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Chng 1: M au

M AU
Ngay nay, toan cau hoa a tr thanh mot xu the tat yeu. The gii ang chuyen mnh manh me. Trong the gii phat trien khong ngng o, con ngi phai oi dien vi nhieu van e ln. Mot trong nhng van e o la nhien lieu. Nhu cau ve nhien lieu ngay mot tang trong khi san lng dau khai thac cac nc khong the ap ng u. Lam sao e co u nhien lieu van hanh guong may the gii ma van am bao khong lam can kiet nguon tai nguyen dau hoa la mot bai toan ln ma con ngi can giai quyet. Sau nhieu nghien cu, ngi ta a tm ra mot nguon nhien lieu mi: con nhien lieu t cellulose mot nguon nhien lieu sach va than thien vi moi trng. Etanol t cellulose la loai nhien lieu etanol c tao ra t cac nguon sinh khoi giau cellulose nh rac thai nong nghiep (rm, ra, phan than cay bap), chat thai go va ngay ca rac thai o th bang con ng len men. Nguon rac thai t nhien giau cellulose nay rat a dang va chiem t le ln, theo c tnh cellulose chiem gan phan na nguon cacbon hu c tren hanh tinh. Mot phan tch gan ay cua Hoi bao ve cac nguon tai nguyen thien nhien (Natural Resources Defense Council) My a ch ra rang etanol t cellulose co the ap ng mot na nhu cau ve nhien lieu van chuyen cua My en nam 2050 [44]. Viec pha tron etanol vao xang dau giup giam viec tieu thu nhien lieu cua xe co ngay nay va giup ot chay nhien lieu sach va hieu qua hn. Cac nha san xuat xe hi bao am cho viec s dung etanol t cellulose c pha tron vao xang dau vi t le 10% Bac My (E10) va 5% Chau Au (E5). Hn na, mot so nha san xuat tren the gii a tao ra loai xe hi va xe tai s dung nhien lieu linh hoat co t le pha tron etanol len en 85% (E85). Theo phan tch cua Vien nang lng My (US Department of Energy), etanol t cellulose lam giam en 90% cac kh gay ra hien tng hieu ng nha knh so vi nhien lieu thong thng [45].

Chng 1

Chng 1: M au Nh vay, viec tan dung nguon cacbon t sinh khoi cellulose e tao ra nang lng cho the gii khong ch giai quyet bai toan ve nhien lieu ma con gop phan x ly rac thai lnh vc moi trng. ieu nay rat thiet thc v no ap ng c yeu cau va muc tieu phat trien ben vng cua toan xa hoi. Vi nhng y ngha thc te neu tren, viec san xuat etanol t cellulose can sm c nghien cu va a vao ng dung Viet Nam. V vay vi viec chon e tai Nghien cu kha nang s dung rm e san xuat etanol chung toi mong muon gop mot phan vao nhng nghien cu khoa hoc nham a etanol tr thanh mot nhien lieu quen thuoc va c s dung rong rai Viet Nam, giam c s le thuoc vao xang dau nhap khau, gop phan phat trien nen kinh te va giam thieu van e o nhiem moi trng do xang dau. Qua trnh san xuat etanol t cellulose bang phng phap len men co cac bc chnh: - Pha v cau truc phc tap cua lignocellulose e giai phong cellulose, hemicellulose, lignin va mot so chat khac. - Thuy phan cellulose, hemicellulose e tao cac ng t do (pentose, hexose). Len men hon hp ng tren e tao etanol. Chng cat thu etanol tinh khiet.

Trong pham vi cua e tai, chung toi xin i vao qua trnh thuy phan tao dch ng va qua trnh len men etanol t dch ng thu c nham: Xac nh ieu kien toi u cho qua trnh thuy phan cellulose bang enzym cellulase e nang cao nong o dch ng thu c . Xac nh ieu kien toi u cho qua trnh len men etanol e thu c nong o etanol cao.

Chng 2: Tong quan

Chng 2

TONG QUAN TAI LIEU


2.1 NGUYEN LIEU CELLULOSE

Nguon nguyen lieu cellulose (lignocellulose) pho bien trong t nhien c tao thanh t cac polysaccharide (cellulose, hemicellulose), lignin va mot so chat khac (tro, cac chat trch ly). Trong o, cellulose va hemicellulose chiem a so va la nhng chat co the thuy phan tao ng.
2.2.1 Cellulose

Cellulose la polyme phong phu nhat trong t nhien, cau tao nen vach te bao thc vat, chiem 39 42% khoi lng kho cua go mem [42]. Mot so vi sinh vat khac, nh vi khuan Acetobacter xylinum, co kha nang tao ra cellulose [32]. Cellulose la mot polysaccharide ong nhat bao gom nhng n v lap lai cellobiose mot dimer cua -D-glucose. o trung hp (DP) cua cellulose thay oi t 2000 en 14000 [37]. Tren hnh 2.1 la ai dien cua cellulose. Hai n v glucose ke tiep nhau (cau truc vong) khong c nh hnh nh nhau. ieu nay giai thch tai sao cellobiose la n v lap lai cua cellulose ma khong phai glucose.

Hnh 2.1. Cau truc ai dien cua cellulose. Cac n v glucose trong cellulose c lien ket vi nhau bi lien ket -1,4 glucoside. ieu nay cho phep hnh thanh nhng chuoi co the xep lai va tng tac vi nhau mc o cao. Trong vach te bao thc vat, cac chuoi cellulose hnh thanh nen cac bo goi la micelle hay si c ban [53]. Chung lan lt tap hp lai thanh nhng vi si vi ban knh t 3-6 nm. Gia cac chuoi cellulose co rat nhieu goc OH va tao ra rat nhieu lien ket hydro giup on nh si cellulose, lam cho no rat ben. Cac vi si hnh thanh nen cac si ln hn va tng tac vi hemicellulose, lignin e tao thanh phc hp thanh te bao thc vat (hnh 2.2). 3

Chng 2: Tong quan

Hnh 2.2. Cau truc thanh te bao thc vat.

Hnh 2.3. Cau truc tinh the cellulose. Cac si cellulose s cap co the ket hp chat che vi nhau e tao thanh dang co trat t, hay cau truc tinh the (hnh 2.3) [30]. Tuy nhien, mot phan nho cua chuoi co the tr nen vo trat t va sap xep mot cach ngau nhien tao nen vung vo nh hnh. Do s tng tac manh cua cac chuoi cellulose trong vung tinh the, nen dung moi va cac chat hoa hoc khac rat kho khan e xam nhap vao cau truc trong khi vung vo nh hnh th xam nhap rat de dang [53].
2.2.2 Hemicellulose

Hemicellulose chiem 35% khoi lng go cng va 25% go mem [53]. Hemicellulose la mot nhom cac polysaccharide khong ong nhat bao gom s ket hp cua glucose, galactose, mannose, xylose va arabinose. Loai chiem u the trong hemicellulose go mem khac go cng. Hemicellulose co cau truc phan nhanh, vi nhng chuoi ngan hn so vi cellulose (DP ~ 150) [40]. Ngoai nhng hp 4

Chng 2: Tong quan phan cua ng, mot so phan cua hemicellulose co the dang acetyl hay dang uronic acid [36]. Hnh 2.4 cho thay hai nhom c ban nhieu nhat cua hemicellulose go mem. Galactoglucomannan (hnh 2.4a) la thanh phan chnh trong go mem c theo sau bi arabinoglucuronoxylan (hnh 2.4b) [21]. oi vi go cng, nhom hemicellulose chnh la glucuronoxylan hay O-acetyl 4-O-methylglucorono- -xylan.

Hnh 2.4. Hemicellulose (a) Galactoglucomannan; (b) Arabinoglucuronoxylan. (1) -D-glucopyranose; (2) -D-galactopyranose; (3) -Dmannopyranose (4) -D-xylopyranose; (5) -L-arabinofuranose. S phan nhanh trong hemicellulose lam cho chung de b tan cong va do o nhay cam vi dung moi va cac loai phan ng hoa hoc. oi vi qua trnh x l nhiet, hemicellulose de b thuy phan nhat trong khoang nhiet o 150 2300C [23]. Mat khac, thuy phan cellulose xay ra nhiet o cao hn la 210 2200C. That vay, qua trnh tien x l co the bang acid hay hi nc, muc ch la loai i thanh phan hemicellulose e co san lng cellulose va ng cao trong cac bc tiep theo [32].

Chng 2: Tong quan


2.2.3 Lignin

Thanh phan cau truc cua lignin, hp chat phong phu th ba trong biomass, van con cha c nghien cu ay u cho ti ngay nay [47]. Ly do la thieu phng phap trch ly e loai bo cau truc lignin ma khong anh hng g ti ham lng cua no trong suot qua trnh trch ly t biomass. Tuy nhien, cac nha nghien cu lignin cho rang o la mot ai phan t vo nh hnh, hau het c tao bi cac n v phenylpropan va ket hp bat ngang bi lien ket carboncarbon [40]. Hnh 2.5 cho thay cau truc e ngh cua lignin. Lignin c tao thanh t ba n v phenylpropane c ban: pcoumaryl, sinapyl va coniferl. Lignin go mem c goi la guiacyl lignin chu yeu la t cac n v coniferyl alcohol. Con go cng cha ca syringyl (t sinapyl alcohol) va guiacyl lignin. Ba tien chat cua lignin c the hien hnh 2.6.

Hnh 2.5. Cau truc e ngh cua lignin go mem.

Chng 2: Tong quan

Hnh 2.6. Cac n v tien chat cua lignin. (a) coniferyl alcohol; (b) p-coumaryl alcohol; (c) syringyl alcohol. Lignin ong mot vai tro quan trong trong viec lam vng chac thanh te bao nh la lien ket ngang noi cac polysacharide, do o ho tr cho cac thanh phan cau truc cua c the thc vat. Ngoai ra, no cung giup thc vat chong lai am va s tan cong sinh hoc. Tuy nhien, nhng tnh chat nay cua lignin lai gay tr ngai cho qua trnh thuy phan bang enzym do can tr s tiep xuc gia enzym va cellulose. Nh vay, cau truc t nhien cua thanh te bao thc vat a han che s xam nhap cua cac chat hoa hoc va enzym en c chat cua chung. Lp lignin-hemicellulose phu ngoai cac vi si cellulose cua thanh te bao a anh hng en s thuy phan cua enzym. Ngoai ra, cau truc tinh the cua cellulose cung han che nhieu en kha nang tan cong cua enzym. ay chnh la tr ngai ln trong phng phap thuy phan cellulose. V vay, e giai quyet van e nay, nguyen lieu cellulose can trai qua qua trnh tien x ly.
2.2.4 Chat trch li

Chat trch ly bao gom cac thanh phan hoa tan trong dung moi hu c hay nc. Chung chiem 2 8% khoi lng kho cua go, phu thuoc vao loai thc vat, v tr cua cay, cung nh nhng tieu chuan khac [46]. Nhng hp chat o rat quan trong trong nhng chc nang sinh hoc cua cay. Terpenoid, steroid, chat beo, nha va nhng phan t phenolic nh stilbene, lignan, tannin va flavonoid eu la nhng 7

Chng 2: Tong quan chat trch ly. Cac phenolic co thuoc tnh diet nam va anh hng en mau cua go. Chat beo va sap, trong nhieu he thong sinh hoc c tan dung nh la nguon nang lng trong khi terpenoid va steroid c biet en la nha dau. Nhom cuoi cung cung co hoat tnh khang vi sinh vat va con trung. Mot so chat trch ly la nhng dc pham quan trong. V du, flavonoid c s dung nh la chat chong tac nhan oxy hoa va chong virus [51]. Mot so cau truc chat trch ly c the hien hnh 2.7.

Hnh 2.7. Cac v du ve chat trch ly. (a) abietic acid (oleoresin); (b) cathechin (flavonoid); (c) palmitic acid (acid beo)
2.2.5 Tro

Tro la thanh phan vo c cua nguyen lieu cellulose. Tro t hn 1% hau het go. Mot so chat vo c t muoi kim loai ton tai trong vach te bao thc vat. Kim loai vet thng ton tai dang phc hp nh magie trong chlorophyll. Canxi thng la kim loai phong phu nhat, sau o la kali va magie. Thanh phan nguyen lieu cellulose

Bang 2.1 trnh bay thanh phan chnh cua mot so nguyen lieu cellulose trong t nhien. Theo o, thanh phan cellulose, hemicellulose va lignin thay oi tuy thuoc vao loai thc vat, v tr va mc o trng thanh cua cay. Tuy nhien, cac thanh phan nay eu nam trong khoang gia tr cua nguon nguyen lieu cellulose noi chung. Cu the, ham

Chng 2: Tong quan lng cellulose trong hau het cac nguon nguyen lieu la 3050%, hemicellulose la 20-40% va lignin 20-30%. Bang 2.1. Thanh phan cua rm ra theo mot so tai lieu. Thanh Cellulose Hemicellulose Lignin Tro TLTK phan (%) (%) (%) (%) Loi ngo 45 35 15 [28] Than 36,4 22,6 16,6 7,3 [43] ngo Ba ma 33,4 30 18,9 [28] ti Rm 32,1 24 18 [28] (rice straw) Rm 30 50 15 [28] (wheat straw) Rm ra 34-48 32-40 12 14-18 [2] Rm 38,2 24,7 23,4 13 [43] (wheat straw) Nguyen 30-50 20-40 20-30 [44] lieu cellulose
2.2 PHNG PHAP LEN MEN ETANOL T CELLULOSE

Phng phap len men etanol t nguyen lieu cellulose gom cac bc chnh: - Pha v cau truc phc tap cua lignocellulose e giai phong cellulose, hemicellulose, lignin va mot so chat khac bang qua trnh tien x ly. - Thuy phan cellulose, hemicellulose e tao cac ng t do (pentose, hexose). 2.2.1

Len men hon hp ng tren e tao etanol. Qua trnh tien x ly (pretreatment process)

Muc ch cua qua trnh tien x ly la pha v cau truc nguyen lieu cellulose e tang kha nang tiep xuc cua cellulose vi enzym hoac acid, tao thuan li cho qua trnh thuy phan tiep theo.

