Thesis Research CompetitionBIODIESEL PRODUCTION USING MICROALGAE Chlorella vulgaris OIL AND HOMOGENEOUS, IMMOBILIZED LIPASE ENZYME AND HETEROGENEOUS CATALYSTS

Presented by:Arindam Haldar

Student ID: 109592Food Engineering and Bioprocess Technology

School of Environment , Resources and DevelopmentAsian Institute of Technology

16 May, 2011

US military have tested successfully 100% algal biodiesel Solajet(TM) HRJ-5 Jet Fuel for its F-18 Superhornet fighter plane

Source: The Guardian

Broadly biodiesel is composed of Carbon - 77%, Hydrogen -12%, Oxygen - 11% and traces of Nitrogen and Sulfur (Tomasevic et al, 2003)

Advantages – Renewable, biodegradable, less emissions, can be produced from cooking oil (Nantiyakul, 2007)

Fuel suitable to be used in compression ignition diesel engines mainly comprising of fatty acid monoalkyl esters derived from biological sources of oils and fats like vegetable oils, animal fats and microalgal oils (CEN, 2008)

WHAT IS BIODIESEL?

Algae are one of the most primitive plants having no roots, stems and leaves, having chlorophyll a as their primary photosynthetic pigment.Advantages of using microalgae: Capable of all year round production Grow in aqueous media but need less water than terrestrial crops Can be cultivated in brackish water on non-arable land Have rapid growth and many species have oil content in the range of 20–50%

dry weight of biomass Microalgae biomass production can effect biofixation of waste CO2

Can produce valuable co-products such as proteins and residual biomass after oil extraction which may be used as feed or fertilizer

Are capable of photobiological production of ‘biohydrogen’Chlorella vulgaris had been reported to produce around 40-45 % oil under optimum conditions, has high growth rate and excellent CO2 sequestration ability

MICROALGAE AS BIODIESEL SOURCE

Table 1: Comparison of the fuel properties of Biodiesel from Microalgae and Diesel (Department of Biological Sciences and Biotechnology, Tsinghua University, Beijing, China, 2004)

MICROALGAE AS BIODIESEL SOURCE

Compare transesterification

efficiency using homogeneous,

immobilized enzyme and heterogeneous

catalyst

Compare oil extraction from biomass using

ultrasonic waves and microwave

heating

Production of oil from Chlorella vulgaris

and study of its growth dynamics

RESEARCH OBJECTIVES

METHODOLOGY

Pure microbial culture of Chlorella vulgaris was procured from TISTR

Pure microbial culture of Chlorella vulgaris was procured from TISTR

Culture grown on Nutrient agar slantsCulture grown on Nutrient agar slants

Working culture prepared by inoculating in Bold’s basal medium (BBM)

Working culture prepared by inoculating in Bold’s basal medium (BBM)

Growth conditions: Temperature – 250C, RPM – 100, pH – 6.7, Air – 1 L/min, CO2 - 0 ml/min, 10 ml/min and 20 ml/min

Growth conditions: Temperature – 250C, RPM – 100, pH – 6.7, Air – 1 L/min, CO2 - 0 ml/min, 10 ml/min and 20 ml/min

Two table lamps with 14 W cool white bulbs were fixed 20 cm away from the bioreactor to provide illumination

Two table lamps with 14 W cool white bulbs were fixed 20 cm away from the bioreactor to provide illumination

Optical Density was checked every day once by UV Spectrophotometer at 682 nm

Optical Density was checked every day once by UV Spectrophotometer at 682 nm

CULTURE PREPARATION AND STUDY OF GROWTH DYNAMICS

BIOMASS PRODUCTION, OIL ACCUMULATION AND HARVEST

C.Vulgaris grown in BBM for 15 days

C.Vulgaris grown in BBM for 20 days

Grown in N-deficient BBM for 10 days

Grown in N-deficient BBM for 15 days

Grown in N-deficient BBM for 10 days

Grown in N-deficient BBM for 15 days

Centrifuged at 5000 rpm for 15

minutes and then thick cell

mass slurry freeze-dried at

-48 deg C overnight

Figure1: Bioreactor set-up along with external illumination

BIOMASS PRODUCTION, OIL ACCUMULATION AND HARVEST

Figure2: Bioreactor vessels containing microalgae C. vulgaris during growth and incubation for oil accumulation phases

