n chuyn ngnh

CHNG 1: GII THIU

Do s gia tng lin tc gi thnh ca du th-ngun nng lng chnh trn th gii hin nay, rt nhiu ti nghin cu gn y quan tm n vic pht trin cc ngun nhin liu mi c gi tr v mt kinh t v mi trng. C th ni nhin liu sinh hc l mt ngun nhin liu y tim nng ha hn thay th cho ngun xng du sp cn kit trong tng lai, c bit l nhng nhin liu c sn xut thng qua qu trnh ln men t cc ngun nguyn liu ti to, in hnh l butanol. Hin nay butanol thng mi c sn xut ch yu t con ng ha du nhng kt qu nghin cu cho thy qu trnh ln men t vi sinh vt thu nhn butanol c rt nhiu trin vng. Qu trnh ln men sn xut butanol hay cn gi l qu trnh ln men Ethanol-Butanol-Acetone (ABE) c thc hin bi gingvi khun Clostridium, c bit l loi Acetobutylicum (Lin v Blaschek, 1983). Loi vi sinh vt ny c xem l lu i nht trong nghnh cng nghip ln men k kh bt buc. N c xp v tr th hai ch sau ln men ethanol t nm men Saccharomyces cerevisiae quy m sn xut. Qu trnh ln men ABE c thc hin rng ri trong cng nghip n na u th k 20 vi 66% sn lng tiu th trn ton th gii ca butanol c sn xut t phng php sinh hc (Drre, 2008). Tuy nhin, vi s ra i ca Th chin th hai v s pht trin leo thang ca ngnh cng nghip ha du, sn xut butanol t phng php sinh hc nhanh chng gim dn. n nm 1960, hiu qu thp ca qu trnh ln men ABE so ngnh cng nghip ha du cng vi cc chi ph cao hn khi s dng cc ngun carbohydrate lm c cht dn ti kt qu l vic xo b hon ton cc hot ng cng nghip t qu trnh ln men ny. Tuy nhin, khi gi du bt u tng t u nhng nm 1970 do "cuc khng hong du th" cng vi s suy gim dn v ngun du v hn na l s thc y x hi trn ton th gii nhn thc v cc vn mi trng ang trong tnh trng cp cu, lm cho con ngi quan tm n ngnh cng nghip sinh hc (Drre, nm 1998; Drre, 2008). K t , nhng n lc nghin cu ng k v qu trnh ln men ABE sn xut butanol c thc hin trong nhiu lnh vc, c th l trong ngnh cng ngh vi sinh vt, to ra cc chng t bin mi nhm nng cao sn lng dung mi, nghin cu thay i cc iu kin ln men nhm ci thin nng sut thp v nng butanol trong dch ln men. Ging nh trong mi qu trnh sinh hc khc, qu trnh ln men ABE th hin mt s hn ch v kinh t v kh nng cnh tranh so vi cc qu trnh sn xut bng phng php ha hc tinh khit. Nhng bt li chnh trong trng hp ny lin quan n con ng trao i cht kh phc tp bi cc vi khun sn xut butanol. Trong mt chu trnh ln men ABE tiu biu t ngun carbohydrate: axit butyric, propionic v acetic trc ht c sinh tng hp bi C. acetobutylicum (acidogenesis) trong giai on tng trng theo cp s nhn ca t bo. Khi vi sinh vt bc vo giai SVTH: Nguyn Phan Duy Nguyn 1

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on n nh s bt u giai on to thnh acetone, butanol v ethanol theo t l 3:6:1 tng ng (solventogenesis) (Fond et al, 1985.). Qu trnhchuyn pha t acidogenesis sang solventogenesis c iu khin bng cch thay i hoc lm gim pH (<5) cui giai on tng trng theo cp s nhn hoc tng nng acid butyric (> 2 gl-1) (Gottschalk v Morris, 1981; Gottwald v Gottschalk, 1985; Monot etal., 1984). Tuy nhin, qu trnh ln men trong thc t kh phc tp cn phi kim sot cht ch cc iu kin ln men t hiu qu cao (Chauvatcharin et al., 1998). Trong qu trnh ln men ABE truyn thng, mt t bo vi sinh vt thp trong mi trng ln men lm hiu sut sinh tng hp butanol t glucose ni chung l kh thp thng khong 15% (w / w) v him khi vt qu 25% (w / w). Ngoi ra, vic sinh tng hp butanol cn b gii hn bi nhiu yu t c ch khc, trong butanol l yu t chnh gy c ch ngc ln s tng trng ca t bo lm ngng qu trnh ln men. Trong thc t, vi khun kh c th duy tr hot tnh khi nng butanol trong dch ln men xp x 10 gl-1. Kt qu l nng butanol trong dch ln men ABE thng xp x 13 gl-1. Do , vic sn xut butanol t qu trnh ln men ABE truyn thng cho hiu qu kinh t rt thp. (Maddox, 1989). Ni dung bi vit ny s gii thiu chung v butanol cng nh cc ng dng ca n trong thc tin. Da trn cc nghin cu gn y v qu trnh ln men tnh sn xut butanol s trnh by mt s chng Clostridium c phn lp c kh nng sinh tng hp v chu c nng butanol cao. Gii thiu cc ngun c cht khc nhau m vi sinh vt c th s dng ln men cng nh cc yu t nh hng ti qu trnh ln men to butanol, qua c th so snh hiu sut sinh tng hp butanol. Trnh by mt s kt qu nghin cu t c khi ln men s dng t bo vi khun c nh trn mt s cht mang. Cc phng php tch chit butanol t dch sau ln men cng s c gii thiu trong bi vit ny.

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CHNG 2: BUTANOL
2.1. Gii thiu chung
Butanol (danh php IUPAC, 1-butanol; CAS no.71-36-3) cn c gi l ru butylic, n-butanol hoc methylolpropane, l mt ru gm 4 cacbon c cng thc phn t C4H9OH (KLPT = 74,12 g.mol-1). Butanol l mt cht lng khng mu, d chy, k lng, c mi thm nh chui hng v c mi cn mnh. Khi tip xc trc tip n c th gy kch ng mt v da.Hi ca n c tc dng kch thch vo nim mcmng v c kh nng gy m khi ht phi nng cao.Butanol hu nh ha tan hon tonvi cc dung mi hu c ph bin, nhng li t ha tan trong nc (Lee v cng s, 2008a).

Hnh 1: Cu trc khng gian ca butanol Bng 1: Tnh cht vt l ca n- butanol

Khi lng phn t Dng tn ti T trng Nhit nng chy Nhit si tan trong nc Nhit nh la Nhit t bc chy Flash point Nhit ha hi Nhit t chy p sut hi 20oC

74.12 g mol-1 Dung dch lng mu vng 0.810 0.812 90 C, 183 K, -130 F 118 C, 391 K, 244 F 9.0 ml/100 ml (7.7 g/100 ml 20C) 3537 oC 343345 oC 2529 oC 43.8 kJ mol-1 198.2 kJ mol-1 0.63 kPa

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 n chuyn ngnh 2.2. ng dng ca butanol trong thc tin

Mt trong nhng ng dng u vit ln ca biobutanol (butanol sinh hc) l c s dng pha trn vo xng du lm nhin liu chnh cho cc ng c. Trong khi ethanol nhn c rt nhiu s quan tm nh l mt ngun nhin liu h tr bi nhiu l do (Hansen et al., 2005 v Niven, 2005) th butanol li cho thy n c th l mt la chn tt hnso vi ethanol v c cc tnh cht ha hc v vt l ph hp.
Bng 2: Bng so snh tnh cht ca butanol vi xng v ethanol

Tnh cht Nhit si (oC) T trng 20C (g/ml) Kh nng tan trong 100g nc Mt nng lng (MJ.l-1) Nhit bay hi (MJ/kg) Nhit dung ring (kJ/kmol.K) Ch s Octane nht (10-3 Pa.s)

Butanol 117118 0.8098 Khng tan 2729.2 0.43 178 96 2.593

Xng 27221 0.70.8 Khng tan 32 0.36 160300 9199 0.240.32

Ethanol 78 0.7851 Tan 19.6 0.92 112.3 129 1.078

Theo Shapovalop v Ashkinazi, (2008) v Drre, (2007) butanol th hin nhiu u im vt tri hn so vi ethanol trong vic thay th ngun nhin liu xng du, c th: - Gi tr nng lng ca butanol l 29.2 MJ/L trong khi gi tr nng lng ca ethanol ch l 19.6 MJ/L v ca xng l 32 MJ/L th nng lng ca butanol nhiu hn ethanol khong 25%. - Da vo p sut hi c th thy butanol s dng an ton hn so vi ethanol do butanol t bay hi hn ethanol v xng. - Butanol t gy n mn hn so vi ethanol, khng nh hngn cc thit b cha ng nh b cha, ng ng, my bm khi s dng lu di. - Kh nng ha tan thp trong nc ca butanol cho php n c th c pha trong xng du nng cao hn bt k loi nhin liu sinh hc no m khng s b tch pha lm xut hin nc, trong khi ethanol ch c th c pha thm vo xng khong 25% vch c gi trong mt thi gian ngn s dng. p sut hi ca butanol (4 mmHg 20C) l thp hn so vi ethanol 11 ln (45 mmHg 20C) cho php n c th pha trc tip vo xng m khng cn quan tm n s bay hi. - Trong qu trnh t chy, butanol khng thi ra bt k hp cht kh no cha sulphur v cc oxit nit, y l mt u im vt tri v mi trng so vi cc ngun nhin liu khc. SVTH: Nguyn Phan Duy Nguyn 4

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Vi nhng u im trn cng vi mt ngun c cht phong ph c th c s dng trong qu trnh ln men ABE, biobutanol s l mt tim nng ln trong vic thay th ngun nhin liu xng du ang cn kit v gp phn lm gim hiu ng nh knh. Bn cnh vai tr l nhin liu sinh hc cho ng c, butanol thc s l mt ha cht quan trng c s dng vi lng ln trong cc ngnh cng nghip. Gn mt na sn lng butanol c sn xut trn ton th gii c s dng lm dung mi dng butyl acrylate v este methacrylate trong sn xut cc loi sn nhenamel,nitrocellulose; keo dn, cht n hi, cng ngh dt may, sn xut si, nha, sn xut knh an ton, cht ty ra. Cc hp cht quan trng c ngun gc t butanol nh butyl ete glycol, butylacetate,amin butyl,nha amin v cht do. N cng c s dng lm dung mi trong ngnh cng nghip m phm nh nc hoa, cc sn phm trang im cho mt, sn mng tayv c dng cho vic sn xut cc loi thuc khng sinh, vitamin v hormone.

