Tgp chi Khoa hgc Tnrdng Dgi hgc Cdn Tho Phdn A: Khoa hoc Tu nhien, Cong nghe vd Mdi U-udng- 26 (2013): 90-96
Tap Chl Khoa hoc Tri/cfng Dai hoc Can Thtf .., website: sj.ctu.edu.vn
TRICH LY VA PHAN TICH THANH P H A N CHAT BEO TRUNG TINH (NEUTRAL LIPIDS) TRONG BUN HOAT TTNH
Huynh Lien Huang' va Ho Qudc Phong^
' Bg mdn Cdng nghe Hda hgc, Khoa Cong nghe, Trudng Bgi hoc Cdn Tha
Thong tin chung:
Ngdy nhdn: 08/01/2013 Ngdy chdp nhdn. 19/06/2013
Title:
Isolation and analysis of Neutral lipids from activated sludge Tirkhda:
Biin hogt tinh, chdt beo trung tinh, trich ly
Keywords:
Activated sludge,, neutral lipids, biodiesel
ABSTRACT
? sludge with its high lipid content is a potential raw material for producing biodiesel Activated sludge, which also contains large amount of phospholipids originated from cell membranes of microorganism, recently has attracted considerable interests as a potential source of raw material for biodiesel production. In this study, oil was firstly extracted from dry sludge which was obtained from Taipei municipal waste water treatment plant, dev\'axed and degummed, and then titration and saponification were employed to separate saponifiable lipids from the unsaponifiables. Compositions of the neutral lipids obtained, as well as fatty acid profiles of the neutral lipids were investigated
TOM T A T
Biin hogt tfnh vdi hdm luang chdt beo tich trie cao Id ngudn nguyen lieu thd Hem ndng cho viic sdn xudt biodiesel. Ngodi ra, trong bim hogt tinh cSng cd chda mgt lugng ldn cdc phospholipids tir mdng ti bdo cua cdc vi sinh vgt Chinh vi vdy, viic su dungcdc ngudn chdt beo trich ly tit biin hoat tinh ldm ngudn nguyen lieu mdi cho sdn xudt biodiesel da vd dang thu hiit nhieu sy quan tdm cua cdc nhd khoa hgc trin thi gidi. Trong nghiin ciht ndy, ddu /chdt beo se dugc trich ly tit bim sau khi da sdy khd. Bitn hoat tinh sir dung cho nghiin cuu duac ldy tit nhd mdy xu ly nude thdi ddn dung a Taipei. Ddu sau khi trich ly se duac xir ly logi sdp vd gum, sau dd chudn do vd xd phdng hda di phdn tdch cdc thdnh phdn xd phdng hda vd cdc thdnh phdn khdng xd phdng hda. Thanh phdn ca bdn cua cdc chdt heo trung tinh cOng nhu thdng tin ve cdu triic mgch cacbon ciia chiing se duac khdo sdt.
1 G i m THIEU
Cung vdi sy phit fridn manh me cua khoa hgc v i ky thuat, nhan Ioai dang ddng frudc nguy e a cgn kiet ngudn nhidn lieu khoing sin.
Chinli vi thd, vide tim kiem cac ngudn nguyen lieu tiiay thd cd y nghia vd cijiig quan trgng.
Gin diy, cac ngudn ning lugng sinh hge cd
ngudn gdc td cic sinh khdi, die biet la biodiesel d i v i dang thu hut nhieu sy quan tim cua hiu hdt t i t ca cae nude frdn thd gidi bdi vi biodiesel cd the dugc su dyng tryc tidp hoic phdi frpn vdi pefro-diesel vdi b i t ky ti le nao m i khdng cin ddi hdi phii cd sy thay ddi frong ket ciu cua dgng ca. Ngoii ra, biodiesel cdn ed
Tap chi Khoa hoc Trudng Dgi hgc Cdn Tho Phdn A' Klioa hoc Ty nhien, Cdng ngh^vd Moi Irirdng: 26 (2013): 90-96
kha nang phan huy sinh hgc, khdng ddc hai va thin thidn vdi mdi trudng (Ma va Hana, 1999).
Biodiesel la hon hg este cua cac axit beo vdi rugu dan, thdng thirdng la methanol.
