Environmental Science and Pollution Research

, Volume 26, Issue 3, pp 3075–3090 | Cite as

Oxidation stability of yeast biodiesel using Rancimat analysis: validation using infrared spectroscopy and gas chromatography–mass spectrometry

  • Anbarasan TamilalaganEmail author
  • Jayanthi Singaram
Research Article


Biodiesel and single cell oils obtained from oleaginous yeasts grown in industrial waste are attractive alternatives to the conventional fuels. However, there are only few articles dealing with the stability of the microbial biofuels. Hence, this study aimed at characterizing the storage time of biodiesels using Rancimat methods. The microbial oil and the biodiesel obtained from microbial oil have been characterized with storage stability due to various oxidizing and thermal damage. Here, the microbial fuels were subject to Rancimat analysis and found to have high thermal-oxidative stability of 18 and 8.78 h for biodiesel and oil, respectively. The storage stability resulting from storage conditions was extrapolated for biodiesel and oil and has been found to be 1.62 and 0.54 years, respectively. The infrared spectroscopic analysis reveals the degree of oxidation found after the induction time was reached and shows the characteristic peaks for degradation products. Gas chromatography revealed the compounds that were responsible for the stability as well as the amount of degradation products left.


Microbial biodiesel Storage stability Thermal oxidative stability Induction time Fourier-transform infrared spectroscopy Gas chromatography–mass spectrometry 



The authors would like to thank the Centre of Excellence for environmental studies for supplying Rancimat for performing studies. Special thanks to Mechanical Research Scholar R. Sakthivel for his timely inputs on article preparation and gas chromatography interpretations.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no competing interests.