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CHNG 3: VI SINH VT TRONG LN MEN SN XUT BUTANOL CLOSTRIDIUM

3.1. c im hnh thi
Clostridium l mt ging trc khun Gram dng, thuc ngnh Firmicutes. y l nhng vi khun k kh bt buc, c kh nng sinh nha bo khi mi trng sng bt li. i din tiu biu l loi Clostridium acetobutylicum c dng hnh que thng, kch thc khc nhau t 0,5-1,5 1,5-6 m (Robinson, 2000). Clostridium acetobutylicum l vi khun Gram dng khi ang pht trin trong giai on logarite trong canh trng,v chuyn thnh Gram m khi bc sang giai on n nh v sinh bo t, ln men d dng, di ng bng roi. Trong qu trnh hnh thnh bo t, t bo phnh ln r rt v tch tr cc ht, mt polysaccharide cung cp ngun carbon v nng lng trong giai on solventogenesis. Bo t c hnh thnh c dng bu dc. Cc vi sinh vt thuc ging Clostridium ln men thng c xp thnh bn nhm khc nhau da trn c tnh sinh ha v di truyn ca chng (Woods, 1995) bao gm: Clostridium acetobutylicum, C.beijerinckii,C.saccharobutylicum, v C. saccharoperbutylacetonicum. Hai loi c bit n vi nhiu u im vt tri l C. acetobutylicum v C. beijerinckii (trc y gi l C. butylicum) v hu ht cc ti liu nghin cu v ln men ABE u da trn hai loi ny.

Hnh 2: Hnh nh ca C.acetobutylicum v C.beijerinckii trn knh hin vi in t (SEM)

3.2. c im sinh l ca vi khun Clostridium

3.2.1. Nhu cu dinh dng - Ngun Carbon: Qu trnh tng hp acetone, butanol, and ethanol t cc loi thuc ging Clostridium u bt ngun t c cht l carbohydrate. Nhm vi sinh vt ny c kh nng s dng ngun dinh dng rt a dng, c th s dng gn nh hon ton cc loi ng nh glucose, fructose, mannose, sucrose, lactose v s dng mt phn cc ng xylose, SVTH: Nguyn Phan Duy Nguyn 6

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galactose, arabinose, raffinose Nhiu nghin cu cho thy glucose vn l loi ng tt nht cho Clostridium s dng. - Ngun Nit: Nit l thnh phn quan trng ca protein v enzyme, c th c cung cp vo mi trng nhn ging v ln men cho vi sinh vt s dng di dng cc mui NH4+ v NO3hay cc hp cht hu c phc tp t cht chit nm men, sn phm thy phn t u nnh Vic b sung nit l cn thit cho vic to sinh khi vi sinh vt, tuy nhin nu b sung qu nhiu c th lm c ch vic sinh tng hp sn phm. - Khong: S pht trin t bo ca vi khun ph thuc vo cc mui khong trong mi trng nh Mg2+, Fe2+, Mn2+ v K+ . Chng c cung cp di dng cc mui nh MgSO4, MnSO4, FeSO4, KCl, KH2PO4 v K2HPO4;Nu nng ca Mg2+ v Mn2+ trong mi trng qu cao s gy c ch n s sinh trng ca t bo. - Yu t sinh trng: Cng ging nh cc loi vi khun khc, qu trnh sinh trng v pht trin ca Clostridium i hi cung cp mt s yu t sinh trng nh biotin, thiamine hydrochloride, p-aminobenzoic, resazurin Cc yu t ny hin din trong mi trng nng tng i thp t 0.1-1 mg/L, nhng nh hng nhiu n hot ng trao i cht ca vi khun trong qu trnh ln men. Cc loi mi trng thng c s dng nui cy v nhn ging vi khun Clostridium: - Mi trng P2: l mi trng ti u cho tt c cc loi ca Clostidium pht trin, c s dng lm mi trng gi ging v ln men vi cc nng glucose khc nhau. Thnh phn Glucose MgSO4.7H2O MnSO4.H2O FeSO4.7H2O KCl KH2PO4 K2HPO4 Ammonium acetate p-aminobenzoic acid Thiamin Biotin Dch trch nm men SVTH: Nguyn Phan Duy Nguyn Hm lng (g/L) 60 20 1 1 1 50 50 220 0.1 0.1 0.001 1 7

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Mi trng Tryptone-yeast extract-sucrose(TYS): Thnh phn Sucrose Na2SO4 K2HPO4 Dch trch nm men Biotin p-aminobenzoic acid Tryptone Antifoam C Hm lng (g/L) 60 0.18 3.48 5.0 0.01 0.01 1.0 0.5 ml

Mi trng CAB: l mi trng c s dng lm mi trng gi ging v ln men ti u cho loi Clostridium acetobutylicum Thnh phn Glucose Dch trch nm men Tryptone K2HPO4 KH2PO4 DL-asparagine MgSO4.7H2O MnSO4.H2O NaCl FeSO4.7H2O resazuin Hm lng (g/L) 50 4 1 0.7 0.7 0.5 0.1 0.1 0.1 0.015 0.002

3.2.2. iu kin nui Nhit hot ng ca Clostridium nm trong khong t 29-37oC, ty thuc vo tng loi m c nhit ti thch khc nhau.Clostridium acetobutylicum c nhit ti thch nm trong khong 35-37 oC, trong khi i vi loi Clostridium beijerinckii li l 31-32 oC v Clostridium saccharobutylicum l 30 oC. a s cc qu trnh nhn ging v ln men s dng vi khun Clostridium acetobutylicum u c duy tr 35 oC. pH l yu t quan trng nh hng ln n kh nng sinh trng v to sn phm ca Clostridium. Khong pH ti thch ca cc loi thuc ging Clostridium nm trong khong t 5.0-6.5, bt k mt s thay i no lm gim pH di 4.5 u c kh nng gy c ch s sinh trng ca vi khun. a s cc mi trng ban u u c chnh v pH=6.5, tuy nhin cng vi qu trnh sinh trng th vi khun li sinh ra cc hp cht SVTH: Nguyn Phan Duy Nguyn 8

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nh axit butyric, propionic v acetic lm gim pH mi trng. V vy, cn kim sot v iu chnh pH trong sut qu trnh sinh trng ca vi khun.

3.3. Cc loi vi sinh vt c dng trong sn xut butanol

Nh gii thiu trn, cc loi c kh nng sinh tng hp butanol bao gm bn loi chnh l: Clostridium acetobutylicum, C. beijerinckii, C. saccharobutylicum v Clostridium saccharoperbutylacetonicum. Clostridium acetobutylicum l loi c c tnh di truyn khc bit nht v t lin quan n ba loi cn li. Nhng chng thuc loi ny pht trin tt trn cc mi trng c cht t dch thy phn tinh bt nn c s dng ph bin quy m cng nghip sn xut dung mi. Clostridium acetobutylicum l loi c nghin cu trong sn xut dung mi nhiu nht, nhiu chng c pht trin t loi ny cho kh nng tng hp butanol kh cao. Cho n nay, mt trong s nhng kt qu ln men tt nht l t chng C. acetobutylicum PCSIR-10 c kh nng khng butanol, s dng c cht l r ng ma c thc hin bi Syed (1994). Theo , tng nng dung mi t c l 19.2 g/L vi hiu sut l 34%. Loi Clostridium beijerinckii lin quan nhiu n hai loi C. saccharobutylicum v C. saccharoperbutylacetonicum. C ba c bit n nh l cc vi sinh vt saccharolytics dng cc loi ng lm c cht ln men v hu ht cc chng ln men ng quy m cng nghip u thuc loi C. beijerinckii. Mc d C.acetobutylicum c nghin cu nhiu nht trong sn xut dung mi nhng C. beijerinckii li cho thy nhiu u im v c tim nng trong vic ng dng sn xut quy m cng nghip. C. beijerinckii c mt khong pH ti thch rng trong vic sinh trng v to dung mi. Bn cnh C. beijerinckii l mt i tng tim nng trong vic ng dng cc k thut chuyn gene nhm to ra cc chng c kh nng s dng a dng cc ngun carbohydrate (Ezeji, et al., 2004). Vi C. beijerinckii, ngi ta cho rng cc chng ny c kh nng chu c tc ng ca acid nng cao nn thch hp trong cc qu trnh ln men lin tc hn so vi cc chng t C.acetobutylicum (theo Grube & Gapes, 2002; Zverlov v cc cng s, 2006). Trong nghin cu ca Ezeji v cc cng s (2004), chng C. beijerinckii BA101 s dng rt linh hot nhiu ngun c cht khc nhau v cho nng dung mi t t 14.8-26.1 g/L vi nng sut t 37-50%. C kh t nghin cu v hai loi C. saccharobutylicum v C. saccharoperbutylacetonicum c s dng ln men ABE. Shaheen v cng s (2000) tp hp nhng chng c s dng ln men v nhn thy rng chng pht trin tt hn trn mi trng glucose v r ng ma so vi trn mi trng dch ng. Kt qu ln men tt nht c thc hin trn chng C. saccharobutylicum BAS/B3/SW/336(S),

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khi s dng mt r ma vi nng dng ln men l 6.5%. Nng trung bnh t 19.6g/L vi hiu sut 30%.
Bng 3: So snh kh nng ln men ca mt s chng c phn lp

Mi trng/c cht ( 6% ng ln men) C. acetobutylicum PCSIR-10b R ng PCSIR-5b R ng a ATCC 4259 R ng a ATCC 824 R ng d ATCC 824 Glucose BA 101c BA 101c NCP P260a NCP P260a Glucose R ng R ng* R ng

Chng

Nng cc dung mi (g/L)

Hiu sut (%)

Nng sut ( g/L.h )

A:B:E

BAS/B3/SW/ R ng* a 336(S) NCP P108a R ng* 18.6 NCP P258a R ng 18.3 C. saccharoperbutylacetonicum N1-504a R ng 15.6

34.0 30.0 15.8 13.0 42.0 C. beijerinckii 24.2 42.0 22.8 39.0 21.9 33.4 18.9 31.5 C. saccharobutylicum 19.6 30.0 28.6 30.5 26.0

