Spectral, In Vitro Biological, Engine and Emission Performances of Biodiesel Production from Chlorella protothecoides: A Sustainable Renewable Energy Source


In this research, microalgae species, Chlorella protothecoides was selected for biodiesel production due to its ability to produce large amount of hydrocarbons and oils with high lipid composition. The extracted bio-oil was characterized systematically by proximate, ultimate, spectral (FT-IR, UV–vis., GC–Mass, 1H NMR and 13C NMR) and thermogravimetric (TG/DTA) techniques. The fuel characterization of the bio-oil was evaluated using standard methods. The bio-oil samples were examined for their notable in vitro antimicrobial as well as antioxidant activities. The engine parameters unlike brake specific fuel consumption and brake thermal efficiency for three fuel samples namely diesel (B100), microalga biodiesel 20% blend (CB20) and microalga biodiesel 50% blend (CB50) along with their emission characteristics towards CO2, NOx, and HCs were measured.

Graphic Abstract

Chlorella protothecoides micro alga was selected for bio-oil extraction. The bio-oil extracted was characterized by proximate, elemental, spectral and thermogravimetric techniques and their biodiesel potentiality, the fuel properties were evaluated using standard methods, consequently compared to the standards. In addition, the bio-oil samples were tested for their in vitro antimicrobial and antioxidant activities. The fuel properties show that the microalgae bio-diesel has a cold filter plugging point (CFPP) around –13 °C with 4.5 h oxidation stability. The micro algal oil produces high efficiency (ηbth), low BSFC with lesser CO, (NO)x and hydrocarbons emissions with a single cylinder, water cooled, DI four stroke diesel engines using algae oil blends which is an alternative to diesel engine. Moreover, CB50 blend has a good combustion and emission characteristics when compared to CB20 and B100 fuels.

This is a preview of subscription content, access via your institution.

Fig. 1
Fig. 2


  1. 1.

    Demirbas, A., Fatih Demirbas, M.: Importance of algae oil as a source of biodiesel. Energy Convers. Manag. 52, 163–170 (2011)

    Google Scholar 

  2. 2.

    Chen, Y.H., Huang, B.Y., Chiang, T.H., Tang, T.C.: Fuel properties of microalgae (Chlorella protothecoides) oil biodiesel and its blends with petroleum diesel. Fuel 94, 270–273 (2012)

    Google Scholar 

  3. 3.

    Al-lwayzy, S.H., Yusaf, T., Al-Juboori, R.A.: Biofuels from the fresh water microalgae Chlorella vulgaris (FWM-CV) for diesel engines. Energies 7(3), 1829–1851 (2014)

    Google Scholar 

  4. 4.

    Cadenas, A., Cabezndo, S.: Biofuels as sustainable technologies: perspectives for less developed countries. Tech. Forecast Soc. Change 58, 83–103 (1998)

    Google Scholar 

  5. 5.

    Ghayal, M.S., Pandya, M.T.: Microalgae biomass: a renewable source of energy. Energy Proced. 32, 242–250 (2013)

    Google Scholar 

  6. 6.

    Boyles, D.T: Bioenergy Technology, Thermodynamics, and Costs. Halsted Press, New York (1984)

    Google Scholar 

  7. 7.

    Demirbas, A.: Production of biodiesel from algae oils. Energy Sources A 31, 163–168 (2009)

    Google Scholar 

  8. 8.

    Lewicki, A., Dach, J., Janczak, D., Czekala, W.: The experimental photoreactor for microalgae production. Proc. Tech. 8, 622–627 (2013)

    Google Scholar 

  9. 9.

    Blair, M.F., Kokabian, B., Gude, V.G.: Light and growth medium effect on Chlorella vulgaris biomass production. J. Environ. Chem. Eng. 2, 665–674 (2014)

    Google Scholar 

  10. 10.

    Khan, S.A., Hussain, M.Z., Prasad, S., Banerjee, U.: Prospects of biodiesel production from microalgae in India. Renew. Sust. Energ. Rev. 13, 2361–2372 (2009)

    Google Scholar 

  11. 11.