Chng 2: Tong quan Ba yeu to chnh e pha v cau truc cua lignocellulose la viec tao ra kch thc lo xop [27], s ket tinh cua cellulose [17] va s loai bo lignin [24]. Kha nang tan cong vao cellulose co the gia tang bang cach loai bo hemicellulose, tao ra lo xop ln hn trong vi si do hemicellulose b thuy phan. The tch lo xop co anh hng ti lng glucose tao thanh trong qua trnh thuy phan. Cu the, kch thc lo xop ln th phan tram glucose cao [39]. S ket tinh cua cellulose la nhan to th hai quyet nh en san lng glucose. Mc o ket tinh cao tng tng vi toc o thuy phan cham [24]. Weimer va cac cong s ch ra rang qua trnh x ly hoa hoc va nhiet co khuynh hng lam gia tang ch so ket tinh tng oi (RCI) cua cellulose vo nh hnh [61]. Do kha nang xam nhap vao nhng vi si cellulose b can tr bi ma tran lignin bao quanh nen viec loai bo lignin se lam gia tang kha nang tac ong vao cac ng a. Hau het cac phng phap tien x ly khong thuy phan ang ke en lng cellulose trong nguyen lieu biomass. Tuy thuoc vao nguyen lieu ma ta s dung phng phap vat ly hoac hoa hoc e tien x ly [29]. Phng phap vat ly: s dung nhiet o va ap suat cao, xay nghien, bc xa hoac lam ong, nham lam giam kch thc va pha v cau truc tinh the. Phng phap sinh hoc: cac giong vi sinh vat phat trien tren biomass se lam thoai bien cellulose va lignin. Phng phap hoa hoc: s dung dung moi pha v va hoa tan cau truc cua cellulose.
2.2.1.1 No hi nc (steam explosion)

No hi nc la mot qua trnh tien x ly theo phng phap vat ly. Qua trnh tien x ly nguyen lieu cellulose

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Chng 2: Tong quan bang no hi nc c gii thieu t nam 1980 bi cong ty Iotech Corporation. Iotech a tien hanh mot vai th nghiem tien phong e tm hieu anh hng cua no hi nc len go cay dng. Trong bao cao len bo nang lng My, Iotech a mo ta anh hng cua thi gian phan ng va ap suat len lng xylose va glucose thu c. mot ap suat nhat nh, vi thi gian lu khac nhau san lng cc ai cua glucose va xylose cung khac nhau, trong o xylose thng at cc ai trc glucose. Tng t, san lng cc ai cua xylose va glucose c tm thay ln nhat nhng ap suat khac nhau. ieu kien phan ng toi u cua holocellulose (xylose + glucose) la 500 550 psi trong thi gian 40 giay [17]. a co vai nghien cu ng dung no hi cho cac loai nguyen lieu cellulose sau khi bao cao cua Iotech c trnh bay. Shultz va cong s a so sanh hieu qua cua no hi len hon hp cac manh go cng, vo trau, rm bap, va ba ma. No hi 240 2500C va 1 phut se lam gia tang toc o thuy phan enzyme cua cac manh go cng, vo trau, va ba ma len ngang bang vi toc o thuy phan giay loc. Nghien cu cung tm thay khong co s khac nhau ve toc o thuy phan cua mau a tr trong 8 thang trc vi toc o thuy phan cua mau c tr trong thi gian ngan hn [52]. Qua trnh no hi la mot qua trnh c hoa nhiet [13]. o la pha v cau truc cac hp phan vi s giup cua nhiet dang hi (nhiet), lc cat do s gian n cua am (c) va thuy phan cac lien ket glycosidic (hoa). Trong thiet b phan ng, hi nc di ap suat cao tham nhap vao cau truc lignocellulose bi qua trnh khuech tan. Hi nc ngng tu di ap suat cao lam am vat lieu. Am trong nguyen lieu thuy phan cac nhom acetyl cua hemicellulose hnh thanh nen cac acid hu c nh acetic va uronic acid. Cac acid nay lan lt xuc tac qua trnh depolymer hoa hemicellulose, giai phong xylan va mot phan glucan. Di ieu kien khac nghiet, vung vo nh hnh cua

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Chng 2: Tong quan cellulose co the b thuy phan en mot mc o nao o. Di ieu kien khac nghiet hn, v du nh nhiet o cao va ap suat cao, co the thuc ay s phan huy xylose thanh furfural va glucose thanh 5-hydroxymethyl furfural. Furfural km ham s phat trien cua vi sinh vat, do o no khong thuan li cho qua trnh len men. Khi ap suat trong thiet b phan ng c giai phong, am trong biomass se no ra. ien hnh la vat lieu c a ra khoi thiet b qua mot lo nho bi lc ep. Mot vai hien tng xay ra tai thi iem nay. au tien, am ngng tu trong cau truc biomass boc hi tc thi do giam ap ot ngot. S gian n cua hi nc gay ra lc cat bao quanh cau truc vat lieu. Neu lc cat nay u ln, hi nc se gay ra s pha huy c hoc len cau truc lignocellulose. S mo ta qua trnh lam noi bat tam quan trong cua viec toi u hai yeu to: thi gian lu va nhiet o. Thi gian nguyen lieu lu lai trong thiet b phan ng giup xac nh pham vi thuy phan hemicellulose bi cac acid hu c, giup cho qua trnh len men thuan li hn. Tuy nhien, thi gian lu dai cung lam gia tang s phan huy san pham. Nhiet o co lien quan en ap suat hi trong thiet b phan ng. Nhiet o cang cao th ap suat cang cao, do o cang lam gia tang s khac nhau gia ap suat trong thiet b phan ng so vi ap suat kh quyen. S chenh lech ve ap suat ty le vi lc cat cua am hoa hi.
2.2.1.2 Phng phap hoa hoc

Tat ca cac qua trnh vat ly eu tieu thu nang lng ln, do o co chi ph cao. Cac phng phap sinh hoc khong at hieu qua cao va oi hoi thi gian x ly dai. Do o, phng phap hoa hoc c quan tam nhieu nhat. Cac phng phap hoa hoc khac nhau ang c nghien cu, trong o co phng phap acid loang va phng phap kiem. Phng phap s dung acid loang (chu yeu s dung acid sunfuric) va hi nc hoac nc nong c gia ap dan en viec thuy phan mot lng ang ke hemicellulose. V vay co the at c ham lng ng hoa tan cao t 12

Chng 2: Tong quan hemicellulose. Mot dang khac cua phng phap tien x ly bang acid loang la qua trnh ra nong, lien quan en s phan tach nhiet o cao va viec ra phan ran a x ly nham ngan chan s tai ket tua cua lignin va xylan a hoa tan di ieu kien x ly. S tai ket tua cua lignin co tac ong xau en qua trnh thuy phan tiep theo cua phan ran a x ly. Phng phap kiem nhn chung co hieu qua hn trong viec hoa tan mot lng ln lignin trong khi van gi lai nhieu hemicellulose dang polyme khong hoa tan. Phng phap AFEX (Ammonia freeze explosion) pha v cau truc cua lignocellulose va giam lng cellulase can s dung nhng lai khong loai bo c ca hemicellulose va lignin. Cac phng phap thng c ket hp vi nhau e at c hieu qua cao khi x ly nguyen lieu cellulose. V du, Eniko va cong s [22] a s dung phng phap tien x ly oxy hoa kiem am (alkaline wet oxidation) oi vi phan nguyen lieu than ngo. ay la mot qua trnh oxy hoa am , nguyen lieu se bien oi di tac dung cua hi nc, oxy va nhiet o cao (195oC, 12 bar O2 trong 15 phut). Ngoai ra, do co ket hp s dung kiem (2 g/L Na2CO3) nen kha nang phan huy lignin cung tang, hieu qua x ly nguyen lieu v the cung tang theo. Hieu suat thu hoi cellulose trong nguyen lieu sau x ly at 88% va chuyen hoa t cellulose sang etanl at 41%. Han che cua nhng phng phap tren la can nhieu von. V du, yeu cau ve vat lieu at tien cho bnh phan ng, cac bc them vao e x ly chat thai va thu hoi cac chat xuc tac a lam gia tang chi ph cho qua trnh thuy phan. Mot vai qua trnh tien x ly, v du nh AFEX, tao ra nhng thuan li trong chi ph van hanh do t phat sinh ra chat thai. V vay, tieu chuan cua qua trnh tien x ly thanh cong co the c thu hep theo kha nang x ly cellulose cao, lng ng t hemicellulose thu hoi cao, von ban au va chi ph nang lng thap, s thoai bien lignin thap va s dung hoa chat co the thu hoi c. 13

Chng 2: Tong quan


2.2.2

Qua trnh thuy phan cellulose tao dch ng

Qua trnh thuy phan cellulose c thc hien nham pha v cau truc polimer cua cac phan t cellulose va hemicellulose, giai phong ra cac monomer la cac ng n, tao dch ng len men c chuan b cho qua trnh len men tiep theo. Co ba phng phap e thc hien qua trnh thuy phan nay: Thuy phan bang acid. Thuy phan bang enzyme. Phng phap nhiet hoa.

2.3.1.1 Thuy phan bang acid (acid hydrolysic)

Phng phap thuy phan bang acid co hai loai: thuy phan bang acid co nong o thap va thuy phan bang acid co nong o cao. thap Thuy phan bang acid co nong o

Qua trnh thuy phan bang acid loang c thc hien di nhiet o va ap suat cao va la qua trnh lien tuc co thi gian phan ng t vai giay en vai phut. V du: qua trnh thuy phan cellulose s dung acid sunfuric 1% trong bnh phan ng lien tuc 237oC (458oF) trong 0,22 phut thu c hn 50% ng. Nh vay, vi 1 tan go kho se thu c khoang 189 lt etanol tinh khiet [29]. S ket hp gia acid vi nhiet o va ap suat cao oi hoi bnh phan ng phai c lam bang vat lieu ac biet, do o chi ph kha cao. Hau het cac qua trnh s dung acid loang co hieu qua thu hoi ng gii han trong khoang 50%. Nguyen nhan la trong qua trnh nay xay ra ca hai phan ng: phan ng chuyen cellulose thanh ng va phan ng chuyen ng thanh cac chat hoa hoc khac. ieu kien xay ra phan ng th nhat cung chnh la ieu kien cho phan ng th hai. Do o, mot khi phan t cellulose b pha v th cung nhanh chong xay ra phan ng chuyen ng thanh cac chat

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Chng 2: Tong quan khac - ang ke nhat la furfural, mot chat c s dung trong cong nghiep chat deo. Khong ch viec bien oi cua ng lam giam hieu suat thu hoi ng ma furfural va cac san pham phan huy khac cung gay oc cho cac vi sinh vat len men. u iem ln nhat cua thuy phan bang acid loang la thi gian thuy phan ngan, qua trnh thuy phan lien tuc. Tuy nhien, nhc iem cua no la hieu suat ng thap. e tang s tiep xuc cua acid va c chat, nguyen lieu can c giam kch thc (en vai mm). Do ng C5 b phan huy nhanh hn ng C6 nen mot cach e giam s phan huy ng la thc hien qua trnh qua hai giai oan. Giai oan au c thc hien trong ieu kien on hoa e thu hoi ng C5 trong khi giai oan sau thu hoi ng C6 c thc hien di ieu kien khac nghiet hn. Tuy nhien, s phan huy ng van xay ra va hieu suat gii han trong khoang 272 lt etanol/tan go [29]. cao Thuy phan bang acid co nong o

Qua trnh thuy phan bang acid ac thng s dung nhiet o on hoa va ch dung ap suat khi bm nguyen lieu. Qua trnh nay c thc hien au tien bi ai hoc Purdue va Tennessee Valley Authority (TVA). Trong qua trnh o, phan vo bap c tron vi acid sunfuric loang (10%) va gia nhiet 100oC trong 2-6 gi trong bnh thuy phan th nhat (hemicellulose). Nhiet o va ap suat thap lam giam s phan huy ng. e thu hoi ng, cac chat sau thuy phan c ngam trong nc roi lam kho nhieu lan. Sau o, phan can ran trong giai oan mot c kh nc va ngam trong acid sunfuric nong o 30-40% trong 1-4 gi. ay c coi nh la giai oan tien thuy phan. Sau o tiep tuc tach nc va lam kho e tang nong o acid len khoang 70% va tien hanh phan ng trong mot bnh khac 100oC trong 1-4 gi. Tiep theo loc va loai bo phan can ran, thu hoi dch ng va acid. Dch nay c tai s dung cho lan thuy phan giai oan mot. ng cua giai oan hai 15

Chng 2: Tong quan nh vay c thu hoi trong phan dch thuy phan cua giai oan au. u iem chnh cua qua trnh thuy phan bang acid ac la hieu qua thu hoi ng cao hn (co the len en 90%, cua ca ng t hemicellulose va cellulose). Nhiet o va ap suat thap nen co the s dung vat lieu lam thiet b khong at nh ong dan va cac bon cha bang si thuy tinh. Tuy nhien, nhc iem la qua trnh cham va ton chi ph cho he thong thu hoi acid hieu qua. Neu khong thu hoi acid th can phai s dung lng ln kiem e trung hoa acid, ket qua la tao ra nhieu canxi sunfat can phai loai bo va ieu nay lam gia tang chi ph cho qua trnh. S dung hieu suat chuyen hoa va len men etanol theo gia thuyet, ngi ta tnh c hieu suat tao etanol t glucose oi vi phan than bap (phan tren mat at cua cay bap) trong bang 2.2. Hieu suat tao etanol t xylose c tnh toan va trnh bay trong bang 2.3. Nh vay, nang suat tong cong la 227 lt etanol/ tan than bap. Nhng con so tren cung cho thay gia tr ti han cua hieu suat chuyen hoa va thu hoi ng, hieu suat len men. Neu ca hai hieu suat bay at en 95% th nang suat co the len en 350 lt/tan. Bang 2.2. Lng etanol t glucose [29]. Than bap 1 tan Ham lng cellulose x 0,45 Hieu suat chuyen hoa va thu x 0,76 hoi cellulose Hieu suat thuy phan x 0,51 Hieu suat len men etanol x 0,75 Lng etanol t glucose 131 kg 151 L Bang 2.3. Lng etanol t xylose [29]. Than bap 1 tan Ham lng hemicellulose x 0,29 Hieu suat chuyen hoa va thu x 0,90 hoi hemicellulose Hieu suat thuy phan x 0,51 Hieu suat len men xylose x 0,50 16

etanol

Chng 2: Tong quan Lng etanol t glucose


2.3.1.2 Thuy

66 kg etanol = 76 L bang enzym (enzymatic

phan hydrolysic)

Mot phng phap c ban khac cua qua trnh thuy phan la phng phap enzym. Enzym la nhng protein thc vat xuat hien mot cach t nhien va la nguyen nhan xay ra cac phan ng hoa hoc. e enzym co the hoat ong, chung can phai phu hp vi phan t c thuy phan. e thuy phan cellulose, ngi ta thng dung che pham enzym cellulase. Enzym cellulase c tao ra bi Trichoderma reesei QM 9414. No la mot phc he enzym bao gom ba loai khac nhau lien quan en s ng hoa cellulose la endoglucanase (E.C.3.2.1.4), cellobiohydrolase (E.C.3.2.1.91) va beta-glucosidase (E.C.3.2.1.21). Cac phan ng ket hp cua ba loai enzym nay trong che pham cellulase se thuy phan hoan toan cellulose thanh D-glucose. Tuy nhien, cellulase khong the phan huy hoan toan cellulose trong nguyen lieu lignocellulose t nhien cha x ly. Hemicellulose va lignin can tr s tiep xuc cua phc he enzym nay vi cellulose, chu yeu do lp vo si cellulose bao ngoai. Hn na, ban than lignin co the ket hp vi cellulase do o lam cho no mat hoat tnh hoac giam hieu qua cua viec phan giai cellulose. V du, oi vi go cng th ch phan giai c khoang 10-20% thanh ng khi s dung dch cellulase. [41]. Do o, e cac qua trnh x ly bang enzym hieu qua can phai co giai oan tien x ly e pha v cau truc cua lignocellulose, loai bo lignin e phi bay ra cac phan t cellulose va hemicellulose. Hau het cac phng phap tien x ly khong thuy phan ang ke en lng cellulose trong biomass. Tuy thuoc vao nguyen lieu ma ta s dung phng phap vat ly hoac hoa hoc e tien x ly. Tiem nang ln nhat trong viec san xuat etanol t biomass la phng phap thuy phan bang enzym s dung enzym cellulase. Mac du a c nghien cu hn mot thap k, gia thanh cua loai enzym nay van rat cao. V vay can

