Nghiên cứu chế độ xử lý bã mía cho mục tiêu lên men bioethanol

TểM TT LUN VN THC S ti: Nghiờn cu ch x lớ bó mớa cho mc tiờu lờn men bioethanol Tỏc gi lun vn: Lờ Duy Khng Khúa: 2009 Ngi hng dn: PGS.TS Tễ KIM ANH Ni dung túm tt: a Lý chn ti: Nm 2007, chớnh ph ó phờ duyt ỏn phỏt trin nhiờn liu sinh hc n nm 2015, tm nhỡn 2025 nhm to dng nng lng tỏi to c thay cho nhiờn liu húa thch, gúp phn bo m an ninh nng lng v bo v mụi trng: n nm 2015 sn lng ethanol v du thc vt t 250 nghỡn tn (pha c triu tn E5 v B5), ỏp ng 1% nhu cu xng du c nc v n 2025 s t 1,8 triu tn, ỏp ng 5% nhu cu xng du Trong cỏc nguyờn liu lignocellulose, bó mớa l mt nhng ngun lignocellulose trung nht Nu lng sinh ny c chuyn húa thnh ng lờn men c, bó mớa s l mt cỏc ngun nguyờn liu quan trng cho mc tiờu sn xut cn nhiờn liu nc ta núi riờng v trờn th gii núi chung b Mc ớch nghiờn cu ca lun vn, i tng, phm vi nghiờn cu ti s trung la chn ch x lý bó mớa bao gm tin x lớ bó mớa, thy phõn bng h enzym cellulase v nghiờn cu thu nhn dch ng cú th lờn men c Hin vic thy phõn hemicellulose cng nh to cỏc chng lờn men t ng 5C ó cú nhng thnh cụng bc u nhng cha thc s sn sng ỏp dng, ú h cellulase c s dng thy phõn bó mớa thnh glucose cho mc tiờu lờn men c.Túm tt cụ ng cỏc ni dung chớnh v úng gúp mi ca tỏc gi o Kho sỏt la chn phng ỏn tin x lớ bó mớa o Kho sỏt kh nng ỏp dng laccase kt hp vi tin x lớ húa-lý o ó la chn ch s dng laccase cho x lớ dch sau tin x lớ, lm tng hiu sut thu hi ethanol o La chn t l cỏc enzym cellulase thy phõn bó mớa d Phng phỏp nghiờn cu: La chn nguyờn liu lignocellulose cho nghiờn cu Kho sỏt cỏc ch tin x lớ nguyờn liu bng tỏc nhõn húa nhit bao gm axớtnhit, kim-nhit v kt hp nhit kim vi laccase Hiu qu tin x lớ c ỏnh giỏ thụng qua hiu qu thy phõn bó mớa v mc gim hm lng lignin bó mớa trc v sau tin x lớ Nghiờn cu loi phenol dch sau tin x lớ bng laccase, nhm gim s c ch t bo nm men Hiu qu loi phenol c ỏnh giỏ thụng qua hiu sut thu hi ethanol v lng phenol trc v sau x lớ Nghiờn cu ti u t l b sung enzym h cellulase v thi gian thy phõn bó mớa, cỏc thớ nghim c c a theo phn mm Design Expert (DX-7) Phng ỏn ti u c kim tra bng thc nghim e Kt lun ó kho sỏt iu kin tin x lớ la chn c ch tin x lớ bó mớa nh sau: + Bó mớa sy 45oC, b sung kim vi 0,1g NaOH/g bó mớa, quỏ trỡnh thc hin nhit 121oC thi gian 60 phỳt, cho hiu qu thy phõn bó mớa cao hn so vi axớt, vụi + Phi hp laccase (40ữ70) U/g bó mớa tin x lớ lm tng hiu qu thy phõn bó mớa lờn 284,154,6 mg ng kh/g bó mớa (tng 8,2% so vi khụng phi hp laccse), lm gim hm lng lignin ti 72% (tng 3,1% so vi khụng phi hp laccase) Bó mớa ó x lớ vi ch la chn cú th c thy phõn hiu qu vi 31,5/53,61/20,47 (CMCase/FPU/CBU)/g bó mớa, thi gian thy phõn 40,5 gi, t 435 mg ng kh/g bó mớa 3.Laccase cú th s dng loi phenol kh c dch thy phõn, lm tng hiu sut thu hi ethanol ti 76,274,67% (tng 68% so vi khụng kh c) Luan Van Tot Nghiep MC LC MC LC LI CM N LI CAM OAN DANH MC CC CH VIT TT DANH MC CC BNG DANH MC CC HèNH V V TH M U CHNG I: TNG QUAN .11 1.1 Nhiờn liu sinh hc 11 1.2 Bó mớa, nguyờn liu trung cho sn xut bioethanol 13 1.2.1 Cellulose .14 1.2.2 Hemicellulose 15 1.2.3 Lignin 18 1.3 Tin x lớ nguyờn liu lignocellulose 19 1.3.1 Tin x lớ nguyờn liu lignocellulose bng phng phỏp vy lý 20 1.3.2 Tin x lớ lignicellulose bng phng phỏp húa hc 20 1.3.3 Tin x