Giao Trinh Cong Nghe TB - Nguyen Hoang Loc(Da Nen Dung Luong)

Tai lieu chia se tai: wWw.SinhHoc.edu.vn Nguyn Hong Lc

Gio trnh

CNG NGH T BO

Nh xut bn i hc Hu Nm 2006

Tai lieu chia se tai: wWw.SinhHoc.edu.vn

Tai lieuPGS. TS.tai: wWw.SinhHoc.edu.vn chia se Nguyn Hong Lc

Gio trnh

CNG NGH T BO

Nh xut bn i hc Hu Nm 2006


Tai lieu chia seLi ni u tai: wWw.SinhHoc.edu.vnCng ngh t bo l mt b phn quan trng ca cng ngh sinh hc, ch yu nghin cu cc qu trnh nui cy t bo ng-thc vt v vi sinh vt sn xut sinh khi, sn xut cc hp cht c hot tnh sinh hc (enzyme, vaccine, cc cht th cp), lm m hnh thc nghim kho st cc tc ng ca ho cht, lm nguyn liu ghp t bo v c quan Mc d, cc k thut nui cy t bo ch c pht trin vo na u th k 20, nhng n nay cc ng dng ca chng c nhng bc tin vt bc nh s ng gp ca cng ngh DNA ti t hp. Bn cnh cc gio trnh nh: sinh hc phn t, nhp mn cng ngh sinh hc, cng ngh DNA ti t hp, cng ngh chuyn gen gio trnh cng ngh t bo s gip sinh vin tip cn thm mt lnh vc khc ca cng ngh sinh hc thng qua vic cung cp nhng kin thc c bn v cc vn sau: - Sinh trng v ng hc sinh trng ca t bo. - Thit k cc h ln men. - Nui cy t bo v cc ng dng ca chng. Gio trnh cng ngh t bo c bin son theo hng kho st mt qu trnh sinh hc mang tnh cng ngh nhiu hn c l qu trnh ln men ng dng cho c t bo vi sinh vt, ln t bo ng-thc vt trong cc thit b nui cy (bioreactor/fermenter). Do , mt s ng dng khc ca cc k thut nui cy m v t bo ni chung chng ti khng a vo gio trnh ny. Lnh vc cng ngh t bo rt rng v a dng, hn na gio trnh ny mi c xut bn ln u tin nn kh trnh khi thiu st hoc cha p ng c yu cu bn c. V th, chng ti rt mong nhn c nhiu kin ng gp ln xut bn sau c hon thin hn.

Tc gi


Tai 1 Chnglieu

chia se tai: wWw.SinhHoc.edu.vnM u

I. Cng ngh sinh hcn nay c rt nhiu nh ngha v cch din t khc nhau v cng ngh sinh hc ty theo tng tc gi v t chc. Tuy nhin, cng ngh sinh hc (biotechnology) c th c nh ngha mt cch tng qut nh sau: Cng ngh sinh hc l cc qu trnh sn xut quy m cng nghip m nhn t tham gia trc tip v quyt nh l cc t bo sng (vi sinh vt, thc vt v ng vt). Mi t bo sng ca c th sinh vt hot ng trong lnh vc sn xut ny c xem nh mt l phn ng nh. Nu cng ngh sinh hc c nh ngha theo hng trn th n khng th c tha nhn l mt lnh vc khoa hc mi. Bi v, t xa xa loi ngi bit s dng cc vi sinh vt ln men bnh m v thc phm, cho d h khng bit c ch ca nhng bin i sinh hc ny l nh th no. Loi ngi cng bit t rt lu vic lai to ng vt v thc vt ci thin nng sut vt nui v cy trng c tt hn. V th, cng ngh sinh hc c nh ngha nh trn c xem nh cng ngh sinh hc truyn thng. Tuy nhin, trong nhng nm gn y thut ng cng ngh sinh hc thng c s dng nhm cp n nhng k thut mi nh DNA ti t hp v dung hp t bo, v c xem l lnh vc cng ngh sinh hc hin i. 1. Cng ngh DNA ti t hp (DNA recombinant technology) L nhng k thut cho php thao tc trc tip nguyn liu di truyn ca cc t bo ring bit, c th c s dng pht trin cc vi sinh vt sn xut cc sn phm mi cng nh cc c th hu ch khc. Nhng k thut ny cn c gi l k thut di truyn (genetic engineering), cng ngh di truyn (genetic technology), thao tc gen (gene manipulation), k thut gen (gene engineering) hay cng ngh gen (geneCng ngh t bo


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technology)... Mc tiu chnh ca cng ngh DNA ti t hp l gn mt gen ngoi lai (foreign gene) m ha cho mt sn phm mong mun vo trong cc dng DNA mch vng (plasmid vector) v sau a chng vo trong mt c th vt ch, sao cho gen ngoi lai c th biu hin sn xut sn phm ca n t c th ny. 2. Dung hp t bo (cell fusion) L qu trnh hnh thnh mt t bo lai n (single hybrid cell) vi nhn v t bo cht t hai loi t bo ring bit t hp cc c im mong mun ca c hai loi t bo ny. Chng hn, cc t bo c bit ca h thng min dch c th sn xut ra cc khng th hu ch. Tuy nhin, cc t bo ny thng kh nui cy v tc sinh trng ca chng rt chm. Mt khc, cc t bo khi u nht nh no c cc c im bt t v phn chia nhanh. Bng cch dung hp hai t bo ny, mt t bo lai hybridoma c th c to ra mang c hai tnh trng trn. Cc khng th n dng (monoclonal antibodies-Mabs) c sn xut t cc t bo lai, c dng chn on, iu tr bnh v tinh sch protein. 3. ng dng ca cng ngh sinh hc hin i Cc ng dng ca cng ngh sinh hc hin i l rt nhiu (Bng 1.1). Cc dc phm him v t trin trc y nh insulin cha bnh i tho ng, hormone sinh trng ngi iu tr bnh ci ca tr em, interferon chng vim nhim, vaccine phng bnh v cc khng th n dng dng chn on... c th c sn xut bng cc t bo c bin i di truyn hoc cc t bo lai r tin vi s lng ln. Cc con ging sch bnh hoc kho mnh hn, cc vt nui dng lm thc phm c sn lng cao c th c pht trin, cc loi cy trng quan trng c th c bin i di truyn c cc tnh trng chng chu stress, chng chu cht dit c v khng cn trng. Hn na, cng ngh DNA ti t hp c th c ng dng pht trin cc vi sinh vt c bin i di truyn (genetically modification) sao cho chng c th sn xut cc hp cht ha hc khc nhau vi sn lng cao hn cc vi sinh vt bnh thng.


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Bng 1.1. Cc ng dng wWw.SinhHoc.edu.vn Tai lieu chia se tai: ca cng ngh sinh hc hin i.

Lnh vc Dc phm

Cc sn phm hoc cc ng dng Khng sinh, khng nguyn (kch thch cc p ng khng th), endorphin (cht dn truyn thn kinh), globulin (ngn cn s vim nhim), hormone sinh trng ngi (iu tr tr em b bnh ci), albumin huyt thanh ngi (iu tr chn thng c th), cc nhn t iu ha min dch, insulin, interferon (iu tr bnh vim nhim), interleukin (iu tr cc bnh nhim trng v ung th), lymphokine (phn ng min dch iu chnh), khng th n dng (chn on hoc phn phi thuc), peptide hot ha thn kinh (bt chc cc peptide iu khin s au ca c th), cc nhn t hot ha plasminogen ca m (ha tan cc cc mu ng), vaccine. Pht trin cc con ging sch bnh v mnh kho hn, cc gia sc cho tht c sn lng cao hn. Chuyn cc tnh trng chng chu stress, khng cn trng v cht dit c vo cc loi cy trng, pht trin cc ging cy trng c kh nng tng qu trnh quang hp v c nh m, pht trin cc thuc tr su sinh hc v cc vi khun nhn khng ng bng (non-ice nucleating). Cc amino acid, enzyme, vitamin, lipid, cc cht thm c hydroxyl ha, cc polymer sinh hc. Ngm chit khong, c c kim loi, kim sot s nhim, phn hy cht thi c v thu hi du loang. Acetic acid, acetone, butanol, ethanol, nhiu sn phm khc t cc qu trnh bin i sinh khi. Biosensor, biochip.

Chn nui-Th y Trng trt

Cc ha cht c bit Cc ng dng mi trng Cc ha cht thng mi in t sinh hc

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II. Tai lieu t bo Cng ngh chia

se tai: wWw.SinhHoc.edu.vn

Cc cng ngh DNA ti t hp hoc dung hp t bo c khi u bi nhng nghin cu thun ty v cc kt qu cui cng c th pht trin thnh mt loi t bo mi c th sn xut sn phm vi s lng t i qui m phng th nghim. Tuy nhin, cc kt qu ni trn li rt c ngha thng mi v v th n i hi phi pht trin thnh quy trnh cng nghip vi mt cng ngh kh thi v c hiu qu kinh t. pht trin mt qu trnh sn xut quy m phng th nghim thnh mt quy trnh cng nghip ln, chng ta khng th ch n thun tng kch thc ca bnh nui cy (vessel) ln. V d: quy m phng th nghim l 100 mL, mt bnh tam gic nh nui trn mt my lc l phng thc l tng nui cy t bo. Nhng i vi hot ng quy m ln 2.000 L, chng ta khng th s dng mt bnh nui khc c th tch ln hn v lc n, m cn phi thit k mt h ln men (fermenter) hay cn gi l ni phn ng sinh hc (bioreactor) hiu qu nui cy t bo trong nhng iu kin ti u nht. V th, cng ngh t bo (mt trong nhng lnh vc chnh ca cng ngh sinh hc) c vai tr rt quan trng trong thng mi ha cc sn phm ca n. minh ha vai tr ca cng ngh t bo, c th xem mt qu trnh sinh hc c trng bao gm cc t bo vi khun nh trnh by hnh 1.1. Cc nguyn liu th (thng l sinh khi) c x l v trn vi cc thnh phn cn thit khc t bo c th sinh trng tt trong mt hn hp dch lng, mi trng nui cy c kh trng loi b tt c cc c th sng v a vo bnh nui cy hnh tr ln, thit b c trng vi cnh khuy, vch ngn, h thng thng kh v cc b phn cm bin iu chnh cc iu kin ln men. Mt chng vi sinh vt thun khit c a vo trong mt bnh nui cy. Cc t bo khi u sinh sn theo hm m sau mt thi gian nht nh ca pha lag v t ti nng t bo cc i khi mi trng b s dng ht. S ln men s dng li v cc thnh phn s c ht ra thu hi sn phm v tinh sch chng. Qu trnh ny c hot ng theo kiu ln men m (batch culture) hoc lin tc (continuous culture). Khi tin hnh mt qu trnh sinh hc (bioprocessing) trn quy m ln cn lu : - Phi thu c cc cht xc tc sinh hc tt nht (vi sinh vt, t bo ng vt, t bo thc vt, hoc enzyme) cho mt qu trnh mong mun.

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cc bioreactor/fermenter thch hp v cho n hot ng trong mt phng thc ti u. - Phn tch cc sn phm mong mun t hn hp phn ng trong mt phng thc kinh t nht.

- To ra mi trng tt nht wWw.SinhHoc.edu.vn Tai lieu chia se tai: c th cho s xc tc bng cch thit k

Cc nhim v t ra bao gm thit k v pht trin mt qu trnh sinh hc. Cc vn c bn c i hi cho cng vic ny nh sau:

Nui cy stock

Nguyn liu th

Nui cy lc H ln men kt ht

Chun b mi trng

Kh trng

H ln men sn xut

Khng kh

Thu hi Tinh sch X l nc thi Cc sn phm

Hnh 1.1. Mt qu trnh sinh hc c trng.

1. Chng ta mong i thay i ci g tr li cu hi ny, cn phi c nhng hiu bit v cc khoa hc c bn ca qu trnh cng ngh. l vi sinh vt hc, ha sinh hc, di truyn hc, sinh hc phn t... Chng ta cn phi tm hiu cc vn ny trong mt phm vi nht nh. iu quan trng y l cc cht xc tc sinh hc c chn lc hoc sa i di truyn phi thch hp cho cc hot ng sn xut quy m ln.Cng ngh t bo


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2. Qu trnh sinh hc xy ra vi mt tc nh th no


Nu mt qu trnh nht nh c th sn xut mt sn phm, th iu quan trng cn bit l qu trnh s xy ra vi tc nh th no. ng hc ca qu trnh s chi phi cc tc phn ng di nh hng ca cc iu kin vt l v ha hc nht nh. Chng ta cn nm vng ha ng hc (chemical kinetics) thit k ni phn ng (reactor) thch hp. Cc k thut tng t c ng dng gii quyt ng hc enzyme (enzyme kinetics) hoc ng hc t bo (cell kinetics). thit k mt h ln men hiu qu cho cc cht xc tc sinh hc hot ng, iu quan trng cn bit l tc phn ng b nh hng nh th no bi cc iu kin hot ng khng ging nhau. iu ny bao gm c nghin cu v nhit ng hc (thermodynamics), cc hin tng vn chuyn, cc tng tc sinh hc, kh nng n nh ca cc dng t bo vi sinh vt (hoc t bo ng vt v thc vt) dng lm nguyn liu sn xut... 3. H thng c hot ng v iu chnh nh th no t c hiu sut ti a s hot ng v iu chnh h thng c ti u, chng ta cn phi pht trin cc b cm bin trc tuyn (on-line sensor) chnh xc. Thut ton ti u trc tuyn cn c xy dng v ti u ha tng cng kh nng hot ng ca cc qu trnh sinh hc v m bo rng nhng qu trnh ny c hot ng mt cch kinh t nht. 4. Cc sn phm c phn tch nh th no c c s tinh sch cc i v gi thnh ti thiu i vi bc ny, qu trnh bio-downstream (phn tch sinh hc), chng ta c th s dng cc k thut phn tch khc nhau c pht trin trong cc qu trnh ha hc nh chng ct, hp th, tch chit, hp ph, sy kh, lc, kt ta v ngm chit. Hn na, song song vi cc k thut phn tch tiu chun ny, chng ta cn thit pht trin cc k thut mi thch hp phn tch cc nguyn liu sinh hc. Nhiu k thut c pht trin phn tch hoc phn tch cc nguyn liu sinh hc quy m phng th nghim, nh l sc k (chromatography), in di (electrophoresis) v thm tch (dialysis). Cc k thut ny cn c nghin cu thm sao cho chng c th hot ng hiu qu trn quy m cng nghip.

