Nghin cu & Trin khai

Nghin cu nui trng vi to Chlorella vulgaris

lm nguyn liu sn xut Biodiesel
NGUYN MINH TUN, L TH BCH YN, NGUYN PHC HI
NGUYN TH THANH XUN, NG KIM HONG, NGUYN HONG MINH, NGUYN NGC TUN
Khoa Ha, Trng i hc Bch Khoa, i hc Nng
inh khi vi to ang l i tng nghin cu ph
bin trn th gii cho mc tiu sn xut nhin liu
sinh hc, trong lipid t vi to l ngun nguyn
liu cho sn xut biodiesel. V vy, cc bin php
nhm nng cao nng sut lipid c c bit quan tm hin
nay. Trong ti ny, chng ti kho st nh hng ca
cc iu kin nui trng trong phng th nghim nh tc
sc kh CO2, nng dinh dng Nit, cng nh
sng n nng sut sinh khi v hm lng lipid trn vi to
Chlorella Vulgaris. y l mt trong cc chng vi to tim
nng cho mc tiu kt hp x l nc thi, tn dng CO2
t kh thi v ph hp vi iu kin kh hu Vit Nam. Kt
qu ban u cho thy, vic tng cng chiu sng v
gim nng Nit c th tng hm lng lipid nhng gim
nng sut sinh khi. ng thi, vic b sung CO2 vo mi
trng cho php tng nng sut sinh khi v lipid. Bn cnh
, vic th nghim nui trng Chlorella Vulgaris trong nc
thi hm biogas c pha long 1,25 ln cho thy, chng
ny c kh nng sinh trng, cho du v gim ng k
nng Nit v Photpho trong nc thi.

1. T VN
Hin nay, nguyn liu sn xut biodiesel Vit Nam l
cc ngun sinh khi (SK) truyn thng (du n ph thi,
m c basa, du ht jatropha) c nhc im thu hoch
kh khn, hm lng du thp v chim nhiu din tch t
canh tc, t rng v cn nhiu nc ti [1]. khc
phc cc nhc im , SK vi to ra i v c xem
l ngun SK y ha hn sn xut biodiesel.
Vi to l cc vi sinh vt n bo, sinh trng bng
quang t dng nh qu trnh quang hp, hoc d dng,
hoc c hai hnh thc [1]. So vi cc ngun SK truyn
thng, vi to c nhng u im ni bt nh tc sinh
trng nhanh, nng sut thu SK v thu du cao hn cc
loi thc vt c du khc; d nui trng, t cnh tranh vi
t nng nghip v khng cn ngun nc sch; thn thin
vi mi trng. Trung bnh sn xut 1 kg SK to th tiu th
c 1,83 kg CO2 [2]. Ngoi ra, c th tn dng CO2 t kh
thi cng nghip cng vi nc thi nui trng vi to
[3], lm gim ng k chi ph cho qu trnh; ngun SK vi to
ngoi mc ch sn xut biodiesel cn c nhiu ng dng
khc,... [3].
Nng sut thu SK v hm lng du ca vi to ph
thuc nhiu vo iu kin nui trng [2]. Trong nhm vi to
lc, Chlorella Vulgaris l loi c tim nng sn xut biodiesel
v tc sinh trng cao, nng sut SK cao v d nui
trng, c bit c th nui trong mi trng nc thi [4].
Hn na, chng c kh nng hp th CO2 [5], c hm

lng du dao ng t 5-58% khi lng [3]. Do vy, vi

nh hng ca ti l tn dng ngun kh thi CO2 v
nc thi t hm biogas nui trng vi to sn xut
biodiesel, chng ti chn Chlorella Vulgaris l chng vi to
nghin cu nh hng ca mt s iu kin nui trng
n nng sut thu hi du, ng thi nh hng nui trong
mi trng nc thi.