lớ lignocellulose bng phng phỏp húa lý 24 1.4 Tin x lớ lignocellulose bng phng phỏp sinh hc .25 1.4.1 Peroxidase 25 1.4.2 Lacase v c ch phõn hy lignin 27 1.5 Thy phõn cellulose .31 1.5.1 Thy phõn lignocellulose bng phng phỏp húa hc 31 1.5.2 Thy phõn lignocellulose s dng enzym 31 1.6 Lờn men ethanol dch thy phõn 34 CHNG II: VT LIU V PHNG PHP NGHIấN CU 37 2.1 Vt liu 37 2.1.1 Bó mớa 37 2.1.2 Enzym v húa cht 37 Le Duy Khuong Vien Cong nghe Sinh hoc & Cong nghe Thuc pham Luan Van Tot Nghiep 2.1.3 Chng vi sinh vt 37 2.1.4 Dng c v thit b 38 2.2 Cỏc phng phỏp phõn tớch 38 2.2.1 Xỏc nh m bó mớa bng phng phỏp sy n trng lng khụng i 38 2.2.2 Xỏc nh hm lng cellulose ng phng phỏp Kurshner 38 2.2.3 Xỏc nh hm lng lignin theo phng phỏp in tớnh ca Komarov 39 2.2.4 Xỏc nh hm lng ng kh dung dch 41 2.2.5 Xỏc nh hot enzym .42 2.2.6 Xỏc nh hm lng phenol tng s dung dch bng phng phỏp Folin Ciocalteau 46 2.3 Phng phỏp nghiờn c u .47 2.3.1 nh hng ca nhit sy bó mớa 47 2.3.2 Nghiờn cu la chn tỏc nhõn tin x lớ húa-nhit bó mớa 48 2.3.3 Kho sỏt vai trũ ca laccase tin x lớ 49 2.3.4 nh hng ca thi gian x lớ húa-nhit 49 2.3.5 Ti u t l cỏc enzym h cellulase 49 2.3.6 Kho sỏt vai trũ kh c dch thy phõn lignocellulose ca laccase 49 CHNG III: KT QU V BN LUN 52 3.1 La chn nguyờn liu lignocellulose s dng nghiờn cu .52 3.2 nh hng ca nhit sy ó mớa n hiu qu trỡnh thy phõn 52 3.3 Tin x lớ bó mớa vi H2 SO4 53 3.4 Tin x lớ mụi trng kim Ca(OH)2 v NaOH .54 3.5 Tin x lớ húa nhit kt hp vi laccase .58 3.6 nh hng ca thi gian x lớ NaOH- nhit n hiu qu thy phõn bó mớa 60 3.7 nh hng ca t l enzym h cellulase n hiu qu thy phõn bó mớa 60 3.8 Nghiờn cu k thut kh phenol ca dch tin x lớ bng laccase 66 3.8.1 nh hng ca nhit ti kh nng kh phenol ca laccase 67 Le Duy Khuong Vien Cong nghe Sinh hoc & Cong nghe Thuc pham Luan Van Tot Nghiep 3.8.2 nh hng ca thi gian ti kh nng kh phenol ca laccase 69 3.8.3 nh hng ca nng laccase ti kh nng loi phenol dch 69 KT LUN 72 KIN NGH 73 TI LIU THAM KHO 74 PH LC 80 Le Duy Khuong Vien Cong nghe Sinh hoc & Cong nghe Thuc pham Luan Van Tot Nghiep LI CM N hon thnh lun ny cn cú mt thi gian di lm vic trung, chm ch, vi s giỳp v h tr ca cỏc thy cụ, gia ỡnh v n bố Trc tiờn, tụi mun núi li cm n sõu sc ti gia ỡnh vỡ nhng h tr, s khuyn khớch v ng viờn tụi thi gian thc hin lun ny Tụi mun cm n c bit ti PGS.TS Tụ Kim Anh, Vin CN sinh hc v CN Thc phm, ngi ó hng n v cho tụi li khuyờn, kin thc v nghiờn cu ca tụi thi gian thc hin lun ny Tụi xin cm n cỏc thy cụ Vin Cụng ngh Sinh hc v Thc phm, Trng i hc Bỏch khoa H Ni vỡ nhng kinh nghim v kin thc m cỏc thy cho em quỏ trỡnh hc v lm nghiờn cu Xin chõn thnh cm n Ths Phựng Th Thy, KS Lờ Tuõn, KS Nguyn Th Huyn, ó to iu kin thun li tụi hon thnh lun Cui cựng, tụi xin cm n Vin o to sau i hc Trng i hc Bỏch Khoa - H Ni vỡ ó to iu kin cho tụi hon thnh cỏc th trong quỏ trỡnh hc v bo v lun H Ni, ngy 20 thỏng 05 nm 2011 Tỏc gi Lờ Duy Khng Le Duy Khuong Vien Cong nghe Sinh hoc & Cong nghe Thuc pham Luan Van Tot Nghiep LI CAM OAN Tụi xin cam oan tt c cỏc s liu nghiờn cu ca ti l hon ton trung thc Cỏc thớ nghim c tin hnh mt cỏch nghiờm tỳc quỏ trỡnh nghiờn cu, khụng cú s chộp t t k ti liu khoa hc no Tỏc gi Lờ Duy Khng Le Duy Khuong Vien Cong nghe Sinh hoc & Cong nghe Thuc pham Luan Van Tot Nghiep DANH MC CC CH VIT TT - ABTS: 