Nghien ci'fu & iBHIgBB
Nghien cuTu nuoi trong vi tao Chlorella vulgaris
lam nguyen lieu san xuat BMIesel
NGUYtN MINH TUAN, LE T H I BICH YEN, N G U Y I N PHUCTC HAI
N G U Y I N T H I THANH XUAN, DANG KIM HOANG, NGUYEN HOANG MINH, NGUYEN NGQC TUAN Khoa Hoa, TrUdng Dai hoc Bach Khoa, Dai hoc Da Ning
S
mh khoi VI tao dang la ddi tUpng nghiln cuU phd bien tren the gidi eho muc tieu d n xuat nhien lieu smh hoc, trong do lipid tirvi tao la ngudn nguyen lieu eho san xuat biodiesel. Vi vay, cac bien phip nham nang eao ning suat lipid dUde dac bilt quan tam hiln nay. Trong de tai nay, chimg tdi khao sat anh hUdng ciia d c dieu kiln nudi trdng trong phdng thi nghiem nhu toe dp sue khi CO2, ndng dd dinh dudng Nitd, cUdng dd anh sang den ning suat sinh khoi va ham lUPng lipid tren vi tao Chlorella Vulgaris. Day la mdt trong d c chimg vi tao tiem nang cho muc tilu ket hPp xii ly nUdc thai, tan dung CO2 t y khi thai va phii hpp vdi dieu kien khi hau Viet Nam. Ket qua ban dau cho thay, vile tang eUdng do chieu d n g va giam ndng dd Nitd cd t h i tang ham IUpng lipid nhung giim nang suat sinh khoi. Odng thdi, viec bo sung CO2 vao mdi trudng eho phep ting nang suat sinh khdi va lipid. Ben canh dd, viec thu' nghiem nudi trong Chlorella Vulgans trong nude thil ham biogas dude pha loang 1,25 lan cho thay, chiing nay ed k h i nang smh trUdng, eho dau va giam ding ke nong do NitP va Photpho trong nUde thai.1. DAT VAN DE
Hien nay, nguyin lieu san xuat biodiesel d Viet Nam la cie nguon sinh khoi (SK) truyen thdng (dau an phe t h i i , md d basa, dau hat jatropha) ed nhUde dilm thu hoach khd khan, ham IUpng dau thap va chiem nhieu diln tfch dat canh tac, dat rimg va d n nhieu nUde tudi [1]. O l khac phue cac nhUdc dilm dd, SK vi t i o da ra ddi va dUPc xem la ngudn SK day hda hen d l d n xuat biodiesel.
Vi tao la eac vi sinh vat dPn bao, sinh trUdng bang quang tU dudng nhd qui trinh quang hdp, hoac di duQng, hoac ca hai hinh thye [1]. So vdi cic ngudn SK truyen thdng, vi t i o ed nhyng uu diem noi bat nhU tdc dp sinh trUdng nhanh, nang suat thu SK va thu dau cao hPn d e loai thUe vat d dau khic; de nudi trdng, ft canh tranh vdi dat ndng nghiip v i khdng d n ngudn nudc sach; than thien vdi mdi trUdng. Trung binh d n xuat 1 kg SK tao thi tieu thy dupc 1,83 kg CO2 [2]. Ngoai ra, ed the tan dung CO2 tii khf t h i i edng nghiep cimg vdi nUde thai d l nudi trong vi tao [3], lam giam dang ke chi phi cho qua trinh; nguon SK vi tao ngoai mue dfch d n xuat biodiesel d n ed nhieu dng dung khic,... [3].
Nang suat thu SK va ham lUdng dau cua vi tao phu thude nhieu vao dieu kiln nudi trdng [2]. Trong nhdm vi tao lye, Chlorella Vulgans la loai ed tiem nang san xuat biodiesel vi tdc dp sinh trUdng cao, nang suift SK eao va de nudi tiong, dac biet cd till nudi trong mdi tnj-dng nUde thai [4].