17

Chng 2: Tong quan giam chi ph san xuat enzym e qua trnh mang tnh kinh te hn. Cellulase la mot enzym tac ong cham, chu yeu bi v s phc tap, khong hoa tan va cau truc ban tinh the cua c chat. Ngoai ra, hoat tnh enzym toi a ch at c vi s co mat cua cac enzym lien quan nh endoglucanase, exoglucanase va beta-glucosidase e chuyen hoan toan cellulose thanh glucose. Khong ch can phai cai thien ve o ben, hieu suat, lam tang hoat tnh enzym ma con phai lam cho chung hieu qua trong moi trng khac nghiet tao ra bi qua trnh tien x ly. Weich va cong s [61] a tien hanh thuy phan si x bap bang hon hp enzym Novozym 188 va Cellulast 1,5L vi t le 1 :1 50oC. Hieu suat thuy phan oi vi nguyen lieu a x ly la 58% ng vi nong o ng glucose la 39 g/L. Mot v du khac la qua trnh thuy phan bang enzym c thc hien bi National Renewable Energy Laboratory (NREL) [29]. Sau khi x ly bang acid loang, hon hp va c kh oc e loai cac chat co the gay oc cho vi sinh vat. Mot phan nho cua hon hp c a vao mot bnh rieng biet e nuoi vi sinh vat san sinh ra enzym cellulase. Mot phan khac c a vao bnh khac e nuoi dng va phat trien giong nam men cho s len men. Sau o, ca enzym va vi sinh vat len men eu c them vao hon hp va cung mot luc e xay ra qua trnh ng hoa va len men ong thi (SSF). Do cau truc ben cua cellulose, cac enzym can thi gian vai ngay e at ket qua tot. V thi gian cua qua trnh dai nen bnh phan ng can phai ln do o gia thanh cua no cao. Ngoai ra, gia enzym cao cung la mot nhc iem cua phng phap nay. Tuy vay phng phap enzym cung co nhng u iem. o la hieu qua qua trnh kha cao va ieu khien c s hnh thanh cac san pham phu, ieu kien phan ng on hoa nen khong oi hoi thiet b co vat lieu at tien va chi ph ve nang lng la tng oi thap.

18

Chng 2: Tong quan


2.3.1.3 Phng phap nhiet hoa (thermochemical

hydrolysic)

Co hai qua trnh san xuat etanol co s dung cac phan ng nhiet hoa trong o. Qua trnh th nhat la he thong pha tron gia nhiet hoa va sinh hoc. Mot v du cua qua trnh nay c thc hien bi Bioengineering Resource Fayetteville, Arkanas. au tien, nguyen lieu sinh khoi c chuyen thanh dang kh bang phan ng nhiet hoa. Sau o, kh tong hp (hon hp cua hidro va cacbon dioxide) c suc vao mot bon len men c thiet ke ac biet. Giong vi sinh vat co the chuyen oi kh tong hp nay c cho vao bon len men di ieu kien ac biet e len men etanol. Qua trnh th hai khong s dung bat k vi sinh vat nao. au tien, nguyen lieu sinh khoi a kh hoa va kh tong hp c a vao bnh phan ng cha cac chat xuc tac chuyen kh thanh etanol. Qua trnh chuyen kh tong hp thanh etanol nay co the at en hieu suat 50%. Mot so cac qua trnh ban au san xuat ra metanol, sau o s dung s thay oi chat xuc tac e san xuat ra etanol co the thu c etanol vi hieu suat trong khoang 80% [29]. Tuy nhien, viec tm ra mot qua trnh nhiet hoa co hieu qua ve kinh te la rat kho.
2.2.3 Qua trnh len men ng thanh etanol 2.2.3.1 C s ly thuyet cua qua trnh len men

Ly thuyet qua trnh len men a c nhieu nha khoa hoc nghien cu t rat lau. Nam 1789, Lavoisier lam th nghiem va a ra ket luan khi len men ng khong ch tao thanh ethanol va CO2 ma con tao ra acid acetic [1]. Nam 1810, Gay-Lussac nhan thay rang c 45 phan khoi lng ng se chuyen thanh 23 phan ethanol va 22 phan CO2 [1]. Tren c s o ong a ra phng trnh tong quat nh sau: C6H12O6 2C2H5OH + 2CO2 + Q

19

Chng 2: Tong quan Nam 1857, Louis Pasteur a ra ket luan ve qua trnh len men: Len men la s song khong co oxi va no ch xay ra khi co mat vi sinh vat. Khi can ung 100 phan trong lng ng saccharose em thuy phan va len men se tao ra 51,1 phan ethanol, 48,4 phan CO2, 3,2 phan glycerin, 0,7 phan acid succinic va hai phan cac san pham phu khac. T o suy ra c 45 phan khoi lng glucose khi len men se tao ra 21.80 phan ethanol ch khong phai 23 phan nh Gaylussac a tnh. Tuy nhien phng trnh len men do Gay- Lussac a ra van ung va dung lam c s ly thuyet e tnh hieu suat thu hoi ru theo ly thuyet. Gay-Lussac con ket luan s len men la qua trnh sinh hoc co lien he mat thiet en s hoat ong cua te bao nam men[1]. Ban chat cua qua trnh len men la qua trnh oxy hoa kh. Qua trnh oxy hoa nay lai xay ra trong c the sinh vat di tac ong cua he thong enzym, cho nen ngi ta goi qua trnh len men la qua trnh oxy hoa sinh hoc [3]. Dien bien cua qua trnh len men: trc tien, ng cung cac chat dinh dng khac cua moi trng len men c hap phu tren be mat cua te bao nam men, sau o khuech tan qua mang, vao ben trong te bao. ng b chuyen hoa qua mot chuoi phan ng enzym e tao thanh san pham cuoi cung la ru etylic va CO2. Ru etylic va CO2 va tao thanh lien thoat ra khoi te bao va khuech tan vao moi trng xung quanh. S tao thanh ru t glucose phai trai qua nhieu giai oan. Glucose se tham gia vao chu trnh ng phan e tao thanh pyruvate. Chu trnh ng phan c the hien hnh 2.8. Sau o, pyruvate se chuyen thanh con theo cac phng trnh sau:
CH3 C O COOH pyruvat decarbonxylase -CO2 CH3CHO

20

Chng 2: Tong quan


alcol- dehydrogenase

CH3CHO NADH

C2H5OH

+ H+

NAD+

Hnh 2.8. Chu trnh ng phan.

21

Chng 2: Tong quan


2.2.3.2 Vi sinh vat s dung trong len men

Vi sinh vat s dung trong len men etanol la nhng vi sinh vat co kha nang tao ra etanol nh la san pham chnh. Nhng vi sinh vat nay c liet ke trong bang 2.4.

22

Chng 2: Tong quan

Bang 2.4. Mot so vi sinh vat s dung trong len men [63].
Chung- Giong 27817Saccharomyces cerevisiae L-041-S. cerevisiae 181-S.cerevisiae (acrobic) UO-1-S. cerevisiae V5-S. cerevisiae Fiso-S. cerevisiae GCB-K5-S. cerevisiae GCA-II-S. cerevisiae 27774Kluyveromyces fragilis 30017-K. fragilis 30016-K. fragilis 30091-Candida utilis ATCC-32691Pachysolen tannophilus Nhiet o (oC) 30 pH 5,5 Nguon cacbon va nong o (g/L) Glucose (50200) Saccharose (100) Glucose (10) Nguon Nit va Thi nong o (g/L) gian (h) Pepton (2) va 18-94 Amoni Sunfat (4) Ure (1) va Amoni 24 Sunfat (4) Pepton (5) 40-160 60-96 36 60 72 Nong o etanol (g/L) 5,1-91,8 25-50 TLTK [58] [34] [56] [10] [58] [15] [33] [33] [58] [58] [58] [58] [49]

30 hoac 35 27 6,0 30 24 30 30 30 30 30 30 30 30 5,0 5,0 6,0 6,0 5,5 5,5 5,5 5,5 4,5

Saccharose (20) Amoni Sunfat (1) Glucose (250) Galactose (20- Pepton, Amoni 150) Sunfat va casamino acid (10) Saccharose (30) Pepton (5)

4,8-60 27 42 48,96 48,96 48,4 48,4 7,8

Saccharose (30) Pepton (5) 72 Glucose (20- Pepton (2) va 18-94 120) Amoni Sunfat (4) Glucose (20- Pepton (2) va 120) Amoni Sunfat (4) Glucose (100) Pepton (2) va Amoni Sunfat (4) Glucose (100) Pepton (2) va Amoni Sunfat (4) Glucose (0-25) Pepton (3,6) va va xylose (0- Amoni Sunfat (3) 25) 18-94 18-94 18-94 100

23

Chng 2: Tong quan

Loai vi sinh vat thong dung nhat c s dung la nam men Saccharomyces cerevisiae. No co the tao ra etanol at ti nong o 18% dch len men. Loai nam men nay co the phat trien trong ca moi trng ng n (glucose) va moi trng ng oi (saccharose). Saccharomyces cerevisiae c xem la an toan cho con ngi do o c s dung trong cong nghiep san xuat thc uong co con va trong len men banh m. Cung nh nhieu loai vi sinh vat, Saccharomyces cerevisiae chuyen hoa glucose theo con ng EmbdenMeyerhof (EM). Zymomonas mobilis la mot vi sinh vat gram am khong thong dung co tnh chat nh mot chat xuc tac sinh hoc cho qua trnh len men etanol. Vi sinh vat nay co kha nang len men homoetanol va chu c ti 120 g/L etanol. No co hieu suat etanol cao hn (hn 5-10% etanol/glucose c len men) va co kha nang san xuat etanol ac biet cao hn Saccharomyces cerevisiae 2,5 lan [63]. Ngoai ra, Zymomonas mobilis co nhu cau dinh dng n gian hn. Tuy nhien, no khong thch hp cho tat ca s chuyen hoa cac nguon sinh khoi bi v no ch len men c glucose, fructose va saccharose. Trong nhng moi trng tong hp cha glucose, fructose hoac saccharose, t le ac trng cua sugar uptake va s san sinh etanol la cc ai khi tan dung moi trng glucose. Saccharomyces cerevisiae van c cong nghiep a dung hn nh kha nang chu ng cua nam men. E. coli k thuat la mot loai vi khuan co gia tr trong viec san xuat etanol. Cau truc cua giong E. coli e san xuat etanol mot cach chon loc la mot trong nhng ng dung thanh cong au tien cua k thuat trao oi chat. E. coli co nhng thuan li cua mot chat xuc tac sinh hoc cho s san sinh etanol, bao gom kha nang len men mot pho rong ng, khong oi hoi nhng yeu to phat trien phc tap va a dc s dung trong cong nghiep trc o (san xuat protein tai to hp). Bat li chnh cua viec s dung E. coli la day pH e phat trien cua no hep va nam trong

24

Chng 2: Tong quan khoang trung tnh (6,0-8,0), kha nang chu ng cua no thap hn nam men va viec nhan thc ve s nguy hiem cua no. Qua trnh chuyen hoa sinh hoc t cellulose thanh etanol co the c ieu khien bi cac vi khuan k kh chu nhiet khac nhau nh Clostridium thermocellum cung nh bi nam si nh Monilia sp., Neurospra crassa, Neurospora sp., Zygosaccharomyces rouxii, Aspergillus sp., Trichoderma viride va Paecilomyces sp.. Tuy nhien nhng nghien cu tren qua trnh len men s dung nhng giong vi sinh vat nay cho thay thi gian len men rat lau (3-12 ngay) va nang suat thap (0,8-60 g/L etanol) [63]. o la do kha nang khang lai nong o con cao cua nhng vi sinh vat nay la rat thap. Ngoai ra, qua trnh len men con tao ra cac san pham phu khac nh acid acetic va acid lactic.
2.2.3.3 Qua trnh len men trong SHF

SHF la qua trnh thuy phan va len men rieng biet (Separate Hydrolysis and Fermentation). Sau khi tien hanh thuy phan, phan dch thu c se c hap tiet trung roi cay giong vi sinh vat e thc hien qua trnh len men. Che o len men (nhiet o, thi gian, pH) cung nh nong o etanol thu c tuy thuoc vao giong vi sinh vat s dung va nong o ng ban au. Weich va cong s [60] a tien hanh len men dch ng thu c t qua trnh thuy phan si x bap. Vi nong o ng ban au 42 g/L, sau thi gian 25 gi len men thu c 20 g/L etanol. Tien hanh chnh pH va nong o xylose trong dch thuy phan ti mc co the len men, nong o glucose ban au la 38 g/L. Nong o etanol thu c lan lt la 24 va 29 g/L ng vi gia tr pH 4,4 va 5,7. Nong o etanol thu c trong cac th nghiem len men SHF co gia tr khong cao. Co nhieu nguyen nhan dan en van e nay. Ben canh ly do nong o ng trong dch thuy phan thap th mot nguyen nhan khong kem phan quan trong la do s co mat cua cac chat c che (lignin, fufural) hnh thanh trong cac giai oan trc. ieu nay ch co the c khac phuc mot phan thong qua viec la chon 25

Chng 2: Tong quan cac phng phap tien x ly va thuy phan. Ngoai ra, con co the han che bang cach s dung phng phap SSF.
2.2.3.4 Qua trnh ng hoa va len men ong

thi (SSF)

Trong phng phap thuy phan bang enzym, cellulase trong dch sinh khoi tien x ly b c che manh bi cac san pham thuy phan (glucose va cac chuoi cellulose mach ngan). Mot cach e khac phuc s c che enzym nay la len men glucose thanh etanol ngay khi no va tao thanh. o chnh la ky thuat ng hoa va len men ong thi (SSF). No ket hp thuy phan bang enzym vi len men etanol e gi nong o glucose thap. Bi v s tch tu cua etanol trong bnh len men khong c che cellulase nhieu bang nong o glucose cao nen SSF la mot cach tot e tang t le cellulose chuyen thanh etanol. Dale va cong s [16] a tien hanh qua trnh SSF tren nguyen lieu la rm a x ly, ket qua thu c nong o etanol sau 95 gi la 40 g/L. Th nghiem tng t oi vi vo cay thch (maple shaving) cho ket qua etanol la 25 g/L sau 72 gi. Eniko va cong s [22] a tien hanh th nghiem tren vo cay bap va thu c 50 g/L etanol vi hieu suat chuyen hoa t cellulose sang etanol la 41% cung bang phng phap SSF. Nh vay, so vi qua trnh ma hai giai oan nay c tach rieng (SHF) ra th phng phap SSF co the at c lng etanol cao hn (len en 40%) nh loai bo s c che cua san pham cuoi. Phng phap SSF con giup giam viec can cac thiet b rieng biet cho qua trnh ng hoa va len men. Ngoai ra, no cung co nhng u iem khac nh thi gian len men ngan hn, giam nguy c nhiem vi sinh vat ben ngoai nh nhiet o cao cua qua trnh, s hien dien cua etanol trong phan ng va ieu kien k kh [62]. Tuy nhien, phng phap SSF cung co mot so nhc em. o la s khac nhau gia nhiet o toi u cua s thuy phan (45-50oC) va s len men (28-35oC) [9]. Ngoai ra, 26

Chng 2: Tong quan ban than etanol va mot so chat oc tang len trong qua trnh tien x ly lignocellulose gay c che hoat ong cua cac vi sinh vat len men va hoat tnh cua cellulase [63]. Viec at c s tng hp gia vi sinh vat va enzym la van e chnh trong SSF bi v co mot so chat sinh ra trong qua trnh phan giai te bao vi sinh vat, v du nh enzym phan giai protein, co the lam thoai hoa cellulase; ngc lai, co co nhng chat trong che pham enzym cung lam giam kha nang song sot cua vi sinh vat dan en s phan giai te bao. Tom lai, oi vi phng phap SSF th mot so ch so cua qua trnh can phai c toi u nh nong o c chat, t le enzym cho vao c chat, lieu lng cac thanh phan co hoat tnh trong che pham enzym (t le anpha-glucosidase: glucanase) va nong o nam men.