2,2' -azinobis-3-ethylbenzthiazoline-6-sulfonate - AFEX- Ammonia filber explosive - CBU: Celobiase unit - CFU: Colony forming unit - CMC: Carboxyl-methyl cellulose - DNS: Dinitro salicylic - DX: Design expert - FAO: Food and agriculture organization - FC: Folin ciocalteau - FPU: Filter paper unit - GJ: Gigajoule - HBT: 1-Hydroxybenzotriazole - HMF: Hydroxy methyl furfural - Lac: Laccase - LiP: Lignin peroxidase - MnP: Manganese peroxidase - OPEC: Organization of the Petroleum Exporting Countries - SHF: Separate hydrolysis and fermentation (thy phõn v lờn men riờng r ) - SSF: Simultaneous saccharification and fermentation (thy phõn v lờn men ng thi) - VA: Veratryl alcohol Le Duy Khuong Vien Cong nghe Sinh hoc & Cong nghe Thuc pham Luan Van Tot Nghiep DANH MC CC BNG Bng 1: Thnh phn ca mt s loi nguyờn liu lignocellulose 13 Bng 3.1 : Cỏc yu t u vo v khong bin i 61 Bng 3.2 : Cỏc ch thc nghim v kt qu hiu qu thy phõn bó mớa thit k theo DX-7 61 Bng 3.3 : Kt qu phõn tớch phng sai c a mụ hỡnh 62 Bng 3.4: Cỏc ch v kt qu hiu qu thy phõn bó mớa theo DX-7 65 Bng 3.5 : So sỏnh hiu qu ca cỏc ch thy phõn bó mớa 70 Le Duy Khuong Vien Cong nghe Sinh hoc & Cong nghe Thuc pham Luan Van Tot Nghiep DANH MC CC HèNH V V TH Hỡnh 1.1: Mụ phng cu trỳc ca nguyờn liu bó mớa 14 Hỡnh 1.2: Cu trỳc ca lignocellulose 15 Hỡnh 1.3 Mch acetyl-4-O-methylglucuronoxylan .16 Hỡnh 1.4: Mch glucomannan 17 Hỡnh 1.5: Mch galactoglucomannan 17 Hỡnh 1.6: Mch arabinoglucuronoxylan .18 Hỡnh 1.7: Cu trỳc ca mng lignin 18 Hỡnh 1.8: Mụ phng cu trỳc lignocellulose trc v sau tin x lớ 19 Hỡnh 1.9: Quỏ trỡnh phõn c t mi liờn kt C-C 26 Hỡnh 1.10: C ch xỳc tỏc ca peroxidase 27 Hỡnh 1.11: C ch laccase tỏc ng n lignin 28 Hỡnh 1.12: C ch phõn hy lignin v cỏc dng bin i ca laccase .30 Hỡnh 1.13: C ch quỏ trỡnh thy phõn cellulose bi h enzym cellulase 33 Hỡnh 1.14: S c ch t bo nm men bi cỏc cht c ch sinh 35 Hỡnh 3.1 : Thnh phn bó mớa nghiờn c u 52 Hỡnh 3.2: nh hng ca nhit sy ti hiu qu thy phõn ó mớa 53 Hỡnh 3.3: Kt qu tin x lớ bng axớt H2 SO4 54 Hỡnh 3.4 : Kt qu tin x lớ ó mớa mụi trng kim 55 Hỡnh 3.5 : So sỏnh kt qu tin x lớ bó mớa 57 Hỡnh 3.6: ỏnh giỏ vai trũ ca laccase tin x lớ bó mớa 59 Hỡnh 3.7: Hiu qu thy phõn bó mớa theo thi gian húa nhit 60 Hỡnh 3.8: Kt qu b mt ỏp ng ca hiu sut thy phõn bó mớa 63 Hỡnh 3.9: Kt qu kho sỏt vai trũ laccase kh c dch tin x lớ bng laccase 69 Le Duy Khuong Vien Cong nghe Sinh hoc & Cong nghe Thuc pham Luan Van Tot Nghiep Kt qu hỡnh 3.9C cho thy nng laccase tng, hiu qu kh phenol tng, nhng mc loi phenol tt nht khong 20ữ80 (U/g bó mớa), nu tng nng laccase lờn cao hn, hiu qu x lớ tng chm Do ú, cỏc nghiờn cu tip theo laccase cú th c s dng nng 20ữ80 (IU/g bó mớa) chng minh tỏc dng loi cht c ch cho quỏ trỡnh lờn men, dch tin x lớ nhit-NaOH x lớ vi laccase (70 IU/g bó mớa, nhit 37 oC, lc 150 vũng/phỳt, thi gian 60 phỳt), c b sung h cellulase thy phõn bó mớa Dch sau thy phõn c b sung nm men S cerevisae t l 10 CFU/ml, cht inh ng v Nit Hiu sut thu hi ethanol c dựng ỏnh giỏ hiu qu x lớ cht chc ch (phenol) M u kim chng c tin hnh song song v khụng s dng laccase, chi tit c mụ t 2.3.6 Kt qu trờn hỡnh 3.9D cho thy, m u thớ nghim x lớ dch thy phõn vi laccase, cho hiu sut thu hi ethanol t 76,274,67 % so vi cellulose, so vi m u khụng kh c bng laccase