19.2 15.2 9.5 7.8 20.6

0.42 0.24 0.58 0.34 0.19 -

1.8:95.3:2.9 5.3:79:15.7 20.6:66.5:26.2 17.8:81:1.2 18.4:80.3:1.3 -

*: 6.5% cc loi ng c th ln men a: Shaheen v cng s (2000) b: Syed (1994) c: Ezeji v cng s (2004) d: Roffler v cng s (1987)

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CHNG 4: QU TRNH LN MEN ACETON-BUTANOLETHANOL

4.1. S trao i cht trong qu trnh ln men ca vi khun Clostridium
Trong qu trnh ln men ca Clostridium, hai giai on sinh trng ring bit xy ra: giai on acidogenic v giai on solventogenic. Giai on acidogenic xy ra u tin, khi Clostridium thc hin qu trnh ln men butyrat in hnh v pht trin trn mi trng c cht l tinh bt hoc ng. Cc sn phm chnh trong giai on ny l butyrat (butyric acid), acetate (acid acetic), carbon dioxide v hydrogen. Ethanol v acetone c hnh thnh vi mt lng nh. Vic sinh ra cc hp cht c tnh acid lm gim pH ca mi trng ln men xung thp, c th lm cht t bo vi khun. trnh b tiu dit, vi khun bt u mt s thay i chuyn ha din ra cui giai on tng trng theo cp s nhn. iu ny cng nh du s kt thc ca giai on acidogenic v bt u giai on solventogenic. Cc acid bi tit c s dng trong giai on ny v c chuyn i thnh cc sn phm trung tnh, bao gm aceton v butanol (theo t l thng thng l 1:2). S chuyn i butyrate v acetate thnh dung mi lm tng pH tr li, ngha l cc t bo c th tr li hot ng trao i cht trong mt thi gian di hn. Tuy nhin, cc dung mi ny li cng gy c ch cc t bo vi khun, c bit l butanol. Dung mi gy bt hot cc protein mng t bo v ph hy cc mng ca t bo, do c mt gii hn v nng dung mi ti a c th t c trong qu trnh ln men (khong 2% v khi lng). Qu trnh trao i cht din ra c th theo s nh sau:

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Hnh 3: S trao i cht trong qu trnh ln men ca Clostidium

Qu trnh tin x l sinh khi cha lignocellulosic hoc thy phn tinh bt c thc hin nh cc enzyme -amylase, -amylase, pullulanase, enzym glucoamylase,glucosidase do vi khun tit ra, (1,2). S thy phn cellulose nh enzyme cellulase, glucosidase to ra glucose . Glucose c hp thu bi h enzyme phophotransferase (PTS) v chuyn i thnh pyruvate theo con ng EMP (Embden-Meyerhof-Parnas), (3,6). S thy phn hemixenlulose to ra cc ng xylose / arabinose c hp thu, di tc dng ca enzyme transketolase-transaldolase to thnh fructose 6-phosphate v glyceraldehydes 3-phosphate theo con ng EMP, (4,5). Sau pyruvate c ferrodoxin oxidoreductase chuyn ha thnh Acetyl-CoA, cht ny sau c chuyn ha thnh Acetoacetyl- CoA nh enzyme thiolase, (7,10). S to thnh acetate t AcetylCoA c thc hin nh cc enzyme phosphate acetyltransferase v acetate kinase, (8) v to ethanol nh cc enzyme l acetaldehyde dehydrogenase v ethanol dehydrogenase, (9). Di tc dng ca enzyme Acetoacetyl-CoA transferase, Acetoacetyl-CoA c chuyn ha thnh acetoacetic. S loi b CO2 ca acetoacetic khi c xc tc bi enzyme acetoacetic decaboxylase dn n s to thnh acetone pH ti u vo khong 5.0. S to thnh acetone qua acetoacetyl-CoA khng tiu th NADPH nn vi khun ch c th to acetone mt mc gii hn, (11). SVTH: Nguyn Phan Duy Nguyn 12

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T nhnh acetoacetyl-CoA nhn H2 nh enzyme - Hydroxibutiryl CoA hydrogenase to thnh - Hydroxibutiryl CoA . Sau loi H2O di tc dng ca enzyme crotanase to thnh crotonyl-CoA. Nh enzyme butyryl-CoA hydrogenase chuyn thnh ButyrylCoA, (12). Butyryl-CoA chuyn nhm Co-A to thnh Butyric nh enzyme Butyric transferase, (13). Mt khc butyryl-CoA va nhn H2 v ng thi tch nhm Co-A to thnh Butyraldehit, cht ny sau nhn H2 to thnh butanol, (14).

4.2. Ln men ca vi khun t do

4.2.1. Kh nng s dng cc ngun c cht trong ln men a. Kho st kh nng s dng cc loi ng n pentose v hexose kho st kh nng s dng cc loi ng khc nhau ca Clostridium, Nasib Qureshi v cc cng s (2006) thc hin cc qu trnh ln men vi cc loi ng l glucose, xylose, arabinose, galactose s dng chng Clostridium acetobutylicum P260. Trong th nghim ny, 60g mi loi ng trn s c pha vo 1L mi trng P2 thc hin ln men song song trong vng 72h nhit 35oC. Kt qu thu c sau 72h ln men, canh trng tng hp c 21.37, 11.12, 10.09, v 15.18 g/L ABE tng ng vi cc dch ln men ca glucose, xylose, galactose v arabinose (bng 4).
Bng 4: Cc thng s ca qu trnh ln men ABE trong 72h s dng cc loi ng glucose, xylose, galactose v arabinose

Nng ABE (g/L) Nng acid (g/L) S tiu th ng (g/L) Hiu sut Nng t bo (g/L) Nng sut tng hp ABE (g/L.h)

Glucose 21.37 3.74 59.4 0.36 2.50 0.30

Xylose 11.12 4.08 36.2 0.31 1.41 0.15

Galactose 10.09 4.34 31.9 0.32 1.88 0.14

Arabinose 15.18 3.30 37.0 0.41 1.94 0.21

Trong sut qu trnh ln men, lng glucose, xylose, galactose v arabinose c s dng tng ng l 59.4, 36.2, 31.9 v 37.0 g/L. Mc d Clostridium acetobutylicum P260 c th s dng c c bn loi ng nhng kt qu cho thy canh trng vi sinh vt s dng glucose nhiu hn nhng loi ng khc. Glucose hu nh khng cn trong dch sau ln men, trong khi galactose c s dng t nht trong bn loi ng (37.0 g/L). Tuy nhin, hiu sut ln lt t 0.36, 0.31, 0.32 v 0.41 tng ng ca cc dung dch glucose, xylose, galactose v arabinose. Nh vy, arabinose d khng ln men c hon ton nhng li cho hiu sut sinh butanol cao nht. Nng t bo vi sinh vt t SVTH: Nguyn Phan Duy Nguyn 13

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dch ln men ca glucose cng cho kt qu cao nht (2.50 g/L), iu ny cho thy glucose to mi trng thun li nht cho s pht trin ca t bo vi khun. nh gi tc s dng glucose, xylose, galactose v arabinose trong qu trnh ln men, Nasib Qureshi v cc cng s thc hin ln men hn hp 15g mi loi ng trn pha trong 1L mi trng P2 35oC. Kt qu c biu din trn th hnh 4:

Hnh 4: Qu trnh ln men t hn hp cha 15 g/L cc loi ng glucose, xylose, galactose v arabinose s dng Clostridium acetobutylicum P260. (A): nng sn phm ca qu trnh ln men cc thi im khc nhau, () Acetone, (): butanol, (): ethanol, ( ): acid acetic, (): acid butylic. (B): nng ng cn li trong dch ln men cc thi im khc nhau, (): glucose, (): xylose, (): arabinose, (): galactose.

Hnh 4B cho thy, glucose c s dng nhanh hn cc loi ng cn li, tt c 15g glucose c s dng ht trong 35h ln men. S tiu th arabinose ngng li sau 53h ln men v cn st li hn 0.6 g/L. Qu trnh s dng xylose v galactose din ra kh chm, sau 70h ln men 8.7 g/L xylose v 9.4 g/L galactose vn cha c s dng ht. Nng ABE t c sau 70h ln men t hn hp ng trn l 16.25 g/L vi nng butanol t xp x 12 g/L, nng sut l 0.23g/L.h v hiu sut l 0.40. Vi nhng kt qu ny cho thy Clostridium acetobutylicum P260 tuy c th s dng c nhiu loi ng khc nhau nhng vic tiu th cc loi ng xylose v galactose din ra kh chm, hm lng ng st cn nhiu lm gim nng butanol. Pht trin cc chng mi t loi Clostridium acetobutylicum c kh nng s dng ng xylose v galactose tng ng vi glucose v arabinose s mang li hiu qu cao hn trong ln men. Mt nghin cu khc trn loi Clostridium beijerincki, kho st kh nng s dng cc loi ng n khc nhau. Ezeji v cng s (2007) s dng chng Clostridium beijerinckii BA101 ln men cc loi ng glucose, xylose, cellobiose, mannose, arabinose v galactose. Dch ln men c chun b trong mi trng P2, 55g mi loi

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ng s c pha vo 1L mi trng P2 35oC khng kim sot pH trong sut qu trnh ln men. Kt qu c biu din trn hnh 5:

Hnh 5: Nng dung mi ABE t c khi ln men cc ng n (55g/L: glucose, xylose, cellobiose, mannose, arabinose v galactose) s dng Clostridium beijerinckii BA101. Qu trnh ln men c thc hin sau 60h th Clostridium beijerinckii ngng to sn phm. Nng dung mi ABE t c cao nht trong dch ln men ca cellobiose 19.1 g/L, t hiu sut 0.35. Trong khi , dch ln men t glucose cho nng dung mi cui l 17.8 g/L t nng sut 0.30 g/L.h, nng cc acid trong dch ln men ch cn 2.5 g/L. Nng sut to dung mi ca cc loi ng cn li dao ng trong khong 0.23-0.32 g/L.h. Galactose tip tc th hin l loi ng cho hiu sut thp khi c s dng ln men ABE khi cho nng dung mi thp nht. Nng acid trong dch ln men ca galactose kh cao (>7 g/L), iu ny cho thy s gim kh nng hp thu v chuyn ha cc acid ca vi khun trong dch ln men ca galactose, t lm gim nng dung mi ABE cui. nh gi kh nng ln men hn hp cc loi ng khc nhau ca Clostridium beijerinckii, Ezeji v cng s thc hin qu trnh ln men hn hp ng gm glucose, mannose, arabinose v xylose b sung vo dch ln men vi t l tng ng l 5:1:2:4. Sau 84h ln men (khi qu trnh ln men dng li), nng dung mi ABE t c l 17.9 g/L vi nng butanol t ti a l 13.9 g/L ( hnh 6). Mc d lng butanol v tng lng ABE c to ra xp x nh trong qu trnh ln men t ng glucose nhng thi gian ln men li di hn ( 84h so vi 60h ca glucose), do dn ti kt qu l tc sinh tng hp sn phm tng i thp (0.21 g/L.h).

