement and c"ntr"l "( eac! %arameter are made %lu* int" a rac) system ("r c"m%act, (le5i&le, instrumentati"n' A &us system in t!e sta&lin* transmits t!e si*nals (r"m t!e m"dule t" an"t!er all"ed measured value t" &e %assed t" c"ntr"llers and ("r "ne c"ntr"ller t" in(luence an"t!er e'*' "5y*en level alterin* s%eed'

Anal"*ue c"ntr"llers7 se%arate m"dule in !"usin* Eac!

measurement? c"ntr"l m"dule !as se%arate !"usin* and essentially "%erates in is"lati"n' T!e m"dules may &e stac)ed "ne "n t"% "( an"t!er and can &e su%%lied it! :Pass t!r"u*!; %"er c"nnecti"n s" t!at "nly "ne %"er s"c)et is needed'

Bec)man (ermenter

Fermenter desi*n

C"mm"n Measurement and C"ntr"l Systems S%eed c"ntr"l S%eed

c"ntr"l relies "n t!e (eed &ac) (r"m a tac!"meter l"cated it!in drive

m"t"r  A di*ital dis%lay s!"s actual s%eed in r%m as determined &y t!e tac!"meter si*nal' A %"er meter is used t" maintain t!e set s%eed de%ends u%"n t!e -isc"sity "r /density3 "( t!e culture (luid' A DC l" v" v"lta*e m"t"r is "(ten used ("r sa(ety reas"ns'

Tem%erature c"ntr"l A

t!erm" circulati"n system ar"und a vessel .ac)et is used' Heater %ad is (erried settin* t!e desired tem%erature and sitc!in* "n3 C""lin* is n"rmally via a c"ld (in*er and (l" "( c""lin* ater is c"ntr"lled via t!e acti"n "( /S"len"id value3' T!e Pt7188 sens"r %r"vides t!e (eed &ac) si*nal t" t!e c"ntr"ller' Heat radiati"n system a%%eared t" t!e c!"ice ("r la&"rat"ry &i"react"rs' T!e s"urce "( sad rati"n is directed t" t!e vessel, t!en t!e !eatin* "uld &e *entle as t!at "( ater &y Sun' t!e "%timal l"cati"n ("r t!e recitati"n is underarm Neat! t!e &"tt"m "( t!e vessel'

C"ntr"l "( *as su%%ly T!e

su%%ly "( *as #n"rmally air$ t" t!e (erment "r vessel is %r"vided (r"m a c"m%ressed air su%%ly s"il (ree !ic! may %r"vide "il ("r m"re t!an "ne vessel de%endin* u%"n its si4e  "ut%ut' A %ressure re*ulati"n value ensures t!at air cras!es t!e vessel at a ma5imum "( 1'= &ar' T!e r"tameter c"ntr"l t!e actual (l" rate "( air t!r"u*! t!e (erment"r' T!is s!"uld n"t e5ceed 1'= vessel v"lumes %er minute "r' T!e air %asses t!r"u*! inlet (ilter !ic! %revent any micr"&es (r"m enterin* t!e vessel' T!e &u&&les are immediately &r")en u% and dis%ersed &y im%ell"r "n t!e drive s!a(t and t!e &a((les !ic! can &e (elted near t!e  all "( t!e vessel'

C"ntr"l "( %H %H

c"ntr"l is ac!ieved &y t!e additi"n "( eit!er acid "r al)ali t" c"rrect c!an*es in t!e %H "( t!e culture durin* *r"t!' T!e c"ntr"ller uses a %H electr"de t" sense t!e %H c!an*es and %r"vide a (eed &ac) si*nal' T!e electr"de must &e in direct c"ntact it! t!e medium and s" must &e (i5ed in t!e (ermenter all'