lờn men rt yu hiu sut thu hi thp hn ỏng k (8,032,67%) iu ny khng nh, vai trũ laccase quỏ trỡnh thu nhn ch thy phõn ó mớa cú th lờn men c Bng 3.5 : So sỏnh hiu qu ca cỏc ch thy phõn bó mớa HQ thy phõn Ch Enzym (mg ng kh/g Ngun bó mớa) -NaOH 0,1g/g bó mớa, 121oC, 1h Celluclast 1.5L, -31,5/53,61/20,47 Novozym N188 (CMCase/FPU/CBU)/g bó mớa, 70U laccase/g bó mớa Celluclast-1.5L, - axớt 0,75% (w/v), 121 oC Novozym N188 - H3PO4 , Cellusoft-L, - 7FPU, 3,5CBU Novozym N188 Cellusoft-L, Novozym N188 - H2SO4 , 7FPU, 3,5CBU Le Duy Khuong 435 Lun ny 485 Badal v cng s [7] 404,5 Gúmez Sandra [17] 414,9 nt Vien Cong nghe Sinh hoc & Cong nghe Thuc pham 70 Luan Van Tot Nghiep - Ca(OH)2 0,1g/g bó mớa, Celluclast-1.5L, 121 oC, 30 phỳt Novozym N188 -35FPU, 61,5CBU Cellusoft-L, Novozym N188 Biocellulase ca Kerry Bioscience Novozym 188 ca Sigma Aldrich 415 Jiele v cng s 21] - 0,1g NaOH/ g tre 568 Ymashita v cng s [48], - H3PO4 1% ,1gi,145 oC - 50FPU/g bó mớa 424 Gỏmez v cng s [34], - H3PO4 1% ,1gi,145 oC - 0,5FPU/g bó mớa 198 nt Bng vic ỏp dng ch tin x lớ: ó mớa sy 45 oC, sung NaOH 0,1g/g bó mớa, t l rn lng S/L=1/10, nhit 121 oC, 60 phỳt Bó mớa sau ú c thy phõn bng h cellulase bao gm 31,5/53,61/20,47 (CMCase/FPU/CBU)/g bó mớa, thu c hiu qu thy phõn ó mớa cú th so sỏnh c vi cỏc nghiờn cu hin Nh vy, cú th kt lun ó xỏc lp c ch x lớ bó mớa cho mc tiờu sn xuõt bioethanol t bó mớa Le Duy Khuong Vien Cong nghe Sinh hoc & Cong nghe Thuc pham 71 Luan Van Tot Nghiep KT LUN Cỏc nghiờn cu cho phộp rỳt kt lun sau: ó kho sỏt cỏc iu kin tin x lớ la chn c ch tin x lớ bó mớa nh sau: + Bó mớa sy 45o C, b sung kim vi 0,1g NaOH/g bó mớa, quỏ trỡnh thc hin nhit 121 oC thi gian 60 phỳt, cho hiu qu thy phõn bó mớa cao hn so vi axớt, vụi + Phi hp laccase 70 IU/g bó mớa tin x lớ lm tng hiu qu thy phõn bó mớa lờn 284,154,6 mg ng kh/g bó mớa (tng 8,2% so vi khụng phi hp laccse), lm gim hm lng lignin ti 72% (tng 3,1% so vi khụng phi hp laccase) Bó mớa ó x lớ vi ch la chn cú th c thy phõn hiu qu vi 31,5/53,61/20,47 (CMCase/FPU/CBU)/g bó mớa, thi gian thy phõn 40,5 gi, t 435 mg ng kh/g bó mớa Laccase cú th s dng loi phenol kh c dch thy phõn, lm tng hiu sut thu hi ethanol ti 76,274,67% (tng 68% so vi khụng kh c) Le Duy Khuong Vien Cong nghe Sinh hoc & Cong nghe Thuc pham 72 Luan Van Tot Nghiep KIN NGH - Ti u húa k thut tin x lớ nguyờn liu nhm gim chi phớ, ng thi trung nghiờn cu sõu hn v cỏc k thut tin x lớ sinh hc v k thut kt hp tin x lớ sinh-lý, sinh-húa, sinh-lý-húa, nhm gim nng liu tiờu hao cng nh chi phớ sn xut - Cn cú thờm cỏc nghiờn cu v k thut quỏ trỡnh nh SSF, nhm to mt ch phi kt hp hng ti tng hiu sut thu hi ca ton quỏ trỡnh - Ch thy phõn hemicellulose, cỏc enzym tham gia thy phõn hemicellulose v to lp cỏc chng lờn men ng cac on tn dng nguyờn liu cng nh tng hiu qu ca c quỏ trỡnh x lớ bó mớa - Vỡ s phỏt trin bn vng tng lai, ngh cỏc nh lónh o, chớnh ph, cỏc t chc cú liờn quan cú s h tr, u t khuyn khớch vi cỏc nghiờn cu nhm to ioethanol núi riờng v nng lng n vng núi chung Le Duy Khuong Vien Cong nghe Sinh hoc & Cong nghe Thuc pham 73 Luan Van Tot Nghiep TI LIU THAM KHO [1] B mụn Cụng ngh cellulose v Giy, i hc Bỏch Khoa H Ni Cỏc bi thớ nghim húa hc g v cellulose, (2009) [2] ng Th Thu, Nguyn Th Xuõn Sõm, Tụ Kim Anh Thớ nghim húa sinh cụng nghip Trng i