Screening and Identification of native algal isolates for ... · Screening and Identification of...
Transcript of Screening and Identification of native algal isolates for ... · Screening and Identification of...
8/16/2010 1
Screening and Identification of
native algal isolates for
Biodiesel production
Krish Jayachandran & Priyanka Narendar
Dept of Earth and Environment
Florida International University
Miami, Florida
USA
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Why Biofuels??
Improve energy security.
High oil price.
Mitigate climate change.
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Third Generation Biofuels
Algal fuel or Oilgae.
30-100 times more oil per acre than corn
and soybean.
No sulfur, non-toxic.
Grown in marginal land .
Biodegradable.
Less water consumption.
Carbon sequestration.
Tolerate brackish and saline waters.
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Source: http://www.dailymarkets.com/stocks/2008/07/02/investing-in-
algae-biofuel/
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Disadvantages:
Not economically feasible .
Major issues with harvesting and labor
costs.
Contamination issues.
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Objectives:
1. To screen various strains of native green
algal strains from the Florida Everglades to
identify those with potential for biodiesel
production.
2. Quantify the lipid content in algal cells
using gravimetric method.
3. Assess the effect of environmental
conditions on accumulation of cell lipid.
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Algae
Algae are eukaryotes having chlorophyll and
other pigments for carrying out oxygen
producing photosynthesis.
Lipid of interest: Neutral lipids ( in the form
of Triacyl Glycerol) – best substrate for
producing biodiesel.
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Materials and Methods Organisms: 31 algal strains from the FIU culture collection
(Dr. M. Gantar) are being studied.
Botyrococcus braunii - reference strain
Genus Strain
Chlamydomonas EV 29
Chlorella EV 2-4,71-4
Selanstrum EV 2-7,34-4
Scenedesmus EV 3-11, 66-1, 79-1, 80-15, 81-5, 103-4
Chlorococcum EV 5-1, 45-3, 55-2, 55-5
Coelenstrum EV 46-4, 108-5
Coccoid Green EV 56-5, 56-4, 81-7, 103-6, 64-12
Stirgeoclonium EV 64-8
Dactylococcus EV 64-10
Pediastrum EV 81-6, 104-6, 108-4
Prochloro EV 104-1a
Kirchneriella EV 104-7
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Culture conditions:
Algal biomass will be produced by growing
algal strains in 3 l flasks in BG11 medium
(Rippka et al. 1979) under cool white light
(30µ E m-2 sec-1) at 27°C with aeration with
sterile air.
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Screening of lipid:
Nile Red Fluorescence technique
(Greenspan et al.,1985).
a lipophilic dye .
Spectroflurometer analysis at
excitation and emission
wavelengths of 530 nm and 575 nm.
Calibration Curve-Lipid standard
Triolein.
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Quantification of Lipid (Johnson & Wen, 2009):
Freeze dried algal biomass (1g)
Solvent-Chloroform-methanol- water
system.
Solvent evaporated using nitrogen gas
Mass of lipid estimated gravimetrically
(Bligh &Dyer, 1959).
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Assessment of the effect of
environmental factors: Biomass and Lipid accumulation:
1. Determined over a 45 day period.
2. Biomass concentration (Dere et al., 1997)
Ca = 15.65A666-7.340 A653 (Methanol)
3. Lipid accumulation: Nile red method
Nitrogen Depletion and Lipid accumulation (Widjaja et al., 2008).
1. Cells washed thoroughly with N free medium before transferred to fresh media.
2. Concentrations:0%,50% and 100%.
Phosphorous Depletion and Lipid accumulation (Rodolfi et al., 2008).
1. Cells washed thoroughly with P free medium before transferred to fresh media.
2. Concentrations: 0%,50% and 100%.
3. Lipid accumulation: Nile red.
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Results for Screening:
0
500
1000
1500
2000
2500
3000
3500
4000
81-6
Pedias
trum
108-
4 Ped
ias tru
m
108-
5 Coelas
trum
2,4
Chlore
lla
EV 2
9 chlam
ydom
onas
56-5
Coc
coid G
reen
34-4
Selans
trum
81-5
Sce
nede
smus
104-
1a P
roch
loro
81-7
Cocc
oid
green
79-1
Sce
nede
smus
64-8
Stir
geoc
lonium
56-4
Coc
coid g
reen
103-
6 Cocc
od g
reen
45-3
Chlor
ococ
cum
64-1
2 Cocc
oid
Gre
en
5-1 C
hloro
cocc
um
66-1
Sce
nede
smus
103-
4 Sce
ned
esm
us
64-1
0 Dactyloc
occu
s
80-1
5 Sce
ned
esm
us
71-4
Chlor
ella
46-4
Coe
lastru
m
55-5
Chlor
ococ
cum
Bot
yroco
ccus br
aun
ii
111-
4
S pec ies
Flu
oro
sc
en
ce
in
ten
sit
y
13 Day C ulture
45 day culture
E ffect of L ipid Accumulation in C ells for 13 Day and 45 Day P eriod
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Standard curve : Triolein
Y= 47.206e0.1148x
Y represents
fluorescence
intensity .