Anti("am electr"de T!e

c"ntr"l "( a ("am is &ased "n its detecti"n &y a c"nductance %r"&e in t!e vessel !ead s%ace, !ic! leads t" t!e c"ntr"ller deliverin* a d"se "( li+uid anti("am rea*ent via %eristaltic %um%' T!e sensitivity "( a %r"&e t" ("am can &e ad.usted &y t!e user t" suit t!e c"nditi"ns %revailin* in t!e (ermenter' N"rmally, metal t"% %late is used t" %r"vide t!e electrical circuit ("r t!e %r"&e t" "%erate ("am can &e c"ntr"lled in t" ays

C!emical c"ntr"l ("am It inv"lves t!e additi"n "( C!emical su&stances !ic! l"er t!e sur(ace tensi"n "( t!e medium "( case &u&&le t" c"lla%se'

Mec!anical ("am c"ntr"l It uses a device called a ("am &racers t" &rea) u% ("am &y mec!anical acti"n' T!e ("am &rea)er must &e %"siti"ned at t!e t"% "( t!e (ermenter in "rder t" deal it! ("am in lead s%ace

Nutrient C"ncentrati"n It

is "(ten advanta*e"us t" limit t!e nutrient c"ncentrati"n in medium, s" t!ey are n"t %resent in e5cess' Electr"de tec!n"l"*y !ave made t!e m"nit"rin* t!e s"me nutrients %"ssi&le' I"ns selective electr"de are n" availa&le ("r t!e measurement "( NH 0 ? NH *luc"se "5ide$ are availa&le ("r t!e ' En4yme electr"des #&ased "n measurement "( *luc"se'

Stirred- Baffled system In

t!e stirred system t!e medium is stirred &y  paddles #im%ellers driven &y an electric motor ' T!e m"t"r is %laced a&"ve t!e (ermenter and it drives a s!a(t !ic! %asses d"n int" a (ermenter and attac!ed t" t!e Impellers. A c"mm"n ty%e "( im%eller is t!e disc -turbine impeller , !ic! c"nsists "( vertical vanes "n a !"ri4"ntal disc'

Sterili4ati"n M"st industrial "%erates usin* %ure and t!us usin* ase%tic c"nditi"ns' T!e seas"n ("r t!is9 a$ C"m%etiti"n ("r su&strate &$ In!i&iti"n "( *r"t! "r %r"duct ("rmati"n c$ Destructi"n "( t!e %r"duct F"r t!ese seas"n %ure culture are usually used' Fermenter and t!e medium must &e sterili4ed &e("re in"culati"n' It is can &e d"ne9

A$ Sterili4ati"n &y !eat9 Heat is used t" sterili4e t!e (ermenter' i$ In

Sterili4ati"n in situ 9

t!is Met!"d, t!e (ermneter is (illed t" t!e re+uired level it! medium and !"le t!in* is !eated' Heatin* is carried "ut &y %assin* %ressuri4ed steam ar"und a .ac)et "r c"il &uilt int" t!e (ermenter all' A .ac)et n"rmally c"vers t!e vessel alls u% t" t!e !ead s%ace' F"r sterili4ati"n, steam under %ressure in %assed ar"und t!e .ac)ed "r c"il and t!e medium is !eated' At &"ilin* %"int all t!e valves "n t!e (ermenter re cl"sed and system &ec"me su%er!eated'

B$ C"ntinu"us !eat sterili4ati"n T"

"verc"me t!e %r"&lems "( sterili4ati"n in situ , c"ntinu"us !eat sterili4ati"n can &e used' In c"ntinu"us !eat sterili4ati"n, medium is %assed in a stream t!r"u*! devices in !ic! it is !eated u% very +uic)ly t" 18 "C, !eld at t!e tem%erature ("r "ne minute, t!en c""l d"n a*ain very +uic)ly'

