A novel approach using low-cost Citrus limetta waste for mixotrophic cultivation of oleaginous microalgae to augment automotive quality biodiesel production

  • Richa KatiyarEmail author
  • Bhola Ram Gurjar
  • Amit Kumar
  • Randhir Kumar Bharti
  • Shalini Biswas
  • Vikas Pruthi
Research Article


The present study reports the use of Citrus limetta (CL) residue for cultivating Chlorella sp. mixotrophically to augment production of biodiesel. The cultivation of Chlorella sp. using CL as media was carried out by employing a fed-batch technique in open tray (open tray+CL) and in software (BioXpert V2)–attached automated photobioreactor (PBR+CL) systems. Data showed the limit of nitrogen substituent and satisfactory organic source of carbon (OSC) in CL, causing > 2-fold higher lipid content in cells, cultivated in both the systems than in control. For the cells grown in both the systems, ≥ 3-fold enhancement in lipid productivity was observed than in control. The total fatty acid methyl ester (FAME) concentrations from lipids extracted from cells grew in PBR+CL and in open tray+CL techniques were calculated as 50.59% and 38.31%, respectively. The PBR+CL system showed improved outcomes for lipid content, lipid and biomass productivity, FAME characteristics and physical property parameters of biodiesel than those obtained from the open tray+CL system. The physical property parameters of biodiesel produced from algal cells grown in PBR+CL were comparable to existing fuel standards. The results have shown lower cold filter plugging point (− 6.57 °C), higher cetane number (58.04) and average oxidative stability (3.60 h). Collectively, this investigation unveils the novel deployment of CL as a cost-effective feedstock for commercialisation of biodiesel production.


Photobioreactor Microalgae Citrus limetta waste Fed-batch technique Lipid content FAMEs 


Funding information

The first author received financial assistance from the MHRD, Government of India, and the Indian Institute of Technology Bombay (IIT Bombay), India, in the form of fellowship for conducting research.

Supplementary material

11356_2019_4946_MOESM1_ESM.docx (142 kb)
ESM 1 (DOCX 141 kb)