Figure3: Freeze dried microalgal biomass

BIOMASS PRODUCTION, OIL ACCUMULATION AND HARVEST

Microwave HeatingMicrowave Heating UltrasonicationUltrasonication

OIL EXTRACTION FROM DRIED BIOMASS

Figure4: Microwave oven

Figure5: Ultrasonic processor

OIL EXTRACTION FROM DRIED BIOMASS

Freeze dried

Chlorella Vulgaris

Microwave @ 450 W2,4,6,8,10 mins

Microwave @ 900 W2,4,6,8,10 mins

Ultrasonication @ Half wave cycle

2,4,6,8, 10 mins

Ultrasonication @ Full wave cycle

2,4,6,8, 10 mins

Oil from Chlorella vulgaris biomass

Density at 150CKinematic viscosity

at 400C

Acid value Fatty acid

composition

CHARACTERIZATION OF ALGAL OIL

Properties were reported according to ASTM specifications and analysis was done using AOCS Official Methods (AOCS, 1997)

Properties were reported according to ASTM specifications and analysis was done using AOCS Official Methods (AOCS, 1997)

TRANSESTERIFICATION

Chemical homogeneous

Catalyst Potassium hydroxide

(KOH)

Biodiesel

Immobilized enzyme catalyst

Novozyme 435

Chemical Heterogeneous catalyst

CaO/Al2O3

TRANSESTERIFICATION OF OIL

Recycled Enzyme and Heterogeneous catalyst :Used catalyst mixed with fresh catalyst in a ratio of 1:1

Chemical and physical properties of biodiesel were determined according to the standard specifications of biodiesel of Department of Energy Business, B.E.2548 (2005)

ANALYSIS OF PROPERTIES OF BIODIESEL

Table 2: Standard method of biodiesel testing according to Department of Energy Business, B.E.2548 (2005)

RESULTS AND DISCUSSION

GROWTH DYNAMICS AND BIOMASS PRODUCTION

0 5 10 15 20 25 300.0

0.5

1.0

1.5

2.0

2.5

3.0

CO2 @ 10 ml/min

CO2 @ 0 ml/min

CO2 @ 20 ml/min

Fermentation Time (day)

OD

(682

nm

)

End of exponential phase of growth - 20 days after inoculationOptimum level of CO2 concentration in delivered air - 20 ml/minThe biomass production after 15 day growth was 0.63 mg of

biomass/ml of culture and that after 20 day growth period was 0.91 mg of biomass/ml of culture.

Figure 6: Growth curves (based on optical density) of Chlorella vulgaris under standard fermentation conditions and external supply of carbon dioxide mixed in air at various concentrations

Effect of growth period and oil accumulation incubation period OIL EXTRACTION FROM ALGAL BIOMASS

Average oil extraction after incubation in normal media15 day – 26 %20 day – 29 %

Average oil extraction after incubation in normal media and N-deficient media15 day growth and 10 day oil accumulation – 37 %20 day growth and 10 day oil accumulation – 37.4 %15 day growth and 15 day oil accumulation – 44.2 %20 day growth and 15 day oil accumulation – 45 %

0 10 150.000

20.000

40.000

Microwave Ultrasonication

Incubation time for oil accumulation in N2 deficient medium

(days)

% o

il ex

trac

tion

Figure 7: Oil production for different oil accumulation incubation period

More incubation in N-deficient medium

More accumulation of oil

2

4

6

8

10

Optimum8

2 4 6 8 1035.500

36.000

36.500

37.000

37.500

38.000

38.500

Microwave (450 W) Microwave (900 W)Ultrasonication (half cycle) Ultrasonication (full cycle)

Time (min)%

oil

extr

actio

n

Figure 8: Oil extraction efficiency after different treatment time for microwave heating and ultrasonication applied on dry biomass after growth in normal medium and incubation in N-deficient medium for oil accumulation

Effect of treatment time OIL EXTRACTION FROM ALGAL BIOMASS

450 W 900 W

A B C D E F0.000

10.000

20.000

30.000

40.000

50.000

450 W900 W

Growth and oil accumulation period (days)

% o

il ex

trcti

on

Figure 9: Oil extraction efficiency by microwave heating at 450 W and 900 W power levels for biomass obtained after different periods of growth and oil accumulation

Effect of treatment method – Microwave heating OIL EXTRACTION FROM ALGAL BIOMASS

HALF CYCLE

FULL CYCLE

A B C D E F0.000

10.000

20.000

30.000

40.000

50.000

Half cycleFull cycle

Growth and oil accumulation period (days)