Biodiesel chu ydu duge tgo thanh tu qua frinh chuyen hda este (fransesterification) hay este hda (esterification) gida cic rugu dan (methanol) vdi dau thyc vit hay cac ma dgngvat. Tuy nhidn, viec su dyng cac ngudn nguyen lieu dat tien cd ngudn gdc thyc pham trdn la nguyen nhan chinh dan ddn gia thanh cda biodiesel cdn kha cao so vdi cae nhien lieu truydn thdng. Gia nguydn lieu cho sin xuat biodiesel tu cac diu ddng thyc vat cd the chidm ddn fren 80% tdng chi phi cua qua frinh san xuat (Ma va Hanna, 1999). Vi vay, dd gia ting tinh canh franh cho biodiesel va dd cd the nhan rdng, phat fridn sin xuit dgi tra biodiesel, vide tim kidm cac ngudn nguyen Iidu thay thd cd gia thanh thap la mdt frong nhung myc tieu quan trgng.
Bun hogt tmh la chit thii d dang rin hoac bin ran dugc tao thanh td cac qua frinh xu ly sinh hgc nude thii. NhiJng nghien cuu trudc diy cho thay bun hogt tinh eo khi nang tich IQy chat beo ldn ddn khoang 20% frgng lugng bdn (Dufireche et al., 2007) va lugng chit beo tren eo the dugc tan dyng de chuydn hda sang biodiesel.
Myc tidu ciJa nghien cuu frdn la trich ly va phin tich thanh phan cac chit beo trung tinh trong bun boat tfnh nham khao sat vi dinh gia kha nang sd dung biin hogt tinh lam ngudn nguydn lieu thay the cho cac ngudn nguyen lieu truyen thong trong sin xuit biodiesel.
2 PHU'OfNG PHAP NGHIEN CUtJ 2.1 Nguyen vit lieu va hda chat
Bun boat tinh da Ioai nude so bd dugc thu gom tu nha may xd ly nude thai dan dyng d thanh phd Taipei, Dii Loan. Cic raiu chuin biodiesel (methyl esters cda cac axit beo) dugc cung cip bdi Cdng ty hda chit Sigma Aldrich (St Louis, MO. USA). Dung mdi va hda chit su dung frong trich ly va phan tich thugc chuin HPLC hogc chuan phan tich theo qui dinh trong hda hgc.
2.2 Xirly mlu
Bun hoat tinh sau khi da dugc loai nude so bg, thdng thudng vin cdn chua tu 80-90%
lugng nude sd dugc tidp tyc phai khd dd logi bdt nude. Mau bim khd sau dd se dugc nghien min bing miy nghidn bi vi nung frong Id say d 105 "C, 24 h dd logi truTiiu hdt nude trudc khi tidn hanh frich ly diu.
2.3 Trich ly va lam tinh khiet diu Miu bun sau khi dugc nghien min va siy khd se dugc su dung dd frich Iy diu bing phuang phap soxhlet. Dung mdi su dung la hexane frong 24h d nhiet do sdi eua hexane.
Sau trich ly, dung moi sd dugc thu hdi bing thiet bi eo quay va tai su dyng. Diu thd sau khi tach logi dung mdi se dugc logi sap va gum trudc khi tien hanh phin tich bing phuong phap ket tua acetone. Dau bun tho sd dugc hoa tan trong acetone d dO^C eho den khi thu dugc dung dich frong sudt. Hdn hgp sau dd duge de nguSi ddn nhiet do phdng va lam lanh d 5 ''Ctrong vdng 24 h. Sau 24 h, hon hgp se dugc lgc lanh frong didu kien chin khdng &k phan tach phin dung dich va phan rin ket tua (chi!l ydu la sap va gum). Diu sau khi logi sip, gum va tach dung mdi se duge luu trft d -20°C.
2.4 Xac dinh thanh phin lipid trung tinh Dau bdn sau khi di Ioai sap va gum se dugc chuan do vdi dung dich KOH trong ethanol (IN) nhim loai cac axit beo ty do (FFA). Sau chuan do, cac axit beo d dang mudi vdi kali (FFA-K) se dugc phan tich bing phuang phap frieh ly Idng- Idng. Diethyl ether va nuac cit se Ian lugt cho vao phlu chidt. Phin diu biin khdng phan ung se hda tan frong diethyl ether va phin tach vdi phan mudi cua axit beo trong pha nude. Axit beo frong pha nude se duge thu hdi va chuydn hda sang dan xuit methyl este bing each cho phan ung lin lugt vdi axit sulfuric dam die va phan dng vdi dung dich boron trifluoride frong methanol (14-15%) d dCC.
