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

8/16/2010 2

Why Biofuels??

Improve energy security.

High oil price.

Mitigate climate change.

8/16/2010 3

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.

8/16/2010 4

Source: http://www.dailymarkets.com/stocks/2008/07/02/investing-in-

algae-biofuel/

8/16/2010 5

Disadvantages:

Not economically feasible .

Major issues with harvesting and labor

costs.

Contamination issues.

8/16/2010 6

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.

8/16/2010 7

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.

8/16/2010 8

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

8/16/2010 9

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.

8/16/2010 10

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.

8/16/2010 11

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).

8/16/2010 12

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.

8/16/2010 13

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

8/16/2010 14

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)

8/16/2010 15

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

8/16/2010 16

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

8/16/2010 17

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.

8/16/2010 18

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

8/16/2010 19

Assessment of environmental

factors on lipid accumulation :

8/16/2010 20

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

8/16/2010 21

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

8/16/2010 22

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.

8/16/2010 23

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

8/16/2010 24

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

8/16/2010 25

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

8/16/2010 26

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.

8/16/2010 28

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