ii$ Sterili4ati"n %r"cess An"t!er

met!"d is t" use !eat e5c!an*es' F"r steam sterili4ati"n a s%iral !eat e5c!an*er is "(ten used' In t!is system, steam under %ressure %asses in "ne directi"n r"und t!e system, !ile t!e medium %asses ar"und t!e s%iral in "t!er c!annel in "%%"site directi"n' Medium t!en travel t!r"u*! a !"ldin* tu&e' T!is is a c"iled %i%e %ac)ed in insulati"n material s" t!at tem%erature d"es n"t dr"%' T!is system is desi*ned s" t!at it ta)es t!e medium "ne minute t" (l" t!r"u*! !"ldin* tu&e' Medium is t!en c""led d"n &y %assin* it t!r"u*! an"t!er !eat e5c!an*e'

iii$ Sterili4ati"n &y (iltrati"n S"me media, es%ecially t!"se !ic! are ric! in %r"teins, can n"t &e !eat sterili4ed at all, in suc! cases medium must &e sterili4ed &y (iltrati"n' T!ere are t" &asic ty%e "( (ilters used in sterile (iltrati"n' 1$

1$

Mem&rane (ilters

>$

De%t! (ilters

Mem&rane (ilters

Mem&rane (ilters are c"m%"sed "( a t!in s!"rt "( material (i5ed in a casin* #"r !"usin*$' 6i+uid is %um%ed t!r"u*! !"usin* and is ("rced t!r"u*! t!e (ilter material' T!e (ilter material suc! as cellul"se, nitrate, cellul"se acetate "r %"ly car&"nate'

>$ De%t!

De%t! (ilters

(ilter c"m%"sed "( a %"d "( (i&r"us material (i5ed in a casin*' T!is ty%e "( (ilter d"es n"t "r) &y sievin*, &ut &y ads"r%ti"n, t!e micr""r*anisms stic) "n t" t!e (i&re !en medium is %assed &eteen (i&re' Filter Desi*n F"r lar*e scale (iltrati"n, (ilter material is n"rmally %ac)a*ed in dis%"sa&le cartrid*es' In t!ese, a rectan*ular %iece "( (ilter material is (luted, ."ined and %ac)a*ed in %er("rated %lastic surr"und'







T!e cartrid*e (its int" a stainless steel !"usin*, !en medium is %um%ed t!r"u*! t!e (ilter !"usin*, t!e medium !as %ass t!r"u*! t!e (ilter material in "rder t" reac! t!e "utlet' De%t! (ilters are s"metime used in %late (rame (ilters' In t!is ty%e "( device dis%"sa&le (ilter s!eets are %laced &eteen %lates, !ic! are l"c)ed t"*et!er in a (rame' medium is %um%ed t!r"u*! (ilter, t!e medium %asses t!r"u*! t!e c"m%artments in %arallel' F"r lar*e scale (iltrati"n, t!e medium is %assed t!r"u*! a de%t! (ilter t" rem"ve m"st micr""r*anisms and der&is'

Filter Desi*n   





F"r lar*e scale (iltrati"n, (ilter material is n"rmally %ac)a*ed in dis%"sa&le cartrid*es' In t!ese, a rectan*ular %iece "( (ilter material is (luted, ."ined and %ac)a*ed in %er("rated %lastic surr"und' T!e cartrid*e (its int" a stainless steel !"usin*' !en medium is %um%ed t!r"u*! t!e (ilter !"usin*, t!e medium !as %ass t!r"u*! t!e (ilter material in "rder t" reac! t!e "utlet' De%t! (ilters are s"metime used in %late (rame (ilters' In t!is ty%e "( device dis%"sa&le (ilter s!eets are %laced &eteen %lates, !ic! are l"c)ed t"*et!er in a (rame' Medium is %um%ed t!r"u*! (ilter, t!e medium %asses t!r"u*! t!e c"m%artments in %arallel' F"r lar*e scale (iltrati"n, t!e medium is %assed

