mitshu

H Quc gia H Khoa hc t nhin TP.HCM

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Chng 1: TNG QUAN

1.1. Ngun rm r Vit Nam Rm r gn lin vi i sng ngi dn Vit Nam t bao i nay. Rm r

cng to nn nt p rt ring ca lng qu Vit Nam vi cy rm, mi r. N l

ngun nhin liu, nguyn liu cho rt nhiu lnh vc sn xut v l vt phm phc

v mun mt i sng sinh hot ca chng ta.

Tuy nhin vi nm tr li y tnh trng t rm r din ra ngy cng ph

bin sau ma gt gy nh hng tiu cc n mi trng v sc khe con ngi.

Nh vy rm r nn gii quyt th no? Hu ht cc nc v ang tm

kim cc phng php tn dng rm r x l theo cch an ton thn thin vi mi

trng.

Rm r l mt trong cc ngun nguyn liu lignocellulose. Hng nm nn

nng nghip Vit Nam to ra mt lng ln rm r nhng phn ln dng bn,

lm cht t hay lm thc n cho gia sc m cha c s dng mt cch hiu qu

trong vn nng lng.

Rm r chim 62,6% cc ngun biomass chnh Vit Nam cho thy vic tn

dng ngun nguyn liu ny trong vic sn xut bioethanol l cch s dng chng

hiu qu trong vn nng lng [3]

Hnh 1.1: Rm r Vit Nam

1.2. Cu trc rm r Thnh phn chnh ca rm r l cellulose, hemicellulose, lignin, cc cht trch

ly v tro. Cellulose, hemicellulose v lignin c quan tm ch yu trong lun vn.


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Cellulose chim 29,68%, hemicellulose chim 17.11% v lignin chim 12.17%

thnh phn rm r [17].

1.2.1. Cellulose

Cellulose l mt polymer mch thng ca cc D- glucose, cc D- glucose lin

kt vi nhau bng lin kt -1,4- glycoside (Hnh 1.2).

Hnh 1.2 : Cc mt xch -D-Glucose trong cellulose [19]

Lin kt -1,4- glycoside l mt lin kt ni phn t nn cc n v glucose

xen k trn v di to nn cellulose c dng chui xon. Mi mt chui celllose

cha hng chc nghn n v -1,4- glycoside, cc chui ny li sp xp song song

nhau to thnh cc si c ng knh 3.5 nm. Cc si ny li kt hp vi nhau

thnh b si c ng knh ln hn. Gia cc chui cellulose thng hnh thnh rt

nhiu cc lin kt hydro, nh m si cellulose rt bn. Cc b si c cu trc

khng ging nhau:

+ Phn cu trc cht gi l cellulose kt tinh

+ Phn cu trc khng cht gi l cellulose v nh hnh.

Hnh 1.3: Vng v nh hnh v vng kt tinh trong vch t bo thc vt.


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Cellulose l ngun nguyn liu sinh hc di do nht trn tri t v c sn

xut vi tc 4x1010 tn/ nm. Cc sn phm thy phn ca cellulose l ngun

nguyn liu ch bin cho rt nhiu qu trnh ha hc, l ngun cung cp glucose ln

nht v c dng sn xut nhin liu ethanol.

1.2.2. Hemicellulose

Hemicellulose cng vi lignin v cellulose to thnh t bo thc vt.

Hemicellulose l polymer ca xylose, galactose, mannose, aribinose, cc ng

khc v cc acid uronic ca chng

Hnh 1.4: Cc thnh phn ca hemicellulose [37]

Thnh phn cu to ca hemicellulose rt khc nhau trong t nhin v ph

thuc vo ngun thc vt nhng c mt vi c im chung sau:

+ Mch chnh c cu to t lin kt 1,4

+ Xylose l thnh phn quan trng nht

+ Nhm th ph bin l nhm acetyl O- lin kt vi v tr 2 hoc 3

+ Mch nhnh cu to t cc nhm n gin, thng thng l disaccharide

hoc trisaccharide. S lin kt ca hemicellulose vi cc polysaccharide khc v vi

lignin l nh cc mch nhnh ny. Cng v hemicellulose c mch nhnh nn tn ti

dng v nh hnh v v th d b thy phn.


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1.2.3. Lignin

Lignin l mt phc hp polymer thm v nh hnh, l polymer phong ph th

hai sau cellulose, thng thng c tm thy g v l mt phn khng th thiu

ca vch t bo thc vt.

Lignin lp y khong trng trong vch t bo gia cellulose, hemicellulose v

cc thnh phn pectin, c bit trong tracheids, sclereids v xylem. N c lin kt

ng ha tr vi hemicellulose to bn c hc cho vch t bo.

Lignin thng thng cha 3 alcohol thm bao gm coniferyl alcohol, sinapyl

v p-coumaryl [33].

p-coumaryl alcohol Coniferyl alcohol Sinapyl alcohol

Hnh 1.5: Cc alcohol thm ca lignin [31]

Nhng nghin cu gn y ch ra rng lignin hon ton khng ng nht trong

cu trc. Lignin dng nh bao gm vng v nh hnh v cc vng c cu trc

hnh thun hoc hnh cu. Lignin trong t bo thc vt bc cao hn khng c vng

v nh hnh. Cc vng phenyl trong lignin ca g mm c sp xp trt t trn

mt phng thnh t bo. Ngoi ra, c cu trc ha hc v cu trc khng gian ca

lignin u b nh hng bi mng polysaccharide. Vic m hnh ha ng hc phn

t cho thy rng nhm hydroxyl v nhm methoxyl trong cc oligomer tin lignin

s tng tc vi vi si cellulose cho d bn cht ca lignin l k nc.