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III. Qu lieu sinh hc Tai trnh chia se

tai: wWw.SinhHoc.edu.vn

Cc ng dng cng nghip ca cc qu trnh sinh hc l s dng cc t bo sng hoc thnh phn ca chng thc hin nhng thay i vt l v ha hc. So vi cc qu trnh ha hc truyn thng, cc qu trnh sinh hc c nhng u im v nhc im nh sau: 1. Cc u im - iu kin phn ng nh nhng. iu kin phn ng cho cc qu trnh sinh hc l nh nhng-n ha. c trng l nhit phng, p sut kh quyn v pH mi trng kh trung tnh. Kt qu, s hot ng t nguy him v iu kin sn xut t phc tp hn so vi cc qu trnh ha hc c bit. - Tnh c hiu. Mt cht xc tc enzyme c tnh c hiu cao v xc tc ch mt hoc mt s t cc phn ng ha hc. S a dng ca cc enzyme hin c c th xc tc cho mt phm vi rt rng cc phn ng khc nhau. - Tnh hiu lc. Tc ca mt phn ng c xc tc bng enzyme thng nhanh hn nhiu so vi khi phn ng ny thc hin nh cc cht xc tc khng phi sinh hc. Ch mt lng nh enzyme c yu cu cng sn xut mt hiu qu mong mun. - Cc ti nguyn c th i mi. Nguyn liu th ch yu ca cc qu trnh sinh hc l sinh khi (biomass) cung cp c b khung carbon ln nng lng cn cho s tng hp cc ha cht hu c. - Cng ngh DNA ti t hp. L nhng k thut sa i h thng di truyn nhm nng cao nng sut sinh hc. S pht trin ca nhng k thut ny ha hn cc kh nng khng l ci thin cc qu trnh sinh hc. 2. Cc nhc im - Cc hn hp sn phm phc tp. Trong cc trng hp nui cy t bo (vi sinh vt, thc vt hoc ng vt). Cc phn ng a enzyme xy ra trong mt chui tun t hoc song song, hn hp sn phm cui cng cha khi lng t bo, nhiu sn phm trao i cht ph, v mt phn cn li ca cc cht dinh dng ban u. Khi lng t bo cng cha cc thnh phn khc nhau ca t bo. - Cc mi trng nc long. Cc thnh phn c gi tr thng mi ch c sn xut vi mt lng nh trong mi trng nc nn s phnCng ngh t bo


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tch chng l rt t tin. Bi v cc sn phm ca cc qu trnh sinh hc thng mn cm vi nhit, do cc k thut phn tch truyn thng khng th s dng m phi pht trin cc k thut phn tch mi cho cc mc ch sn xut trn quy m ln. - S nhim bn. H thng ln men c th d dng b nhim bn, do nhiu vi khun v nm mc c th sinh trng rt mnh trong hu ht cc mi trng nui cy. Vn tr nn kh khn hn khi nui cy t bo ng vt v thc vt bi v chng cn mt thi gian sinh trng di ngy v tc sinh trng ca chng chm hn rt nhiu so vi tc sinh trng ca vi khun v nm mc trong mi trng nhim bn. - Khuynh hng hay bin i. Cc t bo c khuynh hng t bin do s thay i mi trng v c th mt i mt vi c im gy thit hi cho s thnh cng ca qu trnh sn xut. Cc enzyme tng i mn cm hoc l cc phn t khng n nh v i hi s cn thn trong khi s dng chng.


IV. nh ngha s ln menThng thng, s ln men (fermentation) c nh ngha l qu trnh sn xut ethanol hoc lactic acid t glucose (C6H12O6). - Qu trnh sn xut ethanol. L qu trnh m mt s nm men phn gii cc loi ng trong mi trng ym kh sn xut ru ethanol. - Qu trnh sn xut lactic acid. L qu trnh m mt s enzyme nh lactodehydrogenase phn gii cc cht trung gian nh NADH (trong ng phn ym kh) thnh lactic acid ch khng thnh ethanol. Ln men lactic c dng trong cng ngh ch bin sa lm phomt v sa chua.nm men

C6H12O6cc enzyme

2C2H5OH + 2CO2

C6H12O6

2CH3CHOHCOOH

Tuy nhin, ngy nay ngi ta m rng nh ngha cho khi nim ny nh sau: Ln men l qu trnh s dng cc enzyme bin i nhng hp cht hu c theo Websters New College Dictionary (A Merriam-WebsterCng ngh t bo


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1977) v y l nh ngha m chng ti s dng trong gio trnh ny dng m t cc qu trnh nui cy cc t bo vi sinh vt, ng vt v thc vt trong cc h ln men hay cc ni phn ng sinh hc.


Ti liu tham kho/c thm1. Atkinson B and Mavituna F. 1991. Biochemical Engineering and Biotechnology Handbook. 2nd ed. Stockton Press, New York, USA. 2. Flickinger MC and Drew SW. 1999. Encyclopedia of Bioprocess Technology: Fermentation, Biocatalysis and Bioseparation. John Wiley & Sons, New York, USA. 3. Lee JM. 2001. Biochemical Engineering. Prentice Hall, Inc. USA. 4. Ratledge C and Kristiansen B. 2002. Basic Biotechnology. Cambridge University Press, UK. 5. Shuler ML and Kargi F. 2002. Bioprocess Engineering-Basic Concepts. 2nd ed. Prentice Hall, Inc. New Jersey, USA.

Cng ngh t bo

Tai lieu chia se tai: wWw.SinhHoc.edu.vn 10

Tai 10 Chnglieu

chia se tai: wWw.SinhHoc.edu.vnKhuy trn v thng kh

I. M uMt trong nhng nhn t quan trng cn c lu khi thit k h ln men l kh nng khuy trn thch hp cc thnh phn ca n. Cc vn chnh ca s khuy trn trong h ln men l s phn tn ca cc bong bng kh, to huyn ph cc c th vi sinh vt (hoc t bo thc vt v ng vt) v tng cng s chuyn nhit v chuyn khi trong mi trng. Ni chung, hu ht cc cht dinh dng u c kh nng ha tan cao trong nc, do trong thi gian ln men nu ch phn b u mi trng khi cc t bo tiu th cht dinh dng th s khuy trn khng tht cn thit. Tuy nhin, trng hp oxygen ha tan th ngi ta li rt mong mun c mt s khuy trn tt v kh nng ha tan ca n trong mi trng ln men l rt km, trong khi yu cu oxygen cho s sinh trng ca cc vi sinh vt hiu kh (hoc t bo thc vt v ng vt) li rt cao. V d: khi oxygen c cung cp t khng kh, nng cc i c trng ca n trong dung dch nc l t 6-8 mg/L. Nhu cu oxygen ca t bo, mc d c th ph thuc rt ln vo loi t bo, thng l khong 1 g/L gi. Ngay c khi mi trng ln men c bo ha hon ton vi oxygen, th oxygen ha tan s c c th tiu th t hn mt cht nu nh n khng c cung cp lin tc. quy m phng th nghim, s khuy trn c to ra nh my lc (shaker) l thch hp nui cy t bo trong cc bnh thy tinh hoc ng nghim. Cc my lc vng hoc lc ngang to ra mt s phi trn nh v trao i kh b mt rt hiu qu. Trng hp ln men quy m pilot hoc quy m sn xut, s khuy trn thng c to ra bng cch khuy c hc c hoc khng c sc kh. Ph bin nht l s dng loi cnh khuy (impeller) to ra dng chy ta trn vi su cnh khuy mng c gn vo trong mt a, gi l turbine a c cnh khuy mng (flat-blade disk turbine) hoc Rushton turbine (Hnh 10.1 v 10.2). Cc cnh khuy dng ta trn (cc mi cho v turbine) to ra dng chy ta trn t cnh ca turbine hng ti vch ngn ca bnh nui (vessel), trong dng chy chia ra theo hai hng: mt hng i ln dcCng ngh t bo

Tai lieu chia se tai: wWw.SinhHoc.edu.vn169

theo vch, ri i tr vo vng trung tm theo b mt cht lng, v i xung vng cnh khuy dc theo trc khuy. Mt hng khc i xung dc theo vch v y, sau i vo vng cnh khuy.


Hnh 10.1. S Rushton turbine.

4 x vch ngn

Rushton turbine B phn phun kh

Hnh 10.2. S bnh ln men c cnh khuy.

Mt khc, cc cnh khuy dng chy theo trc (cnh qut v cc mi cho khng bng phng) to ra dng chy i xung y bnh, sau i ln dc theo vch v quay xung vng trung tm ca cnh khuy. V th, cc turbine a c cnh khuy mng c u im hn ch on mch (short-

Cng ngh t bo


circuiting) ca kh dc theo trc truyn ng (drive shaft) nh s nn kh, a vo t pha di, dc theo hng vo trong vi thot (discharge jet). 1. Con ng chuyn khi Con ng ca cc cht kh t mt bong bng vo mt c quan t trong t bo c th c phn chia trong mt vi bc nh sau: a. Chuyn t kh nn (bulk gas) trong mt bong bng ti mt lp kh tng i nguyn cht (relatively unmixed gas layer). b. Khuch tn thng qua lp kh tng i nguyn cht. c. Khuch tn thng qua lp cht lng tng i nguyn cht quanh bong bng. d. Chuyn t lp cht lng tng i nguyn cht ti khi cht lng nn (bulk liquid). e. Chuyn t khi cht lng nn ti mt lp cht lng tng i nguyn cht quanh mt t bo. f. Khuch tn thng qua lp cht lng tng i nguyn cht. g. Khuch tn t b mt ca mt t bo ti mt c quan t m trong oxygen b tiu hao. Cc bc c v e l chm nht. S khuy trn v thng kh s tng cng tc chuyn khi trong cc bc ny v tng din tch tng tc gia kh v cht lng. Chng ny trnh by mt s mi tng quan khc nhau i vi s chuyn khi lng-kh, din tch tng tc, kch thc bong bng, s tc nghn kh, s tiu th cng sut khuy v h s th tch chuyn khi, l nhng cng c quan trng thit k v hot ng cc h ln men. S ti hn i vi vic tng quy m sn xut v s khuy trn nhy cm vi lc trt cng c trnh by. u tin, chng ta tm hiu cc khi nim c bn ca s chuyn khi m quan trng l hiu c s chuyn khi lng-kh trong h ln men.


II. Cc khi nim c bn v chuyn khi1. S khuch tn phn t trong cht lng Khi nng ca mt thnh phn bin thin t mt im ny n mt im khc, th thnh phn ny c xu hng chy theo hng lm gim nhng s khc bit cc b trong nng .Cng ngh t bo


Tai lieu chia se t A lin quan vi vn tc phn t trung bnh Dng phn t ca cu tai: wWw.SinhHoc.edu.vnca tt c cu t JA l t l vi gradient nng dC A / dz khi:

J A = D AB

dC A dz

(10.1)

Phng trnh (10.1) l nh lut th nht ca Fick c vit cho chiu z. K hiu DAB trong phng trnh (10.1) biu din kh nng khuch tn cu t A vo B, tc l gi tr o chuyn ng khuch tn ca n. Dng phn t ca A lin quan vi ta c nh (stationary coordinate) NA l bng:

NA =

CA dC A ( N A + N B ) D AB C dz

(10.2)

Trong : C l nng tng s ca cc cu t A v B, v NB l dng phn t ca B lin quan vi ta c nh. i vi dung dch long ca cu t A th:NA JA (10.3)

1.1. S khuch tn L thuyt ng hc cht lng khng c nhiu u im so vi cht kh. V th, mi tng quan cho kh nng khuch tn trong cht lng l khng r rt nh trong cc cht kh. Trong s nhng mi tng quan c cp, th tng quan Wilke-Chang (1955) c s dng rng ri nht cho cc dung dch long ca cc cht khng in phn: Do AB

1,173 10 16 (M B ) 0,5 T = 0 VbA, 6

(10.4)

Khi cc dung mi l nc, Skelland (1974) gii thiu s dng mi tng quan c pht trin bi Othmer v Thakar (1953): Do AB

1,112 10 13 = 0 1,1VbA, 6

(10.5)

Cng ngh t bo


Tai lieu chia se tai: wWw.SinhHoc.edu.vnth Hai mi tng quan cho trc khng ph hp v th nguyn, vcc phng trnh s dng n v SI nh sau:o DAB kh nng khuch tn ca A trong B, trong mt dung dch rt

long, m2/s MB khi lng phn t ca cu t B, kg/kmol T nhit , oK tc ha tan, kg/m/s

VbA th tch phn t ha tan im si bnh thng, m3/kmol (0,0256 m3/kmol cho oxygen) yu t kt hp i vi dung mi: 2,26 i vi nc; 1,9 i vi methanol; 1,5 i vi ethanol; 1,0 cc dung mi khng kt hp nh benzene v ethyl ether. 2. H s chuyn khi Dng chy khi (mass flux), tc chuyn khi qG trn n v din tch, t l vi s chnh lch nng . Nu mt cht ha tan chuyn t pha kh vo pha lng, th dng chy khi ca n t pha kh ti b mt chung NG l:NG = qG = kG (CG CGi ) A(10.6)

Trong : C G v CGi l nng kh mt bin (gas-side concentration) tng ng phn chnh v vng phn gii (b mt chung) (Hnh 10.3). kG l h s chuyn khi ring r cho cho pha kh v A l din tch vng phn gii. Tng t, dng chy khi ca pha lng mt bin (liquid-side phase) NL l:NL = qL = k L (C Li C L ) A(10.7)

Trong : kL l h s chuyn khi ring r i vi pha lng, qL l tc hp th kh. Do lng cht ha tan c chuyn t pha kh ti vng phn gii phi bng lng cht ha tan t vng phn gii ti pha lng, nn:

Cng ngh t bo


Tai lieu chia seN tai: wWw.SinhHoc.edu.vn (10.8) =NG L

Kh

Lng

CG C Gi

C Li

CG C GiCLCL

k L / kG

C Li

Hnh 10.3. Profile nng gn vng phn gii kh-lng v mt ng cong trng thi cn bng.