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Hnh 6: Ln men ABE t hn hp ng glucose, mannose, arabinose v xylose vi t l 5:1:2:4 bi Clostridium beijerinckii BA101. (A): nng cc sn phm t c trong sut qu trnh ln men. (B): nng cc loi ng cn li. HAc: acid acetic, HBu: acid butylic, HAB: tng acid acetic v acid butylic.

Trong sut qu trnh ln men, nng cc acid trong dch ln men tng i thp (hnh 6A), iu ny cho thy qu trnh ln men cho hiu qu tt. Tt c cc loi ng c s dng ng thi trong sut qu trnh ln men mc d tc s dng l khc nhau (hnh 6B). Kh nng s dng ng glucose nhanh tip tc c th hin loi Clostridium beijerinckii, iu ny cho thy glucose l c cht a thch trong qu trnh trao i cht ca hu ht cc loi. Khi nng glucose dn cn kit, loi ng tip theo c Clostridium beijerinckii s dng nhiu hn l xylose. C th sp xp th t u tin s dng cc loi ng trong hn hp ca Clostridium beijerinckii theo th t l glucose > xylose > arabinose > mannose. Kt thc qu trnh ln men, nng cc loi ng cn li khng c s dng trong dch ln men l 0 g/L glucose, 4.0 g/L xylose, 3.3 g/L arabinose v 1.7 g/L mannose. Nh vy, s khc nhau v kh nng s dng cc loi ng gia hai loi C.acetobutylicum v C.beijerincki c ghi nhn: sau glucose th SVTH: Nguyn Phan Duy Nguyn 16

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C.acetobutylicum th hin kh nng s dng ng arabinose tt hn nhiu so vi ng xylose. so snh r hn v kh nng s dng ng glucose v ng pentose (xylose) trn loi Clostridium beijerinckii, N. Qureshi v cng s (2008) tin hnh hai th nghim ln men. Chng C.beijerinckii BA101 c s dng, hai mi trng ln men ln lt cha 25 g/L glucose v 25 g/L xylose. Sau 60 gi ln men, tt c ng glucose c s dng v to ra 9.9 0.4 g/L ABE trong c 6.1 g/L butanol. Nng acetone v ethanol ln lt l 3.9 v 0.4 g/L. Nng sut t c l 0.17 0.006 g/L.h v hiu sut l 0.39 0.025. Trong khi qu trnh ln men t ng xylose cho nng ABE t 9.6 0.4 g/L vi nng sut trong 60 gi ln men t 0.16 0.01 g/L.h v hiu sut l 0.39 0.011.

Hnh 7: Nng cc sn phm thu c trong qu trnh ln men ABE t chng C.beijerinckii BA101, (A): Glucose, (B): Xylose

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Kt qu ln men cho thy nng ABE thu c t glucose v xylose xp x bng nhau, N. Qureshi v cng s kt lun kh nng s dng ng xylose ca C.beijerinckii l tng ng so vi glucose. Nhng kt qu trn cho thy cc loi Clostridium u c kh nng s dng a dng nhiu loi ng khc nhau (hexose v pentose) c bit pht trin rt tt trn c cht l ng glucose. Trn mi trng l hn hp nhiu loi ng khc nhau, Clostridium cng cho thy kh nng ln men kh tt. iu ny chng t vic s dng cc ngun ph phm nng nghip c cha sinh khi lignocellulosic (biomass) lm c cht cho qu trnh ln men ABE l kh thi. Khi thy phn cc ngun sinh khi cha lignocellulosic s cho ra hn hp nhiu loi ng, vic thy phn c th tch ring hoc thc hin ng thi vi qu trnh ln men. Nhiu nghin cu thc hin trn cc ngun c cht khc nhau vi mc ch lm gim chi ph cho qu trnh ln men tng hp butanol. Vic x l cc ngun biomass trc khi s dng lm dch ln men l kh phc tp, c th nh hng v lm gim nng dung mi t c. V vy, vic ti u ha cc iu kin ln men vi tng loi c cht t hiu qu cao nht l tng i phc tp. b. Ln men t bt sn (cassava flour CF) Trong mt nghin cu ca Leonardo v cng s (2010), bt sn c s dng lm c cht cho qu trnh ln men s dng chng Clostridium beijerinckii BA101. Bt sn s c x l bng hai phng php s dng enzyme (-amylase v -glucoamylase)v acid (HCl). Bt sn c pha vo nc ct thnh dung dch huyn ph cc nng 60, 80 g/L. Vi phng php x l bng acid (CT: acid chemical treatment), dung dch HCl 1M c b sung vo dung dch CF n khi pH t 1.5. Qu trnh thy phn thc hin trong 2h nhit 121oC, sau c lm lnh n 4oC ngng qu trnh thy phn. Vi phng php x l bng enzyme (E1), CaCl2 (1g/L) c b sung vo dung dch huyn ph t 40 mgCa/L, iu chnh pH n 6.5 bng NaOH 1M. -amylase c b sung vo ( 1ml/kg bt) v thc hin qu trnh h ha 93oC trong 2h. Sau , b sung glucoamylase (1.7 ml/kg bt) thc hin qu trnh dch ha 60oC, tc khuy o 150 rpm trong 1h. Cui cng hn hp c a v 40oC gi trong 39h trc khi ln men. Qu trnh ln men c thc hin cc nhit 35 v 40oC vi nng CF l 60 v 80 g/L. Kt qu thu c th hin trong bng 5:

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Bng 5: Kt qu ln men ABE ca chng Clostridium beijerinckii BA101 trn c cht bt sn cc iu kin khc nhau.

Nhit (oC)

Nng CF (g/L)

Phng php thy phn CT CT CT CT ET ET ET ET CT CT CT CT ET ET ET ET

Butanol (g/L)

Ethanol (g/L)

Acetone (g/L)

35 40 35 40 35 40 35 40 35 40 35 40 35 40 35 40

60 60 80 80 60 60 80 80 60 60 80 80 60 60 80 80 Trung bnh

21.87 26.73 27.54 17.82 12.53 31.59 19.44 22.68 19.44 25.92 20.25 18.63 16.20 24.30 11.34 18.63 20.81

14.58 1.70 20.25 6.32 2.75 10.21 8.91 2.84 1.54 3.56 9.72 0.01 12.15 7.70 0.01 0.01 6.39

0.01 7.19 0.01 0.01 0.01 2.69 0.01 8.14 6.87 11.85 8.69 5.14 0.01 0.01 8.69 7.51 4.18

Tng lng dung mi (g/L) 36.46 35.62 47.80 24.15 13.29 44.49 28.36 33.66 27.85 41.33 38.66 23.78 28.36 32.01 20.04 26.15 31.38

Nh vy, vi kt qu thu c t bng 5, nng butanol t c cao nht l 31.59 g/L ng vi qu trnh ln men 40oC s dng nng CF l 60g/L x l bng enzyme. Nng butanol t c thp nht l 11.34 g/L khi ln men 25oC, nng CF l 80 g/L x l bng enzyme. Kt qu ny chng t, qu trnh x l c cht trc khi ln men cng nh nng c cht ban u s dng c nh hng ng k n kh nng sinh tng hp butanol ca vi khun. Khi thy phn bng phng php acid, cc tp cht ln trong dung dch HCl c th gy c ch kh nng hot ng ca vi khun lm gim hiu sut tng hp butanol. Nng butanol trung bnh t 20.81 g/L sau 96h ln men, tc sinh tng hp butanol tng ng l 0.22 g/L.h. c. Ln men t X bp ( corn fiber) X bp l ph phm t tri bp, khng c gi tr v mt kinh t c s dng lm thc n gia sc. Tuy nhin trong x bp cha ti 60-70 % cc thnh phn carbohydrate, 30% trong s l arabinoxylan hoc hemicellulose. Nhiu nghin cu s dng x bp nh l ngun c cht cho qu trnh ln men sn xut butanol. Trong nghin cu ca N. Qureshi v cng s (2008) s dng chng Clostridium beijerinckii BA101, x bp trc khi ln men c thy phn bng hai phng php s dng enzyme v acid SVTH: Nguyn Phan Duy Nguyn 19

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sulfuric. Cc tc gi so snh kh nng tng hp butanol t dch thy phn ca hai phng php trn. Kt qu thu c t qu trnh ln men dch thy phn bng acid sulfuric ( nng ng t c sau qu trnh thy phn l 29.8 g/L; glucose chim 4.3 g/L) cho bi bng sau:
Bng 6: Nng sn phm to thnh trong qu trnh ln men ABE t x bp c thy phn bng acid sulfuric.

Thi gian (h) 0 24 54 72 96

Nng t bo (g/L) 0.02 0.58 0.75 0.60 0.31

Acetone (g/L) 0.0 0.2 0.1 0.1

Butanol (g/L) 0.1 1.4 1.0 0.9

Ethanol (g/L) 0.1 0.1 0.2 0.2

Tng ABE (g/L) 0.2 1.7 1.3 1.2

Nng t bo t c ti a trong dch ln men l 0.75 g/L ng vi lng ABE t c l 1.7 0.2 g/L. Kt qu trn cho thy vi khun b c ch mnh bi mt s sn phm ca qu trnh thy phn lm t bo khng th pht trin. Vic s dng acid thy phn to ra nhiu yu t gy c ch ln canh trng. Trong khi , qu trnh ln men t dch thy phn x bp c thy phn bng enzyme cho kt qu tt hn. Nng ng t c sau qu trnh thy phn l 25 g/L. Sau 72 gi ln men 24.6 g/L ng c s dng.

Hnh 8: Nng sn phm to thnh trong qu trnh ln men ABE t dch thy phn x bp bng enzyme.