    Jian-Ming, L.V., Li-Hua, C., Xin-Hua, X., Lin, Z., Huan-Lin, C.: Enhanced lipid production of Chlorella vulgaris by adjustment of cultivation conditions. Bioresour. Technol. 101, 6797–6804 (2010)

    Google Scholar 

  12. 12.

    Hyka, P., Lickova, S., Pribyl, P., Melzoch, K., Kovar, K.: Flow cytometry for the development of biotechnological processes with microalgae. Biotechnol. Adv. 31(1), 2–16 (2013)

    Google Scholar 

  13. 13.

    Juneja, A., Ceballos, R.M., Murthy, G.S.: Effects of environmental factors and nutrient availability on the biochemical composition of algae for biofuels production: a review. Energies 6(9), 4607–4638 (2013)

    Google Scholar 

  14. 14.

    Sharma, K.K., Schuhmann, H., Schenk, P.M.: High lipid induction in microalgae for biodiesel production. Energies 5(5), 1532–1553 (2012)

    Google Scholar 

  15. 15.

    Slade, R., Bauen, A.: Micro-algae cultivation for biofuels: cost, energy balance, environmental impacts and future prospects. Biomass Bioenerg. 5, 29–38 (2013)

    Google Scholar 

  16. 16.

    Li, Y., Horsman, M., Wu, N., Lan, C.Q., Dubois-Calero, N.: Biofuels from microalgae. Biotechnol. Prog. 24(4), 815–820 (2008)

    Google Scholar 

  17. 17.

    Feofilova, E.P., Sergeeva, Y.E., Ivashechkin, A.A.: Biodiesel-fuel: content, production, producers, contemporary biotechnology (Review). Appl. Biochem. Microbiol. 46, 369–378 (2010)

    Google Scholar 

  18. 18.

    Juntila, D.J., Bautista, M.A., Monotilla, W.: Biomass and lipid production of a local isolate Chlorella sorokiniana under mixotrophic growth conditions. Bioresour. Technol. 191, 3–6 (2015)

    Google Scholar 

  19. 19.

    Krohn, B.J., McNeff, C.V., Yan, B., Nowlan, D.: Production of algae-based biodiesel using the continuous catalytic Mcgyan process. Bioresour. Technol. 102, 94–100 (2011)

    Google Scholar 

  20. 20.

    Amin, S.: Review on biofuel oil and gas production processes from microalgae. Energy Convers. Manag. 50(7), 1834–1840 (2009)

    Google Scholar 

  21. 21.

    Saharan, B.S., Sharma, D., Sahu, R., Sahin, O., Warren, A.: Towards algal biofuel production: a concept of green bio energy development. Innov. Rom. Food Biotechnol. 12, 1–21 (2013)

    Google Scholar 

  22. 22.

    Perrin, D.D., Armarego, W.L.F., Perrin, D.R.: Purification of Laboratory Chemicals. Pergamo Press, Oxford (1980)

    Google Scholar 

  23. 23.

    Mistry, B.B.: A Handbook of Spectroscopic Data Chemistry (UV, IR, PMR, 13CNMR and Mass Spectroscopy). Oxford Book Company, Jaipur, pp. 27–63 (2009)

    Google Scholar 

  24. 24.

    Arvindnarayan, S., Sivagnana Prabhu, K.K., Shobana, S., Pasupathy, A., Dharmaraja, J., Kumar, G.: Potential assessment of micro algal lipids: a renewable source of energy. J. Energy Inst. 90(30), 431–440 (2017)

    Google Scholar 

  25. 25.

    Arvindnarayan, S., Sivagnana Prabhu, K.K., Shobana, S., Dharmaraja, J., Pasupathy, A.: Algal biomass energy carriers as fuels: an alternative green source. J. Energy Inst. 90(2), 300–315 (2017)

    Google Scholar 

  26. 26.

    Yaşar, F., Altun, S.: Biodiesel properties of microalgae (Chlorella protothecoides) oil for use in diesel engines. Int. J Green Energy. 15(14–15), 941–946 (2018)

    Google Scholar 

  27. 27.

    Al-Lwazy, S.H., Yusaf, T.: Chlorella protothecoides microalgae as an alternative fuel for tractor diesel engines. Energies 6(2), 766–783 (2013)

    Google Scholar 

  28. 28.