Thay phng trnh (10.6) v (10.7) vo trong phng trnh (10.8) ta c:

CG CGi C L C Li

=

kL kG

(10.9)

Phng trnh (10.9) c dc ca ng cong kt ni ( C L , C G ) v (C Li , C Gi ) nh trnh by hnh 10.3. S dng phng trnh (10.6) hoc (10.7) xc nh h s chuyn khi gp nhiu kh khn do chng ta khng th o nng ca vng phn gii C Li hoc C Gi . V th, thun li cho vic xc nh ton b h s chuyn khi c th dng phng trnh sau:* * N G = N L = K G (C G C G ) = K L (C L C L )

(10.10)

* Trong : C G l nng kh mt bin s cn bng vi nng kh

* hin din trong pha lng. Tng t, C L l nng cht lng mt bin s

cn bng vi nng cht lng hin din trong pha kh. Nhng thng s ny d dng c t ng cong trng thi cn bng trnh by hnh 10.4. KG v KL c nh ngha li l cc h s chuyn khi ton b tng ng cho cc mt bin ca kh v lng.

Cng ngh t bo


Tai lieu chia se tai: wWw.SinhHoc.edu.vnCG CGi* CG

Hnh 10.4. ng cong trng thi cn bng gii thch * * ngha ca C G v C L .

CL3. C ch ca chuyn khi

* CLi C L

Mt vi c ch khc nhau c a ra cung cp c s cho l thuyt chuyn khi gian k (interphase). Ba c ch tt nht c bit l: thuyt hai mng (two-film), thuyt thm qua (penetration) v thuyt phc hi b mt (surface renewal). 3.1. Thuyt hai mng Thuyt ny gi thit rng c tnh kh di chuyn hon ton c bao gm trong hai mng gi bn ny hoc bn kia vng phn gii, trong s di chuyn xy ra nh khuch tn phn t. M hnh ny dn n kt lun rng h s chuyn khi kL t l vi kh nng khuch tn DAB v t l nghch vi dy ca mng zf nh sau: kL = D AB zf(10.11)

3.2. Thuyt thm qua Thuyt ny tha nhn rng xoy nc hn lon i t phn chnh ca pha ti vng phn gii, chng duy tr mt thi gian phi khng i te. Cht ha tan c tha nhn l thm vo trong xoy nc c sn vng phn gii bi mt qu trnh khuch tn phn t trng thi khng n nh. M hnh ny d bo rng h s chuyn khi t l trc tip vi cn bc hai ca kh nng khuch tn phn t:

Cng ngh t bo


Tai lieu chia se tai: (10.12) D wWw.SinhHoc.edu.vn1/ 2

k L = 2 t e AB

Trong : l p sut tuyt i. 3.3. Thuyt phc hi b mt Thuyt ny xut rng c mt gii hn thi gian v tn cho cc nhn t b mt v hm phn b tui b mt (surface age). L thuyt ny d bo mt ln na h s chuyn khi t l vi cn bc hai ca kh nng khuch tn phn t:

k L = ( sD AB )1 / 2

(10.13)

Trong : s l tc phn on ca s phc hi b mt. Tt c l thuyt ni trn i hi phi bit mt s thng s cha xc nh nh: dy mng c tht zf, thi gian phi te hoc tc phn on ca s phc hi b mt s. Ni chung, nhng tnh cht ny t c bit n, n mc c ba l thuyt l khng hon chnh. Tuy nhin, nhng l thuyt ny gip chng ta hnh dung c ch chuyn khi vng phn gii v cng bit s ph thuc hm m ca kh nng khuch tn phn t trn h s chuyn khi.

III. Xc nh vng phn gii tnh ton tc hp th kh qL ca phng trnh 10.7, chng ta cn bit din tch vng phn gii kh-lng l thng s c th o c bng cch ng dng mt vi k thut nh l chp nh, truyn sng v quang ph laser. Din tch vng phn gii (a) trn mt n v th tch c th c tnh ton t ng knh trung bnh Sauter D32 (m) v phn on th tch ca pha kh H, nh sau:a= 6H D32(10.14)

ng knh trung bnh Sauter, mt gi tr trung bnh ca b mt th tch, c th c tnh ton bng cch o cc kch thc git trc tip t cc hnh nh ca phn tn theo phng trnh sau:

Cng ngh t bo


Tai lieu chia se tai: wWw.SinhHoc.edu.vn (10.15)D32 =

n Di =1 n i i =1 i

n

3 i

n D

2 i

Xc nh kch thc cc git bng hnh nh l phng php d lm trong s nhiu k thut xc nh do n khng i hi s nh c trc (calibration). Tuy nhin, chp mt bc nh r rng c th l rt kh khn v c cc bc nh ny l mt cng vic n iu t nht, tn nhiu thi gian. Cc bc nh c th chp thng qua chn hoc thnh bn ca bnh ln men. loi b tnh trng khng r rng do b mt b cong ca thnh bnh, bnh ln men c th c cho ngp chm trong mt ci thng hnh ch nht hoc mt ti nc c gn trn thnh. Nhc im ca phng thc ny l vic o kch thc git b hn ch i vi nhng vng gn thnh bnh, l ni khng th i din cho ton b s phn tn trong h ln men. S phn b kch thc git c th c o gin tip bng cch dng k thut truyn sng. Khi mt chm sng i qua mt vng c phn tn kh-lng, th nh sng c ta ra bi cc bong bng kh. Ngi ta nhn thy rng th ca t l dp tt (hm thun nghch ca truyn sng 1/T) da theo din tch vng phn gii trn mt n v th tch ca phn tn a, to ra mt ng thng, nh sau:1 = m1 + m 2 a T(10.16)

V l thuyt, m1 l phn t n v, cn m2 l mt hng s c lp ca s phn b kch thc git vi iu kin l tt c cc bong bng kh gn nh hnh cu. K thut truyn sng c s dng thng xuyn nht cho vic xc nh kch thc trung bnh ca bong bng kh trong s phn tn kh-lng. K thut ny c mt s u im nh o nhanh v hot ng trc tuyn.

IV. Tc nghn khTc nghn kh l mt trong nhng thng s quan trng nht m t thy ng hc ca h ln men. Tc nghn kh ty thuc ch yu vo vn tc b mt ca kh v s tiu th cng sut, v thng l rt nhy cm vi ccCng ngh t bo


tnh cht vt l ca cht lng. Tc nghn kh c th c xc nh d dng bng cch o mc cht lng c thng kh trong sut thi gian hot ng (ZF) v mc cht lng sch (ZL). Nh vy, vic tc nghn kh trung bnh tiu phn H c tnh theo cng thc sau:


H=

ZF ZL ZF

(10.17)

1. Phun kh (sparging) bng khuy trn khng c hci vi mt h thng hai pha, trong pha lin tc duy tr ch thch hp ca n, th s tc nghn kh s lin quan vi vn tc kh b mt Vs v vn tc tng bong bng kh Vt:

H=

Vs Vs + Vt

(10.18)

Akita v Yoshida (1973) t mi tng quan tc nghn kh i vi vic hp th oxygen cc dung dch nc khc nhau trong ct bong bng nh sau:2 gDC c H = 0,20 (1 H ) 4

1/ 8

3 gDC 2 v c

1 / 12

Vs gD C

(10.19)

Trong : g l gia tc do trng lc, Dc l ng knh ct bong bng, v l p lc b mt, c l th tch cht lng ca pha lin tc, v c l mt ca pha lin tc.

2. Phun kh bng khuy trn c hcCalderbank (1958) t mi tng quan tc nghn kh i vi vic phn tn lng-kh c khuy trn bng turbine dng a c cnh khuy mng nh sau:

V H H = s V t

1/ 2

( P / v) 0, 4 c 0 , 2 Vs + (2,16 10 ) m 0, 6 Vt 4

1/ 2

(10.20)

Trong 2,610-4 c n v (m) v Vt = 0,265 m/s khi kch thc bong bng trong khong 2-5 mm ng knh, Pm l cng sut b tiu haoCng ngh t bo


do cnh khuy trong s phn tn cht lng c thng kh, v v l th tch cht lng. Trng hp vn tc kh b mt cao (Vs > 0,02 m/s), thay Pm v Vt ca phng trnh (10.20) bng cch a vo cng sut hiu qu Pe v Vt + Vs tng ng.


V. Xc nh tc hp th oxygen c lng cc thng s thit k a oxygen vo h ln men, chng ta c th s dng cc mi tng quan c trnh by trong cc phn trc y, ng dng cho mt phm vi rng cc h thng kh-lng b sung vo h thng nc-khng kh. Tuy nhin, phng thc tnh ton ny di dng v cc gi tr d bo t nhng mi tng quan ny c th thay i rt nhiu. Cng c trng hp chng ta cng khng th tm thy cc mi tng quan thch hp p dng cho kiu v th tch ca h ln men mun s dng. Trong nhng trng hp nh th, chng ta c th t o tc chuyn oxygen hoc dng cc mi tng quan da trn nhng th nghim ny. Tc hp th oxygen trn mt n v th tch qa/v c th c c lng nh phng trnh:

qa * * = K L a(C L C L ) = k L a (C L C L ) v

(10.21)

Do oxygen l loi kh t ha tan, nn h s chuyn khi ton b KL bng h s chuyn khi ring r kL. Mc tiu ca chng ta trong thit k h ln men l cc i ha tc chuyn oxygen vi s tiu th cng sut ti thiu cn thit khuy trn cht lng v cng gim thiu lu tc kh. cc i ha tc hp th oxygen, chng ta phi cc i ha kL, a, * C L C L . Tuy nhin, s khc bit nng c hn ch hon ton bi v * gi tr C L c gii hn bi kh nng ha tan cc i rt thp ca n. V th, cc thng s quan tm chnh trong thit k l h s chuyn khi v din tch vng phn gii. Bng 10.1 lit k kh nng ha tan ca oxygen 1 atm trong nc di cc nhit khc nhau. Cc gi tr thu c l nng oxygen ccCng ngh t bo


i trong nc khi n trong s cn bng vi oxygen tinh khit. Kh nng ha tan ny gim khi c b sung acid hoc mui nh trnh by bng 10.2.Bng 10.1. Kh nng ha tan oxygen trong nc 1 atm. Nhit ( C) 0 10 15 20 25 30 35 40o


Kh nng ha tan mmol O2/L 2,18 1,70 1,54 1,38 1,26 1,16 1,09 1,03 mg O2/L 69,8 54,5 49,3 44,2 40,3 37,1 34,9 3,0

Bng 10.2. Kh nng ha tan ca oxygen trong dung dch mui hoc acid 25oC. Nng (mol/L) 0,0 0,5 1,0 2,0 Kh nng ha tan (mmol O2/L) HCl 1,26 1,21 1,16 1,12 H2SO4 1,26 1,21 1,12 1,02 NaCl 1,26 1,07 0,89 0,71

Thng thng, chng ta s dng khng kh cung cp nhu cu oxygen cho h ln men. Nng cc i ca oxgen trong nc trong * s cn bng vi C L khng kh p sut kh quyn l khong mt phn mi lm ca kh nng ha tan c lit k, theo nh lut Henry:

Cng ngh t bo


Tai lieu chia se tai: wWw.SinhHoc.edu.vn p* CL =O2

H O2 (T )

(10.22)

Trong : p O2 l p sut tng phn (partial pressure) ca oxygen vH O2 (T ) l hng s oxygen ca nh lut Henry nhit T. Gi tr ca

hng s nh lut Henry c th thu c t cc kh nng ha tan c * lit k bng 10.2. V d: 25oC, C L l 1,26 mmol/L v p O2 l 1 atm do n l oxygen tinh khit. Bng cch thay th cc gi tr ny vo trong phng trnh (10.22), chng ta thu c H O2 (T ) l 0,793 atm L/mmol. V th, nng oxygen cn bng cho s tip xc nc-kh 25oC s l:* CL =

0,209 atm = 0,264 mmol O 2 /L = 8,43 mg/L 0,793 atm L/mmol

Theo iu kin l tng, tc chuyn oxygen phi c o trong h ln men cha mi trng dinh dng v t bo trong sut qu trnh ln men thc t. Tuy nhin, iu ny kh tin hnh do bn cht phc tp ca mi trng v s thay i lu bin hc (rheology) trong sut qu trnh sinh trng ca t bo. Phng thc chung l s dng mt h thng tng hp xp x nh cc iu kin ca qu trnh ln men.

1. Phng php oxy ha sodium sulfite Phng php oxy ha sodium sulfite da trn nguyn tc oxy ha sodium sulfite thnh sodium sulfate vi s c mt ca cht xc tc (Cu2+ hoc Co2+) nh sau:Cu2+ hoc Co2+

Na2SO3 + 1/2O2

Na2SO4

(10.23)

Phn ng ny c cc c im sau, p ng cho vic o tc chuyn oxygen: - Tc phn ng c lp vi nng ca sodium sulfite trong khong 0,04 n 1 N. - Tc phn ng nhanh hn nhiu so vi tc chuyn oxygen. V th tc oxy ha c iu chnh ch bi tc chuyn khi.Cng ngh t bo


bng dung dch sodium sulfite 1 N cha t nht 0,003 M Cu2+. M b phn sc v bt u bm gi khi bong bng kh u tin xut hin trong h ln men t b phn sc kh. Cho php s oxy ha tip tc t 4-20 pht, sau dng dng kh, b phn khuy trn v timer cng mt thi gian, ri ly mu. Trn mi mu vi mt lng d thuc th iodine chun bng pipette sch. Chun bng dung dch sodium thiosulfate chun (Na2S2O3) ti im cui ca cht ch th tinh bt. Mt khi oxygen a vo c o, th h s th tch chuyn khi kLa c th c tnh ton bng cch dng phng * trnh (10.21), trong CL l bng 0 v C L l nng oxygen trng thi cn bng. K thut oxy ha sodium sulfite c hn ch ca n v trong thc t dung dch khng th xp x vi cc tnh cht vt l v ha hc ca mi trng ln men. Thm mt vn na l k thut ny i hi cc nng ion cao (1-2 mol/L), s c mt ca cc ion ny c th nh hng n din tch vng phn gii v, trong mt mc thp hn, n h s chuyn khi. Tuy nhin, k thut ny hu ch khi so snh vi hiu sut ca cc h ln men v nghin cu nh hng ca s pht trin quy m sn phm v cc iu kin hot ng.