Fundamentals "( Fermenter Desi*n T!e ada%tati"n "( su&mer*ed culture tec!ni+ues ("r t!e %r"ducti"n "( %rimary and sec"ndary meta&"lite, !ave attracted t!e c"m&ined attenti"n "( micr"&i"l"*ist, &i"c!emists and en*ineers t" res"lve a variety "( %r"&lems in "rder t" im%r"ve' A variety "( %r"&lems in "rder t" im%r"ve &"t! yield and %r"cess e((iciency' T!e t" as%ects are9

i$

Fermenter Desi*n and Re+uirement "( t!e micr"&ial system

T!e

material "( c"nstructi"n must &e suc! t!at t!ey ill n"t adversely a((ected &y t!e desired micr"&ial activity, eit!er interacti"n it! (ermentati"n medium "r &y !ar&"urin* unanted "r*anisms' T!e (ermenter must &e resistant t" c"rr"si"n &y t!e nutrient medium and %r"ducts and t" e((ect "( sterili4ati"n' T!ere must &e %r"visi"n ("r t!e re*ulati"n "( tem%erature and air su%%ly ("r c!ar*in* and disc!ar*in* t!e vessel c"ntents' In c"ntinu"us culture system additi"nal (acilities must &e %r"vided t" c"ntr"l culture v"lume and medium (l" rate'





T!e e((ect "( "r*anism "n t!e macr"envir"nment !ic! may &e re(lected in c"nc"mitant c!an*es in t!e micr"envir"nment, arises mainly a$ R!e"l"*ical c!aracter &$ c!an*e in inter(acial tensi"n' It "uld desira&le t" relate (ermenter desi*n and c"ntr"l systems t" micr" envir"nment in %ractice "nly t!e macr"envir"nment can &e in(luenced directly' T!e (act"r it! in t!e c"ntr"l "( t!e desi*ner and "%erat"r are9 a$ &$ c$ d$ e$ ($ *$ !$ i$

System *e"metry Aerati"n rate Intensity "( a*itati"n Tem%erature Pressure Nutrient su%%ly %H and "t!er %arameters inv"lvin* s%eci(ic i"ns Diluti"n rate in c"ntinu"us (l" system and F"amin*'

C!emical en*ineerin* as%ects "( (ermenter desi*n T!e "&.ective "( (ermenter desi*n and "%erati"n ensure t!at t!e desired activity "( t!e micr""r*anisms c"ncerned s!all n"t &e restricted &y t!e c!aracteristics "( t!e e+ui%ment' T!e %!ysical %r"cess mi*!t limit t!e activity are9

A$

Mass Trans(er

T!e

trans(er "( mass it!in t!e system is t!e (undamental t" t!e !"le "%erati"n' Mass Trans(er  More or less uniform distribution of substrate and product molecule in the bulk of fluid

First

Transfer of the bulk of the fluid and microbial cell

as%ect is lar*ely *"verned &y t!e ("rced c"nvecti"n and tur&ulence %r"duced &y a*itati"n and t!e (l" "( *as' Sec"nd as%ect is determined &y di((usi"nal ("rces'

Heat Trans(er  Heat

is *enerated in su&mer*ed micr"&ial systems, %artly &y t!e meta&"lic activity "( t!e "r*anisms and %artially &y t!e mec!anical "r) %er("rmed &y t!e a*itat"r and *as &u&&le' S"me !eat is l"st as a result "( increased !umidi(icati"n "( *as stream in its %assa*es t!r"u*! culture and s"me &y radiati"n (r"m t!e "uter sur(ace "( t!e (ermenter'

Mi5in* e((ects A

mi5in* serves t" minimi4e l"cal variati"n in c"ncentrati"n and tem%erature' It causes rand"m redistri&uti"n "( elements "( t!e culture sus%ensi"n in t!e im%eller 4"ne and (r"m t!e rand"m interacti"n, it! t!e &ul) "( sus%ensi"n, "( stream leavin* t!e im%eller' In a *iven system rate "( mi5in* in direct' T!is may &e e5%resses as mean circulati"n rate' Alternately "ne may c"nsider t!e mean circular time' T!is ill als" re%resent a avera*e mean time'

Scalin* u%  T!e

t!ree %arameters are Ge"metry P"er in%ut %er unit v"lume Su%er(icial air vel"city T!e "verall e((ect is t" reduce t!e avera*e s!ear rate and relative v"lumetric (l" "( t!e sus%ensi"n t!r"u*! im%eller, it!"ut increasin* tur&ulence and ma5imum

