Extraction of Intracellular Lipid as Potential Biofuel Source From Algal Biomass

Fossil fuels occuring as ancient algal deposit is the major source of energy for our engines today. It is a limited non-renewable resource that will eventually run out. It is in this regards that this work was carried out to determine and extract intracellular lipid in algal biomass for subsequent use as biofuel. Four average sized ponds within Federal University Dutse Campus, Nigeria (11.00̊ N to 13.00̊ N and longitude 8.00̊ E to 10.15̊ E and altitude 465.5m). were randomly selected and 20ml pond water were collected from top, middle and bottom of each pond. 60ml pond water was taken to laboratory and were processed by preparing the media. 3ml NPK gel fertilizer was mixed with distilled water and autoclave at 121°C for 15 min. Bold's Basal Medium (BBM) consisting stock solution and BBM consisting of trace elements were separately mixed with distilled water. 10 ml of the stock solution and 1.0 ml of the trace elements were autoclave at 121°C for 15 min. The media (BBM and fertilizer) were cooled at room temperature, antibiotics (penicillin G, di-hydro-streptomycin sulfate and gentamycin sulfate) were added and pH adjusted to 7.5 and the media were kept ready for used. Isolations of species were carried out by serial dilution using BBM and under microscope sterile syringe and needles were used to isolate target cells. Four different algal species; Euglena, Spirogyra, Selenastrum and Chlorella were isolated and identified. Culturing was carried out by transferring isolate into 50 ml growth chamber containing BBM and NPK fertilizer media separately and cultured using sunlight for 10 days under controlled pH. Two successfully grown species; Spirogyra and Selenastrum were transferred aseptically into four constructed photobioreactors containing 400 ml liquid media; two containing BBM and the other fertilizer media. Nile Red was used to stain the algal biomass, observed under microscope and photographed. Residual salts were removed and mass dried using thermostatic drying oven and subsequently grinded. Growths were determined through dry weight determination and growth percentage was obtained. The dried algal biomass was extracted and evaporated and extracts were heated using vacuum rotary evaporator to separate the solvent from the extracts. Transesterification was carried out. Phase separation was carried out along with purification in which soap and other impurities were removed and resulting solution remained flammable biodiesel. While Euglena and Chlorella failed to grow in the initial media, both Spirogyra, and Selenastrum produced significant amount of biodiesel with Selenastrum species producing higher quantity than that obtained from Spirogyra, hence a potential source of the fuel.

Extraction, Intracellular lipid, Algae, Biofuel, Production.

1. Achara, N. (2012). Biofuel from algae. Journal of American Sciences, 8(1), 240–244. Retrieved from http://www.americanscience.org. [Google Scholar] [Crossref]

2. Andersen, R.A. (Ed.). (2005). Algal culturing techniques, (pp. 437), New York (NY), USA: Elsevier Academic Press. [Google Scholar] [Crossref]

3. Bahadar, A. and Khan, M.B. (2013). Progress in energy from microalgae. A review: Renewable and Sustainable Energy Reviews, 27, 128–148. [Google Scholar] [Crossref]

4. Bajhaiya, A.K., Mandotra, S.K., Suseela, M.R., Toppo, K. and Ranade, S. (2010). Algal biodiesel: The next generation biofuel for India. Asian Journal of Experimental Biological Sciences, 1(4), 728–739. [Google Scholar] [Crossref]

5. Batan, E., Carriquiry, M., Xiaodong, D. and Govinda, R. (2010). Second generation biofuels: Economics and policies. Energy Policy, 39(7), 4222–4234. [Google Scholar] [Crossref]

6. Bellinger, E.G. and Sigie, D.C. (Eds.). (2010). A key to the more frequently occuring freshwater. In: Freshwater Algae: Identification and use as Bioindicators (pp. 155–238). [Google Scholar] [Crossref]

7. Bischoff, H.W. and Bold, H.C. (1963). Phycology studies for Some soil algae from Enchanted Rock and related algal species (pp. 1–95). Texas: University of Texas. [Google Scholar] [Crossref]