Tai o tc chia se oxygenwWw.SinhHoc.edu.vn lieu chuyn tai: trong h ln men, lm y h ln men

2. K thut tch khng khK thut ny gim st s thay i nng oxygen trong mt cht lng giu oxygen c kh oxygen bng cch cho nitrogen i qua n. in cc ca php o cc ph (polarography) thng c dng o nng . Cn bng khi trong mt bnh nui cho ra:

dC L (t ) * = k L a C L C L (t ) dt

[

]

(10.24)

Ly tch phn phng trnh cho gia t1 v t2 cho kt qu:* C L C L (t1 ) ln * C L C L (t 2 ) kLa = t 2 t1

(10.25)

Cng ngh t bo


Tai phngchiatrn k tai: th c tnh ton da trn cc gi tr o T lieu trnh se a c wWw.SinhHoc.edu.vnL

c CL(t1) v CL(t2). 3. Xc nh trc tip Trong k thut ny, chng ta o trc tip hm lng oxygen ca dng kh i vo v i ra khi h ln men bng cch s dng thit b phn tch oxygen khng kh. S hp th oxygen c th c tnh ton nh sau:qa = Qin CO 2 , in Qout CO 2 , out(10.26)

Trong : Q l tc dng kh. Mt khi oxygen hp th c o, th kLa c th c tnh ton bng cch dng phng trnh (10.21), trong CL l nng oxygen ca cht * lng trong h ln men v CL l nng ca oxygen s trng thi cn bng vi dng kh. Nng oxygen ca cht lng trong h ln men c th c o bng mt b cm bin oxygen trc tuyn (on-line oxygen sensor). Nu th tch ca h ln men l kh nh (< 50 L), th s bin thin ca * (CL CL ) trong h ln men cng kh nh. Tuy nhin, nu kch thc ca h ln men l rt ln, th s bin thin c th c ngha. Trong trng hp * ny, gi tr trung bnh logarithm (CL CL ) ca dng kh chy vo v chy ra c th c s dng, khi : (C C L ) LM* L * * (C L C L ) in (C L C L ) out = * * ln (C L C L ) in /(C L C L ) out

[

]

(10.27)

4. K thut ng lc hc Bng cch s dng k thut ng lc hc chng ta c th c lng gi tr kLa i vi s chuyn oxygen trong sut qu trnh ln men thc t vi cc t bo v mi trng nui cy thc s. K thut ny da trn nguyn tc ca s cn bng oxygen ca nguyn liu trong mt h ln men m hiu kh trong lc cc t bo ang hot ng sinh trng khi:dC L * = k L a (CL CL ) rO 2 C X dt(10.28)

Cng ngh t bo


Trong : l tc tai: hp t bo (g O /g t bo gi). Tai lieu rchia se ca hwWw.SinhHoc.edu.vnO2

2

Trong khi nng oxygen ha tan ca h ln men l n nh, nu t nhin chng ta ngt s cung cp khng kh, th nng ca oxygen s b gim (Hnh 10.5) vi tc nh sau:dC L = rO 2 C X dt(10.29)

V kLa trong phng trnh (10.28) l bng 0. V th, bng cch o dc ca ng cong CL theo t, chng ta c th c lng rO 2 C X . Nu chng ta m dng kh thm mt ln na, th nng oxygen ha tan s c tng ln theo phng trnh (10.28), phng trnh ny c th c sp xp li cho mt mi quan h tuyn tnh nh sau:* CL = CL

1 dC L + rO 2 C X k L a dt

(10.30)

Ngt khng kh

CL

dCL dt

Hnh 10.5. K thut ng hc cho vic xc nh kLa.

t

th ca CL theo dc

dC L + rO 2 C X s cho kt qu mt ng thng c dt

1 * v mt phng y ca C L . kLa

VI. Cc k hiuA a din tch vng phn gii, m2 din tch vng phn gii kh-lng trn mt n v th tch ca s phn tn cho cc s Reynolds ca cnh khuy thp, m-1

Cng ngh t bo


C DAB

nng , chia Tai lieu kmol/m se tai: wWw.SinhHoc.edu.vn3

kh nng khuch tn ca cu t A vo B, tc l gi tr o ca chuyn ng khuch tn, m2/s

kh nng khuch tn ca cu t A trong mt dung dch B rt long m2/s JA dng phn t ca cu t A lin quan vi vn tc phn t trung bnh ca tt c cu t, kmol/m2s K h s chuyn khi ton phn, m/s k h s chuyn khi ring r, m/s k La h s th tch chuyn khi g gia tc do trng lc, m/s2 H tiu phn th tch (phn on) ca pha kh trong s phn tn, khng c th nguyn NA, NB dng khi ca A v B lin quan vi ta c nh, kmol/m2s NG, NL dng khi t pha kh ti pha lng v t pha lng n pha kh, tng ng, kmol/m2s P p sut tng s, N/m2 Pe cng sut hiu qu c a vo nh phun kh v khuy c hc, W Pm cng sut b tiu hao do cnh khuy trong s phn tn cht lng c thng kh, W q tc chuyn khi, kmol/s Q tc dng kh, m3/s s tc phn on ca s phc hi b mt, s-1 te thi gian phi cho l thuyt thm qua, s Vs vn tc kh b mt, m/s Vt vn tc tng bong bng kh, m/s v th tch cht lng, m3 vc th tch cht lng ca pha lin tc ZF, ZL mc cht lng c sc kh trong sut thi gian hot ng, m zf dy ca mng trong l thuyt hai mng, m nht, kg/m s

o D AB

p sut tuyt i, N/m2 mt , kg/m3

Cng ngh t bo


c

mt ca pha se tai: Tai lieu chialin tc wWw.SinhHoc.edu.vn p lc b mt kg/s2

Ti liu tham kho/c thm1. Asenjo JA and Merchuk JC. 1995. Bioreactor System Design. Marcel Dekker, Inc. New York, USA. 2. Atkinson B and Mavituna F. 1991. Biochemical Engineering and Biotechnology Handbook. 2nd ed. Stockton Press, New York, USA. 3. Chia TF. 2003. Engineering Applications in Biology. Updated 1st ed. McGraw-Hill Education, Singapore. 4. Flickinger MC and Drew SW. 1999. Encyclopedia of Bioprocess Technology: Fermentation, Biocatalysis and Bioseparation. John Wiley & Sons, New York, USA. 5. Lee JM. 2001. Biochemical Engineering. Prentice Hall, Inc. USA. 6. Shuler ML and Kargi F. 2002. Bioprocess Engineering-Basic Concepts. 2 ed. Prentice Hall, Inc. New Jersey, USA.nd

7. Vogel HC and Todaro CL. 1997. Fermentation and Biochemical Engineering Handbook (Principles, Process Design, and Equipment). 2nd ed. Noyes Publications. New Jersey, USA.

Cng ngh t bo


Tai 2 Chnglieu

chia se tai: wWw.SinhHoc.edu.vn

Sinh trng v bt ng ca t boI. Xc nh sinh trng ca t boTrong cc h thng sinh hc, mi s sinh trng u c th c nh ngha l s tng tun t ca cc thnh phn ha hc. Tng n thun khi lng khng th phn nh y s sinh trng, do t bo c th ch tng hm lng cc sn phm d tr ca chng nh l glycogen, poly-hydroxybutyrite. S sinh trng cn bng (balanced growth) c nh ngha l s sinh trng m trong sut qu trnh s nhn i sinh khi xy ra cng vi s nhn i ca tt c cc c tnh xc nh khc ca qun th nh l protein, DNA, RNA v nc ni bo. Mt khc, qu trnh nui cy tri qua s sinh trng cn bng duy tr mt thnh phn ha hc khng i. Trong mt mi trng dinh dng thch hp m sau t bo s tr nn thch nghi th n s trong trng thi sinh trng cn bng. Tip theo qu trnh sinh trng, cn phi xc nh s lng t bo. S sinh trng ca t bo c th c xc nh bng s lng t bo, sinh khi t bo hoc hot tnh t bo. 1. Xc nh s lng t bo 1.1. m bng knh hin vi S lng t bo trong qun lc c th c m di knh hin vi bng cch m cc t bo c a vo trong mt bung m c bit. C hai loi bung m c dng m s lng t bo trong mt mu dch lng: - Haemocytomete. Bung m t bo mu dng cho nhng t bo c ng knh 3 m (Hnh 2.1). - Petroff-Hausser counting chamber. Bung m Petroff-Hausser c dng ch yu cho vi khun. C hai loi bung m c cc ng k vung c trng trn b mt ca tm knh (lam knh). Khung trn mi mt ca tm m (grid) gi mt tm knh ph (cover glass) cch tm m mt khong cch bit (v d: 0,1 mm) sao cho th tch ca vung c bit chnh xc. Mt mu dch huyn ph t bo cn m c cho chy qua di tm ph v lmCng ngh t bo


y bung m. Sau , m s lng t bo trn mt n v din tch ca ng k di knh hin vi. Cc dch huyn ph m c cng c th m c nu chng c pha long thch hp. Mt s u im ca phng php m trc tip: - Ch cn cc thit b ti thiu. - Cc kt qu thu c nhanh. - C th quan st cc c im hnh thi ca c th.a mu vo


Tm knh ph

su ca mu 0,1 mm Bung m Khung tm knh

Hnh 2.1. Bung m haemocytometer.

Mt s nhc im ca phng php m trc tip: - Thng rt kh phn bit cc t bo cht v t bo sng. - Khng thch hp cho cc dch huyn ph c mt thp. - Cc t bo c kch thc nh thng kh quan st di knh hin vi v c th khng thy khi m. - Phng php m thc t gy mi mt v nhm ln trong qu trnh m. - Khng thch hp i vi cc t bo xp thnh cm nh l mycelium (th si nm). 1.2. m cc t bo pht trin trn a nui cy (petri dish) Cc t bo pht trin c nh ngha l t bo c th phn chia v to ra khun lc. C hai cch thc hin phng php m trn a petri: phng php a tri (spread plate) v phng php a rt (pour plate). Vi phng php a tri, mt th tch nh hn 100 L c tri khp b mt agar. Vi phng php a rt, mu vt c trn vi agar nngCng ngh t bo


chy ( ngui n khong 60oC) v c rt trn a v trng. Cc a sau c nui cho n khi xut hin khun lc, v s lng khun lc c m trc tip. iu quan trng l s lng cc khun lc pht trin trn a phi khng qu ln hoc khng qu nh. thu c mt s lng t bo thch hp trn mt n v din tch th mu vt phi c pha long. Trng hp cn pha long nhiu, ngi ta thng s dng k thut pha long tun t (serial dilution). V d: thc hin pha long 1/106, th c th thc hin ba ln pha long lin tip 1/100 hoc su ln pha long lin tip 1/10. 1.3. m bng my m trnh s n iu khi m trc tip bng knh hin vi, c th s dng phng php m bng my m. K thut ny cho php khng ch m c s lng t bo, m cn o c kch thc t bo. Nhc im ca phng php ny l n khng th phn bit gia t bo v cc phn t bn khc. K thut ny cng kh s dng vi cc c th dng chui v khng em li kt qu tt vi cc c th dng h si (v d nh nm). 2. Xc nh sinh khi t bo 2.1. Trng lng kh ca t bo Trng lng kh t bo c th c xc nh trc tip bng cch ly mt lng ti thiu ca dch huyn ph t bo ly tm. Sau khi th ni, t bo c ra bng nc ct loi b tt c cc cht ha tan. Dch huyn ph c ly tm mt ln na v cc t bo sau khi kt c li c sy kh trong t sy v cn xc nh trng lng. y l phng thc trc tip nht xc nh s lng sinh khi t bo. Tuy nhin, cch xc nh nh th tn nhiu thi gian v d b qua nhng thay i nh ca sinh khi t bo. K thut ny ch c th s dng i vi nhng dch huyn ph dy c v t bo phi c ra sch hon ton khi nhng cht ngoi sinh bm vo. 2.2. c ca dch huyn ph t bo Sinh khi t bo cng c th c xc nh bng phng php quang hc thng qua xc nh lng nh sng b tn x bi dch huyn ph t bo. K thut ny da trn c s lp lun rng cc phn t nh tn x nh sng mt cch tng xng, trong cc gii hn nht nh, ti nng ca chng. Khi tia sng xuyn qua dch huyn ph ca t bo, th lng nh sng truynCng ngh t bo



qua b gim i do kt qu ca s tn x, nh vy chnh l phng php xc nh mt t bo. Vic o c ca dch huyn ph t bo thng c thc hin trn my quang ph c cc n v hp th (A). Kh nng hp th (absorbency) c nh ngha l s logarithm ca t l gia cng nh sng va chm vo dch huyn ph t bo (Io) v cng nh sng c truyn qua bi dch huyn ph (I):


A = log

Io I

ng cong chun (standard curve) c th thu c bng cch o hp th (A) ca mu vi nng t bo bit trc. Vic o thng c thc hin bc sng t 600-700 nm. 3. Cc phng php gin tip Phng php gin tip xc nh sinh khi t bo da trn php tnh h s t lng ton phn (overall stoichiometry) cho s sinh trng v to thnh sn phm, c th c trnh by trong mt dng chung nh sau:ngun carbon + ngun nitrogen + phosphate + O2 sinh khi t bo + CO2 + H2O + sn phm + nhit

S thay i sinh khi t bo c th c kim sot gin tip bng cch xc nh s tiu th cht dinh dng, to thnh sn phm, cc thnh phn t bo, gii phng nhit hoc cc tnh cht vt l khc ca dch nui cy t bo. 3.1. S tiu th cht dinh dng Cn chn mt cht dinh dng m cht ny khng bao gi c s dng tng hp sn phm trao i cht. Phosphate, sulfate hoc magnesium c th l nhng chn la tt. Khi sinh khi t bo l sn phm chnh, th nng ca ngun carbon cn li trong mi trng c th c o nh gi sinh khi t bo.