Unknown x is the
lipid
concentration in
the algal cells.
Triolein standard curve y = 47.206e
0.1148x
R2 = 0.9329
0
2000
4000
6000
8000
10000
12000
14000
16000
0 20 40 60
Concentration ug/ml
Flu
ore
sc
en
ce
in
ten
sit
y
Series1
Expon.(Series1)
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Strain Lipid concentration on13th
day (ug)
Lipid concentration on 45th day
(ug)
81-6 Pediastrum 10.4374154819987 15.2742197328845
108-4 Pediastrum 11.5478990293633 14.7902409291716
108-5 Coelastrum 10.8782524317431 16.8931598545643
2-4 Chlorella10.3231310670479 10.7881125153986
EV 29 Chlamydomonas 10.5579447163199 11.8504042799696
56-5 Coccoid green 9.89767174415345 14.214319533937
34-4 Selanstrum 10.4375505490238 14.2959393185065
55-2 Chlorococcum9.79642908926664 12.1779334529304
55-5 Chlorococcum 10.1043498095117 16.1912263161184
64-10 Dactylococcus9.66323740781956 20.7521647457002
46-4 Coelastrum 10.2788334785514 32.7072335595228
103-4 Scenedesmus9.92390643824308 13.5432801267255
45-3 Chlorococcum 10.2058177595784 15.2267692289687
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64-12 Coccoid Green10.3235521636551 28.8980659619241
66-1 Scenedesmus10.7890128247306 11.9264850756913
5-1 Chlorococcum 11.1657055972044 16.0240790059667
80-15 Pediastrum9.71383311489515 11.5957867692921
71-4 Chlorella9.622160922159 12.1040128421204
81-5 Scenedesmus9.72632286620263 11.1127898854546
104-1a Prochloro 9.77726455585745 11.4201424820779
81-7 Coccoid Green 9.88243685284034 12.8819237441846
79-1 Scenedesmus 9.74505749316385 16.0508912015053
64-8 Stigeoclonium 10.2839646211756 18.5071770989367
56-4 Coccoid Green 10.1848625273624 11.3956883053848
103-6 Coccoid green 11.0580814551602 12.8410195017856
104-7 Kircherniella9.97844779925473 12.4077787987834
Botryococcus braunii (Control)24.033823691502 16.5273695160237
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Results:
46-4 Coelastrum,64-12 Coccoid green, 64-8
Stirgeoclonium, 64-10 Dactylococcus and 108-5
Coelastrum showed higher amount of lipid
accumulation on the 45th day.
Due to depletion of nutrients like phosphorous and
nitrogen.
Botryococcus showed a decrease in lipid content.
Due to decrease in intensity of light that can alter the
lipid composition when high cell densities are reached.
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Neutral lipid extraction by
gravimetric technique:
Neutral lipid extraction using gravimetric technique
0%10%20%30%40%50%60%70%80%90%
64-12 C
occoid
gre
en
46-4 C
oelast
rum
108-5 C
oelastru
m
64-8 S
tigeocl
onium
64-10 D
actyloco
ccus
Botyro
cocc
us bra
unii
Strain
Ne
utr
al
lip
id (
% d
ry w
eig
ht)
Series1
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Assessment of environmental
factors on lipid accumulation :
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Biomass and Cellular accumulation
of neutral lipid over a 45 day period.Biomass and cellular accumulation of neutral lipid in
64-12 coccoid green over a 45 day cultivation period
0
500
1000
1500
2000
2500
3000
3500
Control0 5 10 15 20 25 30 35 40 45
Cultivation time (Days)
Flu
ore
scen
ce
inte
nsit
y n
orm
ali
zed
wit
h c
hlo
rop
hyll
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
Bio
mass (
ug
/ml
ch
loro
ph
yl) 64-12
Fluorscence
intensity
64-12 Biomass
Biomass and cellular accumulation of neutral lipid in 64-
10 Dactylococcus over a 45 day cultivation period
0
200
400
600
800
1000
1200
1400
1600
ControlD0 D5 D10D15D20D25D30D35D40D45
Cultivation time (days)
Flu
ors
cen
ce i
nte
nsit
y
mo
rmali
zed
wit
h
ch
loro
ph
yll
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
Bio
mass (
ug
/ml
ch
loro
ph
yll
)
64-10
Fluorscence
intensity 64-10
Amount of
Biomass
Biomass and cellular accumulation of neutral lipid in
64-8 stirgeoclonium over a 45 day cultivation period
0
200
400
600
800
1000
1200
1400
Control0 5 10 15 20 25 30 35 40 45
Cultivation time(Days)
Flu
ors
cen
ce i
nte
nsit
y
no
rmali
zed
wit
h
ch
loro
ph
yll
0
0.2
0.4
0.6
0.8
1
1.2
1.4
Bio
mass (
ug
/ml
ch
loro
rph
yll
)
64-8
Fluorscence
intensity
64-8 Biomass
Biomass and cellular accumulation of neutral lipid of
46-4 Ceolastrum over a 45 day cultivation period
0
200
400
600
800
1000
1200
1400
Control0 5 10 15 2025 30 35 40 45
Cultivation time (Days)
Flu
ors
cen
t in
ten
sit
y
no
rmali
zed
wit
h
ch
loro
ph
yll
0
0.5
1
1.5
2
2.5
3
3.5
Bio
mass (
ug
/ml
ch
loro
ph
yll
)
46-4
Fluorescence
intensity
46-4 Biomass
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Results continued..
