Microalgae Lipid Extraction For Biodiesel Production Neha Arora1, Richa Katiyar2, Parul A. Pruthi3, Vikas Pruthi4 and B. R. Gurjar5 1Research
Scholar, Department of Biotechnology, I.I.T. Roorkee-247 667, Uttarakhand, India. 2Research Scholar, Centre for Transportation Systems, I.I.T. Roorkee-247 667, Uttarakhand, India 3Professor, Department of Biotechnology, I.I.T. Roorkee- 247 667, Uttarakhand, India. 5Professor, Department of Civil Engineering and Centre for Transportation Systems, I.I.T. Roorkee- 247 667,Uttarakhand, India Email Id:
[email protected],
[email protected].
MICROALGAE BIODIESEL
OBJECTIVES
• As the world’s petroleum reserves are depleting there is a great demand for alternative sources of petroleum-based fuel, including diesel and gasoline fuels. • Biodiesel is a mixture of fatty acid methyl esters (FAME), has been considered as the best candidate for commercial diesel fuel substitution due to its renewability and decreased carbon dioxide emissions. • Microalgae Triacylglycerol (TAG’s) are viable alternative for biodiesel production owing to their high growth rate and lipids accumulation as compared to other conventional used feed stocks. • Transesterification converts TAG’s into non-toxic and biodegradable biodiesel for direct consumption by unmodified diesel engines.
Biodiesel 2020: Global market survey, feed stock trends and forecasts
Chlorella sp.
Lipid content (% dry weight biomass)
Lipid Productivity (mg/L/day)
Chlorella emersonii
25.0–63.0
10.3–50.0
Chlorella protothecoides
14.6–57.8
1214
Chlorella sorokiniana
19.0–22.0
44.7
Chlorella vulgaris
5.0–58.0
11.2–40.0
Chlorella pyrenoidosa
2.0
-
• • • •
Batch culture of Chlorella sp. autotrophically Estimation of lipid accumulation by Nile red staining Lipid extraction Triacylglycerol (TAG) detection by Thin layer chromatography
Culture of Chorella sp
Nile red staning
Lipid extracted
EXPERIMENTAL DESIGN
• Chlorella sp. was grown autotrophically in batch culture with photoperiod ( ) of 24h at 28 ºC; 130 rpm (O.D.650nm= 0.25).
Nile red staining
• Harvested cells dried at 80ºC for 2h. • Lipid was extracted by modified Bligh and Dyer method.(chloroform: methanol ; 2:1). • TAG’s aws detected by TLC; Hexane: Diethyether:: acetone (70:30:1)
• Lipid accumulation in microalgal cells were estimated by measuring absorbance at 650nm and Nile red staining (0.1mg/ml in DMSO) every alternative day. • Cells in their early stationary phase (O.D.650nm= 1.558) were centrifuged at 5000rpm for 5minutes at room temperature to obtain wet biomass
• Microalgae was grown for 10 days .
Autotrophic batch culture
Lipid extraction and TLC
RESULTS Batch culture of Chlorella sp. Absorbance (650nm)
1
Lipid extraction
2
• Dry weight of cells: 75.30mg/100ml
3
1.5
Thin layer Chromatography 6
• Dry weight of total lipids: 42.80mg
1
6 Lane 1
Lane 2
0.5 0 0
50
100
150
Time (hours)
Figure1: Growth curve of Chlorella sp.
200
250
Nile red staining
• Total lipid extracted = Lipid weight X 100 Normalized biomass 300 =56.83% 4
5
4
2
TAG DAG MAG
Triolein control
Figure 2: Chlorella cells at early stationary phase observed under light microscope (60X).
Figure 3: Chlorella cells stained with Nile red showing lipid accumulation as observed under fluorescent microscope (60X).
Figure 4: Lipid extracted in chloroform Figure 5: Air dried total lipid
CONCLUSION • • • •
This study demonstrated that senescent phase (early stationary phase) is appropriate for algal • lipid recovery. Dried biomass can be used to extract lipid. 56.83% of lipid was extracted from dried biomass using two solvent system : Chloroform: • Methanol (2:1). Thin layer chromatography (TLC) separation of lipids indicated the presence of mono (MAG), di (DAG) and triacylglycerol (TAG). These serve as feedstock for biodiesel. •
Lipid Sample
Figure 6: TLC of extracted lipid Lane 1: Control, Lane 2: Sample
REFERENCES Rios DS, Castaneda J, Torras C, Farriol X and Salvado J. (2013). Lipid extraction methods from microalgal biomass harvested by two different paths: Screening studies toward biodiesel production. Bioresource Technology, 133:378–388. Sakthivel R, Elumalai S and Mohommad M. (2011). Microalgae lipid research, past, present: A critical review for biodiesel production, in the future. Journal of Experimental Sciences. 2: 29-49. Sharma K and Schuhmann P M. (2012). High Lipid Induction in Microalgae for Biodiesel Production. Energies. 5, 1532-1553.