HPn nu^, Chung co kha ning hap thy CO2 [5], ed ham
iUdng dau dao ddng tif 5-58% khdi lUdng [3], Do vay, vdi dinh hudng cua de tai l i tan dyng nguon khf thai CO; va nUdc thai tii' ham ii biogas de nudi trong vi tao sin xuat biodiesel, chiing tdi chon Chlorella Vulgans la chiing vi t i o d l nghiln euU anh hUdng ciia mot so dieu kien nudi trong den ning suat thu hdi dau, dong thdi dinh hudng nudi trong mdi trUdng nudc thai.
2. VAT LIEU VA P H U O N G PHAP 2 . 1 . Moi tru'dng nuoi trong
sydung mdi trUdng nudi t i o cd thanh phan nhUsau:
2.2. Nguon giong vi tao: Lay t y Vien Cdng nghe Sinh hoc, Ha Ndi, la mdt trong cae chimg vi tao dUpe nghien cull nhieu nhat cho muc tieu san xuat biodiesel [9].
Bang 1: Thanh phan moi tru'dng nudi trdng
NH4CI Caa2.2H20 MgS04.7H20 KH2PO4 NaHCOj Khoang vi li/ong
725 25 140 350 840 0,5 ml/1 2.3. Cac phu'tfng phap do dac
2.3.1. PhuUng phap do mat do quang OD (Optical Den- sity)
Muc dich dmh tinh ndng do SK trong mdi trUdng, du^
vao dd hap thu quang ciia cac sac td Chlorophyll trong te bao, Cie sac to niy hap thu ehii yeu 2 bUde sdng tUdng img la 420 nm va 665 nm [7].
2.3.2. PhWdng phap do nong do Chlorophylla DUa vao khi nang hap thu inh d n g eua d c loai sac td d nhijng budc sdng nhat dinh d l xac djnh ham lUPng ciia chimg cd trong mau. Mau dUpc ly tam 15,000 vdng/phiit d 40c trong 5 phiit de thu SK mau. Phan SK nay dUcfc bo sung 1ml acetone vao dng eppendorf, Sau do, dem ly tam 13.000 vdng/phiit d 4°C trong 5 phiit. Lay djch ndi v i do d bUdc d n g 664 nm, 647 nm. Ap dung cdng thye xac dmh ndng dp chlorophyll a:
Chlorophyll a (pg/ml) = 11,93.E664 - 1,93.E647 2.4. Phu'dng phap bao quan gidng
Trong sd d c phUdng phap da thy nghiem nhU bio quan tren dia thach, thach nghieng, giy gidng bang Glycerol, giff gidng bang ddng kho, k i t qua eho thay, thdi gian giy giong bing phUdng phip bao quan tren dia thach dai hPn, ddng thdi viec hoat hda gidng de dang hdn.
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Nghien ci'fu cK:iB^nia;ni
2 . 5 . Phu'dng phap nhan gidng
De bao dam dii lUdng gidng nudi trong nhieu dieu kien khic nhau, ta can tien hanh nhan gidng theo timg d p : cap 1, cap 2, tiiy thudc vao y l u d u lUdng gidng can cho moi lln nudi.
2 . 6 . Phu'dng phap nuoi trong
Tien hanh cac thi nghiem nudi trdng vi t i o trong binh Erien, dung tich nudi 1 lit. The tfch gidng eho vao chiem 10% the tfch mdi trUdng, Gidng sii dung la gidng cap 1 dupe nhan trong vdng 7 ngay, Cic mSu thf nghiim dupc chieu sang bSng dng den huynh quang 20W, chu ky chilu sing 12hdng:12h tdi, quang thdng dUpc do bing may Ad- vance Light Meter Nhiet dp thi nghiem dUde duy tri d 27- 28°C. Khao sat sinh trudng bSng cich do OD ciing mpt thdi dilm trong timg ngiy nudi trong. Tien hinh thu ho^ch SK khi sinh trUdng di v i o giai doan suy vong (tUdng ijng vdi 2 ngay giim tren dUdng eong OD).
2.7. Phu'dng phap t h u hoach SK
Mau dupe ly tam bing may ly tam Hettich ZENTRIFU- GEN vdi tdc dp 6.000 vdng/phiit, trong 10 phiit. Sau khi ly tam, mau dupc d y khd d 50°C den khdi lupng khdng ddi.