Biomass and cellular accumualtion of neutral lipid for
108-5 Coelastrum over a 45 day period
0
200
400
600
800
1000
1200
1400
1600
Control0 5 10 15 20 25 30 35 40 45
Cultivation time(days)
Flu
orscen
t in
ten
sit
y
no
rm
ali
zed
wit
h
ch
loro
ph
yll
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
Bio
mass (
ug
/ m
l
ch
loro
ph
yll
)
108-5
Fluorscence
intensity
108-5
Biomass
Biomass and cellular accumulation of neutral lipid of
B.braunii over a 45 day period cultivation period
0
500
1000
1500
2000
2500
3000
3500
Control0 5 10 15 20 25 30 35 40 45
Cultivation time(days)
Flu
orscen
ce i
nte
nsit
y
no
rm
ali
zed
wit
h
ch
loro
ph
yll
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
2
Bio
mass (
ug
/ml
ch
loro
ph
yll
)
B.braunii
Fluor
intensity
B.b Biomass
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Results
As the biomass concentration decreases the
amount of lipid concentration increases.
64-12 coccoid green has the maximum
amount of lipid.
Botryococcus braunii showed a decrease in
lipid content as the biomass concentration
increases.
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Effect of nitrogen depletion on
neutral lipid accumulation lipid accumulation under nutreint replete and deplete
conditions for 64-8
0.00
500.00
1000.00
1500.00
2000.00
2500.00
1 2 3 4 5no of days
Fu
ors
ce
nc
e in
ten
sit
y
Control Day 5 Day 10
100%
N
0% N
50%N
lipid accumualtion under nutrient replete and deplete
conditions for 64-12
0
200
400
600
800
1000
Control Day 0 Day 5 Day 10
duration ( Days)
Flu
ors
ce
nc
e in
ten
sit
y
0%N
50%N
100%
N
Lipid accumulation in 64-12 Coccoid
green under nitrogen replete and deplete
conditions
0
5
10
15
20
Control Day 0 Day 5 Day 10
Cultivation (in days)
ug
lip
id p
resen
t
N conc 0
N conc 50
N conc 100
Lipid accumulation in 108-5 Coelastrum under
Nitrogen replete and deplete conditions
0
2
4
6
8
10
12
14
16
Control Day 0 Day 5 Day 10
Cultivation (in days)
ug
of
lip
id p
resen
t
N conc 0
N conc 50
N conc 100
Lipid accumulation in 64-8 Stirgeoclonium under
Nitrogen replete and deplete conditions
0
5
10
15
20
25
30
Control Day 0 Day 5 Day 10
Cultivation time (in days)
ug
of
lip
id p
resen
t
N conc 0
N conc 50
N conc 100
Lipid accumulation in 46-4 Coelastrum under
nitrogen replete and deplete conditions
0
2
4
6
8
10
12
14
Control Day 0 Day 5 Day 10
Cultivation time (in days)
ug
of
lip
id p
resen
t
N conc 0
N conc 50
N conc 100
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Results contd…
lipid accumulation under nutrient replete and deplete
conditions for B.braunii
0
500
1000
1500
2000
2500
3000
3500
Nmae of the
strain
Control Day 0 Day 5 Day 10
Number of days
flu
ors
ce
nc
e in
ten
sit
y 0%N
50%
N
100%
64-8 Stirgeoclonium responded to depletion of
nitrogen by showing the maximum concentration of
lipid.
64-10 Dactylococcus and Botyrococcus braunii did
not seem to respond to Nitrogen depleted
conditions.