2. VT LIU V PHNG PHP

2.1. Mi trng nui trng
S dng mi trng nui to c thnh phn nh sau:
2.2. Ngun ging vi to: Ly t Vin Cng ngh
Sinh hc, H Ni, l mt trong cc chng vi to c nghin
cu nhiu nht cho mc tiu sn xut biodiesel [9].
Bng 1: Thnh phn mi trng nui trng
Thnh phn

Nng , g/l

NH4Cl

725

CaCl2.2H2O

25

MgSO4.7H2O

140

KH2PO4

350

NaHCO3

840

Khong vi lng

0,5 ml/l

2.3. Cc phng php o c

2.3.1. Phng php o mt quang OD (Optical Density)
Mc ch nh tnh nng SK trong mi trng, da
vo hp th quang ca cc sc t Chlorophyll trong t
bo. Cc sc t ny hp th ch yu 2 bc sng tng
ng l 420 nm v 665 nm [7].
2.3.2. Phng php o nng Chlorophylla
Da vo kh nng hp th nh sng ca cc loi sc t
nhng bc sng nht nh xc nh hm lng ca
chng c trong mu. Mu c ly tm 15.000 vng/pht
40C trong 5 pht thu SK mu. Phn SK ny c b sung
1ml acetone vo ng eppendorf. Sau , em ly tm 13.000
vng/pht 40C trong 5 pht. Ly dch ni v o bc
sng 664 nm, 647 nm. p dng cng thc xc nh nng
chlorophyll a:
Chlorophyll a (g/ml) = 11,93.E664 1,93.E647

2.4. Phng php bo qun ging

Trong s cc phng php th nghim nh bo qun
trn a thch, thch nghing, gi ging bng Glycerol, gi
ging bng ng kh, kt qu cho thy, thi gian gi ging
bng phng php bo qun trn a thch di hn, ng
thi vic hot ha ging d dng hn.

(S 10 - 6/2012) t KHOA HC & CNG NGH 29

Nghin cu & Trin khai

2.5. Phng php nhn ging
bo m lng ging nui trong nhiu iu kin
khc nhau, ta cn tin hnh nhn ging theo tng cp: cp
1, cp 2, ty thuc vo yu cu lng ging cn cho mi ln
nui.

2.6. Phng php nui trng

Tin hnh cc th nghim nui trng vi to trong bnh
Erlen, dung tch nui 1 lt. Th tch ging cho vo chim
10% th tch mi trng. Ging s dng l ging cp 1
c nhn trong vng 7 ngy. Cc mu th nghim c
chiu sng bng ng n hunh quang 20W, chu k chiu
sng 12h sng:12h ti, quang thng c o bng my Advance Light Meter. Nhit th nghim c duy tr 27280C. Kho st sinh trng bng cch o OD cng mt thi
im trong tng ngy nui trng. Tin hnh thu hoch SK
khi sinh trng i vo giai on suy vong (tng ng vi 2
ngy gim trn ng cong OD).

2.7. Phng php thu hoch SK

Mu c ly tm bng my ly tm Hettich ZENTRIFUGEN vi tc 6.000 vng/pht, trong 10 pht. Sau khi ly
tm, mu c sy kh 500C n khi lng khng i.

2.8. Phng php trch ly lipid

p dng phng php Bligh v Dyer (1959) v c ci
tin bi Benemann v Tillett (1987). y l phng php
trch ly du hiu qu nht theo Attilio [7,2]. Mu SK kh
c b sung hn hp CH3OH, CHCl3, sau c siu m
ph v mng t bo tn s sng 20 kHz trong 10 pht
ri c b sung nc v ly tm 6.000 vng/pht trong 10
pht phn thnh 3 lp: lp MeOH v nc trn, lp SK
gia, di cng l lp Chloroform v lipid ha tan. Phn
Chloroform v lipid di c chit v chng ct thu
lipid. Th tch tng dung mi b sung vo mu SK c ly
theo quy tc 100 ml dung mi trn 1 gam SK. T l th tch
dung mi b sung l CH3OH:CHCl3:H2O = 10:10:9.