    Gülyurt, M.O., Özçimen, D., İnan, B.: Biodiesel production from Chlorella protothecoides oil by microwave-assisted transesterification. Int. J. Mol. Sci. 17(4), 579–587 (2016)

    Google Scholar 

  29. 29.

    Holbrook, G.P., Davidson, Z., Tatara, R.A., Ziemer, N.L., Rosentrater, K.A., Scott Grayburn, W.: Use of the microalga Monoraphidium sp. grown in wastewater as a feedstock for biodiesel: cultivation and fuel characteristics. Appl. Energy 131, 386–393 (2014)

    Google Scholar 

  30. 30.

    Nautiyal, P., Subramanian, K.A., Dastidar, M.G.: Production and characterization of biodiesel from algae. Fuel Process. Technol. 120, 79–88 (2014)

    Google Scholar 

  31. 31.

    Sivakumar, G., Xu, J., Thompson, R.W., Yang, Y., Randol-Smithd, P., Weathers, P.J.: Integrated green algal technology for bioremediation and biofuel. Bioresour. Technol. 107, 1–9 (2012)

    Google Scholar 

  32. 32.

    Xu, H., Miao, X., Wu, Q.: High quality biodiesel production from a microalgae Chlorella protothecoides by heterotrophic growth in fermenters. J. Biotech. 126(4), 499–507 (2006)

    Google Scholar 

  33. 33.

    Vlachos, N., Skopelitis, Y., Psaroudaki, M., Konstantinidou, V., Chatzilazarou, A., Tegou, E.: Applications of fourier transform-infrared spectroscopy to edible oils. Anal. Chimica Acta 573–574, 459–465 (2006)

    Google Scholar 

  34. 34.

    Indhumathi, P., Syed Shabudeen, P.S., Shoba, U.S.: A method for production and characterization of biodiesel from Green Micro Algae. Int. J. Bio-Sci. Bio-Technol. 6(5), 111–122 (2014)

    Google Scholar 

  35. 35.

    Stansell, G.R., Gray, V.M., Sym, S.D.: Microalgal fatty acid composition: implications for biodiesel quality. J. Appl. Phycol. 24(4), 791–801 (2012)

    Google Scholar 

  36. 36.

    Dash, A., Banerjee, R.: In silico optimization of lipid yield utilizing mix-carbon sources for biodiesel production from Chlorella minutissima. Energy Convers. Manage. 164, 533–542 (2018)

    Google Scholar 

  37. 37.

    Tang, H., Chen, M., Garcia, M.E.D., Abunasser, N., Simon Ng, K.Y., Salley, S.O.: Culture of microalgae Chlorella minutissima for biodiesel feedstock production. Biotechnol. Bioeng. 108(10), 2280–2287 (2011).

    Google Scholar 

  38. 38.

    Song, M., Pei, H., Hu, W., Ma, G.: Evaluation of the potential of 10 microalgal strains for biodiesel production. Bioresour. Technol. 141, 245–251 (2013)

    Google Scholar 

  39. 39.

    Singh, S.K., Bansal, A., Jha, M.K., Jain, R.: Production of biodiesel from wastewater grown Chlorella minutissima. Ind. J. Chem. Tech. 20, 341–345 (2013)

    Google Scholar 

  40. 40.

    Dash, A., Banerjee, R.: Enhanced biodiesel production through phyco-myco co-cultivation of Chlorella minutissima and Aspergillus awamori: an integrated approach. Bioresour. Technol. 238, 502–509 (2017)

    Google Scholar 

  41. 41.

    Chakraborty, S., Mohanty, D., Ghosh, S., Das, D.: Improvement of lipid content of Chlorella minutissima MCC 5 for biodiesel production. J. Biosci. Bioeng. 122(3), 294–300 (2016)

    Google Scholar 

  42. 42.

    Prartono, T., Kawaroe, M., Katili, V.: Fatty acid composition of three diatom species Skeletonemaco statum, Thalassiosira sp. and Chaetoceros gracilis. Int. J. Environ. Bioenergy 6(1), 28–43 (2013)

    Google Scholar 

  43. 43.

