Tap chi Cdng ngh^ Sinh hgi IU(2) 371 377 2012 N G H I E N C O u A P DI ING P H U O N * ; P H A P C H U M N V | E S T E R TAI ( M O DP SAN X U A T D I E S E L S I N H H O C T i l VI T A O UIEN A t WOCl[.]
Tap chi Cdng ngh^ Sinh hgi IU(2): 371 -377 2012 N G H I E N C O u A P D I I N G P H U O N * ; P H A P C H U M N V | E S T E R T A I ( M O DP SAN X U A T D I E S E L S I N H H O C T i l VI T A O U I E N A t.WOCllLOROPSIS OCULAT ( Dinh Thj Nggc Mai, ftinh Dtic Hos^nfi, I.e Thj 1110111, Itiii Dinh Lii I Nguyen Ciim Hii, Danj; Diem llfiiig Vien Cong nghe.wih hgc Vien Khoa lioi yd Cdng nglu,- Vicl Nam TOM TAT Van de bao x^- moi truong \a su eiin kiei ngudn nlueii li Cd phan chan nudi C P Vii;t Nam cd ham lugng niirogcn >IS.4''(, phosphor > 2.1% va chat mang dang long vira dii lit Nuoc bien nhan t^o dugc pha tu nude 6t 30%o va nuac mudi 30%o \di t\ le 1:1 (nude dt va mu6i bien dugc lay tir MJng lam muoi tai Hai Hau tinh Nam Dinh) tiio khd vdi 10 ml hon hgp dung mdi chloroform: methanol (2:1), B.l sinh khoi dugc chiet tiip \in chloroform - lim de thu toi da lipid chura sinh khoi tt)o, Djch chici dugc irgn deu vdi nhau, Igc qua gifiy Igc Whatman No.I, Ud sung them 15 ml dung djch NaCl 0,9",,, trgn deu v^ de yen d nhiet dg phong qua ddiii, Lt!rp dung mdi hihi ca phia dudi chiia cdc ihfinh ph^n lipid dugc thu n h ^ Sau do, dung mdi dugc logi bd hoAn loAn iron^ b i on nhi?t d 60"C \k lAin khd desiccator Tiep Hic hda tan sAn ph^rn thu dugc irong n-hexan Igc qua giay Igc dc logi bd c(iii va l^m bay hai hexan de thu hgi lipid I'hiro'nK phiip nhu^m lipid hfing Nile Red (Doan daL.2010) Nhugm te b^o tao bang Nile Red (9(Diethylainino)-5II benzo [u] phenoxazin-5-one) nhilm myc dich phat h i ^ cac hgi lipid ti bao Iao ml djch nudi tao cd mgt liii 40x10* le bao/ml dugc bd sung them glycerol tinh khict de dgt ndng dg cudi cimg ciia glycerol la 0.1 g/ml Sau S pi dung djch Nile Red ndng 0.25 mg/ml aceton dugc bd sung vao djch tao Hdn hgp dugc \ortex nh? nhang va ii toi trtmg 10 phiit d nhi?t dg phdng Cac te bao tao da dugc nhugm Nile Red dugc quan sal ducri kinh hien vi huynh quang (NIKON eclipse 80j-Nhal Ban) vdi knh sang kich thich co budc song 450-490 nm va anh sang phat huynh quang ciia Nile Red co budc sdng 540-640 nm Thu hoach sinh khoi tao Chu>en hda diesel sinh hgc tir sinh khoi tao (.tohnson lien 2009) Khi tao dang sinh trudng d giira pha logarit khoang 30" o the tich djch nudi tao dugc liy khdi binh nudi va smh khdi tao dugc thu hoach bing each sir dung chat tao bdng sat(IM) clorua (FeCli) nong dg 0,21 g/l (ket qua chi tiel ve ndng dg tdi uu ciia FeCl] len hi^u qua tgo bdng sinh khdi tao nAy khdng dugc chi o day) Sau I gio de lang, djch phia tren dugc loai bd bang xy phdng smh khdi tao lang phia dudi dugc thu nh^n bang each ly tSm d 4000 v/p_ h-ong 10 phiit nia nuoc cat \kn Sau dd, sinh khdi tao dugc say khd d 80''C va dugc bao quan desiccator neu sir dyng cho chuyin hoa diesel sinh hgc hoac bao quan d -20°C cho su dung lau dai (tir 