Tng nng ABE t c l 8.6 1.0 g/L trong butanol t 6.5 g/L tng ng vi tc sinh tng hp 0.1 0.011 g/L.h v hiu sut 0.35. Kt qu cho thy phng SVTH: Nguyn Phan Duy Nguyn 20

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php thy phn x bp bng enzyme hn ch cc cht c ch so vi phng php thy phn bng acid. Nng t bo t c trong dch ln men l 1.65 0.40 g/L. Hm lng ng st ch cn 0.4 g/L cho thy s bp c thy phn bng enzyme l ngun c cht tim nng cho sn xut butanol quy m ln. Trong mt nghin cu khc ca Xin-Liang Li v cng s (2006), xylan ly t x bp ( corn fiber xylan) c tin hnh ng ha v ln men ng thi s dng chng C.acetobutylicum P260. Mc ch ca nghin cu l xc nh kh nng t thy phn cc hp cht carbohydrate phc tp thnh ng n ca C.acetobutylicum.Th nghin tin hnh vi mi trng ban u cha 60 g xylan trong 1 lt mi trng P2, ln men 35oC. Tuy nhin, qu trnh ln men khng din ra, canh trng vi sinh vt khng pht trin v khng tng hp c bt c dung mi no. iu ny chng t C.acetobutylicum khng c kh nng thy phn xylan. Xin-Liang Li v cng s (2006) tip tc qu trnh ln men nh trn c b sung 0.5 ml enzyme xylanase h tr cho qu trnh thy phn. Kt qu cho thy vn khng c s pht trin ca t bo trong canh trng, iu ny c gii thch l do enzyme thy phn xylan khng nhanh v ln vi sinh vt s dng. Th nghim tip theo, 5 g/L xylose c b sung vo mi trng trn nhm cung cp c cht cho vi sinh vt c th sng st trong qu trnh xylanase thy phn xylan trong mi trng. Kt qu l vi khun pht trin v tng hp c 9.6 g/L ABE (hnh 9).

Hnh 9: Nng cc sn phm t c trong qu trnh ln men ABE cha 60 g/L xylan + 5g xylose + 0.5ml xylanase.

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c. Ln men t dch bo bo ( sweet sorghum juice) A.Areesirisuk v cng s (2010) s dng dch ng ly t vic thy phn thn cy bo bo lm c cht cho qu trnh ln men sn xut butanol. Ht v thn cy ca bo bo c th c s dng sn xut ng, ethanol, syrup, nhin liu, giy(Rajvanshi v Nimbkar, 2005). Mc ch nghin cu ca A.Areesirisuk v cng s (2010) l kho st kh nng pht trin v tng hp butanol ca C.beijerinckii JCM 1390 t dch bo bo m khng cn b sung thm cc ngun dinh dng t ng n khc. Dch thy phn t thn cy bo bo s c ly tm, b sung vo mi trng P2 iu chnh pH v 6.0 bng dung dch NaOH 1.0M. Qu trnh ln men c thc hin trong thit b ln men ym kh 2-L cha 1500ml dch ln men 37oC. Cc thng s trong sut qu trnh ln men c biu din trn th hnh 10:

Hnh 10: Cc thng s ca qu trnh ln men t dch bo bo do chng C.beijerinckii JCM 1390 thc hin.

T th c th thy sau 60 gi ln men, kh nng s dng c cht ca vi khun bt u gim dn, sau 80 gi th qu trnh ln men gn nh khng cn xy ra. pH trong sut qu trnh ln men c s thay i khng ng k, iu ny chng t cc acid sinh ra trong giai on acidogenesis c vi khun chuyn ha thnh dung mi. Nng cc sn phm t c sau qu trnh ln men c biu din trn hnh 11:

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Hnh 11: Nng dung mi trong sut qu trnh ln men dch bo bo ca C.beijerinckii JCM 1390

Nh vy nng butanol t c sau 60 gi ln men l 8.0 g/L v ti a l 8.8 g/L sau 120 gi ln men, hiu sut t 0.30. Nh vy so vi kt qu t nghin cu ca N. Qureshi v cng s (2008) trn c cht l dch thy phn x bp ( nng butanol t c sau 60 gi ln men l 6.5 g/L, hiu sut 0.35), dch bo bo c kh nng cho nng butanol cao hn. Tuy nhin, hm lng ng st trong dch sau ln men ca bo bo li kh cao lm cho hiu sut sinh tng hp sn phm gim ng k. Tc tng hp dung mi ch t 0.09 g/L.h. d. Ln men t b nho (grape pomace) B nho l mt ph phm trong cc ngnh cng nghip sn xut ru vang, c 100 kg nho s cho khong 18-20 kg b nho sau qu trnh p ly dch. Ngoi cc ng dng trong thc tin kh ph bin sn xut acid tartaric, trch ly cht mu anthocyan, lm thc n gia sc b nho cn cha mt lng ng st kh ln 7.81-10.81 % w/w (Hang v Woodams 2008) c th c s dng lm c cht cho cc qu trnh ln men t vi sinh vt. Laurent Law (2010) tin hnh nghin cu kh nng ln men tng hp butanol t b nho ly t ging nho trng Chardonnay s dng ging vi khun Clostridium acetobutylicum P262. chun b cho dch ln men, b nho s c pha vo nc ct theo t l 12.5% w/v, b sung dung dch m kali phosphate 1M. Ton b mi trng s c thanh trng 121oC trong 15 pht. Qu trnh ln men s c thc hin 35oC, pH mi trng ban u 5.5.

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Hnh 12: Nng ng v nng t bo trong qu trnh ln men t b nho s dng chng Clostridium acetobutylicum P262

C th thy trong 24 gi u ca qu trnh ln men, kh nng s dng c cht ca vi sinh vt cn tng i thp, nng t bo tng khng ng k. C th y l qu trnh thch nghi vi mi trng ca vi khun, canh trng cha bc vo giai on sinh trng. Tuy nhin, trong 48 gi ln men tip theo, tc s dng c cht ca vi khun tng rt nhanh. Sau 72 gi ln men, 97.6% hm lng ng trong dch ln men c s dng, nng t bo tng u trong 72 gi ln men v t nng 0.81 g/L. Nh vy, Clostridium acetobutylicum P262 pht trin kh tt trn mi trng ln men t b nho, nng ng st cn li kh thp ( 2.4%). Nng dung mi t c trong qu trnh ln men c biu din trn hnh 13:

Hnh 13: Nng cc dung mi t c trong qu trnh ln men b nho s dng chng Clostridium acetobutylicum P262

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Tc sinh tng hp cc acid acetic v butylic trong 24 gi u ln men tng kh cao 0.59 g/L.h, chng t canh trng ang trong giai on acidogenesis ch yu tng hp acid. Sau nng cc acid gim nhanh trong 48 gi tip theo, ngha l canh trng bc sang giai on solventogenesis, vi khun bt u s dng cc acid sinh ra trong giai on trc tng hp nn dung mi. Nng cc dung mi t c trong sut qu trnh ln men c tm tt trong bng sau:
Bng 7: Nng cc dung mi t c trong qu trnh ln men t b nho pH Acetone Butanol Ethanol Acid Acid Nng (g/L) (g/L) (g/L) Acetic butylic t bo (g/L) (g/L) (g/L) 5.49 0 0 0 0 0 0.59 5.06 0 0.53 1.55 6.13 3.39 0.65 4.67 0.87 2.99 0.93 5.7 2.98 0.80 4.75 2.83 5.96 0.51 3.03 2.49 0.81

Thi gian ln men (h) 0 24 48 72

Sau 24 gi ln men, acetone v butanol mi bt u c hnh thnh v t nng ln lt l 2.83 v 5.96 g/L sau 72 gi ln men. Nh vy t l nng cc dung mi l 6:3:1 (butanol:acetone:ethanol), kt qu ny tng t vi kt qu t c trong nghin cu ca Jones v Woods (1986). Tng nng dung mi t c l 9.08 g/L, hiu sut v tc tng hp sn phm ln lt l 0.36 g/g v 0.16 g/L.h. Hiu sut t c kh cao gn bng vi hiu sut t c khi ln men bng ng glucose ( 0.40 g/g) theo Nasib Qureshi v cc cng s (2006). Kt qu ny chng minh b nho l mt ngun c cht y tim nng cho qu trnh ln men sn xut butanol.
Bng 8: Bng tng hp mt s nghin cu thc hin trn cc cht mang khc c ngun gc t nhin
C cht Chng vi sinh vt Nng ABE (g/L) 17.6 20.1 24.6 18.1 13.0 17.7 25.8 Hiu sut (g/g) 0.36 0.31 0.34 0.33 0.28 0.18 0.34 Nng sut (g/L.h) 0.73 0.84 1.03 0.30 0.15 0.47 1.08 Thc hin

G thng Cy hon dip ( Aspen) Xc ma V la go V la m Rm t thn bp

C. acetobutylicum P262 C. acetobutylicum P262 C. saccharoperbutylacetonicum ATCC 27022 C. saccharoperbutylacetonicum ATCC 27022 C. acetobutylicum IFP 921 C. acetobutylicum P262

Parekh et al.(1988) Parekh et al.(1988) Soni et al. (1982) Soni et al. (1982) Marchalet al. (1984) Parekh et al.(1988)

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4.2.2. iu kin ln men a. pH pH ban u ca mi trng ln men ph thuc nhiu vo pH ti thch ca chng vi sinh vt s dng tng hp butanol. Cc th nghim c thc hin bng cch iu chnh v pH ti thch v khng kim sot trong sut qu trnh ln men. Cc loi Clostridium c kh nng hot ng tt trong khong pH t 5.0-6.5. Vi mi loi nht nh cn tin hnh ln men mt gi tr pH ban u xc nh nhm t c hiu sut tng hp cao nht. Trong nghin cu ca Shamsudin v cng s (2006), loi C.Saccharoperbutylacetonicum N1-4 c s dng ln men to butanol. Th nghim tin hnh cc gi tr pH ban u khc nhau ca mi trng ln men nhm xc nh gi tr pH ti u ca loi ny. Mi trng s dng l mi trng TYA cha 40 g glucose, pH c iu chnh bng dung dch NaOH 1M. Kt qu t c trn hnh 14:

Hnh 14: nh hng ca pH ln nng dung mi (), hiu sut () v tc sinh tng hp () butanol ca chng C.Saccharoperbutylacetonicum N1-4.

