Bioprocess and Biosystems Engineering

, Volume 43, Issue 2, pp 217–232 | Cite as

Medium optimization and kinetic modeling for the production of Aspergillus niger inulinase

  • Mustafa Germec
  • Hilal Nur Gürler
  • Ali Ozcan
  • Selime Benemir Erkan
  • Ercan Karahalil
  • Irfan TurhanEmail author
Research Paper


The goals of this study were to optimize the medium formulation for enhanced production of Aspergillus niger inulinase using Plackett–Burman Design (PBD) and to model the fermentation in optimal medium formulation. Results indicated that (NH4)2SO4 (negative effect), yeast extract and peptone (positive effect) were determined as significant factors affecting the inulinase production. Different media including Medium A (non-enriched), Medium B (contains both negative and positive factors) and Medium C (contains only positive factors) were formed and inulinase fermentations were performed. Findings showed that the best nutritional formulation was Medium C, which yielded to be 1011.02 U/mL, 834.28 U/mL, 1.22, 4383.44 U/mg, 4186 U/mg, 158.49 U/mL/day, 128.60 U/mL/day and 94.54% of PInulinase, SInulinase, I/S ratio, SInulinase, SSucrase, QInulinase, QSucrase and SUY, respectively. Additionally, fungal growth, enzyme or protein production and substrate consumption were modeled using the logistic model, Luedeking–Piret model, and modified Luedeking–Piret model, respectively, and found that enzyme or protein production was non-growth associated. Besides, maintenance value (Z) was lower than γ value, indicating that A. niger mainly utilizes the sugars for enzyme production and fungal growth. Consequently, optimum medium composition was successfully determined by PBD and also the kinetic models fitted the experimental data very well with high regression coefficient.


Inulinase Sucrase Aspergillus niger Sugar beet molasses Medium optimization Kinetic modeling 



This work was supported by the Akdeniz University Research Foundation [Grant number #FDK-2019-4761]

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.