1-2 nkm) Hem hgp phan ling gdm 15 gam sinh khdi khoM octdata, 51 ml methanol ml acid sulfuric dgm dSc va 60 ml chloroform Hdn hgp dugc tri dnhi?t dg V gid va khuay trgn lien tgc Sau phan img neu tren hdn hgp dugc lam ngugi den nhiet dp phoig, Sau do, bd sung them 60 ml nude cat va trgn deu hdn hgp trcHig 45 giay Hdn hgp se phan Idp Idp dung mdi hihi co chira metyl ester ciia acid beo (FAME) dugc thu nhgn Dung mdi dugc loai bo hoan toin bang may co quay chan khdng Sau do, san phSm tiip tgc dugc lam sgch bang hexan, loai bo hexan de thu dugc diesel sinh hgc T^ch chiet lipid PhSn (ich thdnh phiin yk ham lirgng c i c acid h^o Tach chiet lipid tir sinh khdi tao dugc tien hanh theo phuoTig phap Bligh va Dyer (1959) vdi mgt sd cai tien de phii hgp vdi dieu ki?n phdng Ihi nghiem cua Viet Nam, Lipid tong so dugc tach chiet tir bgt Thanh phan va ham lugng acid beo dugc xac djnh bang phucmg phap sac ky tai Vi?n Hda hgc cac hgp chat thien nhien theo tieu chuan ISO/FDIS 5590:1998, Lien Bang Diic theo phuong phdp dS m6 Tgp chi Cdng nghi- Sinh hoc 10(2): 371 -377, 2012 ta cdng bd ciia Dang Dilm Hdng va ddng liic gia (2007) Xac dinh cac dfic linh ciia diesel sinh hQC Cac dac tinh ciia diesel sinh hgc thu dugc trgng_ lugng rieng d I5"c diim chdp chay coc chi sd lot, dg nhdl dgng hgc, Irj so celane dugc dinh thdng qua cac phuong trinh ly thuyil Hoekman va ddng tac gia (2011) xay dgng nhu kin xiic KET QUA Phat hi^n nhanh lipid tront> (c han \i\ \ac djnh ham lugiig lipid sinh khoi tiio Bang each nhugm te bao tao vdi Nile Red va soi mau dudi kinh hien \ i huynh quang anh sang kich thich cd budc sdng 450-490 nm ciie hat lipid ti bao n tao cd the dugc quan s;il kha nang tham nhgp cua Nile Red qua mang te bao va gin kit vdi cac hai lipid trung tinh ngi bao phai anh sang huynh quang mau vang Ket qua nhudni Nile Red cua ,\ oculala dugc cht tren hinh 1B da cho thSy, hiu hel cac te bao tao deu cd cac \ung nhd mau vang d ben trong, dieu chting td su co mat ciia cac hat lipid te bao vi tao Ddng thdi bang each tach chiit lipid tir sinh khdi tao theo phuang phap Bligh va Dyer (1959) cai bien nhu da neu phan phucmg phap nghien cuu chung tdi cung xac dmh dugc ham lugng lipid tdng sd sinh khdi tao A' oculala la 6,7% so vdi trgng lugng khd Nhu vay, so vdi ket qua ve ham lugng dau ciia cac loai vi tao khac Chisti cdng bd (2007), ham lugng lipid tdng sd ciia loai N oculala ciia chimg tdi cd gia trj thap hon Nhieu cdng bd da cho thay ham lugng lipid tdng sd ciia tao khdng chi phu thugc vao loai vi tao ma cdn phy thugc rat nhieu vao cac dieu kien nudi cay (Ehimen et 2010) Do dd, viec nang cao ham lugng lipid sinh khdi tag bang each thay ddi cac dieu kien nudi cay nhu moi truong dinh duong, cuong anh sang (Qiang et al 2008)., la diiu cin thiet frong thdi gian tdi de nang cao tiem nang ung dung lam nguyen lieu san xuat diesel sinh hgc ciia loai Vl tao bien Nhu vay, phuong phap nhugm lipid bang Nile Red va tach chiet lipid bang dung moi deu la hai phuong phap hi?u