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I HC QUC GIA THNH PH H CH MINH TRNG I HC BCH KHOA KHOA K THUT HA HC B MN CNG NGH SINH HC ------------------oOo-------------------

N MN HC

SN XUT BIODIESEL T VI TO: K THUT NUI CY VI TO THU LIPID


SVTH: BI NGC OAN CHIU MSSV: 60604048 GVHD: KS. HUNH NGUYN ANH KHOA

Tp HCM, Thng 6/2010


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LI CM N n mn hc Sn xut biodiesel t vi to: K thut nui cy vi to thu lipid c thc hin t thng 2 n thng 7/2010. Trong sut qu trnh , hon tt tt cc ni dung nghin cu, em nhn c rt nhiu s gip nhit tnh v ci m. Em xin c gi n: KS. HUNH NGUYN ANH KHOA, l cn b trc tip hng dn em lm n ny, li cm n su sc v chn thnh. Chnh nh s hng dn tn tnh v mt kin thc v gip v mt ti liu hc thut ca thy, em hon thnh tt cc ni dung ra. TS. NGUYN TH HUYN, l cn b lm vic ti phng 117B2, lng bit n tri n. C lun to khng kh thoi mi khi em vo phng trao i hc tp, iu c tc dng ng vin em rt nhiu. Tp th qu thy c thuc b mn Cng ngh sinh hc ni ring v khoa K thut ha hc ni chung lng bit n tht nhiu. Thy c l nhng ngi cung cp cc kin thc c s cng nh chuyn ngnh cho em trong sut qu trnh lu di lm nn tng cho nhng nghin cu v sau.

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TM TT N Thc trng nhim mi trng v s thiu ht ngun nng lng trong tng lai chnh l mi quan tm hng u ca cc quc gia hin nay, v biodiesel c xem l mt gii php kh thi nhm thay th cho ngun nhin liu diesel du ha s dng cho cc phng tin giao thng vn ti ln trong cng nghip. Trong khi cc ngun nguyn liu sn xut biodiesel thng thng: du thc vt, m ng vt v ngun du m ph thi u t ra khng th p ng nhu cu biodiesel trn ton th gii, vi to li th hin l mt i tng rt tim nng cho lnh vc ny nh vo kh nng sn xut sinh khi ln v ngun lipid thu nhn t cc loi vi to cng kh ph hp iu ch biodiesel. Tnh hnh nghin cu v p dng nui cy vi to trn quy m ln ngy cng ph bin trong nhiu lnh vc nh: sn xut thc phm chc nng v x l mi trng, do vic ng dng nui cy vi to trn cc mi trng sa i nng cao nng sut lipid phc v sn xut biodiesel l hon ton c kh thi. Nghin cu cc c im sinh l v sinh ha ca loi vi to Nannochloropsis oculata c th d on c y l mt loi vi to rt ph hp vi mc tiu sn xut biodiesel: nng sut sinh khi cao trong mi trng quang t dng, hm lng lipid nhiu v thnh phn lipid d iu chnh theo iu kin nui cy. T xut kho st cc yu t mi trng nhm nui cy Nannochloropsis oculata thu lipid vi nng sut cao: mi trng f/2, mn trong khong 22-49g NaCl/L, nhit mi trng 25-270C, chiu sng bng n hunh quang t ta nhit 400-500mol/m2s, hn ch nng nitrogen v phosphorus.

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ABSTRACT Pollution and limitation of energy are the most important concerns of nations, and biodiesel is considered such as a possible solution to replace petroleum diesel, which is used for transportation and industry. While plant oil, animal fat and waste oil which are common for biodiesel production cannot satisfy biodiesel demand all over the world, microalgae express to be an essential candidate for this task due to high yield of biomass and compatible lipid component for biodiesel production. Researchs and applications in microalgae culturing on large scale become more and more common in many fields: functional food production and environment treatment, so using modified medium culture to improve lipid content in microalgae cells is really feasible. Studying in physiological and biochemical characteristics of Nannochloropsis oculata gives suggestion that N. oculata should be a suitable source for biodiesel production: produces high yield of biomass in autotrophic culture, accumulates high lipid content and easily be controlled lipid composition under different conditions. Hence some nutriments factors, temperature and light regimes are proposed to grow N. oculata with high lipid productivity: f/2 medium, salinity at 22-49g NaCl/L, temperature at 25-270C, irradiance at 400-500mol/m2s with cool white fluorescent tubes, limited nitrogen and phosphorus concentrations.

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MC LC mc Trang

LI CM N .....................................................................................................ii TM TT N ............................................................................................ iii MC LC .......................................................................................................... v DANH SCH BNG ....................................................................................... vii DANH SCH HNH .......................................................................................... ix DANH SCH T VIT TT ............................................................................. x

1. TNG QUAN V BIODIESEL ................................................................... 1 1.1. nh ngha cc dng nng lng v biodiesel ......................................... 1 1.1.1. Nng lng khng ti sinh ............................................................... 1 1.1.2. Nng lng ti sinh ......................................................................... 2 1.2. Tm quan trng ca biodiesel v kh nng thay th cho ngun nhin liu ha thch ......................................................................................................... 4 1.3. Nguyn tc iu ch v nhu cu v lipid trong sn xut biodiesel ........... 7 1.4. Cc ngun nguyn liu giu lipid phc v cho vic sn xut biodiesel v tim nng ca vi to ........................................................................................ 9 2. NNG SUT LIPID V NH HNG CA MI TRNG LN S TCH LY LIPID CA MT S LOI VI TO ........................................ 15 2.1. Cc loi vi to c cha nhiu lipid ........................................................ 15 2.2. Mt s nghin cu v nh hng ca iu kin mi trng ln s tch ly lipid vi to ............................................................................................ 16 2.2.1. Yu t nhit .............................................................................. 16 2.2.2. Yu t thnh phn mi trng ....................................................... 19 3. NUI VI TO NANNOCHLOROPSIS OCULATA THU LIPID NHM SN XUT BIODIESEL ................................................................................ 25 3.1. Nannochloropsis oculata ...................................................................... 25 3.1.1. Phn loi........................................................................................ 25 3.1.2. c im hnh thi......................................................................... 25 v

3.1.3. c im sinh l ............................................................................ 26 3.1.4. c im sinh ha ......................................................................... 26 3.2. xut m hnh th nghim nghin cu s tch ly lipid theo iu kin mi trng Nannochloropsis oculata ......................................................... 27 3.2.1. Yu t nhit .............................................................................. 27 3.2.2. Yu t nh sng ............................................................................. 32 3.2.3. Yu t mn ............................................................................... 36 3.2.4. Yu t thnh phn mi trng ....................................................... 38 4. KT LUN.................................................................................................. 48 TI LIU THAM KHO ............................................................................... 51

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DANH SCH BNG Bng 1. 1: Cc acid bo thng c trong cc ngun nguyn liu dng sn xut biodiesel .............................................................................................................. 9 Bng 1. 2: So snh vi to vi cc ngun nguyn liu sn xut biodiesel ............ 11 Bng 1. 3: So snh cc thuc tnh gia du t vi to, diesel thng thng v tiu chun ASTM biodiesel [28]............................................................................... 12 Bng 2. 1: Hm lng v nng sut lipid ca cc loi to khc nhau ................. 15 Bng 2. 2: S sinh trng v sn xut lipid ca C. vulgaris ti cc nhit khc nhau [5] ............................................................................................................. 17 Bng 2. 3: Nng sut sinh khi v nng sut lipid ca Botryococcus braunii, Chlorella vulgaris v Scenedesmus sp. khi nui 10% CO2 trong 14 ngy ....... 20 Bng 2. 4: Thnh phn cc acid bo ca Botryococcus braunii, Chlorella vulgaris v Scenedesmus sp. khi nui cy 10% CO2 trong 14 ngy................. 21 Bng 2. 5: Tham s sinh trng v s sn xut lipid ca C. vulgaris cc nng NaNO3 khc nhau ......................................................................................... 23 Bng 3. 1: S sinh trng v sn xut lipid ca N. oculata ti cc nhit khc nhau .................................................................................................................. 28 Bng 3. 2: Thnh phn acid bo (%w/w acid bo tng) ca Nannochloropsis sp. vo ngy th 10 ti cc nhit khc nhau........................................................ 30 Bng 3. 3: Sn lng sinh khi v thnh phn hp cht ha sinh ca Nannochloropsis sp. vo ngy th 10 ti cc nhit khc nhau ....................... 31 Bng 3. 4: Sn lng sinh khi v hm lng lipid ca Nannochloropsis sp. vo ngy th 10 ti cc mn khc nhau ............................................................... 36 Bng 3. 5: Thnh phn acid bo (%w/w TFA) ca Nannochloropsis sp. vo ngy th 10 ti cc mn khc nhau ........................................................................ 37 Bng 3. 6: Nng sut sinh khi v lipid ca N. oculata trong h thng nui cy bn lin tc vi cc hm lng CO2 khc nhau .................................................. 41 Bng 3. 7: Tham s sinh trng v s sn xut lipid ca N. oculata cc nng NaNO3 khc nhau .............................................................................................. 42 Bng 3. 8: Sn lng sinh khi v thnh phn hp cht ha sinh ca Nannochloropsis sp. vo ngy th 10 ti cc nng NaNO3 khc nhau .......... 44

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Bng 3. 9: Thnh phn acid bo (%w/w TFA) ca Nannochloropsis sp. vo ngy th 10 ti cc nng NaNO3 khc nhau .......................................................... 44 Bng 3. 10: Sn lng sinh khi v thnh phn hp cht ha sinh ca Nannochloropsis sp. vo ngy th 10 ti cc nng NaH2PO4 khc nhau ....... 46 Bng 3. 11: Thnh phn acid bo (%w/w TFA) ca Nannochloropsis sp. vo ngy th 10 ti cc nng NaH2PO4 khc nhau .............................................. 47

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DANH SCH HNH Hnh 1. 1: S hnh thnh cc lp nhin liu ha thch ......................................... 1 Hnh 1. 2: Cc dng nng lng ti sinh .............................................................. 2 Hnh 1. 3: Bng thng k s dng cc ngun nng lng .................................... 3 Hnh 1. 4: Phn ng chuyn v ester .................................................................... 7 Hnh 2. 1: Phn trm cc loi FAME trn tng lng FAME (g/100gFAME) ca C. vulgaris ti cc nhit sinh trng khc nhau ................................................. 17 Hnh 2. 2: Phn trm cc loi FAME trn tng lng FAME (g/100gFAME) ca C. vulgaris ti cc nng NaNO3 khc nhau........................................................ 23 Hnh 3. 1: Thnh phn acid bo cc lipid chnh trong Nannochloropsis sp. ....... 27 Hnh 3. 2: Phn trm cc loi acid bo methyl ester trn tng lng acid bo methyl ester (g/100gFAME) ca N. oculata ti cc nhit khc nhau ................. 29 Hnh 3. 3: nh hng ca mc chiu sng trn s sinh trng ca t bo v hm lng chlorophyll a i vi Nannochloropsis sp. ...................................... 33 Hnh 3. 4: S phn phi cc acid bo chnh trong Nannochloropsis sp. c nui cy theo m di nh hng ca mc chiu sng ......................................... 33 Hnh 3. 5: Thnh phn acid bo ca Nannochloropsis sp. khi nui cy trong iu kin n nh lin tc ti ba mc chiu sng................................................... 35 Hnh 3. 6: nh hng nng kh CO2 ln s sinh trng ca N. oculata ........ 39 Hnh 3. 7: S sinh trng ca N. oculata khi nui cy bn lin tc trong mi trng sc kh c cha 2%, 5%, 10%, 15% CO2 ............................................... 40 Hnh 3. 8: Phn trm cc loi FAME trn tng lng FAME (g/100gFAME) ca N. oculata ti cc nng NaNO3 khc nhau ......................................................... 43

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DANH SCH T VIT TT DW: dry weight FAME: fatty acid methyl ester f/2AW: f/2 artificial seawater EPA: eicosapentaenoic acid PUFA: polyunsatured fatty acid TFA: total fatty acid v/v: volume/volume vvh: volume/ volume/hour vvm: volume/ volume/minute w/w: weight/weight khi lng kh ester ca acid bo v methyl mi trng f/2 nc bin nhn to acid 3 (C20:5) acid bo cha no mang nhiu ni i hm lng acid bo tng th tch/th tch th tch/th tch/gi th tch/th tch/pht khi lng/khi lng

1. TNG QUAN V BIODIESEL

1. TNG QUAN V BIODIESEL


1.1. nh ngha cc dng nng lng v biodiesel
1.1.1. Nng lng khng ti sinh Ngun nng lng khng ti sinh l nhng ngun nng lng thin nhin m con ngi khng c kh nng can thip vo s hnh thnh cng nh qu trnh tch ly. l dng nng lng khng th phc hi, khng th ti to, hay khng th ti s dng. Tuy nhin dng nng lng ny ang c s dng trn phm vi rng ln khp ton cu, v cho thy rng khng th p ng c nhu cu s dng ca con ngi trong tng lai. Nng lng khng ti sinh c chia thnh hai nhm: nng lng ha thch v nng lng ht nhn. Nng lng ha thch: l dng nng lng c hnh thnh da trn cc qu trnh a cht di hng triu nm xy ra i vi xc ng thc vt, nh mt dng ha thch. Bao gm than , du m v kh thin nhin. Do qu trnh hnh thnh lu di nh vy, nn khi b con ngi khai thc ht s khng c kh nng phc hi c. Mt trong s cc nhin liu ha thch ngy cng c s dng rng ri l diesel. Diesel l mt loi nhin liu lng, l sn phm thuc phn on nh ca qu trnh chng ct trc tip du m vi khong nhit si p sut kh quyn t 250 n 3500C (cao hn du ha v xng) bao gm cc hydrocarbon khc nhau t C14 n C20. Nhin liu diesel c s dng ch yu cho ng c diesel v mt phn c s dng trong cc tuabin c kh.

Hnh 1. 1: S hnh thnh cc lp nhin liu ha thch 1

1. TNG QUAN V BIODIESEL Nng lng ht nhn: l dng nng lng c hnh thnh do kh nng phng x ca mt vi nguyn t. C hai kiu phn ng hnh thnh nn nng lng ht nhn, l phn ng phn hch v phn ng nhit hch. 1.1.2. Nng lng ti sinh Nng lng ti sinh l dng nng lng thu c t cc ngun m con ngi xem l v hn. S v hn y ngoi ngha nhiu n mc khng th cn kit, nn c hiu theo ngha rng hn l c kh nng ti to trong mt thi gian ngn v lin tc. Dng nng lng ny bao gm: Nng lng mt tri Nng lng gi Nng lng sng Nng lng thy triu Nng lng a nhit Nng lng sinh khi

Hnh 1. 2: Cc dng nng lng ti sinh Ngun nng lng ti sinh ang c s dng nhiu nht l thy nng. Song, ngun nng lng sinh khi cng l mt trong nhng ngun c con ngi khai thc v s dng hiu qu. Sinh khi c xem l ngun nng lng ln th t, c tnh chim khong 14-15% tng lng nng lng tiu th trn 2

1. TNG QUAN V BIODIESEL th gii. cc nc ang pht trin, sinh khi thng l ngun nng lng ln nht, trung bnh ng gp khong 35% trong tng cung cp nng lng. V vy, trong chin lc s dng cc ngun nng lng cch hiu qu th nng lng sinh khi lun c xem l u tin hng u v mang tnh quyt nh trong vic p ng nhu cu nng lng ca th gii trong tng lai [1]. Hin nay, theo thut ng v nhin liu, th sinh khi (biomass) c xem l nhin liu dng rn, nhin liu sinh hc (biofuel) l nhng nhin liu di dng lng thu nhn t sinh khi v cui cng kh sinh hc (biogas) l sn phm ca qu trnh phn gii ym kh cc cht hu c [1]. Hai dng nhin liu sinh hc ph bin nht l biodiesel v bio-ethanol, l hai dng nhin liu tng ng c th thay th c cho diesel v gasoline m khng cn ci tin nhiu hoc khng cn ci tin ng c cc phng tin giao thng cng nh my mc sn xut. Chng c sn xut ch yu t sinh khi hay cc ngun nng lng ti sinh khc v gp phn gim thiu kh thi t vic t chy nhin liu so vi nhin liu ha thch tnh trn cng mt n v hiu sut [80]. nh ngha Biodiesel Biodiesel l hn hp cc alkyl monoesters thu nhn c t qu trnh chuyn v ester du thc vt v m ng vt [80], c kh nng thay th cho diesel t du m.