27

Chng 3: Nguyen lieu va phng phap nghien cu

NGUYEN LIEU VA PHNG PHAP NGHIEN CU


3.1 NGUYEN LIEU 3.1.1 Rm

Chng 3

Nguyen lieu s dung trong nghien cu nay la rm c lay t xa Thai My, huyen Cu Chi, thanh pho Ho Ch Minh. Trc khi tien hanh th nghiem, rm tho c xac nh thanh phan cac chat bao gom: ham lng cellulose, ham lng lignin, hemicellulose va ham lng tro. Rm c trai qua qua trnh tien x ly bang cach cho no hi (steam explosion). Phan rm thu hoi sau khi loai bo phan dch long c s dung lam nguyen lieu cho cac th nghiem.
3.1.2 Enzym cellulase

Trong nghien cu nay, chung toi s dung enzym Cellusoft L cua hang Novo Nordisk do cong ty TNHH Nam Giang, 133/11 Ho Van Hue, quan Phu Nhuan phan phoi tai Viet Nam. Cellusoft L la ten thng mai cua enzyme cellulase. ay la mot che pham cellulase co dang long, mau nau o, c ieu che bang phng phap len men chm vi hoat tnh bieu th 1500 NCU/g. Hoat tnh bieu th nay c xac nh theo phng phap phan tch cua Novo Nordisk, AF 187.2. Cac thong so cua enzyme Cellusoft L: Nhiet o hoat ong: 50 - 550C

pH hoat ong toi u: 4,5 5,5

3.1.3 Hoa chat Moi trng nuoi cay

Danh sach cac hoa chat s dung c trnh bay trong bang 3.1. Bang 3.1. Cac hoa chat s dung. Ten hoa chat Acid sulfuric 98% Thuoc th glucose CII Ni san xuat Trung Quoc Centronic GmbH - c 28

Chng 3: Nguyen lieu va phng phap nghien cu Thuoc th ethanol Cong ty Diasys c

Thanh phan moi trng nuoi cay vi sinh vat c trnh bay trong bang 3.2. Bang 3.2. Thanh phan moi trng Hansen dung e nuoi cay nam men. Ten hoa chat Glucose K2HPO4 Peptone MgSO4.7H2O Agar Nc cat Khoi lng 50g 3g 10g 2,5g 20g 1000ml Ni san xuat Trung Quoc Trung Quoc Difco Trung Quoc Viet Nam Viet Nam

3.1.4 Giong vi sinh vat

Chung toi s dung giong nam men Saccharomyces cerevisiae vi nhieu chung khac nhau, t o chon ra chung thch hp e tien hanh len men etanol. Cac chung nam men s dung c trnh bay trong bang 3.3. Bang 3.3. Cac chung nam men s dung Chun Ni cung cap g KHTN Bo mon Cong Nghe Sinh Hoc ai hoc Khoa Hoc T Nhien TP HCM V1 Bo mon Cong Nghe Thc Pham ai hoc Bach Khoa TP HCM V6 Bo mon Cong Nghe Thc Pham ai hoc Bach Khoa TP HCM BT Bo mon Cong Nghe Thc Pham ai hoc Bach Khoa TP HCM RD Bo mon Cong Nghe Thc Pham ai hoc Bach Khoa TP HCM TG Bo mon Cong Nghe Thc Pham ai hoc Bach Khoa TP HCM Cac chung nay eu a c hoat hoa va cay chuyen tren thach nghieng, bao quan 40C va c gi giong bang cach cay chuyen hang thang.
3.2 PHNG PHAP NGHIEN CU 3.2.1 Muc ch cua qua trnh nghien cu

29

Chng 3: Nguyen lieu va phng phap nghien cu Muc ch cua nghien cu nay la xac nh cac thong so toi u cho qua trnh thy phan rm va qua trnh len men nham nang cao ham lng con thu c.
3.2.2 S o nghien cu

Kh s g y lie a a nue o t n u

Kh s u n t u p a a a q at h h h o t r y n To h m ot o gs u u o o s oc i a t hn a q at n t u p a u hh h r y n

Kh s u n le mn a a q at h e o t r n To h m ot o gs u u o o s oc i a t hn a q at n le mn u h e r n

Hnh 3.1. S o nghien cu.


3.2.3 Bo tr th nghiem 3.2.3.1 Khao sat nguyen lieu

Khao sat thanh phan rm tho Th nghiem khao sat thanh phan nguyen lieu rm tho c thc hien tai trung tam cong nghe Loc Hoa dau, ai Hoc Bach Khoa Thanh Pho Ho Ch Minh. Mau rm tho c xac nh ham lng cellulose, lignin, tro theo phng phap hoa ly trnh bay muc 3.2.5.1, t o xac nh ham lng hemicellulose theo cong thc: % hemicellulose = 100 - (% cellulose + % tro + % lignin) (3-1) Tien x ly rm tho Rm tho c cat nho (khoang 3 cm) va tien hanh cho no hi trong thiet b no hi nc. Nc c them vao rm vi t le la 100 gam rm :1 lt nc. Thong so cua qua trnh no hi: Nhiet o: 200-230oC. 30

Chng 3: Nguyen lieu va phng phap nghien cu Ap suat:1 atm. Thi gian: 1 phut.

Sau no hi thu c phan dch set (slurry) gom rm a no hi va phan dch no hi. Tien hanh tach rieng phan rm va dch no hi, sau o say kho phan rm e s dung trong cac th nghiem. Phan dch c xac nh nong o ng trc khi loai bo. Khao sat thanh phan rm a x ly Th nghiem khao sat thanh phan rm a qua qua trnh tien x ly (qua trnh no hi) c thc hien tai trung tam cong nghe Loc Hoa dau, ai Hoc Bach Khoa Thanh Pho Ho Ch Minh. Mau rm a x ly c xac nh ham lng cellulose, lignin, tro theo phng phap hoa ly trnh bay muc 3.2.5.1, t o xac nh ham lng hemicellulose theo cong thc 3-1.
3.2.3.2 Khao sat qua trnh thuy phan

Cac th nghiem thuy phan c thc hien tai phong th nghiem Cong nghe sinh hoc 3 cua Bo mon Cong Nghe Sinh Hoc, ai Hoc Bach Khoa Thanh Pho Ho Ch Minh. Khao sat cac phng phap thuy phan Cac mau rm (rm tho va rm a qua no hi) c thuy phan bang acid va enzym. Phng an bo tr th nghiem c trnh bay trong bang 3.4. Thong so ban au cua cac qua trnh thuy phan c trnh bay trong bang 3.5 va 3.6. Moi mau th nghiem c thc hien 3 lan va lay ket qua trung bnh. Bang 3.4. Phng an bo tr th nghiem khao sat cac phng phap thuy phan Mau Loai rm s dung Che o thuy phan 1 Rm tho Acid 2 Rm a x ly Acid 3 Rm tho Enzym 4 Rm a x ly Enzym

31

Chng 3: Nguyen lieu va phng phap nghien cu Bang 3.5. Thong so cua qua trnh thuy phan rm bang acid. Thong so Gia tr Loai acid H2SO4 Nong o acid 4% o Nhiet o ( C) 121 Ap suat (atm) 1 T le rm (%) 10 Thi gian (h) 1,5 Bang 3.6. Thong so cua qua trnh thuy phan rm bang enzym. Thong so Gia tr Loai enzym Cellusoft L (1500 NCU/g) T le enzym 5% o Nhiet o ( C) 50 pH 5,5 T le rm (%) 10 Thi gian (h) 24 Sau thi gian thuy phan, cac mau th nghiem c xac nh nong o glucose theo phng phap glucose GOD PAP. T o tnh hieu suat chuyen hoa cellulose theo cong thc:
H = C.V .100 m. A

(3-2)

Trong o: H: hieu suat chuyen hoa cellulose (%). C: nong o glucose (g/L). V: the tch dch (L). m: khoi lng rm s dung (g). A: % cellulose trong rm s dung. Khao sat t le enzym Tien hanh th nghiem khao sat anh hng cua t le enzym s dung en nong o ng glucose thu c. Cac mau rm a qua qua trnh tien x ly (no hi) c tien hanh thuy phan bang enzym Cellulsoft. Tien hanh thuy phan trong becher 100ml vi the tch dch la 50ml. Mau thuy

32

Chng 3: Nguyen lieu va phng phap nghien cu phan c cho vao be u lac vi toc o 100 rpm. Gi co nh cac thong so cua qua trnh thuy phan nh bang 3.6, thay oi lan lt t le enzym la 1%, 3%, 5%, 7% va 9%. Sau thi gian thuy phan, vo hoat enzym 100oC va tien hanh loc dch ng e o nong o glucose bang phng phap glucose GOD PAP. Tnh hieu suat chuyen hoa cellulose theo cong thc (3-2).

33

Chng 3: Nguyen lieu va phng phap nghien cu

Khao sat t le rm Cac mau rm a qua qua trnh tien x ly (no hi) c tien hanh thuy phan bang enzym. Tien hanh thuy phan trong becher 100ml vi the tch dch la 50ml. Mau thuy phan c cho vao be u lac vi toc o 100 rpm. Gi co nh cac thong so cua qua trnh thuy phan nh bang 3.6, thay oi lan lt t le rm la 1%, 5%, 10%, 15% va 20%. Sau thi gian thuy phan, vo hoat enzym 100oC va tien hanh loc dch ng e o nong o glucose bang phng phap glucose GOD PAP. Tnh hieu suat chuyen hoa cellulose theo cong thc (3-2). Ham muc tieu c tnh theo cong thc:
Y = C.H 100

(3-3)

Trong o: Y: ham muc tieu. C : nong o ng (g/L). H : hieu suat chuyen hoa cellulose (%). Khao sat thi gian thuy phan Rm a qua qua trnh tien x ly (no hi) c tien hanh thuy phan bang enzym Cellusoft. Tien hanh thuy phan trong erlen 500ml vi the tch dch la 250ml. Mau thuy phan c cho vao tu lac on nhiet vi toc o 100 rpm. Gi co nh cac thong so cua qua trnh thuy phan nh bang 3.6, lay mau thuy phan lan lt theo thi gian. Cac mau thuy phan c vo hoat enzym 100oC va tien hanh loc dch ng e o nong o glucose bang phng phap glucose GOD PAP.
3.2.3.3 Toi u hoa mot so thong so cua qua trnh

thuy phan

Cac mau rm a qua qua trnh tien x ly (no hi) c tien hanh thuy phan bang enzym. Tien hanh thuy phan trong becher 100ml vi the tch dch la 50ml. Mau thuy phan 34

Chng 3: Nguyen lieu va phng phap nghien cu c cho vao be u lac vi toc o 100 rpm. Gi co nh cac thong so cua qua trnh thuy phan nh bang 3.6, hai yeu to thay oi la ty le rm (Z1) va ty le enzym (Z2). S dung phng an trc giao cap 2 vi so th nghiem toi u la 9, trong o co mot th nghiem tam phng an, ong thi thc hien them 3 th nghiem tam e kiem tra y ngha cua cac he so hoi quy. Phng an tien hanh th nghiem c tom tat trong bang 3.7. Bng 3.7. Phng an tien hanh th nghiem toi u hoa qua trnh thuy phan. Mau T le rm (%) T le enzym (%) 1 5 1 2 5 3 3 5 5 4 10 1 5 10 3 6 10 5 7 15 1 8 15 3 9 15 5 10 10 3 11 10 3 12 10 3 Bng 3.8. Mc c s, khoang bien thien cua yeu to ty le rm va ty le enzym. Yeu to Ky Z1 Z2 Cong thc Ghi hieu chu max Mc tren Zj 15 5 Mc di Mc c s Zjmin Zjo 5 10 1 3
Z max +Z min j j 2

Z =
o j

(3-4)

Khoang bien thien

Zj

j = 1, 2, 3 k
Z =
o j

Z max Z min j j 2

(3-5)

T he toa o Z1,,Zk; chuyen sang he toa o mi khong th nguyen x1,xk.


xj = Zj Zo j Z j

(3-6)

35

Chng 3: Nguyen lieu va phng phap nghien cu Trong he toa o khong th nguyen, mc tren bang +1, mc di bang -1, toa o cua tam phng an bang 0 va trung vi goc toa o. Sau thi gian thuy phan, vo hoat enzym 100oC va tien hanh loc dch ng. ng vi moi mau th nghiem, o nong o glucose thu c bang phng phap glucose GOD PAP, tnh hieu suat chuyen hoa cellulose theo cong thc (3-2), t o tnh c ham muc tieu theo (3-3).
3.2.3.4 Khao sat qua trnh len men

Khao sat giong nam men Th nghiem khao sat cac chung nam men s dung cho qua trnh len men etanol t dch ng thu c qua trnh thuy phan c thc hien tai phong th nghiem Vi sinh Bo mon Cong nghe thc pham trng ai hoc Bach Khoa TpHCM. Cac chung nam men c khao sat c trnh bay trong bang 3.3. Cac chung nay c khao sat s bo ve s thay oi nong o ng trong qua trnh len men, nong o ng sot va nong o etanol tao thanh. Dch ng thu c t qua trnh thuy phan c s dung lam nguyen lieu cho th nghiem nay. Moi mau th nghiem gom 100 ml dch ng cha trong erlen 250 c chnh ve pH 4,5 va hap tiet trung 121oC, 1 at trong 20 phut. Cac chung nam men trc khi a vao len men can c nhan giong qua hai cap nham tang sinh khoi, tang hoat lc giong. Phng phap nhan giong hai cap c trnh bay muc 3.2.4.2 B. 10 ml canh trng giong cua moi chung nam men c them vao cac erlen cha dch ng a hap tiet trung. Bang 3.9 trnh bay tong so te bao trong 1 ml canh trng cua cac chung nam men s dung. Cac thong so cua qua trnh len men c trnh bay trong bang 3.10. Cac mau len men c tien hanh lay mau theo thi gian e o nong o glucose theo phng phap glucose GOD PAP. Khi nong o ng glucose con lai trong mau giam 36