2.8. Phu'dng phap trich ly lipid
Ap dung phUPng phip Bligh v i Dyer (1959) va dUpe d i tien bdi Benemann va Tillett (1987). Day la phUPng phap trfeh ly dau hieu qua nhat theo Attilio [7,2], Mau SK khd dupc bd sung hon hdp CH3OH, CHCI3, sau do dUPc sieu am de phi v3 mang te bao d tan sd sdng 20 kHz trong 10 phiit roi dupe bd sung nUde va ly tam 6,000 vdng/phiit trong 10 phut de phan thanh 3 Idp: ldp MeOH v i nude d t r i n , Idp SK d giij^, dudi cung la Idp Chloroform va lipid hda tan. Phan Chloroform va lipid d dUdi dupc chiet va chung d t d l thu lipid. T h i ti'ch tdng dung mdi bd sung v i o mau SK dupe lay theo quy t i e 100 ml dung mdi tren 1 gam SK. Ty le t h i tieh dung mdi bo sung la CH30H:CHCl3:H20 = 10:10:9.
3. KET QUA VA THAO LUAN
3 . 1 . Khao sat su* anh hu'dng cua CO2 den sinh tru'dng va tich luy lipid
Dau tien, ta khao d t vai trd eiia CO2 ddi vdi sinh trUdng bang d c h t i l n hinh thUe nghiem trin 3 mau: (1) hoan toan khdng sue khi; (2) chi sue khdng khf; (3) sue khdng khf va CO2, dUdi inh sing 4,000 lux. Kit qua tren hinh 1 cho thay su cd m i t eiia CO2 trong mdi trudng lam tang tdc dd sinh trUdng cijng nhU nang suat thu SK v i lipid.
O l khao sat chi tilt anh hudng ciia toe dp sue khf CO2, ta tien hanh 3 thf nghiem dUdi anh sang 7.000 lux, tdc do sue khdng khf la 650 ml/phiit. Cac mau khic nhau ve tdc dd sue khf CO2: 20, 40, 60 ml/phut. Kit qua eho thay ehu ki sdng ciia tao la 17 ngay. Mat dd SK Idn nhat vao ngay thy 14, ung vdi gia tri eUc dai ciia pha d n bang.
Kit qua tren bang 1 cho tiiay, SK thu dUpc va ham IUpng lipid deu giam dan khi tdc dp syc CO2 tang bj" 20ml/phut den 60ml/phut. Dieu nay cung cho tiiay COj cung cd anh hudng u'c che den sinh trUdng d nong dp qui Idn.
Khi phan ti'ch thinh phan eiia mau lipid thu dUpc tir mau
Hinh I: Vai tro cua CO2 doi vdi sUsinh trudng cua vi tao sue CO2 bing miy phan tich s3e ky Idng cao ap HPLC, ket qua ri hinh ? rho thay car peak triglyceride xuat hien c) limn I 'lA V.I hjiKl lihi l.iixliiiu ;inh huang cud toe do sue khiCO2
Miu DFftu klfn
1 ?n ml COj/phut 2 40 ml C02/phOt 3 50 ml COj/phut
SKkhd (fl/l) 1,3862 1,1344 1,0964
upid (Q/i)
0,:517 0,1186 0.1157
k M ^ 10,945 10,451 m 10,549 1
khoang thdi gian lull 10,42 phiit va thanh phan d a tiiglyx- eride trong hon hpp khi eao.
Odng thdi, ket qua phan tich thanh phan triglicende bang phUPng phap GC-MS theo hinh 3 cho thay d 2 peak die trung eiia metylester ciia axit beo C16:0 (31,53) va axit oleic C18:l (35,47). Day la 2 loai axit beo rat phii hpp de d n xuat biodiesel vi dam bao dUpc b l u chuan dilin chiy va dd on djnh oxy hda ciia d n pham, cho thay lipkl trich ly tif vi t i o Chlorella Vulgaris rat d tnIn vong cho muc tieu sin xuat biodiesel.