Pha diu frong hexane sau phin tach khdi pha nude se dugc Ioai tru dung mdi vi xa phdng hda bing dung dich KOH 9 N trong 24 h d dO^C. Sau phin ung, thanh phin xa phdng
Tgp ehi Khoa hgc Trudng Dgi hgc Cdn Tho Phdn A: Khoa hgc Ty nhien. Cong nghi vd Moi trudng- 26 (2013): 90-96
hda vi khdng xa phdng hda ciing se dugc phin tich bing phuang phap frich ly long-long vdi diethyl ether va nude. Thanh phin xi phdng hda frong pha nude chii ydu la mudi cda cic glyceride frong diu bun vdi kali se dugc thu hdi va chuydn hda sang din xuit methyl este cfing bang each cho phin ung lin lugt vdi axit sulfuric dim dac va phin ung vdi dung djch boron trifluoride 14 - 15% trong methanol d dCC.
Thanh phin mach cacbon cau true ndn eae axit beo cung nhu cua cac glyceride trong mau diu se duge phan tich gian tidp thdng qua dan xuat methyl este tuong ung eua chung bing phuang phip sic ky.
2.5 Phan tich bang sac ky ban mdng va sac ky khi
Sicky bin mdng dugc sd dyng de dinh tinh cic thinh phin ca bin trong miu dau frieh ly tu bun. Pha dpng sd dyng eho bin mdng la hdn hgp dung mdi bao gdm hexane: ethyl acetate:
acetic acid vdi ti Id la 80:20: 1 (v / v / v). Cac thanh phin frong diu sau khi phin tach se dugc tiid hien fren bin mdng bing tac nhin hidn mau die trung. Ferric chloride dugc su dung trong trudng hgp nay.
Dau bun va cau true mach cacbon cua cac axit beo ty do va cie glyceride trong dau bdn se dugc xac dinh bang phuang phap sic ky khi.
Ddi vdi diu bdn, phan tieh sac ky dugc thyc hien fren may sic ky Shimadzu GC 2010 (sin xuit bdi Nhat Ban) vdi diu do FID; su dyng cgt sic ky DB - 5HT (5% phenyl - methylpolysiloxane (15 m x 0.32 mm i.d., Aligent, CA, USA). Didu kien nhiet cho qua frinh phan tieh la: nhidt do diu dd va dau tidm li 370°C. Nhiet do ban diu cda cgt dugc cai dit d 80°C, sau do gia nhiet len 1 SO^C vdi tdc do I5°C / phut, tidp tuc gia nhidt ddn 185''C vdi tdc do I^C / phut, den 215*^0 vdi tdc do I5°C / phut; 250''C vdi tdc do 2°C / phut;
325T vdi toe do 1 5 ^ / phut; 365°C vdi tdc do
50°C / phut va giu d nhidt dp tren frong 6,83 phut. Tdng thdi gian phan tich la 51 phut.
Dd xic dinh vdi ciu frue mach cacbon cua cac axit beo ty do va cae glyceride, phin tich sac ky duge thyc hidn tren may sac ky hidu China 8700F (san xuit bdi Dai Loan) vdi dau dd FID; sd dung cgt sic ky Rtx-2330 - 10%
cyanopropylphenyl 90% biscyanopropyl polysiloxane (30 m x 0.25 mm i.d., Supleco, Bellefonte, PA). Didu kidn nhiet cho qua trinh phin tich li: nhidt do dau dd va dau tiem la 250°C. Nhidt do ban dau cda cpt dugc cii dit d 150''C, giu frong vdng 2 phut sau dd dug"e gia nhidt ldn ddn 245°C vdi tdc do 5''C / phut va gid d 245''C frong vdng 10 phut.