Hnh 1. 3: Bng thng k s dng cc ngun nng lng 3

1. TNG QUAN V BIODIESEL

1.2. Tm quan trng ca biodiesel v kh nng thay th cho ngun nhin liu ha thch
Gn y, con ngi ang quan tm n hai vn quan trng, l mi trng v s khng hong nng lng. i vi mi trng, s nng dn ln ca tri t chnh l tm im. Mi ngi u bit rng s dng nhin liu ha thch chnh l nguyn nhn gy ra s nng dn ln ca ton cu, v vy ngun nng lng sch v c kh nng ti sinh sn xut t sinh khi nhm thay th cho nhin liu ha thch l rt cp thit gim thi CO2. Ngoi ra, s khng hong nng lng khin cho gi du th trn th gii ngy cng tng, nh hng n tnh hnh nng lng s dng trong gia nh cng nh trong khu vc [67]. Xt trn lnh vc mi trng, giao thng vn ti v sn xut cng nghip l nhng ngun thi ch yu ca con ngi. Kho st ti lin minh Chu u, ngi ta nhn thy c n 20% kh thi nh knh l do giao thng vn ti v 60% kh thi nh knh xut pht t cc khu cng nghip [20]. Nng nghip l ngun thi ln th ba, tng ng khong 9% kh thi nh knh, trong quan trng nht l cc kh nitrous oxide N2O v kh methane CH4 [21]. Theo d on, s pht trin ca cc nn kinh t mi ang tng trng nh n v Trung Quc s lm gia tng s tiu th nng lng trn ton cu, dn n thm nhiu mi nguy hi cho mi trng [35]. Kh nh knh khng ch gp phn gy ra hin tng nng dn ln ca tri t m cn gy ra nhiu nh hng khc ti mi trng v i sng nhn loi. Cc i dng hp th khong mt phn ba lng CO2 thi ra mi nm do cc hot ng ca con ngi. Khi nng CO2 tng ln trong khng kh, lng ha tan trong nc bin cng tng, dn n gim pH v nc bin c tnh acid hn. S gim pH l mt iu kin bt li c th gy ra nhng hy hoi nhanh chng i vi cc rng san h cng nh s a dng sinh thi i dng, vn c quan h to ln trong i sng cc sinh vt i dng v trn cn [55]. Chnh s nng dn ln ca tri t , ang v s tip tc nh hng n nhiu kha cnh khc nhau ca i sng nhn loi v mi trng trn ton cu, chng ta cn khng phi ch ring l mt m l mt lot cc gii php c th ci thin v khc phc vn bc bch ny [40]. Xt trn lnh vc khng hong nng lng, s st gim ngun nguyn liu du th d tr v nhng kh khn trong vic trch ly v tinh luyn chng dn n s gia tng v gi thnh [40]. y thc s l nhng tr ngi ln i vi vn giao thng

1. TNG QUAN V BIODIESEL vn ti, v cho ti hin nay vn cha tm ra c ngun nguyn liu no c th thay th cho ngun nguyn liu ha thch vi gi thnh tng ng [80]. Tm kim nhng ngun nguyn liu sch v c kh nng ti sinh l mt trong nhng vn thch thc nht m con ngi ang phi i mt trong hin ti ln v lu di. y l vn lin quan mt thit vi s pht trin kinh t v s thnh vng cng nh cht lng cuc sng, s bn vng ton cu do i hi cc nh u t phi c nhng quyt nh lu bn v nhng gii php di hn [80]. Hin nay, nhiu la chn ang c nghin cu v a vo thc nghim, t c nhng mc thnh cng khc nhau trong cc giai on nghin cu v thc nghim khc nhau, bao gm cc ngun nng lng nh nng lng mt tri, bao gm c nhit nng v quang nng, thy in, a nhit, gi, nhin liu sinh hc, v s c lp carbon, cng nhng loi khc [19, 27]. Mi loi u c nhng u im cng nh nhng vn vng mc, ty thuc vo khu vc p dng, nhng iu kin khc nhau s c mc ph hp tt hn i vi tng loi phng php. Mc tiu quan trng l gim thiu cc kh thi t cc phng tin chuyn ch, nh l thay th dn tng bc mt ngun nguyn liu ha thch bng nhng ngun nguyn liu ti sinh, trong nhin liu sinh hc c xem l mt cng tc vin thc s t c nhng mc tiu , c bit trong tng lai gn [80]. Tri t nng dn ln, do s gia tng nng ca kh nh knh trong khng kh, v ang l mi lo ngi quan trng nht v mi trng [85]. Gim bt s hnh thnh CO2 trong kh quyn, nhim v hng u ca vic lm gim hiu ng nh knh, c th thc hin bi ba phng php sau [6]: Gim bt s dng nhin liu ha thch Loi b CO2 trong kh quyn Thu hi v c lp hoc tn dng CO2 t vic t nhin liu ha thch trc khi n c thi ra mi trng.

Sn xut nhin liu sinh hc c th em li nhng c hi mi gp phn gia tng s a dng v cc ngun thu nhp cng nh cc ngun cung cp nng lng, c th l y mnh vic lm cc vng nng thn, pht trin s thay th di hn i vi ngun nhin liu ha thch, v gim bt lng kh thi nh knh, y mnh vic loi b Carbon t cc nhin liu vn ti v nng cao s an ton trong vic cung cp nhin liu [80].

1. TNG QUAN V BIODIESEL Tm kim nhng ngun nguyn liu thay th ang l vn nng bng i vi nhiu nc, c bit l nhng nc thiu cc ngun ti nguyn nng lng thng thng. Vo nhng thp nin 1930 v 1940, du thc vt c s dng nh l nhin liu diesel trong nhng trng hp khn cp. Trong nhng nm gn y, khi nn cng nghip hin i pht trin ngy mt rng ri v quy m, nhu cu v nng lng gia tng t bin, v vy nhng ngun nng lng thay th ang c thm d v nghin cu ro rit. Trong tng s cc ng c phng tin hin nay, t l phng tin s dng nhin liu diesel ngy cng ph bin v c s dng nhiu hn. Thm vo , ng c diesel cng c s dng rng ri trong cng nghip [75]. V vy, nhin liu diesel l ngun s dng ln nht trong s cc nhin liu du m s dng cho ng c, v t l ny tng ln lin tc ngy qua ngy [11]. T , nhu cu i vi biodiesel cng tr nn cp thit, v thut ng biodiesel xut hin mt cch thng xuyn trong cc bi bo co gn y [24]. Biodiesel c sn xut t sinh khi du (biomass oils), ch yu l t du thc vt. Biodiesel tr thnh ngun nng lng ng c ch v nhng l do nh sau. Th nht, biodiesel l ngun nng lng ti sinh c th p ng cung v cu. Ngi ta c tnh c rng lng du m d tr s hon ton cn kit trong vng 50 nm tr li tnh theo mc tiu th hin nay [70]. Th hai, biodiesel cho thy thn thin vi mi trng hn cc loi nhin liu ha thch. S dng biodiesel khng lm gia tng lng kh thi carbon dioxide nhng li c hm lng cc kh sulfur thp hn hn [4, 82]. Thnh phn kh thi sulfur v carbon monoxide gim thiu ln lt l 30% v 10% khi s dng biodiesel. Lng kh to thnh trong qu trnh t chy nhin liu c th t li, v s gim bt carbon monoxide l nh vo hm lng oxygen cao c trong biodiesel. Hn na, biodiesel khng cha cc hp cht d bay hi v cc hp cht ha hc khc gy hi cho mi trng. Gn y, cc cuc iu tra cho thy rng s dng biodiesel gim thiu c 90% cht c hi trong khng kh v 95% cc bnh ung th khi so snh vi diesel thng thng [69]. Th ba, biodiesel em li tim nng kinh t ng k bi v trong tng lai, cc dng nng lng khng ti sinh nh nhin liu ha thch s ngy mt gia tng gi thnh l mt iu khng th trnh khi [10]. Cui cng, biodiesel tt hn diesel thng thng nhit pht chy v kh nng phn hy sinh hc [45].

1. TNG QUAN V BIODIESEL Ngun sinh khi khng nhng s tr thnh ngun cung cp nng lng bn vng m cn gp phn vo cuc sng x hi trong khu vc nh gip gia tng cc hot ng thng mi v to nhiu c hi vic lm cho nng dn. Vi to, thu nhn di dng sinh khi, l mt ngun nng lng ti sinh rt tim nng, v chng c th c chuyn ha thnh nng lng di dng nhin liu sinh hc bao gm du v gas. V vi to c hm lng nc nhiu, do khng phi ton b lng sinh khi c th p dng thnh nng lng. S dng phng php nhit ha hc c th sn xut ra du v gas, v bng cc phng php ha sinh sn xut ra ethanol v biodiesel. Sn phm t vi to c cc c tnh tng t nh cc sn phm t du thc vt v du c, v vy, n c th thay th c cho du m [67].

1.3. Nguyn tc iu ch v nhu cu v lipid trong sn xut biodiesel


Biodiesel l hn hp cc ester gia acid bo v alkyl thu c thng qua qu trnh chuyn v ester ca du thc vt hay m ng vt. Ngun nguyn liu lipid du thc vt hay m ng vt bao gm 90-98% khi lng l cc triglyceride v mt lng nh cc mono v diglyceride, acid bo t do chim khong 1-5%, phn cn li l cc phospholipid, phosphatide, carotene, tocopherol, hp cht sulphur, v mt t nc [7]. Chuyn v ester l mt phn ng phc tp, bao gm ba bc chuyn i lin tip: triglyceride c chuyn ha thnh diglyceride, sau diglyceride chuyn ha thnh monoglyceride,v monoglyceride s chuyn ha thnh cc ester (biodiesel) v glycerol (ph phm). Phn ng chuyn v ester tng qut c m t trong hnh sau, trong R1, R2, R3 tng trng cho cc hydrocarbon mch di, chnh l cc acid bo [80].

Hnh 1. 4: Phn ng chuyn v ester 7

1. TNG QUAN V BIODIESEL Trong phn ng chuyn v ester, tc nhn phn ng l du hay m cng cc alcohol c mch carbon ngn (thng l methanol), c s hin din ca cht xc tc (thng l NaOH). Mc d t l phn t gam gia alcohol : du theo l thuyt l 3:1 nhng thng thng ngi ta p dng theo t l 6:1 phn ng xy ra hon ton. Mi quan h gia nguyn liu u vo v sn phm biodiesel u ra t t l khong 1:1, ngha l theo l thuyt, 1kg du s to ra c 1 kg biodiesel [80]. C th s dng cht xc tc ng th hay d th, acid, base hoc enzyme lipase tng mc phn ng chuyn v ester, mc d i vi vi qu trnh s dng cc cht lng siu ti hn (methanol hoc ethanol) c th khng cn dng n cc cht xc tc [84]. Trong cng nghip, thng s dng nht l cc cht xc tc ng th c tnh kim (v d nh NaOH hay KOH) trong cc bnh phn ng c khuy o, thc hin theo m [80]. Cc phn ng chuyn v ester c xc tc bi cc cht c tnh kim c tc phn ng nhanh hn 4000 ln so vi xc tc bi acid [25]. Trong phn t triglyceride, khi lng glycerol l khong 41 g/mol, trong khi khi lng cc gc acid bo dao ng t 650-790 g/mol. Chnh thnh phn cc gc acid bo to ra cc nhm hot ng trong phn t glyceride, v chng c tm nh hng ng k n tnh cht ca du thc vt v m ng vt [11]. Trong qu trnh sn xut biodiesel, ngi ta c th s dng ngun lipid t du thc vt, m ng vt hoc cc loi du thi. Tuy nhin, sn xut biodiesel t chun nhin liu, tnh cht ca ngun nguyn liu rt quan trng, ngay t qu trnh nghin cu ban u cho ti giai on sn xut, v cht lng ca biodiesel ph thuc vo cht lng ngun nguyn liu [11]. Nhng ngun nguyn liu giu cc acid bo cha bo ha cha nhiu ni i (polyunsaturated) th d mc phi qu trnh oxy ha hn cc ngun lipid c nhiu acid bo bo ha hay ch c mt ni i trong mch (monounsaturated) [11]. Tuy nhin, biodiesel t ngun nguyn liu cha mt lng ln cc acid bo bo ha s c nhit im sng (nhit biodiesel bt u xut hin tinh th) v nhit dng chy (nhit thp nht m biodiesel vn c th bm v chy c trong ng dn) cao. Thc t cho thy, biodiesel t du thc vt c nhit im sng v nhit dng chy thp hn biodiesel t m ng vt [11]. Nu ngun nguyn liu lipid cha mt lng ln cc acid bo t do v nc th qu trnh chuyn v ester s gp nhiu kh khn v chng khng th chuyn ha c thnh biodiesel thng qua phn ng xc tc vi kim. y l vn vng 8

1. TNG QUAN V BIODIESEL mc khi s dng du qua ch bin, du thi nh hng, du cn, m ng vt lm ngun nguyn liu sn xut biodiesel d y l nhng ngun nguyn liu c gi thnh r [11]. Thnh phn ester ph bin nht trong biodiesel l ester ca cc acid palmitic, acid stearic, acid oleic, acid linoleic v acid linolenic. Cc loi nguyn liu nh u nnh, hoa hng dng, ht ci du (canola), c v lc c thnh phn lipid kh ph hp nh trn [26]. Trong s , methyl oleate c xem l thnh phn chnh l tng sn xut biodiesel. Mt s ester khc nh methyl palmitoleic cng c nhng u im, khi so vi methyl oleate th tri hn v nhit ng c thp [26]. Bng 1. 1: Cc acid bo thng c trong cc ngun nguyn liu dng sn xut biodiesel [26]