hc Bỏch Khoa H Ni, 1997 [3] Lờ Thanh Mai, Nguyn Th Hin, Phm Thu Thy, Nguyn Thanh Hng, Lờ Th Lan Chi Cỏc phng phỏp phõn tớch ngnh cụng ngh lờn men (2009) NXB Khoa hc v K thut [4] Phan ỡnh Tun, Nguyn Duy (2009), Thy phõn enzym bó mớa n hi nc v lờn men glucose cho sn xut ethanol i hc Bỏch khoa TP H Chớ Minh [5] A Rodriguez-Chong, J.A Ramirez, G Garrote et M Vazquez (2004), Hydrolysis of sugarcane bagasse using nitric acid: a kinetic assessment, J Food Eng, 61, pp 143152 [6] A Pandey, C.R Soccol, P Nigam and V.T Soccol (2000), Biotechnological potential of agro-industrial residues I Sugarcane bagasse, Bioresour Technol, 74(2), pp 6980 [7] Badal C Saha , Loren B Iten, Michael A Cotta, Y Victor Wu (2005) Dilute acid pretreatment, enzymatic saccharification and fermentation of wheat straw to ethanol Biochem Eng, 40, pp 36933700 [8] Ballerini, D, Desmarquest, J.P, Pourquie, J, Nativel, F, et Rebeller, M (1994), Ethanol production from lignocellulosics: Large scale experimentation and economics Bioresour Technol, 50, pp 17-23 [9] C Carrasco, H.M Baudela, J Sendeliusa, T Modiga, C Roslandera, M Galbea, B Hahn-Họgerdalb, G Zacchia and G Lidộn (2010), SO2catalyzed pretreatment and fermentation of enzymatique hydrolyzed sugarcane bagasse, Enzym Microb Technol, 46, pp 64-73 Le Duy Khuong Vien Cong nghe Sinh hoc & Cong nghe Thuc pham 74 Luan Van Tot Nghiep [10] Carmen, S (2009), Lignocellulosic residues: Biodegradation and bioconversion by fungi Biotechnology Advances, 27, pp 185-194 [11] Carlos, M., Mette, Moniruzzaman, M (2008), Lignocellulose biomass processing pretreatment, enzymatic hydrolysis and simultaneous saccharification and fermentation of cloverery grass mixtures Bioresource Technology, 99, pp 8777-8782 [12] Charles E.Wyman (1996), Handbook on Bioethanol: Product and Utilization,Taylor & Francis, pp 119-285 [13] C.H Kuo and C.K Lee (2009), Enhanced enzymatic hydrolysis of sugarcane bagasse by N-methylmorpholine-N-oxide pretreatment, Bioresour Technol, 100, pp 866871 [14] Dekker R F H (1991) Steam explosion: an effective pretreatment metho for use in the ioconversion of lignocelluloses materials In: Steam Explosion Techniques: Fun amentant Principles an In ustrial Application, (B Focher, A Marzetti & V Crescenzi, editors), Gordon and Breach Scientific Publisher, Philadelphia (USA), pp 277-305 [15] Ernesto J del Rosario, Ph.D (2008), Cellulosic Ethanol: Biofuel of the Future, Institute of Chemistry and National Institute of Molecular Biology & Biotechnology, University of the Philippines Los Baủos, Laguna [16] Food and Agriculture Organization of the United Nation (FAO), (2008) State of food insecurity in the world, Rome [17] Gúmez Sandra M R, Andrade Rafael R, Santander Carlos G, Costa Aline C, Maciel Filho Rubens (2009), Pretreatment of sugar cane bagasse with phosphoric and sulfuric diluted acid for fermentable sugars production by enzymatic hydrolysis [18] Hans E Schoemaker and Klaus Piontek (1996), On the interaction of lignin peroxidase with lignin, Pure & Appl Chem, 68(11), pp 2089-2096 Le Duy Khuong Vien Cong nghe Sinh hoc & Cong nghe Thuc pham 75 Luan Van Tot Nghiep [19] Himmel, M E., Ding, S Y., Johnson, D K., DNA, W C., Nimbus, MR., Brady, J W., Foust, T D (2007), Biomass recalcitrance : engineering