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Nhm chc nh hng n hot tnh ca lignin l nhm phenolic hydroxyl t

do, methoxyl, benzylic hydroxyl, ether ca benzylic vi cc ru thng v nhm

carbonyl.

Lignin c lin kt ha hc vi thnh phn hemicellulose v c vi cellulose,

bn ha hc ca nhng lin kt ny ph thuc vo bn cht lin kt v cu trc ha

hc ca lignin v nhng n v ng tham gia lin kt.

Cu trc ha hc ca lignin d b thay i trong iu kin nhit cao v pH

thp.

Lignin kh c tiu ha bi enzyme do bi cc lin kt cho vi cc thnh

phn khc ca t bo, n cng lm gim s tip cn ca cellulose v hemicellulose

ti mt vi enzyme ca vi khun. Do , lignin thng thng gip bo v thc vt

khi mm bnh v vt gy hi. Tuy nhin, vi loi nm v vi khun c kh nng

phn hy cc polymer ny: Nm mc trng Coriolus versicolor, P. chrysosporium

and T. versicolor [34].

Hnh 1.6 : Cu trc lignin [31]


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Tm li, cc thnh phn ca rm r c th c minh ha trong hnh di y

Hnh 1.7: Cu trc cc thnh phn rm r [38]

1.3. Nm rm 1.3.1. Tng qut v nm rm

Nm rm l loi nm kh ph bin ng Nam , nm thng mc trn

nguyn liu ph bin l rm nn c tn chung l nm rm, (straw mushroom), tn

khoa hc l Volvariella volvacea, thuc h nm rm Plutaceae. Nm cng c th

mc trn mt vi loi nguyn liu khc nh: lc bnh, b chui kh, bng gn, mt

ca, b ma,.

V phn loi, nm rm d kin c hn 100 loi nhng s phn bit gia chng

thng khng r rng. V d mt vi loi nm nh: V.diplasia, m nm c mu nu

t, hay nm rm la bc V.bombycina,.

iu kin sinh trng ca nm rm c tm tt trong bng 1.1.


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Bng 1.1: iu kin sinh trng ca nm rm [17]

Yu t Nui t nm Ra qu th

Khong bin

thin

Ti thch Khong bin

thin

Ti thch

Nhit 14-400C 350C 25-300C 280C

m 50-70% 60% 80-100% 90%

pH 6-7 6.5 6-7 6.5

1.3.2. Cu to hnh thi qu th nm: Qa th nm rm gm:

- Bao gc: Bao gc di v cao, lc nh bao ly m nm, khi m nm trng thnh

gy nt bao, bao gc ch cn li phn trm ly gc chn cung nm. Bao nm cha

sc t melanin to ra mu en bao gc. m nht ty thuc vo nh sng, nh

sng cng nhiu th bao gc cng en.

- Cung nm: Khi cn non th mm v gin. Khi gi x cng li v kh b gy.

- M nm: M nm hnh nn cng c cha melanin nhng nht dn t trung tm ra

ra mp. Bn di m c nhiu phin nm.

Hnh 1.8: Qu th nm rm [17]

Tai nm rm c bit lc cn non c bao trong mt lp v c mu trng

xm n en, khi trng thnh x rch v bc v vn m nm ln cao, cung c

bao gc v khng vng c. m bo t hnh thnh hai mt phin nm bn di


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m nm. m bo t nm rm c mu hng tht nn khi tai nm trng thnh phin

c mu , vn m nm ln cao.

T lc hnh thnh n nm n khi pht trin thnh tai trng thnh, qu th

nm rm tri qua nhiu giai on. Ngi ta gi tn qu th da theo hnh dng ca

chng mi giai on: Hnh inh ghim, hnh nt, hnh trng, hnh ko di v hnh

d hay trng thnh.

Dng nt Dng trng Dng ko di Dng trng thnh

Hnh 1.9: Cc giai on pht trin khc nhau ca tai nm rm [17]

1.3.3. Vng i ca nm rm :

Bt u t cc m bo t v c xem l kt thc khi hnh thnh tai nm hon

chnh. m bo t l bo t hu tnh, khi rng s bay i khp ni, gp iu kin

thun li ny mm v cho li h si nm. H si nm ny thng ch c mt nhn

nn gi l si nm s cp. i vi nm rm, ch cn h si s cp t mt bo t ny

mm cng c th t phi hp cho ra t th cp. T h si t th cp cha hai nhn,

nm pht trin thnh mng si, lan ra khp ni trn c cht rt ly dinh dng.

Trong trng hp b t khc, cc si nm t lm lnh vt thng v ti lp li h

si, tng t nh cy trng trong gim chit cnh. H si nm pht trin thnh

mng si. Gp nhng iu kin nht nh, h si nm s bn li v to thnh hch

nm. Hch nm tip tc pht trin cho qu th trng thnh.


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Hnh 1.10: Chu trnh sng ca nm rm [17]

Nm rm cn mt dng bo t th hai gi l hu bo t. y l bo t v tnh

c dng hnh cu v l mt dng sng c bit ca chng nhm trnh qua cc giai

on mi trng khc nghit. Hu bo t hnh cu, c hai nhn v c mu hng tht

nh m bo t. Khi ny mm, hu bo t s cho li h si nm.