    Casas, A., Ramos, M.J., Perez, A., Simon, A., Lucas-Torres, C., Moreno, A.: Rapid quantitative determination by 13C NMR of the composition of acetylglycerol mixtures as byproduct in biodiesel synthesis. Fuel 92(1), 180–186 (2012)

    Google Scholar 

  44. 44.

    Peng, W., Wu, Q., Tu, P., Zhao, N.: Pyrolytic characteristics of microalgae as renewable energy source determined by thermogravimetric analysis. Bioresour. Technol. 80, 01–07 (2001)

    Google Scholar 

  45. 45.

    da Silva, V.M., Silva, L.A., de Andrade, J.B., Veloso, M.C., Santos, G.V.: Determination of moisture content and water activity in algae and fish by thermoanalytical techniques. Quimi. Nova 31(4), 901–905 (2008).

    Google Scholar 

  46. 46.

    Gui, M.M., Lee, K.T., Bhatia, S.: Supercritical ethanol technology for the production of biodiesel: process optimization studies. J. Supercrit. Fluid. 49(2), 286–292 (2009)

    Google Scholar 

  47. 47.

    Patil, P.D., Reddy, H., Muppaneni, T., Mannarswamy, A., Schuab, T., Holguin, F.O., Hammers, P., Nirmalakhandan, N., Cooke, P., Deng, S.: Power dissipation in microwave-enhanced in situ transesterification of algal biomass to biodiesel. Green Chem. 14, 809–818 (2012)

    Google Scholar 

  48. 48.

    Lopez, C.E., Castro, J.M., Gonzalez, V., Gonzalez, E., Perez, J., Seco, H.M., Fernandez, J.M.: Determination of metal ions in algal solution samples by capillary electrophoresis. J. Chromatogr. Sci. 36, 352–356 (1998)

    Google Scholar 

  49. 49.

    Drora, K.: Adsorptions and adsorptions of heavy metal by microalgae. In: Richmond, A., Hu, Q. (eds.) Ch.32: Handbook of Microalgae Culture: Applied Phycology and Biotechnology, 2nd ed. Wiley, New York (2013)

    Google Scholar 

  50. 50.

    Blois, M.S.: Antioxidant determinations by the use of a stable free radical. Nature 181, 1199–1200 (1958)

    Google Scholar 

  51. 51.

    Gumus, M.: A comprehensive experimental investigation of combustion and heat release characteristics of a biodiesel (hazelnut kernel oil methyl ester) fueled direct injection combustion engine. Fuel 89(10), 2802–2814 (2010)

    Google Scholar 

  52. 52.

    Ho, S.H., Chen, C.Y., Lee, D.J., Chang, J.S.: Perspectives on microalgal CO2 emission mitigation systems: a review. Biotechnol. Adv. 29(2), 189–198 (2011)

    Google Scholar 

  53. 53.

    Atmanli, A., Iieri, E., Yuksel, B.: Effects of higher ratios of n–butanol addition to diesel-vegetable oil blends on performance and exhaust emissions of a diesel engine. J. Energy Inst. 88(3), 209–220 (2015)

    Google Scholar 

Download references


This work was supported by the Korea Institute of Energy Technology Evaluation and Planning (KETEP) and the Ministry of Trade, Industry & Energy (MOTIE) of the Republic of Korea (Grant No. 20194110300040 and Grant No. 20173010092470). The authors would also like to acknowledge STIC, CUSAT, Cochin for giving the analytical facilities.

Author information



Corresponding authors

Correspondence to Gopalakrishnan Kumar or Kandasamy K. Sivagnana Prabhu.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Arvindnarayan, S., Shobana, S., Dharmaraja, J. et al. Spectral, In Vitro Biological, Engine and Emission Performances of Biodiesel Production from Chlorella protothecoides: A Sustainable Renewable Energy Source. Waste Biomass Valor 11, 5809–5819 (2020). https://doi.org/10.1007/s12649-019-00888-3

Download citation


  • Chlorella protothecoides
  • GC–mass
  • In vitro biological
  • Engine parameters
  • Emission characteristics