T kt qu trn c th thy r pH ti thch ca loi C.Saccharoperbutylacetonicum trong qu trnh ln men l 6.0. Trong khi ti pH l 5.5 li cho hiu sut tng hp thp nht. Trong nghin cu ca Madihah (2002) cng cho kt qu tng t khi pH ban u ca dch ln men t loi C.Saccharoperbutylacetonicum cho hiu sut cao nht. so snh cc qu trnh ln men c thc hin bng cch ch iu chnh pH ban u mt ln v duy tr pH n nh trong sut qu trnh ln men. George v Chen (1983) s dng chng C.beijerinckii VPI 13436 tin hnh ln men trn mi trng TYS. Th nghim c tin hnh pH ban u l 6.8, s dng dung dch KOH 8N iu chnh pH trong sut qu trnh ln men. SVTH: Nguyn Phan Duy Nguyn 26

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Hnh 15: Kh nng tng hp dung mi ca C.beijerinckii VPI 13436 trn mi trng TYS khng kim sot pH v duy tr pH 6.8. butanol (), acetate (), butyrate ( ), mt t bo ()

Trong mi trng khng kim sot pH, vi sinh vt bt u sinh tng hp dung mi sau 5 gi ln men khi pH mi trng t 4.5-4.6, nng butanol bt u tng nhanh sau 10 gi ln men v t ti a 100 n 120 mM butanol sau 120 gi ln men ( khng hin th trn hnh). Trong khi mi trng c duy tr pH 6.8, c th tng hp dung mi sau 2 gi ln men v nng butanol t ti a l 90 mM sau 120 gi ln men. Vic kim sot pH ca mi trng ln men lm gim kh nng tng hp dung mi ca vi khun, trong khi nng ca cc acid (acetate) trong dch ln men li tng lin tc. Nh vy vic duy tr pH c nh trong mi trng ln men khng c nhiu ngha, do hu ht cc nghin cu u thc hin cc gi tr pH ti thch ban u ca vi sinh vt v khng iu chnh trong sut qu trnh ln men. SVTH: Nguyn Phan Duy Nguyn 27

 n chuyn ngnh
b. Nhit Tng t nh iu kin v pH, gi tr nhit ln men s phc thuc nhiu vo chng vi sinh vt s dng. Ty vo tng loi c nhit ti thch khc nhau m ta s la chn nhit ln men thch hp. Khong nht ti thch ca cc loi Clostridium nm trong vng t 30-40oC, nhiu th nghim trn cc loi Clostridium acetobutylicum v C. beijerinckii u thc hin nhit trung bnh l 35oC. Shamsudin v cng s (2006) nghin cu trn loi C.Saccharoperbutylacetonicum li cho thy kh nng tng hp butanol cao nht 30oC.

Hnh 15: nh hng ca nhit ln kh nng ln men tng hp dung mi ca C.Saccharoperbutylacetonicum.

30oC, kh nng tng hp dung mi ca vi khun t cao nht (15g/L). Trong khi , 40oC hu nh vi khun u b c ch lm mt kh nng tng hp dung mi. Nh vy c th thy, yu t nhit nh hng rt ln n hiu sut ln men tng hp dung mi, cn kim sot v duy tr n nh nhit trong sut qu trnh ln men. c. Nng cc mui trong mi trng tng hp Ngoi cc ng pentose v hexose l c cht chnh c Clostridium s dng ln men tng hp butanol, mi trng ln men cn cha nhiu loi mui khong (Fe2+, Mn2+, K+, Mg2+, NH4+..) gip cho vi sinh vt c th sinh trng v to sn phm. Nhiu nghin cu cho thy nng cc mui ban u trong mi trng ln men c th nh hng rt ln n kh nng sinh trng v tng hp sn phm ca vi khun. F. Monot v cng s (1982) tin hnh nghin cu nh hng nng cc hp cht ca mi trng tng hp n qu trnh ln men ABE s dng chng Clostridium acetobutylicum ATCC 824. Cc th nghim c tin hnh bng cch ln lt thay i

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nng ban u ca cc mui MgSO4, FeSO4, KCl, ammonium acetate trong mi trng tng hp. - KCl: kho st nh hng ca nng mui KCl, cc th nghim c tin hnh vi cc nng KCl ban u l 0, 3, 6, 9, 15, 30, 60, 120, 360, 600 mg/l v 1.2, 2, 4, 8, 12 g/l. Kt qu nng dung mi thu c trong cc th nghim cho bi bng sau:
Bng 9: nh hng ca nng KCl ban u ti qu trnh ln men ABE. Nng Nng Buta Aceto Etha Tng (mg/l) t nol (g/l) ne (g/l) nol (g/l) ABE (g/l) bo* 0 0.8 0 0 0 0 3 0.59 0.48 0.48 6 0.72 0.78 0.78 KCl 9 0.88 4.22 1.39 0.23 5.84 15 1.28 4.26 1.28 0.25 5.79 30 1.88 4.26 1.05 0.28 5.59 602.60 4.32 1.03 0.50 5.85 600 0.6-8 2.94 4.72 0.81 0.48 6.01 (g/l) 12 3.10 3.89 0.90 0.37 5.16 *: Nng t bo tnh theo mt quang.

Kt qu trn cho thy qu trnh ln men ABE chu nh hng kh ln bi nng ca mui K+. Nng t bo trong mi trng ln men tng lin tc khi nng mui tng t 0 n 60 mg/l v gi n nh khi nng mui tip tc tng t 60-600 mg/l. Nng dung mi bt u tng nhanh khi nng mui t 9 mg/l, trong khong nng mui t 0.6-8 g/l nng dung mi vn duy tr khng i 6.01 g/l. Khi nng mui tng cao 12 g/l gy s c ch lm gim hiu sut tng hp dung mi ca vi khun (nng dung mi ch t 5.16 g/l). - Ammonium acetate: Mui ammonium acetate c kho st cc nng 1.1, 2.2, 3.3, 4.4, 5.5, 6.6 g/l. Kt qu cho bi bng sau:

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Bng 10: nh hng ca nng ammonium acetate ban u ti qu trnh ln men ABE.

Ammonim acetate

Nng (g/l) 1.1 2.2 3.3 4.4 5.5 6.6

Nng t bo 2.14 1.83 1.84 1.80 1.86 1.80

Butano l (g/l) 3.11 2.78 2.04 1.48 1.04 1.04

Aceton e (g/l) 0.99 1.10 0.65 0.47 0.35 0.35

Ethano l (g/l) 0.12 0.12 0.09 0.09 -

Tng ABE (g/l) 4.22 4.00 2.78 2.04 1.39 1.39

Kt qu cho thy ammonium acetate c nh hng ln n kh nng pht trin ca vi sinh vt. Nng ammonium acetate 1.1 g/l cho kt qu tt hn cc nng khc, nng cao hn hu nh vi sinh vt u b c ch. Do vic tng hp dung mi cng b nh hng khi tng nng mui ln cao hn 1.1 g/l. Ti nng ammonium acetate 1.1 g/l tng lng dung mi cng t kh thp 4.22 g/l. - MgSO4: Mui MgSO4 c kho st cc nng 0, 50, 100, 200, 350 v 500 mg/l. Kt qu cho bi bng sau:
Bng 11: nh hng ca nng MgSO4 ban u ti qu trnh ln men ABE.

MgSO4

Nng (mg/l) 0 50-200 350-500

Nng t bo* 0.65 2.99 2.64

Butanol (g/l) 2.22 5.28 4.48

Acetone (g/l) 0.87 1.48 1.28

Ethanol (g/l) 0.14 0.74 0.61

Tng ABE (g/l) 3.23 7.5 6.37

C th thy rng nng t bo vi sinh vt ph thuc kh nhiu vo s hin din ca Mg2+ trong dch ln men. Khi khng c mt Mg2+ trong mi trng, s sinh trng ca vi khun b c ch nhng vn tng hp c dung mi vi nng kh thp 3.23 g/l. Khi c mt MgSO4 trong mi trng (50-200 mg/l), vi khun pht trin rt tt ng thi kh nng tng hp dung mi cho hiu sut cao nht (7.5g/l). C th kt lun nng mui MgSO4 ti u cho vi khun pht trin nm trong khong 50-200 mg/l. Khi tng nng ln 350-500 mg/l, t bo vi khun bt u b c ch v kh nng tng hp dung mi cng gim r rit (6.37 g/l). - FeSO4: S hin din ca Fe2+ trong mi trng ln men nh hng n kh nng pht trin ca vi sinh vt. Khi mi trng tng hp cha 1 mg FeSO4 trong 1L dung dch, t bo vi khun pht trin kh tt, vi khun s dng ht c cht (glucose) v tng hp c SVTH: Nguyn Phan Duy Nguyn 30

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5.5 g/l dung mi. Khng c s khc bit ng k v kh nng sinh trng cng nh tng hp sn phm khi tng nng FeSO4 t 1-50 mg/l. Tuy nhin, khi mi trng khng c cung cp FeSO4, s pht trin ca vi khun b yu i, tng lng dung mi ch t 2.08 g/l. d. S c ch ngc ca butanol trong qu trnh ln men Trong qu trnh ln men ABE, butanol c sinh ra vi t l cao nht v c xem l sn phm chnh ca qu trnh ln men. Tuy nhin, butanol cng nh nhng alcohol khc u c nh hng xu n thnh ca t bo vi khun. nng cao, butanol c th ngn chn s vn chuyn cht dinh dng qua thnh t bo, ph hy chc nng ca thnh t bo. Trong nghin cu c thc hin bi Bowles v Ellefson (1985) kho st nh hng ca butanol n kh nng s dng ng glucose ca loi Clostridium acetobutylicum. Kh nng s dng ng ca vi khun c nh gi thng qua s g glucose c tiu th trn mg protein t t bo vi khun ti mt thi im ca qu trnh ln men. Th nghim c tin hnh cc nng butanol ban u ln lt l 0, 139, 167, , 236, 278 mM.

Hnh 16: nh hng ca butanol ln kh nng s dng glucose ca C. acetobutylicum. 0 mM butanol(),139 mM butanol (), 167 mM butanol (), 208 mM butanol (), 236 mM butanol (), 278 mM butanol ( )

Kt qu cho thy r s c ch ca butanol ln kh nng s dng c cht ca vi khun. Nguyn nhn chnh c th l do butanol lm cho t bo mt kh nng duy tr pH bn trong t bo lm ri lon chc nng phn gii c cht, cc ATPase ca mng t bo b c ch, ATP ni bo gim. SVTH: Nguyn Phan Duy Nguyn 31

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Trong mt nghin cu khc ca Zhijie.S v Shijie.L (2010) tin hnh kho st nh hng ca butanol n kh nng ln men ABE ca chng C. acetobutylicum ATCC 824. Qu trnh ln men ABE c thc hin 37oC, nng glucose ban u l 50 g/l, nng butanol ban u trong dch ln men ln lt l 3, 6, 9, 12 g/l.