3 . 2 . Anh hu'dng cua cu'dng dp dnh sang den sinh tru'dng va tich luy lipid
Chu ky d n g tdi thfch hdp nhat cho sU sinh trudng va tfch Itiy lipid ddi vdi Chlorella Vulgaris la 16h d n g : 8 h ^
Hinh 2: KSt qua phin tidi thanh phin lipid tai Cong ty Danapha Da Ning
30| KHOA HQC ft CONG NGHE >• (S6 10 - 6/2012]
Nghien ci'fu &
Bang 3: SK va lipid cua tao dUdi anh hUdng eiia ndng dp NH4CI
Hinh 3: Kit qua phan b'ch thinh phin tnglyxende bSng GC-MS d Cue Hii quan TP Di Nang
[8]. CUdng dp anh sing la mdt trong nhOng yeu td anh hudng quan trpng nhat den sU quang hpp eiia t i o .
Khio d t tren 3 mau vdi eUdng dp chieu d n g lan lUdt la 4.000,7.000 v i 13.000 lux. Cac mau thi nghiem deu khdng syc khdng khf va CO2. Kit qui dUdc ting hpp trong bang 2 Kit qua t r i n cho thay, hien tUpng "photoinhibition" xiy ra dudi cUdng dd anh d n g qui cao. Tuy nhien, sy tieh luy lipid tiong te bao tang theo eUdng dp chilu d n g . Ning suat thu lipid dat cao nhat tai 7.000 lux. Ket qua nay cijng phii hdp vdi nghien culj ciia G.Gacheva [10].
Bang 2: Ket q u i SK va lipid dUdi anh hUdng CLia anh sang
Mau Khoi lu'dng SK khd Khoi IUdng lipid ( g / l ) (9/1)
7.000 lux 0,265 13,000 lux 0,083
0,071 0,051
3.3. Anh hu'dng cua nong do N i t d den sinh tru'dng va tich luy lipid
Viec tim ra nong dp NitP toi Uu d y nghTa quan ti-png trong qua trinh d n xuat biodiesel t y vi t i o [6]. Thuc hien 5 mau cd ndng dp NH4CI khic nhau dUdi cUdng dp anh sang 7.000 lux, b l sung CO2 theo phUPng thirc gian doan vdi tdc dp 30ml/phut trong 1 phut vao buoi d n g , ket qua t h i hiln d bing 3.
Ket qua eho thay, sU tiiieu hut Nitd cung nhU sU dU tiii;^
qua nhieu Nitd (dang NH4CI) cung u'c c h l sinh b-Udng. Dieu nay phii hpp vdi k i t qua nghiln cuU ve sU iJfc c h l cua ion amoni b'ong mdi tru'dng mang ti'nh kilm (d khoing pH Idn hdn 8) [11]. Do vay, khi nudi trdng Chlorella Vulgans trong nuidc thai cung nhU d c mdi trUdng cd tfnh bazP, d n lUu y d i n v i n de nay. Ben canh dd, k i t qua cho thay sy thilu hut NibJ trong mdi tnibng lam tSng tich luy lipid ti-ong te bao.
3.4. ThuT nghiem nudi trong dieu ki£n nu'dc thai
NUdc thai sd'dung nudi ti-dng dudc lay tuT ham ii biogasMlu (mg NH4CI/I)
475 600 725 850 975
SKkh6 (g/l) 0,763 0,807 0,798 0,801 0,725
Lipid (mg/1) 71 74 64 61 53
% iipld/SK kho 9,31%
9,16%
8,02%
7,63%
7,31%
d ndng trai chin nudi Hda Phii, Da Ning. SU pha loang nudc thai cho ph^p giim dd due nhUng lim giim ndng dd chat dinh dudng trong mdi trUdng. Tien hanh nudi cay vi tao trong 5 mlu nUdc thai vdi myc do pha loang bSng nUde eat tir 1/5 (1 t h i tieh nUde thii pha thinh 5 t h i tfch) den 5/5 (khdng pha loang). Xac djnh mii'e dp sinh trUdng ciia vi tao trong cic mau thdng qua do gia tri nong dd Chlorophyll a (hinh 4).
Hinh 4: Smh tru'dng cua w (30 trong nddc thai pha loang Nhan thay rang, trong mau nUdc thai khdng pha loang (mau 5/5), tao smh trUdng rat cham, thdi gian nudi trdng keo dai, dong thdi IUpng lipid thu dUPe rat thap. Trong d c mau edn lai, mau ed kha nang ket hpp xii ly nUdc thai (vdi yeu cau miic dp pha loang khdng qui Idn) v i sin xuat biodiesel la mau 4/5 (4 phan nudc thai -i-1 phan nUdc d t ) (bing 4).