  1. Ali T, Dina H (2014) El-Ghonemy. Optimization of culture conditions for the highest lipid production from some oleaginous fungi for biodiesel preparation. Asian J Appl Sci 02(05)Google Scholar
  2. Ami D, Posteri R, Mereghetti P, Porro D, Doglia SM, Branduardi P (2014) Fourier transform infrared spectroscopy as a method to study lipid accumulation in oleaginous yeasts. Biotech for Biofuels 7:12CrossRefGoogle Scholar
  3. Anbarasan T, Regina Y (2015) Investigation on synthesis of biodiesel from distillery spent wash using oleaginous yeast Metschnikowia pulcherrima. Inter J of Appl Engg Res 10(67):310–314 (2015) Research India Publications;Google Scholar
  4. Athenaki M, Gardeli C, Diamantopoulou P, Tchakouteu SS, Sarris D, Philippoussis A (2017) Lipids from yeasts and fungi: physiology, production and analytical considerations. Google Scholar
  5. Bellou S, Triantaphyllidou I, Aggeli D, Elazzazy AM, Baeshen MN, Aggelis G (2016) ScienceDirect Microbial oils as food additives : recent approaches for improving microbial oil production and its polyunsaturated fatty acid content. Curr Opin Biotechnol 37:24–35.
  6. Bligh, Dyer (1959) Neutral lipid extraction by the method of Bligh–Dyer. Can J Biochem Physiol 37:922CrossRefGoogle Scholar
  7. Bondioli P, Gasparoli A, Lanzani A, Fedeli E, Veronese S, Sala M (2001) Storage stability of biodiesel. Ibid 72:699Google Scholar
  8. Brennan L, Owende P (2010) Biofuels from microalgae—a review of technologies for production, processing, and extractions of biofuels and co-products. Renew Sust Energ Rev 14:557–577CrossRefGoogle Scholar
  9. BSI, BSEN14214 (2008) Automotive fuels—fatty acid methyl esters (FAME) for diesel engines—requirements and test methods. 2008.Google Scholar
  10. Bucy HB, Baumgardner ME, Marchese AJ (2012) Chemical and physical properties of algal methyl ester biodiesel containing varying levels of methyl eicosapentaenoate and methyl docosahexaenoate. Algal Res 1(1):57–69CrossRefGoogle Scholar
  11. Dean AP, Sigee DC, Estrada B, Pittman JK (2010) Using FTIR spectroscopy for rapid determination of lipid accumulation in response to nitrogen limitation in freshwater microalgae. Bioresour Technol 101:4499–4507CrossRefGoogle Scholar
  12. Doshi VA, Vuthaluru HB, Bastow T (2005) Investigations into the control of odour and viscosity of biomass oil derived from pyrolysis of sewage sludge. Fuel Process Technol 86(8):885–97CrossRefGoogle Scholar
  13. Dunn RO (2006) Oxidative Stability of Biodiesel By Dynamic Mode Pressurized−Differential Scanning Calorimetry (P−Dsc). Am Soc Agric Biol Eng 49(5):1633–1641Google Scholar
  14. Engines (2013) Requirements and test methods data on cold flow properties pour points. fuelGoogle Scholar
  15. Fakas S, Papanikolaou S, Batsos A, Panayotoumg, Malloucho A, Aggelis G (2009) Evaluating renewable carbon sources as substrates for single cell oil production by Cunninghamella echinulata and Mortierella isabellina. Biomass Bioenergy 33:573–580CrossRefGoogle Scholar
  16. Falk O, Meyer-Pittroff R (2004) The effect of fatty acid composition on biodiesel oxidative stability. Eur J Lipid Sci Technol 106(12):837–843CrossRefGoogle Scholar
  17. Fame En 14214 (2012) + a1 : 2014. 2014;14214.Google Scholar
  18. Forough GN, Thomas-Hall SR, Ratnam RD, Pratt S, Schenk PM (2014) Comparative effects of biomass pre-treatments for direct and indirect transesterification to enhance micro algal lipid recovery. Front. Energy Res
  19. Galafassi S, Cucchetti D, Pizza F, Franzosi G, Bianchi D, Compagno C (2012) Lipid production for second generation biodiesel by the oleaginous yeast Rhodotorula graminis. Bioresour Technol 111:398–403CrossRefGoogle Scholar
  20. Gao C, Zhai Y, Ding Y, Wu Q (2010) Application of sweet sorghum for biodiesel production by heterotrophic Chlorella protothecoides. Appl Egy 87:756–761CrossRefGoogle Scholar
  21. Gao Q, Cao X, Huang YY, Yang JL, Chen J, Wei LJ, Hua Q (2018) ACS. Overproduction of fatty acid ethyl esters by the oleaginous yeast Yarrowia lipolytica through metabolic engineering and process optimization. Synth Biol 7(5):1371–1380CrossRefGoogle Scholar
  22. Giles HH (2003) Methods for assessing stability and cleanliness of liquid fuels. In: Rand SJ (ed). Significance of Tests for Petroleum Products, 7th edn. American Society for Testing and Materials, West Conshohocken, pp 108−117Google Scholar
  23. Gouda MK, Omar SH, Aouad LM (2008) Single cell oil production by Gordonia sp. DG using agro-industrial wastes. World J Microbiol Biotech 24:1703–1711CrossRefGoogle Scholar