plants and enzyms for biofuels production Science, 315 : 804-807 [20] Hofrichter, M., Vares., Maksim., Galkin, S., Scheibner, K., Fritche, W and Hatakka, A (1999), Production of mangannase peroxidase and organic acids and mineralization fermentation of of C14-labelled lignin (C14-DHP) during solid-state Wheat straw with the white-rot fungus Nematoloma frowardii, Appl env Microbiol 65, pp 1864-1870 [21] Jiele Xu, Jay J Cheng a, Ratna R Sharma-Shivappa, Joseph C Burns (2010), Lime pretreatment of switchgrass at mild temperatures for ethanol production Bioresource Technology 101, pp 29002903 [22] Jurado, M., Prieto, A., Martinez, A.T., Martinez, M.J (2009), Laccase detoxification of steam-exploded wheat straw for second generation bioethanol Bioresource Technology, 100, pp 6378-6384 [23] J.M Hernỏndez-Salas, M.S Villa-Ramớrez, J.S Veloz-Rendún, K.N Rivera-Hernỏndez, R.A Gonzỏlez-Cộsar, M.A Plascencia-Espinosa et S.R TrejoEstrada (2009), Comparative hydrolysis and fermentation of sugarcane and agave bagasse Bioresour Technol 100, pp 12381245 [24] Kim, S., Holtzpple, M T (2006), Effect of structural feature and enzym digestibility of cellulose Bioresource Technol 95, pp 583-591 [25] K.K Cheng, B.Y Cai, J.A Zhang, H.Z Ling, Y.J Zhou, J.P Ge and J.M Xu (2008), Sugarcane bagasse hemicellulose hydrolysate for ethanol production by acid recovery process, Biochem Eng J 38, pp 105109 [26] Larissa Canilha &Walter Carvalho & Maria Das Graỗas De Almeida Felipe & Joaxo Batista De Almeida E Silva & Marco Giulietti (2009), Ethanol Production from Sugarcane Bagasse Hydrolysate Using Pichia stipitis, Applied Biochemmistry and Biotechnology Le Duy Khuong Vien Cong nghe Sinh hoc & Cong nghe Thuc pham 76 Luan Van Tot Nghiep [27] Martin, C., Klinke, H.B., Thomsen, A.B (2007), Wet oxidation as a pretreatment method for enhancing the enzymatic convertibility of sugarcane bagasse, Enzym Microb Technol 40, pp 426432 [28] Marie- Paule Le Traon- Masson and Patrice Pellerin (1998), Purification an Characterization of Two -d-Glucosidases from an Aspergillus Niger Enzym Preparation: Affinity and Specificity Toward Glucosylated Compounds Characteristic of the Processing of Fruits Enzym and Microbial Technology, 22, pp 374-382 [29] Majcherczyk, A.; Johannes, C.; Huăttermann, A (1999), Oxidation of aromatic alcohols by laccase from Trametes versicolor mediated by the 2,2Â-azinobis-(3-ethylbenzothiazoline-6-sulphonic acid) cation radical and decation Appl Microbiol Biotechnol, 51, 267-276 [30] P Ander, K E Erikson and H S Yu (1976), The importance of phenol oxidase activity in lignin degradation by white rot fungus Sporotrichum pulverulentum, Arch Microbiol 109(1), pp.1-11 [31] P Rezayati-Charani, J Mohammadi-Rovshandeh, S.J Hashemi and S Kazemi-Najafi, Influence of dimethyl formamide pulping of bagasse on pulp properties, Bioresour Technol 97 (2006), pp 24352442 [32] Rachi al , Bour onnais, Jurasek, Paice, M.g & Rei (1997), Kraft pulp leaching an elignification y Trametes versicolor, J Biotechnol, 53 , pp 215- 236 [33] Saha BC, Iten LB, Cotta Ma, Wu YV (2008), Diluted acid pretreatment, enzymatic saccharification and fermentation of wheat straw to ethanol Biochem Eng, 40, pp 3693 [34] S Gỏmez, J.J Gonzỏlez-Cabriales, J.A Ramớrez, G