Journal of Applied Phycology

, Volume 30, Issue 6, pp 3483–3492 | Cite as

Heterotrophic growth and oil production from Micractinium sp. ME05 using molasses

  • Iskin Kose Engin
  • Deniz Cekmecelioglu
  • Ayse Meral Yücel
  • Huseyin Avni OktemEmail author


In this study the thermo-resistant green alga Micractinium sp. ME05 was cultivated in media containing molasses as a carbon source. Shake flask experiments and 2-L bioreactor experiments were conducted at different inoculum ratios, aeration rates, and agitation speeds. The experimental condition which resulted in the highest biomass concentration (3.73 ± 0.45 g L−1) with 10% inoculum in 500-mL flasks was scaled up to 2-L flasks at two aeration rates (0.25 and 0.5 L min−1). An increase in biomass concentration from 2.35 ± 0.53 to 3.06 ± 0.21 g L−1 was observed with an increase of aeration rate from 0.25 to 0.50 L min−1, which demonstrated significant effect of aeration rate on biomass concentration (p = 0.000 < 0.05). In 2-L bioreactor experiments, highest biomass productivity (0.53 ± 0.076 g L−1 day−1) and lipid productivity (7.7 ± 1.6 g L−1 day−1) were obtained with 5% (v/v) inoculum and 50 rpm agitation speed. The principal fatty acids were palmitic acid (C16:0) and linoleic acid (C18:2) comprising 30.2 ± 1.01 and 45.2 ± 1.32% of the total fatty acid content, respectively. Thus, the present study highlights the possibility of using molasses for biomass and lipid production with Micractinium sp. ME05 under different cultivation conditions. Using low cost feedstock such as molasses would be valuable in terms of evaluating waste materials for further biodiesel production.


Chlorophyta Micractinium sp. Molasses Heterotrophic growth Bioreactor 



This study was carried out at the Middle East Technical University (METU) Biology Department Plant Biotechnology Laboratory and METU Food Engineering Department Bioprocess Laboratory. We would like to thank Asst. Prof. Dr. Melih Onay for his isolation and characterization of microalgal species used in this study.

Funding information

This study was funded by the Scientific and Technological Research Council of Turkey (TUBITAK) Project Number 114Z487).

Supplementary material

10811_2018_1486_MOESM1_ESM.docx (14 kb)
ESM 1 (DOCX 13 kb)