A%%licati"n Fermenter %re%arati"n and use Disassem&ly "( t!e vessel Fermenter

is s!utd"n (r"m t!e c"ntr"l units ca&le c"nnecti"n and trans(er lines t" &e rem"ved' -essel s!"uld &e reaut"claved a(ter (ermentati"n Inlets and "utlets t" %re%ared ("r t!at Culture s!"uld &e dis%"sed "( as %er sa(ety reas"n C!i%s, &"lts?clam%s "( vessel t"% %late t" und"ne t" turn !"le assem&ly s%rin* (ree  T!e t"% %late can &e li(ted u%ards aay (r"m t!e vessel ta)in* care t!at air s%ar*er? drive s!a(t? im%ell"rs and Pt7188 tem%erature %r"&es c"m%letely clear t!e vessel'

Cleanin* %H

and diss"lved O> electr"des s!"uld &e rem"ved and st"red in a suita&le rea*ents acc"rdin* t" t!e manu(actures instructi"ns  Peri"dic

cleanin* and re*enerati"n "( t!e electr"des is als" im%"rtant ("r t!eir maintaince and c"st e((ective' -essel s!"uld &e rinsed several times in distilled ater t" rem"ve any l""se

    

Cleanin* "( *r"t! "( culture "n alls "( culture "n alls "( vessels it! li*!t &rus!in* is d"ne' C!i%%in*? crac) in *lass vessel s!"uld &e av"ided? re%lacement is t" &e d"ne' -essel s!"uld &e st"red clean and dry' C"ntact &eteen t"% %late and li+uid it! !i*! c!l"ride c"ntents s!"uld &e av"ided t" %revent c"rr"si"n  Pum% !ead and must t!"r"u*!ly clean'

Pre%arati"n ("r aut"clavin*       

-essel seal s!"uld &e rem"ved and li*!tly *reased it! suita&le silic"n *rease' -essel is (illed it! medium t" ma5imum "( 8 (ull' #s%ace vital ("r *as e5c!an*e$ Ma5imum am"unt "( medium v"lume is t" &e c"ver electr"des ade+uately' Re%lacin* "( t!e t"% %late "n vessel' Clam%s?&"lts t" ti*!ten (irmly' Electr"des t" &e %us!ed in %"rt (ittin* directly and %i%es are (itted in same ay' %H electr"des s!"uld &e cali&rated in a%%r"%riate &u((ers i'e' in %H  t!en eit!er %H  "r %H J ("r !i*! re(erence7 l" re(erence

       

%H and DO electr"de t" &e (itted it! utm"st care n"t t" dama*e t!em' B"t! must &e ti*!tly ca%%ed t" %revent m"isture *ettin* int" electrical c"ntacts' Pt7 188 must &e (itted and ca%%ed' Outlet %i%es "( rea*ent &"ttles s!"uld &e clam%ed and s!ut' In"culati"n %"rt is cl"sed &y ca% E5it *as c""ler must &e le(t "%en' Drive !u& is c"vered it! ("il' Tu&in* is clam%ed s" t!at n" li+uid can esca%e durin* aut"clavin*'

Aut"clavin*     

A (inal c!ec) u% is made t!at at least "ne r"ute is availa&le ("r air t" enter and leave t!e vessel' All t!e lines di%%in* int" li+uid are clam%ed cl"sed' Aut"clavin* at 1>1 "C ("r 08 minute' -essel and access"ry must &e all"ed t" c""l, c"m%letely &e("re !andlin*' Reduce t!e v"lume "( t!e medium u%t" 18  "( t!e t"tal