Hnh 17: nh hng ca nng butanol ln qu trnh ln men ABE s dng chng C. acetobutylicum ATCC 824. HAB: tng acid acetic v butylic.

T kt qu trn c th thy nng butanol ban u nh hng n nng cc acid v dung mi trong dch sau ln men. Khi nng butanol ban u tng ln, nng cc acid tng ln v nng dung mi li gim xung. Khi nng butanol ban u ln hn 6 g/l, vi khun s dng glucose ch yu tng hp acid lm cho hiu sut tng hp cc acid tng mnh v t cc i 0.23 g/g trong khi hiu sut tng hp butanol gim nhanh chng. Nh vy vic tng nng butanol nh hng ln n qu trnh sinh trng ca vi sinh vt lm cho vi sinh vt tr v giai on solventogenic ch yu sn sinh acid. C th thy butanol c nh hng ln n hot ng ca vi sinh vt trong qu trnh ln men, t hiu sut ln men cao cn c cc bin php tch butanol ngay trong qu trnh ln men gim s c ch ca butanol n vi sinh vt. Cc phng php tch butanol trong qu trnh ln men nhm nng cao hiu sut ln men s c cp trong chng sau.

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 n chuyn ngnh 4.3. ng dng t bo vi khun c nh trong ln men sn xut butanol

Trong nhng nm gn y, k thut c nh t bo trn cht mang ng dng trong cc qu trnh ln men c nghin cu rng ri. ng dng t bo vi khun c nh trong ln men sn xut butanol c th mang li nhiu u im vt tri nh: Cht mang ng vai tr nh mt tc nhn bo v t bo chng li cc nh hng bt li t mi trng nh nng c cht cao, s thay i pH, nhit v lm gim i s c ch t bo bi sn phm cui (butanol). Mt t bo trn mi n v th tch ca bnh phn ng sinh hc cao hn, t dn n nng sut th tch cao hn, rt ngn thi gian ln men. Tng kh nng s dng c cht v tc sinh tng hp sn phm, t ci thin hiu sut ln men. C th s dng cho qu trnh sn xut lin tc. D dng thu hi sn phm, gim bt cng on phn ring canh trng sau ln men, do gim chi ph v thit b v nng lng. C th ti s dng nm men nhiu ln trong khi vn m bo c hot tnh sinh hc n nh. Nhng nghin cu v t bo c nh trong ln men sn xut butanol u da trn hai k thut chnh l hp th v nht t bo vi khun Clostridium ln cht mang. R. Sallam v cng s (2003) tin hnh nghin cu c nh t bo C. acetobutylicum ln cht mang l Ca-alginate v than hot tnh qua so snh kh nng ln men sn xut butanol ca t bo c nh v t bo t do. Sau 4 ngy ln men thu c kt qu sau:
Bng 12: Kt qu ln men t t bo t do v c nh trn Ca-alginate v than hot tnh

T do

C nh trn Ca-alginate

C nh trn than hot tnh

Thi gian ln men (ngy) 0.0 1.0 2.0 3.0 4.0 0.0 1.0 2.0 3.0 4.0 0.0 1.0 2.0 3.0 4.0

Nng ng (kg/m3) 70.0 46.3 35.0 23.0 14.0 70.0 45.0 30.0 20.0 9.3 70.0 30.0 19.5 13.0 12.0

Acetone (kg/m3) 0.0 1.7 1.9 3.6 6.3 0.0 1.9 3.4 6.3 8.4 0.0 1.0 1.2 2.0 2.9

Butanol (kg/m3) 0.0 3.3 4.3 8.5 15.7 0.0 3.3 8.0 15.0 20.5 0.0 2.3 3.0 4.6 6.9 33

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Kt qu t bng 12 cho thy nng c cht trong dch ln men gim nhanh chng trong giai on u ca qu trnh ln men. T bo vi khun c hp th trn than hot tnh c tc s dng c cht cc i t rsm= 100 kg/m3.ngy, trong khi ca t bo t do v t bo c nh trn Ca-alginate ln lt l 50 v 80 kg/m3.ngy. Tuy nhin, t bo c nh trn Ca-alginate li c kh nng tng hp butanol cao nht 20.5 kg/m3 t hiu sut 0.34. Trong khi t bo c nh trn than hot tnh li cho kh nng tng hp butanol thp hn c t bo t do (6.9 kg/m3 < 15.7 kg/m3). Nh vy, Ca-alginate th hin l mt cht mang c nhiu u im vt tri hn so vi than hot tnh. Vic nht cc t bo vi khun C. acetobutylicum trong gel alginate gip cho cc t bo t b nh hng bi cc yu t c ch t mi trng hn so vi t bo t do lm tng hiu sut tng hp butanol. Trong mt nghin cu khc ca S. Shamsudin v cng s (2006) thc hin c nh trn 5 loi cht mang khc nhau bao gm : stainless steel scrubber, nylon scrubber, cc ht polyurethane c kch thc mao qun bng nhau, cc ht polyurethane c kch thc mao qun khng ng u v fruit bunch fiber. S dng chng C.Saccharoperbutylacetonicum N1-4 c nh, ln men trn mi trng TYS cha 40 g glucose. Tin hnh kho st nh hng ca cc loi cht mang khc nhau ln nng dung mi thu c sau qu trnh ln men, hiu sut v tc sinh tng hp sn phm trn mi trng ln men TYA 30oC. Kt qu thu c t qu trnh ln men th hin trn th sau:

Hnh 18: Nng cc sn phm thu c t cc qu trnh ln men ABE s dng t bo vi khun c nh trn cc loi cht mang. 1, stainless steel scrubber. 2, nylon scrubber. 3, cc ht polyurethane c kch thc mao qun ging nhau. 4, cc ht polyurethane c kch thc mao qun khng ng u. 5, fruit bunch fiber. 6, t bo t do.

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Tin hnh so snh cc qu trnh ln men ABE thc hin bi t bo vi khun c nh vi t bo vi khun t do sau 24 gi ln men. Kt qa cho thy t bo vi khun c nh trn cc ht polyurethane c kch thc mao qun khng ng u c kh nng tng hp dung mi cao nht (26.679 g/l dung mi) sau 24 gi ln men vi 19.228 g/l butanol. Cht mang stainless steel scrubber cho kt qu ln men cao th hai vi 16.243 g/l dung mi, tip theo l fruit bunch fiber vi 15.650 g/l dung mi, cc ht polyurethane c kch thc mao qun ging nhau cho kt qu l 10.606 g/l dung mi v cui cng l nylon scrubber cho kt qu thp nht vi 3.863 g/l. Tng nng acid thu c t qu trnh ln men s dng cc ht polyurethane c kch thc mao qun khng ng u cho kt qu thp nht vi 1.373 g/l acetate v 0.235 g/l butyrate. iu ny hon ton ph hp khi qu trnh ln men s dng cc ht polyurethane c kch thc mao qun khng ng u cho nng dung mi cao nht. Hiu sut v tc sinh tng hp dung mi ca cc qu trnh ln men s dng t bo c nh so vi t bo t do c biu din trn th hnh 19:

Hnh 19: Hiu sut v tc sinh tng hp sn phm sau 24 gi ln men ABE 30oC trn mi trng TYA. 1, stainless steel scrubber. 2, nylon scrubber. 3, cc ht polyurethane c kch thc mao qun ging nhau. 4, cc ht polyurethane c kch thc mao qun khng ng u. 5, fruit bunch fiber. 6, t bo t do.

Kt qu cho thy tc sinh tng hp dung mi t c t 5 loi cht mang khc nhau ln lt l 1.084, 0.648, 0.633, 0.406 v 0.122 g/l.h tng ng ca cc ht polyurethane c kch thc khng ng u, stainless steel scrubber, fruit bunch fiber, cc ht polyurethane c kch thc ging nhau v nylon scrubber. T nhng kt qu trn, c th kt lun rng qu trnh ln men t t bo vi khun c nh trn cht mang l cc ht polyurethane c kch thc khng ng u cho kt qu tt nht. So vi qu trnh ln men t vi khun t do, cc ht polyurethane c kch SVTH: Nguyn Phan Duy Nguyn 35

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thc khng ng u gip cho qu trnh ln men c th tng hiu sut gp 1.9 ln v tng tc sinh tng hp dung mi hn 3.9 ln. iu ny c c l do cu to ca cc ht polyurethane c cu trc xp rng, bao gm rt nhiu mao qun nh c ng knh t 20-900 m (hnh 20). Nh cc t bo vi khun d dng c nht trong cc mao qun, c th tip xc v trao i cht d dng vi mi trng. Bn cnh , polyurethane cn c nhiu u im hn so vi cc loi cht mang khc nh gi thanh r, qu trnh c nh n gin v nhanh chng, khng cn s dng thm ha cht h tr no. Do , polyurethane th hin l mt cht mang tim nng trong vic ng dng c nh vi khun s dng trong ln men quy m cng nghip.

Hnh 20: Cu trc mao qun ca cc ht polyurethane c kch thc mao qun khng ng u.

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CHNG 5: CC K THUT PHN TCH BUTANOL T DCH LN MEN

Trong qu trnh ln men sn xut butanol quy m cng nghip, qu trnh phn tch butanol t dch sau ln men l mt qu trnh phc tp v tiu tn kh nhiu nng lng. Chng ct l phng php truyn thng c s dng thu hi butanol t dch sau ln men. Tuy nhin, nng nng dung mi trong dch sau ln men kh thp trong khong t 18-33 g/l trong butanol ch c khong 13-18 g/l. Ngoi nng trong dch ln men thp, butanol cn c im si cao hn so vi nc (118oC). Tt c nhng yu t trn lm cho qu trnh chng ct thu hi butanol i hi phi tiu tn rt nhiu nng lng, c th chim ti 40% gi thnh sn phm. Philips v Humphrey (1985) cho rng nu nng butanol trong dch sau ln men tng t 10 g/l ln 40 g/l th qu trnh tch chit butanol bng phng php chng ct s tit kim nhiu chi ph. Theo , ti nng 10 g/l butanol, t l nhin liu du cn tiu tn thu hi 100% butanol l 1.5, trong khi ti nng 40 g/l t l ny ch cn 0.25. iu ny cho thy, chi ph s c tit kim rt nhiu nu nh nng butanol trong dch sau ln men tng gp ba. ci thin hiu sut thu hi v gim chi ph, nhiu k thut mi c nghin cu v ng dng nh: gas stripping, pervaporation, hp th, trch ly lng-lng v thm thu ngc.