3. KT QU V THO LUN
3.1. Kho st s nh hng ca CO2 n sinh
trng v tch ly lipid
u tin, ta kho st vai tr ca CO2 i vi sinh trng
bng cch tin hnh thc nghim trn 3 mu: (1) hon ton
khng sc kh; (2) ch sc khng kh; (3) sc khng kh v
CO2, di nh sng 4.000 lux. Kt qu trn hnh 1 cho thy
s c mt ca CO2 trong mi trng lm tng tc sinh
trng cng nh nng sut thu SK v lipid.
kho st chi tit nh hng ca tc sc kh CO2,
ta tin hnh 3 th nghim di nh sng 7.000 lux, tc
sc khng kh l 650 ml/pht. Cc mu khc nhau v tc
sc kh CO2: 20, 40, 60 ml/pht. Kt qu cho thy chu k
sng ca to l 17 ngy. Mt SK ln nht vo ngy th
14, ng vi gi tr cc i ca pha cn bng.
Kt qu trn bng 1 cho thy, SK thu c v hm lng
lipid u gim dn khi tc sc CO2 tng t 20ml/pht
n 60ml/pht. iu ny cng cho thy CO2 cng c nh
hng c ch n sinh trng nng qu ln.
Khi phn tch thnh phn ca mu lipid thu c t mu

30 KHOA HC & CNG NGH u (S 10 - 6/2012)

Hnh 1: Vai tr ca CO2 i vi s sinh trng ca vi to

sc CO2 bng my phn tch sc k lng cao p HPLC, kt

qu hnh 2 cho thy cc peak triglyceride xut hin
Bng 1: SK v lipid ca to di nh hng ca tc sc kh CO2
Mu

iu kin

SK kh
(g/l)

Lipid (g/l)

% lipid/SK
kh

20 ml CO2/pht

1,3862

0,1517

10,945

40 ml CO2/pht

1,1344

0,1186

10,451

60 ml CO2/pht

1,0964

0,1157

10,549

khong thi gian lu 10,42 pht v thnh phn ca triglyxeride trong hn hp kh cao.
ng thi, kt qu phn tch thnh phn trigliceride
bng phng php GC-MS theo hnh 3 cho thy c 2 peak
c trng ca metylester ca axit bo C16:0 (31,53) v axit
oleic C18:1 (35,47). y l 2 loi axit bo rt ph hp
sn xut biodiesel v m bo c tiu chun im chy v
n nh oxy ha ca sn phm, cho thy lipid trch ly t
vi to Chlorella Vulgaris rt c trin vng cho mc tiu sn
xut biodiesel.

3.2. nh hng ca cng nh sng n sinh

trng v tch ly lipid
Chu k sng ti thch hp nht cho s sinh trng v
tch ly lipid i vi Chlorella Vulgaris l 16h sng: 8h ti

Hnh 2: Kt qu phn tch thnh phn lipid ti Cng ty Danapha

Nng

Nghin cu & Trin khai

Bng 3: SK v lipid ca to di nh hng ca nng
NH4Cl
Mu
(mg NH4Cl/l)

Hnh 3: Kt qu phn tch thnh phn triglyxeride bng GC-MS

Cc Hi quan TP Nng

[8]. Cng nh sng l mt trong nhng yu t nh

hng quan trng nht n s quang hp ca to.
Kho st trn 3 mu vi cng chiu sng ln lt l
4.000, 7.000 v 13.000 lux. Cc mu th nghim u khng
sc khng kh v CO2. Kt qu c tng hp trong bng 2
Kt qu trn cho thy, hin tng photoinhibition xy
ra di cng nh sng qu cao. Tuy nhin, s tch ly
lipid trong t bo tng theo cng chiu sng. Nng sut
thu lipid t cao nht ti 7.000 lux. Kt qu ny cng ph
hp vi nghin cu ca G.Gacheva [10].
Bng 2: Kt qu SK v lipid di nh hng
ca nh sng
Mu

Khi lng SK kh
(g/l)

Khi lng lipid

(g/l)

4.000 lux

0,293

0,058

7.000 lux

0,265

0,071

13.000 lux

0,083

0,051

3.3. nh hng ca nng Nit n sinh

trng v tch ly lipid
Vic tm ra nng Nit ti u c ngha quan trng
trong qu trnh sn xut biodiesel t vi to [6]. Thc hin 5
mu c nng NH4Cl khc nhau di cng nh sng
7.000 lux, b sung CO2 theo phng thc gin on vi tc
30ml/pht trong 1 pht vo bui sng, kt qu th hin
bng 3.
Kt qu cho thy, s thiu ht Nit cng nh s d tha
qu nhiu Nit (dng NH4Cl) cng c ch sinh trng. iu
ny ph hp vi kt qu nghin cu v s c ch ca ion
amoni trong mi trng mang tnh kim ( khong pH ln
hn 8) [11]. Do vy, khi nui trng Chlorella Vulgaris trong
nc thi cng nh cc mi trng c tnh baz, cn lu
n vn ny. Bn cnh , kt qu cho thy s thiu ht
Nit trong mi trng lm tng tch ly lipid trong t bo.