Set u% F"ll"in* Aut"clavin* Air

s%ar*er line uncli%%ed and c"nnects t" r"tameter' E5it *as c""ler is c"nnects t" H >O su%%ly Tu&in* (r"m t!e rea*ent &"ttle c"nnected t" multiay inlet and relevant %eristaltic %um%' Drive m"t"r "n t"% %late is c"nnected t" drive s!a(t' Pt' 188 is c"nnected t" c"ntr"l m"dule and %H electr"de &y rem"vin* ca% and DO electr"de t" a%%r"%riate ca&le' Main electricity t" mentati"n m"dule' Is sitc!ed "n all"in* s"me !"urs #> minute and > !"urs ma5$ ("r OD electr"des t" &e %"lari4ed %r"%erly a(ter t!at' DO is cali&rated and air su%%ly is turned "n ma5imum stirred s%eed t" &e used, ma5imum air (l" is needed "n r"tameter are set' Fermenter is ready t" in"culate'

In"culati"n "( vessel P"rt

(itted it! a mem&rane it! in ca%%ed "(( &e("re aut"clavin*' In"culum =718  "( t"tal culture v"lume ase%tically trans(erred t" a sterile dis%"sa&le syrin*e "( suita&le si4e' P"rt (ittin* is rem"ved Needle is +uic)ly %us!ed t!r"u*! t!e mem&rane and in"culum is trans(erred in t!e vessel' Needle is +uic)ly it!dran and silic"n mem&rane is reseals'



F"r added security 8  et!an"l can &e %laced "n mem&rane sur(ace &e("re %iercin*'

Sam%lin* (r"m vessel          

Sam%lin* starts it! a sam%le %i%e !ic! s!"uld &e di% int" t!e &ul) "( culture li+uid' Sam%le %i%e %lu**ed and c"vered at "%en end' A(ter aut"clavin*, %lu* is rem"ved and "%ened end di%%ed in a c"ntainer "( a 8  et!an"l !en sam%le is t" &e ta)en syrin*e is +uic)ly c"u%led t" t!e tu&in* and sam%le is it!dran int" it' Aerati"n is st"%%ed durin* t!is %r"cess t" %revent /sur*in*3 "( culture int" syrin*e' Sec"nd a%%r"ac! is t" use sam%lin* device' T!is is a &"ttle it! t" metal needles? %ermanently (i5ed "( its ca% One %i%e !as 8'>> @m air (ilter and > nd is lin)ed t" sam%le %i%e Syrin*e is li(ted t" air (ilter a(ter aut"clavin*' Sam%le device is attac!ed t" t!e %late'

Additi"nal sens"rs Red"5 T!is

re(ers t" reducti"n?"5idati"n %"tential "( t!e system and can &e a%%lied t" &i"l"*ical cultures as ell as c!emical reacti"ns' Red"5 values are *iven in milliv"lt and it re(ers t" a standard !ydr"*en !al( cell'

Air (l" A

sim%le system "uld use a ma*netic sens"r and varia&le area (l"meter t" measure t!e current air (l" and ad.ustant am"t"ri4ed valve &eteen 8 and 188  "%en "r cl"sed t" c"ntr"l t!e air (l"

ei*!t 

It is %"ssi&le t" m"unt !"le (ermenter "n a &alance and /tare3 "ut its ei*!t'   A m"re %recise a%%r"ac! is t" use a system it! a small l"ad cell m"unted in suc! a t!at t!at t!e "nly t!e vessel and its c"m%"nent are measured'

Pressure T!e

use "( t!e %ressure as a c"ntr"l is m"re c"mm"n in situ sterli4a&le (ermenter' T!e %ressure measurement is %r"vided &y %ie4" electric sens"r !ic! can &e m"unted int" a %"rt cl"sure and (itted t" t!e vessel t"% %late' T!e c"ntr"l element is %r"%"rti"nal valve !ic! restricts t!e (l" "( t!e *as "ut "( t!e (ermenter and t!ere&y crate a &ac), %ressure'