Garrote et M Vỏzquez (2006), Study of the hydrolysis of sugar cane bagasse using phosphoric axớt, J Food Eng 74, pp 7888 Le Duy Khuong Vien Cong nghe Sinh hoc & Cong nghe Thuc pham 77 Luan Van Tot Nghiep [35] S Gỏmez, J.A Ramớrez, G Garrote et M Vỏzquez (2004), Manufacture of fermentable sugar solutions from sugar cane bagasse hydrolyzed with phosphoric acid at atmospheric pressure, J Agric Food Chem 52, pp 41724177 [36] Shahab Sokhansanj, Sudhagar Mani, Xiaotao Bi, Parisa Zaini, Lope Tabil (2005), Binderless Pelletization of Biomass, American Society of Agricultural and Biological Engineers, St Joseph, Michigan [37] Slinkard, K; Singleton, VL (1977), Total Phenol Analysis: Automation and Comparison with Manual Methods AJEV, 28: pp 49-55 [38] S Pattra, S Sangyoka, M Boonmee et A Reungsang (2008), Biohydrogen production from the fermentation of sugarcane bagasse hydrolysate by Clostritium butyricum, Int J Hydrogen Energy 33, pp 52565265 [39] Sun,Y et Cheng, J (2002), Hydrolysis of lignocellulosic materials for ethanol production: a review Bioresour Technol, 83, pp 1-11 [40] Sun F, Cheng (2008), Comparison of atmospheric aqueous glycerol and stream explosion pretreatment of wheat straw for enhanced enzymatic hydrolysis, chemical technology and biotechnology, 83, pp 707-714 [41] Taiz, L., Zeiger, E (2006), Plant physiology, Sinauer Associates, Inc [42] T K Ghose (1987), Measurement of cellulose activities Pure and Appl 59(2) : pp 257-268 [43] United States Department of Agriculture (2007), The Future of Biofuels: A Global Perspective, Economic Research Service [44] Vincent Chang et al (1998), Lime pretreatment of crop residues bagasse and wheat straw, Texas Univerzity [45] Watanabe, T (2007), Analysis of native bonds between lignin and carbohydrate by specific chemical reaction, in: Koshijima, T., Watanabe, T (Eds.), Asociation between lignin and carbohydrates in wood and other plant issues, Springer-Verlag, Heidelberg, pp 91-131 Le Duy Khuong Vien Cong nghe Sinh hoc & Cong nghe Thuc pham 78 Luan Van Tot Nghiep [46] Sloneker, J.H (1971), Determination of cellulose and apparent hemicellulose in plant tissue by gas-liquid chromatography Anal Biochern 43, pp 539 [47] Xu Q, Singh A, Himmel ME (2009), Perspectives and new direction for the production of bioetahonol using consolidated bioprocessing of lignoce llulose Curr Opin Bioethanol 20(3), pp 364-71 [48] Yamashita YShono M, Sasaki C, Nakamura Y (2010), Alkaline peroxide pretreatment for efficient enzymatic saccharification of bamboo Carbohydr Polym, 79, pp 91420 Le Duy Khuong Vien Cong nghe Sinh hoc & Cong nghe Thuc pham 79 Luan Van Tot Nghiep PH LC DUNG DCH THUC TH DNS - Nc ct : 1416 ml - 3,5 axớt dinitro salycilic : 10,6g - NaOH : 19,8g Hũa tan hon ton sau ú thờm vo ung ch : - Mui K, Na tartrate: 306g - Phenol : 7,6ml - Natri metabisulfite: 8,3g DUNG DCH RODZEVICH - Fehling (I): 34,64g CuSO4.5H2O/500ml H2O - Fehling (II): 173g Kali, Na tartrate + 50g NaOH/500ml H2O XY DNG NG CHUN GLUCOSE Chun b dung dch gc glucose 2g/l: cõn 200mg glucose v nh mc n 100ml Bng 1: Xõy dng ng chun glucose G H2O 0,8 0,6 0,4 0,2 Glucose 0,2 0,4 0,6 0,8 C(mg/ml) 0,4 0,8 1,2 1,6 0,007 0,216 0,462 0,695 0,910 1,132 OD540nm Ly ml dung dch vo ng nghim, thờm ml thuc th DNS, lc u un