  24. Graboski MS, McCormick RL (1998) Combustion of fat and vegetable oil derived fuels in diesel engines. Prog Egy Combust Sci 24:125–164CrossRefGoogle Scholar
  25. Hara A, Radin NS (1978) Lipid extraction of tissues with a low-toxicity solvent. Anal Biochem 90(1):420–426CrossRefGoogle Scholar
  26. Hardon H, K Zürcher Dtsch. Lebensm.-Rudsch (1974) 70:57 Google Scholar
  27. Hasenhuetti GL, Wan PJ (1992) Temperature effects on the determination of oxidative stability with the Metrohm Rancimat. J Am Oil Chem Soc 69:525CrossRefGoogle Scholar
  28. Hiruta O, Yamamura K, Takebe H, Futamura T, Iinuma K, Tanaka H (1997) Application of Maxblend fermenter for microbial processes. J Ferment Bioeng 83(1):79–86CrossRefGoogle Scholar
  29. Hoekman SK, Robbins C (2012) Review of the effects of biodiesel on NOx emissions. Fuel Process Technol 96:237–249CrossRefGoogle Scholar
  30. Hossain ABMS, Salleh A (2008) Biodiesel fuel production from algae as renewable energy. Am J Biochem Biotech 4(3):250–254CrossRefGoogle Scholar
  31. Hu Q, Sommerfeld M, Jarvis E, Ghirardi M, Posewitz M, Seibert M, Darzins A (2008) Micro algal triacylglycerols as feedstocks for biofuel production: perspectives and advances. Plant J 54:621–639CrossRefGoogle Scholar
  32. International Energy Organization 2013 and 2016 (2015)Google Scholar
  33. Khot M, Kamat S, Zinjarde S, Pant A, Chopade B, Ravi Kumar A (2012) Single cell oil of oleaginous fungi from the tropical mangrove wetlands as a potential feedstock for biodiesel. Microbe Cell Fact 11:71CrossRefGoogle Scholar
  34. Klensporf D, Jeleñ HH (2005) Analysis of volatile aldehydes in oat flakes by SPME-GC/MS. Pol J Food Nutr Sci 14/55(4):389–395Google Scholar
  35. Knothe G, Dunn RO (2001) Biofuels derived from vegetable oils and fats, in oleo chemical manufacture and applications. pp. 106–163Google Scholar
  36. Knothe G, Dunn RO, Bagby MO (1997) Biodiesel: the use of vegetable oils and their derivatives as alternative diesel fuels. In: Saha BC, Woodward J (eds) ACS Symposium Series No. 666: Fuels and Chemicals from Biomass. ACS, Washington, DC, pp 172–208CrossRefGoogle Scholar
  37. Kolouchová, Maťátková O, Sigler K, Masák J, Řezanka T (2016) Production of palmitoleic and linoleic acid in oleaginous and nonoleaginous yeast biomass. Int J Anal Chem 2016:1–8CrossRefGoogle Scholar
  38. Koutinas AA, Chatzifragkou A, Kopsahelis N, Papanikolaou S, Kookos IK. (2014) Design and techno-economic evaluation of microbial oil production as a renewable resource for biodiesel and oleochemical production. FUEL 116:566–77.
  39. Ling J, Nip S, Cheok WL, de Toledo RA, Shim H (2014) Lipid production by a mixed culture of oleaginous yeast and microalga from distillery and domestic mixed wastewater. Bioresour Technol 173:132–139CrossRefGoogle Scholar
  40. Loha S, Chewb S, Chooa Y (2006) Oxidative stability and storage behavior of fatty acid methyl esters derived from used palm oil. J Am Oil Chemists’ Soc 83(11):947–952CrossRefGoogle Scholar
  41. Lucarini M, Durazzo A, Sánchez del Pulgar J, Gabrielli P, Lombardi-Boccia G (2018) Determination of fatty acid content in meat and meat products: the FTIR-ATR approach. Food Chem 267:223–230CrossRefGoogle Scholar
  42. Mandal S, Patnaik R, Singh AK, Mallick (2013) Comparative assessment of various lipid extraction protocols and optimization of transesterification process for micro algal biodiesel production. Env Tech 34(13–16):2009–2018CrossRefGoogle Scholar
  43. Marinkovic DM, Stankovic MV, Velickovic AV, Avramovic JM, Miladinovic MR, Stamenkovic OO, Veljkovic VB, Jovanovic DM (2016) Calcium oxide as a promising heterogeneous catalyst for biodiesel production: current state and perspectives. Renew Sus Egy Rev 56:1387–1408CrossRefGoogle Scholar
  44. McCormick RL, Ratcliff M, Moens L, Lawrence R (2007) Several factors affecting the stability of biodiesel in standard accelerated tests. Fuel Proces Tech 88(7):651–657CrossRefGoogle Scholar
  45. Metrohm AG (2008). Quality control of biofuelsGoogle Scholar
  46. Natarajan E (2012) Stability studies of biodiesel. IJES 2(4):152–155Google Scholar
  47. Papanikolaou S, Aggelis G (2011) Review article lipids of oleaginous yeasts. Part I: biochemistry of single cell oil production. 1031–1051.
  48. Patel D, Sindhu K, Arora N, Singh RP, Pruthi V, Pruthi PA (2015) Biodiesel production from non-edible lignocellulosic biomass of Cassia fistula L. fruit pulp using oleaginous yeast Rhodosporidium kratochvilovae HIMPA1. Bioresour Technol 197:91–98CrossRefGoogle Scholar
  49. Peer MS, Kasimani R, Rajamohan S, Ramakrishnan P (2017) Experimental evaluation on oxidation stability of biodiesel/diesel blends with alcohol addition by Rancimat instrument and FTIR spectroscopy†, 31(1), 455–463.