Chng 3: Nguyen lieu va phng phap nghien cu en mc thap, tien hanh ngng qua trnh len men va o nong o etanol tao thanh theo phng phap Etanol FS. Bng 3.9. Tong so te bao trong 1 ml canh trng cua cac chung nam men s dung. Chung nam V1 V6 KHTN RD TG BT men So te bao 15,14 15,15 15,10 15,12 15,14 15,12 (trieu TB/mL) Bng 3.10. Cac thong so cua qua trnh len men. Thong so Gia tr Nhiet o (oC) 30 pH 4.5 T le giong (%) 10 Thi gian (ngay) 5 Van toc lac (rpm) 50 Khao sat thi gian len men Th nghiem khao sat thi gian len men c thc hien tai phong th nghiem Vi sinh Bo mon Cong nghe thc pham trng ai hoc Bach Khoa TpHCM. Dch ng thu c t qua trnh thuy phan c s dung lam nguyen lieu cho th nghiem nay. Mau th nghiem gom 200 ml dch ng cha trong erlen 250 c chnh ve pH 4,5 va hap tiet trung 121oC, 1 at trong 20 phut. Chung nam men s dung la chung KHTN c nhan giong qua hai cap nham tang sinh khoi, tang hoat lc giong. Phng phap nhan giong hai cap c trnh bay muc 3.2.4.2 B. 20 ml canh trng giong cap 2 cua nam men c them vao erlen cha dch ng a hap tiet trung. Cac thong so cua qua trnh len men c trnh bay trong bang 3.10. Tien hanh lay mau theo thi gian, xac nh nong o glucose va etanol trong mau ong thi em tong so te bao nam men.
3.2.3.5 Toi u hoa thong so cua qua trnh len men

Dch ng thu c t qua trnh thuy phan c s dung lam nguyen lieu cho th nghiem nay. Moi mau th nghiem gom 100 ml dch ng cha trong erlen 250 c 37

Chng 3: Nguyen lieu va phng phap nghien cu chnh ve pH 4,5 va hap tiet trung 121oC, 1 at trong 20 phut. Chung nam men s dung la chung KHTN c nhan giong qua hai cap nham tang sinh khoi, tang hoat lc giong. Phng phap nhan giong hai cap c trnh bay muc 3.2.4.2 B. Gi co nh cac thong so cua qua trnh thuy phan nh bang 3.10, hai yeu to thay oi la pH (Z1) va ty le giong (Z2). S dung phng an trc giao cap 2 vi so th nghiem toi u la 9, trong o co mot th nghiem tam phng an, ong thi thc hien them 3 th nghiem tam e tnh phng sai tai hien, kiem tra y ngha cua cac he so hoi quy. Phng an tien hanh th nghiem c tom tat trong bang 3.11. Mc c s, khoang bien thien cua cac yeu to khao sat c trnh bay trong bang 3.12.

38

Chng 3: Nguyen lieu va phng phap nghien cu

Bng 3.11. Phng an tien hanh th nghiem toi u hoa qua trnh len men. Mau pH T le giong (%) 1 4 10 2 4 15 3 4 20 4 4,5 10 5 4,5 15 6 4,5 20 7 5 10 8 5 15 9 5 20 10 4,5 15 11 4,5 15 12 4,5 15 Bng 3.12. Mc c s, khoang bien thien cua yeu to pH va ty le giong. Yeu to Ky hieu Z1 Z2 Ghi chu max Mc tren Zj 5 20 Mc di Mc c s Khoang bien thien Zjmin Zjo Zj 4 4,5 0,5 10 15 5 (3-4) (3-5)

T he toa o Z1,,Zk; chuyen sang he toa o mi khong th nguyen x1,xk bang cong thc (3-6). Trong he toa o khong th nguyen, mc tren bang +1, mc di bang -1, toa o cua tam phng an bang 0 va trung vi goc toa o. Sau thi gian len men, o nong o etanol thu c bang phng phap etanol FS. Chon nong o etanol lam ham muc tieu. Hieu suat qua trnh len men c tnh theo cong thc:
H = CE .100 C G .0,51

(3-7)

Trong o: CE: nong o etanol thc te (g/L).

39

Chng 3: Nguyen lieu va phng phap nghien cu CG: nong o glucose ban au (g/L). 0,51: he so chuyen hoa t glucose thanh etanol.
3.2.4 Quy trnh cong nghe
Rm

Tie x (no n ly hi)

Thu pha y n

Enzym / Acid Giog na n m men

Le men n

Chng ca t

Etanol

Hnh 3.2. Quy trnh cong nghe.


3.2.5 Cac phng phap phan tch 3.2.5.1 Phng phap hoa ly

A. Phan tch nguyen lieu a) Phan tch ham lng cellulose Ap dung phng phap cua Nha may giay ong Nai TAPPI TESST METHODS. Cach tien hanh - Can 1-2g mau a say kho, cho vao bnh cau dung tch 500ml. Them vao 200ml dung dch NaOH 0,5%, lap ong sinh han ngc (hoi lu) va un nhe trong 30 phut (khong e bot trao len). Ky thuat nay dung e hoa tan tinh bot, cac pectin, lignin co tnh chat acid va t ket hp vi cellulose, khong anh hng g en ham lng cellulose ngay ca nhng phan t thap nhat. 40

Chng 3: Nguyen lieu va phng phap nghien cu - Loc qua giay loc, ra can con lai vi dung dch NaOH nong, cho can cellulose tac dung vi 10ml dung dch HCl 10% trong bnh cau nhiet o thng. Them 10ml dung dch nc javel tng giot mot, va cho va khuay eu. e yen trong 5 phut roi loc. Cho can tac dung vi dung dch NaOH 0,5% nhiet o 40oC e hoa tan lignin (a b clo hoa), e yen vai phut roi loc. Lap lai 2 lan na e co cellulose that trang. Ra that ky bang nc soi, say kho en khoi lng khong oi , at trong bnh ut am va can. Tnh toan ket qua Ham lng cellulose = Trong o: C: khoi lng cua cellulose (g). W: khoi lng kho cua mau (g). b) Phan tch ham lng lignin Ap dung phng phap cua Nha may giay ong Nai TAPPI TESST METHODS. Dung acid sulfuric 72% 24N e hydro hoa va hoa tan cac cacbonhydrat trong nguyen lieu; lignin khong hoa tan trong acid c loc va can. Cach pha hoa chat - Acid sulfuric 72% 24N, trong lng rieng 1,6338 c chuan b nh sau: rot 655ml acid sulfuric am ac vao 300ml H2O, lam lanh, pha thanh 1000ml. ieu chnh nong o thanh 24N bang cach chuan o vi kiem. - Hon hp etanol-benzen: 1 the tch etanol 95% va 2 the tch benzen. Cach tien hanh - Loai resin: lay 2g rm ra, cho 25ml hon hp etanol- benzen vao, e yen trong 30 phut, gan bo hon hp tren, cho them 25ml etanol e trong 15 phut, gan bo etanol tha, ra lai bang nc nong, loc e loai nc. Phan ran c say kho trong tu say 60oC.
C x100 (%) W

(3-8)

41

Chng 3: Nguyen lieu va phng phap nghien cu - Can lay 1g rm ra a loai resin cho vao becher 250ml, cho t t 15ml H2SO4 72% vao, dung ua khuay that ky, gi mau 2oC cho en khi hoa tan. - Sau khi mau tan, bao mieng becher lai va gi nhiet o thng trong 2 gi. Thng xuyen khuay cho en khi mau trong becher tan thanh dch. - Cho khoang 300-400ml nc vao becher 1000ml va chuyen mau t becher 250ml vao, ra va pha loang ti the tch 575ml (o H2SO4 khoang 3%). - Lap ong sinh han ngc (hoi lu) va un trong 4 gi ke t luc soi. - e yen cho lang tua, hut bo phan dch noi. Ra acid lan trong lignin vi nc nong. Lam kho lignin bang cach say 105oC en trong lng khong oi (A). - at trong bnh hut am va can. Tnh toan ket qua Ket qua tnh ra ham lng lignin (%) trong 1g mau. Cong thc tnh: Ham lng lignin = Trong o: A: khoi lng cua lignin (g). W: khoi lng kho cua mau (g). c) Phan tch ham lng tro Thanh phan tro c xac nh theo quy trnh ASTM E172195 [8]. Tien hanh Acid sulfuric 72% c dung e hydro hoa 0.3g mau trong 2 gi nhiet o 300C. San pham sau hydro hoa c loc qua giay loc. Ba loc c say nhiet o 1050C trong thi gian dai va sau o em can. Lng ba loc nay sau o c tro hoa trong lo nung nhiet o 5750C trong vong 3 gi va em can. 42
A x100 (%) W

(3-9)

Chng 3: Nguyen lieu va phng phap nghien cu Tnh toan ket qua Ham lng tro
ash Ash = * 100 % rawmat ' l

(3-10)

Trong o: Ash: phan tram tro (%) ash: khoi lng tro thu c sau khi nung 5750C (g) rawmatl: khoi lng kho ban au (g). B. Xac nh ham lng ng glucose trong dch thuy phan Xac nh ham lng glucose bang phng phap glucose GOD-PAP [11]. a) Nguyen ly Glucose b oxy hoa bi glucose oxidase vi s hien dien cua oxygen e tao thanh gluconolactone. Hydrogen peroxide c hnh thanh phan ng vi phenol va 4-aminophenazone di xuc tac cua peroxidase e tao thanh thuoc nhuom tm-o nh la chat ch th. Cng o mau cua thuoc nhuom o ty le vi nong o glucose.
O Glucose + O2 + H2O G D gluconolactone + H2O2 O 2H2O2+4-aminophenazone+phenol P D

POD

4-(p-benzo-chinonemonoimino)phanozone + 4H2O b) Xay dng ng chuan - Pha cac dung dch glucose chuan vi nong o 50, 100, 200, 300, 400 mg/dL. - Lay 10 l cac dung dch glucose chuan vao cac ong nghiem khac nhau. - Cho 1000 l thuoc th vao cac ong nghiem cha dung dch glucose chuan tren. - Chuan b mau trang: cho 1000 l thuoc th vao ong nghiem co san 10 l nc cat. 43

Chng 3: Nguyen lieu va phng phap nghien cu - Lac eu, u cac ong nghiem chuan va mau trang nhiet o 370C trong 10 phut. - o o hap thu cua ong chuan va mau trang bc song 500nm. - Ve o th OD=f(C), ta co ng chuan glucose. c) Xac nh ham lng ng glucose trong mau - Pha loang cac dung dch mau en nong o thch hp sao cho nong o ng glucose nam trong khoang nong o cua ng chuan. - Tien hanh nh tren vi cac dung dch mau a pha loang thch hp. - o o hap thu cua mau bc song 500nm. - Xac nh nong o ng glucose trong mau bang phng phap noi suy t ng chuan. C. Xac nh ham lng con trong dch len men Nong o ethanol c xac nh bang phng phap Etanol FS [18]. ay la phng phap nh lng etanol do hang Holzheim cua c cung cap. a) Nguyen ly Phng phap nay s dung thuoc th cha NAD va Alcohol dehydrogenase (ADH).
ADH Ethanol + NAD+ Acetaldehyde + NADH + H+

Vi s hien dien cua NAD, ethanol se b chuyen hoa bi enzyme Alcohol dehydrogenase. o hap thu cua NADH va hnh thanh ty le vi nong o ethanol trong mau. o o hap thu nay bc song thch hp (360-380nm) se xac nh c ham lng etanol trong mau. b) Hoa chat

44

Chng 3: Nguyen lieu va phng phap nghien cu Etanol chuan 3 (g/L). Thuoc th 1. Thuoc th 2.

Thanh phan thuoc th c trnh bay trong bang 3.13. Bang 3.13. Thanh phan thuoc th trong phng phap Etanol FS Loai Thanh phan Ham lng Thuoc th 1 Dung dich em pH 9,0 240 Chat on nh va chat mmol/L bao quan Thuoc th 2 Dung dich em pH 6,6 8 mmol/L NAD 2 mmol/L Alcohol dehydrogenase (ADH) 40 kU/L Chat on nh va chat bao quan c) Phng phap o - Pha dung dch ethanol chuan vi nong o 3g/l. - Chuan b mau trang: cho 1000 l thuoc th 1 vao ong nghiem cha san 10l nc cat. - Mau chuan: cho 1000l thuoc th 1 vao ong nghiem cha san 10l dung dch ethanol chuan. - Chuan b mau e o nong o ethanol: pha loang cac mau can xac nh nong o ethanol en nong o thch hp sao cho nong o ethanol gan bang nong o cua mau chuan. Cho 1000l thuoc th Reagent 1 vao ong nghiem cha san 10l dung dch mau a pha loang. - Lac eu, u cac mau trong 5 phut 370C. oc o hap thu A1 bc song 376 nm tren may o UV - VIS. - Them 250l thuoc th 2 vao tat ca cac mau tren, lac eu, u trong 5 phut 370C. oc o hap thu A2 ngay sau o bc song 376 nm. d) Tnh toan ket qua A = A2 A1 (3-11)

45

Chng 3: Nguyen lieu va phng phap nghien cu Nong o ethanol c xac nh theo phng phap mot chuan Ethanol (g/l) = D. o pH pH cua cac mau th nghiem c xac nh bang may o pH hieu Mettler Toledo.
3.2.5.2 Phng phap sinh hoc

Amau % Achuan %

x 3 (g/l)

(3-12)

A. Phng phap cay chuyen gi giong [6] Tien hanh gieo cay giong nam men t ong giong goc cua phong th nghiem sang ong moi trng thach nghieng a c chuan b san t trc (moi trng Hansen co bo sung thach vi ham lng 2%). Cong viec nay c tien hanh trong tu cay vo khuan e tranh b nhiem cac vi sinh vat khac co anh hng xau en nam men giong va qua trnh len men sau nay. Phng phap gieo cay: thng dung que cay vong va lam theo trnh t sau: ot en con len. Tay trai cam hai ong nghiem: ong canh trng vi sinh vat ngoai, ong moi trng trong, gi ca hai ong hi nghieng. Dung tay phai xoay long cac nut bong. Tay phai cam que cay va vo khuan tren en con. Kep nut bong gia cac ngon tay roi rut ra va gi nh vay tren tay en luc ay lai. ot nong mieng 2 ong nghiem tren en con. a que cay vao ong canh trng va lay mot t canh trng nam men. a au que cay vao ay ong moi trng, hoa giot canh trng vao giot nc ngng ay. Chu y khong e au que cay cham vao thanh ong nghiem.

46

Chng 3: Nguyen lieu va phng phap nghien cu Nhe nhang va t t keo au que cay len tren mat thach, t ay len theo cac kieu: hnh ch chi, hnh vong xoan, cac vach ngang song song Rut que cay ra, vo khuan tren en con, ot mieng ong nghiem va nut bong roi ay lai, e vao gia ong nghiem. Dan nhan len ong moi trng va gieo cay.

