World Journal of Microbiology and Biotechnology

, Volume 29, Issue 8, pp 1421–1429 | Cite as

Scale-up from shake flasks to bioreactor, based on power input and Streptomyces lividans morphology, for the production of recombinant APA (45/47 kDa protein) from Mycobacterium tuberculosis

  • Ramsés A. Gamboa-Suasnavart
  • Luz D. Marín-Palacio
  • José A. Martínez-Sotelo
  • Clara Espitia
  • Luis Servín-González
  • Norma A. Valdez-Cruz
  • Mauricio A. Trujillo-Roldán
Original Paper


Culture conditions in shake flasks affect filamentous Streptomyces lividans morphology, as well the productivity and O-mannosylation of recombinant Ala-Pro-rich O-glycoprotein (known as the 45/47 kDa or APA antigen) from Mycobacterium tuberculosis. In order to scale up from previous reported shake flasks to bioreactor, data from the literature on the effect of agitation on morphology of Streptomyces strains were used to obtain gassed volumetric power input values that can be used to obtain a morphology of S. lividans in bioreactor similar to the morphology previously reported in coiled/baffled shake flasks by our group. Morphology of S. lividans was successfully scaled-up, obtaining similar mycelial sizes in both scales with diameters of 0.21 ± 0.09 mm in baffled and coiled shake flasks, and 0.15 ± 0.01 mm in the bioreactor. Moreover, the specific growth rate was successfully scaled up (0.09 ± 0.02 and 0.12 ± 0.01 h−1, for bioreactors and flasks, respectively), and the recombinant protein productivity measured by densitometry, as well. More interestingly, the quality of the recombinant glycoprotein measured as the amount of mannoses attached to the C-terminal of APA was also scaled- up; with up to five mannose residues in cultures carried out in shake flasks; and six in the bioreactor. However, final biomass concentration was not similar, indicating that although the process can be scaled-up using the power input, others factors like oxygen transfer rate, tip speed or energy dissipation/circulation function can be an influence on bacterial metabolism.


Scale-up Power input APA 45/47 kDa O-mannosylation Mycobacteriumtuberculosis Streptomyceslividans 



Impeller diameter (m)


Volumetric gas flow rate (m3/min)


Gravitational acceleration (g/m2)


Agitation speed (rev/min)


Power number (Dimensionless)


Power input by agitation (W)


Gassed power input (W)


Corrected gassed power input by bioreactor geometry (W)


Reynolds number (Dimensionless)


Operation volume (m3)


Impeller blade width (m)


Fluid viscosity (mPa.s)


Density (kg/m3)



This work was financed by CONACYT-INNOVAPYME 181895, CONACYT 178528, 104951-Z and PAPPIT-UNAM IN-209113, IN-210013. RGS thanks CONACyT scholarship (316929). Authors thank Celia Flores, M. Sc., and Enrique Galindo, PhD. (Instituto de Biotecnología, UNAM), and Marisol Córdova, PhD. (Centro de Ciencias Aplicadas y Desarrollo Tecnológico, UNAM) for their technical assistance in image analysis. Authors thank Erendida Garcia, Chem. (Instituto de Química, UNAM) for MALDI-TOF analysis. We also thank Ana Delgado for reviewing the English version of the manuscript.

Conflict of interest

The authors declare that they have no conflict of interest.