  1. Abou-Shanab RA, Raghavulu SV, Hassanin NMA, Kim s KYJ, Oh SU, Jeon B-H (2012) Manipulating nutrient composition of microalgal growth media to improve biomass yield and lipid content of Micractinium pusillum. Afr J Biotechnol 11:16270–16276CrossRefGoogle Scholar
  2. Azma M, Mohamed MS, Mohamad R, Rahmin RA, Ariff AB (2011) Improvement of medium composition for heterotrophic cultivation of green microalgae, Tetraselmis suecica, using response surface methodology. Biochem Eng J 53:187–195CrossRefGoogle Scholar
  3. Barbosa MJ, Albrecht M, Wijffels RH (2003) Hydrodynamic stress and lethal events in sparged microalgae cultures. Biotechnol Bioeng 83:112–120PubMedCrossRefGoogle Scholar
  4. Borowitzka MA (2013) High-value products from microalgae—their development and commercialisation. J Appl Phycol 25:743–756CrossRefGoogle Scholar
  5. Borowitzka MA, Moheimani NR (2013) Open pond culture systems. In: Borowitzka MA, Moheimani NR (eds) Algae for biofuels and energy. Springer, Dordrecht, pp 133–152CrossRefGoogle Scholar
  6. Borowitzka MA, Vonshak A (2017) Scaling up microalgal cultures to commercial scale. Eur J Phycol 52:407–418CrossRefGoogle Scholar
  7. Bouarab L, Dauta A, Loudiki M (2004) Heterotrophic and mixotrophic growth of Micractinium pusillum Fresenius in the presence of acetate and glucose: effect of light and acetate gradient concentration. Water Res 38:2706–2712PubMedCrossRefGoogle Scholar
  8. Carvalho AP, Meireles LA, Malcata FX (2006) Microalgal reactors: a review of enclosed system designs and performances. Biotechnol Prog 22:1490–1506PubMedCrossRefGoogle Scholar
  9. Chen F (1996) High cell density culture of microalgae in heterotrophic growth. Trends Biotechnol 14:421–426CrossRefGoogle Scholar
  10. Chen G-Q, Chen F (2006) Growing phototrophic cells without light. Biotechnol Lett 28:607–616PubMedCrossRefGoogle Scholar
  11. Cheng Y, Lu Y, Gao C, Wu Q (2009a) Alga-based biodiesel production and optimization using sugar cane as the feedstock. Energy Fuel 23:4166–4173CrossRefGoogle Scholar
  12. Cheng Y, Zhou W, Gao C, Lan K, Gao Y, Wu Q (2009b) Biodiesel production from Jerusalem artichoke (Helianthus Tuberosus L. ) tuber by heterotrophic microalgae Chlorella protothecoides. J Chem Technol Biotechnol 84:777–781CrossRefGoogle Scholar
  13. Chisti Y (2007) Biodiesel from microalgae. Biotechnol Adv 25:294–306PubMedCrossRefGoogle Scholar
  14. Chiu SY, Kao CY, Chen CH, Kuan TC, Ong SC, Lin CS (2008) Reduction of CO2 by a high-density culture of Chlorella sp. in a semicontinuous photobioreactor. Bioresour Technol 99:3389–3396PubMedCrossRefGoogle Scholar
  15. Contin A, Van Der Heijden R, Ten Hoopen HJG, Verpoorte R (1998) The inoculum size triggers tryptamine or secologanin biosynthesis in a Catharanthus roseus cell culture. Plant Sci 139:205–211CrossRefGoogle Scholar
  16. De Swaaf ME, Sijtsma L, Pronk JT (2003) High-cell-density fed-batch cultivation of the docosahexaenoic acid producing marine alga Crypthecodinium cohnii. Biotechnol Bioeng 81:666–672PubMedCrossRefGoogle Scholar
  17. Miller GL (1959) Use of dinitrosalicyclic acid reagent for determination of reducing sugar. Anal Chem 31:426–428Google Scholar
  18. Gao C, Zhai Y, Ding Y, Wu Q (2010) Application of sweet sorghum for biodiesel production by heterotrophic microalga Chlorella protothecoides. Appl Energy 87:756–761CrossRefGoogle Scholar
  19. Gaurav K, Srivastava R, Sharma JG, Singh R, Singh V (2015) Molasses based growth and lipid production by Chlorella pyrenoidosa: a potential feedstock for biodiesel. Int J Green Energy 13:320–327CrossRefGoogle Scholar
  20. Gorman DS, Levine RP (1965) Cytochrome f and plastocyanin: their sequence in the photosynthetic electron transport chain of Chlamydomonas reinhardi. Proc Natl Acad Sci U S A 54:1665–1669PubMedPubMedCentralCrossRefGoogle Scholar
  21. Graverholt OS, Eriksen NT (2007) Heterotrophic high-cell-density fed-batch and continuous-flow cultures of Galdieria sulphuraria and production of phycocyanin. Appl Microbiol Biotechnol 77:69–75PubMedCrossRefGoogle Scholar
  22. Griffiths MJ, Harrison STL (2009) Lipid productivity as a key characteristic for choosing algal species for biodiesel production. J Appl Phycol 21:493–507CrossRefGoogle Scholar
  23. Gurkok S, Cekmecelioglu D, Ogel ZB (2011) Optimization of culture conditions for Aspergillus sojae expressing an Aspergillus fumigatus α-galactosidase. Bioresour Technol 102:4925–4929PubMedCrossRefGoogle Scholar