On line measurement A

direct measurement "( num&er "( t!e "r*anisms in a culture is t" &e desira&le ("r  Feed rate O5y*enati"n Pr"cess "%timi4ati"n  Measurement "( t"tal num&er "( cells, et ei*!t, and via&le cells c"unts "uld &e made &y ta)in* sam%le "( culture at set time and analysis is %er("rmed in la&"rat"ry' Flu"rescence Micr"sc"%y !as &een used ("r t!e measurement "( t!e via&le cell density eit!er &y a %r"&e "r e5ternally t!r"u*! t!e vessel *lass'  Metteler7 T"lde" FCS tur&idity system Ca%acitance?c"nductance7&ased &i"mass m"nit"r 

T!e main c"m%"nents "( t!e (ermenter and t!eir Kse Pr"vide

aerati"n (ree (r"m c"ntaminati"n maintain a s%eci(ic tem%erature Pr"vide ade+uate mi5in* and aerati"n C"ntr"l t!e %H "( t!e culture All" m"nit"rin* and "r? C"ntr"l "( diss"lved "5y*en All" (eedin* "( nutrients and rea*ents  Privides acess %"ints ("r in"culati"n and sam%lin* Kse (ittin* and *e"metry relevant t" scale u% Minimi4e li+uid l"ss (r"m t!e vessel Facilitate t!e *r"t! "( a ide ran*e "( t!e "r*anism

 Fermenter c"nstructi"n A

c"m%lete descri%ti"n "( met!"d"l"*ies used ("r its devel"%ment, installati"n and "%erati"n -alidiati"n "( t!e system it! res%ect t" t!e !ardare, "%erati"n and its a%%licati"n D"cumentati"n "( s%eci(ic valida&le activities Evidence c"n(irmin* t!at eac! element "( !ardare an s"(tare %er("rm its (uncti"n relia&ly and in acc"rdance it! d"cumentati"n s%eci(icati"ns

T!e ("ll"in* c"m%anies "((er s"(tare %r"*rammes it! a ide ran*e "( a%%licati"ns APP6ILON B' BRAKN BIOTECH INCE6TECH AND INCE6TECH SOFT NE BRKNSICL SCIENTIFIC

Configuration and software  All

are fully object oriented softwares running under WINDW! "# The data files can be processed under different operating systems $M!%Dos& WINDW! '()& WINDW! "#& *NI+& etc((,

Data ac+uisiti"n "n t!e micr"c"m%uter

E5am%le "( ind" availa&le t" t!e user

E5am%le "( t!e c"n(i*urati"n "n t!e dis%lay unit durin* a (ermentati"n

E5am%le "( t!e screen vie durin* a (ermentati"n in t!e %r"ducer 

Summary and c"nclusi"n Ferment"rs

are c"m%"sed "( a num&er "( di((erent c"m%"nents !ic! can &e *r"u%ed &y t!eir (uncti"ns, i'e' tem%erature c"ntr"l, s%eed c"ntr"l, c"ntinu"us culture access"ries, etc' A ide ran*e "( %eri%!eral devices can en!ance t!e &asic (acilities "( t!e (erment"r, e'*' (eed %um%s, e5it *as analysers, etc' Ferment"r instrumentati"n can &e "( several di((erent ty%es, de%endin* "n t!e a*e, c"st and s"%!isticati"n "( t!e (erment"r' Micr"%r"cess"r7&ased system "((er advanta*es in terms "( t!e (le5i&ility "r c"ntr"l and t!e a&ility t" st"re "%erati"nal %r"t"c"ls' Many di((erent ty%es "( vessels e5ist suc! as air li(t, (luidi4ed &ed, !"ll" (i&re and s%ecially m"di(ied stirred tan) react"rs ("r c"ntainment "( in situ  sterili4ati"n' Practical ste%s ("r aut"clavin* use, in"culati"n and sam%lin* ensure sa(e and relia&le "%erati"n ("r any ma)e "r ty%e "( (erment"r' R"utine maintenance "( electr"des, vessel, *lass, etc' is re%aid in l"n*evity "( c"m%"nents and sa(e "%erati"n' T!e ran*e "( "r*anisms !ic! can &e ada%ted ("r *r"t! in (erment"rs is ide and includes micr"&es, %lant and animal cells'