1.4. Cc ngun nguyn liu giu lipid phc v cho vic sn xut biodiesel v tim nng ca vi to
Thng thng, biodiesel ch yu c sn xut t du ca u nnh v cc loi rau qu [9], c [2], hoa hng dng [4], ht cy ci du [58] cng nh du ph thi t cc nh hng [8]. S lng nguyn t carbon trong chui mch carbon ca phn t du diesel vo khong 15C, v iu ny gn nh tng ng vi du thc vt vi chui carbon vo khong 14-18C. Cc c im v cu trc ca biodiesel cho thy rng biodiesel c th ng dng thay th c cho nng lng thng thng. Hin ti, gi thnh ca biodiesel th cao gp 2 ln so vi diesel thng. Chi ph sn xut biodiesel bao gm 2 phn chnh, l chi ph cho ngun nguyn liu th (du thc vt v m ng vt) v chi ph qu trnh. Chi ph cho ngun nguyn liu th chim khong 60-75% tng chi ph nhin liu biodiesel [39]. D vy, vn c th tm c mt lng ln du m gi thnh thp t cc ch phm ph thi nh 9

1. TNG QUAN V BIODIESEL hng v t ng vt [30], vn chnh khi s dng nhng ngun du m ny l chng thng cha nhiu acid bo t do gy kh khn cho qu trnh sn xut biodiesel thng qua qu trnh chuyn v ester [17]. Ngun nguyn liu th c t l triglyceride cao th ph hp hn. V d nh du thc vt l loi nguyn liu c t l triglyceride rt cao, do n c s dng sn xut biodiesel trong vi nm qua [18]. Tuy nhin, du thc vt cn l ngun tiu th ca con ngi. V vy s dng du thc vt sn xut biodiesel c th lm cho gi thnh du thc vt dng trong thc phm tng cao, t dn n gi thnh biodiesel cao v ngi tiu dng hn ch s dng d rng biodiesel c nhiu u im hn so vi diesel [80]. Du thc vt c s dng trong nhiu mc ch thng mi v kh nng cung ng du thc vt cho vic sn xut biodiesel th khng th p ng ni tim nng thng mi ca biodiesel trn mt khu vc. Mt v d c th l p ng c 10% ch tiu ti EU t vic sn xut quy m h gia nh, th ngun nguyn liu thc s cung cp khng cho nhu cu hin ti, v s i hi v din tch canh tc th vt xa tim nng pht trin t trng i vi cc loi cy trng sn xut nng lng sinh hc [68]. M rng trng trt, p lc trong s thay i s dng t trng v s gia tng cc cnh ng canh tc c th dn ti cnh tranh t ai v lm mt i s a dng sinh hc do s ln chim rng v cc vng sinh thi quan trng [64]. Biodiesel s khng cn c xem l mt gii php u vit khi n chim dng ngun cy trng phc v cho nhng li ch khc ca con ngi hay ngun nguyn liu ca n xm ln vo din tch rng v cc mi trng trng yu khc c lin quan mt thit n s a dng sinh hc [80]. Gi thnh v nhng kh khn trong vic thu nhn v tinh ch cc nhin liu ha thch cng nhng chnh sch cp khu vc cho ti quc gia s ngy cng thc y gia tng sn xut biodiesel hoc cc loi nhin liu ti sinh khc. V biodiesel c th tr thnh quen thuc i vi ngi tiu dng th gi c ca n phi cnh tranh c vi diesel thng thng. Do mc tiu t ra l phi gim bt c chi ph cho ngun nguyn liu th vn chim 60-70% tng chi ph nhin liu biodiesel [11]. khng phi cnh tranh vi ngun du thc phm, biodiesel nn c sn xut t cc ngun nguyn liu gi thnh r nh l cc ngun du phi thc phm, s dng du rn, m ng vt, du cn, v du nhn. Tuy nhin, khi lng du thi v m ng vt sn c th khng p ng nhu cu biodiesel hin nay. V vy, 10

1. TNG QUAN V BIODIESEL cng ngh sn xut biodiesel chuyn tip qua mt th h mi, th h th hai, ly ngun nguyn liu l sinh khi vi to, c th gp phn gim bt c nhng i hi bc thit v mt bng nh c hiu sut nng lng cao hn trn mi n v din tch t cng nh khng cnh tranh nh hng ti t nng nghip. Hn na, biodiesel cn phi c mc tc ng mi trng thp hn v bo m c cng cht lng so vi cc ngun nhin liu hin nay [61]. Thc t, vi to c hiu sut du cao nht trong s cc cy trng ly du a dng khc. C th so snh hiu sut nui trng vi to vi vic khai thc cc ngun sinh khi khc qua bng s liu sau: Bng 1. 2: So snh vi to vi cc ngun nguyn liu sn xut biodiesel khc [80] Hm lng t s Nng sut Nng sut du (% khi dng biodiesel (kg lipid (L lng sinh (m2.nm/kg biodiesel/ du/ha.nm) khi) biodiesel) ha.nm) 44 33 18 28 42 41 40 48 36 30 50 70 172 363 636 741 915 974 1070 1307 5366 58700 97800 136900 66 31 18 15 12 12 11 9 2 0.2 0.1 0.1 152 321 562 656 809 862 946 1156 4747 51927 86515 121104

Ngun

Bp/Ng (Zea mays L.) Gai du (Cannabis sativa L.) u nnh (Glycine max L.) Jatropha (Jatropha curcas L.) Camelina (Camelina sativa L.) Canola/Ci du (Brassica napus L.) Hng dng (Helianthus annuus L.) Hi ly (Ricinus communis) C du (Elaeis guineensis) Vi to (lng du thp) Vi to (lng du trung bnh) Vi to (lng du cao)

11

1. TNG QUAN V BIODIESEL gii quyt vn thiu ht nng lng ton cu, tm kim cc ngun nguyn liu sinh hc giu lipid sn xut biodiesel t hiu qu cao ang thu ht rt nhiu s quan tm. Cc vi sinh vt c cht du c k vng nh c chu k sinh trng ngn, hm lng lipid cao v d dng c ci to ging bi cc phng tin cng ngh sinh hc. Vi loi vi to c lit vo nhm vi sinh vt giu cht du ph hp vi nhu cu sn xut lipid [15]. Vi to c xem l i tng tim nng sn xut nhin liu bi rt nhiu u im nh l hiu sut quang hp cao, sinh khi ln v mc sinh trng cao hn khi so snh vi cc loi cy trng sn xut nng lng [50]. Trong s cc ngun sinh khi, vi to c xem l mt ngun sinh khi hin i v c nhiu tim nng nht hin nay bi v vi to c kh nng quang hp hiu qu hn bt k loi sinh khi thc vt no khc, m theo Hall in Ref, qu trnh quang hp ca vi sinh vt l qu trnh ti sinh s dng nng lng mt tri chuyn ha thnh mt dng nng lng d tr mi di dng cc lin kt ha hc, ngoi ra con ngi hon ton c th ch ng sn xut sinh khi vi to vi s lng ln. V vy nu c th thu hi nng lng vi nng sut cao t chng th sinh khi vi to c xem l mt ngun ti nguyn y ha hn sn xut nhin liu [67], v vi to c th c xem nh mt ngun nng lng thay th cho ngun nng lng ha thch ang cn kit dn [48]. Hn na, theo tiu chun biodiesel ca American Society for Testing Materials (ASTM), biodiesel t du vi to c cc thuc tnh tng t vi biodiesel tiu chun, v n cng an ton hn v c nhit pht chy cao. Bng 1. 3: So snh cc thuc tnh gia du t vi to, diesel thng thng v tiu chun ASTM biodiesel [28] Thuc tnh Khi lng ring (kg/L) nht (mm2/s, cSt ti 400C) im pht chy (0C) im ng c (0C) Ch s acid (mg KOH/g) Gi tr nhit nng T l H/C Biodiesel t vi to 0.864 5.2 115 -12 0.374 41 1.81 12 Diesel 0.838 1.9-4.1 75 -50-10 Ti a 0.5 40-45 1.81 Tiu chun biodiesel ASTM 0.84-0.90 3.5-5.0 Ti thiu 100 Ti a 0.5 -

1. TNG QUAN V BIODIESEL Nhng u im khi nui cy vi to nh ngun ti nguyn sinh khi: To c xem l cc c th sng c kh nng thu nhn nng lng mt tri to ra cc hp cht hu c rt hiu qu [83]. To c xp vo loi thc vt khng c h mch dn, a phn u thiu c quan sinh sn phc tp [83]. Vi to d dng nui cy sn xut mt s hp cht c th chn lc, c gi tr kinh t vi nng cao nh protein, carbohydrate, lipid v cc sc t da vo cc iu kin sinh trng a dng [83]. T c th ti u ha mi trng thu c sinh khi vi hm lng lipid cao. Vi to thuc vo nhm vi sinh vt sinh sn theo chu k phn i t bo [83]. Vi to c th sng c mi trng nc bin, nc l hoc nc ngt. Mc d vi to sng trong mi trng nc, nhng khng i hi cn nhiu nc nh cc loi cy trng khc [43]. Mt khc, vi to cng c th thch nghi vi mi trng nc thi, do khng cn thit phi tn chi ph s dng nc sch [80]. H thng sn xut sinh khi to d dng thch nghi cc quy m v k thut khc nhau [83]. Vic nui thu sinh khi vi to khng i hi nhiu din tch nh khi trng cc loi cy ly du khc [15], v nng sut sinh khi vi to cng khng ph thuc vo thi tit hay nh hng ca mi trng [43]. Biodiesel sn xut t vi to khng lm nh hng n vic sn xut thc phm v cc sn phm khc t thc vt [28]. Vi to c mc sinh trng rt nhanh, chu k sinh trng hon tt ch trong vi ngy [71], v c rt nhiu loi to cha nhiu du [28], nng sut du trn mi n v nui cy vi to c th cao vt tri hn so vi nng sut du ca cy c ht cha hm lng du nhiu nht [43]. Thng thng cc loi vi to c hm lng du vo khong 20-50% [15]. V d nh loi to Chlorella protothecoides khi nui theo phng thc d dng c th tch ly lipid t 55% khi lng kh ca t bo sau 144h nui cy trong mi trng c b sung bt cao ng trong fermenter [87]. Sn xut sinh khi vi to c th c xem l mt phng php c nh trc tip kh thi CO2 v vi to s dng CO2 nh ngun Carbon nh kh nng quang hp (1kg sinh khi kh i hi cn c 1.8kg CO2) [43]. Nui cy vi to khng cn dng cc loi thuc xt c hay thuc tr su [43].

13

1. TNG QUAN V BIODIESEL Cc phn sinh khi vi to cn d li sau qu trnh trch ly du c th c dng nh ngun thc n cho gia sc, hoc lm phn bn, hoc qua qu trnh ln men to cc sn phm ethanol hay methane [43]. Ton b qu trnh t vic nui thu sinh khi c hm lng lipid cao n sn xut biodiesel t du vi to u c nghin cu. iu kin phng th nghim, hm lng lipid l tng c th t ti 5660% trn tng sinh khi kh nh vo k thut di truyn hoc k thut nui d dng. Nhng tin b k thut cho thy rng trong tng lai vic sn xut biodiesel t vi to c th c thc hin trn quy m cng nghip [28].

14

2. NUI VI TO SN XUT BIODIESEL

2. NNG SUT LIPID V NH HNG CA MI TRNG LN S TCH LY LIPID CA MT S LOI VI TO


2.1. Cc loi vi to c cha nhiu lipid
Mt s loi vi to c th tch ly lipid t n 75% (w/w) sinh khi kh nhng nng sut thp, nh l Botryococcus braunii. Mt s loi vi to khc rt ph bin cho vic sn xut lipid l Chlorella, Crypthecodinium, Cylindrotheca, Dunaliella, Isochrysis, Nannnochloris, Nannochloropsis, Neochloris, Nitzschia, Phaeodactylum, Porphyridum, Schizochytrium, Tetraselmis v c nng sut sinh khi cao nn nng sut lipid cao hn nhiu mc d hm lng lipid ch t t 2050% sinh khi kh [80]. Ngoi ra, thnh phn lipid trong t bo vi to cng khc nhau ty theo chng loi. y thc s cng l mt im ng lu khi la chn cc loi vi to v s nh hng ng k n c tnh ca biodiesel c sn xut t lipid ca chng. Thnh phn lipid trong vi to gm c cc acid bo bo ha v cha bo ha, cha 12-22 nguyn t carbon, v mt s thuc h 3 v 6 [80]. Bng 2. 1: Hm lng v nng sut lipid ca cc loi to khc nhau [80] Cc loi vi to nc mn v nc ngt Chlorella emersonii Chlorella sorokiniana Chlorella sp. Chlorococcum sp. Nannochloris sp. Nannochloropsis oculata Nannochloropsis sp. Neochloris oleoabundans Scenedesmus sp. Spirulina maxima Tetraselmis suecica Tetraselmis sp. Hm lng lipid (% sinh khi kh) 25.0-63.0 19.0-22.0 10.0-48.0 19.3 20.0-56.0 22.7-29.7 12.0-53.0 29.0-65.0 19.6-21.1 4.0-9.0 8.5-23.0 12.6-14.7 Sinh khi kh (g/L/ngy) 0.036-0.041 0.23-1.47 0.02-2.5 0.28 0.17-0.51 0.37-0.48 0.17-1.43 0.03-0.26 0.21-0.25 0.12-0.32 0.30 Nng sut lipid (mg/L/ngy) 10.3-50.0 44.7 42.1 53.7 60.9-76.5 84.0-142.0 37.6-90.0 90-134.0 40.8-53.9 27.0-36.4 43.4

15

2. NUI VI TO SN XUT BIODIESEL Theo s liu trn, ta thy trong s cc loi vi to c hm lng lipid cao, Nannochloropsis oculata l mt tim nng sng gi. Mc d hm lng lipid tnh theo phn trm sinh khi kh ch trong khong 22.7-29.7%, thp hn mt s loi nh Chlorella emersonii, Chlorella sp., Nannochloris sp., Neochloris oleoabundans, nhng do Nannochloropsis oculata nui theo phng php t dng c sc sinh sn cao, lng sinh khi trong ngy ln, nng sut lipid ca loi ny c gi tr cao ng k. V vy c th xem Nannochloropsis oculata l mt loi vi to rt c tim nng trong vic sn xut biodiesel.