Cht x ca thc vt l cellulose, hemicellulose v lignin trong khi cht x ca

nm l chitin.[28]. Chitin l vt liu cu trc c bn trong nm v c chng minh

c gi tr nh cht x. N cng c thy phn thnh glucosamine.

1.3.4. Nguyn liu trong mi trng trng nm

Trong thin nhin, hu nh tt c cc xc b thc vt u c th trng nm.

Tuy nhin, khng phi nguyn liu no nm cng s dng ngay m cn tri qua qu

trnh ch bin thch hp. Qu trnh ny gm nhiu cng on vi s tham gia ca

cc yu t khc nhau, va sinh hc va khng sinh hc [17].

Ty loi nm, c loi sn xut c cc loi enzym phn gii mnh nn

nguyn liu s dng tng i th hn. Ngc li, cng c nhng loi cn c

nguyn liu gn nh mn ha. Di y l mt s ngun nguyn liu trng nm

(bng 1.2)


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Bng 1.2: Thnh phn mt s ngun nguyn liu trng nm [7]

Rm r la kh

Nc 13,4% Cellulose v lignin 28%

Protein th 1,8% Hydrat cacbon 42,9%

Lipid th 1,5% Cht khong 12,4%

B ma phi kh

Nc 8,7% Cellulose v lignin 28,2%

Protein th 2% Hydrat cacbon 58,4%

Lipid th 0,7% Cht khong 2,1%

C kh

Nc 14,14% Hydrat cacbon 3,45%

Protein th 4,15% Cht khong 9,88%

1.4. Qa trnh sn xut bioethanol t rm r 1.4.1. Bioethanol: Vic cung cp v s dng nng lng ng mt vai tr trong tm trong t

chc x hi, t phc li c nhn cho n s pht trin x hi v cng nghip. Kh

nng tip cn nng lng v chi ph cho nng lng c nh gi l yu t quyt

nh cho mi quan h kinh t v chnh tr gia cc quc gia. Xem xt cc ngun

nng lng, x hi loi ngi s dng nhin liu ha thch tng ln ng k

trong 50 nm qua . Vic s dng xng cho vn giao thng vn ti chim 1/3

lng kh thi gy hiu ng nh knh (Wyman, 1996) nh hng nghim trng n

mi trng dn n mt vn thit yu l gim nhu cu tiu th. Tht vy, cc

nn kinh t mnh nht th gii k cam kt su sc i vi vic pht trin cc k

thut cng ngh nhm vo vic s dng cc ngun nng lng ti to. Trong

chng trnh ngh s ny, vic thay th xng nhin liu lng bng ethanol ti to l

c tm quan trng hng u.


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Nhin liu bioethanol ch yu c sn xut t qu trnh ln men ng.

Ngun ng chnh sn xut ethanol l cc cy trng cho mc ch nng lng

bao gm ng cc, ng, la m, mn ca, rm,. Cng c nhng nghin cu vic s

dng rc thi th sn xut nhin liu ethanol.

Ethanol l mt cht lng khng mu, c th b phn hy do vi khun, tnh

c thp v t gy nhim mi trng nu b trn. Khi chy s to ra CO2 v H2O.

Bioethanol c mt s thun li hn cc ngun nhin liu khc: C th ti to, gim

lng kh nh knh, c th b phn hy v t c hn cc nhin liu ha thch khc,

gim lng carbon monoxide do ci thin cht lng khng kh.

Ethanol c th c sn xut t biomass thng qua qu trnh thy phn v ln

men ng. Biomass cha mt phc hp carbohydrate nh cellulose, hemicellulose

v lignin. sn xut ng t biomass, biomass phi c tin x l bng acid

hay enzyme lm gim kch thc vt liu th v m cu trc thc vt. Cc phn

ca cellulose v hemicellulose b ph v bi enzyme hay acid thnh ng v sau

s ln men thnh ethanol.

Brazin l nc tin phong trong vic s dng nhin liu ti to. Vic thay th

xng bng ethanol bt u t nm 1975, khi chnh ph Brasil pht ng chng

trnh Prolcool Program". Ti thi im cuc khng hong du la u tin vo

nhng nm 1970, c nc phi nhp khu 85% du th tim nng sn xut ethanol

t ma nh mt nhin liu vn chuyn ph hp vi chnh sch ca chnh ph v

cung cp nng lng. Chng trnh Prolcool Program" u i cho cc nh my

ru v cc cng ty t sn xut xe hi ch chy bng ethanol.

Gn y, ngnh cng nghip ethanol Brazin khng c s khuyn khch

ca chnh ph v nhin liu sinh hc c phn phi bi cng ty du Petrobras. Cc

hm i Brazin ca 20 triu xe hi (gm xe hi, nh sng qung co, xe ti v xe

but khong 24 triu) chy c xng pha trn c cha 22-24% ethanol hay 100%

ethanol. Kh t t nhin cng c s dng s lng t. S tiu th ethanol c

d bo s tng ln khi s lng xe chy bng c xng pha trn hay bng ethanol,

c d bo s tng t 4 n 15 triu vo 2013.


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M cng u t mnh vo sn xut nhin liu sinh hc. Lin minh chu u

cng tin ti vic khuyn khch nng lng ti sinh cho tng lai vi nhng o

lut v iu khon s dng phng tin giao thng ti thiu cho cc nc thnh

vin.