3 KET QUA VA THAO LUAN 3.1 Sac ky ban mdng va sic ky khi cua dau
bun sau khi loai sap va gum Vide dinh tinh cac thinh phan ca ban trong mau diu bun da loai sap va gum dugc thye hidn thdng qua sic ky khi va sac ky bin mdng.
Sic ky ban mdng va sic ky khi cho phep nhan danh dugc radt sd thinh phan ca bin ma ta quan tim frong didu kidn phan tfch nhat dinh.
Kdt qua phan tich cho thay co sy hien didn cua rit nhidu thanh phin vi lugng ben cgnh cac chat beo trung tinh. Mdt so thinh phan die thu CO thd xac dinh dugc thdng qua mau sac die tnmg nhu cic phytosterols, cac axit beo ty do hay eae friglyceride tren bing mong. Tuy nhidn co thd nhan thiy ring vide phan tieh va dinh danh tung chi tidt tdng thanh phan ddi hdi nhidu thdi gian va khdng can tiiiet vi mue tidu chinh CLia nghien cdu li tip frung vao cic thanh phin chit beo trung tfnh co kha nang chuydn hda thanh biodiesel. Tu kdt qua phin tich cd thd nhan thay dugc eo sy hien didn eua cac chat beo trung tinh frong miu dau bun.
Day chfnh la ddi tugng mi ta quan tim va dd dinh gii duge tfnh khi thi frong vide su dyng cac chit beo fren thi vide phin tich dinh lugng vi ciu true mach cua cic thanh phan frdn la ein thidt. Hinh la va lb thd hidn ket qui sic ky khi (a) va sic ky ban mdng (b) cua diu bun trich ly sau khi da logi sap va gum.
Tgp chi Khoa hgc Trudng Dgi hgc Cdn Tho Phan A: Khoa hgc Ty nhiin, Cong nghi vo Mdi trudng: 26 (2013): 90-96
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Hinh I: Ket qui sac k^ khf (a) va sac 1^ ban mong (b) cua dau bun frich ly sau khi da lo^i sap va gum 3.2 Thanh phan eg ban cua cac lipid
trung tinh
Bang 1 frinh bay thanh phin ca ban cua chit beo frung tinh frong dau bun frfch ly bao gdm cae axit beo ty do, cac glyceride (monoglyceride, diglyceride, triglyceride) va cae thanh phan khdng bi xa phdng hoa. Kdt qui dinh lugng cho thiy frdn cie thanh phin beo trung tinh chidm frdn 65% tdng cac thanh phin frong diu bun trich Iy frong do 50% la cic axit beo ty do, 50% cdn Igi l i cic glyceride. Diy cung l i mdt trong nhiing didm khac biet co ban giua diu bun va cac diu thuc vat, thdng thudng d cae diu thyc vat: 90% diu la cac glyceride, phin cdn Igi Ii cic axft beo ty do v i cac thanh phan mang boat tinh khac. Su hidn didn cua chit beo frong bun hoat tfnh duge giai thieh la td su hip thy va tich luy cic chit beo cua vi sinh vit frong giai doan xu ly sinh hgc cua nude thai, tu cic thanh phin eiu tao nen mang td bao hoic la cac sin phira tao thinh td cac qua frinh sinh tdng hgp frong td bao vi sinh vgt (Chipasa v i Medrzycka, 2008;
Seltmann va Hoist. 2002). Ngoai ra, ham lugng cua eae axft beo trong diu chidm kha eao CO the dugc giai thfch dya fren thuydt
chuydn hda chat beo trong biJn sinh hge eua Duehoira et al. (2001) va Hvra et al. (1998):
axit beo ty do l i sin phim ciia q u i trinh thdy phan cac glyeerides bang cac enzyme ngoai bao chd ydu la lipase tidt ra td cac vi sinh vat va eae axft beo trdn cd k h i ning phin hdy kem han cac hgp chat huu co khac nhu dudng hoac cac amino axit do do chung cd khuynh hudng ti'ch luy tang dan frong sudt qua frmh xd ly nude thai.