Giong nam men sau khi c gieo cay sang moi trng thach nghieng c at vao tu am va gi nhiet o 300C. Sau 48h giong a phat trien tot, cac ong nghiem c at vao tu lanh (nhiet o 4-60C) e bao quan giong, phuc vu cho qua trnh nghien cu. B. Phng phap nhan giong Giong nam men trc khi a vao len men can c nhan giong nham tang sinh khoi, tang hoat lc giong. quy mo phong th nghiem, chung toi tien hanh nhan giong qua hai cap: - Cap 1: Nam men sau khi a phat trien tren moi trng thach nghieng c cay vao ong nghiem cha 10ml dch moi trng Hansen a hap tiet trung va c nuoi nhiet o thng (28-300C) trong 24 gi. - Cap 2: cho ong nghiem a nuoi giong (cap 1) vao 100 ml dch moi trng cap 2 gom 50 ml dch moi trng Hansen cong vi 50 ml dch thuy phan rm ra cha trong erlen 250 a hap tiet trung. Canh trng giong cap 2 c nuoi ieu kien 300C, 130 rpm trong 24 gi. C. em tong so te bao nam men trong 1ml canh trng e anh gia chat lng canh trng nam men can em so te bao trong 1ml. Trong 1ml canh trng nam men phat trien bnh thng phai co t nhat 12-14 trieu te bao. Chung toi em trc tiep bang buong em hong cau. Tien hanh

47

Chng 3: Nguyen lieu va phng phap nghien cu Lac eu canh trng, dung pipet pha loang canh trng vi H2SO4 10%. Dung bong tham nc tam nc cat va phet nhe len 2 khoang ben cua buong em, at la knh len roi dung ngon tay an nhe cho la knh dnh chat vao phien knh. Lac eu dch te bao nam men va dung pipet pasteur e lay mot giot cho vao khe h gia li em va la knh, chu y khong e canh trng ri xuong cac ranh va tranh tao bot kh. at buong em vao ban knh hien vi va e yen trong 3-5 phut. Chnh knh hien vi vi vat knh x40, tm mang o em khu vc buong em. Chnh th trng sao cho mot th trng cha tron mot o ln (4 x 4 = 16 o nho). em so te bao trong 5 o vuong ln ai dien cho 25 o vuong ln trong o trung tam.

Cach tnh So lng te bao trong 1 ml mau nghien cu c tnh bang cong thc:
X = a.4000 .b .1000 c

(3-13)

Trong o: X a b c 1000
3.3

: so lng te bao trong 1 ml mau nghien cu. : so te bao trong 5 o vuong ln. : o pha loang mau : so o vuong nho trong 5 o vuong ln (80 o). : so chuyen mm3 thanh ml.

THIET B S DUNG

Cac thiet b s dung trong nghien cu gom: Thiet b no hi tai trung tam Loc hoa dau. Be lac on nhiet BS 11. 48

Chng 3: Nguyen lieu va phng phap nghien cu Tu u lac SHAKING INCUBATOR JISICO. Noi hap tiet trung HIRAYAMA AUTOCLAVE HV 85. Knh hien vi KRUSS. Buong em hong cau. May o quang GENESIS 20. pH ke Mettler Toledo. Can k thuat SARTORIUS CP2202S. Can phan tch SATORIUS CP224S. Buong cay vi sinh ESCO. May o UV VIS. May o quang HATCH.

49

Chng 4: Ket qua va ban luan

Chng 4

KET QUA VA BAN LUAN


4.1 KHAO SAT THANH PHAN NGUYEN LIEU 4.1.1 Thanh phan rm tho

Ket qua phan tch thanh phan rm tho c trnh bay bang 4.1. Bang 4.1. Thanh lng chat kho). Thanh phan Cellulose Hemicellulose Lignin Tro phan cua rm tho (tnh tren ham Ham lng (%) 43,05 23,82 18 15,13

Tro 15.13% Lignin 18.00% Cellulose 43.05%

Hemicellulose 23.82%

Hnh 4.1. Thanh phan cua rm tho. Ket qua phan tch cho thay rm tho s dung trong th nghiem co ham lng cellulose tng oi cao (43,05%) so vi hemicellulose (23,82%) va lignin (18%). ieu nay phu hp vi thc te v cellulose la thanh phan chu yeu trong rm ra. Ham lng cellulose cao se dan en kha nang thu c nong o ng glucose trong dch thuy phan cao. Ngoai ra, gia tr cua ham lng cellulose trong th nghiem cung phu hp vi ket qua cac tai lieu khac. Bang 2.2 trnh bay thanh phan cua rm ra theo mot so tai lieu trong va ngoai nc. Thanh phan cellulose trong rm la 34-48% theo Ho S Trang [2] va la 32,1% theo Howard [28]. Ket qua th

50

Chng 4: Ket qua va ban luan nghiem ham lng cellulose la 43,05% co chenh lech so vi 32,1% theo Howard, tuy nhien no van nam trong khoang 3448% oi vi nguyen lieu rm ra va nam trong khoang t 30-50% cua nguyen lieu lignocellulose noi chung. o la do ham lng cac chat trong nguyen lieu cellulose co the thay oi phu thuoc vao nhieu yeu to nh loai cay, ni trong va mc o trng thanh cua cay. Hemicellulose la thanh phan nhieu th hai trong rm, no chiem 23,82%. Ket qua nay phu hp vi 24% theo Howard va tng oi thap so vi 32-40% theo Ho S Trang. Hemicellulose ton tai vung vo nh hnh, ket cau tren phan t trang thai lon xon va ri rac, de tham nc nen xuc tien qua trnh nghien bot giay. Do o, ham lng hemicellulose cao rat thuan li trong cong nghiep giay nhng lai khong co li trong viec san xuat cac chat hoa hoc t cellulose. Trong cac qua trnh san xuat etanol t cellulose neu cha tan dung c lng ng 5C t hemicellulose th khong can thanh phan nay chiem t le cao. Hau het hemicellulose b thuy phan trong qua trnh no hi va c loai bo trong cac giai oan sau. Lignin trong rm tho s dung trong nghien cu nay chiem 18%. Ham lng nay neu so vi nguyen lieu lignocellulosic biomass noi chung la thap. Lng lignin thap th qua trnh phan huy lignin e thu nhan cellulose dien ra de dang hn, ong thi se giam nhng chat c che qua trnh thuy phan va len men ethanol. Rm tho s dung co ham lng tro kha cao (15.13%). ay la mot bat li khi muon thu cellulose. Tuy nhien trong qua trnh tien x l (no hi) tiep theo th lng tro nay se b phan huy cung vi hemicellulose. Nhn chung, ket qua phan tch thanh phan rm tho tng oi phu hp vi cac tai lieu. Ham lng cellulose, hemicellulose va lignin gia cac tai lieu va th nghiem co s chenh lech, tuy nhien eu nam trong khoang dao ong cua nguyen lieu lignocellulose noi chung.

51

Chng 4: Ket qua va ban luan


4.1.2 Thanh phan rm a qua x ly

T 300 g rm tho ban au vi lng nc them vao la 3 lt, sau qua trnh no hi thu c 140 g rm a x ly va 2,9 lt dch no hi. Ket qua phan tch thanh phan rm a x ly c trnh bay bang 4.2. T ket qua phan tch, chung toi xac nh c ham lng cac chat trong rm trc va sau qua trnh no hi. Ket qua c trnh bay trong bang 4.3.

52

Chng 4: Ket qua va ban luan

Bang 4.2. Thanh phan cua rm a no hi (tnh tren ham lng chat kho). Thanh phan Ham lng (%) Cellulose 77,45 Hemicellulose 5,60 Lignin 8,96 Tro 7,99
Cellulose 77.45%

Tro 7.99%

Lignin 8.96%

Hemicellulose 5.60%

Hnh 4.2. Thanh phan cua rm a no hi.

90 80 70
Thah pha (%) n n
77.45

60 50 40 30 20 10 0
Ce llulos e Hemicellulos e Lig nin Tro
5.6 23.82 18 8.96 15.13 7.99 43.05

Rm tho Rm a ly x

53

Chng 4: Ket qua va ban luan Hnh 4.3. Thanh phan cua rm trc va sau qua trnh tien x ly. Nh vay, sau qua trnh tien x ly (no hi) thanh phan rm a thay oi. Ham lng cellulose tang trong khi lignin, tro va hemicellulose eu giam. Trong o, cellulose, hemicellulose va lignin la cac thanh phan co s thay oi ang ke. Ham lng cellulose t 43,05% trc no hi tang len 77,45%, tang gan hai lan so vi trc. Lignin giam t 18% xuong con 8,96%, giam mot na so vi trc. Tng t, ham lng tro giam t 15,13% xuong 7,99%. ac biet, hemicellulose giam 4 lan so vi trc, t 23,82% xuong con 5,6%. Nguyen nhan cua s thay oi nay la do di tac ong cua nhiet o cao va s thay oi ap suat ot ngot trong qua trnh no hi, hemicellulose, lignin va tro a b phan huy cac mc o khac nhau. Theo so lieu th nghiem th mot lng ln hemicellulose a b thuy phan, giai phong cac ng C5 va mot phan ng C6. Lignin va tro cung b phan huy mot phan lam giam ham lng cua chung trong rm sau no hi. Ham lng cellulose b thuy phan khong ang ke, trong khi cac thanh phan khac eu giam do o phan tram cellulose trong rm sau no hi tang len. Xet ve mat can bang vat chat, khoi lng cac chat trong rm sau qua trnh no hi eu giam. Phan khoi lng giam nay la do cac chat b thuy phan va i vao dch no hi, ngoai ra con co mot phan that thoat ra moi trng. Bang 4.3 cho thay s thay oi cua cellulose trc va sau no hi. Theo o, sau qua trnh tien x ly ham lng cellulose thu c la 83,96% so vi ban au. Ket qua nay kha gan so vi gia tr 88% cua hieu suat thu hoi cellulose trong nghien cu cua Eniko va cong s [22]. Vi nguyen lieu la phan than ngo, tac gia a s dung phng phap tien x ly oxy hoa kiem am (alkaline wet oxidation). ay la mot phng phap x ly kha hu hieu at c hieu suat thu hoi tng oi cao. Bang 4.3. Ham lng cac chat trong rm trc va sau qua trnh no hi (tnh cho 300g rm tho).

54

Chng 4: Ket qua va ban luan Thanh phan Cellulose Hemicellulo se Lignin Tro Khoi lng rm trc no hi (g) 129,15 71,46 54,00 45,39 Khoi lng rm sau no hi (g) 108,43 7,84 12,54 11,19 % so vi ban au 83,96 10,97 23,23 24,64

Nh vay, qua trnh no hi la mot qua trnh can thiet va co hieu qua. No a mang lai mot so li ch sau: Lam giam phan tram tro, tang phan tram cellulose trong rm s dung cho qua trnh thuy phan, do o nang cao nong o ng glucose thu c trong dch thuy phan. Giam ham lng lignin nen giam c mot phan cac chat c che qua trnh len men, lam cho cac tac nhan thuy phan tiep xuc de dang hn vi c chat. Giam ang ke lng hemicellulose khong can thiet cho qua trnh thuy phan.
4.1.3 Phan tch dch sau no hi

Nong o ng glucose trong dch sau no hi la 0,317 (g/L). Ket qua phan tch dch no hi cho thay trong phan dch co cha glucose. Lng glucose nay la do mot phan cellulose b thuy phan di tac ong cua nhiet o cao (220oC) trong qua trnh no hi. Tuy nhien, nong o ng glucose nay rat thap, ch at 0,317 g/L. Neu tnh tren the tch dch no hi thu hoi c la 2,9 lt th lng ng trong o la 0,92g. ieu o chng to ch mot lng nho cellulose trong rm b thuy phan. Nh vay, viec loai bo phan dch sau no hi khong lam giam ang ke lng ng thu c trong qua trnh thuy phan.
4.2 QUA TRNH THUY PHAN

55

Chng 4: Ket qua va ban luan


4.2.1 Khao sat cac phng phap thuy phan

Ket qua th nghiem khao sat cac phng phap thuy phan c the hien bang 4.4. Bang 4.4. Ket qua nong o glucose va hieu suat chuyen hoa cellulose cua mau rm tho va rm a no hi cac che o thuy phan. Ma Loai rm u 1 2 3 4 Rm tho Rm a x ly Rm tho Rm a x ly Che o thuy phan Acid Acid Enzym Enzym Nong o Hieu suat ng (g/l) chuyen hoa cellulose (%) 0,357 1,66 1,957 1,310 37,540 5,05 3,04 48,47

56

Chng 4: Ket qua va ban luan

Anh hng cua qua trnh tien x ly (no hi) en qua trnh thuy phan
60 Nog o n glucose (g/L) Hie suachuye ho (%) u t n a 50 40 30 20 10 0
0.357 1.66 5.05 1.310 3.04 37.540 48.47

1.957

Rm tho

Rm a ly x

Rm tho

Rm a ly x

Thu pha acid y n

Nog o n glucose (g/L) Hie suachuye ho cellulose (%) u t n a

Thu pha enzym y n

Hnh 4.4. Ket qua thuy phan bang acid va thuy phan bang enzym cua rm tho va rm a x ly. Hnh 4.4 cho thay s vt troi ro ret ve ham lng ng glucose cung nh ve hieu suat chuyen hoa cellulose cua mau rm a no hi so vi mau rm tho. oi vi thuy phan bang enzyme, mau rm a no hi, nong o ng at c sau 24 gi thuy phan la 37,54 g/L, hieu suat thuy phan la 48,47%, trong khi o rm tho, nong o ng va hieu suat thuy phan lan lt la 1,31 g/l va 3,04% (bang 4.4). Con oi vi mau thuy phan bang acid, nong o ng at c sau 1,5h thuy phan la 1.957 g/l oi vi rm a no hi va 0,357g/l oi vi rm tho, hieu suat chuyen hoa cellulose tren rm a no hi la 5,05% va tren rm tho la 1,66%. Sau khi no hi, kch thc lo xop se ln v hemicellulose a b thuy phan, ong thi cau truc cua cellulose a b bien tnh, nen kha nang tan cong cua cac tac nhan thuy phan se cao hn la mau cha c x ly. Trong cellulose cha x ly (no hi), ch co mot phan nho cua lo tiep can c vi tac nhan thuy phan [26]. Phng phap 57

Chng 4: Ket qua va ban luan no hi c nhan thay la co hieu qua oi vi go cng va chat thai nong nghiep [14]. Han che cua no hi la lam bien tnh tng phan xylan, phan huy khong hoan ma tran lignin carbohydrate, lam gia tang o ket tinh cua cellulose va co the tao ra nhng hp chat c che s phat trien cua vi sinh vat len men [35]. Nhng nhng ieu o hoan toan co the khac phuc c thong qua viec ra nguyen lieu that sach bang nc e loai bo nhng chat c che trong nguyen lieu ong thi hoa tan hemicellulose [39]. Nh vay, qua trnh no hi la qua trnh tien x ly can thiet e nang cao hieu qua thuy phan. Do o, rm s dung trong cac th nghiem tiep theo se la rm a qua x ly (no hi). So sanh thuy phan bang acid va thuy phan bang enzym
60
Nog o n glucose (g/L) hie suachuye ho (%) u t n a