Vit Nam, cng ngh sn xut ethanol c hnh thnh t rt lu, ch yu

sn xut t r ng ma lm ethanol cho ngnh thc phm v cng nghip.

Cn c nhng nghin c lm sao tng hiu qu ca qu trnh sn xut

ethanol v tng qu trnh chuyn ha biomass thnh nhin liu bn vng thay th

nhin liu ha thch.

1.4.2. Qu trnh sn xut bioethanol t rm r 1.4.2.1. Tin x l

Tin x l lignocellulose l cn thit bi v qu trnh thy phn cc nguyn

liu khng c tin x l xy ra chm v kt qu l sn lng sn phm thp. Vi

phng php tin x l lm gim s kt tinh ca cellulose. Tin x l cng lm cho

cellulose n gn hn vi cc enzyme phn hy cellulose, iu ny lm gim chi

ph qu trnh x l.

Nhiu phng php tin x l khc nhau c s dng, c th chia thnh ba

nhm chnh:

+ Ha hc ( acid hay kim)

+ Vt l/ ha l

+ Phng php sinh hc bng vi sinh vt

Trong phng php tin x l bng phng php ha hc, v d dng acid,

hemicellulose s b tn cng v phn ln lignin b loi b.

Khng c mt nguyn l tin x l chung no v cc nguyn liu khc nhau s

c phng php tin x l khc nhau. V nhiu enzyme phn hy cellulose ca nm

hot ng pH thp ( 4-5), tin x l bng acid dng nh c u tin trong khi

enzyme nm c chn cho thy phn.

Tin x l sinh hc dng vi sinh vt v cc enzyme ca chng c nhng thun

li nh: Nhu cu nng lng thp, to cht thi thp v t tc ng n mi trng.


Trang 16

Vi loi nm m, v d P. chrysosporium, Streptomyces viridosporus, Phelebia

tremellosus, Pleurotus florida v Peurotus cornucopiae c chng minh c kh

nng tn cng lignin v carbohydrate, n cng c chng minh c kh nng bin

i nguyn liu lignicellulose thnh ethanol.

1.4.2.2. Thy phn

Sau khi tin x l, cellulose v hemicellulose c thy phn thnh ng n

( hexose v pentose) bng cch dng cellulase v hemicellulase. Enzyme chu ng

nhit cao v pH thp c dng cho thy phn v hu ht cc k thut tin x l

hin nay da vo acid v nng. Hn na, enzyme chu nhit c vi thun li nh

hot tnh chuyn bit v tnh n nh cao, ci thin ton b qu trnh thy phn, t

lm gim chi ph. Do bi nhng ha hn v enzyme nm chu nhit v acid nn

chng c tim nng tt dng thy phn lignocellulose quy m cng nghip.

Enzyme cellulase

Hnh 1.11 : Cellulase

- Cellulase l mt phc hp gm nhiu enzyme. Cc loi enzyme trong phc hp

ny s ln lt phn hy cellulose thnh sn phm cui cng l glucose.

Theo Wood v Mc Crae (1979) qu trnh thy phn cellulose c thc hin

bi ba nhm enzyme sau :

+ Endocellulase hay endoglucanase : Phn gii lin kt -1,4-glycosid bng cch

tn cng vo vng khng kt tinh ca cellulose, pH thch hp 4-5, nhit thch

hp t 50 n 700C. Sn phm ca qu trnh ny l cellodextrin, cellobiose v

glucose


Trang 17

+ Exocellulase hay exobiohydrolase : Enzyme ny thy phn cellobiose t u

khng kh ca chui glucan v cellodextrin, pH thch hp 4-5, nhit thch hp

37-600C.

+ -glucosidase hay cellobiase : Enzyme ny thy phn cellobiose v cellodextrin

to thnh glucose, nhit thch hp 45-750C.

- C ch tc ng ca cellulase

Qu trnh tc dng ca cellulase c th chia thnh nhng giai on sau :

+ Qu trnh hp ph enzyme ln x si

C hai yu t quyt nh nng lng hp ph ca protein ln b mt phn pha

rn/ lng l bn cht ca b mt v lc lin kt gia cc phn t. Nhng tng tc

ny thng khng mang bn cht cng ha tr, ni cch khc, cc tng tc ny

thng to thnh do lin kt hydro, lc tnh in hoc l tng tc gia cc nhm

k nc. Cc phn t protein hay cc ion khi lng phn t thp hp ph trc

trn b mt s c nh hng n s hp ph mi. Lc tnh in gp phn vo vic

hp ph ca enzyme ln b mt phn pha, tuy nhin khng phi l yu t quyt nh

qu trnh hp ph ca protein.

Protein l mt polymer lng cc, cha c in tch dng v m, iu ny

lm cho protein c bn cht ca mt phn t hot ng b mt. Phn k nc trong

phn t protein l nhng nhm cha nhn thm nh trong tryptophane,

phenylalanine v tyrosine. Thng thng, khi phn t protein b gp khc, cc

amino acid mang nhm k nc s c giu vo bn trong. Tuy nhin cng c mt

vi phn k nc c sp xp ti b mt ngoi ca phn t protein. Chnh cc

phn ny to lin kt vi cc c cht mang bn cht k nc nh vo tng tc gia

cc phn k nc v lin kt hidro. i lc ca protein s tng khi bn cht k nc

trong phn t protein tng mc d iu ny s nh hng ti tng tc tnh in.