2.2. Mt s nghin cu v nh hng ca iu kin mi trng ln s tch ly lipid vi to


Nng sut du ca vi to l khi lng du c sn xut trn mi n v th tch canh trng vi to mi ngy, ph thuc vo mc sinh trng ca to v hm lng lipid c trong sinh khi. Xem xt qua mt vi kt qu nghin cu, ta c th thy rng hm lng v c tnh ca lipid trong t bo vi to rt a dng, ty thuc vo s thay i ca iu kin sinh trng (nh nhit v cng chiu sng) hay thnh phn dinh dng trong mi trng nui cy (nh nng nitrogen, phosphate v st) [34, 44]. 2.2.1. Yu t nhit Nhit nh hng su sc n s sinh trng v s tch ly cc thnh phn ha sinh trong t bo vi to, c bit l s tch ly lipid v s thay i thnh phn lipid ni bo. Mt v d c th l Chlorella vulgaris trong th nghim ca Attilio v cc cng s: loi vi to ny c nui trong mi trng c bn Bold, s dng CO2 c sn trong khng kh (khong 300ppm) v NaNO3 l nhng ngun cung cp carbon v nitrogen duy nht trong sut qu trnh th nghim, nui cy theo phng thc quang t dng trong 14 ngy di nh sng lin tc c cng 70E/m2s, cc nhit 25, 30, 35 v 380C [5]. S sinh trng ca C. vulgaris b nh hng mnh m khi nhit trn 30 C. 350C, loi vi to ny bt u b c ch, c th l mc sinh trng c trng gim 17% so vi 300C. Khi tng nhit ln cao hn na (380C), s
0

sinh trng ca C. vulgaris b ngng t ngt, sau cc t bo vi to b cht. iu ny rt d nhn bit v cc t bo s thay i mu sc, chuyn t mu xanh sang mu nu, v mc sinh trng ca t bo vi to c kt qu l s m [5]. 16

2. NUI VI TO SN XUT BIODIESEL Khi nhit sinh trng gim t 300C xung cn 250C th hm lng lipid trong t bo C. vulgaris tng, t 5.9 ln 14.7%, trong khi mc sinh trng vn duy tr khng i. Kt qu nng sut lipid tng t 8 ln n 20mg/L.ngy [5]. Bng 2. 2: S sinh trng v sn xut lipid ca C. vulgaris ti cc nhit khc nhau [5] Nhit (0C) 25 30 35 38 - Tc sinh trng c trng (1/ngy) 0.14 0.00 0.14 0.00 0.12 0.01 -0.01 0.01 Sn lng lipid (glipid/100gsinh khi kh) 14.71 0.30 5.90 0.42 5.60 0.59 11.32 0.20 Nng sut lipid (mglipid/L.ngy) 20.22 0.60 8.16 0.65 8.21 0.17 -2.72 1.62

Hnh 2. 1: Phn trm cc loi FAME trn tng lng FAME (g/100gFAME) ca C. vulgaris ti cc nhit sinh trng khc nhau [A4]

(FAME: fatty acid methyl ester) Sau qu trnh chuyn v ester, phn tch cc FAME th thy lng acid palmitic (C16:0) trong C. vulgaris chim 60% (mol/mol) trn tng lng lipid [5]. Mt iu ng ghi nhn na l khi tng nhit vt qu nhit nui cy ti u (34-380C), hm lng acid oleic trong C. vulgaris s tng cao ng k [5]. Xem xt thm cc kt qu t nhng nghin cu khc cng c th chng minh rng nhit c mt tm tc ng ng k trong qu trnh nui cy cc loi vi to. Mi loi vi to thch ng vi mt ngng nhit nht nh v cc nhit khc nhau th thnh phn ha sinh trong t bo l khc nhau. 17

2. NUI VI TO SN XUT BIODIESEL Nh trong th nghim ca Susan M. Renaud v cc cng s, bn loi vi to nhit i Australian bao gm to ct Chaetoceros (CS256), 2 loi cryptomonad: Rhodomonas (NT15) v Crytomonas (CRF101) v 1 loi prymnesiophyte khng xc nh (NT19) c nui 5 nhit khc nhau. Th nghim c thit k theo mi trng f/2 nhit 25, 27, 30, 33 v 350C (nng mui 25 , pH 8.3, mt photon 80 mol/m2.s; chu k sng : ti l 12 : 12 h) [62]. Ba loi Cryptomonas sp., Rhodomonas sp., v prymnesiophyte (NT19) t mc sinh trng ti u vo khong nhit t 250C n 300C, mc sinh trng s chm hn v s lng t bo gim khi nhit vt trn 300C. iu ny th tng ng vi cc nghin cu trc y trn cc loi to khc nh Isochrysis sp., Nitzshia closterium, N. paleacea [63], Ochoromas danica, Chaetocerso gracilis, Porphyridium cruentum, Chlorella sp., v Nannochloropsis sp. [62]. C th l nhit ti u vo khong 25-270C i vi loi Rhodomonas sp. (mc sinh trng c trng, =0.27/ngy) v 27-300C i vi prymnesiophyte NT19, Cryptomonas sp., Chaetoceros sp., v Isochrysis sp. (=0.56, 0.33, 0.87 v 0.97/ngy). Khi nhit vt qu 300C hoc 330C th tng ng Cryptomonas sp. v Rhodomonas sp. s ri vo phase suy vong, cc qu trnh chuyn ha trong t bo b ngng t ngt v t bo vi to s cht. Ch c Chaetoceros sp. c kh nng chu c nhit cao, vn duy tr mc sinh trng bnh thng 33 v 350C (>0.78/ngy). Ti 300C Chaetoceros sp. c mt t bo cao nht, do lng sinh khi cng t cao nht (0.17g/L) [62]. Khi tng nhit mi trng nui cy, s tng hp lipid ca ba loi Chaetoceros sp., Rhodomonas sp., v prymnesiophyte NT19 c xu hng gim, lng lipid gim tuy nh, nhng xt trn t l th li rt ng k. Kt qu ny tng t i vi Chaetoceros calcitrans, C. simplex [81] v Nitzschia sp. [63]. Tuy nhin cng c nhng kt qu phn nh ngc li: i vi mt s loi vi to khc s tng hp lipid s tng khi nhit cao [81, 54]. Chaetoceros sp. c t l v lipid cao nht (16% DW; P<0.01) khi nui cy 25C, trong khi Rhodomonas sp., Cryptomonas sp., NT19 v Isochrysis sp. c hm lng lipid cao ng k nhit trong khong 27-300C (ln lt l 15.5, 12.7, 21.4 v 21.7% DW, P<0.05) [62]. T thy rng vic la chn nhit ph hp cho s sinh trng v tch ly lipid khi nui cy vi to nhm sn xut biodiesel l rt quan trng.

18

2. NUI VI TO SN XUT BIODIESEL 2.2.2. Yu t thnh phn mi trng Hm lng lipid trong vi to c th tng ln di cc iu kin nui cy khc nhau nh s thiu ht nitrogen [34, 79, 42], cng chiu sng mnh [37], nhit thp [62], nng mui cao [78], v nng st cao [44]. Di cc iu kin kch thch, nhiu loi vi to c th p ng bng cch gia tng hm lng lipid, thng thng t 30% ti 60% (w/w) t bo kh [14]. 2.2.2.1. Thnh phn Carbon C hai loi ngun carbon phi hu c (CO2) ln carbon hu c (glucose, acetate) u c vi to s dng sn xut lipid. Do vi to c kh nng s dng CO2 nh ngun carbon chnh trong thnh phn dinh dng nh vo kh nng quang hp, con ngi hng n vic c lp CO2 t cc ngun kh thi tn dng cho qu trnh quang hp ca vi to, mt mt gim c chi ph trong qu trnh nui cy vi to, mt khc l gim thiu CO2 trong khng kh, p ng nhim v ci thin mi trng v khc phc hin tng nng dn ln ca tri t do cc loi kh nh knh [12]. Theo c tnh, sn xut c 100 tn sinh khi vi to th c khong 180 tn CO2 c s dng trong qu trnh quang hp di iu kin nh sng t nhin hoc nhn to [5]. vic sn xut biodiesel v lm gim CO2 trong khng kh t hiu qu, cc loi vi to phi c chn la tha mn cc yu cu: sinh trng mnh, hm lng lipid nhiu v c kh nng thch nghi tt khi mc CO2 cao [12]. Khi kho st yu t nng CO2 i vi mc sinh trng v s tch ly lipid trn mt s loi to c la chn nhm sn xut lipid, Chan Yoo v cc cng s t c mt s kt lun nh sau [12]: Ba loi to Scenedesmus sp., Chlorella sp., v Botryococcus braunii c nui trong iu kin nhit 25 10C, chiu sng lin tc 150mol/m2s trong 2 tun. rt ngn thi gian phase lag, vi to trc c nui trong mi trng sc khng kh c 2% CO2 trong 1 tun trc khi cy mu. Mi trng nui cy thc s th s dng khng kh cha 10% CO2 hoc kh thi t ng khi vi vn tc 0.3v/v/m, nng CO2 l 5.5%. i vi mc sinh trng ca t bo khi nng CO2 l 10% Trong ba loi c kho st th Scenedesmus sp. l loi t nng sut sinh khi cao nht. Sinh khi ti a t c l 3.13g/L vo ngy th 14, nng sut sinh khi t 217.50 11.24 mg sinh khi kh/L.ngy. Trong mt nghin 19

2. NUI VI TO SN XUT BIODIESEL cu khc ca Morais v Costa [51], i vi hai loi Chlorella sp. v Scenedesmus sp. c lp t nh my nhit in than , nng sut sinh khi ca chng cng kh cao khi nng CO2 t 6-12%. Nng sut ti a ca hai loi ny vi 6% CO2 ln lt l 87 v 85mg sinh khi kh/L.ngy. Hn na, hai loi to ny cng c th sng trong mi trng cha CO2 vi nng ln ti 18%, v nng sut sinh khi cao nht ca loi Scenedesmus obliquus l 140mg sinh khi kh/L trong mt ngy vi nng CO2 l 12% [50]. Qua thy rng, khi nui 10% CO2, nng sut sinh khi ca Scenedesmus sp. cao gp 2-3 ln so vi cc nghin cu nng khc, v c ba loi to c kho st nng CO2 ny u khng gp phi bt k nh hng xu no. Tuy nhin, B. braunii li c mc thch nghi chm vi nng CO2 10%, do mc sinh trng ca loi ny thp hn. Sc kh nng cao hoc sc kh lin tc CO2 2% s lm gim ng k pH ca mi trng [60]. Lee v cc cng s [41] xut rng khi tng mt t bo ban u th s tng c sc chu dng ca vi to i vi nng CO2 cao v gim c thi gian thch nghi. Nh vy, nu B. braunii c cho thch nghi vi nng CO2 thp trc , th mc sinh trng v sn lng sinh khi s c th gia tng m khng cn qua tri qua thi k thch nghi [12]. i vi lipid v acid bo khi nng CO2 l 10% Hm lng lipid tng trong vi to theo nghin cu ca Chan Yoo v cc cng s t t 6.25% ti 25.79% khi lng kh. T ngy th 7 n ngy th 14, cc gi tr c xu hng gim. Hm lng lipid trong Scenedesmus sp. th thp hn so vi B. braunii, nhng do Scenedesmus sp. c nng sut sinh khi cao nn c nng sut lipid tng cao nht trong ba loi kho st. Bng 2. 3: Nng sut sinh khi v nng sut lipid ca Botryococcus braunii, Chlorella vulgaris v Scenedesmus sp. khi nui 10% CO2 trong 14 ngy [12]. Loi B. braunii C. vulgaris Scenedesmus sp. Nng sut sinh khi (mg sinh khi kh/L.ngy) 26.55 7.66 104.76 10.73 217.50 11.24 Nng sut lipid tng (mg/L.ngy) 5.51 1.53 6.91 0.03 20.65 0.13

Trong khi hm lng lipid ca c hai loi Scenedesmus sp. v C. vulgaris u thp hn 11.92%, hm lng lipid tng ca B. braunii vo ngy th 7 v ngy th 14 ln lt l 25.79% v 21.10%. Cc kt qu khc li cho thy 20

2. NUI VI TO SN XUT BIODIESEL rng, hm lng lipid tng c th t c t 20-50% l kh ph bin, thm ch mt s loi vi to c th tch ly lipid ti 80% (w/w) sinh khi kh. V nui cy trn cc iu kin mi trng khc nhau cng c th gia tng c hm lng lipid tch ly trong t bo vi to [44]. Hm lng lipid tng ca B. braunii trong phase cn bng sm th cao hn trong phase sinh trng theo hm s m (phase log). Mc d hm lng lipid ca c ba loi to c Chan Yoo kho st vi nng 10% CO2 c thp hn mt cht so vi kt qu nghin cu trong cc bn bo co khc, nhng ch xt trn phase cn bng th hm lng ny li cao hn [12]. Bng 2. 4: Thnh phn cc acid bo ca Botryococcus braunii, Chlorella vulgaris v Scenedesmus sp. khi nui cy 10% CO2 trong 14 ngy [12]. C16:0 C16:1 C18:0 C18:1 C18:2 (% w/w acid (% w/w acid (% w/w acid (% w/w acid (% w/w acid bo) bo) bo) bo) bo) 29.5 0.8 24.0 0.1 36.3 7.1 3.4 0.3 2.1 0.0 4.0 0.8 1.0 0.2 1.3 0.2 2.7 0.7 44.9 0.4 24.8 1.5 25.9 4.5 21.2 0.4 47.8 1.6 31.1 11.5

Loi B. braunii C. vulgaris Scenedesmus sp.

Trong s cc acid bo, th acid palmitic, acid oleic, acid linoleic c xem l thnh phn chnh vi t l t 21% ti 45% tn tng hm lng lipid trong ba loi to, cc acid palmitoleic v acid stearic ch l th yu, t l chim rt thp. c bit, acid oleic, l acid bo c xem nh l thnh phn chnh v rt l tng sn xut biodiesel, th li chim ti 44.9% hm lng lipid tng trong B. braunii [12]. T l ny l rt cao khi so snh vi t l acid oleic t c l 28% trong bn bo co ca Ranga Rao v cc cng s [60]. V vy, c th kt lun rng, nng CO2 10% l mt nng kh ti u nui cy vi to thu lipid nhm mc ch sn xut biodiesel. khc phc nhc im hm lng lipid tng hi thp, ta c th s dng nng 10% CO2 kt hp vi mt s yu t dinh dng ti u khc, vn chnh l nng ny c th thu c thnh phn lipid ph hp cho yu cu sn xut biodiesel cht lng cao, nht l trn mt s chng c th c la chn. Vn sinh trng chm ca mt s loi nh B. braunii, ta c th p dng mt s bin php ci thin nh tin thch nghi ging vi nng CO2 thp rt ngn thi gian thch nghi khi nui cy nng CO2 cao. 21

2. NUI VI TO SN XUT BIODIESEL Khi nui bng kh thi t ng khi, nng CO2 5.5% Hai trong ba loi to nu trn, B. braunii v Scenedesmus sp. c nui hai ch so snh, l sc khng kh v dng kh thi t ng khi c nng CO2 l 5.5%. Nng sut sinh khi ca B. braunii v Scenedesmus sp. vi kh thi t nh my ln lt l 77 v 203mg/L.ngy. Nng sut lipid ca B. braunii v Scenedesmus sp. t 21 v 39mg/L.ngy, tng ng vi hm lng lipid l 24 v 18% [12]. Qua chng t rng B. braunii vn gi c hm lng lipid cao khi nui bng kh thi nh khi nui bng kh cha 10% CO2. Sinh khi v hm lng lipid trong cc chng Scenedesmus sp. th tng ng khi nui 5% CO2 [66]. Thnh phn acid bo ca B. braunii v Scenedesmus sp. khi nui sc khng kh v kh thi ng khi th gn nh khng thay i, c trng cho mi loi. T l acid oleic trong B. braunii ln lt l 56 v 59% lng acid bo tng khi nui bng khng kh v kh thi. V vy kt lun rng c th s dng kh thi t ng khi nui B.braunii nhm sn xut biodiesel v c hm lng lipid nhiu v trong thnh phn acid oleic chim t l rt cao [12]. 2.2.2.2. Thnh phn Nitrogen Nitrogen l mt yu t nh hng su sc n s chuyn ha v tch ly lipid trong nhiu loi vi to. Thm vo , iu khin nng nitrogen kh d dng v y l mt yu t c chi ph thp khi so snh vi cc yu t khc [14]. V vy, thnh phn v hm lng nitrogen b sung vo mi trng nui cy vi to tng nng sut lipid c nhiu nh khoa hc nghin cu. Nh i tng Chlorella vulgaris, khi c nui trong mi trng c bn Bold, s dng CO2 c trong khng kh (khong 300ppm) v NaNO3 l nhng ngun cung cp carbon v nitrogen duy nht trong sut qu trnh th nghim [5], nui cy theo phng thc quang t dng trong 14 ngy di nh sng lin tc c cng 70E/m2s, nhit 300C. Nng nitrogen trung bnh trong mi trng nui cy Chlorella vulgaris theo Guillard l 1.50g/L, do khi kho st s gim nng nitrogen kch thch s tch ly lipid, Attilio v cc cng s nghin cu 2 nng khc l 0.750 v 0.375g/L so snh vi nng trung bnh [5]. iu kin hm lng nitrogen thp trong mi trng nui cy thc s lm gia tng ng k s tch ly lipid trong vi to [34]. V vy hm lng nitrate ln lt c gim i cn v

22

2. NUI VI TO SN XUT BIODIESEL so vi nng tiu chun trong khi cng chiu sng v tc sc kh vn gi nguyn trong sut cc th nghim. nh hng ca vic gim thiu nng NaNO3 trn s sinh trng ca C. vulgaris c tm tt trong bng sau: Bng 2. 5: Tham s sinh trng v s sn xut lipid ca C. vulgaris cc nng NaNO3 khc nhau [5] NaNO3(g/L) 1.500 0.750 0.375 -Tc sinh trng c trng (1/ngy) 0.140.00 0.140.01 0.140.00 Sn lng lipid (glipid/100gsinh khi kh) 5.900.42 14.370.64 15.310.51 Nng sut lipid (mglipid/L.ngy) 8.160.65 20.440.75 20.300.40

Qua ta thy, trong khi tc sinh trng ring ca C. vulgaris thay i khng ng k th hm lng lipid tng gp 3 ln khi gim nng nitrate t 1.500g/L cn 0.375g/L. Khi thay i nng NaNO3, hm lng acid palmitic khng thay i ng k [5]. Tuy nhin, hm lng acid linolenic acid (C18:3) trong C. vulgaris th ph hp vi tiu chun biodiesel s dng cho cc phng tin vn chuyn Chu u (12%, mol/mol) [38].