Bang 1: Thanh phin chit beo frung finh frong dau bun
Thanh pha Axit beo tu do Glyceride (Thanh phin xa phong hoa) Thanh phin khdng xa phong hoa
Phan tram khoi Imrag ( " t % ) 36.38±1.31 30.37 ±,0.47
3.3 Cau triic m^ch cacbon ciia chat beo frung tinh trong dau biin
Kit qua sSc ky cau true mach cacbon cila cac thanh phSn trung tinh trong dSu bun bao g6m cac axit beo tir do va cac glyceride duoc minh hga trong Hinh 2 va 3 va the hien chi tiSt trong Bang 2. It nhat 21 loai mach carbon
Tgp chi Khoa hoc Trudng Dgi hoc Cdn Tha Phdn A: Khoa hgc Ty nhien, Cong nghi vd Moi tiudng: 26 (2013): 90-96
tirang dng ciu true ndn cic axit beo ty do va cac glyceride dugc xic dinh. Cie axit beo tu do va glyeerides frong dau bun cd mgch cacbon tu CIO cho ddn C20 vdi muc do bao hda khac nhau trong dd thinh phan chiem uu the la eic axit beo hay cae glyceride cd mach cacbon 16 C (palmitic C16:0 va pahnitoleic axit C16:l) vdi him Iugng tuang dng la 47%
vi 38% cho axft beo va glyceride. Tidp theo la cae thanh phin cd mach cacbon 18 C (stearic va cae ddng phin) vdi thinh phin tuang ung li 33% vi 32% cho axit beo va glyceride. Cae thanh phin co mach cacbon khae hidn didn chu ydu d dgng vi lugng,
Sy da dang trong mach cacbon cua cic chit beo trdn duge giai thich dya trdn ngudn gdc cua nude thai dugc xu ly. Ngudn nude thii sd dung d day cd ngudn gdc tur nude thai din dyng ndn la sy tap hgp cua nhidu ngudn chit thai cd thanh phan khae nhau. Su chidm uu the cda cac thinh phin cd mach cacbon CI6 va C18 dugc cho la xuit phat tu cac diu thyc vat hay cae md ddng vat frong cac thuc an thua, tu nude thii niu an hoac tu cic sin pham chuydn hoa frong co the (Quemeneur vi Marty, 1994).
Ben canh cac thanh phin chinh, sy hien dien 15 16 21
¥.
cda cac thanh phan vi lugng ttong dau bun la hoan loan hgp ly dya trdn thuydt da dang smh hgc trong he bun sinh hgc: "sy hidn didn cua cac chat beo trong bun vdi mach cacbon tu 12 ddn 20C tham chi ldn ddn 28C khdng phai la ngoai Id ma la can thidt cho hogt ddng sdng va sy tdn tai cua cae vi sinh vgt frong biin"
(Seltman vi Hoist, 2002). Ngoii ra, qua ket qua phin tfeh ta cung nhin thiy ring cac axft beo va cic glyceride frong diu bun cd mgch cacbon chu ydu la mach chin. Didu nay la do qua frinh phan hdy sinh hgc didn ra frong bun hogt ddng theo ca chd p- oxi hda frong dd mach cacbon se fai cit dut 2C frong mdi lin din ddn mach cacbon eua cac chit beo fren se ngin di 2C (Chipasa va Medrzycka, 2008).
Nhin chung, cic chit beo trung tinh frong diu bun chu yeu co mach cacbon C16 vi CIS bao hda hogc chua bao hda mdt ndi ddi. Vdi ciu tao tren, lipid frung tinh frong diu bun duge danh gia cho sin phim biodiesel cd do nhdt hgp Iy vi mdc do oxi hda thap. Day la mdt trong nhung uu diem eua diu bun khi duge lya chgn lam ngu6n nguydn lieu eho sin xuat biodiesel.
Ky^rU^^jA^-^^
5 -n 6 3 ntilB
Hinh 2: D6 thi sac ky ciu tao m^ch carbon cua cac axit beo tijr do
25
Tap chi Khoa hoc Tritang Bai hoc Cdn Thcr Phdn A. Khoahoc Tti nhien, Cdng nghe va Moi iruang: 26(2013): 90-96^
WXoiJ^
12
Hinh 3: D6 thi s^c k j cSu tao mach carbon ciia cac glyeerides trong dan biin dS loai sdp va gum Bang 2: Thanli phSn mach carbon eiu tao nen cae axit beo vS glyeerides trong dau biin sau 156 khi da
loai sdp va gum
Thanh phan Phan tram dien tieh (%)
Ajtit beo Glyceride
AxitCapric(C10:0) Axft Undecanoic (C11:0) Axit Laurie (C12:0) Axit Tridecanoic (C13:0) Axit Myristic (C14:0) Axit Myristoleic (C14:l) Axit Pentadecanoic (C15:0) Axit Palmitic (C16:0) Axit Palmitoleic (C16;l) Axit Heptadecanoic {C17:0) Axit Stearic (Cl 8;0)
Dong phan cua Axit Octadecenojc (C18:l) Axit Linoleie (C18:2n6c)
Axit Cis-11-eicosenoic (C20:l) Tiianh phan khac
n.d.
