Bioethanol Production Using Saccharomyces cerevisiae Immobilized in Calcium Alginate–Magnetite Beads and Application of Response Surface Methodology to Optimize Bioethanol Yield

  • Snehal Ingale
  • Venkata Anand Parnandi
  • Sanket J. JoshiEmail author
Part of the Biofuel and Biorefinery Technologies book series (BBT, volume 7)


We studied the bioethanol production in molasses-based medium by yeast Saccharomyces cerevisiae immobilized in calcium alginate magnetite beads (CAMB). The yeast was isolated from soil samples collected near a local sugar mill, and identified as S. cerevisiae. We synthesized magnetite nanoparticles and immobilized yeast in CAMB. The media components and environmental parameters were statistically screened and optimized for better ethanol production, using statistical design methodologies—factorial designs and response surface methodology. The factors of molasses concentration, temperature and incubation time were found to have significant effect on ethanol production. The immobilized cells could be reused for more than 120 days, retaining its original activity. The CAMBs with immobilized yeast cells were analysed by ESEM with EDAX, after 96 h of fermentation to observe the surface structure of the beads. It can be observed that yeast was immobilized in the beads and actively growing. Further ethanol production was carried out in packed-bed column reactor using yeast immobilized in CAMB, under fed-batch mode. The average ethanol produced by fed-batch fermentation was 1.832 g% ± 0.103, and the average ethanol yield was 81.420% ± 4.6. Further studies using yeast immobilized in CAMB are recommended to carry out continuous fermentation, and further scale up bioethanol production in a magnetically stabilized fluidized bed reactor (MSFBR), where the position of the beads in the system can be controlled and maintained by the application of oscillating electric field.



SI and VP would like to thank ARIBAS and CVM, and SJ would like to acknowledge Sultan Qaboos University for providing the research facility.