Hnh 2. 2: Phn trm cc loi FAME trn tng lng FAME (g/100gFAME) ca C. vulgaris ti cc nng NaNO3 khc nhau [A4]

(FAME: fatty acid methyl ester) Tuy nhin trong k thut nui cy vi to, ngun nitrogen c th c s dng di nhiu dng khc nhau, nh l ammonia, nitrate, nitrite v urea. Ty 23

2. NUI VI TO SN XUT BIODIESEL thuc vo c tnh loi, vic s dng cc ngun nitrogen khc nhau em n nhng hiu qu khc nhau. Nh i vi Ellipsoidion sp, s dng ammonium lm ngun cung cp nitrogen s em li mc sinh trng v hm lng lipid cao hn khi s dng urea v nitrate [88]. Neochloris oleoabundans khi nui bng nitrate s sinh trng nhanh hn v tch ly lipid nhiu hn khi nui vi urea [42], nhng t bo li sinh trng yu trong mi trng s dng ngun nitrogen l ammonium. Thm vo , nhiu nghin cu cho thy rng c ba loi ngun nitrogen l nitrate, ammonium v urea u c th p ng tt i vi Spirulina platensis [16, 74]. Trong s cc ngun nitrogen hu c, urea ng mt vai tr quan trng v thng c s dng trong nui cy vi to trn quy m ln bi v gi thnh ca urea th thp hn nhiu khi so vi cc ngun nitrogen khc [14]. Ngoi ra, ch nui cy nh hng ti s sinh trng v thnh phn ha sinh trong t bo vi to. S dng phng php nui cy fed-batch iu chnh mc b sung mi trng nhm gia tng nng sut [14]. Ci thin nng sut lipid c th thc hin bng bin php nui cy fed-batch b sung gin on ngun nitrogen [79]. Thm vo , trong phng php nui cy bn lin tc, nng cht dinh dng ban u v mc khi phc cng c s dng bin i cc thnh phn ha sinh trong t bo vi to [56, 22].

24

3. NUI N. OCULATA SN XUT BIODIESEL

3. NUI VI TO NANNOCHLOROPSIS OCULATA THU LIPID NHM SN XUT BIODIESEL


3.1. Nannochloropsis oculata
3.1.1. Phn loi Ngun gc: thut ng Nannochloris bt ngun t ch Nannochlorid, v vy th chnh xc l Nannochloridopsis. V Nannochloropsis l mt cm t vit tt t thut ng trn [32]. Nannochloropsis oculata thuc vo lp to Eustigmatophyceae,

b Eustigmatales, h Monodopsidaceae, chi Nannochloropsis [32]. S phn loi Nannochloropsis tng mc phi nhiu nhm ln. y l loi to c kch thc rt nh (nh hn 5 m), mu hi xanh, khng c giai on giao t chuyn ng sinh sn hu tnh. Khi phn loi hnh thi c th sinh vt ca chi to ny, cc nh khoa hc xem xt da trn nhng nhm chnh l Chlorophyceae, Tribophyceae v Eustigmatophyceae. S dng knh hin vi quang hc phn bit hnh thi gia cc loi to trn gp phi nhiu hn ch, do dn n cc nhn nh sai lm [32]. Hu ht cc to c hnh dng chung chung nh thuc to n bo, c lp lc lp n ti vch t bo, vch t bo trong sut v cu trc thiu cht nhy bao quanh, v ch sinh sn theo kiu chia i thnh 2 phn bng nhau u c lit vo nhng loi ca chi Nannochloris Naumann thuc Chlorophyceae [53]. Nannochloropsis c Droop pht hin ra vo nm 1955 ti cc vng nc l b mt trong cc h gia nhng rng . Antia v cc cng s [3] pht hin ra nhng im khng tng thch trong s phn loi ca Droop [32] da trn c s thnh phn cc hp cht trong lc lp v vi cu trc, qua N. coccoides c xp vo lp Chlorophyceae trong khi N. oculata c xp vo Eustigmatophyceae. Nannochloropsis l mt chi mi bao gm cc loi rt nh thuc lp Eustigmatophyceae, trong ph bin nht l loi Nannochloropsis oculata. 3.1.2. c im hnh thi Nannochloropsis oculata l loi to n bo, t ni trn mi trng lng. T bo c dng hnh cu vi ng knh khong 2-4m, hoc hnh tr vi kch thc l 3-4x1.5m [32]. 25

3. NUI N. OCULATA SN XUT BIODIESEL Nannochloropsis oculata c mt lp mng ngoi mng hin din cc giai on nht nh trong chu k sinh trng ca t bo, thnh t bo trong sut v khng to hnh dng xc nh, mang mt lp n cc sc t dip lc mu vng xanh, l sc t c trng ca nhm Eustigmatophyceae, khng c lp mng nhy bn ngoi [32]. 3.1.3. c im sinh l Nannochloropsis sp. l mt chi thuc dng phiu sinh vt t dng, trong t bo c cc hp cht chlorophylls [32]. Nannochloropsis oculata l loi vi to sng ti u trong mi trng nc mn, tuy nhin i khi cng hin din trong mi trng nc ngt hay nc l v s thay i v hnh thi l khng ng k my [36]. Nannochloropsis oculata c kh nng sng trong mt di nhit kh rng. Thch nghi tt trong iu kin mi trng c nhit thp, thm ch l gn 00C [36]. iu kin sinh trng tt nht ca Nannochloropsis oculata khong 210C, mc chiu sng 52mol photon/m2s, pH 8.4, mc sc kh 14.7 vvh [57]. Nannochloropsis oculata thuc vo th n bi [36], sinh sn v tnh theo kiu phn i theo chiu ngang [32]. 3.1.4. c im sinh ha Khi iu kin dinh dng y , Nannochloropsis sp. thng c khuynh hng u tin s chuyn ha carbon thnh protein [52]. Tuy nhin, di cc iu kin thay i khc nhau, t bo vi to b kch ng, s c nhiu carbon c chuyn ha thnh lipid v carbohydrate. nh hng ca cc yu t mi trng s tc ng ln thnh phn lipid v cc acid bo c trong t bo vi to [77]. Cc acid bo ch yu c trong Nannochloropsis sp. l C14:0, C16:0, C16:1, C20:4 v C20:5. Ngoi ra cng c s hin din ca mt s acid bo ph khc nh C18:0, C18:1, C18:2 v C18:4. Cc acid bo ny tn ti ba dng lipid trong t bo vi to, l: galactolipid, phospholipid v lipid trung tnh. Galactolipid ca Nannochloropsis sp. giu cc acid bo C20:5 v C20:4, kt hp vi C16:0 v C16:1, mt lng nh C14:0. Lipid trung tnh ca Nannochloropsis sp. th ch yu l cc triacylglycerol, gm cc acid bo C16:0 v C16:1, mt t acid bo C14:0 v C18:0. Phospholipid ca Nannochloropsis sp. giu C16:1, mt lng ng k C18:1 v C18:4, hm lng C16:0 thp [77]. 26

3. NUI N. OCULATA SN XUT BIODIESEL

45 40

35
%w/w lipid tng 30 25 20 15 10 5 C14:0 C16:0 C16:1 C18:0 C18:1 C18:2 C18:4 C20:4 C20:5 Lipid tng Lipid trung tnh Galactolipid Phospholipid

Hnh 3. 1: Thnh phn acid bo ca cc nhm lipid chnh trong Nannochloropsis sp. [77] th trn chng t rng Nannochloropsis sp. l loi vi to quang t dng ph hp cho vic sn xut acid eicosapentaenoic (C20:5; EPA). V vy, Nannochloropsis sp. c xem l ngun acid bo cha bo ha 3 quan trng cho con ngi [13]. Ngoi ra, t l acid palmitoleic (C16:1) cng kh cao. Di cc iu kin mi trng khc nhau, t l cc thnh phn lipid v acid bo trong t bo vi to c th thay i [77]. Ngha l ta hon ton c kh nng iu khin c qu trnh sn xut lipid trong t bo Nannochloropsis sp. khi tm c mi trng ti u tng ng. V vy, c th xem Nannochloropsis oculata l mt loi tim nng dng sn xut lipid phc v cho biodiesel.

3.2. xut m hnh th nghim nghin cu s tch ly lipid theo iu kin mi trng Nannochloropsis oculata
3.2.1. Yu t nhit 3.2.1.1. Nghin cu ca Attilio Converti, 2009 Attilio Converti v cc cng s lm mt nghin cu v s nh hng ca nhit ln mc sinh trng v hm lng lipid i vi Nannochloropsis oculata nhm ng dng cho vic nui cy loi vi to ny phc v cho sn xut biodiesel. Trong nghin cu ny, Attilio Converti v cc cng s thit lp cc th nghim nui cy N. oculata ti cc nhit 15, 20 v 250C trong mi trng f/2 ca Guillard, s dng ngun cung cp 27

3. NUI N. OCULATA SN XUT BIODIESEL carbon l CO2 c sn trong khng kh vi tc sc kh khong 300ppm, v ngun cung cp nitrogen l NaNO3. Cc m nui cy t dng thc hin trong 14 ngy di nh sng lin tc c mt dng photon l 70E/m2s. Mi th nghim lp li hai ln [5]. Mc sinh trng v s sn xut lipid Thay i iu kin sinh trng ti u ca N. oculata (200C) dn n kt qu lm bin i ng k mc sinh trng ca loi vi to ny. Bng 3. 1: S sinh trng v sn xut lipid ca N. oculata ti cc nhit khc nhau [5] Nhit (0C) 15 20 25 - Tc sinh trng c trng (1/ngy) 0.06 0.00 0.13 0.00 0.07 0.01 Sn lng lipid (glipid/100gsinh khi kh) 14.92 0.82 7.90 0.21 13.89 0.61 Nng sut lipid (mglipid/L.ngy) 9.11 0.30 10.01 0.22 10.10 2.09

Khi nhit di mc ti u, mc tng trng gim hn mt na, t 0.13 gim cn 0.06/ngy. S gim r nt ny cng c ghi nhn khi nui cy nhit cao, 250C [5]. S gim thiu ng k ny chng t rng N. oculata mc d c th thch nghi trong mt ph rng nhit nh nu trong phn trn, nhng l mt loi kh nhy cm khi sinh trng ti cc nhit khc nhau. V vy, nu nhit cng h thp hoc cng tng cao th mc sinh trng ca loi vi to ny cng b hn ch. S thay i nhit li c nh hng tch cc ln s tch ly lipid i vi N. oculata. Nhit ti u cho s sinh trng ca loi vi to ny l 200C, nhng khi tng hoc gim nhit nui cy, sn lng lipid tch ly trong vi to tng ln ng k, gn nh gp i so vi 200C [5]. Nhit thay i nh hng khng tt n mc sinh trng c trng nhng li c tc ng tch cc ln s tng hp v tch ly lipid trong t bo vi to. Chnh v th nng sut lipid ca N. oculata khi nui cy ti cc nhit khc nhau li gn nh tng ng nhau, khng hn km nhau nhiu lm. Kt qu cho thy, ti 250C, nng sut lipid ca N. oculata t gi tr cao nht, c th ln n 12.19mg/L.ngy [5]. S thay i hm lng cc acid bo trong thnh phn lipid 28

3. NUI N. OCULATA SN XUT BIODIESEL

Hnh 3. 2: Phn trm cc loi FAME trn tng lng FAME (g/100gFAME) ca N. oculata ti cc nhit khc nhau [A4]

(FAME: fatty acid methyl ester) Qua th trn ta thy, ti 150C, hm lng C16:0 v C18:3 kh cao. Cn hm lng C18:1 li rt thp. Khi nhit tng ln 250C, hm lng C18:1 tng ln nhiu (khong 20%), trong khi C16:0 v C18:3 li c xu hng gim [5]. 3.2.1.2. Nghin cu ca Hanhua Hu v Kunshan Gao, 2006 Hanhua Hu v Kunshan Gao cng kho st s sinh trng v thnh phn lipid ca Nannochloropsis sp. ti ba mc nhit khc nhau: 140C, 220C v 300C. Trong nghin cu ca mnh, Hanhua Hu v Kunshan Gao thit lp th nghim nui cy Nannochloropsis sp. ti cc iu kin nh sau: mi trng nui cy f/2 nc bin nhn to (f/2AW: f/2 artificial seawater), mc sc kh 200mL/pht vi hm lng CO2 cao (2800L/L), mc chiu sng 50mol/m2s, thu mu vo ngy th 10 sau khi cy ging vo mi trng. Mi th nghim lp li 3 ln [31]. Mc sinh trng v thnh phn hp cht ha hc trong t bo Nannochloropsis sp. c kh nng sinh trng tt trong khong nhit 14-300C [31]. Sn lng sinh khi kh (mg/L) ca c ba ch nhit khng c s thay i ng k, ngoi tr 300C mc sinh trng c s gim nh so vi nhit ti u l 220C [31]. Khi nhit mi trng thay i ra khi khong nhit ti u, d cao hn hay thp hn u cho kt qu hm lng lipid tng tng ng k. C th l 220C, hm lng lipid trong t bo vi to ch t 9%, trong khi 140C 29