Hnh 21: th so snh nng lng cn thit dng trong cc k thut tch chit butanol.

Thm thu ngc c cho l phng php em li hiu qu kinh t cao nht tuy nhin n li c nhc im kh khc phc l d b tc nghn trong qu trnh hot ng. Qu trnh trich ly lng-lng th hin tnh chn lc cao v kh nng tch chit

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kh tt nhng qu trnh thc hin li tn nhiu chi ph. V vy, cn phn tch r nhng u v nhc im ca tng phng php c th t c hiu qu cao nht. a. Gas stripping: l phng php n gin v hiu qu thu hi butanol t dch ln men. Nguyn tc c bn ca phng php ny l sc kh qua mi trng ln men, kh s li ko dung mi trong dch ln men i vo thit b thu hi dung mi. Kh sau khi c thu hi li tip tc dn vo bnh ln men v tip tc qu trnh n khi c cht trong mi trng ln men c s dng ht. Vic p dng phng php gas stripping c th gip cho qu trnh ln men ABE thc hin nng ng kh cao (Qureshi v Blaschek, 2001) v lm gim s c ch ca butanol i vi vi sinh vt trong qu trnh ln men (Maddox v cng s, 1995). Trong nghin cu ca Ezeji v cng s (2003), gas stripping c s dng tch chit dung mi trong qu trnh ln men s dng chng C. beijerinckii BA101. Kt thc qu trnh ln men, 161,7 g/l ng c vi sinh vt s dng v thu c 75.9 g/l dung mi. Trong mt nghin cu khc ca Ezeji v cng s (2004), qu trnh ln men c b sung c cht c tch hp vi qu trnh gas stripping nhm lm gim s c ch ca nng ng ln men cao v gip tng mt vi sinh vt trong canh trng ln men. Trong h thng ln men ny, 500 g glucose c vi sinh vt tiu th ht v 233 g dung mi c thu hi. Hiu sut qu trnh ln men t 0.47 g/g vi tc thu hi dung mi t 1,16 g/l.h.

Hnh 22: S h thng tch chit butanol t qu trnh ln men ABE bng phng php gas stripping. A: bm iu chnh tc kh vo bnh ln men, B: bnh ln men c thit b sc kh, C: thit b ngng t, D: hi ABE ngng t ra, E: thit b lm mt.

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b. Trch ly lng-lng: l mt phng php hiu qu khc tch dung mi t dch ln men. Phng php ny s dng dung mi hu c tch chit butanol t dch ln men. u im ca phng php l butanol c kh nng ha tan rt tt trong cc dung mi hu c hn so vi nc trong dch ln men. Phng php trch ly lng-lng thng s dng dung mi l ru decanol v oleyl (Evans v Wang, 1988). Trong cc qu trnh ln men tnh theo chu k, phng php trch ly lng-lng thng c kt hp vi qu trnh chng ct tch chit cc dung mi thnh ring bit.Theo Korbinian Kraemer v cng s (2010) qu trnh tch chit s dng phng php kt hp ny c biu din theo m hnh sau:

Hnh 23: S h thng tch chit butanol t dch sau ln men ABE bng phng php trch ly lng-lng kt hp chng ct.

Theo s ny, nu s dng oleyl alcohol lm dung mi trch ly th lu lng dch sau ln men i vo ct trch ly c th t 738 kg/h vi 87.5% dung mi c trch ly. Tng nng lng tiu tn cho ton b qu trnh l 15MJ/kg butanol. Nh vy, so vi nng lng m butanol c th to ra (36 MJ/kg) th qu trnh tch chit trn mang li li ch kh ln v mt nng lng. Tuy nhin, phng php ny s c nhiu mt hn ch nu p dng tch dung mi t qu trnh ln men lin tc, dung mi trch ly c kh nng gy c ch ca n t bo vi sinh vt trong qu trnh ln men v c th to thnh h nh tng trong dch ln men hn ch s trao i cht ca vi sinh vt. Nhng vn ny c th c khc phc nu dch ln men v dung mi trch ly c ngn cch bi mt mng bn thm trao i butanol gia hai pha, phng php ny c gi l perstraction (Ezeji v cng s, 2007) c. Pervaporation: c th c xem l k thut tch chit butanol t dch ln men tt nht so vi cc phng php nu trn. Pervaporation th hin u im vt tri v mt khng gy nh hng xu n vi sinh vt trong dch ln men, chi ph cho qu trnh tch chit l kh thp. Nguyn l c bn ca k thut ny l s dng mt mng SVTH: Nguyn Phan Duy Nguyn 39

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membrane xp, dch ln men s c tip xc vi mt mt ca membrane, to chn khng mt i din. Dung mi s i qua membrane theo dng permeate v chuyn thnh pha hi sau c ngng t thu hi.

Hnh 24: Nguyn l tch cu t trong hn hp ca phng php pervaporation.

Cc loi membrane c th c s dng trong k thut ny c th lm t vt liu rn nh silicalitesilicone, polypropylene, zeolite.. Ngoi ra, mt s nghin cu cn s dng membrane lng tch chit butanol, dung mi lng oleyl alcohol s c gi trong cc mao qun ca polypropylene hnh thnh nn loi membrane lng c bit oleyl- polypropylene. Tnh n nh ca mng, kh nng chn lc cao v lu lng dng qua mng l nhng yu t quan trng trong qu trnh tch chit butanol bng phng php pervaporation. Trong mt nghin cu ca Matsumura v cng s (1988), s dng membrane lng oleyl- polypropylene tch butanol t dch ln men nng thp. Kt qu cho thy membrane oleyl- polypropylene th hin tnh chn lc tt hn so vi membrane bng silicone. Nhu cu v nng lng t hn 10 ln so vi phng php chng ct. Tuy nhin, tnh n nh ca membrane lng oleyl- polypropylene li tng i thp. pht trin mt loi membrane c th gi c tnh n nh khi nng cao mc chn lc ca membrane, Qureshi v cng s (1999) nghin cu tng hp mt loi membrane t phc hp silicon-silicalite.

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Hnh 25: S qu trnh ln men tnh c b sung c cht kt hp qu trnh tch chit dung mi bng phng php pervaporation s dng membrane silicon-silicalite. 1: bn cha nc thi, 2: bn cha c cht, 3: bn ln men, 4: membrane, 5: bn lm lnh ngng t, 6: bm chn khng.

Khi kt hp phng php pervaporation s dng membrane silicon-silicalite vi qu trnh ln men ABE s dng chng C. beijerinckii BA101, Qureshi v cng s (1999) nhn thy tng nng dung mi t c tng gp hai ln ( t 24.2 g/l vi qu trnh ln men thng ln 51.5 g/l khi kt hp vi qu trnh pervaporation). Tc sinh tng hp dung mi tng t 0.35 ln 0.98 g/l.h, qu trnh pervaporation khng nh hng ti s pht trin ca C. beijerinckii BA101. Tnh chn lc ca membrane t phc hp silicon-silicalite cao gp 2.2 ln so vi membrane lng oleyl- polypropylene. T nhng kt qu trn cho thy, khi kt hp cc phng php tch chit dung mi t dch ln men u nh hng n kh nng sinh tng hp sn phm ca vi khun trong canh trng ln men.Khi dung mi c tch mt phn ra khi mi trng trong qu trnh ln men, s c ch ca dung mi ln vi sinh vt gim dn lm tng kh nng hot ng ca vi khun t lm tng hiu sut ca qu trnh ln men. Ty vo quy m v cc iu kin t qu trnh ln men ABE, vic la chn v s dng cc phng php tch chit dung mi s mang li hiu qu cao hn so vi cc qu trnh ln men thng thng.

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Chng 6: Kt Lun
Cc nghin cu v qu trnh ln men ABE a s u tp trung s dng hai loi vi khun l Clostridium beijerinckii v Clostridium acetobutylicum. Cc chng t bin t hai loi ny th hin u im vt tri v kh nng sinh tng hp dung mi nng cao. C hai loi u ln men cng nhit v pH ti thch tng ng l 35oC v 5.5. Trong khi , loi C.Saccharoperbutylacetonicum li ln men tt nht iu kin 30oC v ph 6.0. Tuy nhin kh nng sinh tng hp dung mi ca C.Saccharoperbutylacetonicum li km xa so vi hai loi C. acetobutylicum v C. beijerinckii. Khi s dng cc loi ng pentose v hexose lm c cht cho qu trnh ln men th glucose th hin l c cht c a thch nht ca cc loi Clostridium. Nng dung mi cao nht t c khi s dng glucose lm c cht ca chng C. acetobutylicum P260 l 21.37 g/l (hiu sut 0.36), trong khi ca chng C. beijerinckii BA101 l 17.8 g/l (hiu sut 0.30). Mc a thch cc loi ng khc nhau ca loi Clostridium beijerinckii theo th t l glucose>xylose>arabinose>mannose v ca loi Clostridium acetobutylicum l glucose>arabinose>xylose>galactose. Cc nghin cu v s dng cc ngun c cht carbohydrate r tin c sn trong t nhin cho nhng kt qu kh quan. Qu trnh ln men ABE t bt sn s dng chng C. beijerinckii BA101 t c nng butanol trong dch ln men kh cao 20.81 g/l, tc sinh tng hp butanol l 0.22 g/l.h. Ngoi ra, cc ngun c cht r tin khc nh x bp, b nho, dch thy phn bo bo cng cho kt qu ln men kh tt. Vic c nh t bo vi khun c nghin cu trn cc loi cht mang khc nhau nh: Ca-alginate, than hot tnh, stainless steel scrubber, nylon scrubber, cc ht polyurethane, fruit bunch fiber Trong , Ca-alginate v cc ht polyurethane th hin l nhng cht mang tt, mang li hiu qu cao hn so vi qu trnh ln men bng vi khun t do. Kt hp vi cc phng php tinh chit dung mi t dch ln men, cc qu trnh ln men ABE cho hiu sut cao hn, gip lm gim s c ch ca dung mi ln hot ng ca vi khun. C th thy qu trnh ln men sn xut butanol t vi sinh vt c nghin cu kh nhiu v cho thy tim nng ln ng dng sn xut quy m cng nghip.

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