  1. Abdul Rahman I, Ayob MTM, Radiman S (2014) Enhanced photocatalytic performance of NiO-decorated ZnO nanowhiskers for methylene blue degradation. J Nanotechnol 212694:8.
  2. Al-Bahry SN, Al-Wahaibi YM, Elshafie AE, Al-Bemani AS, Joshi SJ, Al-Makhmari HS, Al-Sulaimani HS (2013) Biosurfactant production by Bacillus subtilis B20 using date molasses and its possible application in enhanced oil recovery. Int Biodeterior Biodegradation 81:141–146CrossRefGoogle Scholar
  3. Alexandre H, Rousseaux I, Charpentier C (1994) Ethanol adaptation mechanisms in Saccharomyces cerevisiae. Biotechnol Appl Biochem 20(2):173–183Google Scholar
  4. Amin G, De Mot R, Van Dijck K, Verachtert H (1985) Direct alcoholic fermentation of starchy biomass using amylolytic yeast strains in batch and immobilized cell systems. Appl Microbiol Biotechnol 22(4):237–245CrossRefGoogle Scholar
  5. Bajaj BK, Taank V, Thakur RL (2003) Characterization of yeasts for ethanolic fermentation of molasses with high sugar concentrations. J Sci Ind Res 62(11):1079–1085Google Scholar
  6. Bajaj BK, Yousuf S, Thakur RL (2001) Selection and characterization of yeasts for desirable fermentation characteristics. Indian J Microbiol 41(2):107–110Google Scholar
  7. Bajpai PK, Margaritis A (1985) Kinetics of ethanol production by immobilized cells of Zymomonas mobilis at varying D-glucose concentrations. Enzyme Microb Technol 7(9):462–464CrossRefGoogle Scholar
  8. Baptista CMSG, Cóias JMA, Oliveira ACM, Oliveira NMC, Rocha JMS, Dempsey MJ, Benson PS (2006) Natural immobilisation of microorganisms for continuous ethanol production. Enzyme Microb Technol 40(1):127–131CrossRefGoogle Scholar
  9. Beaven MJ, Charpentier C, Rose AH (1982) Production and tolerance of ethanol in relation to phospholipid fatty-acyl composition in Saccharomyces cerevisiae NCYC 431. J Gen Microbiol 128(7):1447–1455Google Scholar
  10. Bekers M, Ventina E, Karsakevich A, Vina I, Rapoport A, Upite D, Linde R (1999) Attachment of yeast to modified stainless steel wire spheres, growth of cells and ethanol production. Process Biochem 35(5):523–530CrossRefGoogle Scholar
  11. Berry CC, Curtis AS (2003) Functionalization of magnetic nanoparticles for applications in biomedicine. J Phys D Appl Phys 36(13):R198CrossRefGoogle Scholar
  12. Bisson LF (1999) Stuck and sluggish fermentations. Am J Enol Vitic 50(1):107–119Google Scholar
  13. Black GM, Webb C, Matthews TM, Atkinson B (1984) Practical reactor systems for yeast cell immobilization using biomass support particles. Biotechnol Bioeng 26(2):134–141CrossRefGoogle Scholar
  14. Borzani W (2001) Variation of the ethanol yield during oscillatory concentrations changes in undisturbed continuous ethanol fermentation of sugar-cane blackstrap molasses. World J Microbiol Biotechnol 17(3):253–258CrossRefGoogle Scholar
  15. Borzani W, Gerab A, De La Higuera GA, Pires MH, Piplovic R (1993) Batch ethanol fermentation of molasses: a correlation between the time necessary to complete the fermentation and the initial concentrations of sugar and yeast cells. World J Microbiol Biotechnol 9(2):265–268CrossRefGoogle Scholar
  16. Cassidy MB, Lee H, Trevors JT (1996) Environmental applications of immobilized microbial cells: a review. J Ind Microbiol 16(2):79–101CrossRefGoogle Scholar
  17. Chen JC, Chou CC (1993) Cane sugar handbook: a manual for cane sugar manufacturers and their Chemists. Wiley, New YorkGoogle Scholar
  18. Cohen Y (2001) Biofiltration–the treatment of fluids by microorganisms immobilized into the filter bedding material: a review. Biores Technol 77(3):257–274MathSciNetCrossRefGoogle Scholar
  19. De Carvalho JCM, Aquarone E, Sato S, Brazzach ML, Moraes DA, Borzani W (1993) Fed-batch alcoholic fermentation of sugar cane blackstrap molasses: influence of the feeding rate on yeast yield and productivity. Appl Microbiol Biotechnol 38(5):596–598CrossRefGoogle Scholar
  20. Del Borghi M, Converti A, Parisi F, Ferraiolo G (1985) Continuous alcohol fermentation in an immobilized cell rotating disk reactor. Biotechnol Bioeng 27(6):761–768CrossRefGoogle Scholar
  21. Dubois M, Gilles KA, Amilton JK (1956) Colorimetric determination of sugars and related substances. Anal Chem 28:350–356CrossRefGoogle Scholar
  22. El Ghandoor H, Zidan HM, Khalil MM, Ismail MIM (2012) Synthesis and some physical properties of magnetite (Fe3O4) nanoparticles. Int J Electrochem Sci 7:5734–5745Google Scholar
  23. El-Gendy NS, Madian HR, Amr SSA (2013) Design and optimization of a process for sugarcane molasses fermentation by Saccharomyces cerevisiae using response surface methodology. Int J Microbiol 2013Google Scholar
  24. Fujimura T, Kaetsu I (1985) Nature of yeast-cells immobilized by radiation polymerization activity dependence on the molecular-motion of polymer carriers. Zeitschrift Fur Naturforschung Ca J Biosci 40(7–8):576–579Google Scholar