50 40 30 20 10 0 Rm tho
1.957 5.05 37.540

48.47

Rm a ly x

Nog o n glucose (g/L) Hie sua chuye ho (%) u t n a

Hnh 4.5. Ket qua thuy phan bang acid va thuy phan bang enzym cua rm a x ly. Hnh 4.5 va bang 4.4 cho thay s chenh lech ro rang ve nong o ng glucose thu c va hieu suat chuyen hoa cellulose gia hai phng phap thuy phan. Ro rang vi phng phap thuy phan bang acid, hieu qua at c khong cao. Khi thuy phan bang acid, nong o ng va hieu suat thuy phan lan lt la 1,957g/l va 5,05%. Trong

58

Chng 4: Ket qua va ban luan khi o, oi vi phng phap thuy phan bang enzyme th nong o ng va hieu suat chuyen hoa tang len rat nhieu, tng ng la 35.26g/l va 31.74%. Nong o ng cua phng phap thuy phan bang acid khong cao la do ieu kien khac nghiet (nhiet o cao, ap suat cao, hau het ng tao thanh eu b phan huy thanh cac hp chat khac nh furfural va hydroxymethyl furfural) [39]. Do o khong the thu c hoan toan lng glucose khi thuy phan cellulose. Do ty le rm cho vao la nh nhau nen lng ng glucose giam keo theo hieu suat chuyen hoa cellulose giam. e thu hoi c lng ng cao th qua trnh thuy phan acid phai tien hanh ieu kien em du hn. Thong thng ngi ta s dung acid am ac, bm nguyen lieu t thung nay sang thung khac. Tuy nhien, qua trnh nay dien ra cham, rat kho thu hoi acid va ton kem do phai dung mot lng ln voi e trung hoa acid trong dung dch ng. Phng phap thuy phan bang enzyme c coi la co hieu qua hn thuy phan bang acid do enzyme cellulase mang tnh ac hieu cao oi vi c chat la cellulose, ong thi viec thuy phan c tien hanh trong ieu kien em du (pH 5,5 va nhiet o 500C) nen khong gay ra van e an mon [20]. Nh vay, do co hieu qua cao nen phng phap thuy phan bang enzym se c chon cho cac th nghiem tiep theo.
4.2.2 Khao sat anh hng cua t le enzym

Ket qua th nghiem c trnh bay trong bang 4.5. Bng 4.5. Ket qua khao sat t le enzym. Ma T le enzym C (g/L) H (%) u (%) 1 1 6,67 9,47 2 3 21,27 30,21 3 5 43,80 62,21 4 7 45,12 64,08 5 9 46,81 66,48

59

Chng 4: Ket qua va ban luan Ket qua ve anh hng cua ty le enzyme en nong o glucose va hieu suat chuyen hoa cellulose (hieu suat thuy phan) c trnh bay trong bang 4.5 cho thay nong o glucose gia tang cung vi s gia tang cua ty le enzym. Trong khoang ty le enzym t 1% en 5% th nong o glucose va hieu suat thuy phan co xu hng tang nhanh: t 1% en 3% tang 3,19 lan, t 3% en 5% tang 2,06 lan. Khi nong o enzyme tang len 7%, 9% th nong o ng va hieu suat thuy phan cung tang theo nhng khong manh, ch at tng ng la 1,03 va 1,04 lan.
70
Nog o n glucos (g/L) e

70 60 50 40 30 20 10 0 0 2
T enzym(%) le Hie s t thu pha (%) u ua y n

60 50 40 30 20 10 0 4 6 8 10

Nog o n glucose (g/L) Hie sua(%) u t

Hnh 4.6. Ket qua khao sat t le enzym. Nh vay, khi t le enzym tang th nong o ng thu c tang, nhng khi ty le nay qua cao th nong o ng khong tang ang ke. ieu nay the hien ro qua hnh 4.6. o la do khi ng a tao ra c nhieu th no se c che qua trnh thuy phan theo c che km ham bi san pham cuoi, ngha la glucose tao ra se c che hoat ong cua enzym lam cho enzym kho tiep xuc vi c chat [55]. Da vao ket qua thu c, ty le enzyme 5% c chon cho cac th nghiem tiep theo.
4.2.3 Khao sat anh hng cua t le rm

Ket qua th nghiem c trnh bay trong bang 4.6. 60

Chng 4: Ket qua va ban luan Bng 4.6. Ket qua th nghiem khao sat t le rm. Ma Ty le rm Nong o glucose Hieu suat chuyen u (%) (g/l) hoa (%) 1 1 1,19 16,90 2 5 10,56 30,00 3 10 36,56 51,92 4 15 40,78 38,61 5 20 34,69 24,63
45 40 35 30 25 20 15 10 5 0 0 5 10
T rm(%) le

60 50 40 30 20 10 0 15 20 25
Hie s t thu pha (%) u ua y n

Nog o n glucos (g/L) e

Ha mu tie m c u

Nog o ng (g/L) n Ha mu tie m c u Hie suachuye ho (%) u t n a

Hnh 4.7. Ket qua khao sat t le rm. Anh hng cua t le rm en nong o ng glucose thu c Bang 4.6 va hnh 4.7 cho thay nong o ng tang theo ty le rm t 1% en 15%. Nong o ng tang nhanh trong giai oan au khi t le rm tang t 1% en 5%, tang cham khi t le rm t 10% en 15% va tang nhanh nhat trong giai oan t le rm t 5% en 10%. ty le rm 15%, nong o ng at c cc ai la 40.78 g/L. Khi t le rm tiep tuc tang en 20% th nong o ng bat au giam xuong. ieu nay co the c giai thch da theo phng trnh Michaelis Menten ve anh hng cua nong o c chat en qua trnh thuy phan [3]:

61

Chng 4: Ket qua va ban luan


Vmax .[ S ] Km + [S ]

V=

Nong o c chat co the hieu ay la t le rm. Trong giai oan au, ng vi nong o c chat thap (ty le rm 1%, 5%, 10%), van toc phan ng tang va gan nh phu thuoc tuyen tnh vao nong o c chat [12]. Khi toc o phan ng tang th nong o ng at c cung tang theo. Khi nong o c chat ln, van toc phan ng a at en cc ai roi th sau o, neu tiep tuc tang nong o c chat , van toc phan ng khong tang na, tham ch co the giam xuong [12]. Do o, khi ty le rm tang len en gia tr 20% th nong o ng khong tang na ma giam xuong. Bang 4.6 cho thay ty le rm 15% va 20%, nong o ng at c lan lt la 40,78 g/L va 34,68 g/L. ieu nay con co the c giai thch la do ty le rm 20% lng c chat qua tha so vi enzym, kha nang tiep xuc cua enzym vi c chat tr nen kho khan, v vay kha nang thuy phan cua enzym giam nen lng ng thu c cung giam. Anh hng cua ty le rm en hieu suat chuyen hoa cellulose Hieu suat thuy phan (hieu suat chuyen hoa cellulose) c tnh bng lng ng tao thanh tren lng cellulose ban au. Hnh 4.7 va bang 4.6 cho thay vi ty le rm la 1%, 5% th hieu suat chuyen hoa cellulose tang dan vi cac gia tr lan lt la 16,9%, 30% va at cc ai ty le rm 10% vi hieu suat chuyen hoa la 51,92%. Nh vay, ty le rm 10%, kha nang tan cong cua enzym cellulase vao c chat la cao nhat, cellulose b chuyen hoa nhieu nhat. Khi nong o c chat cao (ty le rm >10%), hieu suat chuyen hoa cellulose b giam xuong. Cu the ty le rm 15% va 20% hieu suat chuyen hoa lan lt la 38,61% va 24,63%, giam so vi 51,92% cua t le rm 10%. o la do khi lng c chat cao, kha nang tiep xuc cua enzym va c

62

Chng 4: Ket qua va ban luan chat b han che, khong the thuy phan hoan toan lng cellulose. Muc tieu cua qua trnh thuy phan la dch thu c co nong o ng glucose cao va hieu suat chuyen hoa cellulose cao. Do o, chung toi chon tch cua hai thong so nay lam ham muc tieu, ay la mot thong so ai dien cho nong o ng va hieu suat chuyen hoa. Hnh 4.7 cho thay mac du lng ng tao thanh ty le rm 15% cao hn 10%, nhng ty le rm 10% th ham muc tieu at gia tr ln nhat. V vay, ty le rm c chon la 10%.
4.2.4 Khao sat thi gian thuy phan

Ket qua th nghiem c trnh bay trong bang 4.7. Bng 4.7. Ket qua thuy phan theo thi gian. Mau Thi gian (h) C (g/L) 1 0 0,00 2 3 8,52 3 6 16,60 4 9 20,10 5 12 24,40 6 15 27,51 7 21 39,79 8 24 41,28 9 27 42,16 10 30 43,96 11 33 44,26 12 36 44,66 13 39 45,11 14 45 45,68 15 48 45,80

63

Chng 4: Ket qua va ban luan

50 45 40 35 30 25 20 15 10 5 0 0 5 10 15 20 25 30 35 40 45 50 Thg (h) i ian

Hnh 4.8. Ket qua thuy phan theo thi gian. Ket qua thuy phan theo thi gian c the hien trong hnh 4.8. Trong giai oan au cua qua trnh thuy phan (khoang 21 gi au), nong o ng tang nhanh theo thi gian, o th gan nh tuyen tnh. T 24 gi tr i, nong o ng co tang nhng khong ang ke. Cu the la sau 24 gi nong o ng at 41,28 g/L nhng sau 27 gi, 30 gi va 48 gi ch at lan lt la 43,16 g/L, 43,96 g/L va 45,8 g/L. Khi lng c chat con u cho enzyme hoat ong, thi gian cang tang th enzyme thuy phan cang triet e hn nen nong o ng cung tang theo. Tuy nhien, thi gian tac dung lau se cang giam kha nang chu nhiet cua enzym [59]. Do o, neu tiep tuc keo dai thi gian thuy phan, nong o ng tang len khong ang ke trong khi chi ph san xuat tang, khong mang lai hieu qua kinh te. Mat khac, vi th nghiem thuy phan ba hoa hng dng, Sanjeev va cac cong s a rut ra ket luan ve nong o glucose trong dch thuy phan co the len men ethanol la 35 40g/L [50]. V vay, thi gian thuy phan c chon la 24 gi.
4.2.5 Quy hoach thc nghiem qua trnh thuy phan

e khao sat s anh hng ong thi cua t le rm va t le enzym cung nh s tng tac gia chung en 64

C (g /L)

Chng 4: Ket qua va ban luan hieu qua cua qua trnh thuy phan, chung toi s dung phng phap quy hoach thc nghiem. Hieu qua cua qua trnh thuy phan c the hien qua nong o glucose thu c va hieu suat chuyen hoa cellulose, do o tch cua hai thong so nay c chon lam ham muc tieu. Ket qua th nghiem c trnh bay trong bang 4.8. Bng 4.8. Ket qua th nghiem toi u hoa thuy phan . CAC YEU HIEU HAM CAC YEU TO TO TRONG NONG SUAT MUC THEO TY LE MA HE MA O CHUYE TIEU XCH T NHIEN U HOA N N Y G (G/L) HOA Z1 Z2 X1 X2 (%) 1 5 1 16,911 48,03 0,81 2 5 3 0 24,553 69,74 1,71 3 5 5 + 28,130 79,90 2,25 4 10 1 0 23,740 33,72 0,80 5 10 3 0 0 44,715 63,51 2,84 6 10 5 0 + 48,780 69,28 3,38 7 15 1 + 30,894 29,25 0,90 8 15 3 + 0 44,309 41,95 1,86 9 15 5 + + 51,626 48,88 2,52 10 10 3 0 0 36,382 51,67 1,88 11 10 3 0 0 35,163 49,94 1,76 12 10 3 0 0 38,008 53,98 2,05 Vi ket qua thu c, chung toi tm ham hoi quy cua ham muc tieu Y theo hai bien so ty le rm ra (X1) va ty le enzym (X2), sau o kiem tra s tng thch cua ham hoi quy vi thc nghiem. Tiep theo chung toi xac nh thong so toi u cua qua trnh thuy phan da vao ham hoi quy. Theo phng phap trc giao hai yeu to, ham hoi qui cua ham muc tieu Y theo bien so ty le rm ra (X1) va ty le enzym (X2) co dang: Y1 = b0 + b1X1 +b2X2 + b12X1X2 + b11(X12 X*12) + b22(X22 X*22) Thong qua viec tnh toan hoi quy thc nghiem, mo hnh ap ng cua ham muc tieu nh sau: Y = 1,8976 + 0,9391 X2 0,6635 ( X 12 2/3)

65

Chng 4: Ket qua va ban luan Thc hien phep quy oi ve he toa o th nguyen, ta co: y = - 1,7226 + 0,5308 Z1 0.0265Z12+ 0,4695 Z2 Kiem tra bang tieu chuan Fisher, phng trnh hoi quy tm c tng thch vi thc nghiem.
3.000-3.500

4
2.500-3.000

2.000-2.500 1.500-2.000

3
1.000-1.500

Ha m c tie m u u

0.500-1.000 0.000-0.500

1
4 3.4 2.8

4.6

le m 2.2 T Enzy (%)

0
5 6 7 8 9 10 11 12 13 14 15 1

1.6

T r (%) le m

Hnh 4.9. Mo hnh mat cong the hien anh hng cua t le rm va t le enzym en qua trnh thuy phan. Hnh 4.9 va phng trnh hoi quy cho thay: - Khi ty le enzyme khong oi, moi quan he gia ty le rm va ham muc tieu (nong o ng va % cellulose chuyen hoa) la mot ng parabol. - Khi ty le rm khong oi, moi quan he gia ty le enzyme va ham muc tieu la mot ng thang. Nh vay, ca hai yeu to ty le rm ra va ty le enzym eu co anh hng en nong o ng va hieu suat 66

Chng 4: Ket qua va ban luan chuyen hoa cellulose (ham muc tieu). Trong o, yeu to ty le rm ra anh hng en ham muc tieu nhieu hn ty le enzym. Vi ket qua cua bai toan quy hoach thc nghiem, chung toi nhan thay s anh hng ong thi cua ty le rm ra va ty le enzym tuan theo quy luat nh a khao sat. Mo hnh hoi quy tnh toan c ve bang MS excel nh hnh 4.9. Theo ket qua tnh toan, iem cc tr cua phng trnh la t le rm 10%. ieu nay phu hp vi thc nghiem, khi tang ty le rm t 10% len 15% th nong o ng co tang len, nhng hieu suat chuyen hoa cellulose lai giam xuong. Muc tieu cua chung toi la thu c lng ng cao u e len men ong thi chuyen hoa c mot lng ln cellulose nen ty le rm c chon la 10%. Khi tang ty le enzym, lng ng va lng cellulose chuyen hoa eu tang len. Tuy nhien, neu tang ham lng enzym len cao qua th se tang chi ph cua qua trnh thuy phan. Do o, chung toi chon ty le enzym 5%. ay la ty le enzym can thiet e va thu c lng ng cao, va at hieu suat chuyen hoa cao ong thi chi ph cung hp l. Nh vay, qua ket qua cua phng phap quy hoach thc nghiem, chung toi nhan thay ty le rm thch hp cho qua trnh thuy phan la 10%, va ty le enzym thch hp la 5%. Khi o, ham lng ng thu c t dch thuy phan at khoang 48 g/L, u e thc hien qua trnh len men etanol cac th nghiem tiep theo. Tong ket qua trnh thuy phan Che o thuy phan thch hp c xac nh la: T le rm: 10% the tch. T le enzym: 5% the tch. Nhiet o thuy phan: 500C. Thi gian thuy phan: 24 gi. pH thuy phan: 5,5.