  1. 1.
    Bairoch A (2000) The ENZYME database in 2000. Nucleic Acids Res 28:304–305PubMedPubMedCentralGoogle Scholar
  2. 2.
    Fleming SE, GrootWassink JW, Murray ED (1979) Preparation of high-fructose syrup from the tubers of the Jerusalem artichoke (Helianthus tuberosus L.). Crit Rev Food Sci Nutr 12:1–28Google Scholar
  3. 3.
    Ettalibi M, Baratti JC (1987) Purification, properties and comparison of invertase, exoinulinases and endoinulinases of Aspergillus ficuum. Appl Microbiol Biotechnol 26:13–20Google Scholar
  4. 4.
    Singh R, Singh R (2017) Inulinases. Current developments in biotechnology and bioengineering. Elsevier, AmsterdamGoogle Scholar
  5. 5.
    Kango N, Jain SC (2011) Production and properties of microbial inulinases: recent advances. Food Biotechnol 25:165–212Google Scholar
  6. 6.
    Singh RS, Chauhan K, Kennedy JF (2016) A panorama of bacterial inulinases: production, purification, characterization and industrial applications. Int J Biol Macromol 96:312–322PubMedGoogle Scholar
  7. 7.
    Singh RS, Singh RP (2010) Production of fructooligosaccharides from inulin by endoinulinases and their prebiotic potential. Food Technol Biotechnol 48:435Google Scholar
  8. 8.
    Barthomeuf C, Regerat F, Pourrat H (1991) Production of inulinase by a new mold of Penicillium rugulosum. J Ferment Bioeng 72:491–494Google Scholar
  9. 9.
    Chen H-Q, Chen X-M, Li Y, Wang J, Jin Z-Y, Xu X-M, Zhao J-W, Chen T-X, Xie Z-J (2009) Purification and characterisation of exo-and endo-inulinase from Aspergillus ficuum JNSP5-06. Food Chem 115:1206–1212Google Scholar
  10. 10.
    Dinarvand M, Ariff B, Moeini H, Masomian M, Mousavi SS, Nahavandi R, Mustafa S (2012) Effect of extrinsic and intrinsic parameters on inulinase production by Aspergillus niger ATCC 20611. Electron J Biotechnol 15:5Google Scholar
  11. 11.
    Nakamura T, Kuramori K, Zaita N, Akimoto H, Ohta K (2001) Purification and properties of intracellular exo-and endoinulinases from Aspergillus niger strain 12. Bull Fac Agric Miyazaki Univ 48:49–58Google Scholar
  12. 12.
    Pandey A, Soccol CR, Selvakumar P, Soccol VT, Krieger N, Fontana JD (1999) Recent developments in microbial inulinases. Appl Biochem Biotechnol 81:35–52PubMedGoogle Scholar
  13. 13.
    Sheng J, Chi Z, Li J, Gao L, Gong F (2007) Inulinase production by the marine yeast Cryptococcus aureus G7a and inulin hydrolysis by the crude inulinase. Process Biochem 42:805–811Google Scholar
  14. 14.
    Ohta K, Akimoto H, Moriyama S (2004) Fungal inulinases: enzymology, molecular biology and biotechnology. J Appl Glycosci 51:247–254Google Scholar
  15. 15.
    Mazutti MA, Skrowonski A, Boni G, Zabot GL, Silva MF, de Oliveira D, Di Luccio M, Maugeri Filho F, Rodrigues MI, Treichel H (2010) Partial characterization of inulinases obtained by submerged and solid-state fermentation using agroindustrial residues as substrates: a comparative study. Appl Biochem Biotechnol 160:682–693PubMedGoogle Scholar
  16. 16.
    Mandenius CF, Brundin A (2008) Bioprocess optimization using design-of-experiments methodology. Biotechnol Prog 24:1191–1203PubMedGoogle Scholar
  17. 17.
    Brereton RG (2003) Chemometrics: data analysis for the laboratory and chemical plant. Wiley, New YorkGoogle Scholar
  18. 18.
    Poorna V, Kulkarni P (1995) A study of inulinase production in Aspergillus niger using fractional factorial design. Bioresour Technol 54:315–320Google Scholar
  19. 19.
    Abd El Aty A, Wehaidy H, Mostafa F (2014) Optimization of inulinase production from low cost substrates using Plackett–Burman and Taguchi methods. Carbohyd Polym 102:261–268Google Scholar
  20. 20.
    El-Naggar NE-A, Metwally E, El-Tanash A, Sherief A (2016) Statistical optimization of culture conditions and overproduction of inulinase using low cost, renewable feedstocks by a newly isolated Aspergillus sclerotiorum under solid-state fermentation conditions: inulin hydrolysis by partially purified inulinase. J Pure Appl Microbiol 10:991–1014Google Scholar
  21. 21.
    Mavituna F, Sinclair CG (2008) Modelling the kinetics of biological activity in fermentation systems. Practical fermentation technology. Wiley, ChichesterGoogle Scholar