n.d.
0,82 ±0,00 0,09 ±0,03 1,94 ±0,02 1,00 ±0,04 1,05 ±0,08 27,12 ±0,00 19,83 ±0,07 0,85 ±0,01 2,95 ±0,07 26,80 ±1,12 3,08 ±0,10 0,17 ±0,00 14,3 ±0,09
0,22 ±0,01 0,13 ±0,00 0,80 ±0,02 0,21 ±0,00 1,86 ±0,11 0,87 ±0,02 1,45 ±0,09 21,79 ±0,30 16,86±0,16 1,13 ±0,01 3,80 ±0,11 28,27 ±0,89 n.d.
3,78 ±0,07 18,83 ± 1,09 n.d. : rdt nho. hdu nhtr Ichortg co
4 MET LUAN
Phan tich dau trich ly tir biin boat tinh bang hexane cho thay tren 66% kh6i lugng d^u thu dugc la chat beo trung tinh trong do 50% la cac axit beo tu do va 50% la cac glyceride.
Phan tich sac ky cho thay cac chat beo trung tinh CO cau true mach cacbon tir CIO din C20 trong do cac thanh phan co mach cacbon C16 va C18 la chu yeu; cac thanh ph§n khac chi hien dien duoi dang vi lugng. Ngoai ra, cac axit beo va cac glyceride trong dku bun da
phan a dang bao hoa hoac chua bao hoa co mpt n6i doi. Dira tren cau triic mach cacbon va mirc do chua bao hoa cua cac chat beo trung ti'nh trong diu bun co the nhan xet rang cac din xuSt methyl este - biodiesel tir diu bOn cho dfi nhdt hgp li va co mirc do oxi hoa thap phit hgp voi tieu chuan quy dinh cho biodiesel.
Tir do CO thS kit luan rang dau bun la mOt trong nhimg ngii6n nguyen lieu tidm nang thay thS cho cac nguon nguyen lieu truySn thfing tit d4u/m8 dgng thuc vJt cho qua trinh san xuat biodiesel.
Tap chi Khoa hgc Tnmig Dai hgc Cdn Tha Phdn A: Khoa hoc Ttr nliien. Cong nglii vd Mdi It-irdng: 26 (2011): 90-96
TAI LffiU THAM KHAO
1 • Chipasa. K. B. and Mednycka, K. 2008.
Characterization of the fate of lipids in activated sludge. Journal of Environmental Science. 20: 536-542.
2. Dueholm, T. E., Andreasen. K. H. and Nielsen, P. H. 2001. Transfoimation of lipids in activated sludge. Water Science Technology.
43: 165-172.
3. Dutreche, S., Hernandez, R., French, T..
Sparks, D., Zappi, M. and Alley, E. 2007.
Extraction of lipids from municipal wastewater plant microorganisms for production of biodiesel. Joumai of the American Oil Chemists' Society. 84: 181-187.
4. Hvra, C.-S., Tseng, S.-K., Yuan, C.-Y., Kulik, Z. and Lettinga, G. 1998. Biosorption of long- chain fatty acids in UASB treatment process.
Water Research. 32: 1571-1579.
5. Ma, F. and Hanna, M. A. 1999. Biodiesel production: a review. Bioresource Technology.
70:1-15.
6. Quemeneur, M. and Marty, Y. 1994. Fatly acids and sterols in domestic wastewaters.
Water Research. 28: 1217-1226.
7. Seltmann, G. and Hoist, O., 2002. The bacteria cell wall, second ed. Springer-Verlag, Berlin.
283 pp.