qua d i danh gia so bg nhung chiing vi tao bien A', oculala tiem nang cd ham lugng lipid cao lam nguyen lieu cho san xuat diesel sinh hpc Trong nhu^m Nile Red la k^ thugt sang Igc rSt nhanh va cd hi?u qua cao thi tdch chiet lipid bang dung mdi giiip djnh lugng chinh xac hiim lugng lipid sinh khdi tao de tii' dd cd nhirng bi^n phap h(,Tp ly nham dicu khien va nang cao kha nang tich luy lipid ciia loai vi tao bien nay, Tinh chiit cua diesel sinh hc stin xuat tii' vi (ao bien Nannochloropsis oculala Kel qua ehuyen hda diesel sinh hgc tir vi tao N ocidata_ bang phuong phap chuyin vi ester lai ehi) cho thay, hipu suat ciia qua trinh chuyin hda dgt dugc la 89"t (in press) Dili' 111 1007/s lOSI I-tllD-'H.UN-'; I lumen \ Sun /1- Carrmgton C( CdlllJ VaiiJibles alVciung the in situ Inmscsterillcation ol'microaltiae lipids (iouveia L, Oli\eiij AC (20()'») Mieioalgae as a raw material for biofuel.s production J Ind Murihiot Biotechnol ib 2M-27A Haas MJ Scott KM Foglia I A M.irmer U N (2(107) The general applicability of in situ transesienllcation for the production of laity aeid esien; Irom a \anety offeedsioLks J Am Oil Chem Soe HA 963-970 Hoekman K Broeh A Robbins C Cenieero I (2011) ln\esiigation of biodiesel chemistry, carbon footprint and regional lliel quality Coordinating Research Council ReponNo AVFL-t7a Dang Diem Hdng, Hoang Minh Hien, Nguyen Dinh Hung Hoang S5 \ j m Hoang Lan Anh Ngo Hoiii Thu, Dinh Khanh Chi |2O07) Nghien eim \e qua tnnh smh long hgp DHA til cac loiu \i tao bien di dudng mdi Labyiintbula Schizoclninum \a img dung Tap chi Kiioa hpc vd Cdng l^ng X Dalai AK, Uakh.shi NN, Reaney MJ Hertz PB (2001) Preparation and ehiiraeien/Liiion of hio-dtesels from various bio-oils Biore.soiir Technot S- $3-l>2 Lardon I Ileshas A, Sialve H, Sleycr JP and Bernard (2009) [,ile-tyi.le assessment of biodiesel production from mieioalgae invironSei Te(hnolAM\l) (>47.';-64SI, I (^ Ilorsman M, Wu N Lan CQ Dubois-Calero \ (20IIS) Hiofuels I'rom mieroalgae Biotechnol Prog 24 Xl'i-S20 Miao X, Wu Q (2(MKi) Biodiesel production from helcrolrophiL mieroalgal ral Bioresour 7tf/HJH/97:84l-X46 (.)i,iii J, V\,ing F, Liu S, Yun Z (20(m) In situ alkaline transeMeriJkalion ol cottonseed oil lor production of biodiesel and nontoxic eoitonsecd meal Bioresonr Technot 99'91)09.901 Qiang H Soinmerfeld M Jams E, dhirjdi \l Posewitz M Siebcn M Darzins A (200>-) Microaleal in.iLylgKeerols as feedstock for biofuel produaion perspectives and advances P/6r«/7 54 621-639 SLhenk I'M iTiomas-Hall SR Stephens E, Marx UC Mussgnug JH, Posicn C Kruse O Hankamer B (2008) Second generation biofuels: high-efficiency mieroalgae for bitxliesel production Bioenerg res 1:20-43, ShmPJ,Guna«anS, llsieh WH, Kasim \ S , Ju YH(2010) Biodiesel production from rice bran by a two-step in situ proctiii Bioresoiir Teehiiol 101 9S4-999 T C \ S 7717 (2007) Nhiin lieu diesel sinh hoc goc (BlOO) 'I'i-u cau ky thuat STLDY ON IN S I T l TRANSESTERIFICATION METHOD FOR BIODIESEL P R O D U C T I O N F R O M T H E M A R I N E M I C R O A U \ \.4.