sụi phỳt, lm lnh bng nc thng nh mc dung dch lờn 10 ml, lc u o mt quang c súng 540nm Le Duy Khuong Vien Cong nghe Sinh hoc & Cong nghe Thuc pham 80 Luan Van Tot Nghiep Hỡnh 1: ng chun glucose PHA M NATRI CITRATE PH 4,8 Cõn 21,6 g axớt citric hũa tan lớt nc ct, iu chnh pH ng ung ch NaOH 60% ti pH 4,8 MễI TRNG LấN MEN - Mụi trng Hansen: + Glucose 50g + Peptone 10g + KH2 PO4 3H20 3g + MgSO4.7H2O 3g + Agar 10ữ15g Hũa tan 1lớt nc ct CHNG CT ETHANOL Lp thit b chng ct * Thit b: - B ct cn n gin - Bp in hoc bp gas * Trỡnh t thớ nghim: - Lp b ct cn nh hỡnh - Ly 150 ml dch lờn men vo bỡnh ct, thờm khong 100ml nc ct - M nc lm mỏt vo sinh hn Le Duy Khuong Vien Cong nghe Sinh hoc & Cong nghe Thuc pham 81 Luan Van Tot Nghiep - Bt bp in, quỏ trỡnh chng ct bt u Kt thỳc chng ct mc dung dch ỡnh thu t xp x 80 90 ml - nh mc bng nc ct n 100ml Hỡnh 2: B ct cn n gin O CN BNG MY O IM SễI [4] Thit b: Mỏy o im sụi * Trỡnh t thớ nghim: - Lp nhit k, bt vũi nc lm mỏt - Trỏng h thng bng nc ct ln - Trỏng li vi dung dch o - Rút dung dch cn o vo ỡnh n vch nh mc, bt in, c giỏ tr nhit sụi ca dung dch trờn nhit k * Kt qu Da vo nhit sụi ca dung dch v nhit sụi ca nc ct v thang im sụi tớnh toỏn nng ethanol dung dch Xoay thang im sụi cho vch 0% (v/v) trựng vi giỏ tr nhit sụi ca nc ct c giỏ tr nng cn tng ng vi nhit sụi ca dung dch Le Duy Khuong Vien Cong nghe Sinh hoc & Cong nghe Thuc pham 82 Luan Van Tot Nghiep XC NH HM LNG PHENOL TNG S TRONG DUNG DCH BNG PHNG PHP FOLIN CIOCALTEAU * Chun b: - Thuc th Folin ciocalteau 2N (FC) (Sigma; Singleton & Rossi, 1965): pha loóng 10 ml dung dch gc FC n 50ml c thuc th folin 0,4N, bo qun iu kin 4C v trỏnh ỏnh sỏng - Dung dch natri cacbonat: hũa tan 200g natri cacbonat khan vo 800ml nc ct, un sụi Sau lm ngui n nhit phũng, thờm vo mt vi tinh th Na2CO3 v yờn 24 gi Lc dung dch vi giy lc Whatman no1 v nh mc bng nc ct n lớt Bo qun vụ thi hn nhit phũng - Dung dch chun acid gallic: hũa tan hon ton 0,05 g acid gallic 1ml ethanol sau ú nh mc lờn 10ml bng nc ct ta c dung dch gc acid gallic mg/ml (GA) T dung dch chun GA mg/ml, pha loóng n cỏc nng khỏc theo bng sau : Bng 2: Xõy dng ng chun aclid gallic STT H2 O (l) 1000 980 950 900 850 GA (l) 20 50 100 150 Vtotal (ml) 1000 1000 1000 1000 1000 C (mg/ml) 0,1 0,25 0,5 0,75 Dung dch chun GA bo qun iu kin 4C v s dng vũng tun k t ngy pha Le Duy Khuong Vien Cong nghe Sinh hoc & Cong nghe Thuc pham 83 Luan Van Tot Nghiep Bng 3: Hiu chnh hm lng phenol theo nng ng m u Hm lng phenol Hm lng ng m u (mg GAE / ml) 2,5% 5% 10% 100 10 20 200 10 15 40 500 20 30 50 1000 30 60 100 2000 60 120 200 * Giỏ tr thc tớnh c tr i giỏ tr tng ng tra bng Le Duy Khuong Vien Cong nghe Sinh hoc & Cong nghe Thuc pham 84 ... góp phần vào trình nghiên cứu nâng cao hiệu xử lí lignocellulose chung, đề tài chọn Nghiên cứu chế độ xử lí bã mía cho mục tiêu lên men bioethanol Nội dung vấn đề nghiên cứu bao gồm: o Khảo... điều kiện tiền xử lí lựa chọn chế độ tiền xử lí bã mía sau: + Bã mía sấy 45oC, bổ sung kiềm với 0,1g NaOH/g bã mía, trình thực nhiệt độ 121oC thời gian 60 phút, cho hiệu thủy phân bã mía cao so với... tiền xử lí bã mía o Khảo sát khả áp ụng laccase kết hợp với tiền xử lí hóa -lý o Khảo sát khả khử độc dịch tiền xử lí enzym laccase o Lựa chọn tỷ lệ enzym cellulase thủy phân bã mía o Đƣa chế độ xử