Mt cch tng qut, s hp ph ca phn t protein ln b mt k nc l bt thun

nghch hn s hp ph ln b mt a nc. iu ny bi v phn t protein s c

mt vi thay i mt khi hp ph ln b mt k nc.


Trang 18

B mt cellulose mang bn cht k nc do gia cc mch to lin kt hydro.

Cellulose tinh khit khng cha nhm mang in. Trong thc t, in tch b mt

ca cc cht s c to thnh khi c s phn b cc ion t b mt .

Ty thuc vo tnh cht ca protein v b mt hp th m tng tc in tch-

in tch hay tng tc ca cc nhm k nc s ng vai tr quyt nh cho qu

trnh.

in tch ca cellulase v s hin din ca nhm khng phn cc c th dn

ti s hp ph khng chn lc ln c cellulose v lignin. Nhiu nghin cu ch ra

rng cellulase hp ph ln c lignin trong vt liu lignocellulose. Ngoi ra, i vi

- glucosidase khng ging cc enzyme cellulase khc khng c trung tm to lin

kt CBD, enzyme ny hp ph kh mnh ln lignin c tch ring.

Rt cn thit hn ch ti a vic hp ph khng chn lc ca cellulase ln

lignin nu mun ng dng qu trnh thy phn enzyme mt cch kinh t. V mt l

thuyt, cng ngh protein c th lm thay i cu trc ca cellulase lm cho

enzyme ny hp ph chn lc hn. Tuy nhin, kh nng hp ph ca cellulase ln

vng cellulose kt tinh l yu t quan trng trong qu trnh thy phn, v vy cc tc

ng ln cu trc cn c trnh. Hin ti, vn cha c nghin cu no thnh cng

trong vic thay i chui acid amin ca cellulase lm cho enzyme ny thy phn

vt liu lignocellulose hiu qu.

Cellulase tng tc vi b mt cellulose thng qua trung tm lin kt cellulose

-CBD v trung tm hot ng chnh -CD. Cellulase to c lin kt vi cellulose

l nh vo trung tm to lin kt CBD. i vi cc chng T. reesi, CBD l trung

tm gm cc phn t nh, gm 36 amino acid. Thng thng trung tm lin kt c

3 amino acid tyrosine, chu trch nhim cho vic lin kt vi cellulose. Trong CBD

I, mt tyrosine c thay th bng tryptophane.

Khc bit trong cu trc ca cc loi enzyme (CBD I, CBDII, EG,) s dn

n i lc khc nhau trong qu trnh hp ph ca cc enzyme ny ln c cht. c

nhiu nghin cu v cu trc ca cc enzyme v s lin h n kh nng to lin kt

vi cellulose.


Trang 19

+ C ch tc ng hip ng ca cc enzyme

T nhng nm 1954, Gilligan v Reese ch ra rng vic pha trn cellulose

t cc ngun nm mc khc nhau c th lm tng lng ng kh to thnh trong

qu trnh thy phn. T lc , nhiu nghin cu c tin hnh nhm m t qu

trnh tc ng hip ng ca cc enzyme endo, exo cellulase.

Nghin cu v tc ng hip ng ca endo-exo cng b u tin vo 1980. S

tc ng hip ng ca endo-exo c th ni l nhm mc ch to b mt tn cng

mi cho exoglucanse. Enzyme exo thng tn cng vo nhng cellulose b ct

mch (cellodextrin) c u l nhm kh C (C cha OH hemiacetal) hoc khng

kh. Trong khi , enzyme endo li tn cng ngu nhin vo gia mch cellulose,

to thnh cc cellodextrin, l cc c cht thch hp cho enzyme exo. Exoglucanase

c nhng loi enzyme tn cng c trng ty theo tnh cht kh c hay khng

kh c ca nhm u mch. Tht vy, theo nhng nghin cu ca Teeri v cng

s, nhm kh u mch ca cellodextrin s c nh hng vo trung tm xc tc

ca CBHI. Trong khi , CBH II li u tin tn cng vo u khng c nhm kh

ca cellodextrin.

Ngoi ra cng c nhiu nghin cu v s tc ng hip ng ca cc enzyme

endo, exo c ngun gc nm mc v vi khun. Da trn cc nghin cu ny,

enzyme cellulase c th c pha trn vi cc thnh phn t cc ngun gc khc

nhau nhm to c mt h enzyme c tc ng mong mun.

Tm tt qu trnh thy phn (Hnh 1.12)

Enzyme endo-cellulase tn cng ngu nhin vo mch cellulose nh to lin kt bng tng tc gia CBD vi cellulose, to thnh cc

oligosaccharide.

Enzyme exo-cellulase tn cng vo cellulose v c oligomer t u ng kh v khng kh thng qua tng tc ca CBD vi cellulose, to

thnh cellobiose v c glucose.

-glucosidase tn cng cellobiose v oligosaccharide tan, to glucose.


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Hnh 1.12: Tm tt qu trnh thy phn ca cellulase [38]

- Cc yu t nh hng n qu trnh thy phn

+ nh hng ca cu trc nguyn liu

Cu trc t nhin ca lignocellulose to ra nhiu cn tr n qu trnh tn cng

ca cc tc nhn thy phn. Ngay c qu trnh thy phn cellulose tinh khit, tc

thy phn cng gim theo thi gian. Tc phn ng gim dn theo thi gian v

mt s l do sau : S c ch enzyme do sn phm, s gim ca cc phn c cht d

thy phn, enzyme b bt hot hoc b gi li trong cc l xp ca cellulose.