\.\OCHLOROPSIS OCL L.4TA Dinh Thi Ngoc Mai, Dinh Due Hoang, Lc Thi Thom Bui Dinh Lam Nguyen C a m Ha Dang Diem Hong" Inslilule if Biotechnology Vielnam h ademy ol Si iciu c and Technotogi' In the world energy crisis context, finding sufficient supplies of cheap and clean energy for the fijUire is one of society's biggest challenges and is related to global stability, economic prosperity and quality of human's life Environmental protection and depletion of fossil fuel supplies are becoming the overriding challenges faced lo oil industries Recently, biodiesel has attracted considerable attention as a renewable, biodegradable, nontoxic and environmenially friendly fuel lo\\e\cr the lack of oil feedstocks limits the large- ' Author for c respondence: Tel: +84-4-379! 1059; E-mail: ddhong60vn(d).vahoo.com 376 Tgp chi Cong nghe Sinh hgc 10(2) 37\-377,2012 scale development of biodiesel to a large extenl Mieroalgae have long been lecognized as a potenlial Icedstock due to their many advantages for biodiesel production such as their high oil conieni and rapid biomass production Oil yield per acre could be 100 times higher than oleaginous plants They iirc able lo giow extremely rapidly, generally doubling iheir biomass with in a day They can be Lulti\ iited in large open ponds or in closed photobioreactors located on non-arahle land, avoiding compelilion with conventional agriculture for land resources and consume less water than terrestrial crops Moreover, mieroalgae can absorb carbon dioxide as the carbon source for growlh and this eonlributes to both atmospheric CO: emission mitigation and high biomass production In this sud\, we present the results of research on biodiesel production from a marine microalga Nannochloropsis oculata through the technology of in situ transesterificalion, which lesulled in a biodiesel yield of 89% based on the algal oil On the other hand, the prediction values of deiisii\ at 15 0, viscosity al 0"C, flashpoint, iodine value, celane numciie value meet the BlOO biodiesel standard of Vielnam, The obtained results suggested that the in situ transesierificaiion technology \sas a feasible and elTeetive meihod for the production of high qualily biodiesel from microalga Keywords Biodiesel in situ li an.sestei iflcation lipid, marine microalga, Nannochloropsis oculata ... loai vi tao bien nay, Tinh chiit cua diesel sinh hc stin xuat tii'' vi (ao bien Nannochloropsis oculala Kel qua ehuyen hda diesel sinh hgc tir vi tao N ocidata_ bang phuong phap chuyin vi ester. .. vii lij so lelane m a ,siiii phiim diesel sinh hgi dugc siiii xual lir sinh klu''n vi Uio bien \ oeulala deu iiiun Irong mirc elm phep cua san pham diesel sinh hgc gdc HlOO then lieu chu.iii Viv-I... pham diesel sinh hgc san xuat tir vi Tap chi Cong nghe Sinh hoc 10(2), 371-377,2012 tao N oculata tinh toan dugc theo phuang trinh ly thuyet deu nam mirc cho phep ciia san phiim diesel sinh hgc