  1. Athira U (2017) Evaluation of carbohydrate and phenol content of citrus fruits species. Int J Appl Res 3(9):160–164Google Scholar
  2. Barbera E, Sforza E, Kumar S, Morosinotto T, Bertucco A (2016) Cultivation of Scenedesmus obliquus in liquid hydrolysate from flash hydrolysis for nutrient recycling. Bioresour Technol 207:59–66CrossRefGoogle Scholar
  3. Bermudez SPC, Garcia-Perez JS, Rittmann BE, Saldivar RP (2015) Photosynthetic bioenergy utilizing CO2: an approach on flue gases utilization for third generation biofuels. J Clean Prod 98:53–65CrossRefGoogle Scholar
  4. Bharti RK, Srivastava S, Thakur IS (2014) Production and characterization of biodiesel from carbon dioxide concentrating chemolithotrophic bacteria, Serratia sp. ISTD04. Bioresour Technol 153:189–197CrossRefGoogle Scholar
  5. Bhatnagar A, Chinnasamy S, Singh M, Das KC (2011) Renewable biomass production by mixotrophic algae in the presence of various carbon sources and wastewaters. Appl Energy 88(10):3425–3431CrossRefGoogle Scholar
  6. Bligh EG, Dyer WJ (1959) A rapid method of total lipid extraction and purification. Biochem Physio 8:911–917Google Scholar
  7. Bridgewater L, Rice EW, Baird RB, Eaton AD, Clesceri LS (eds) (2012). Standard methods: for the examination of water and wastewater. American Public Health Association, APHAGoogle Scholar
  8. Cheirsilp B, Torpee S (2012) Enhanced growth and lipid production of microalgae under mixotrophic culture condition: effect of light intensity, glucose concentration and fed-batch cultivation. Bioresour Technol 110:510–516CrossRefGoogle Scholar
  9. Chen GQ, Chen F (2006) Growing phototrophic cells without light. Biotechnol Lett 28:607–616CrossRefGoogle Scholar
  10. Chu FF, Chu PN, Cai PJ, Li WW, Lam PKS, Zeng RJ (2013) Phosphorus plays an important role in enhancing biodiesel productivity of Chlorella vulgaris under nitrogen deficiency. Bioresour Technol 134:341–346CrossRefGoogle Scholar
  11. Deng X, Chen B, Xue C, Li D, Hu X, Gao K (2019) Biomass production and biochemical profiles of a freshwater microalga Chlorella kessleri in mixotrophic culture: effects of light intensity and photoperiodicity. Bioresour Technol 273:358–367CrossRefGoogle Scholar
  12. Dubois M, Gilles KA, Ton JKH, Rebers PA, Smith F (1956) Colorimetric method for determination of sugars and related substances. Anal Chem 28:350–356CrossRefGoogle Scholar
  13. Farooq W, Lee YC, Ryu BG, Kimd BH, Kimd HS, Choie YE, Yang JW (2013) Two-stage cultivation of two Chlorella sp. strains by simultaneous treatment of brewery wastewater and maximizing lipid productivity. Bioresour Technol 132:230–238CrossRefGoogle Scholar
  14. Feng Y, Li C, Zhang D (2011a) Lipid production of Chlorella vulgaris cultured in artificial wastewater medium. Bioresour Technol 102:101–105CrossRefGoogle Scholar
  15. Feng D, Chen Z, Xue S, Zhang W (2011b) Increased lipid production of the marine oleaginous microalgae Isochrysis zhangjiangensis (Chrysophyta) by nitrogen supplement. Bioresour Technol 102:6710–6716CrossRefGoogle Scholar
  16. Fields FJ, Ostrand JT, Mayfield SP (2018) Fed-batch mixotrophic cultivation of Chlamydomonas reinhardtii for high-density cultures. Algal Res 33:109–117CrossRefGoogle Scholar
  17. Franscisco EC, Neves DB, Lopes EJ, Franco TT (2010) Microalgae as feedstock for biodiesel production: carbon dioxide sequestration, lipid production and biodiesel quality. J Chem Technol Biotechnol 85:395–403CrossRefGoogle Scholar
  18. Gouveia L, Oliveira AC (2009) Microalgae as a raw material for biofuels production. J Ind Microbiol Biotechnol 36:269–274CrossRefGoogle Scholar
  19. Hempel N, Petrick I, Behrendt F (2012) Biomass productivity and productivity of fatty acids and amino acids of microalgae strains as key characteristics of suitability for biodiesel production. J Appl Phycol 24(6):1407–1418CrossRefGoogle Scholar
  20. Islam MA, Magnusson M, Brown RJ, Ayoko GA, Nabi MN, Heimann K (2013) Microalgal species selection for biodiesel production based on fuel properties derived from fatty acid profiles. Ener. 6:5676–5702Google Scholar
  21. Jaaskelainen H (2009) Biodiesel standards & properties. Copyright © Ecopoint Inc. Revision. 01f, (accessed January 2017).
  22. Katiyar R, Gurjar BR, Biswas S, Pruthi V, Kumar N, Kumar P (2017a) Microalgae: an emerging source of energy based bio-products and a solution for environmental issues. Renew Sust Energ Rev 2(C):1083–1093CrossRefGoogle Scholar
  23. Katiyar R, Kumar A, Gurjar BR (2017b) Microalgae based biofuel: challenges and opportunities. In: Biofuels: technology, challenges and prospects. Springer, pp 157–175.
  24. Katiyar R, Gurjar BR, Bharti RK, Kumar A, Biswas S, Pruthi V (2017c) Heterotrophic cultivation of microalgae in photobioreactor using low cost crude glycerol for enhanced biodiesel production. Renew Energy 113:1359–1365CrossRefGoogle Scholar
  25. Katiyar R, Bharti RK, Gurjar BR, Kumar A, Biswas S, Pruthi V (2018) Utilization of de-oiled algal biomass for enhancing vehicular quality biodiesel production from Chlorella sp. in mixotrophic cultivation systems. Renew Energy 122(C):80–88CrossRefGoogle Scholar
  26. Kiss AS, Markus MT, Sass M (2004) Chemical composition of citrus fruits (orange, lemon, and grapefruit) with respect to quality control of juice products, Chapter 3. ACS Sym Series 871:24–34CrossRefGoogle Scholar
  27. Knothe G (2006) Analyzing biodiesel: standards and other methods. J Amer Oil Chem Soc 83:823–833CrossRefGoogle Scholar