Bang 4: Ket qua SK va lipid trong cac mSu nu'dc thai pha loang
Mau 1/5 2/5 3/5 4/5 5/5
Luljng SK (g/i) 1,263 0,743 0,357 0,705 1,265
Lipid (g/i) 0,111 0,061 0,029 0,063 0,016
%llpid/SK kho 8,77 8,15 8,21 8,99 1,26 Dong thdi, k i t qua phan tieh nUdc thai ciia mau 4/5 eho thay vi t i o d kha ning hap thu mdt IUpng Idn N, P (bang 5).
4. KET L U A N V A D I N H HiStiUG DE TAI SUthay doi d e dieu kiln nuoi trong nhUeUdng dp chieu sing, tdc do sue khf CO2 va ndng dp N (NH4*) inh hUdng ro ret d i n ket qua thu SK va lipid. Kit qua ban dau cho thay.
b10-6/£012)'4 KHOA HQC & C 0 N G N G H | | 3 1
Nghien ci^i ^. IfiB^MffiB
Bang 5: Ket qua phan tich m§u nu'dc thdi dUdc pha loang 1,25 ^ n trUdc va sau 14 ngdy nudi Chlorella Vulgaris, do t ^ i Trung t a m Nghien cuii Bao v^ mdi tru'dng, OH fia NSng
Kfitqui M 3 u 4 / 5 ( t r u ' 6 c M S u 4 / 5 ( s a u Thong s6'
phan tich pH NitP tong hotpho tong
Phu'dng phip TCVN 6492-2000
TCVN 6202-1996
TCVN 5987-1995
khi n u 6 l )
35,39 mg/1
khi nudi) 8,2 3,5 mg/1 31,05 mg/1 d e dieu kiln nudi trong: Anh sing 7.000 lux, 600 mg NH4CI/lft va toe dp sue khf 20 ml C02/phut k i t hpp vdi sue 650 ml khdng khf/phiit eho k i t qui khi quan. Chat IUpng cua lipid thu dUpc khi phii hpp eho mMC tieu d n xuit biodiesel. Ngoii ra, ket q u i thy nghiim nuoi trdng trong nUdc thai hUm biogas cho thay Chlorella Vulgaris cd the sinh trUdng tdt, dong thdi cd khi ning xy ly nUde t h i i v i cho lipid nham mue tilu d n xuat biodiesel.
Dinh hudng eua de tai trong thdi gian tdi la toi Uu d c dieu kien nudi trdng nhim t ^ da ning suat thu lipid, xay dUng md hinh nudi tao bang he thdng dng ndi tiep va t i l n hanh nudi trong mdi tnj'dng nUdc thai.-:-
TAI LIEU THAM KHAO
) Uam Brennan, Philip Owende (2009). 'Blofuels from microal- gae-A review of technologies for production, processing, and extrac- tions of blofuels and co-products'. Renewable and Sustainable Energy Reviews. ELSEVIER, RSER-805.
2. Atdlio Converti (2009), "Effect of temperature and nitrogen con- centration on the growth and lipid content of Nannochloropsisoculata and Chlorella vulgaris for biodiesel production", Chemical Engineering and Processing: Process lntensification,ELSEVIER. 48 1148-1151.
3. Teresa M. Mata, "Microalgae for biodiesel production and other applications: A review". Renewable and Sustainable Energy Reviews,EL- SEVIER, RSER-757.
4. Zhi-Yuan Uu, Quong-Ce Wang, Bai-Cheng Zhou (2008), 'Effect of iron on growth and lipid accumulation in Chlorella vulgaris', Biore- source TechnologyELSEVIER, 994717-4722.
5. Mijeong Lee Jeong, James M. Giltis (2003) 'Carbon Dioxide fiAit- igation by Microalgat Photosynthesis", Bulletin of the Korean Chemi- cal Society, Vol 24, No 12, page 1763.
[6. Subhosha Nigam (2011), "Effect of Nitrogen on Growth and Lipid Content of Chlorella pyrenoldosa", American Journal of Bio- chemistry and Biotechnology, vol 7, No3 124-129.