8. Chen, W., Sommerfeld, M. and Hu, Q. (2011). Microwave-assisted Nile red method for in vivo quantification of neutral lipids in microalgae. Bioresource Technology, 102, 135–141 [Google Scholar] [Crossref]

9. Chen, W., Zhang, C., Song, L., Sommerfeld, M. and Hu, Q. (2009). A high throughput Nile red method for quantitative measurement of neutral lipids in microalgae. Journal of Microbiological Methods, 77, 41–47. doi:10.1016/j.mimet.2009.01.001 [Google Scholar] [Crossref]

10. Chisti, Y. (2007). Biodiesel from microalgae. Biotechnology Advance, 25(3), 294–306. [Google Scholar] [Crossref]

11. Chisti, Y. (2008). Biodiesel from microalgae beats bioethanol. Trends in Biotechnology, 26(3), 126–131. [Google Scholar] [Crossref]

12. Daily Trust on Face-Book (2012, April 22). Nigeria’s source of revenue, crude oil, will dry up in 41 years’ time unless exploration is stepped up and new oil wells found and exploited. Daily Trust. Retrieved from http://mobile.facebook.com/dailytrust/posts/3913888907550970?_rdc=1&_rdr [Google Scholar] [Crossref]

13. Dhakal, N., Lama, S., Shrestha, A., Karki, T.B., Timilsina, P.M. and Rai, S.K. (2014). Comparative study of microalgae samples from Nepal for biofuel potentials. Journal of Algal Biomass Utilization, 5(3), 8–15. [Google Scholar] [Crossref]

14. Drennan, C. (2016, January 28). STEM Flicks: From algae to biofuel [Video file]. Retrieved from https://m.youtube.com/watch? [Google Scholar] [Crossref]

15. Eze, C.N., Ogbonna, J.C., Ndu, O.O., Ochiogu, I.S. and Charles, O.N. (2017). Evaluation of some biological activities of Euglena gracilis biomass produced by a fed-batch culture with some crop fertilizers. African Journal of Biotechnology, 16(8), 337–345. doi: 10.5897/AJB2016.15651 [Google Scholar] [Crossref]

16. Gartner, G., Uzunov, B., Ingolic, E., Kofler, W., Gacheva, G., Pilarski, P. and Stoyneva, M. (2015). Мicroscopic investigations (LM, TEM and SEM) and identification of Chlorella isolate R-06/2 from extreme habitat in Bulgaria with a strong biological activity and resistance to environmental stress factors. Biotechnology and Biotechnological Equipments, 29(3), 536–540. doi: 10.1080/13102818.2015.1013283. [Google Scholar] [Crossref]

17. Guillard, R.R.L. (Ed.). (2005). Purification methods for microalgae. In: Algal culturing techniques. (pp. 23). New York (NY), USA: Elsevier Academic Press. [Google Scholar] [Crossref]

18. Halim, R. and Webley, P.A. (2015). Nile red staining for oil determination in microalgal cells: A new insight through statistical modelling. International Journal of Chemical Engineering. Retrieved from http://dx.doi.org/10.1155/2015/695061. [Google Scholar] [Crossref]

19. Hill, D.R.A. (2015). What is the quick and fast method for isolation of microalgae strain in order to get the pure strain from the bulk one? [Blog post]. Retrieved from http://www.researchget.net/post/what_is_the_quick_and_fast_method_for_isolation_of_microalgae_strain_in_order_to_get_pure_strain_from_the_bulk_one [Google Scholar] [Crossref]

20. Hossain, A.B.M. (2015). Duel biofuel and bio-solvent as biodiesel and bioethanol from algal oil and its biomass residues using bioprocess technology. Asian Journal of Biotechnology, 7, 23–31. doi: 10.3923/ajbkr.2015.23.31. [Google Scholar] [Crossref]

21. Hossain, A.B.M.S., Nasrulhaq, A.B., Majid, H.A.M., Chandran, S. and Zuliana, R. (Eds.). (2007). Biodiesel production from waste cooking oil as environmental benefits and recycling process. A review: Asian Biofuel Conference Book. (2007, Dec. 11–13). Singapore. [Google Scholar] [Crossref]