67

Chng 4: Ket qua va ban luan

4.3 QUA TRNH LEN MEN 4.3.1 Khao sat cac chung nam men

Ket qua o nong o glucose theo thi gian cua cac chung nam men c trnh bay trong bang 4.9. Ket qua o nong o etanol cua cac mau len men c trnh bay trong bang 4.10. Bng 4.9. Ket qua o nong o glucose theo thi gian cua cac chung nam men. Thi gian (h) 0 24 48 72 96 120 V1 V6 KHTN 39,66 0 0,571 0,099 0,076 0,052 0,050 RD 39,66 0 2,595 0,148 0,062 0,061 0,061 TG BT

39,660 39,660 4,511 0,162 0,123 0,106 0,081 6,723 0,102 0,098 0,077 0,060

39,660 39,660 2,709 0,626 0,064 0,063 0,062 6,404 0,184 0,115 0,114 0,114

Bng 4.10. Ket qua o nong o etanol tao thanh cua cac mau len men. Mau Nong o con (g/L) V1 14,222 V6 10,815 KHTN 17,266 RD 17,006 TG 11,414 BT 15,284 Ket qua th nghiem trnh bay trong bang 4.9 cho thay nong o glucose trong cac mau len men eu giam theo thi gian, giam manh trong 24h au va giam nhe trong giai oan sau. Trong 24h au, chung V6 va BT giam t nong o 39,660 g/L xuong con 6,723 g/L va 6,404 g/L. Trong khi o, chung V1, RD va TG giam xuong lan lt la 4,511 g/L, 2,595 g/L va 2,709 g/L. ac biet, chung KHTN giam manh, t 39,660 g/L xuong con 0,571 g/L. Trong 24h tiep theo, nong o ng tiep tuc giam manh. (hnh 4.10). Lng ng sot hau 68

Chng 4: Ket qua va ban luan het cac mau ch con lai khoang 0,1%, ngoai tr chung TG la 0,626 g/L. Trong cac ngay tiep theo, s giam nong o ng cua cac mau len men la khong ang ke. Nh vay, sau hai ngay lng ng trong cac mau len men hau nh a het.
8 7
Nog do lucose (g n g /L)

6 5 4 3 2 1 0 V1 V6
Chug na men n m

KHTN 24h

RD

TG

BT

48h

Hnh 4.10. Nong o glucose cua cac mau len men sau 24h va 48h. Hnh 4.11 va bang 4.10 the hien ket qua nong o etanol at c cac mau len men. Theo o, chung KHTN va RD cho ket qua cao nhat, tng ng la 17,266 g/L 17,006g/L. Tiep theo la chung BT va V1 vi nong o etanol la 15,284 g/L va 14,222 g/L. Chung TG at nong o 11,414 g/L va thap nhat la chung V6 vi nong o 10,815 g/L.

69

Chng 4: Ket qua va ban luan

20 18 16 14 12 10 8 6 4 2 0 V1 V6 KHTN RD TG BT
Chug na m n m en

Hnh 4.11. Nong o etanol cua cac chung nam men s dung. Nh vay, trong cac chung nam men khao sat, chung KHTN va RD co u the ve kha nang tao etanol. Trong o, chung KHTN chiem u the hn do kha nang tieu thu ng. ieu nay the hien qua viec giam nhanh nong o ng trong 24h au (0,571 g/L so vi 2,595 g/L cua RD). Ngoai ra, lng ng sot cua chung KHTN la 0,050 g/L, cung thap hn so vi 0,061 g/L cua chung RD. V vay, chung KHTN c chon lam giong nam men s dung cho cac th nghiem tiep theo.
4.3.2 Khao sat thi gian len men

Ket qua c trnh bay bang 4.11. Bng 4.11. Ket qua khao sat thi gian len men. Tong so te Thi gian Nong o Nong o bao (trieu (h) glucose (g/L) etanol (g/L) TB/ml) 0,5 48,38 2,04 15 2 35,24 2,37 18 4 24,67 3,34 21,89 21 6,81 12,09 76,48 24 5,29 18,85 79,14 42 0,11 21,72 81,59 48 0,07 23,24 78,62 70

Nog o n etanol (g/L)

Chng 4: Ket qua va ban luan 54 72 0,06 0,05 22,72 22,46 69,12 30,15

Bang 4.11 va hnh 4.12 cho thay s thay oi cua nong o glucose, nong o etanol va mat o te bao nam men theo thi gian. Nong o glucose giam dan trong qua trnh len men. Trong giai oan au, glucose c tieu thu rat nhanh, t 48,38 g/L ban au giam con 24,67 g/L sau 4h. Nong o glucose tiep tuc giam manh trong 17 gi ke tiep, ch con 6,81 g/L. Trong khi o, so te bao nam men va nong o etanol lai tang nhanh trong 20 gi au. Nh vay, nam men a s dung glucose e tang sinh khoi va tao ra etanol. Tong so te bao nam men a tang t 15-76,48 trieu te bao/ml. Nong o etanol cung tang 12,09 g/L. T gi th 21 en gi th 42, nong o ng giam cham, t 6,81 g/L tai thi iem 21 gi en 0,11 g/L tai 42 gi. Giai oan nay vi lng ng con lai rat thap, nam men chu yeu s dung ng vao viec tao etanol. Sinh khoi nam men tang nhe, t 76,48 trieu te bao/ml len81,59 trieu te bao/ml. Trong khi o, lng etanol tiep tuc tang t 12,09 g/L en 21,72 g/L va at gia tr cc ai la 23,24 g/L tai thi iem 48 gi.

71

Chng 4: Ket qua va ban luan

60

100 Mt t bo nm men (triu TB/mL)

Nng glucose v etanol (g/L)

50

80

40 60 30 40 20 20

10

0 0 10 20 30 40 50 60 70 80

Nng glucose Nng ethanol Mt t bo nm men

Thi gian (h)

Hnh 4.12. ong hoc cua qua trnh len men. T sau 48 gi, nong o glucose giam hau nh khong ang ke, t 0,07 g/L en 0,05 g/L. Trong khi o, nam men a bc vao giai oan suy vong, so lng te bao nam men giam manh, t 81,59 trieu te bao/ml xuong con 30,15 te bao/ml. Nong o etanol trong giai oan nay bat au giam nhe, t 23,24 g/L xuong con 22,46 g/L. o la do mot phan etanol a b chuyen hoa thanh cac hp chat hu c khac nh acid acetic Nh vay, thi iem ket thuc qua trnh len men thch hp la 48 gi. Khi o, nong o glucose gan nh a het, nong o etanol a at en gia tr cc ai. Neu tiep tuc keo dai thi gian len men se khong mang lai hieu qua do nam men bat au chet, lng etanol tao thanh khong ang ke so vi lng mat i do b chuyen hoa.
4.3.3 Toi u hoa pH va t le giong

e khao sat s anh hng cua pH va t le giong cung nh s tng tac gia chung en hieu qua cua qua trnh len men, chung toi s dung phng phap quy hoach thc nghiem. Ket qua th nghiem c trnh bay trong bang 4.12. 72

Chng 4: Ket qua va ban luan Bng 4.12. Ket qua th nghiem toi u hoa qua trnh len men. CAC YEU TO CAC YEU TO NONG O MA THEO TY LE TRONG HE MA ETANOL U XCH T NHIEN HOA (G/L) Z1 Z2 X1 X2 1 4 10 18,310 2 4 15 0 22,417 3 4 20 + 18,310 4 4,5 10 0 21,904 5 4,5 15 0 0 23,016 6 4,5 20 0 + 20,364 7 5 10 + 17,283 8 5 15 + 0 19,040 9 5 20 + + 18,642 10 4,5 15 0 0 23,128 11 4,5 15 0 0 23,427 12 4,5 15 0 0 22,629 Vi ket qua thu c, chung toi tm ham hoi quy cua ham muc tieu Y theo hai bien so pH (X1) va t le giong (X2), sau o kiem tra s tng thch cua ham hoi quy vi thc nghiem. Tiep theo chung toi xac nh thong so toi u cua qua trnh len men da vao ham hoi quy. Theo phng phap trc giao hai yeu to, ham hoi qui cua ham muc tieu Y theo bien so pH (X1) va t le giong (X2) co dang: Y1 = b0 + b1X1 +b2X2 + b12X1X2 + b11(X12 X*12) + b22(X22 X*22) Thong qua viec tnh toan hoi quy thc nghiem, mo hnh ap ng cua ham muc tieu nh sau: Y = 19,9207 2,7606 ( X 12 2/3) 2,3560 ( X 22 2/3) Thc hien phep quy oi ve he toa o th nguyen, ta co: y = - 221,4806 + 99,38248Z1 + 2,826888Z2 11,0425Z12 0,09423Z22

73

Chng 4: Ket qua va ban luan Kiem tra bang tieu chuan Fisher, phng trnh hoi quy tm c tng thch vi thc nghiem. T ket qua quy hoach thc nghiem, chung toi nhan thay:

- Khi pH khong oi, moi quan he gia t le giong va


ham muc tieu (nong o etanol) la mot ng parabol.

- Khi t le giong khong oi, moi quan he gia pH va


ham muc tieu (nong o etanol) cung la mot ng parabol. Nh vay, ca hai yeu to pH va t le giong eu co anh hng en nong o etanol. Mo hnh hoi quy tnh toan c ve bang MS excel nh hnh 4.13.
25

20

Nog o n (g/L) n co

15

10

14

Hnh 4.13. Mo hnh mat cong the hien anh hng cua pH va t le giong en qua trnh len men.

T giog (%) le n

18

19

20

4.2

15

16

4.4

11

12

13

17

pH

4.6

10

4.8

74

Chng 4: Ket qua va ban luan Theo ket qua tnh toan, iem cc tr cua phng trnh la pH 4,5 va t le giong 15%. Nh vay, qua ket qua cua phng phap quy hoach thc nghiem, chung toi nhan thay pH thch hp cho qua trnh len men la 4,5 va t le giong thch hp la 15%. Khi o, ham lng con thu c t dch len men at khoang 23 g/L, at hieu suat len men tng oi cao 95%. Nong o etanol thu c trong th nghiem nay tng oi cao so vi ket qua cac th nghiem khac. Ladisch va cong s [60] a tien hanh len men dch ng thu c t qua trnh thuy phan si x bap. Vi nong o ng ban au 42 g/L, sau thi gian 25 gi len men thu c 20 g/L etanol. Tien hanh chnh pH va nong o xylose trong dch thuy phan ti mc co the len men, nong o glucose ban au la 38 g/L th nong o etanol thu c la 24 g/L ng vi gia tr pH 4,4. Tuy nhien, so vi ket qua cua cac th nghiem len men SSF th gia tr nay van thap hn. Dale va cong s [16] a tien hanh qua trnh SSF tren nguyen lieu la rm a x ly, ket qua thu c nong o etanol sau 95 gi la 40 g/L. Th nghiem tng t oi vi vo cay thch (maple shaving) cho ket qua etanol la 25 g/L sau 72 gi. Eniko va cong s [22] a tien hanh th nghiem tren vo cay bap va thu c 50 g/L etanol bang phng phap SSF. ieu nay hoan toan phu hp vi ly thuyet v ban than len men SHF van con nhieu han che so vi SSF. Do o, hng nghien cu tiep theo can tap trung vao qua trnh ng hoa va len men ong thi (SSF). Tong ket qua trnh len men

Che o len men thch hp c xac nh la: Giong nam men: Saccharosemyces cerevisiae, chung do trng KHTN cung cap. T le giong: 15% the tch. Nhiet o len men: 300C. Thi gian len men: 48 gi.

75

Chng 4: Ket qua va ban luan pH len men: 4,5.

76

Chng 5: Ket luan va kien ngh

KET LUAN VA KIEN NGH


5.1 Ket luan

Chng 5

Qua qua trnh nghien cu thc nghiem, chung toi rut ra mot so ket luan nh sau:

- Qua trnh no hi (steam explosion) la qua trnh tien x


ly can thiet e nang cao hieu suat thuy phan cellulose. - Phng phap thuy phan bang enzym mang lai hieu qua cao hn so vi thuy phan bang acid ca ve nong o ng glucose thu c va hieu suat chuyen hoa cellulose.

- Cac thong so cua qua trnh thuy phan: t le rm 10%,


loai enzym Cellusoft L, t le enzym 5%, nhiet o thuy phan 500C, thi gian thuy phan 24 gi, pH thuy phan 5,5. - Cac thong so cua qua trnh len men: Giong nam men Saccharosemyces cerevisiae, chung do trng KHTN cung cap, t le giong 15% the tch, nhiet o len men 300C, thi gian len men 48 gi, pH 4,5. 5.2 Kien ngh Chung toi xin e ngh cac hng nghien cu tiep theo cua e tai nay gom cac noi dung sau: Tm hieu cac phng phap tien x ly e loai them lignin, hemicellulose, nang cao hieu suat cua qua trnh thuy phan. Nghien cu ng dung phng phap ng hoa va len men ong thi (SSF), so sanh vi phng phap ng hoa va len men rieng biet (SHF).

77

TAI LIEU THAM KHAO Tai lieu tham khao trong nc [1] Bui Ai, Cong nghe len men thc pham, Nha xuat ban ai Hoc Quoc Gia Thanh Pho Ho Ch Minh, 2003, trang.

[2] Ho S Trang, C s hoa hoc go va xenlulo, Nha


xuat ban khoa hoc va k thuat Ha Noi, 2004, trang 30 81.

[3] Le Ngoc Tu, Hoa sinh cong nghiep, Nha xuat ban
khoa hoc va k thuat Ha Noi, 2000, trang 348 357 [4] Nguyen Canh, Quy hoach thc nghiem, Nha xuat ban ai Hoc Quoc Gia Thanh Pho Ho Ch Minh, 2004, trang 2 68. [5] Nguyen nh Thng, Nguyen Thanh Hang, Cong nghe san xuat va kiem tra con etylic, Nha xuat ban khoa hoc va k thuat Ha Noi, 2005, trang 244 255.

[6] Nguyen

c Lng, Nguyen Chuc, Le Van Viet Man, Thc tap vi sinh vat hoc thc pham, Nha xuat ban ai Hoc Quoc Gia Thanh Pho Ho Ch Minh, 2004.

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[7] Akpan et al, The production of ethanol from Maize Cobs and
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[8] ASTM, Standard Test Methods For Determination Of Acid

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