0%N 50%N 100%N
Lipid accumulation in 64-10 Dactylococcus
under nitrogen replete and deplete conditions
0
5
10
15
20
Control Day 0 Day 5 Day 10
Cultivation (in days)
ug
of
lip
id p
resen
t
N conc 0
N conc 50
N conc 100
Lipid accumulation in Botyrococcus
braunii under nitrogen replete and
deplete conditions
0
5
10
15
20
25
30
35
Control Day 0 Day 5 Day 10
Cultivation time (in days)
ug
of
lip
id p
rese
nt
N conc 0
N conc 50
N conc 100
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Effect of Phosphorous depletionEffect on lipid accumulation under phosphorous replete
and deplete conditions in 64-12 Coccoid green
0
5
10
15
20
25
30
35
Control Day0 Day 5 Day 10
Cultivation time (days)
ug
of
lip
id p
resen
t
0% P Conc
50% P Conc
100% P Conc
Effect of lipid accumulation under phosphorous replete
and deplete conditions in 108-5 Coelastrum
0
2
4
6
8
10
12
14
16
18
20
Control Day 0 Day 5 Day 10
Cultivation time (days)
ug
of
lip
id p
resen
t
0% P Conc
50% P Conc
100% P Conc
Effect of lipid accumulation under phosphorous replete
and deplete conditions in 64-10 Dactylococcus
0
2
4
6
8
10
12
14
16
18
20
Control Day 0 Day 5 Day 10
Cultivation time (days)
ug
of
lip
id p
resen
t
0% P Conc
50% P Conc
100% P Conc
Effect of lipid accumulation under phosphorous replete
and deplete conditions in 64-8 Stirgeoclonium
0
5
10
15
20
25
Control Day 0 Day 5 Day 10
Cultivation time (days)
ug
of
lip
id 0% P Conc
50% P Conc
100% P Conc
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Results continued:
Influence of phosphorous deprivation on lipid
accumulation of 46-4 Coelastrum
0
500
1000
1500
2000
Control D0 D5 D10
Time(Days)
Flu
ore
sc
en
ce
in
ten
sit
y
0%P
50%P
100%P
Influence of phosphorous deprivation on lipid accumulation
of B.braunii
0
500
1000
1500
2000
Control D0 D5 D10
Time(Days)
Flu
ors
ce
nc
e in
ten
sit
y
0%P
50%P
100%P
64-12 Coccid green showed a maximum
fluorescence intensity over a 10 day period.
64-10 Dactylococcus showed no response.
0%P 50%P100%P
108-5 Coelastrum
Effect on lipid accumulation under phosphorous replete
and deplete conditions in 46-4 Coelastrum
0
5
10
15
20
25
Control Day 0 Day 5 Day 10
Cultivation time (in days)
ug
of
lip
id p
resen
t
0% P Conc
50% P Conc
100% P Conc
Effect of lipid accumulation under phosphorous
replete and deplete conditions in Botyrococcus
braunii
0
5
10
15
20
25
Control Day 0 Day 5 Day 10
Cultivation time (days)
ug
of
lipid
pre
sen
t
0% P Conc
50% P Conc
100% P Conc
8/16/2010 27
Limitations:
Lag in algal taxonomic definition.
Lipid contents and biomass and lipid productivities, not the only characteristics to ensure a cost effective and feasible biodiesel production.
Contamination, tolerance to conditions like light, temperature, ionic strength, nutrient requirements, ease of harvesting and downstream processing impacts the success of large scale production.
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Selected References: Bligh,E.G. and Dyer,W.J. 1959. A rapid method for total lipid
extraction and purification.Can.J.Biochem.Physiol. 37:911-917.
Borowitzka M.A (1992) Algal Biotechnology products and processes – matching science and economics. J Appl Phycology. 4:267-279.
Chisti Y (2008) Biodiesel from microalgae beats Bioethanol. Trends Biotechnology 26:126-131.
Kulkarni,M.G. and A.K Dalai , 2006.Waste cooking oil-an economical source for biodiesel.A review.Ind.Eng.chem.res.,45:2901-2913.
Lang, X., Dalai, A. K., Bakshi,N.N.,Reaney,M.J.,Hertz,P.B.,2001.Preparation and characterization of biodiesel from various bio-oils.Bioresourc.Technol.80,53-62.
Sawayama S, Inoue S, Dote Y, Yokoyama S-Y CO2 fixation and oil production through microalga (1995) . Energy convers Mang 36: 729-31.
Orcutt, D.M., Patterson, G.W(1974). Effect of light intensity upon Nitzschia cloisternium Lipids 9: 1000-1003.
8/16/2010 29
Thank you