3.4. Th nghim nui trong iu kin nc thi

Nc thi s dng nui trng c ly t hm biogas

SK kh
(g/l)

Lipid
(mg/l)

% lipid/SK
kh

475

0,763

71

9,31%

600

0,807

74

9,16%

725

0,798

64

8,02%

850

0,801

61

7,63%

975

0,725

53

7,31%

nng tri chn nui Ha Ph, Nng. S pha long nc

thi cho php gim c nhng lm gim nng cht
dinh dng trong mi trng. Tin hnh nui cy vi to
trong 5 mu nc thi vi mc pha long bng nc ct
t 1/5 (1 th tch nc thi pha thnh 5 th tch) n 5/5
(khng pha long). Xc nh mc sinh trng ca vi to
trong cc mu thng qua o gi tr nng Chlorophyll a
(hnh 4).

Hnh 4: Sinh trng ca vi to trong nc thi pha long

Nhn thy rng, trong mu nc thi khng pha long

(mu 5/5), to sinh trng rt chm, thi gian nui trng
ko di, ng thi lng lipid thu c rt thp. Trong cc
mu cn li, mu c kh nng kt hp x l nc thi (vi
yu cu mc pha long khng qu ln) v sn xut
biodiesel l mu 4/5 (4 phn nc thi + 1 phn nc ct)
(bng 4).
Bng 4: Kt qu SK v lipid trong cc mu nc thi
pha long

Mu

Lng SK (g/l)

Lipid (g/l)

%lipid/SK kh

1/5

1,263

0,111

8,77

2/5

0,743

0,061

8,15

3/5

0,357

0,029

8,21

4/5

0,705

0,063

8,99

5/5

1,265

0,016

1,26

ng thi, kt qu phn tch nc thi ca mu 4/5

cho thy vi to c kh nng hp th mt lng ln N, P
(bng 5).

4. KT LUN V NH HNG TI
S thay i cc iu kin nui trng nh cng chiu
sng, tc sc kh CO2 v nng N (NH4+) nh hng
r rt n kt qu thu SK v lipid. Kt qu ban u cho thy,

(S 10 - 6/2012) t KHOA HC & CNG NGH 31

Nghin cu & Trin khai

Bng 5: Kt qu phn tch mu nc thi c pha
long 1,25 ln trc v sau 14 ngy nui Chlorella
Vulgaris, o ti Trung tm Nghin cu Bo v mi
trng, H Nng
Thng s
phn tch

Phng
php

TCVN
pH
6492-2000
TCVN
Nit tng
6202-1996
TCVN
Photpho tng
5987-1995

Tng cng ty in lc
H Ni (EVN HANOI)

Kt qu
Mu 4/5 (trc
khi nui)

Mu 4/5 (sau
khi nui)

7,6

8,2

24,7 mg/l

3,5 mg/l

35,39 mg/l

31,05 mg/l

cc iu kin nui trng: nh sng 7.000 lux, 600 mg

NH4Cl/lt v tc sc kh 20 ml CO2/pht kt hp vi sc
650 ml khng kh/pht cho kt qu kh quan. Cht lng
ca lipid thu c kh ph hp cho mc tiu sn xut
biodiesel. Ngoi ra, kt qu th nghim nui trng trong
nc thi hm biogas cho thy Chlorella Vulgaris c th sinh
trng tt, ng thi c kh nng x l nc thi v cho
lipid nhm mc tiu sn xut biodiesel.
nh hng ca ti trong thi gian ti l ti u cc
iu kin nui trng nhm ti a nng sut thu lipid, xy
dng m hnh nui to bng h thng ng ni tip v tin
hnh nui trong mi trng nc thi.v