Hiu sut qu trnh thy phn b nh hng mnh bi tnh cht ca ngun

nguyn liu. Mt cch tng qut, g mm thng kh thy phn hn g cng. Cu

trc ca nguyn liu cng vi c ch tc ng ca cc enzyme v c cht l hai yu

t chnh lm hn ch hiu sut qu trnh thy phn.

Kh nng tip cn vt liu lignocellulosic ca cellulase ng vai tr quan trng

trong qu trnh thy phn. Cellulose c b mt trong v ngoi, b mt ngoi bao

gm b mt bao quanh cc x si, b mt trong l b mt do cc mao qun bn

trong x si to thnh. Nu cellulose khng c tin x l, hiu qu thy phn

thp. X l loi b hemicellulose (bng tin x l acid) s lm tng kh nng thy


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phn cellulose ( theo nghin cu ca Grohmann). Grethlein gii thch rng loi

b hemicellulose s lm tng cu trc xp ng thi tng b mt cellulose, lm cho

enzyme d tn cng hn. Kch thc ca cc l xp li lin quan n trng n

ca vt liu. Tht vy, nhiu nghin cu ch ra rng, khi lm kh vt liu

lignocellulose, cc mao qun s b mt i, iu ny lm gim kch thc l xp v

v vy hiu sut qu trnh thy phn gim r rt. Hm lng v s phn b ca

lignin trong cu trc vt liu c nh hng ti kh nng thy phn ca vt liu .

Hiu sut thy phn thu c kh cao i vi cc nguyn liu c loi b gn

ht lignin.

+ nh hng ca nhit

Ging nh nhiu phn ng enzyme khc, phn ng thy phn cellulose bng

enzyme cellulase chu nh hng ln ca nhit . Tc phn ng thy phn tng

theo nhit , tuy nhin n mt nhit nht nh, tc phn ng s gim dn v

n mc trit tiu.

Ngi ta thng s dng h s nhit Q10 biu th nh hng ca nhit

n tc phn ng. Nhit tng ng vi tc phn ng enzyme cao nht

c gi l nhit ti u. Phn ln enzyme hot ng mnh nht nhit 40-

500C. Ring i vi enzyme cellulase, nhit ti u l 500C. Nhng enzyme khc

nhau u c nhit ti u khc nhau.

Nu a nhit cao hn nhit ti u, hot tnh enzyme s b gim, khi

enzyme khng c kh nng phc hi hot tnh. Ngc li khi nhit 00C,

enzyme s b hn ch hot ng rt mnh, nhng khi a nhit ln t t, hot

tnh ca enzyme s tng dn u n mc ti u.

+ nh hng ca pH

pH mi trng thng nh hng n mc ion ha c cht, enzyme v c

bit nh hng n bn ca enzyme. Nhiu enzyme hot ng rt mnh pH

trung tnh. Tuy nhin cng c nhiu enzyme hot ng mi trng acid yu. Mt

s enzyme khc li hot ng mnh pH kim v c pH acid. i vi enzyme

cellulase, khong pH thch hp l 4-5 trong tt nht l 4.8.


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+ Nng enzyme

Khi nng enzyme tng, tc phn ng tng theo ng thng. Tuy nhin,

khi nng enzyme t n mt ngng no , nng c cht s tr thnh yu

t hn ch tc phn ng. Khi tc phn ng s khng tng na m l mt

ng nm ngang.

+ nh hng ca nng c cht

Khi nng c cht tng, tc phn ng enzyme tng v s c nhiu c cht

va chm vi enzyme tin hnh phn ng. Khi nng c cht ln, cc enzyme

b bo ha c cht, v vy tng nng c cht th tc phn ng s khng thay

i ng k.

+ nh hng ca cc cht km hm

Cc cht km hm hot ng ca enzyme thng l cc cht c mt trong cc

phn ng enzyme, lm gim hot tnh enzyme nhng li khng b enzyme lm thay

i tnh cht ha hc, cu to ha hc v tnh cht vt l ca chng.

Cc cht gy km hm hot ng ca enzyme bao gm cc ion, cc phn t v

c, cc cht hu c v c protein. Cc cht km hm c ngha rt ln trong iu

khin cc qu trnh trao i t bo sinh vt. C ch km hm ca cc cht km

hm c th l thun nghch hoc khng thun nghch. Ty thuc vo bn cht cht

km hm ngi ta chia nhng cht km hm: Cht km hm cnh tranh v cht km

hm khng cnh tranh. Cc cht km hm khng cnh tranh thng gm hai loi:

Km hm do sn phm phn ng v km hm do tha c cht.

i vi enzyme cellulase, ngi ta nhn thy, sn phm ca phn ng thy

phn, bao gm c cellobiose v glucose u c tc ng km hm hot tnh

cellulase, c bit l cellobiose.

Enzyme xylanase Hai loi enzyme ch yu tham gia vo qu trnh phn hy hemicellulose l

endo-1,4-D-xylanase v endo-1,4--D-mannase trong endo-1,4-D-xylanase l

tn thng gi ca xylanase. Xylanase l mt tc nhn sinh hc thuc nhm

enzyme thy phn, ch yu xc tc cc phn ng ni thy phn cc cu ni 1,4--


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D-xylosidic xylan v to ra xylose, nh phn hy mt phn hemicellulose gip

qu trnh thy phn hiu qu hn. Xylanase hot ng tt pH 5.3, nhit ti

thch l 550C.