  50. Pullen J, Saeed K (2012) An overview of biodiesel oxidation stability. Renew Sust Energ Rev 16(8):5924–5950CrossRefGoogle Scholar
  51. Rajamohan S, Kasimani R (2018) Analytical characterization of products obtained from slow pyrolysis of Calophyllum inophyllum seed cake: study on performance and emission characteristics of direct injection diesel engine fuelled with bio-oil blends. 25(10), 9523–9538.
  52. Ramos MJ, Maria Fernandez CM, Casas A, Rodriguez L (2009) Perez A. Influence of fatty acid composition of raw materials on biodiesel properties. Bioresour Technol 100(1):261–268CrossRefGoogle Scholar
  53. Ranjith Kumar R, Hanumantha Rao P, Arumugam M (2015) Lipid Extraction Methods from Microalgae: A Comprehensive Review. Front Energy Res [Internet] 2(January):1–9. Available from:
  54. Robert O. Dunn (2008) Antioxidants for improving storage stability of biodiesel. Biofuels, Bioproducts and Biorefining 2 (4):304–318Google Scholar
  55. Saha NK, Balakrishnan M, Batra VS (2005) Improving industrial water use: case study for an Indian distillery. Res. Conserv. Recycl. 43:163–174CrossRefGoogle Scholar
  56. Sakthivel R, Ramesh K, Shameer PM, Purnachandran R (2018) Experimental investigation on improvement of storage stability of bio-oil derived from intermediate pyrolysis of Calophyllum inophyllum seed cake, (5). J Energy Inst xxx 2018.
  57. Santamauro F, Whiffin FM, Scott RJ, Chuck CJ (2014) Low-cost lipid production by an oleaginous yeast cultured in non-sterile conditions using model waste resources. Biotech for Biofuels 7:34CrossRefGoogle Scholar
  58. Selvakumar P, Sivashanmugam P (2018) Study on lipid accumulation in novel oleaginous yeast Naganishia liquefaciens NITTS2 utilizing pre-digested municipal waste activated sludge: a low-cost feedstock for biodiesel production. Appl Biochem Biotech 186(3):731–749. CrossRefGoogle Scholar
  59. Sheehan GJ, Greenfield PF (1980) Utilization, treatment and disposal of distillery wastewater. Water Res 14(3):257–277CrossRefGoogle Scholar
  60. Siddharth J, Sharma MP (2011) Study of oxidation stability of Jatropha curcas biodiesel/diesel blends. Int J Egy Env 2(3):533–542Google Scholar
  61. Stefania V, Castellote AI, Pizzale L, Conte LS, Buxaderasb S, Lo’pez-Tamames E (2003) Analysis of virgin olive oil volatile compounds by headspace solid-phase micro extraction coupled to gas chromatography with mass spectrometric and flame ionization detection. J Chromatography A 983:19–33CrossRefGoogle Scholar
  62. Tewari PK, Batra VS, Balakrishnan M (2007) Water management initiatives in sugarcane molasses based distilleries in India. Res Conserv Recycl 52:351–367CrossRefGoogle Scholar
  63. Thliveros P, Uçkun Kiran E, Webb C (2014) Microbial biodiesel production by direct methanolysis of oleaginous biomass. Bioresour Technol. 157:181-7. CrossRefGoogle Scholar
  64. Turton GC, Wardlaw AC (1987). Pathogenicity of the marine yeasts Metschnikowia zobelli and Rhodotorula rubra for the sea urchin Echinus esculentus. Aquaculture. 67(1–2):199–202.Google Scholar
  65. Vongsvivut J, Heraud P, Gupta A, Puri M, McNaughton D, Barrow CJ (2013) FTIR micro spectroscopy for rapid screening and monitoring of polyunsaturated fatty acid production in commercially valuable marine yeasts and protists. Analyst 138:6016–6031CrossRefGoogle Scholar
  66. Westbrook SR (2003) Fuels for land and marine diesel engines and for non-aviation gas turbines. In: Significance of tests for petroleum products, 7th edn. ASTM International, West Conshohocken, PA, pp 63–81 11Google Scholar
  67. Whiffin F (2015). A palm oil substitute and care product emulsions from a yeast cultivated on waste resources. Thesis (doctor of philosophy (PhD)). University of Bath.
  68. Willian TW, Bariccatti RA, Martins GI, Secco D, de Souza SNM, Rosa HA, Chaves LI (2013) Study of the methyl crambe (Crambe abyssinica Hochst) and soybean biodiesel oxidative stability. Ind Crops Prod 43:207–212CrossRefGoogle Scholar
  69. Xiao-ying LI, Xiao-an NIE, Jie C, Yi-gang W (2015) The development tendency and research statues of microbial oil for biodiesel production. 4(4):137–43Google Scholar
  70. Zuleta EC, Baena L, Riosa LA, Calderón JA (2012) The oxidative stability of biodiesel and its impact on the deterioration of metallic and polymeric materials: a review. J Braz Chem Soc 23(12):2159–2175CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Government College of TechnologyCoimbatoreIndia
  2. 2.Government College of EngineeringBodinayakkanurIndia

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