7. OrellOlivo (2007). "Thise de Doctoral. Conception et etude d'un photobioreacteur pour la production en continu de microalgues en ecloseries aquacoles", £cole polytechnique de I'Universiti de Nante, page UO.
8. D.Sasi and G.A.Hill (2009), "Effect of light intensity on growth of Chlorella Vulgaris In a novel Circulating loop photobioreactor" De- partment of Chemical Engineering, University of Saskatchewan, Canada, page 2.
9. http://www.chlorellavulgaris.com/
10. G.Gacheva (2008), The resistance of a new strain Chlorella sp R-06/2 isolated from an extreme habitat to environnementat stress fac- tors'. General and Applied Plant Physiology. Special Issue, vol 34,3-4, page 347-360.
11. Y.Azov and Joel C. Goldman (1982), "Free Ammonia Inhibition of Algal Photosynthesis in Intensive Cultures", Applied and Environ- mental fiAicrobiology. vol 43, No4, page 735-739.
Pong cong ty Dien lUc I la Noi (i:VN HANOI)
Di dau trong ufng dung KHCN vao san xuat kinh doanh dien
NGUYEN H l / a N G
EVN HANOI au<fc thanh l$p tti ndm 1954 vdi t«n ban ddu Id Nhd may d^n Bd Hd. Sau nhi^u Idn thay d6i, ddn thang 2/2010, theo Quy6t djnh sd 738/QD-BCT ngdy 0 5 / 0 2 / 2 0 1 0 cua Bd trUdng B$ Cong ThiTcrng, Cdng ty Dl^n lUc TP. Hd Npi chinh thurc aiipc ddi tdn thdnti Tdng cdng ty Di^n li^c TP. Hd N Q I . Tdng cdng ty hoat ddng theo md hinh Cdng ty m^-Cdng ty con va la ddn vj thdnh vi6n cua Tdp dodn Di^n \\fc Vidt Nam.
L
a trung tam ehinh tri - xa hdi, kinh t l - van h d cua ea nudc, cd tdc dp t i n g trUdng binh quan phu tii khoang 12%-15%/ n3m, Thii dd H i Ndi ludn d nhu d u su" dung diln I n dinh vdi chat lul^ng eao nhat.Oe dap yng nhu d u do, EVN HANOI da trien khai cae bien phap ddng bd, khoa hpc nham cung d p du dien phue vu nhan dan. Dang ke nhat l i vile tiln hanh day manh dau tU ddi mdi, img dung tien bd khoa hpc ky thuat, d n g nghe mdi vao hoat ddng d n xuat kinh doanh, d i tiln ky thuat va bao ve mdi trUdng sinh thai. Trong nhOtig nim qua, EVN HANOI da danh hang ty dong d l ting thUdng cho d c can bp nghiln cifu, eic ky sU, d n g nhan khi tham gia vao cac cupc thi ve d n g kiln, d i t i l n ky thuat trong nginh Diln.
Nhd do, phong trao phat huy d n g kiln d i tien, ap dung khoa hpc ky thu|t khdng ngCmg phat trien. Ting so sang kiln d i tiln (SKCT) ti'nh tiJ" nam 2008 - 2011 la 1.918 SKCT, trong do, 566 SKCT duWc cdng nhan d p Hdi ddng KHCN eiia Tdng cdng ty; 1.352 SKCT dMc cdng nhan cap tieu ban. Dac biet, trong 09 d l tai NCKH, d 02 de tai d p Tip doan va 07 de tai d p Ting cdng ty duflc d^nh gia tdt. Tong d n g ty da bao ve tiianh cdng de tai NCKH d p Tip doan Diln lye Viet Nam vdl de tai "Nghien cuTu img dung day s i ^ nhilt" dat lo^i gidi va dUdc Tap doan Oiln lyc Viet Nam chpn dy thi giai tiiuidng VIFOTEC cua Bd Khoa hpc va Coni nghi va Hdi Uln hi^p d c hdi Khoa hpc Viet Nam nim 2t^! |
Trong nhiJng nim qua, EVN HANOI dS l i ^ chpn ap dgng d c cdng ngh§ phii hdp vdi dieu Win khf h|u, mdi ti-ydng
2{ KHDA HQC ft C O N G N G H | »' ( S 6 10 - 6/2012)