3. NUI N. OCULATA SN XUT BIODIESEL v 300C hm lng lipid ln lt t 19% v 15%. Hm lng carbohydrate v protein cng c s thay i nh [31]. Thnh phn cc acid bo trong t bo vi to Khi nhit tng, t l acid palmitic tng ng k, tuy nhin hm lng EPA gim. Di cc iu kin stress nhit (nhit cao hoc thp), vi to u p ng gia tng hm lng TFA. Nhng hm lng ca cc PUFA gim dn khi nhit mi trng tng, c th l hm lng cc PUFA ln lt t 40%, 35% v 27% tng ng vi cc nhit 14%, 22% v 30% [31]. Trong khi , ti c 3 ch nhit khc nhau, hm lng cc acid palmitoleic (C16:1) v acid oleic (C18:1) thay i khng ng k. y l hai loi acid bo l tng sn xut biodiesel. Vn chnh l la chn ch nhit nui cy sao cho cc loi acid bo khc chim t l ph hp vi tiu chun biodiesel. Hm lng acid palmitic qu cao s lm tng im ng c ca biodiesel, cn hm lng EPA qu cao s lm cho biodiesel d b oxy ha. Bng 3. 2: Thnh phn acid bo (%w/w acid bo tng) ca Nannochloropsis sp. vo ngy th 10 ti cc nhit khc nhau [31] Nhit (0C) 22 64 1.7 4.1 0.2 25.4 0.6 25.6 0.6 Tr 7.1 0.3 4.8 0.1 1.2 0.1 3.4 0.7 25.3 1.0 1.4 0.2 1.4 0.2

TFA (mg/g DW) Acid bo C14:0 C16:0 C16:1 C18:0 C18:1 C18:2 C20:1 C20:4 C20:5 C22:6 Cc loi khc

14 88 1.3 4.7 0.7 23.3 0.9 21.3 1.1 Tr 8.4 0.4 4.3 0.1 1.3 0.1 3.2 0.6 31.7 1.8 Tr Tr

30 143 3.7 5.5 0.5 40.1 1.4 20.5 1.3 1.0 0.2 4.2 0.2 6.8 0.4 Tr 2.7 0.4 16.4 0.4 Tr 1.7 0.3

Tr: trace, gi tr vt, di 1%. 30

3. NUI N. OCULATA SN XUT BIODIESEL Bng 3. 3: Sn lng sinh khi v thnh phn hp cht ha sinh ca Nannochloropsis sp. vo ngy th 10 ti cc nhit khc nhau [31] Nhit (0C) 14 22 30 Sinh khi kh (mg/L) 388 17.8 403 15.2 318 10.8 Lipid (%w/w) 19 0.8 9 0.5 15 0.7 Carbohydrate (%w/w) 12 0.9 11 1.0 11 0.5 Protein (%w/w) 55 4.1 41 2.9 44 2.7

3.2.1.3. Lin h mc tiu sn xut biodiesel Nguyn liu trong sn xut biodiesel chnh l lipid t vi to. Do nng sut lipid l mc tiu chnh khi nui cy N. oculata cho mc ch sn xut biodiesel. Ngoi ra, thnh phn cc acid bo trong ngun lipid sn xut biodiesel l rt quan trng. T l acid bo bo ha nhiu s lm cho biodiesel d b lng cn do acid bo bo ha c kh nng xon cun v to thnh kt ta, do nhit ng c ca biodiesel giu acid bo bo ha s cao. Cn khi t l acid bo cha bo ha mang nhiu ni i cao, biodiesel li d b oxy ha lm thay i tnh cht ca biodiesel. Hai loi acid bo l tng nht sn xut biodiesel chnh l C16:1 v C18:1. Chnh v vy trong th nghim ca Attilio, khi nui cy N. oculata 250C, ta thy hm lng acid bo bo ha v acid bo cha bo ha mang nhiu ni i c xu hng gim, trong khi C18:1 l mt nguyn liu rt ph hp sn xut biodiesel th li tng ln nhiu. Thm vo , ti 250C, nng sut lipid ca N. oculata tuy khng khc bit ng k so vi mc 150C v 200C, nhng vn l t gi tr nng sut cao nht. Cn trong th nghim ca Hanhua Hu v Kunshan Gao, ta thy sn lng sinh khi c 3 nhit nui cy khng c s khc bit ng k, nhng hm lng lipid tng khi nhit cao hay thp th tng gn nh gp i so vi 220C. Hm lng acid bo bo ha tng nh khi nhit t 140C ti 220C, nhng tng mnh khi nhit tng t 220C ti 300C v hm lng acid bo mang nhiu ni i gim ng k. V vy khi kt hp kt qu ca 2 th nghim, ta thy cn tng nhit gim hm lng EPA trong t bo Nannochloropsis oculata, tuy nhin khng nn tng nhit qu cao so vi nhit ti u hm lng C16:0 ch tng mt lng nh. Do , iu kin nhit thch hp nui cy N. oculata nhm sn xut biodiesel chnh l 250C 270C. 31

3. NUI N. OCULATA SN XUT BIODIESEL 3.2.2. Yu t nh sng Tc sinh trng ca to t dng l mt hm khng tuyn tnh theo mc chiu sng. Ti mt dng photon thp, tc sinh trng gn nh tuyn tnh theo mc chiu sng; tuy nhin, ti cng sng cao, tc sinh trng tr nn bo ha (cn bng) so vi mc chiu sng. Cc t bo sinh trng ti cc mc chiu sng khc nhau c cc c im sinh trng khc nhau v cho thy c s thay i ng k trong thnh phn ha hc tng qut, hm lng sc t v hot ng quang hp [23, 65, 59, 76]. Theo kt qu kho st ca Assaf Sukenik v Yael Carmeli v s iu ha hot ng tng hp cc thnh phn acid bo ca Nannochloropsis sp. thng qua ch chiu sng [77], ta c th d on rng khi nui t bo vi to Nannochloropsis oculata cc iu kin chiu sng khc nhau bao gm nh sng gii hn sinh trng, nh sng bo ha sinh trng v nh sng gy km hm, thnh phn cc hp cht ha hc v s lng t bo trong dch nui cy cng s c s thay i. Trong nghin cu ca Assaf Sukenik v Yael Carmeli, huyn ph Nannochloropsis sp. c nui trong mi trng f/2, nc bin thin nhin, 250C [29]. Trong sut phase sinh trng logarith, cc mu c ly o mc sinh trng v thnh phn sc t. Cui phase sinh trng thu mu xc nh thnh phn lipid. nh hng ca mc chiu sng c xc nh bng cch duy tr trng thi n nh iu kin mi trng trong thit b turbidostat 2.6L (l mt thit b nui cy lin tc), nh sng c cung cp thng qua cc n hunh quang trng t ta nhit, 20W (Osram, W. Germany) theo ba mc c k hiu l: growth-limiting (GLL) 35mol /m2s; growth-saturating (GSL) 290 mol/m2s v photoinhibiting (PIL) 550 mol /m2s. Assaf Sukenik v Yael Carmeli thu c mt s kt qu nh sau [77]: Mc sinh trng v thnh phn ni bo

Mc chiu sng c mi lin h mt thit vi mc sinh trng ca huyn ph Nannochloropsis sp.. Mc sinh trng t bo s t bo ha khi mt dng photon trn 200 mol/m2s. Khi nh sng vt qua mt ngng no , s tr thnh yu t c ch s sinh trng ca t bo, c th Nannochloropsis sp. l 500 mol/m2s. Hm lng chlorophyll ni bo gim theo hm s m khi mc chiu sng tng, c th hin qua th sau:

32

3. NUI N. OCULATA SN XUT BIODIESEL

Hnh 3. 3: nh hng ca mc chiu sng trn s sinh trng ca t bo v hm lng chlorophyll a [S8] Mc sinh trng ca t bo v hm lng chlorophyll a c xc nh trong phase sinh trng hm s m, thnh phn acid bo c xc nh khi bt u phase cn bng. Hm lng chlorophyll a trong t bo gim hn 85% khi mc chiu sng tng 20 ln t 30 ti 600 mol/m2s. Nhng thay i v sc t ni bo c mi lin h vi thnh phn acid bo.

Hnh 3. 4: S phn phi cc acid bo chnh trong Nannochloropsis sp. c nui cy theo m di nh hng ca mc chiu sng [77] 33

3. NUI N. OCULATA SN XUT BIODIESEL Tuy nhin, xc nh nh hng ca mc chiu sng trn s hnh thnh cc hp cht ha hc ni bo, thit b nui cy turbidostat cn c iu chnh sao cho ch chiu sng (bao gm thi gian chiu sng, gc chiu sng) khng gy nh hng ti s sinh trng ca t bo. Khi c nui cy di mc chiu sng GLL (35mol /m2s), mi trng nui cy Nannochloropsis b gii hn v nh sng, v mc nhn i t bo ch t 57% so vi khi nui iu kin nh sng bo ha (GSL, 290 mol /m2s). iu kin nh sng PIL (550mol /m2s) li c tc dng c ch, gy gim mc nhn i ca t bo mt phn. Khi mc chiu sng tng ln n 850mol /m2s, mc nhn i t bo gim nghim trng. iu cho thy, Nannochloropsis sp. kh nhy cm vi dng photon c mt cao. Nng cc sc t quang hp gim i khi cng nh sng tng. Hm lng carotenoid gim khng tng xng vi lng chlorophyll a. Kt qu l t l gia carotenoid:chlorophyll a tng ln khi mc chiu sng tng. Nng protein vn duy tr khng i, trong khi carbohydrate, lipid v cc acid bo tng khi tng mc chiu sng. T l khi lng acid bo trong tng lng lipid tng khi t bo c nui GSL, iu ny cho thy c s tng tng ng v triacylglycerol. Nhng khi nui cy ch PIL th hm lng lipid v cc acid bo ni bo khng gia tng thm na so vi ch GSL. Thnh phn acid bo

Thnh phn acid bo trong Nannochloropsis sp. ch yu l C20:5, tip theo l C16:1, nh nu phn trn. Cc kt qu nghin cu khi nui cy theo m cho thy rng mc chiu sng thc s nh hng nhiu n thnh phn acid bo trong t bo vi to. Khi tng cng chiu sng, hm lng cc PUFA nh C20:4 v C20:5 gim theo hm s m, ng thi hm lng C16:0 v C16:1 tng ln. Nhng kt qu ny c kim tra thng qua nui cy iu kin n nh lin tc cc mc chiu sng khc nhau. Khi nui iu kin nh sng GLL, hm lng acid bo C20:4 v C20:5 t kh cao, ln lt l 7.8 v 37.6%, hm lng C16:0 v C16:1 thp. Khi nui iu kin nh sng GSL v PIL th hm lng C16:0 v C16:1 cao hn, trong khi phn trm C20:4 v C20:5 gim mnh. S tng hp lipid

Qu trnh ng ha CO2 to thnh cc loi lipid trong t bo vi to cng ph thuc nhiu vo mc chiu sng. Di iu kin nh sng yu, ch c

34

3. NUI N. OCULATA SN XUT BIODIESEL 26% l triacylglycerol v ti 40% l cc galactolipid, trong 26% l monogalactosyl diacylglycerol v 14% l digalactosyl diacylglycerol. Di iu kin nh sng bo ha (GSL), s tng hp triacylglycerol tng ln v hm lng tng galactolipid gim xung. Xu hng ny vn tip tc khi tng mc chiu sng n mc c ch (PIL), c th l chiu sng 550mol /m2s th Nannochloropsis sp. s tng hp 50% triacylglycerol v ch c 24% l galactolipid, trong hm lng digalactosyl diacylglycerol ch gim nh, cn hm lng monogalactosyl diacylglycerol gim mnh. Phn carbon lipid cn li chim khong 24-36% hm lng carbon lipid chnh l cc phospholipid, sc t v mt s loi lipid khc cha xc nh.

Hnh 3. 5: Thnh phn acid bo ca Nannochloropsis sp. khi nui cy trong iu kin n nh lin tc ti ba mc chiu sng: GLL 35mol /m2s, GSL 290mol /m2s v PIL 550mol /m2s [S8] Lin h mc tiu sn xut biodiesel

Cht lng ca biodiesel c lin quan mt thit vi tnh cht ngun cung cp lipid. Nh nu phn trn, ngun lipid ph hp sn xut biodiesel l ngun lipid c thnh phn lipid ch yu l cc triacylglycerol vi hm lng cc acid bo C16:1 v C18:1 cng cao cng l tng, hm lng cc acid bo cha no mang nhiu ni i nh C20:4 v C20:5 nm trong mt t l gii hn nht nh, thnh phn acid bo li ph thuc vo mc chiu sng. Do , khi nui cy Nannochloropsis oculata nhm mc ch cung cp lipid sn xut biodiesel cn phi thit lp ch chiu sng sao cho nh sng ti thiu 35

3. NUI N. OCULATA SN XUT BIODIESEL t mc bo ha sinh trng (GSL). Ti mc chiu sng GSL (290 mol /m2s), sinh khi thu c l hiu qu nht, hm lng C16:1 v C18:1 cao, tuy nhin triacylglycerol cha cao lm. Nhng nu chiu sng mc PIL (550 mol /m2s), ta thy hm lng C16:1 v C18:1 c xu hng gim cn C20:5 li bt u tng tr li mc d lng triacylglycerol c tng hp nhiu hn. Chnh v vy, t hiu qu ti u trong nui cy Nannnochloropsis oculata, hng chiu sng c ngh s dng l trong khong nh sng bo ha cao, gn t n mc c ch, ngha l vo khong 400 - 500 mol /m2s. 3.2.3. Yu t mn Hanhua Hu v Kunshan Gao kho st s nh hng ca mn mi trng ln s sinh trng v thnh phn cc acid bo ca Nannochloropsis sp. Th nghim c thit k nh sau: Nannochloropsis sp. c nui cy trong mi trng f/2AW, nhit 220C, mc sc kh 200mL/pht vi hm lng CO2 cao (2800L/L), mc chiu sng 50mol/m2s, thu mu vo ngy th 10 sau khi cy ging. iu chnh mn ca nc bin nhn to bng NaCl cc nng nh sau: 0.20, 0.36, 0.72, 1 hoc 1.5M. Mi th nghim lp li 3 ln [31]. S sinh trng v hm lng lipid trong t bo Nannochloropsis sp. pht trin tt nht trong mi trng c mn l 31g/L (0.36M NaCl). Qua cc nng mui kho st, loi vi to ny th hin kh nng duy tr tt s sinh trng trong khong mn 22 49g/L (0.2-0.72M NaCl) [31]. Hm lng lipid trong Nannochloropsis sp. khi c nui cy khong mn t 22 49g/L xp x khong 11%. Tuy nhin, khi nng mui tng ln n 64g/L th hm lng lipid tng ln 18%, ngha l tng hn 50% so vi cc mn thp hn [31]. Bng 3. 4: Sn lng sinh khi v hm lng lipid ca Nannochloropsis sp. vo ngy th 10 ti cc mn khc nhau [31] NaCl (M)/ mn (g/L) 0.20/22 0.36/31 0.72/49 1.00/64 1.50/88 Sinh khi kh (mg/L) 275 13.9 308 15.4 237 10.3 36 5.1 10 2.3 Lipid (%w/w) 12 0.4 11 0.3 11 0.4 18 0.6 -