  22. 22.
    Keskin Gündoğdu T, Deniz İ, Çalışkan G, Şahin ES, Azbar N (2016) Experimental design methods for bioengineering applications. Crit Rev Biotechnol 36:368–388PubMedGoogle Scholar
  23. 23.
    Thilakavathi M, Basak T, Panda T (2007) Modeling of enzyme production kinetics. Appl Microbiol Biotechnol 73:991–1007PubMedGoogle Scholar
  24. 24.
    Germec M, Turhan I (2019) Evaluation of carbon sources for the production of inulinase by Aspergillus niger A42 and its characterization. Bioprocess Biosyst Eng. CrossRefPubMedGoogle Scholar
  25. 25.
    Ongen-Baysal G, Sukan SS, Vassilev N (1994) Production and properties of inulinase from Aspergillus niger. Biotechnol Lett 16:275–280Google Scholar
  26. 26.
    Michael T, Madigan JMM, Bender KS, Buckley DH, Stahl DA (2014) Brock biology of microorganisms, 14th edn. Benjamin Cummings, San FranciscoGoogle Scholar
  27. 27.
    Bhargavi PL, Prakasham RS (2016) Enhanced fibrinolytic protease production by Serratia marcescens RSPB11 through Plackett–Burman and response surface methodological approaches. J Appl Biol Biotechnol 4:006–014Google Scholar
  28. 28.
    Hensing M, Vrouwenvelder H, Hellinga C, Baartmans R, Van Dijken H (1994) Production of extracellular inulinase in high-cell-density fed-batch cultures of Kluyveromyces marxianus. Appl Microbiol Biotechnol 42:516–521Google Scholar
  29. 29.
    Rech R, Ayub MAZ (2007) Simplified feeding strategies for fed-batch cultivation of Kluyveromyces marxianus in cheese whey. Process Biochem 42:873–877Google Scholar
  30. 30.
    Viswanathan P, Kulkarni P (1995) Enhancement of inulinase production by Aspergillus niger van Teighem. J Appl Microbiol 78:384–386Google Scholar
  31. 31.
    Miller GL (1959) Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal Chem 31:426–428Google Scholar
  32. 32.
    Bender JP, Mazutti MA, de Oliveira D, Di Luccio M, Treichel H (2006) Inulinase production by Kluyveromyces marxianus NRRL Y-7571 using solid state fermentation. Appl Biochem Biotechnol 132:951–958Google Scholar
  33. 33.
    Kalil S, Suzan R, Mougeri F, Rodrigues M (2001) Optimization of inulinase production by Kluyveromyces marxianus using factorial design. Appl Biochem Biotechnol 94:257–264PubMedGoogle Scholar
  34. 34.
    Germec M, Turhan I, Karhan M, Demirci A (2015) Ethanol production via repeated-batch fermentation from carob pod extract by using Saccharomyces cerevisiae in biofilm reactor. Fuel 161:304–311Google Scholar
  35. 35.
    Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254PubMedGoogle Scholar
  36. 36.
    Karahalil E, Demirel F, Evcan E, Germeç M, Tari C, Turhan I (2017) Microparticle-enhanced polygalacturonase production by wild type Aspergillus sojae. 3 Biotech 7:361PubMedPubMedCentralGoogle Scholar
  37. 37.
    Cemeroğlu B (2010) Gıda Analizleri. Gıda Teknolojileri Derneği 34:1Google Scholar
  38. 38.
    Don M, Shoparwe N (2010) Kinetics of hyaluronic acid production by Streptococcus zooepidemicus considering the effect of glucose. Biochem Eng J 49:95–103Google Scholar
  39. 39.
    Mohammad F, Badr-Eldin S, El-Tayeb O, El-Rahman OA (1995) Polysaccharide production by Aureobasidium pullulans III. The influence of initial sucrose concentration on batch kinetics. Biomass Bioenergy 8:121–129Google Scholar
  40. 40.
    Ross T (1996) Indices for performance evaluation of predictive models in food microbiology. J Appl Microbiol 81:501–508Google Scholar
  41. 41.
    Ross T (1999) Predictive food microbiology models in the meat industry. Meat and Livestock, North SydneyGoogle Scholar
  42. 42.
    Feng J, Zhang J-S, Jia W, Yang Y, Liu F, Lin C-C (2014) An unstructured kinetic model for the improvement of triterpenes production by Ganoderma lucidum G0119 based on nitrogen source effect. Biotechnol Bioprocess Eng 19:727–732Google Scholar
  43. 43.
    Shuler ML, Kargi F (2017) Major metabolic pathways. In: Shuler ML, Kargi F (eds) Bioprocess engineering: basic concepts, 2nd edn. Prentice Hall, Upper Saddle RiverGoogle Scholar
  44. 44.