Cng ngh t bo


3.2. To thnh cc sn phm


iu quan trng l cn kim tra sn phm to thnh c c kt hp vi s sinh trng hay khng. Mt s sn phm c to thnh sau khi sinh khi t bo t ti pha tnh (stationary phase) ca chu k sinh trng v v th, khng c kt hp vi s sinh trng. S gii phng CO2 c th c xc nh v n c quan h t lng i vi s sinh trng ca t bo. Mt sn phm hon ton chung cho mi phn ng ln men l ion H+. Lng kim b sung vo dch ln men s duy tr pH thch hp cho sinh trng. 3.3. Cc thnh phn t bo i vi cc nui cy tri qua s sinh trng cn bng, th cc thnh phn t bo thuc nhm i phn t nh l protein, RNA v DNA c th c xc nh thay cho sinh khi t bo. Tuy nhin, cn phi thn trng do t l ca nhng nguyn liu ny trong t bo c th thay i theo thi gian nu nui cy khng tri qua s sinh trng cn bng. 3.4. Gii phng nhit S sinh trng ca t bo pht ra nhit. Lng nhit pht ra ty thuc vo hiu sut s dng nng lng carbon. V th, vic xc nh nhit ca h ln men c th kt hp gin tip vi s sinh trng ca t bo. Tuy nhin, lng nhit ton phn tch ly trong h ln men ph thuc vo hiu qu phi hp ca cc ngun sinh nhit v mt nhit khc nhau nh: nhit t qu trnh khuy v bay hi, nhit tiu hao xung quanh thnh ca h ln men v nhit nhy (sensible heat) trong lung khng kh. V th, xc nh sinh trng bng s gii phng nhit, cn phi thit lp cn bng nng lng ca h ln men mc d y mt cng vic khng d dng. 3.5. nht Sinh trng ca c th h si (nm) hoc s to thnh polysaccharide lm tng nht ca dch ln men (fermentation broth). V th, vic xc nh nht ca dch ln men rt hu ch trong cc qu trnh ln men quy m cng nghip. nht biu kin o c tc dch chuyn c nh c th c dng nh gi nng t bo hoc nng sn phm.

Cng ngh t bo


II. Taing t chia Bt lieu bo

se tai: wWw.SinhHoc.edu.vn

Phng php bt ng (immobilization) cc t bo hon chnh c nhiu u im hn cc k thut nui cy truyn thng. Bng cch bt ng t bo, vic thit k quy trnh n gin hn khi cc t bo c gn vi cc phn t ln hoc trn cc b mt c phn tch d dng khi dng sn phm. iu ny m bo hot ng lin tc ca h ln men khng b nguy c ra tri t bo. S bt ng cng c th cung cp cc iu kin c li cho s phn ha t bo v s truyn t thng tin gia cc t bo, bng cch y thc y sn phm c sn lng cc cht trao i th cp cao. S bt ng c th bo v t bo bng cch lm gim cc s c lin quan ti lc trt (shear forces) gy tn thng t bo. Cc phng php bt ng t bo c th c phn chia thnh bn nhm chnh c tm tt bng 2.1. 1. Gn ln b mt Cc t bo c th gn ln b mt ca mnh g nh, collagen, microcarrier, hoc cc nha tng hp trao i ion (resin). Mt v d ca kiu bt ng ny l s dng cc microcarrier cho cc t bo ng vt. u im chnh ca microcarrier l n cung cp mt din tch b mt ln gn t bo. Vt liu cho microcarrier bao gm cc nha tng hp trao i ion, cc ht nh da trn c s dextran c bc bng gelatin, cc ht polyacrylamide, cc ht polystyrene, cc ht thy tinh rng, cc ht cellulose hnh tr, v cc git floruacarbon nh c n nh bng polylysine. Hin nay, cc microcarrier da trn dextran c s dng rng ri nht bt ng t bo ng vt. 2. To th xp Phng php ny cho php cc t bo khuch tn trong cc th xp c sn nh cordierite v pore glass (ht thy tinh c nhiu l rng nh), trong chng s sinh trng v c gi li. u im chnh ca phng php ny l cc vt liu to th xp c sn chng chu c s phn hy trong cc bnh nui c khuy t hn cc loi vt liu to th xp khc (alginate, acrylamide), v th xp thng khng c hi i vi t bo. Tuy nhin, phng php ny gp kh khn trong vic hng ti nng cao ca t bo do th tch l thy tinh (pore) b gii hn bi chng c lm sn bng cc loi vt liu to th xp c trng.Cng ngh t bo


Bng se tai: wWw.SinhHoc.edu.vn Tai lieu chia2.1. Cc phng php bt ng t bo.

Phng php Gn ln b mt To th xp Bao vi th T kt khi

Vt liu Mnh g mng, collagen1, microcarrier2, cc nha trao i ion, cc oxide kim loi Cordierite, pore glass, acrylamide, alginate, collagen, -carrageenan Polylysine, ethylcellulose, emulsion3, mng tng hp Cc tiu th h si, polyelectrolytes4, nhn t lin kt ngang

Mt phng thc khc l bc cc t bo bng th xp c to thnh trong iu kin in situ. Cc vt liu thuc gelatin khc nhau nh acrylamide, alginate, collagen, v -carrageenan, c th c trn vi dch huyn ph t bo v to gel trong cc dng v kch thc khc nhau. Mt phng thc n gin khc l to cc ht hnh cu dng gel alginate-calcium l nh sau: Cc t bo dch huyn ph sau khi c li s c trn vi alginate to ra mt nng alginate cui cng t 1-3% (w/v) v hn hp alginate-t bo c bm bng kim tim thuc vo dung dch calcium chloride. Cc ht c to thnh ngay tc thi c ng knh t 1-5 mm ty thuc vo nng t bo v alginate ca dung dch v kch thc ca mi kim tim. Cn lu thm l phi duy tr s v trng trong sut qu trnh bt ng t bo. Nhc im chnh ca vic s dng alginate bt ng t bo l lt cc t bo t s phn chia t bo xut hin bn trong cc ht ring r. Vic lt t bo c th c gim thiu hoc bng cch tng nng ca alginate hoc calcium chlorite trong cc ht hoc bng cch to ra cc htCollagen: cht to keo. Microcarrier: mt tiu th c kch thc hin vi (thng l mt ht polymer ng knh khong 200 m) cc t bo trong nui cy dch huyn ph gn vo v sinh trng. 3 Emulsion: dng nh tng. 4 Polyelectrolytes: cc cht a in phn.2 1

Cng ngh t bo


nh. Tuy nhin, vic tng nng ca alginate hoc calcium chloride trong ht c th lm gim tc khuch tn c cht thng qua gel v c th nh hng n kh nng sng st ca cc t bo c bao bc. 3. S dng bao vi th Cc t bo c th c bt ng bng cc bao vi th (microcapsule) c mng hoc mng bn thm khng c nh hoc c nh. u im ca k thut ng v bao l to mt din tch b mt ln cho s tip xc ca c cht v t bo. Mng bn thm ch cho i qua mt cch chn lc nhng thnh phn c trng lng phn t thp. Cc mng dng si rng to thnh mt cu trc hnh ng thng c sp hng nh l cc b si song song bn trong mt bung hnh tr. Cc t bo c gi li trn thnh ca cc si rng trong khi mi trng dinh dng c thng kh lun chuyn quanh cc si. Loi mng ny c th cung cp thm s bo v chng nhim bn mi trng. Tuy nhin, nhc im chnh ca h thng mng ny l gi thnh cao, s tc nghn ca mng lm tr ngi cho vic chuyn khi v gy kh khn trong vic thng kh. 4. T kt khi Cc t bo t kt khi hoc kt thnh cm nh len cng c th c xem nh l cc t bo c bt ng do kch thc ln ca chng c u im tng t nh s bt ng bng cc phng php khc. Trong khi nm mc s to ra cc tiu th t nhin, th cc t bo vi khun hoc nm men li cn n s kt cm. Cc nhn t kt thnh cm nhn to hoc cc nhn t lin kt ngang (cross-linkers) c th c b sung tng cng qu trnh.


III. Mt s th nghim in hnh1. ng cong sinh trng ca nm men Trong th nghim ny, chng nm men s c nui cy trong bnh tam gic thu tinh, v s thay i nng t bo s c kim sot bng cch dng ba k thut khc nhau: m di knh hin vi, xc nh khi lng kh, c ca dch huyn ph t bo.Cng ngh t bo


1.1. Nguyn liu


- Mt chng nm men bt k sinh trng trong nui cy dch huyn ph. Chng ta c th thu chng nm men t mt phng th nghim vi sinh vt hoc mua mt chng c bit t cng ty. - Glucose, dch chit nm men, NH4Cl, MgSO4, CaCl2 v cht chng to bt pha mi trng. - Hai bnh tam gic 125 mL - Pipette v trng - n Bunsen - Ni kh trng - T m - Hemocytometer - Ly tm - Cn ha cht - My quang ph 1.2. Phng thc tin hnh - Chun b mi trng nui cy theo bng 2.2. Rt 50 mL/bnh vo 2 bnh tam gic loi 125 mL.Bng 2.2. Mi trng sinh trng c trng ca nm men. Glucose Dch chit nm men NH4Cl MgSO4 CaCl2 Cht chng to bt Nc 100 g 8,5 g 1,32 g 0,11 g 0,06 g 0,2 mL b sung va 1 L

Cng ngh t bo


plastic chu nhit, np inox, hoc bng khng thm nc. - Kh trng bnh tam gic ng mi trng 121oC trong 20 pht. - Tip mu (inoculate) 1 mL dch nui cy nm men trc vo bnh tam gic cha mi trng v trng. Tin hnh cn thn khi b nhim bn pipette, np y v bnh tam gic trong sut qu trnh tip mu. gim thiu c hi nhim bn, nn t np y v c ca bnh tam gic sau khi ly np ra cy nm men vo. - t bnh tam gic vo trong t m 37oC. - Ly 2 mL mu cc khong thi gian khc nhau trong qu trnh nui cy xc nh nng t bo bng cch m trn knh hin vi, xc nh trng lng kh v o c (mt quang) bng my quang ph. Thi gian ly mu phi c sp xp sao c th thu c cho ng cong sinh trng tt th hin c ba phase sinh trng (xem chng 3). Trc khi ly mu phi trn tt c cc thnh phn trong bnh tam gic bng cch lc. 2. ng cong sinh trng ca thc vt 2.1. Nguyn liu - Mt dng t bo dch huyn ph ca thc vt sinh trng tt. Phng thc sau y da trn c s nui cy t bo thuc l. Nu chn dng t bo khc th cn thay i mi trng. - Hn hp mui khong ca Murashige-Skoog (1962) (Bng 7.2), 2,4dichlorophenoxyacetic acid (2,4-D), KH2PO4, inositol, thiamine.HCl, v sucrose pha ch mi trng. - Hai bnh tam gic 125 mL - Pipette loi ming rng v trng (10 mL) - T nui t bo thc vt km my lc - Cn ha cht - Ly tm - Eppendorf tube 2.2. Phng thc tin hnh - Chun b mi trng mui khong ca Murashige-Skoog, 0,2 mg/L 2,4-D, 0,18 g/L KH2PO4, 0,1 g/L inositol, 1 mg/L thiamine.HCl v 30 g/L

- Nt bnh tam gic bng mt trong s vt liu sau: giy nhm, Tai lieu chia se tai: wWw.SinhHoc.edu.vnnt

Cng ngh t bo


sucrose5. iu chnh pH ti 5,8 bng KOH 1N v phn phi mi trng vo hai bnh tam gic loi 125 mL, sao cho mi bnh tam gic cha 30 mL. - y np bnh tam gic v kh trng 121oC trong 20 pht. - Cy vo bnh tam gic cha 30 mL mi trng vi 1,5 mL ca dch huyn ph t bo 7 ngy tui v t trong t nui t bo thc vt c my lc v lc 150 vng/pht, nhit nui 27oC, chiu sng 8 gi/ngy cng 2.000 lux. - Ly 1,5 mL dch nui cy mi ngy c trn k v cho vo Eppendorf tube cn, ly tm tube 9.000 vng/pht trong 4-5 pht v loi th ni. Cn li tube v tnh ton phn trm trng lng t bo m. t tube trong t sy 70oC trong hai ngy v cn li tnh ton trng lng kh. - V th s thay i nng t bo kh v ti (m) theo thi gian. 3. Bt ng t bo thc vt 3.1. Nguyn liu - 30 mL t bo dch huyn ph thuc l sinh trng bng phng php m t trong th nghim trc. - Alginate - CaCl2 - Bm nhu ng - Ni p sut - Cn ha cht 3.2. Phng thc tin hnh - Trn 0,875 g alginate, 5 mL mi trng nui cy thc vt v 25 mL nc v kh trng. - i cho 30 mL dch nui cy huyn ph ca t bo thc vt lng xung, loi b th ni. Bc ny thng mt khong 10 pht. - B sung hn hp alginate v trn vi cc t bo c c li.


Mi trng ny ch c tnh cht tham kho. Thng thng cc loi thc vt khc nhau vi cc mc ch nui cy khc nhau, s c cc nhu cu dinh dng khc nhau. Khi , chng ta phi thit k cc mi trng nui cy c tnh c hiu cao hn. Cng ngh t bo

5


ID) v cung cp tng git vo trong bnh tam gic cha 200 mL dung dch v trng ca CaCl2 0,12 M. Cc git nh s phn ng ngay lp tc vi CaCl2 to ra cc ht hnh cu c ng knh khong 3,75-4,5 mm. - Gi cc ht trong dung dch CaCl2 khong 1 gi m bo phn ng kt ta xy ra hon ton. - Cy vo bnh tam gic cha 30 mL mi trng thc vt vi khong 30 ht t bo thc vt c bt ng v t n trong t nui t bo thc vt c my lc v lc 150 vng/pht, nhit nui 27oC, chiu sng 8gi/ngy cng 2.000 lux. - Chng ta c th xc nh nng t bo kh v m ca cc t bo t do trong dch huyn ph v cc t bo c bt ng trong sut qu trnh nui cy m. xc nh nng t bo ca cc t bo c bt ng, cn phi ha tan cc ht trong potassium phosphate 1 M trong 24 gi.

- Bm hn hp alginate-t bo qua mt ng silicon v trng (1,6 Tai lieu chia se tai: wWw.SinhHoc.edu.vnmm

Ti liu tham kho/c thm1. Atkinson B and Mavituna F. 1991. Biochemical Engineering and Biotechnology Handbook. 2nd ed. Stockton Press, New York, USA. 2. Flickinger MC and Drew SW. 1999. Encyclopedia of Bioprocess Technology: Fermentation, Biocatalysis and Bioseparation. John Wiley & Sons, New York, USA. 3. Lee JM. 2001. Biochemical Engineering. Prentice Hall, Inc. USA. 4. Ratledge C and Kristiansen B. 2002. Basic Biotechnology. Cambridge University Press, UK. 5. Shuler ML and Kargi F. 2002. Bioprocess Engineering-Basic Concepts. 2 ed. Prentice Hall, Inc. New Jersey, USA.nd

6. Vogel HC and Todaro CL. 1997. Fermentation and Biochemical Engineering Handbook (Principles, Process Design, and Equipment). 2nd ed. Noyes Publications. New Jersey, USA.