22. Hossain, A.B.M.S., Salleh, A., Boyce, A.N., Chowdhury, P. and Naqiuddin, M. (2008). Biodiesel fuel production from algae as renewable energy. American Journal of Biochemistry and Biotechnology, 4(3), 250–254. [Google Scholar] [Crossref]

23. Imase, M., Watanabe, K., Aoyagi, H. and Tanaka, H. (2008). Construction of an artificial symbiotic community using a Chlorella-symbiont association as a model. FEMS Microbiology & Ecology, 63(3), 273–282. [Google Scholar] [Crossref]

24. Janse, v.V.S., Taylor, J., van Ginkel , C. and Gerber, A. (2006). Easy identification of the most common freshwater algae. A guide for identification of microscopic algae in South African freshwater. (pp. 111–181), ISBN: 0-621-354a71-6. [Google Scholar] [Crossref]

25. Khola D. and Ghazala (2012). The sustainability of ethanol production from sugarcane. Energy Policy, 36(6), 2086–2097. [Google Scholar] [Crossref]

26. Konga, A.K., Muchandi, A. S. and Ponnaiah, G.P. (2016): Soxhlet extraction of Spirogyra sp. algae: an alternative fuel. Biofuels. doi: 10.1080/17597269.2016.1196328 [Google Scholar] [Crossref]

27. Lee, R., Lin, R., Xing, B. and Cai, W. (1998). Rapid detection of microalgal lipid content through Sudan black B staining. Journal of Oceanography of Taiwan, 2, 23 [Google Scholar] [Crossref]

28. Mayfield, S. (2010, January 14a). Algae expert discusses latest research in algae biotechnology for fuel and pharmaceuticals. Sieries: on beyond science [Video file]. Retrieved from https://m.youtube.com/watch?v=jPFYjOMNGX0 [Google Scholar] [Crossref]

29. Mayfield, S. (2010, May 26b). The potential of microalgae for the production of bio-fuels and bio-products. Rutgers Energy Institute’s Fifth Annual Symposium [Video file]. Retrieved from https://m.youtube.com/watch?v=PAxokGKu4KU [Google Scholar] [Crossref]

30. Munir, N., Imtiaz, A., Sharif, N. and Naz, S. (2015). Optimization of growth conditions of different algal strains and determination of their lipid contents. Journal of Animal & Plant Sciences, 25(2), 546–553. [Google Scholar] [Crossref]

31. . [Google Scholar] [Crossref]

32. Nazry, M.C., Yahya, L, Kamarudin, K.F., Tao, D.G. and Takriff, M.S. (2017). Isolation, purification and identification of microalgae from coal-fired power plant environment. Malaysian Journal of Analytical Sciences, 21(2), 2017–2102. doi: https://doi.org/10.17576/mjas-2017-2102-zz [Google Scholar] [Crossref]

33. Oniemola, P.K. & Sanusi, G. (2009). The Nigerian Bio-Fuel Policy and Incentives: a need to follow the Brazilian pathway. OH, USA: International Association for Energy Economics. [Google Scholar] [Crossref]

34. Palmer, J.D., Douglas, E.S., and Mark, W.C. (2004). The plant tree of life: An overview and some Points of view. American Journal of Botany, 91(10), 1437–1445. doi: 10.3732/ajb.91.10.1437. [Google Scholar] [Crossref]

35. Pati, P.C. and Meti, B.S. (2014). A brief review on algal lipid. (IJRSB) International Journal of Research Studies in Biosciences, 2(9), 55–61. [Google Scholar] [Crossref]

36. Sokan-Adeaga, A.A. and Ana, G.R.E.E (2015). A comprehensive review of biomass resources and biofuel production in Nigeria: potential and prospects. Review on Environmental Health, 30(3), 143–162. doi: 10.1515/reveh-2015-0015 [Google Scholar] [Crossref]

37. Storms, Z.J., Cameron, E., Siegler, H.H. and McCaffrey, W.C. (2014). A simple and rapid protocol for measuring neutral lipids in algal cells using fluorescence. Journal of Visual Experiment. 87, e51441. doi: 10.3791/51441. [Google Scholar] [Crossref]