  25. Ghareib M, Youssef KA, Khalil AA (1988) Ethanol tolerance of Saccharomyces cerevisiae and its relationship to lipid content and composition. Folia Microbiol 33(6):447–452CrossRefGoogle Scholar
  26. Göksungur Y, Zorlu N (2001) Production of ethanol from beet molasses by Ca-alginate immobilized yeast cells in a packed-bed bioreactor. Turk J Biol 25(3):265–275Google Scholar
  27. Gray WD (1941) Studies on the alcohol tolerance of yeasts. J Bacteriol 42(5):561Google Scholar
  28. Gray WD (1948) Further studies on the alcohol tolerance of yeast: its relationship to cell storage products. J Bacteriol 55(1):53Google Scholar
  29. Gupta AK, Gupta M (2005) Synthesis and surface engineering of iron oxide nanoparticles for biomedical applications. Biomaterials 26(18):3995–4021MathSciNetCrossRefGoogle Scholar
  30. Hansen AC, Zhang Q, Lyne PW (2005) Ethanol–diesel fuel blends—a review. Biores Technol 96(3):277–285CrossRefGoogle Scholar
  31. Haynes WC, Wickerham LJ, Hesseltine CW (1955) Maintenance of cultures of industrially important microorganisms. Appl Microbiol 3(6):361Google Scholar
  32. Huang SH, Liao MH, Chen DH (2003) Direct binding and characterization of lipase onto magnetic nanoparticles. Biotechnol Prog 19(3):1095–1100CrossRefGoogle Scholar
  33. Ibeas JI, Jimenez J (1997) Mitochondrial DNA loss caused by ethanol in Saccharomyces flor yeasts. Appl Environ Microbiol 63(1):7–12Google Scholar
  34. Ingale S, Joshi SJ, Gupte A (2014) Production of bioethanol using agricultural waste: banana pseudo stem. Braz J Microbiol 45(3):885–892CrossRefGoogle Scholar
  35. Ingram LO (1976) Adaptation of membrane lipids to alcohols. J Bacteriol 125(2):670–678Google Scholar
  36. Ivanova V, Petrova P, Hristov J (2011) Application in the ethanol fermentation of immobilized yeast cells in matrix of alginate/magnetic nanoparticles, on chitosan-magnetite microparticles and cellulose-coated magnetic nanoparticles. Int Rev Chem Eng 3(3):289–299Google Scholar
  37. Joshi S, Yamazaki H (1984) Film fermenter for ethanol production by yeast immobilized on cotton cloth. Biotech Lett 6(12):797–802CrossRefGoogle Scholar
  38. Joshi S, Bharucha C, Jha S, Yadav S, Nerurkar A, Desai AJ (2008) Biosurfactant production using molasses and whey under thermophilic conditions. Biores Technol 99(1):195–199CrossRefGoogle Scholar
  39. Joshi S, Yadav S, Nerurkar A, Desai AJ (2007) Statistical optimization of medium components for the production of biosurfactant by Bacillus licheniformis K51. J Microbiol Biotechnol 17(2):313Google Scholar
  40. Kshirsagar SD, Waghmare PR, Loni PC, Patil SA, Govindwar SP (2015) Dilute acid pretreatment of rice straw, structural characterization and optimization of enzymatic hydrolysis conditions by response surface methodology. RSC Adv 5(58):46525–46533CrossRefGoogle Scholar
  41. Limtong S, Sringiew C, Yongmanitchai W (2007) Production of fuel ethanol at high temperature from sugar cane juice by a newly isolated Kluyveromyces marxianus. Biores Technol 98(17):3367–3374CrossRefGoogle Scholar
  42. López A, Lázaro N, Marqués AM (1997) The interphase technique: a simple method of cell immobilization in gel-beads. J Microbiol Methods 30(3):231–234CrossRefGoogle Scholar
  43. Mallick N (2002) Biotechnological potential of immobilized algae for wastewater N, P and metal removal: a review. Biometals 15(4):377–390CrossRefGoogle Scholar
  44. Marcelle A, de Vos Betty-Jayne, Visser MS (2007) The preparation, assay and certification of aqueous ethanol reference solutions. Accred Qual Assur 12:188–193CrossRefGoogle Scholar
  45. McGhee JE, Carr ME, St Julian G (1984) Continuous bioconversion of starch to ethanol by calcium-alginate immobilized enzymes and yeasts. Cereal Chem (US) 61(5)Google Scholar
  46. Miller GL (1959) Use of DNS reagent for the measurement of reducing sugar. Anal Chem 31(1):426–428CrossRefGoogle Scholar
  47. Mino AK (2010) Ethanol production from sugarcane in India: viability, constraints and implications, Doctoral dissertation, University of Illinois at Urbana–ChampaignGoogle Scholar
  48. Mishra P, Prasad R (1989) Relationship between ethanol tolerance and fatty acyl composition of Saccharomyces cerevisiae. Appl Microbiol Biotechnol 30(3):294–298CrossRefGoogle Scholar
  49. Moreno-Garrido I (2008) Microalgae immobilization: current techniques and uses. Biores Technol 99(10):3949–3964CrossRefGoogle Scholar
  50. Muthukrishnan S, Bhakya S, Kumar TS, Rao MV (2015) Biosynthesis, characterization and antibacterial effect of plant-mediated silver nanoparticles using Ceropegia thwaitesii–An endemic species. Ind Crops Prod 63:119–124CrossRefGoogle Scholar
  51. Nagashima M, Azuma M, Noguchi S (1983) Technology developments in biomass alcohol production in Japan: continuous alcohol production with immobilized microbial cells. Ann NY Acad Sci 413(1):457–468CrossRefGoogle Scholar