36

3. NUI N. OCULATA SN XUT BIODIESEL Thnh phn cc acid bo Cc acid bo chim u th trong t bo Nannochloropsis sp. l acid palmitic (C16:0), acid palmitoleic (C16:1) v EPA (C20:53), bt k l sinh trng trong mi trng no. T l phn trm acid oleic (C18:1) c xu hng tng khi tng mn mi trng nui cy. Trong khi , TFA li c xu hng gim [31]. T l % ca PUFAs gim khi tng nng mui, c th t gi tr l 39%, 36%, 35%, v 17% tng ng vi cc mn 22, 31, 49 v 64g/L. Bng 3. 5: Thnh phn acid bo (%w/w TFA) ca Nannochloropsis sp. vo ngy th 10 ti cc mn khc nhau [31] NaCl (M)/ mn (g/L) 0.36/31 0.72/49 78 2.5 3.3 0.1 24.9 1.5 26.0 1.2 Tr 4.6 0.3 7.8 0.8 3.5 0.2 4.0 0.1 23.6 0.9 Tr 1.5 0.5 46 0.9 4.1 0.2 22.1 1.0 27.8 0.9 Tr 6.2 0.5 6.3 0.1 3.2 0.1 4.9 0.2 23.7 1.1 Tr 1.5 0.6

0.20/22 TFA (mg/g DW) Acid bo C14:0 C16:0 C16:1 C18:0 C18:1 C18:2 C20:1 C20:4 C20:5 C22:6 Cc loi khc 90 3.3 3.1 0.3 25.3 1.1 24.0 0.7 Tr 4.5 0.2 6.7 0.3 2.6 0.3 4.1 0.1 27.0 0.5 Tr 1.7 0.3

1.00/64 11 0.7 9.0 0.3 29.8 1.9 23.6 0.8 Tr 17.8 1.1 4.7 0.2 Tr 3.9 0.4 8.4 0.4 Tr 1.7 0.1

Tr: trace, gi tr vt, di 1%. Lin h mc tiu sn xut biodiesel

Mc tiu sn xut biodiesel gn lin vi nng sut lipid trong qu trnh nui cy vi to. V vy, mc d ti nng mui cao (64g/L), hm lng C18:1 tng cao ng k v C20:5 gim mnh, hm lng C16:0 v C16:1 khng c s bin i mnh, nhng sn lng sinh khi kh Nannochloropsis sp. li qu thp (36mg/L) nn khng th p ng nhu cu lipid trong sn xut. T bo sinh trng trong mi trng c mn 31 v 49g/L khi so vi mn 22g/L th hm lng C20:5 c xu hng gim nh, C18:1 v C16:1 li

37

3. NUI N. OCULATA SN XUT BIODIESEL tng. Mc sinh trng trong mi trng mn 31g/L l cao nht (sinh khi kh t 308mg/L). Mc sinh trng ca t bo trong mi trng mn 49g/L cng kh cao (237mg/L). Do c th la chn hai mc mn ny nui cy Nannochloropsis oculata nhm thu lipid. c th chn c nng mui ph hp nht, mi trng nui cy cn c kho st hai nng mui trn kt hp vi cc yu t khc nh nng nitrogen, phosphorus v CO2, nhit v mc chiu sng. 3.2.4. Yu t thnh phn mi trng 3.2.4.1. Thnh phn carbon Cc loi vi to sng trong i dng c kh nng quang hp cao v d dng c nui cy trong mi trng nc bin vn ha tan mt lng ln CO2 [72]. S c nh CO2 nh vo qu trnh quang hp ca vi to ng thi chuyn ha sinh khi thnh dng nhin liu lng c xem l mt qu trnh n gin v rt c gi tr i vi s lun chuyn CO2 hin nay, to nn mt gii php an ton cho mi trng [79]. Nannochloropsis oculata l mt loi vi sinh vt ng c quan tm trong lnh vc cng ngh sinh hc v cc i tng thuc i dng v N. oculata c hm lng lipid cao. Sheng Yi Chiu v cc cng s nghin cu nh hng ca nng CO2 trong dng kh b sung vo mi trng nui cy ln sn lng sinh khi v s tch ly lipid N. oculata, qua nh gi hiu qu nng sut lipid khi nui cy theo m v bn lin tc N. oculata [72]. Vi to N. oculata c nui cy trong mi trng f/2 [72], thit b quang phn ng hnh tr bng thy tinh, nhit 26 10C, di nh sng 300mol/m2s lin tc t cc n hunh quang t ta nhit. Kh cung cp vo (c lc qua mng lc 0.22m) c cc nng CO2 khc nhau: 2%, 5%, 10% v 15%. Kh c sc t y thit b vi tc sc l 200mL/pht (tng ng 0.25vvm) [72]. Sau , Sheng Yi Chiu v cc cng s rt ra c mt s kt lun sau: Sinh trng ca N. oculata nui cy theo m [72] di cc nng CO2 khc nhau khi

Dch nui cy theo m c t 26 10C, chiu sng lin tc 300mol/m2s, sc kh bi khng kh (nng CO2 xp x 0.03%), 2%, 5%,

38

3. NUI N. OCULATA SN XUT BIODIESEL 10% v 15% CO2. Mi trng c ly mu sau mi 8h. ng cong sinh trng ca mi th nghim c th hin trong th sau:

Hnh 3. 6: nh hng ca nng sc kh CO2 ln s sinh trng ca N. oculata Mc sinh trng c trng khi sc khng kh v 2% CO2 ln lt l 0.194/ngy v 0.571/ngy. Qua th ta thy mc sc kh 2% CO2 kch thch s sinh trng ca N. oculata cao nht. Khng ch c sinh khi, m mc sinh trng c trng khi nui cy 2% CO2 cng cao hn khi nui N. oculata bng khng kh. Kt qu ny cng ph hp vi nghin cu ca Hu v Gao [33]. N. oculata sinh trng tt nht trong mi trng giu CO2 hn trong khng kh, c l l nh ngun carbon cung cp cho vi to khng b hn ch. Tuy nhin, khi hm lng CO2 qu cao, 5 15%, s gy ra s c ch ng k [72]. Hm lng lipid ca vi to ti cc phase sinh trng khc nhau [72] Hm lng lipid trong vi to c o phase sinh trng, phase cn bng sm v phase cn bng. Cc kt qu cho thy hm lng lipid c mi lin h cht ch vi phase sinh trng. C th l hm lng lipid trong N. oculata ti phase sinh trng, phase cn bng sm v phase cn bng ln lt l 30.8, 39.7 v 50.4%. Kt qu ny cho thy hm lng lipid tch ly trong t bo tng ln khi N. oculata bc vo phase cn bng. Lin h vi nng nitrate trong dch nui cy, cc mu c em xc nh s gim hm lng nitrate ti cc phase khc nhau, trong hm lng nitrate gim dn t phase sinh trng tr i, iu ny m ch rng lng nitrate b thiu ht khi dch nui cy bc vo phase cn bng. S thiu ht cht dinh dng, c th l thiu ht nitrogen lm gia tng mc tng hp lipid.

39

3. NUI N. OCULATA SN XUT BIODIESEL nh hng ca nng CO2 ln s sinh trng t bo trong nui cy bn lin tc [72] Trc khi nui cy trong mi trng sc kh vi nng CO2 cao, N. oculata c tri qua giai on thch nghi trong mi trng 2% CO2 trc khi cy vo thit b nui cy bn lin tc. Thm vo , lng ging cy vo mi trng c mt t bo cao (xp x 0.4g/L). H thng bn lin tc c vn hnh trong 8 ngy v s sinh trng ca t bo c gi n nh bng cch thay lng mi trng mi ngy, duy tr dch nui cy lun phase sinh trng. Kt qu l mc sinh trng ca N. oculata khi sc kh vi nng CO2 2%, 5%, 10% v 15% l tng ng nhau. Mc sinh trng c trng trung bnh v mt t bo ti a (nng sinh khi) ln lt t t 0.683 n 0.733/ngy v 0.745 v 0.928g/L ti cc nng CO2 khc nhau. Khi sc kh vi hm lng CO2 cao (5 15%) c th gy nh hng khng tt ln s sinh trng t bo nh th hin trong th nu trn. Nhng khi tng mt t bo dch cy v tri qua qu trnh thch nghi trc vi nng CO2 2% th c th ci thin c kh nng sinh trng ca vi to trong mi trng sc kh vi hm lng CO2 cao.

Hnh 3. 7: S sinh trng ca N. oculata khi nui cy bn lin tc trong mi trng sc kh c cha 2%, 5%, 10%, 15% CO2 [72] 40

3. NUI N. OCULATA SN XUT BIODIESEL Kt qu ny chng t rng khi gia tng mt t bo trong dch cy v to iu kin cho t bo thch nghi trong mi trng c nng CO2 l tng th qu trnh nui cy vi nng CO2 cao sau s khng gp phi nhng tc dng tiu cc ln s sinh trng ca t bo vi to. Sinh khi v nng sut lipid trong nui cy bn lin tc Trong h thng nui cy bn lin tc, N. oculata c thu mu vo thi gian trc khi dch nui cy c thay mi trng mi ngy xc nh sinh khi v nng sut lipid. Bng 3. 6: Nng sut sinh khi v lipid ca N. oculata trong h thng nui cy bn lin tc vi cc hm lng CO2 khc nhau [72] Nng CO2 2% 5% 10% 15% Tng nng sut sinh khi (khi lng t bo kh, g/L.ngy) 0.480 0.029 0.441 0.044 0.398 0.069 0.372 0.022 Tng nng sut lipid (g/L.ngy) 0.142 0.049 0.113 0.035 0.097 0.026 0.084 0.021 Phn trm hm lng lipid (%) 29.7 2.0 26.2 1.9 24.6 1.7 22.7 1.9

(H thng nui cy bn lin tc thc hin trong 8 ngy, mi trng mi c thay mi ngy. Th tch thit b quang phn ng l 800mL, th tch dch thi mi ngy l 400mL. S liu SD c o mi ngy t ngy 1 ti ngy 8). Khi tng nng CO2 t 2 ti 15%, c sinh khi ln nng sut lipid u c xu hng gim. Khi nng CO2 l 2%, 5%, 10%, 15% th pH ca mi trng ln lt tng ng l 7.8, 7.7, 7.3 v 7.0. Qu trnh ng ha carbon tng hp lipid s gim khi pH mi trng gim [90]. iu ny c th l do khi mi trng c pH cng cao th lng bicarbonate c th dng c cho s tng hp mch carbon trong lipid cng nhiu. iu ny ni ln rng s tch ly lipid trong N. oculata c th ch yu b nh hng bi pH v hm lng lipid trong t bo vi to s gim khi pH gim [72]. Lin h mc tiu sn xut biodiesel Nannochloropsis oculata nhy cm vi nng CO2 sc vo mi trng nui cy. Mi trng giu CO2 (2%) so vi sc khng kh s lm tng s sinh trng ca N. oculata, nhng khi mi trng cha nhiu CO2 hn na 41

3. NUI N. OCULATA SN XUT BIODIESEL (5-15%) th loi vi to ny li b c ch mnh. Tuy nhin, khi nui cy bn lin tc li cho thy rng s c ch ny hon ton c th khc phc c nu cho N. oculata tri qua qu trnh thch nghi vi nng CO2 2% trc khi sc kh cc ch t 5-15%. V vy, nui cy N. oculata nhm thu nng sut lipid ln, ti u nht nn nui cy nng CO2 2%, lng sinh khi v lipid u t gi tr cc i so vi cc nng khc. Tuy nhin, nu mun kt hp nui cy N. oculata va phc v mc ch sn xut biodiesel va li dng c tnh quang hp cao ca loi ny c nh CO2, iu chnh chu trnh tun hon CO2 nhm ci thin tnh trng nhim mi trng, N. oculata c th c nui cy nng CO2 cao khi qua qu trnh thch nghi vi mi trng giu CO2 so vi khng kh. 3.2.4.2. Thnh phn nitrogen Th nghim ca Attilio v cc cng s [5] Nannochloropsis oculata c nui trong mi trng Guillard f/2, ngun cung cp carbon l CO2 trong khng kh (khong 300ppm), ngun cung cp nitrogen l NaNO3, nui trong 14 ngy, di iu kin nh sng lin tc 70.0E/m2s, nhit mi trng l 200C. Nng nitrogen thch hp nui N. oculata theo Guillard l 0.300g/L. Do , kho st nh hng ca s thiu ht nitrogen trong mi trng nui cy ln s sinh trng v thnh phn lipid trong t bo vi to, Attilio v cc cng s th nghim vi cc nng NaNO3 l 0.300, 0.150, v 0.075g/L [5]. Bng 3. 7: Tham s sinh trng v s sn xut lipid ca N. oculata cc nng NaNO3 khc nhau [5] -Tc sinh trng c trng (1/ngy) 0.130.00 0.100.00 0.100.00 Sn lng lipid (glipid/100gsinh khi kh) 7.880.21 13.010.39 15.860.59 Nng sut lipid (mglipid/L.ngy) 10.010.16 13.611.10 16.410.11

NaNO3(g/L) 0.300 0.150 0.075

42

3. NUI N. OCULATA SN XUT BIODIESEL

Hnh 3. 8: Phn trm cc loi acid bo methyl ester trn tng lng acid bo methyl ester (g/100gFAME) ca N. oculata ti cc nng NaNO3 khc nhau [5]

(FAME: fatty acid methyl ester) Th nghim ca Hanhua Hu v Kunshan Gao Mt th nghim khc ca Hanhua Hu v Kunshan Gao nhm kho st nng nitrate i vi s sinh trng, tch ly lipid, v thnh phn lipid trong t bo cng cho thy rng s thiu ht nitrogen trong mi trng nui cy c tc dng ng k ln cc c im ha sinh ca loi vi to ny. Th nghim ca Hu v Gao c thit lp cc ch nh sau: t bo vi to Nannochloropsis sp. c nui cy trong mi trng 220C, sc kh 200mL/pht vi hm lng CO2 l 2800L/L, mc chiu sng 50mol/m2s, hm lng nitrate c iu chnh da theo mi trng f/2AW c bn: 150 (hm lng N thp), 600 (hm lng N trung bnh) v 3000M NO3- (hm lng N cao) vi 36M PO43-. Dng NaNO3 iu chnh hm lng N. Mi th nghim c lp li 3 ln [31]. Khi tng nng nitrate t 150M n 600M, sn lng sinh khi ca dch nui cy tng 39%. Tuy nhin, nu tng nng ny ln cao 3000M th lng sinh khi li c xu hng gim nh [31]. Hm lng lipid trong vi to tng cao khi nng nitrate gim mnh. Lng acid bo tng tng ng k vi nng nitrate thp. Trong , hm lng C18:1, C16:1 v C16:0 c xu hng tng khi nng nitrate gim, cn acid C20:5 th li gim khi nng nitrate trong mi trng thp. Hm lng ca PUFAs ln lt t 12%., 23%, 41% tng ng vi cc nng N thp, trung bnh v cao [31].