  28. Kong WB, Yang H, Cao YT, Song H, Hua SF, Xia CG (2013) Effect of glycerol and glucose on the enhancement of biomass, lipid and soluble carbohydrate production by Chlorella vulgaris in mixotrophic culture. Food Technol Biotechnol 51(1):62–69Google Scholar
  29. Liang YN, Sarkany N, Cui Y (2009) Biomass and lipid productivities of Chlorella vulgaris under autotrophic, heterotrophic and mixotrophic growth conditions. Biotechnol Lett 31:1043–1049CrossRefGoogle Scholar
  30. Liu J, Huang J, Fan KW, Jiang Y, Zhong Y, Sun Z (2010) Production potential of Chlorella zofingienesis as a feedstock for biodiesel. Bioresour Technol 101:8658–8663CrossRefGoogle Scholar
  31. Mahapatra DM, Chanakya HN, Ramachandra TV (2014) Bioremediation and lipid synthesis through mixotrophic algal consortia in municipal wastewater. Bioresour Technol 168:142–150CrossRefGoogle Scholar
  32. Mahmoud R, Ibrahim M, Ali G (2016) Closed photobioreactor for microalgae biomass production under indoor growth conditions. J Algal Bio Utln 7(1):86–92Google Scholar
  33. Mukabane BG, Mutwiwa U, Njogu P, Njog S (2019) Microalgae cultivation systems for biodiesel production: a review. J Sustain Res Eng 4:144–151Google Scholar
  34. Okoro VO, Suna Z, Birchb J (2017) Meat processing waste as a potential feedstock for biochemicals and biofuels—a review of possible conversion technologies. J Clean Prod 142(4):1583–1608CrossRefGoogle Scholar
  35. Park WK, Moon M, Kwak MS, Jeon S, Choi GG, Yang JW, Lee B (2014) Use of orange peel extract for mixotrophic cultivation of Chlorella vulgaris increased production of biomass and FAMEs. Bioresour Technol 171:343–349CrossRefGoogle Scholar
  36. Perez-Garcia O, Escalante FME, De-Bashan LE, Bashan Y (2011) Heterotrophic cultures of microalgae: metabolism and potential products. Water Res 45:11–36CrossRefGoogle Scholar
  37. Ramos MJ, Fernández CM, Casas A, Rodríguez L, Perez A (2009) Influence of fatty acid composition of raw materials on biodiesel properties. Bioresour Technol 100:261–268CrossRefGoogle Scholar
  38. Rashid N, Rehman MSU, Han JI (2013) Recycling and reuse of spent microalgal biomass for sustainable biofuels. Biochem Eng 75:101–107CrossRefGoogle Scholar
  39. Rohman A, Che Man YB (2010) Fourier transform infrared (FTIR) spectroscopy for analysis of extra virgin olive oil adulterated with palm oil. Food Res Int 43:886–892CrossRefGoogle Scholar
  40. Spolaore P, Joannis-Cassan C, Duran E, Isambert A (2006) Commercial applications of microalgae. J Biosci Bioeng 101:87–96CrossRefGoogle Scholar
  41. Sydney EB, Novak AC, de Carvalho JC, Soccol CR (2014) Respirometric balance and carbon fixation of industrially important algae. In: Biofuels from algae. Elsevier, pp 67–84Google Scholar
  42. Talebi AF, Mohtashami SK, Tabatabaei M, Tohidfar M, Bagheri A, Zeinalabedini M, Mirzaei HH, Mirzajanzadeh M, Shafaroudi SM, Bakhtiari S (2013) Fatty acids profiling: a selective criterion for screening microalgae strains for biodiesel production. Algal Res 2(3):258–267CrossRefGoogle Scholar
  43. Wei CY, Huang TC, Yu ZR, Wang BJ, Chen HH (2014) Fractionation for biodiesel purification using supercritical carbon dioxide. Energy 7:824–833Google Scholar
  44. Wu H, Miao X (2014) Biodiesel quality and biochemical changes of microalgae Chlorella pyrenoidosa and Scenedesmus obliquus in response to nitrate levels. Bioresour Technol 170:421–427CrossRefGoogle Scholar
  45. Xi C, He G, Deng Z, Wang N, Jiang W, Chen S (2014) Screening of microalgae for biodiesel feedstock. Adv in Micro 4:365–376CrossRefGoogle Scholar
  46. Yang F, Hanna MA, Sun R (2012) A review: Value-added uses for crude glycerol—a byproduct of biodiesel production. Biotechnol Bioeng 5:13Google Scholar
  47. Yoo C, Jun SY, Lee JY, Ahn CY, Oh HM (2010) Selection of microalgae for lipid production under high levels carbon dioxide. Bioresour Technol 101:71–74CrossRefGoogle Scholar
  48. Zhang J, Hu B (2012) A novel method to harvest microalgae via co-culture of filamentous fungi to form cell pellets. Bioresour Technol 114:529–535CrossRefGoogle Scholar
  49. Zhang BY, Geng YH, Li ZK, Hu HJ, Li YG (2009) Production of astaxanthin from Haematococcus in open pond by two-stage growth one-step process. Aqua 295:275–281CrossRefGoogle Scholar
  50. Zheng Y, Chi Z, Lucker B, Chen S (2012a) Two-stage heterotrophic and phototrophic culture strategy for algal biomass and lipid production. Bioresour Technol 103:484–488CrossRefGoogle Scholar
  51. Zheng H, Gao Z, Yin F, Ji X, Huang H (2012b) Lipid production of Chlorella vulgaris from lipid extracted microalgal biomass residues through two-step enzymatic hydrolysis. Bioresour Technol 117:1–6CrossRefGoogle Scholar
  52. Zheng Y, Li T, Yu X, Bates PD, Dong T, Chen S (2013) High-density fed-batch culture of a thermotolerant microalga Chlorella sorokiniana for biofuel production. Appl Energy 108:281–287CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Centre for Technology Alternatives for Rural Areas (CTARA)Indian Institute of Technology BombayMumbaiIndia
  2. 2.Centre for Transportation SystemsIndian Institute of Technology RoorkeeRoorkeeIndia
  3. 3.Department of Civil EngineeringIndian Institute of Technology RoorkeeRoorkeeIndia
  4. 4.Department of Civil EngineeringMalaviya National Institute of Technology JaipurJaipurIndia
  5. 5.Center for Rural Development and TechnologyIIT DelhiNew DelhiIndia
  6. 6.Department of BiotechnologyIndian Institute of Technology RoorkeeRoorkeeIndia

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