  52. Najafpour G, Younesi H, Ismail KSK (2004) Ethanol fermentation in an immobilized cell reactor using Saccharomyces cerevisiae. Biores Technol 92(3):251–260CrossRefGoogle Scholar
  53. Nigam JN, Gogoi BK, Bezbaruah RL (1998) Alcoholic fermentation by agar-immobilized yeast cells. World J Microbiol Biotechnol 14(3):457–459CrossRefGoogle Scholar
  54. Nofemele Z, Shukla P, Trussler A, Permaul K, Singh S (2012) Improvement of ethanol production from sugarcane molasses through enhanced nutrient supplementation using Saccharomyces cerevisiae. J Brew Distilling 3(2):29–35Google Scholar
  55. Nyirő-Kósa I, Rečnik A, Pósfai M (2012) Novel methods for the synthesis of magnetite nanoparticles with special morphologies and textured assemblages. J Nanopart Res 14(10):1–10CrossRefGoogle Scholar
  56. Okita WB, Bonham DB, Gainer JL (1985) Covalent coupling of microorganisms to a cellulosic support. Biotechnol Bioeng 27(5):632–637CrossRefGoogle Scholar
  57. Osawemwenze LA, Adogbo GM (2013) Ethanol synthesis using yeast anchored on calcium alginate and clay support. Int J Sci Eng Res 4(4):479–484Google Scholar
  58. Padman AJ, Henderson J, Hodgson S, Rahman PK (2014) Biomediated synthesis of silver nanoparticles using Exiguobacterium mexicanum. Biotech Lett 36:2079–2084CrossRefGoogle Scholar
  59. Pankhurst QA, Connolly J, Jones SK, Dobson J (2003) Applications of magnetic nanoparticles in biomedicine. J Phys D Appl Phys 36(13):R167CrossRefGoogle Scholar
  60. Prakasham RS, Kuriakose B, Ramakrishna SV (1999) The influence of inert solids on ethanol production by Saccharomyces cerevisiae. Appl Biochem Biotechnol 82(2):127–134CrossRefGoogle Scholar
  61. Pretorius IS (2000) Tailoring wine yeast for the new millennium: novel approaches to the ancient art of winemaking. Yeast 16(8):675–729CrossRefGoogle Scholar
  62. Priyadarshini E, Pradhan N, Sukla LB, Panda PK (2014) Controlled synthesis of gold nanoparticles using Aspergillus terreus IF0 and its antibacterial potential against gram negative pathogenic bacteria. J Nanotechnol 653198:9.
  63. Raheem A, Wakg WA, Yap YT, Danquah MK, Harun R (2015) Optimization of the microalgae Chlorella vulgaris for syngas production using central composite design. RSC Adv 5(88):71805–71815CrossRefGoogle Scholar
  64. Raju SS, Shinoj P, Joshi PK (2009) Sustainable development of biofuels: prospects and challenges. Econ Polit Wkly 65–72Google Scholar
  65. Ray S, Goldar A, Miglani S (2012) The ethanol blending policy in India. Econ Political Wkly 47(1):23–35Google Scholar
  66. Roy K, Sarkar CK, Ghosh CK (2014) Photocatalytic activity of biogenic silver nanoparticles synthesized using yeast (Saccharomyces cerevisiae) extract. Appl Nanosci 1–7Google Scholar
  67. Šafařı́k I, Šafařı́ková M (1999) Use of magnetic techniques for the isolation of cells. J Chromatogr B Biomed Sci Appl 722(1):33–53CrossRefGoogle Scholar
  68. Šafařík I, Šafaříková M (2002) Magnetic nanoparticles and biosciences. Springer, Vienna, pp 1–23Google Scholar
  69. Šafaříková M, Šafařik I (2001) Immunomagnetic separation of Escherichia coli O26, O111 and O157 from vegetables. Lett Appl Microbiol 33(1):36–39CrossRefGoogle Scholar
  70. Singh R, Shedbalkar UU, Wadhwani SA, Chopade BA (2015) Bacteriagenic silver nanoparticles: synthesis, mechanism, and applications. Appl Microbiol Biotechnol 99:4579–4593CrossRefGoogle Scholar
  71. Tartaj P, Morales MP, Veintemillas-Verdaguer S, Gonzalez-Carreño T, Serna CJ (2006) Synthesis, properties and biomedical applications of magnetic nanoparticles. Handb Magn Mater 16(5):403–482Google Scholar
  72. Turhan O, Isci A, Mert B, Sakiyan O, Donmez S (2015) Optimization of ethanol production from microfluidized wheat straw by response surface methodology. Prep Biochem Biotechnol 45(8):785–795CrossRefGoogle Scholar
  73. Vanaja M, Paulkumar K, Baburaja M, Rajeshkumar S, Gnanajobitha G, Malarkodi C, Sivakavinesan M, Annadurai G (2014) Degradation of methylene blue using biologically synthesized silver nanoparticles. Bioinorg Chem Appl 742346.
  74. Wang LH, Hsie MC, Chang CY, Kuo YC, Sang SL, Hsiao HD, Chen HC (1984) Improvement of ethanol productivity from cane molasses by a process using a high yeast cell concentration. ASPAC, Food & Fertilizer Technology CenterGoogle Scholar
  75. Wyman CE, Hinman ND (1990) Ethanol. Appl Biochem Biotechnol 24(1):735–753CrossRefGoogle Scholar

Copyright information

© Springer International Publishing AG, part of Springer Nature 2019

Authors and Affiliations

  • Snehal Ingale
    • 1
  • Venkata Anand Parnandi
    • 1
  • Sanket J. Joshi
    • 2
    Email author
  1. 1.Ashok and Rita Patel Institute of Integrated Study& Research in Biotechnology and Allied Sciences (ARIBAS)Sardar Patel UniversityAnandIndia
  2. 2.Central Analytical and Applied Research Unit, College of ScienceSultan Qaboos UniversityMuscatOman

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