% o

il ex

trac

tion

Figure 10: Oil extraction efficiency by ultrasonication treatment at half cycle and full cycle wave modes for biomass obtained after different periods of growth and oil accumulation

Effect of treatment method – Ultrasonication OIL EXTRACTION FROM ALGAL BIOMASS

Ultrasonication

Microwave heating

Effect of treatment method OIL EXTRACTION FROM ALGAL BIOMASS

41.18% 41.07%

0.388 kWh/min

0.432 kWh/min

0.487 kWh/min

0.789 kWh/min

UltrasonicationHalf cycle

UltrasonicationFull cycle

Microwave450 W

Microwave900 W

MicrowaveTreatment more

economic in terms of power

consumption

Liquid at room temperature

Yellowish in color with a tinge of green, no odor

Appearance and consistencyof normal vegtable oil

Acid value=0.08 mgKOH/g Limit – 0.5 mgKOH/g max.

Viscosity = 32.28 cStLimit – 3.5-5.0 cSt

Density = 914 kg/m3

Limit – 860-900 kg/m3

CHARACTERIZATION OF OIL OBTAINED FROM CHLORELLA VULGARIS

Major fatty acid present in the algal oil – Linoleic acid (69.1 %)Average molecular weight of the oil - 874.86 g/mole

ANALYSIS OF FATTY ACID METHYL ESTERS• European standard EN 14103:2003 was used• Methyl heptadecanoate was used as internal standard• Biodiesel from refined palm oil obtained from Bioenergy Laboratory at National

Metals and Materials Technology Center (MTEC), Thailand was used as reference to determine the retention time of free fatty acid methyl ester

• N-heptane was used as washing solvent• The time for each run was determined to be 20 min

Table 3: Comparison of the different methods of transesterification on basis of catalyst used, reaction conditions and the resultant FAME % obtained

Algal oilDensity - 914 kg/m3

Viscosity – 32.28 cSt

Acid value – 0.08 mgKOH/g

Algal BiodieselDensity – 852 kg/m3

Viscosity – 4.5 cSt

Acid value – 0.08 mgKOH/g

All the properties were within specifications of the Department of Energy Business B.E. 2548 (2005)

Highest methyl ester content obtained was 93.44 % which is less than the specified limit of 96.5% for B-100. Biodiesel obtained from transesterification of the algal oil can thus only be used by blending with normal petroleum diesel in lieu of its slightly less FAME content

ANALYSIS OF BIODIESEL PROPERTIES

Optimum concentration of CO2 in air delivered - 20 ml/min

Optimum time period of growth - 20 days Biomass production (mg of biomass/ml of culture) - 0.63 mg/ml (15 day growth) and 0.91 mg/ml (20 day growth). Main factor influencing oil extraction - Treatment time. No effect of different power and wave cycle modes on oil extraction.Oil extraction increased with increasing processing time. Optimal time for treatment - 8 mins.The average oil extraction by microwave heating - 41.07 % and ultrasonication - 41.18 %.On basis of power consumption microwave heating is much economical compared to ultrasonication.Major fatty acid in the oil Linoleic acid (69.1 wt. %) and the average molecular weight of the oil - 874.86 g/mole. Density of the oil at 150C was 914 kg/m3, Kinematic Viscosity at 400C was 32.28 cSt and acid value was 0.08 mg KOH/g.Homogeneous catalyst gave maximum FAME conversion Biodiesel obtained from transesterification of the algal oil can be used only by blending with normal petroleum diesel in lieu of its slightly less FAME contentRecycled catalysts mixed with fresh catalysts can give considerable amount of FAME conversion which add to the commercial viability of those catalysts.Density of oil was reduced to 852-854 kg/m3 from 914 kg/m3. Viscosity of oil was reduced to about 4.5 cSt from 32.28 cSt. Both the properties were in range of DOEB specifications

CONCLUSIONS

Scale up of biomass production by microalgae Chlorella vulgaris in order to improve the economics of the process Optimization of reaction conditions for transesterification of algal oil using various catalysts in order to obtain high (> 96.5 %) methyl ester contentTransesterification reaction of algal oil different catalysts assisted by ultrasonication treatment. Study of transesterification oil using CaO/γ-Al2O3 catalyst prepared by sol-gel method as compared to results obtained by using CaO/γ-Al2O3 catalyst prepared by impregnation method

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