Cng ngh t bo


Tai 3 Chnglieu

chia se tai: wWw.SinhHoc.edu.vn

ng hc sinh trng ca t boI. M uHiu bit y ng hc sinh trng ca cc t bo thc vt, ng vt v vi sinh vt l rt cn thit thit k v hot ng cc h ln men. ng hc t bo c quan h vi tc sinh trng t bo v chu nh hng ca cc iu kin vt l v ha hc. ng hc t bo l kt qu ca h thng cc phn ng ha sinh v cc qu trnh vn chuyn phc tp, bao gm nhiu pha v cc h thng nhiu thnh phn. Trong sut thi gian sinh trng, hn hp khng ng nht ca cc t bo gi v non thay i lin tc v t thch nghi vi mi trng dinh dng l yu t cng thay i lin tc trong cc iu kin vt l v ha hc. Ni chung, m hnh ton hc chnh xc ca ng hc sinh trng l khng c th c c. Thm ch mt m hnh thc t cng kh tip cn bi v n c th cha nhiu thng s khng th xc nh. V th, chng ta cn gi nh c th t c nhng m hnh n gin nh vy s hu ch hn cho vic thit k h thng ln men (xem chng 4) v d bo hiu sut. Cc m hnh khc nhau c th c pht trin trn c s cc gi nh v cc thnh phn v qun th t bo nh trnh by trong bng 3.1. Ngoi cc gi nh i vi t bo, mi trng c thit k sao cho ch mt thnh phn c th gii hn tc phn ng, cn tt c cc thnh phn khc hin din cc nng cao m nhng thay i nh ca chng khng nh hng r rt n tc phn ng. Cc h thng ln men cng c kim sot sao cho cc thng s mi trng nh pH, nhit v nng oxygen ha tan c duy tr mt mc khng i. Trong chng ny, cc phng trnh ng hc t bo bt ngun t m hnh c phn phi, khng cu trc. Cc phng trnh ny c ng dng thit k v phn tch cc h ln men l tng.

Cng ngh t bo


Bng 3.1. Cc m hnh wWw.SinhHoc.edu.vn Tai lieu chia se tai:khc nhau ca ng hc t bo. Cc thnh phn t bo Qun th Khng cu trc (unstructured) c phn phi (distributed) Cc t bo c i din bi mt thnh phn n, phn phi khng ng u trong qu trnh nui cy. Cc t bo c i din bi mt thnh phn n, v to thnh mt hn hp khng ng nht. c cu trc (structured) Cc t bo bao gm nhiu thnh phn phc tp phn phi khng ng u trong qu trnh nui cy. Cc t bo bao gm nhiu thnh phn phc tp v to thnh hn hp khng ng nht.

B c lp (segregated)

II. nh nghaTrc tin, chng ta hy nh ngha mt s thut ng dng cho sinh trng ca t bo. Nu cp n nng t bo m khng km theo bt k mt ghi ch c im no, th n c th c hiu theo nhiu ngha khc nhau. c th l s lng t bo, trng lng ti t bo, hoc trng lng kh t bo trn mt n v th tch. Trong chng ny, chng ta thng nht cc thut ng sau: CX: nng t bo, trng lng kh t bo trn mt n v th tch. CN: mt s lng t bo, s lng t bo trn mt n v th tch.

: mt t bo, trng lng ti t bo trn mt n v th tch cakhi lng t bo. T , c th nh ngha tc sinh trng ca t bo theo mt s cch khc nhau nh sau: dCX/dt: s thay i nng kh ca t bo theo thi gian. rX: tc sinh trng ca t bo trn c s trng lng kh. dCN/dt: s thay i mt s lng t bo theo thi gian. rN: tc sinh trng ca t bo trn c s s lng. : tc phn chia ca t bo trn c s s lng d log 2 C N / dtCng ngh t bo


Dng nh dC se r lun lun ging nhau, nhng thc ra khng Tai lieu chia/ dt v tai: wWw.SinhHoc.edu.vnX X

phi nh vy. Gi tr dC X / dt l s thay i nng t bo trong h ln men, l yu t c th bao gm hiu qu ca tc dng chy i vo v i ra, s ti sinh t bo, v cc iu kin hot ng khc ca h ln men. Trong khi rX l tc sinh trng thc t ca t bo. Hai gi tr ny ch ging nhau trong trng hp hot ng ln men m. Tc sinh trng da trn s lng t bo v tc sinh trng da trn trng lng t bo khng nht thit phi ging nhau, bi v kch thc trung bnh ca cc t bo c th rt khc nhau khi chuyn t pha sinh trng ny n pha sinh trng khc. Khi sinh khi ca mt t bo ring bit tng ln m khng c s phn chia, th tc sinh trng da trn trng lng t bo cng tng ln, trong khi tc sinh trng da trn s lng t bo li gi nguyn. Tuy nhin, trong sut thi gian sinh trng theo hm m, pha sinh trng m chng ta quan tm nht di quan im ca cng ngh, th tc sinh trng da trn s lng t bo v tc sinh trng da trn trng lng t bo c th c gi nh l tng ng nhau. Trong mt s trng hp tc sinh trng b nhm ln vi tc phn chia, l khi nim c nh ngha nh l tc phn chia t bo trn mt n v thi gian. Nu tt c t bo trong bnh nui cy thi im t = 0 (CN = CNo) phn chia ch sau mt thi gian nht nh, th qun th t bo s tng ln C N 0 2 ln. Nu cc t bo c phn chia n ln sau thi gian t, th s lng tng s ca t bo s l:

C N = C N 0 2nV tc phn chia trung bnh l:n t

(3.1)

=

(3.2)

V n = log 2 C N log 2 C N 0 theo phng trnh 3.1, nn tc phn chia trung bnh s l: = 1 log 2 C N log 2 C N 0 t

(

)

(3.3)

V tc phn chia thi gian t l:

Cng ngh t bo


Tai lieu chia se tai: wWw.SinhHoc.edu.vn d log C=2 N

dt

(3.4)

V th, tc sinh trng (c nh ngha l s thay i s lng t bo theo thi gian) chnh l dc ca ng cong CN theo t. Trong khi , tc phn chia l dc ca ng cong log2CN theo t. Nh gii thch, tc phn chia l hng s trong sut thi gian sinh trng theo hm m, trong khi tc sinh trng li khng nh vy. V th, hai khi nim ny khng c nhm ln vi nhau.

III. Chu k sinh trng ca nui cy mNu nui cy cc vi sinh vt n bo trong mi trng v trng sch v o mt s lng t bo theo thi gian th trn th ca n ta c th thy c su pha sinh trng v cht ca t bo (Hnh 3.1), l: - Pha lag. L thi gian khi s thay i s lng t bo bng khng. - Pha sinh trng nhanh. S lng t bo bt u tng v tc phn chia t n cc i. - Pha sinh trng theo hm m. S lng t bo tng theo hm m khi t bo bt u phn chia, tc sinh trng tng ln trong sut pha ny, nhng tc phn chia t l vi d ln C N / dt , l hng s gi tr cc i ca n, nh c minh ha hnh 3.1. - Pha sinh trng chm. Khi tc sinh trng t n cc i, th giai on tip theo l pha sinh trng chm trong c hai tc sinh trng v tc phn chia u gim. - Pha tnh. Qun th t bo t n gi tr cc i v s khng tng thm na. - Pha cht. Sau khi cc cht dinh dng ca t bo cn kit, t bo s bt u cht v s lng t bo sng st s gim. 1. Pha lag Pha lag (hoc pha tnh khi u hoc tim tng) l thi k khi u ca qu trnh nui cy, trong sut thi k ny s thay i s lng t bo l bng khng hoc khng ng k. Mc d s lng t bo khng tng ln,

Cng ngh t bo


nhng t bo c th sinh trng bng cch tng kch thc trong sut thi k ny.A 12 B C D E F


ln C N10

8

t1,5 1,0

C N 105

0,5 0 0,3 0,2 0,1

t

d ln C N dt

0 -0,1 -0,2

t

Hnh 3.1. ng cong sinh trng c trng ca cc c th n bo. (A) pha lag, (B) pha sinh trng nhanh, (C) pha sinh trng theo hm m, (D) pha sinh trng chm, (E) pha tnh, (F) pha cht.

di ca pha lag ty thuc vo nhiu nhn t, chng hn nh loi v tui ca c th vi sinh vt (hoc t bo ng-thc vt), v cc iu kin nui cy. Pha lag thng xut hin do t bo phi iu chnh vi mi trng mi trc khi s sinh trng c th bt u. Nu vi sinh vt c cy t mi trng c nng cht dinh dng thp vo mi trng c nng cht

Cng ngh t bo


dinh dng cao, th pha lag thng ko di. Nu n c chuyn t ni c nng cao n ni c nng thp th thng khng xut hin pha lag. Mt nhn t quan trng khc nh hng n di ca pha lag l lng mu c a vo nui cy (inoculum size). Nu mt lng nh t bo c a vo mt th tch ln th chng s c mt pha lag di. trng hp nui cy t bo trn quy m ln, th thi gian ca pha lag cng ngn cng tt. V th, a mu vo (cn gi l tip mu) quy trnh ln men cng nghip, chng ta cn phi c mt dy cc ni ln men c lng mu ln dn gim thiu nh hng ca pha lag. Vo giai on kt thc pha lag, khi s sinh trng ca t bo bt u, th tc phn chia t bo tng ln t t v t n gi tr cc i thi k sinh trng theo hm m, nh trnh by bng s tng ln gc un cong B trong hnh 3.1. Thi k chuyn tip ny c gi chung l pha sinh trng nhanh v thng c xem nh l mt phn ca pha lag. 2. Pha sinh trng theo hm m (pha logarithm) cc c th n bo, s nhn i tng dn ca s lng t bo cho kt qu tc sinh trng tng ln lin tc trong qun th. Nui cy vi khun tri qua s sinh trng cn bng kiu nh phn ng ha hc bc mt t xc tc. V th, tc tng trng ca qun th t bo mi thi im t l vi mt s lng (C N ) ca t bo hin din ti thi im .rN = dC N = C N dt(3.5)


Trong : hng s c bit nh l tc sinh trng c trng (gi ). Khng nn nhm ln tc sinh trng c trng vi tc sinh trng (c cc n v v ngha khc hn). Tc sinh trng l s thay i ca mt s lng t bo theo thi gian, trong khi tc sinh trng c trng l:-1

=

1 dC N d ln C N = CN dt dt

(3.6)

l s thay i theo logarithm t nhin ca mt s lng t bo theo thi gian. So snh phng trnh (3.4) v (3.6) cho thy:

Cng ngh t bo


Tai lieu chia se tai: log C d ln C d wWw.SinhHoc.edu.vn=N

dt

= ln 2

2

N

dt

= ln 2

(3.7)

V vy, tc sinh trng c trng bng ln2 ln tc phn chia . Nu l hng s theo thi gian trong sut thi k sinh trng theo pha hm m, th phng trnh (3.5) c th c ly tch phn t t0 ti t khi :CN

C N0

dC N = dt CN t0

t

(3.8)

Hay:C N = C N 0 exp[(t t 0 )](3.9)

Trong : C N 0 l mt s lng t bo t0 khi s sinh trng hm m bt u. Phng trnh (3.9) cho thy s tng ln ca s lng t bo theo hm m i vi thi gian. Thi gian cn thit gp i qun th, c gi l thi gian nhn i (td), c th c lng t phng trnh (3.9), bng cch t C N = 2C N 0 v t0 = 0 , gii theo t ta c: td = ln 2 1 = (3.10)

Thi gian nhn i t l nghch vi tc sinh trng c trng v bng s nghch o ca tc phn chia. 3. Cc nhn t nh hng n tc sinh trng c trng 3.1. Nng c cht Mt trong nhng phng trnh c s dng rng ri nht th hin nh hng ca nng c cht (cht dinh dng) ln l phng trnh Monod:

=

max C SK S + CS

(3.11)

Cng ngh t bo


Trong : C l se wWw.SinhHoc.edu.vn Tai lieu chia nngtai:ca c cht gii hn (limiting substrate)S

trong mi trng v K S l h s h thng. Mi quan h ny c trnh by bng th trong hnh 3.2. Gi tr ca K S tng ng vi nng ca cht dinh dng khi tc sinh trng c trng bng mt na gi tr cc i ca n (max). Theo phng trnh Monod, s tng ln v sau ca nng cht dinh dng khi t n max khng nh hng n tc sinh trng c trng, nh trnh by hnh 3.2. Tuy nhin, tc sinh trng c trng s gim xung khi nng cht dinh dng tng ln vt qu mt mc nht nh. 3.2. Nng sn phm Khi cc t bo sinh trng, chng s sn xut ra cc sn phm trao i cht v c th tch ly trong mi trng. Sinh trng ca cc vi sinh vt thng b c ch bi cc sn phm ny, nh hng ca cc sn phm ny c th c b sung vo phng trnh Monod nh sau:

= max hoc:

CS K S + CS

KP K +C P P

(3.12)

CS = max K +C S S

C 1 P C Pm

n

(3.13)

Trong : KP l h s nng sn phm v CP l nng sn phm. C hai phng trnh trn m t s c ch sn phm kh tt. CPm c k hiu l nng cc i ca sn phm, l yu t lm cho cc t bo khng th sinh trng do s c ch sn phm. 3.3. Cc iu kin khc Tc sinh trng c trng ca cc t bo cng b nh hng bi pH mi trng, nhit v s cung cp oxygen. Nhit v pH ti u ca cc loi t bo khc nhau (ng-thc vt v vi sinh vt) l cng khc nhau.

Cng ngh t bo


Tai lieu chia se tai: wWw.SinhHoc.edu.vn1,2

max0,8

0,4

Hnh 3.2. S ph thuc ca tc sinh trng c trng vo nng ca cht dinh dng gii hn sinh trng. max = 0,935/gi; KS = 0,2210-4 mol/L.