43

3. NUI N. OCULATA SN XUT BIODIESEL Tuy nhin, hm lng C18:1 trong kt qu ca Hu v Gao c cht khc bit so vi trong th nghim ca Attilio v cc cng s (C18:1 gim khi gim nng nitrate). S khc bit ny c th l do hai nghin cu thit lp ti iu kin nui cy khc nhau (nh sng v nhit ). Thm vo c th do Nannochloropsis sp. c vi im khng tng ng vi Nannochloropsis oculata. Thc t, C18:1 ch l thnh phn th yu trong thnh phn lipid ca loi vi to ny. V vy, s khc bit ny c xem l nh hng khng ng k trong qu trnh nghin cu. Bng 3. 8: Sn lng sinh khi v thnh phn hp cht ha sinh ca Nannochloropsis sp. vo ngy th 10 ti cc nng NaNO3 khc nhau [31] NaNO3 (M) 150 600 3000 Sinh khi kh (mg/L) 220 10.4 305 20.5 296 15.6 Lipid (%w/w) 62 2.8 23 0.9 13 0.6

Bng 3. 9: Thnh phn acid bo (%w/w TFA) ca Nannochloropsis sp. vo ngy th 10 ti cc nng NaNO3 khc nhau [31] NaNO3 (M) 600 136 5.7 3.7 0.2 33.9 0.8 23.7 0.6 Tr 13.4 0.6 4.2 0.1 Tr 2.5 0.2 15.7 0.8 Tr 1.1 0.2

TFA (mg/g DW) Acid bo C14:0 C16:0 C16:1 C18:0 C18:1 C18:2 C20:1 C20:4 C20:5 C22:6 Cc loi khc

150 403 8.5 3.3 0.2 38.2 1.2 28.3 0.9 Tr 16.4 0.8 2.7 0.5 Tr 1.1 0.1 7.9 0.3 Tr Tr

3000 105 5.3 3.6 0.3 22.7 1.4 22.7 1.1 Tr 4.1 0.1 7.0 0.8 3.3 0.5 3.6 0.1 29.9 0.9 Tr 2.3 0.3

Tr: trace, gi tr vt, di 1%.

44

3. NUI N. OCULATA SN XUT BIODIESEL Lin h mc tiu sn xut biodiesel Qua s liu thu c ta thy rng, N. oculata c mc sinh trng gim khi gim nng NaNO3 v hm lng lipid th gn nh tng gp i. Kt qu ny chng t rng y l mt loi vi to ph hp vi mc tiu nui cy sn xut biodiesel, nhim v ca cc nh nghin cu chnh l tm ra iu kin mi trng ti u sao cho mc sinh trng ca vi to ch gim nh song hm lng lipid trong t bo li gia tng cch ng k [5]. Thnh phn lipid trong t bo N. oculata c t l cc acid bo cha no mang nhiu ni i kh cao, v vy biodiesel sn xut t loi vi to ny t tiu chun s dng cho cc phng tin ng c th biodiesel cn phi tri qua qu trnh x l b sung nh qu trnh hydro ha hoc s dng di dng hn hp vi biodiesel giu cc acid bo bo ha. Mt khc cng c th khc phc bng cch p dng s gim nng nitrogen trong mi trng nui cy kt hp vi cc yu t khc c tc dng tc ng lm thay i thnh phn lipid trong t bo vi to nh yu t nh sng bo ha v yu t nhit mi trng. Khi hm lng cc acid bo bo ha v acid bo cha bo ha mang mt ni i nh C16:1 s tng ln r rt, v biodiesel sn xut t vi to N. oculata s t cht lng nh tiu chun. Trong th nghim ca Hu v Gao, hm lng nitrate trong mi trng nui cy c kho st mc thp hn trong th nghim ca Attilio. Mc thp nht l 150M, tng ng 0.013g/L. Trong khi mc thp nht theo kho st ca Attilio l 0.075g/L. V kt qu ca Hu v Gao cho thy hm lng lipid trong t bo vi to tng cao hn rt nhiu so vi th nghim ca Attilio, vo ngy th 10 nui cy, nng sut lipid t tng ng khong 136.6mg/L. Thnh phn lipid trong trng hp nng nitrate t 150M cng kh ph hp vi yu cu sn xut biodiesel (hm lng C16:1, C18:1 cao, hm lng C20:5 gim mnh). 3.2.4.3. Thnh phn phosphorus Th nghim nghin cu s nh hng ca yu t dinh dng phosphorus trong mi trng nui cy Nannochloropsis sp. c Hanhua Hu v Kunshan Gao thit k nh sau: t bo vi to Nannochloropsis sp. c nui cy trong mi trng 220C, sc kh 200mL/pht vi hm lng CO2 l 2800L/L, mc chiu sng 50mol/m2s, hm lng phosphate c iu chnh da

45

3. NUI N. OCULATA SN XUT BIODIESEL theo mi trng f/2AW c bn: 6 (hm lng P thp), 25 (hm lng P trung bnh) v 120M PO43- (hm lng P cao) vi 882M NO3-. Dng NaH2PO4 iu chnh hm lng P. Mi th nghim c lp li 3 ln [31]. S sinh trng v hm lng lipid trong t bo Khi tng nng phosphate trong mi trng nui cy t 6M n 25M th sn lng sinh khi tng 34%. Tuy nhin nu tng nng phosphate ln cao hn na, 120M th sn lng sinh khi c s gim nh [31]. Hm lng lipid tng khi gim nng phosphate trong mi trng nui cy [H4]. Khi nng phosphate cao (120M), hm lng lipid gim mnh, ch cn 11% w/w sinh khi kh. Hm lng lipid gim 24% khi tng nng phosphate t 6M n 25M. Bng 3. 10: Sn lng sinh khi v thnh phn hp cht ha sinh ca Nannochloropsis sp. vo ngy th 10 ti cc nng NaH2PO4 khc nhau [31] NaH2PO4 (M) 6 25 120 Thnh phn acid bo Khi mi trng nui cy c nng phosphate cao, lng acid bo tng c xu hng gim mnh. Khi hm lng phosphate tng t 6M n 25M, lng acid oleic (C18:1) gim ng k, t 21.6% gim cn 4.4%. Khi tng hm lng phosphate ln 120M, C18:1 li c xu hng tng, nhng tng rt t khng ng k. iu ny cho thy s tng C18:1 trong hn hp acid bo bng cch tng nng phosphate trong mi trng nui cy l khng hiu qu. Hm lng acid palmitic v acid palmitoleic (C16:0 v C16:1) khng c s thay i ng k khi thay i nng phosphate trong mi trng. Trong khi , hm lng EPA (C20:5) gia tng nhiu khi nng phosphate tng t 6M n 25M, c th l t 12.8% tng ln 27.9%. Nhng khi nng phosphate tip tc tng t 25M n 120M th hm lng EPA vn gi nguyn gn nh khng i. 46 Sinh khi kh (mg/L) 238 15.3 318 17.2 308 19.7 Lipid (%w/w) 25 3.1 19 0.8 11 0.3

3. NUI N. OCULATA SN XUT BIODIESEL Bng 3. 11: Thnh phn acid bo (%w/w acid bo tng) ca Nannochloropsis sp. vo ngy th 10 ti cc nng NaH2PO4 khc nhau [31] NaH2PO4 (M) 25 88 3.5 4.3 0.4 25.3 0.5 24.0 0.8 Tr 4.4 0.5 5.1 0.2 3.3 0.2 3.5 0.2 27.9 1.3 Tr 0.7 0.3

TFA (mg/g DW) Acid bo C14:0 C16:0 C16:1 C18:0 C18:1 C18:2 C20:1 C20:4 C20:5 C22:6 Cc loi khc -

6 148 6.0 3.8 0.1 29.8 1.5 23.2 0.2 1.2 0.1 21.6 1.3 3.2 0.4 Tr 2.5 0.4 12.8 1.0 Tr 1.1 0.1

120 29 2.1 3.3 0.1 23.1 0.7 23.0 0.9 Tr 6.3 0.7 7.6 0.6 1.9 0.1 4.4 0.2 27.4 0.5 Tr 1.7 0.1

Lin h mc tiu sn xut biodiesel Kt qu trn cho thy rng ti nng phosphorus 6M, s sinh trng ca Nannochloropsis sp. thp hn khi nui cy trong mi trng cha nhiu phosphorus nhng hm lng lipid trong t bo li cao hn (25%), do nng sut lipid khng b nh hng ng k. Mt khc, ti nng phosphorus thp, thnh phn lipid trong t bo vi to l ph hp nht vi mc tiu sn xut biodiesel: hm lng C20:5 thp, hm lng C16:0, C16:1 v C18:1 mc cao v n nh. S gii hn nng phosphorus trong mi trng cng gip gim bt chi ph dinh dng trong qu trnh nui cy. V vy, vic nui cy Nannochloropsis oculata nhm sn xut biodiesel s t hiu qu cao khi vi to c nui cy trong mi trng hn ch hm lng phosphorus, nng khong 6M NaH2PO4.

47

4. KT LUN

4. KT LUN
Vn nhim mi trng v s thiu ht ngun nng lng trn ton cu ang l vn thu ht rt nhiu s quan tm. Ngun nhin liu ha thch s dng cho giao thng vn ti v cc phng tin c kh theo d tnh s cn kit trong vng 50 nm ti. Trong cc loi nhin liu xng du, diesel c mt vai tr quan trng v ngy cng c s dng rng ri. Chnh v vy nhim v tm ra mt ngun nhin liu mi c kh nng ti sinh v thay th ngun nhin liu ha thch l mt nhim v cp bch mang tnh thi s v lch s i vi cc nh khoa hc. V biodiesel c xem l mt gii php kh thi, p ng c cc yu cu hin ti. Biodiesel c th c sn xut t nhiu ngun nguyn liu nh thc vt, ng vt hay ngun du ph thi. Nhng p ng c nhu cu s dng, chi ph gi thnh v khng nh hng ti s a dng sinh hc cng nh cc hot ng khc ca con ngi (s xm ln rng v s phn b li t trng), ch c vi to cho thy c kh nng tr thnh ngun cung cp lipid ti u cho vic sn xut biodiesel. Trong s cc loi vi to c kho st cho mc ch nui cy thu lipid phc v cho biodiesel ca nhiu nh khoa hc, cng nh tham kho qua nhiu ti liu v c tnh ca cc loi vi to, c th d on rng Nannochloropsis oculata l mt i tng rt c tim nng nh vo kh nng sinh trng mnh trong mi trng quang t dng, s tch ly lipid c th tng cao ng k di cc iu kin stress mi trng v thnh phn lipid d dng iu khin thng qua iu kin nui cy. Hm lng carbon cung cp cho mi trng nui cy vi to l rt quan trng. i vi iu kin sc kh l khng kh th Nannochloropsis oculata s b thiu ht lng carbon nn mc sinh trng khng tt. Khi hm lng CO2 trong khng kh t 2% th mc sinh trng ca Nannochloropsis oculata t gi tr rt cao. Trong sut cc khong nng CO2 kho st (5-15%), nng 2% CO2 chnh l nng ti u nui cy N. oculata. Tuy nhin, khi loi vi to ny c tri qua qu trnh hun luyn thch nghi vi nng CO2 2% trc, sau nui cy trong mi trng c hm lng CO2 cao th vn c th duy tr mc sinh trng kh tt. V vy c th kt hp vic nui cy N. oculata va thu lipid sn xut biodiesel va c tc dng c nh CO2 ci thin s nhim mi trng. Khi c nui dng trong mi trng sc kh lin tc vi hm lng CO2 thch hp, s sinh trng ca Nannochloropsis oculata khng b gii hn bi ngun cung cp carbon. Lc ny, chnh hm lng nitrogen v phosphorus l yu t 48

4. KT LUN quan trng mang tch cht quyt nh n mc sinh trng ca vi to. S gia tng nng nitrogen v phosphorus trong mi trng nui cy kch thch s tng trng mt t bo trong dch nui cy. Tuy nhin, qua kt qu nghin cu ca cc nh khoa hc i trc, c th d on rng mc sinh trng ca Nannochloropsis oculata tng ng k khi iu kin dinh dng (N v P) tng t nng thp n trung bnh, nhng khi nng ny tng cao hn na, mi trng nui cy tr thnh mi trng giu dinh dng th mc sinh trng ca vi to gn nh khng tng thm m li c xu hng gim nh. iu ny cho thy rng, Nannochloropsis oculata ph hp khi sng trong mi trng c hm lng nitrogen v phosphorus mc trung bnh. Gia tng hm lng mt trong hai hoc c hai nguyn t ny u gy nh hng n s cn bng ca t l C:N hoc N:P, do tc ng n s sinh trng ca t bo. Khi hm lng nitrogen hoc phosphorus trong mi trng nui cy b hn ch, vi to Nannochloropsis sp. c xu hng gia tng s tch ly lipid trong t bo. Xu hng ny khng ng i vi tt c cc loi vi to, nhng theo nghin cu ca Shifrin and Chisholm cc loi to xanh thng cho thy kh nng gia tng s tch ly lipid trong iu kin nui cy thiu ht nitrogen [S11]. Hm lng lipid trong t bo Nannochloropsis sp. tng gn gp 4 ln khi nui trong mi trng c mc N thp so vi nui trong mi trng c mc N cao [H4]. Tuy nhin cha tm thy ti liu no kho st ng thi s thiu ht ca c hai yu t dinh dng N v P ln qu trnh sinh trng v tch ly lipid i vi vi to. Nannochloropsis oculata l loi vi to c th sng trong mt khong mn rng v loi to ny c th sinh sng trong mi trng nc mn, nc l hoc thm ch nc ngt. Tuy nhin, qua kt qu kho st ca Hu v Gao i vi Nannochloropsis sp. c th d on rng, Nannochloropsis oculata s b c ch sinh trng ng k trong mi trng c mn qu cao (trn 60g NaCl/L), sinh trng tt trong mi trng c mn t 20-50g/L, sinh trng ti u khi mn khong 30g/L. Trong khong mn ph hp cho Nannocloropsis oculata pht trin sinh khi nhiu th dng nh khng nh hng ng k ti s tch ly lipid trong t bo vi to, dao ng trong khong 11-12%w/w. Theo nghin cu ca Renaud v cc cng s i vi mt s loi vi to [R7] th nhit khng th hin bt k s nh hng no ln s tng hp lipid trong t bo. Trong nghin cu ca Zhu v cc cng s [Z1], s tng hp lipid trong vi loi vi to s tng khi tng nhit , i vi loi Nitzschia paleacea [R7]. Tuy nhin, 49

4. KT LUN Nannochloropsis sp. li cho thy rt nhy cm vi nhit mi trng: ngay c khi nhit thp hn hoc cao hn nhit sinh trng ti u, lipid tch ly trong t bo u c xu hng tng cao ng k, gn nh hm lng lipid tng gp i trong t bo vi to. V vy, c th d on rng khi Nannochloropsis oculata c nui trong mi trng c nhit 25-270C th mc sinh trng ca t bo vn duy tr tt v hm lng lipid trong t bo cao hn mc nhit ti u. iu kin nh sng trong qu trnh nui cy c bit c vai tr quan trng trong mc tiu nui cy Nannochloropsis oculata nhm sn xut biodiesel. Ch chiu sng nh hng su sc n thnh phn lipid ni bo. T bo N. oculata trong iu kin nh sng bnh thng (50mol/m2s) rt giu acid EPA (C20:5). Acid EPA v mt dinh dng c gi tr cao nhng li khng ph hp sn xut biodiesel. Tuy nhin, thnh phn lipid ca N. oculata li d dng bin i ty thuc vo mc chiu sng. Qua nghin cu ca Assaf Sukenik v Yael Carmeli, ch chiu sng c xc nh l ph hp vi mc tiu sn xut biodiesel chnh l chiu sng cng cao, ti thiu l mc nh sng bo ha s sinh trng, ti a l khi dch nui cy bt u c du hiu b c ch sinh trng. V vy, mt dng photon nn nm trong khong gi tr 400-500mol/m2s.

50

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