    Shuler ML, Kargi F, DeLisa M (2017) Bioprocess engineering: basic concepts, 3rd edn. Prentice Hall, Upper Saddle RiverGoogle Scholar
  45. 45.
    Pandey A (2004) Concise encyclopedia of bioresource technology. Food Products Press, New YorkGoogle Scholar
  46. 46.
    Sandhya C, Pandey A (2006) Inulinase. In: Pandey A, Soccol CR, Larroche C (eds) Enzyme technology. Asiatech Publishers Inc, New DelhiGoogle Scholar
  47. 47.
    Vandamme EJ, Derycke DG (1983) Microbial inulinases: fermentation process, properties, and applications. Adv Appl Microbiol 29:139–176PubMedGoogle Scholar
  48. 48.
    Gill PK, Sharma AD, Harchand RK, Singh P (2003) Effect of media supplements and culture conditions on inulinase production by an actinomycete strain. Bioresour Technol 87:359–362PubMedGoogle Scholar
  49. 49.
    Singh R, Dhaliwal R, Puri M (2006) Production of inulinase from Kluyveromyces marxianus YS-1 using root extract of Asparagus racemosus. Process Biochem 41:1703–1707Google Scholar
  50. 50.
    Singh R, Sooch BS, Puri M (2007) Optimization of medium and process parameters for the production of inulinase from a newly isolated Kluyveromyces marxianus YS-1. Bioresour Technol 98:2518–2525PubMedGoogle Scholar
  51. 51.
    Zhang L, Zhao C, Ohta WY, Wang Y (2005) Inhibition of glucose on an exoinulinase from Kluyveromyces marxianus expressed in Pichia pastoris. Process Biochem 40:1541–1545Google Scholar
  52. 52.
    Ge X-Y, Zhang W-G (2005) Effects of octadecanoylsucrose derivatives on the production of inulinase by Aspergillus niger SL-09. World J Microbiol Biotechnol 21:1633–1638Google Scholar
  53. 53.
    Kampen WH (2014) Nutritional requirements in fermentation processes. In: Vogel HC, Todaro CM (eds) Fermentation and biochemical engineering handbook, 3rd edn. Elsevier, AmsterdamGoogle Scholar
  54. 54.
    Ongen-Baysal G, Sukan SS (1996) Production of inulinase by mixed culture of Aspergillus niger and Kluyveromyces marxianus. Biotechnol Lett 18:1431–1434Google Scholar
  55. 55.
    Kowalska A, Antecka A, Owczarz P, Bizukojć M (2017) Inulinolytic activity of broths of Aspergillus niger ATCC 204447 cultivated in shake flasks and stirred tank bioreactor. Eng Life Sci 17:1006–1020Google Scholar
  56. 56.
    Cheng K-C, Demirci A, Catchmark JM, Puri VM (2010) Modeling of pullulan fermentation by using a color variant strain of Aureobasidium pullulans. J Food Eng 98:353–359Google Scholar
  57. 57.
    Saat MN, Annuar MSM, Alias Z, Chuan LT, Chisti Y (2014) Modeling of growth and laccase production by Pycnoporus sanguineus. Bioprocess Biosyst Eng 37:765–775PubMedGoogle Scholar
  58. 58.
    Tavares A, Coelho M, Coutinho J, Xavier A (2005) Laccase improvement in submerged cultivation: induced production and kinetic modelling. J Chem Technol Biotechnol Int Res Process Environ Clean Technol 80:669–676Google Scholar
  59. 59.
    Tevatia R, Demirel Y, Blum P (2012) Kinetic modeling of photoautotropic growth and neutral lipid accumulation in terms of ammonium concentration in Chlamydomonas reinhardtii. Bioresour Technol 119:419–424PubMedGoogle Scholar
  60. 60.
    Mazutti MA, Corazza ML, Maugeri Filho F, Rodrigues MI, Corazza FC, Treichel H (2009) Inulinase production in a batch bioreactor using agroindustrial residues as the substrate: experimental data and modeling. Bioprocess Biosyst Eng 32:85–95PubMedGoogle Scholar
  61. 61.
    Zeng AP, Ross A, Biebl H, Tag C, Günzel B, Deckwer WD (1994) Multiple product inhibition and growth modeling of Clostridium butyricum and Klebsiella pneumoniae in glycerol fermentation. Biotechnol Bioeng 44:902–911PubMedGoogle Scholar
  62. 62.
    Sinclair C, Kristiansen B, Bu’Lock J (1987) Fermentation kinetics and modeling. Open University Press, MaidenheadGoogle Scholar
  63. 63.
    Liu Z, Qi W, He Z, Wang H, Feng Y (2008) PSO-based parameter estimation of nonlinear kinetic models for β-mannanase fermentation. Chem Biochem Eng Q 22:195–201Google Scholar

Copyright information

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

Authors and Affiliations

  1. 1.Department of Food EngineeringAkdeniz UniversityAntalyaTurkey

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