KS0 2 4 6

CS 104 , M4. Pha tnh v pha cht Sinh trng ca qun th t bo thng b hn ch hoc do s dng ht ton b cc cht dinh dng c sn hoc do s tch ly cc sn phm c ca s trao i cht. Kt qu l tc sinh trng gim v s sinh trng cui cng dng li. thi im ny nui cy c gi l pha tnh. Giai on chuyn tip gia pha hm m v pha tnh bao gm mt thi k sinh trng khng cn bng v trong sut thi k ny cc thnh phn khc nhau ca t bo c tng hp cc tc khng bng nhau. Kt qu l cc t bo trong pha tnh c mt thnh phn ha hc khc vi cc t bo trong pha hm m. Pha tnh thng c tip theo bi pha cht m trong cc c th trong qun th b cht. S cht xut hin hoc do s suy yu ca vic bo qun nng lng ca t bo, hoc do s tch ly cc sn phm c t. Ging nh s sinh trng, s cht l mt hm m. Trong mt s trng hp, c th khng ch cht m cn phn hy, mt qu trnh cn c gi l s phn gii.

IV. Cc k hiuC CNC N0

nng , khi lng trn mt n v th tch nui cy, kg/m3 mt s lng t bo, s lng t bo/m3 mt s lng t bo ti thi im t0, s lng t bo/m3 nng sn phm nng cc i ca sn phm

CP CPm

Cng ngh t bo


CS CX dCX/dt dCN/dt KP KS n r rX rN t td

nng chia Tai lieu c cht se tai: wWw.SinhHoc.edu.vn nng t bo, trng lng kh t bo trn th tch kg/m3 s thay i nng kh ca t bo theo thi gian s thay i mt s lng t bo theo thi gian h s nng sn phm h s h thng cho ng hc Monod, kg/m3 s lng t bo tc tc sinh trng ca t bo trn c s trng lng kh tc sinh trng ca t bo trn c s s lng thi gian, s thi gian nhn i, s tc phn chia ca t bo, s-1 tc phn chia t bo trung bnh, s-1 tc sinh trng c trng, s-1 hoc kg/m3/s tc sinh trng cc i, s-1 hoc kg/m3/s mt t bo, kg/m3

max

Ti liu tham kho/c thm1. Asenjo JA and Merchuk JC. 1995. Bioreactor System Design. Marcel Dekker, Inc. New York, USA. 2. Atkinson B and Mavituna F. 1991. Biochemical Engineering and Biotechnology Handbook. 2nd ed. Stockton Press, New York, USA. 3. Chia TF. 2003. Engineering Applications in Biology. Updated 1st ed. McGraw-Hill Education, Singapore. 4. Flickinger MC and Drew SW. 1999. Encyclopedia of Bioprocess Technology: Fermentation, Biocatalysis and Bioseparation. John Wiley & Sons, New York, USA. 5. Lee JM. 2001. Biochemical Engineering. Prentice Hall, Inc. USA. 6. Ratledge C and Kristiansen B. 2002. Basic Biotechnology. Cambridge University Press, UK. 7. Shuler ML and Kargi F. 2002. Bioprocess Engineering-Basic Concepts. 2nd ed. Prentice Hall, Inc. New Jersey, USA. 8. Vogel HC and Todaro CL. 1997. Fermentation and Biochemical Engineering Handbook (Principles, Process Design, and Equipment). 2nd ed. Noyes Publications. New Jersey, USA. Cng ngh t bo


Tai 4 Chnglieu

chia se tai: wWw.SinhHoc.edu.vnThit k h ln men

I. H ln men thng khuyNi phn ng sinh hc (bioreactor) hay cn gi l h ln men (fermenter) l loi thit b m trong n s bin i ha sinh c tin hnh bi cc t bo sng hoc cc thnh phn t bo in vivo (enzyme). Trong chng ny, ni phn ng sinh hc nui cy cc t bo sng c gi l h ln men phn bit cc ni phn ng sinh hc dng cho cc enzyme. Trong phng th nghim, cc t bo thng c nui cy trong cc bnh tam gic trn my lc. Lc nh bnh tam gic rt hiu qu to ra dch huyn ph t bo, tng cng s oxy ha thng qua b mt cht lng v tr gip s chuyn khi (mass transfer) ca cc cht dinh dng m khng gy nguy him cho cu trc t bo.

bt

vch ngn un nng lm lnh turbin dt

khng kh v trng

Hnh 4. 1. S h ln men dng cho sn xut penicillin.

i vi hot ng sn xut quy m ln, th h thng ln men thng khuy (stirred-tank fermenter, STF) c s dng rng ri nht thit k cho qu trnh ln men cng nghip. N c th c dng cho c hai trng hp ln men hiu kh (aerobic) v ym kh (anaerobic) trong mt phm vi rng cc loi t bo khc nhau bao gm vi sinh vt, ng vt v thc vt.

Cng ngh t bo


Tai lieugii thiuse tai: wWw.SinhHoc.edu.vn Hnh 4.1 chia s h ln men dng trong sn xut penicillin.Cng pha trn (mixing intensity) c th rt khc nhau bng cch chn loi cnh khuy (impeller) thch hp v cc tc khuy khc nhau. Vic sc kh v khuy c hc trong h ln men rt tt cho nui cy dch huyn ph t bo, s oxy ha, s pha trn mi trng v truyn nhit. STF cng c th c dng cho cc mi trng c nht cao. N l mt trong nhng h ln men quy m ln u tin c pht trin trong cng nghip dc. c im v tim nng ca STF c nghin cu rng ri. Do h ln men thng khuy thng c lm bng thp khng r v hot ng trong iu kin n ha nn tui th ca thit b rt lu. Nhc im ca h ln men thng khuy bt ngun t u im ca n. B phn (cnh) khuy rt hiu qu trong vic pha trn cc thnh phn ca h ln men, nhng li tiu th mt lng ln cng sut v c th gy nguy him cho nhng h thng t bo nui cy mn cm vi lc trt (shear force) nh t bo ng vt c v hoc t bo thc vt. Lc trt ca cht lng trong hn hp c to ra bi gradient tc ca cc thnh phn tc (hng tm v tip tuyn) ca cht lng khi ri khi vng cnh khuy. Khi cht lng ri khi vng trung tm, th tc ca n v tr trn v di cnh khuy (c khong cch bng chiu rng cnh khuy) s gim khong 85% v to ra mt vng trt cao. Khi t l chiu rng cnh khuy trn ng knh ca n tng th profile tc t c dng c trng ca parabol m tr nn t hn v n to ra lc trt t hn do gradient tc ln dn ln. V th, bng cch tng chiu rng cnh khuy, c th ng dng thnh cng STF trong nui cy t bo ng vt hoc t bo thc vt. Nhiu h ln men quy m phng th nghim c lm bng thy tinh c np bng thp khng r. Cc thng ln men ln hn c lm bng thp khng r. T l chiu cao trn ng knh ca thng ln men (vessel) hoc l 2/1 hoc l 3/1 v thng c khuy bng hai hoc ba turbine khuy (cnh khuy). Trc cnh khuy c gn trn np hoc t y ca thng bng gi . T l ng knh cnh khuy (DI) trn ng knh ca thng (DT) thng l t 0,3-0,4. Trong trng hp h ln men c hai cnh khuy, th khong cch gia cnh khuy th nht vi y ca vessel v khong cch gia hai cnh khuy bng 1,5 ng knh cnh khuy. Khong cch ny gim xung cn 1,0 so vi ng knh cnh khuy trong trng hp h ln men c ba cnh khuy. Bn vch ngn (baffles) cch u nhau thng

Cng ngh t bo


c thit k ngn cn s hnh thnh dng xoy lm gim hiu sut pha trn. Chiu rng ca vch ngn thng bng 1/10 ng knh ca thng (tank). trng hp h ln men hiu kh (aerobic fermenter), th mt b phun l n (single orifice sparger) hoc mt b phun vng c s dng sc kh cho h ln men. B phn phun c t v tr gia cnh khuy cui cng v y ca vessel. pH trong h ln men c th c duy tr bng cch dng dung dch m hoc b iu chnh pH (pH controller). Nhit c iu chnh bng h thng gia nhit v lm lnh t ng. 1. H ln men dng nt (plug-flow fermenter, PFF) hoc m (batch fermenter) Mt h ln men khuy l tng phi c kh nng pha trn tt sao cho cc thnh phn ng nht trong mt kt cu mi thi im. Mt h ln men l tng khc l h ln men dng nt, mt dng tng ng ca h ln men m. Trong h ln men dng ng (tubular-flow fermenter), cht dinh dng (c cht) v t bo i vo mt u ca ng hnh tr v t bo s sinh trng trong khi chng i qua ng ny. Do ng di v thiu b phn khuy nn ngn cn s pha trn hon ton ca cht lng, v th tnh cht ca dng chy thay i trong hai chiu tip tuyn v hng tm. Tuy nhin, s bin thin trong chiu hng tm nh hn chiu tip tuyn. Mt h ln men dng ng m khng c nhng bin thin hng tm th c gi l h ln men dng nt (PFF). Thc t, h ln men PFF rt kh xy dng. Cho d h ln men PFF trng thi n nh (steady state) c hot ng trong mt kiu lin tc, th nng t bo ca h ln men m l tng sau thi gian t s ging nh nng t bo ca h ln men PFF trng thi n nh v tr chiu dc ni m thi gian lu (residence time) bng t (Hnh 4.2). V th, s phn tch sau y ng dng cho c hai, h ln men m l tng v PFF trng thi n nh. Nu mi trng lng c tip mu bng nui cy kt ht (seed culture), th t bo s bt u sinh trng theo hm m sau pha lag. Trong h ln men m, s thay i nng t bo bng tc sinh trng t bo:


dC X = r X = C X dt

(4.1)

Cng ngh t bo


Tai lieu chia se tai: wWw.SinhHoc.edu.vnCXo Cso to t CX Cs(b)CX f CS f

FV, CX , CS

F

p

C X = CX o Cs = Cso(a)

t = to

Hnh 4.2. S (a) h ln men thng khuy m v (b) h ln men dng nt.

thu c phng trnh hiu sut ca ln men m, chng ta cn ly tch phn phng trnh (4.1) s c:CX

CX 0

t X dC X dC X = = dt = t t 0 rX C X t0 CX0

C

(4.2)

Cn lu rng, phng trnh (4.2) ch c ng dng khi rX > 0. V th, t 0 (trong phng trnh 4.2) khng phi l thi gian ca nui cy ban u sau khi tip mu, m l thi gian t bo khi ng sinh trng, l giai on pha sinh trng bt u tng nhanh. Theo phng trnh (4.2), thi gian sinh trng tng m t t 0 chnh l din tch pha di ng cong 1/rX theo C X gia C X 0 v C X (Hnh 4.3). ng cong lin tc hnh 4.3 c tnh ton bng phng trnh Monod v vng c mu ti bng t t 0 . Thi gian sinh trng tng m t khi c c lng bng th ny v xc nh n th da vo ng cong t theo C X l n gin hn. Tuy nhin, biu din bng th s thun tin trong vic so snh tim nng ca cc cu hnh h ln men khc nhau (s c tho lun sau). Lc ny ch lu rng, ng cong c mu ti dng ch U l c trng ca cc phn ng xc tc t ng: S+XX+XCng ngh t bo


Tai lieu chia se tai: wWw.SinhHoc.edu.vn3 2

1 rX1

0

2

4

6

8

CXHnh 4.3. th ca thi gian sinh trng tng m t t 0 (vng ti). ng cong lin tc biu din m hnh Monod vi max = 0,935/gi; K S = 0,71 g/L;

YX/S = 0,6; C X 0 = 1,6 g/L; v CS 0 = 10 g/L. Tc khi u ca phn ng xc tc t ng chm do nng ca X thp. Tc phn ng tng ln khi cc t bo sinh sn v sau s t n tc ti a. Khi lng c cht gim v cc sn phm c c tch ly, th tc phn ng gim xung gi tr thp hn. Nu ng hc Monod (Monod kinetics) biu din thch hp tc sinh trng trong sut pha hm m, th chng ta c th thay th phng trnh (3.11) chng 3 vo phng trnh (4.2) c c:CX

CX 0

t ( K S + C S )dC X = dt max C S C X t0

(4.3)

Phng trnh (4.3) c th tnh c tch phn nu chng ta bit mi quan h gia CS v CX. Ngi ta quan st thy rng s lng sinh khi t bo c sn xut t l vi lng c cht gii hn c tiu th. Hiu sut sinh trng ( Y X/S ) c nh ngha nh sau:

YX/S =

C X CX0 C X = C S (C S C S0 )

(4.4)

Thay phng trnh (4.4) vo phng trnh (4.3), tch phn ca phng trnh tng hp ny s a ra mi quan h gia nng t bo v thi gian:Cng ngh t bo


(t t 0 ) max

Tai lieu chia se tai: wWw.SinhHoc.edu.vn C K Y K Y CS S X /S S X /S = + 1 ln X + ln 0 C X + C S YX / S CX C X 0 + C S0 YX / S CS 0 0 0

(4.5)

2. H ln men thng khuy lin tc (continuous stirred-tank fermenterCSTF) l tng Qun th t bo c th tip tc giai on sinh trng hm m trong mt thi gian di bng cch duy tr h thng nui cy lin tc. Hnh 4.4 trnh by s h ln men thng khuy lin tc (CSTF). Bung sinh trng (thng ln men hay bnh nui) c kt ni vi bnh cha mi trng v trng. Khi qu trnh sinh trng bt u th mi trng sch c cung cp lin tc t bnh cha mi trng. H thng nui cy lin tc c th hot ng nh l mt chemostat (th n nh ha tnh) hoc turbidostat (th n nh c). Trong chemostat tc dng chy c ci t mt gi tr c bit v tc sinh trng ca nui cy s iu chnh tc dng chy ny. Ni chung, hot ng chemostat d dng hn turbidostat, do n c th c thc hin bng cch t my bm mt tc dng chy khng i, trong khi turbidostat i hi mt thit b cm quang (optical sensing device) v mt b iu chnh (controller). Tuy nhin, turbidostat c gii thiu khi h ln men lin tc cn c tin hnh cc tc pha long cao gn vi im ra tri (washout point), khi ta c th ngn cn s ra tri bng cch iu ha tc dng chy trong trng hp tht thot t bo thng qua dng chy ra ngoi vt qu s sinh trng t bo trong h ln men.

F

CXi Csi

V, CX , CS

CX F C s

Hnh 4.4. S h ln men thng khuy lin tc (CSTF).