Xylan l thnh phn phong ph nht ca hemicellulose chim trn 70% cu

trc ca chng. Xylan l c cht ca xylanase, l mt polymer lin kt 1,4 ca

xylose. Di tc dng ca xylanase, xylan s chuyn ha thnh nhiu sn phm

khc nhau v cui cng to thnh ng xylose.

C ch tc ng ca xylanase : Xylanase c hai tc ng chnh :

+ Tc dng trc tip ln lin kt lignin- xylan.

+ Xc tc qu trnh phn gii v ha tan cc xylan trong vch t bo, lm cho lignin

d khuch tn ra ngoi x si, ng thi gii phng cc xylose. (Hnh 1.13)

Hnh 1.13: C ch hot ng ca xylanase


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Laccase Ngoi ra trong nm rm cn c enzyme laccase c kh nng phn gii lignin,

lm cho s gn kt ca lignin vi cc thnh phn cellulose v hemicellulose lng

lo, to iu kin cho enzyme cellulase v hemicellulase tc ng tt hn.

Laccase l enzyme rt ph bin trong t nhin, chng c tm thy thc vt,

nm, mt s vi khun v cn trng, chng lin quan n nhiu qu trnh khc nhau

trong c tng hp v phn hy lignin. Laccase ln u tin c pht hin u

tin cy Rhus vernicifera vo 1883. Laccase t nm c nghin cu v kho st

rt k c bit l laccase t ngnh nm m Basidomyces. Ngoi ra cc cc loi

nm nh Ascomyces, Deuteromyces, basidomyces v cc loi nm c kh nng phn

hy lignocellulose nh: Agaricus bisporus, Botrytis cinerea, Chaetomium

themophilum,Coprius cinereus...

Laccase l enzyme xc tc s oxi ha ca c cht v s dng electron m

chng thu c kh phn t O2 thnh nc. Laccase c 4 phn t ng v c th

oxi ha nhiu loi c cht c bit l cc hp cht phenolic.

- Cu trc phn t laccase : Ba loi nguyn t ng c th phn bit : Loi 1

(T1), loi 2 (T2) v loi 3 (T3).

+ T1 to cho laccase c mu xanh nc bin c trng khi c hp th

bc sng xp x 600nm, thng lin kt 2 nguyn t N2 t 2 gc histidines v

sulphur t gc cystein, lin kt gia ng T1 v gc histidine chu trch nhim vic

to mu xanh ca laccase.

+ T2 th khng to mu, lin kt vi 2 gc histidine bo th v c tnh cht

hp ph in t to ph in t mnh.

+ Trong khi T3 cha mt cp nguyn t ng to lin kt vi 6 gc

histidine bo th v khng to ph hp th in t, c th c hot ha khi lin kt

vi anion mnh.


Trang 25

- C ch xc tc ca laccase

Trung tm nguyn t ng mt (T1) l ni din ra phn ng oxy ha c cht.

C cht chuyn mt in t cho nguyn t ng T1, bin nguyn t ng T1

(Cu2+) tr thnh dng Cu+, hnh thnh phn t laccase c c 4 nguyn t ng u

trng thi kh (Cu+). Mt chu k xc tc lin quan n s vn chuyn ng thi 4

electron t nguyn t ng T1 sang cm nguyn t ng T2/T3 qua cu tripeptide

bo th His-Cys-His. Phn t oxy sau oxy ha laccase dng kh, to thnh hp

cht trung gian peroxy, v cui cng b kh thnh nc.(Hnh 1.14)

Hnh 1.14. C ch xc tc ca laccase [2]


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1.4.2.3. Ln men

Trong qu trnh ln men, cc sn phm thy phn bao gm monomer hexoses (

glucose, mannose v galactose) v pentoses ( xylose v arabinose) s c ln men

thu sn phm ethanol. Trong cc sn phm ca qu trnh thy phn, glucose

phong ph nht, tip theo l xylose hay mannose v ng c nng thp.

Saccharomyces cerevisiae l vi sinh vt thng dng ln men ethanol.

S.cerevisiae c vi thun li nh c chp nhn rng ri, tc ln men cao v

kh nng chu ethanol cao. Tuy nhin, S.cerevisiae khng th s dng hiu qu

xylose nh ngun Carbon hay ln men n thnh ethanol [26].

Hnh 1.15: Nm men Saccharomyces cerevisiae

Pichia stipitis l nm men ph bin nht trong cc chng nm men c th ln

men ng 5C. P.stipitis c u im: Hiu sut tiu th xylose cao, chu nhit v

nng c cht cao. Tuy nhin b c ch bi ethanol nng cao.