TI LIU THAM KHO

1. Liam Brennan, Philip Owende (2009), Biofuels from microalgae - A review of technologies for production, processing, and extractions of biofuels and co-products, Renewable and Sustainable Energy
Reviews, ELSEVIER, RSER-805.
2. Attilio Converti (2009), Effect of temperature and nitrogen concentration on the growth and lipid content of Nannochloropsis oculata
and Chlorella vulgaris for biodiesel production, Chemical Engineering
and Processing: Process Intensification,ELSEVIER, 48 1148-1151.
3. Teresa M. Mata, Microalgae for biodiesel production and other
applications: A review, Renewable and Sustainable Energy Reviews,ELSEVIER, RSER-757.
4. Zhi-Yuan Liu, Guang-Ce Wang, Bai-Cheng Zhou (2008), Eect of
iron on growth and lipid accumulation in Chlorella vulgaris, Bioresource Technology,ELSEVIER, 99 4717-4722.
5. Mijeong Lee Jeong, James M. Gillis (2003) Carbon Dioxide Mitigation by Microalgal Photosynthesis, Bulletin of the Korean Chemical Society, Vol 24, No 12, page 1763.
[6. Subhasha Nigam (2011), Effect of Nitrogen on Growth and
Lipid Content of Chlorella pyrenoidosa, American Journal of Biochemistry and Biotechnology, vol 7, No3 124-129.
7. Orell Olivo (2007), Thse de Doctorat: Conception et etude dun
photobioreacteur pour la production en continu de microalgues en
ecloseries aquacoles, cole polytechnique de lUniversit de Nante,
page 110.
8. D.Sasi and G.A.Hill (2009), Effect of light intensity on growth of
Chlorella Vulgaris in a novel Circulating loop photobioreactor, Department 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 environnemental stress factors, 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 Environmental Microbiology, vol 43, No4, page 735-739.
32 KHOA HC & CNG NGH u (S 10 - 6/2012)

i u trong ng dng
KHCN vo sn xut
kinh doanh in
NGUYN HNG

EVN HANOI c thnh lp t nm 1954

vi tn ban u l Nh my n B H. Sau
nhiu ln thay i, n thng 2/2010, theo
Quyt nh s 738/Q-BCT ngy
05/02/2010 ca B trng B Cng
Thng, Cng ty in lc TP. H Ni chnh
thc c i tn thnh Tng cng ty in
lc TP. H Ni. Tng cng ty hot ng
theo m hnh Cng ty m-Cng ty con v l
n v thnh vin ca Tp on in lc
Vit Nam.

a trung tm chnh tr - x hi, kinh t - vn ho cua

ca nc, co tc tng trng bnh qun ph ti
khong 12%-15%/ nm, Th H Ni lun c nhu
cu s dng in n nh vi cht lng cao nht.
p ng nhu cu , EVN HANOI trin khai cc bin
php ng b, khoa hc nhm cung cp in phc v
nhn dn. ng k nht l vic tin hnh y mnh u t
i mi, ng dng tin b khoa hc k thut, cng ngh
mi vo hot ng sn xut kinh doanh, ci tin k thut v
bo v mi trng sinh thi. Trong nhng nm qua, EVN
HANOI dnh hng t ng tng thng cho cc cn
b nghin cu, cc k s, cng nhn khi tham gia vo cc
cuc thi v sng kin, ci tin k thut trong ngnh in.
Nh , phong tro pht huy sng kin ci tin, p dng
khoa hc k thut khng ngng pht trin. Tng s sng
kin ci tin (SKCT) tnh t nm 2008 2011 l 1.918 SKCT,
trong , 566 SKCT c cng nhn cp Hi ng KHCN
ca Tng cng ty; 1.352 SKCT c cng nhn cp tiu
ban. c bit, trong 09 ti NCKH, c 02 ti cp Tp
on v 07 ti cp Tng cng ty c nh gi tt. Tng
cng ty bo v thnh cng ti NCKH cp Tp on
in lc Vit Nam vi ti "Nghin cu ng dng dy siu
nhit" t loi gii v c Tp on in lc Vit Nam
chn d thi gii thng VIFOTEC ca B Khoa hc v Cng
ngh v Hi Lin hip cc hi Khoa hc Vit Nam nm 2011.
Trong nhng nm qua, EVN HANOI la chn p dng
cc cng ngh ph hp vi iu kin kh hu, mi trng