  24. Heidari M, Kariminia HR, Shayegan J (2016) Effect of culture age and initial inoculum size on lipid accumulation and productivity in a hybrid cultivation system of Chlorella vulgaris. Process Saf Environ Prot 104:111–122CrossRefGoogle Scholar
  25. Heredia-Arroyo T, Wei W, Hu B (2010) Oil accumulation via heterotrophic/mixotrophic Chlorella protothecoides. Appl Biochem Biotechnol 162:1978–1995PubMedCrossRefGoogle Scholar
  26. Huang G, Chen F, Wei D, Zhang X, Chen G (2010) Biodiesel production by microalgal biotechnology. Appl Energy 87:38–46CrossRefGoogle Scholar
  27. Huang J, Li Y, Wan M, Yan Y, Feng F, Qu X, Wang J, Shen G, Li W, Fan J, Wang W (2014) Novel flat-plate photobioreactors for microalgae cultivation with special mixers to promote mixing along the light gradient. Bioresour Technol 159:8–16PubMedCrossRefGoogle Scholar
  28. Ji MK, Abou-Shanab RAI, Kim SH, Salama E, Lee SH, Kabra AN, Lee Y-S, Hong S, Jeon B-H (2013) Cultivation of microalgae species in tertiary municipal wastewater supplemented with CO2 for nutrient removal and biomass production. Ecol Eng 58:142–148CrossRefGoogle Scholar
  29. Karpagam R, Raj KJ, Ashokkumar B, Varalakshmi P (2015) Characterization and fatty acid profiling in two fresh water microalgae for biodiesel production: lipid enhancement methods and media optimization using response surface methodology. Bioresour Technol 188:177–184PubMedCrossRefGoogle Scholar
  30. Knothe G (2009) Improving biodiesel fuel properties by modifying fatty ester composition. Energy Environ Sci 2:759–766CrossRefGoogle Scholar
  31. Knothe G (2013) Production and properties of biodiesel from algal oils. In: Borowitzka MA, Moheimani NR (eds) Algae for biofuels and energy. Springer, Dordrecht, pp 207–221CrossRefGoogle Scholar
  32. Kose Engin I, Cekmecelioglu D, Yücel AM, Oktem HA (2018) Enhancement of heterotrophic biomass production by Micractinium sp.ME05. Waste Biomass Valorization 9:811–820CrossRefGoogle Scholar
  33. Kumar V, Muthuraj M, Palabhanvi B, Ghoshal AK, Das D (2014) High cell density lipid rich cultivation of a novel microalgal isolate Chlorella sorokiniana FC6 IITG in a single-stage fed-batch mode under mixotrophic condition. Bioresour Technol 170:115–124PubMedCrossRefGoogle Scholar
  34. Liang Y, Sarkany N, Cui Y (2009) Biomass and lipid productivities of Chlorella vulgaris under autotrophic, heterotrophic and mixotrophic growth conditions. Biotechnol Lett 31:1043–1049PubMedCrossRefGoogle Scholar
  35. Lin Z, Raya A, Ju LK (2014) Microalga Ochromonas danica fermentation and lipid production from waste organics such as ketchup. Process Biochem 49:1383–1392CrossRefGoogle Scholar
  36. Liu J, Huang J, Jiang Y, Chen F (2012) Molasses-based growth and production of oil and astaxanthin by Chlorella zofingiensis. Bioresour Technol 107:393–398PubMedCrossRefGoogle Scholar
  37. Liu J, Sun Z, Zhong Y, Gerken H, Huang J, Chen F (2013) Utilization of cane molasses towards cost-saving astaxanthin production by a Chlorella zofingiensis mutant. J Appl Phycol 25:1447–1456CrossRefGoogle Scholar
  38. Lu L, Wang J, Yang G, Zhu B, Pan K (2017) Heterotrophic growth and nutrient productivities of Tetraselmis chuii using glucose as a carbon source under different C/N ratios. J Appl Phycol 29:15–21CrossRefGoogle Scholar
  39. Ma Q, Wang J, Lu S, Lv Y, Yuan Y (2013) Quantitative proteomic profiling reveals photosynthesis responsible for inoculum size dependent variation in Chlorella sorokiniana. Biotechnol Bioeng 110:773–784PubMedCrossRefGoogle Scholar
  40. Moheimani NR, Isdepsky A, Lisec J, Raes E, Borowitzka MA (2011) Coccolithophorid algae culture in closed photobioreactors. Biotechnol Bioeng 108:2078–2087PubMedCrossRefGoogle Scholar
  41. Morales-Sánchez D, Tinoco-Valencia R, Kyndt J, Martinez A (2013) Heterotrophic growth of Neochloris oleoabundans using glucose as a carbon source. Biotechnol Biofuels 6:100PubMedPubMedCentralCrossRefGoogle Scholar
  42. Najafpour GD, Poi Shan C (2003) Enzymatic hydrolysis of molasses. Bioresour Technol 86:91–94PubMedCrossRefGoogle Scholar
  43. Onay M, Sonmez C, Oktem HA, Yucel AM (2014) Thermo-resistant green microalgae for effective biodiesel production: isolation and characterization of unialgal species from geothermal flora of central Anatolia. Bioresour Technol 169:62–71PubMedCrossRefGoogle Scholar