S to thnh ethanol t glucose phi tri qua nhiu giai on, c ch qu trnh

c th hin trong hnh 1.16


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Glucose Hexokinase

Glucose 6 Phosphat Photphoglucoza isomerase

Fructose 6 Phosphat PhosphoFructokinase

Fructose 1,6 Diphosphat Aldolase

Triophosphat izomerase

Glyceraldehyd 3 Phosphat Dihydro aceton photphat

Glyceral dehyd phosphatdehydrogenase Acid 1,3 Diphosphoglyceric

Phosphoglyceratkinase Acid 3 Phosphoglyceric

Phosphoglycerat-mutase Acid 2 Phosphoglyceric

Enoiase Acid phosphoenolpyruvic

Pyruvat kinase Acid enol Pyruvic Acid pyruvic

Pyruvate Decarboxylase Ethanal

Aldodeshydrogenase Ethanol

Hnh 1.16: C ch qu trnh ln men [11]


Trang 28

Cc yu t nh hng n qu trnh ln men

+ Nhit

Mi vi sinh vt u c mt khong nhit ti u cho s pht trin ca chng.

i vi nm men Saccharomyces, nhit ti u nm trong khong 28-320C.

nhit cao, hot tnh nm men gim nhanh nhng ch yu l d b nhim vi sinh

vt nh vi khun lactic v nm men hoang di. nhit 300C, nm men hoang di

pht trin nhanh hn Saccharomyces 2-3 ln, cn nhit 35-380C chng pht

trin gp 6-8 ln. Mt khc khi ln men nhit cao s to nhiu sn phm ph

nh ester, aldehyd v tn tht ethanol theo CO2 s tng.

+ pH

Nng ion H+ trong canh trng s nh hng ln n hot ng ca nm

men. Chng c kh nng lm thay i in tch ca v t bo, lm tng hoc gim

mc thm thu ca cc cht dinh dng cng nh chiu hng ca qu trnh ln

men. Mi vi sinh vt ch c th hot ng tt trong trng thi ion nht nh, trng

thi ny ph thuc vo pH ca canh trng.

Trong iu kin ln men ethanol, pH ti u to ethanol l 4.5-5.0. Nu pH

thp khong 3.0-4.0 nm men cn hot ng c v vi khun b c ch. pH hi

cao hn th s to ra sn phm c chua thp, sn phm d b nhim khun v cc

sn phm ph trong qu trnh ln men s to nhiu hn, ln men c hiu sut thp.

+ Nng ca dch ln men

Nu nng dch ng qu cao s dn n lm tng p sut v lm mt cn

bng trng thi sinh l ca nm men. Kt qu l ethanol nhiu s c ch khng

nhng cc tp khun m c nm men. Mt khc ng nhiu s dn n tn hao

ngun nguyn liu v phi ko di thi gian ln men. Hn na nu nng ng

ca dch ln men thp th s lm gim nng sut thit b ln men v lm cho nm

men khng cht dinh dng pht trin.

Thng thng nng dch ng c gii hn 22% khi lng hoc t

hn (nu cao hn thnh t bo c th b v).


Trang 29

+ Thi gian

Thi gian cng l yu t quan trng nh hng n cht lng ca sn phm.

Thi gian ln men ph thuc vo nhiu yu t nh nhit ln men, nng

ng, chng nm men,.

Thi gian ln men c tnh bt u t khi cy chng nm men vo mi

trng ln men, nhng thi gian kt thc th ty thuc vo tng mi trng ln men

c th v ty thuc vo mc ch ln men m ta dng qu trnh ln men.

Trong giai on u ca qu trnh ln men phi cho dch ng tip xc vi

oxy. Lc ny nm men cn oxy tch ly mt lng sinh khi cn thit cho qu

trnh ln men.

Tip theo chuyn ha ng thnh ethanol, nm men phi c pht trin

trong iu kin ym kh hon ton. V trong mi trng ny, s h hp ca nm

men b c ch v bt u phi tm li nng lng bng con ng ln men. p

ng nng lng cn thit th nm men cn phn hy mt lng ng ln v ng

s chuyn ha thnh ethanol v CO2. l nguyn nhn nu mun c ethanol nhiu

th khng c thong kh mi trng. Nu c oxy th trong giai on ny ru s

tip tc chuyn ha thnh acid lactic lm sn phm b chua.

+ Nng CO2 trong mi trng

CO2 c hnh thnh trong qu trnh ln men ru t ng. Mt phn s tn

ti trong mi trng; mt phn tch ln trn b mt mi trng; phn cn li tch t

thnh mt lp ngn cch gia khng kh v mi trng. CO2 tch t trong mi

trng ch lm gim kh nng sinh sn ca nm men, nhng khng th lm cho kh

nng ln men ca nm men yu i.

Trong mi trng c hm lng ng cao s cn tr CO2 thot ra ngoi, dn

n c ch sinh sn ca nm men v s ln men c hiu sut thp. CO2 nm trong

khong khng gia b mt mi trng v khng kh c tc dng kim ch s pht


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trin ca nhng vi sinh vt hiu kh gy hi. Do vy, cc thng ln men phi c nt

c bit ch cho php CO2 bay ra m khng cho khng kh vo.

+ Thnh phn cc cht dinh dng trong mi trng ln men

Mi trng nui cy cn phi c y cc thnh phn dinh dng ch yu l

glucid dng monosaccharide v disaccharide, nit dng acid amin, cc mui v

c, tr dng mui nitrit, nitrat, cc vitamin v mui khong.

+ Hm lng ging nm men

Nm men l nhn t to ra qu trnh ln men, chuyn ha ng thnh ethanol

v kh carbonic. Mi loi nm men c kh nng ln men khc nhau. Cng loi nm

men, nhng nhng iu kin ln men khc nhau th kh nng ln men khc nhau

v sn phm ca qu trnh ln men cng khc nhau. Vic b sung t l ging ln

men cng phi c la chn. Thng thng lng men ging cy vo 15 triu t

bo/ml dch ln men.

Tm li qu trnh sn xut bioethanol t nguyn liu lignocellulose c tm

tt trong hnh 1.17


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Hnh 1.17: Qa trnh sn xut ethanol t nguyn liu lignocellulose [26]

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