Cng ngh t bo


Cn bng nguyn liu cho t bo trong CSTF (Hnh 4.4) c th c vit nh sau:

Tai lieu chia se tai: wWw.SinhHoc.edu.vnFC X i FC X + VrX = V dC X dt

(4.6)

Trong : rX l tc sinh trng t bo trong h ln men v dC X / dt biu din s thay i nng t bo trong h ln men theo thi gian. i vi CSTF hot ng trng thi n nh, th s thay i nng t bo theo thi gian l bng khng (dCX / dt = 0) do cc t bo trong bnh nui ch sinh trng nhanh thay th nhng t bo b hao ht theo dng chy ra ngoi, v phng trnh (4.6) tr thnh:

m =

V C X C Xi = F rX

(4.7)

Phng trnh (4.7) cho thy thi gian lu cn thit (m) bng din tch hnh ch nht c chiu rng C X C X i v chiu cao 1 / rX trn ng cong

1 / rX theo CX.Hnh 4.5 biu din ng cong 1 / rX theo CX. Din tch hnh ch nht c t m trong hnh bng thi gian lu trong CSTF khi dng chy vo l v trng. Minh ha thi gian lu bng th c th gip chng ta so snh hiu qu ca cc h ln men. H ln men c thi gian lu ngn hn ( t ti mt nng t bo nht nh) l hiu qu hn. Hot ng ti u ca h ln men da trn s minh ha th ny s c tho lun trong phn tip theo.4

1 rX

3 2 1

0

2

CX

4

6

Hnh 4.5. Minh ha bng th c lng thi gian lu cho CSTF. ng biu din m hnh Monod vi max = 0,935/gi; K S = 0,71 g/L; Y X/S = 0,6;

CS i = 10 g/L; v C X i = 0.Cng ngh t bo


Nu dng chy se v wWw.SinhHoc.edu.vn Tai lieu chiavo ltai:trng (C = 0), v t bo trong CSTFXi

ang sinh trng theo hm m (rX = C X ) th phng trnh (4.7) s tr thnh: 1 1 D

m =

=

(4.8)

Trong : D c bit nh l tc pha long v c gi tr bng nghch o ca thi gian lu ( m ). V th, i vi CSTF trng thi n nh c cht dinh dng v trng, th tc sinh trng c trng bng tc pha long. Mt khc, tc sinh trng c trng ca t bo c th c iu chnh bng cch thay i tc dng chy mi trng. Nu tc sinh trng c th c biu din bng phng trnh Monod, th sau :

D==

1

m

=

max C SK S + CS

(4.9)

T phng trnh (4.9), CS c th c tnh ton bng thi gian lu bit v cc thng s ng hc Monod nh sau:

CS =

KS

m max 1

(4.10)

Tuy nhin, cn ch rng phng trnh (4.10) ch c gi tr khi m max > 1 . Nu m max < 1 , tc sinh trng ca t bo s thp hn tc t bo tht thot theo dng chy ra ngoi. Do , tt c t bo trong h ln men s b ra tri, v phng trnh (4.10) s khng c gi tr. Nu hiu sut sinh trng (YX / S ) l hng s, th sau :

C X = YX / S (C Si CS )

(4.11)

Thay phng trnh (4.10) vo phng trnh (4.11) s cho hiu sut tng quan i vi CX nh sau:

KS C X = Y X / S C Si m max 1

(4.12)

Cng ngh t bo


Tai lieu chia se tai: wWw.SinhHoc.edu.vn Tng t: KS CP = CPi + YP / S CS i m max 1 (4.13)

Trong : CP l nng sn phm, CPi l nng sn phm a vo. Mt ln na, phng trnh (4.12) v (4.13) ch c gi tr khi m max > 1 . Trong phn ny, chng ta t cn bng nguyn liu cho nng t bo v thu c cc phng trnh khc nhau cho CSTF. Cc phng trnh tng t cng c th thu c bng cch t cc cn bng nguyn liu cho nng c cht v nng sn phm. 3. c lng cc thng s ng hc Monod ng thc tc sinh trng c trng v tc pha long ca CSTF trng thi n nh (phng trnh 4.9) tin li trong nghin cu nh hng ca cc thnh phn khc nhau ca mi trng ln tc sinh trng c trng. Bng cch o nng c cht trng thi n nh vi cc tc dng chy khc nhau, cc m hnh ng hc khc nhau c th c th nghim v gi tr ca cc thng s ng hc c th c c lng. Sp xp li phng trnh (4.9) c th thu c mi quan h tuyn tnh nh sau:

1

=

max

KS

1 1 + CS max

(4.14)

Trong : bng tc pha long (D) cho chemostat. Nu mt t bo nht nh tun theo ng hc Monod, th th 1 / theo 1 / CS s em li gi tr max v KS (bng cch c phn b chn v dc ca ng thng). th ny c u im cho thy mi quan h gia bin c lp (CS) v bin ph thuc . Tuy nhin, 1 / s tin ti nu nng c cht gim dn n trng lng vt qu mc o khi nng c cht thp v trng lng khng o khi cc nng c cht cao. Phng trnh (4.9) c th sp xp li a ra cc mi quan h tuyn tnh ng dng thay cho phng trnh (4.14) nhm c lng tt hn cc thng s trong nhng trng hp nht nh:Cng ngh t bo


Tai lieu chia C tai: wWw.SinhHoc.edu.vn se K CS

=

S

max

+

S

max

(4.15)

= max K S

CS

(4.16)

Tuy nhin, gii hn ca php tnh gn ng ny ( xc nh cc thng s ng hc) gp kh khn khi s dng CSTF. i vi trng hp vn hnh theo tng m, chng ta thm ch c th dng bnh tam gic lc trn my lc vn hnh nhiu m vi cc iu kin khc nhau trong cng mt thi gian. Vn hnh theo tng m trong ni ln men c khuy cng khng kh khn lm, do khng c cc kt ni i vo v i ra (ngoi tr b phn cung cp khng kh) v thi gian vn hnh ngn, t c nguy c ca s nhim bn h ln men. vn hnh CSTF, chng ta cn c cc ngun cung cp dinh dng v tch tr sn phm c kt ni v trng vi h ln men. Tc ca cc dng chy vo v ra khi h ln men cn c kim sot mt cch chnh xc. Thnh thong, vic kim sot tc dng chy ra c th gp kh khn do s to bt v kt khi ca cc t bo. Do thi gian vn hnh t nht mt vi ngy hoc thm ch c tun t ti trng thi n nh (cng gy ra s bin i tc pha long), cho nn lun c ri ro cao i vi h ln men do b nhim bn. Thng xuyn gp kh khn trong vic t ti trng thi n nh bi t bin ca t bo v kh nng thch nghi vi mi trng mi ca chng. Hn na, do hu ht cc h ln men quy m ln c tin hnh trong kiu tng m, cho nn cc thng s ng hc c xc nh bi nghin cu chemostat phi d bo c s sinh trng trong kiu ln men ny. Tuy nhin, bng chng (kim tra v xc minh) m hnh ng hc v c lng cc thng s ng hc bng cch vn hnh chemostat l phng php ng tin cy nht do iu kin mi trng khng thay i ca n. Cc s liu ca vn hnh theo tng m c th c dng xc nh cc thng s ng hc, cho d n khng phi l phng thc c gii thiu cao. Tc sinh trng c trng trong sut qu trnh vn hnh theo tng m c th c c lng bng cch o dc ca ng cong nng t bo theo thi gian cc im khc nhau. Nng c cht cn thit c o cng cc im ni m dc c c. Sau cc th theo cc phng trnh (4.14), (4.15) v (4.16) c th c xy dng xc nhCng ngh t bo


cc thng s ng hc. Tuy nhin, gi tr ca cc thng s thu c trong phng php ny cn thit c kho st cn thn xem chng c trong phm vi hp l cho cc t bo c kim tra hay khng. 4. Hiu sut ca CSTF Thng thng, hiu sut ca h ln men c hiu nh l s lng sn phm c sn xut trn mt n v thi gian v th tch. Nu dng chy vo l v trng (C X i = 0) th hiu sut sinh khi t bo bng C X / m , chnh l dc ca ng thng OAB ca ng cong CX theo m (Hnh 4.6).


10 8 6

CX4 2

C

B

A O0

0

D

2

m

4

6

Hnh 4.6. S thay i nng t bo v c cht nh l mt hm ca thi gian lu. Hiu sut bng dc ca ng thng OAB . ng cong c v bng m hnh Monod vi max = 0,935/gi; KS = 0,71 g/L; YX/S = 0,6; v C S i = 10 g/L.

Hiu sut im A bng hiu sut im B. im A nng t bo ca dng chy ra thp nhng thi gian lu li ngn, v th mi trng c th chy qua d dng hn. Ngc li, im B nng t bo ca dng chy ra cao nhng thi gian lu li di v th ch c mt lng nh ca mi trng chy qua. im A l vng khng n nh v rt gn vi im ra tri D, v v ch cn mt s dao ng nh trong thi gian lu cng c th em li mt s thay i ln trong nng t bo. Khi dc ca ng thngCng ngh t bo


tng ln th hiu sut s tng v di ca AB gim. dc ca ng thng s t gi tr cc i khi n l ng tip tuyn ca ng cong CX. V th, gi tr hiu sut cc i bng dc ca ng OC . Hiu sut cc i s t c im D. iu kin hot ng t hiu sut cc i CSTF c th c lng theo th bng cch dng ng cong 1 / rX theo CX. Hiu sut cc i c th thu c khi thi gian lu l ti thiu. V thi gian lu bng din tch ca hnh ch nht vi chiu rng CX v chiu cao 1 / rX trn ng cong 1 / rX theo CX, cho nn n s t ti thiu khi 1 / rX l ti thiu (Hnh 4.7).4


1 rX

3 2 1

0

2

CX

4

6

Hnh 4.7. Minh ha bng th CSTF vi hiu sut cc i. ng lin tc biu din cho m hnh Monod vi max = 0,935/gi; KS = 0,71 g/L; YX/S = 0,6; C Si = 10 g/L; v C X i = 0 .

iu cn lu l iu chnh cc phng trnh cho nng t bo v thi gian lu sao cho hiu sut t bo t cc i. Hiu sut t bo cho CSTF trng thi n nh vi cht dinh dng v trng l:

CX

m

= rX =

max C S C XK S + CS

(4.17)

Hiu sut t cc i khi drX / dC X = 0, sau khi thay th CS = CS i C X / YX / S vo phng trnh (4.17), ly tch phn theo CX v t phng trnh tng hp bng 0, chng ta thu c nng t bo ti u (C X ,opt ) cho hiu sut cc i nh sau:Cng ngh t bo


Tai lieu chia se tai: wWw.SinhHoc.edu.vn (4.18)C X , opt = YX / S CS i

+1

Trong :

=

K S + C Si KS

(4.19)

V: C S = C Si

CX nn nng c cht ti u (CS, opt) s l: YX / SC S ,opt = C Si(4.20)

+1

Thay phng trnh (4.20) vo phng trnh (4.17) thu c mt thi gian lu ti u (m,opt) nh sau:

m ,opt =

max ( 1)

(4.21)

5. So snh nui cy ca h ln men m v h ln men thng khuy lin tc Nh cp, thi gian lu cn thit nui cy m hoc PFF trng thi n nh t ti mt nng t bo nht nh l:

b = t0 +

CX

CX0

dC X rX

(4.22)

Trong : t0 l thi gian cn thit t ti pha sinh trng theo hm m. Din tch bn di ca ng cong 1 / rX theo CX, gia C X i v CX l bng b t1 nh c trnh by hnh 4.3. Mt khc, thi gian lu CSTF c biu din bi phng trnh (4.17) bng din tch hnh ch nht vi chiu rng C X C X i , v chiu cao

1 / rX .

Cng ngh t bo


V lieu chia theo C c dng hnh ch U nn chng ta c Tai ng cong 1/ rse tai: wWw.SinhHoc.edu.vnthX

X

c mt vi nhn xt cho h ln men n nh sau: - Hu ht cc h ln men sn xut l mt CSTF hot ng vi nng t bo m gi tr ca 1 / rX l ti thiu (Hnh 4.8 a) do n i hi thi gian lu ngn nht. - Nu nng cui cng ca t bo c hng ti trong pha tnh, th h ln men m l chn la tt hn CSTF, v thi gian lu cn thit cho nui cy m (Hnh 4.8 b) l ngn hn ca CSTF.

a

b

1 rX

1 rX

CX

CX

Hnh 4.8. Minh ha bng th thi gian lu c yu cu (vng ti) cho: (a) CSTF v (b) h ln men m.

II. Thu hi t boi vi hot ng lin tc ca PFF v CSTF, cc t bo tht thot cng vi dng chy ra (outlet) hn ch hiu sut ca h ln men. V th, hiu sut c th c ci thin bng cch thu hi (recycling) t bo t dng chy ra a tr li h ln men. 1. Thu hi t bo PFF PFF i hi s hin din ban u ca t bo trong dng chy vo (inlet) nh l mt h ln men m i hi a mu vo ban u. Phng thc kinh t nht cung cp t bo trong dng chy vo l thu hi mt phn ca

Cng ngh t bo


dng chy ra a tr li dng chy vo vi (hoc khng c) thit b tch ri t bo. Hnh 4.9 m t s thu hi t bo PFF. Khng ging nh CSTF, PFF khng i hi thit b tch ri t bo thu hi, v s hin din ca n khng lm tng ng k hiu sut ca h ln men. Phng trnh hiu sut ca PFF vi ng hc Monod c th c vit nh sau:


p V = = (1 + R ) F 1 + R

CX f

' CX

dC X = rX

CX f

' CX

( K S + C S )dC X max C S C X

(4.23)

Trong : p l thi gian lu da trn tc dng chy ca ton b h thng. Thi gian lu thc t trong h ln men ln hn p do tc dng chy tng ln nh thu hi t bo. (1 + R)F CX Cs RF CXR , Csf

CXi F Csi

B

CXf Csf CXL = 0 Csf

L

Hnh 4.9. S thu hi t bo PFF.

Nu hiu sut sinh trng l khng i th:' CS = CS

1

Y X/S

' (C X C X )

(4.24)

Thay phng trnh (4.24) vo trong phng trnh (4.23) cho CS v ly tch phn ta s c kt qu sau:

p max

CX K Y K Y C' = ' S X/S + 1 ln ' f + ' S X' / S ln S C C + C' Y 1+ R C X + C S YX / S C S f S X/S X X

(4.25)

Cng ngh t bo


Tai lieu C v C c th c c lng t s cn bng t bo v Trong : chia se tai: wWw.SinhHoc.edu.vn' X' S

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Giao Trinh Cong Nghe TB - Nguyen Hoang Loc(Da Nen Dung Luong)