  44. Onay M, Sonmez C, Oktem HA, Yucel M (2016) Evaluation of various extraction techniques for efficient lipid recovery from thermo-resistant microalgae, Hindakia, Scenedesmus and Micractinium species. Am J Anal Chem 7:141–150CrossRefGoogle Scholar
  45. Pahl SL, Lewis DM, Chen F, King KD (2010) Growth dynamics and the proximate biochemical composition and fatty acid profile of the heterotrophically grown diatom Cyclotella cryptica. J Appl Phycol 22:165–171PubMedCrossRefGoogle Scholar
  46. Park KC, Whitney C, McNichol JC, Dickinson KE, MacQuarrie S, Skrupski BP, Zou J, Wilson KE, O'Leary JB, McGinn PJ (2012) Mixotrophic and photoautotrophic cultivation of 14 microalgae isolates from Saskatchewan, Canada: potential applications for wastewater remediation for biofuel production. J Appl Phycol 24:339–348CrossRefGoogle Scholar
  47. Perez-Garcia O, Escalante FME, de-Bashan LE, Bashan Y (2011) Heterotrophic cultures of microalgae: metabolism and potential products. Water Res 45:11–36PubMedCrossRefGoogle Scholar
  48. Piasecka A, Krzemińska I, Tys J (2017) Enrichment of Parachlorella kessleri biomass with bioproducts: oil and protein by utilization of beet molasses. J Appl Phycol 29:1735–1743PubMedPubMedCentralCrossRefGoogle Scholar
  49. Posten C (2009) Design principles of photo-bioreactors for cultivation of microalgae. Eng Life Sci 9:165–177CrossRefGoogle Scholar
  50. Anderson RA (2005) Photobioreactors and fermentors: the light and dark sides of growing algae. In: Andersen Robert (ed) Algal culturing techniques. Elsevier Academic Press, NY pp 189–203Google Scholar
  51. Singhasuwan S, Choorit W, Sirisansaneeyakul S, Kokkaew N, Chisti Y (2015) Carbon-to-nitrogen ratio affects the biomass composition and the fatty acid profile of heterotrophically grown Chlorella sp. TISTR 8990 for biodiesel production. J Biotechnol 216:169–177PubMedCrossRefGoogle Scholar
  52. Smith RT, Bangert K, Wilkinson SJ, Gilmour DJ (2015) Synergistic carbon metabolism in a fast growing mixotrophic freshwater microalgal species Micractinium inermum. Biomass Bioenergy 82:73–86CrossRefGoogle Scholar
  53. Sobczuk TM, Camacho FG, Grima EM, Chisti Y (2006) Effects of agitation on the microalgae Phaeodactylum tricornutum and Porphyridium cruentum. Bioprocess Biosyst Eng 28:243–250PubMedCrossRefGoogle Scholar
  54. Sonmez C, Elcin E, Akin D, Oktem HA, Yucel M (2016) Evaluation of novel thermo-resistant Micractinium and Scenedesmus sp. for efficient biomass and lipid production under different temperature and nutrient regimes. Bioresour Technol 211:422–428PubMedCrossRefGoogle Scholar
  55. Stephenson AL, Dennis JS, Howe CJ, Scott SA, Smith AG (2010) Influence of nitrogen-limitation regime on the production of Chlorella vulgaris of lipids for biodiesel feedstocks. Biofuels 1:47–58CrossRefGoogle Scholar
  56. Wei A, Zhang X, Wei D, Chen G, Wu Q, Yang ST (2009) Effects of cassava starch hydrolysate on cell growth and lipid accumulation of the heterotrophic microalgae Chlorella protothecoides. J Ind Microbiol Biotechnol 36:1383–1389PubMedCrossRefGoogle Scholar
  57. Yan D, Lu Y, Chen YF, Wu Q (2011) Waste molasses alone displaces glucose-based medium for microalgal fermentation towards cost-saving biodiesel production. Bioresour Technol 102:6487–6493PubMedCrossRefGoogle Scholar
  58. Yeh KL, Chang JS (2012) Effects of cultivation conditions and media composition on cell growth and lipid productivity of indigenous microalga Chlorella vulgaris ESP-31. Bioresour Technol 105:120–127PubMedCrossRefGoogle Scholar
  59. Yen HW, Chang JT (2015) Growth of oleaginous Rhodotorula glutinis in an internal-loop airlift bioreactor by using lignocellulosic biomass hydrolysate as the carbon source. J Biosci Bioeng 119:580–584PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V., part of Springer Nature 2018

Authors and Affiliations

  • Iskin Kose Engin
    • 1
    • 2
  • Deniz Cekmecelioglu
    • 3
  • Ayse Meral Yücel
    • 1
    • 4
  • Huseyin Avni Oktem
    • 1
    • 4
    • 5
    Email author
  1. 1.Department of BiotechnologyMiddle East Technical UniversityAnkaraTurkey
  2. 2.Central Laboratory, Molecular Biology and BiotechnologyMiddle East Technical UniversityAnkaraTurkey
  3. 3.Departmenf of Food EngineeringMiddle East Technical UniversityAnkaraTurkey
  4. 4.Department of Biological SciencesMiddle East Technical UniversityAnkaraTurkey
  5. 5.Nanobiz Ltd. METU-TechnopolisAnkaraTurkey

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