  1. Anné J, Maldonado B, Van Impe J, Van Mellaert L, Bernaerts K (2012) Recombinant protein production and streptomycetes. J Biotechnol 158(4):159–167CrossRefGoogle Scholar
  2. Buchs J, Maier U, Milbradt C, Zoels B (2000a) Power consumption in shaking flasks on rotary shaking machines: I. Power consumption measurement in unbaffled flasks at low liquid viscosity. Biotechnol Bioeng 68:589–593CrossRefGoogle Scholar
  3. Buchs J, Maier U, Milbradt C, Zoels B (2000b) Power consumption in shaking flasks on rotary shaking machines: II. Nondimensional description of specific power consumption and flow regimes in unbaffled flasks at elevated liquid viscosity. Biotechnol Bioeng 68:594–601CrossRefGoogle Scholar
  4. Chapple D, Kresta M, Wall A, Afacan A (2002) The effect of impeller and tank geometry on power number for a pitched blade turbine. TransIChemE 80(4):364–372Google Scholar
  5. Dickey DS, Bittorf KJ, Ramsey CJ, Johnson KE (2004) Understand flow patterns in glass-lined reactors. Chem Eng Prog 100:21–25Google Scholar
  6. Dobos K, Swiderek K, Khoo K, Brennan PJ, Belisle JT (1995) Evidence for glycosylation sites on the 45-Kilodalton glycoprotein of Mycobacteria tuberculosis. Infect Immun 63:2846–2853Google Scholar
  7. Dobos K, Khoo K, Swiderek K, Brennan P, Belisle J (1996) Definition of the full extent of glycosylation of the 45-kilodalton glycoprotein of Mycobacterium tuberculosis. J Bacteriol 178:2498–2506Google Scholar
  8. Dobson LF, O’Cleirigh CC, O’Shea DG (2008) The influence of morphology on geldanamycin production in submerged fermentations of Streptomyces hygroscopicus var. geldanus. Appl Microbiol Biotechnol 79:859–866CrossRefGoogle Scholar
  9. el-Enshasy HA, Farid MA, el-Sayed S (2000) Influence of inoculum type and cultivation conditions on natamycin production by Streptomyces natalensis. J Basic Microbiol 40:333–342CrossRefGoogle Scholar
  10. Gamboa-Suasnavart RA, Valdez-Cruz NA, Cordova-Davalos LE, Martinez-Sotelo JA, Servín-González L, Espitia C, Trujillo-Roldán MA (2011) The O-mannosylation and production of recombinant APA (45/47 KDa) protein from Mycobacterium tuberculosis in Streptomyces lividans is affected by culture conditions in shake flasks. Microb Cell Fact 10:110CrossRefGoogle Scholar
  11. Giudici R, Pamboukian CR, Facciotti MC (2004) Morphologically structured model for antitumoral retamycin production during batch and fed-batch cultivations of Streptomyces olindensis. Biotechnol Bioeng 86:414–424CrossRefGoogle Scholar
  12. Hoopen HJG, Gulik WM, Schlatmann JE, Moreno PRH, Vinke JL, Heijnen JJ, Verpoorte R (1994) Ajmalicine production by cell cultures of Catharanthus roseus: from shake flask to bioreactor. Plant Cell Tissue Organ Culture 38:85–91CrossRefGoogle Scholar
  13. Horn C, Namane A, Pescher P, Riviere M, Romain F, Puzo G, Barzu O, Marchal G (1999) Decreased capacity of recombinant 45/47-kDa molecules (Apa) of Mycobacterium tuberculosis to stimulate T lymphocyte responses related to changes in their mannosylation pattern. J Biol Chem 274:32023–32030CrossRefGoogle Scholar
  14. Hugmark G (1980) Power requirements and interfacial area in gas-liquid turbine agitated systems. Ind Eng Chem Process Des Dev 19:638–641CrossRefGoogle Scholar
  15. Junker BH, Hesse M, Burgess B, Masurekar P, Connors N, Seeley A (2004) Early phase process scale-up challenges for fungal and filamentous bacterial cultures. Appl Biochem Biotechnol 119:241–277CrossRefGoogle Scholar
  16. Kieser T, Bibb MJ, Buttner MJ, Chater KF, Hopwood DA (2000) Practical Streptomyces genetics. The John Innes Foundation, NorwichGoogle Scholar
  17. Klockner W, Buchs J (2012) Advances in shaking technologies. Trends Biotechnol 30:307–314CrossRefGoogle Scholar
  18. Lara M, Servin-Gonzalez L, Singh M, Moreno C, Cohen I, Nimtz M, Espitia C (2004) Expression, secretion, and glycosylation of the 45-and 47-kDa glycoprotein of Mycobacterium tuberculosis in Streptomyces lividans. Appl Environ Microbiol 70:679–685CrossRefGoogle Scholar
  19. Mehmood N, Olmos E, Goergen JL, Blanchard F, Ullisch D, Klockner W, Buchs J, Delaunay S (2011) Oxygen supply controls the onset of pristinamycins production by Streptomyces pristinaespiralis in shaking flasks. Biotechnol Bioeng 108:2151–2161CrossRefGoogle Scholar
  20. Peter CP, Suzuki Y, Buchs J (2006) Hydromechanical stress in shake flasks: correlation for the maximum local energy dissipation rate. Biotechnol Bioeng 93:1164–1176CrossRefGoogle Scholar
  21. Roubos JA, Krabben P, Luiten RG, Verbruggen HB, Heijnen JJ (2001) A quantitative approach to characterizing cell lysis caused by mechanical agitation of Streptomyces clavuligerus. Biotechnol Prog 17:336–347CrossRefGoogle Scholar
  22. Rushton JH, Costich EW, Everett HJ (1950a) Power characteristics of mixing impellers. Part I. Chem Eng Progr 46:395–404Google Scholar
  23. Rushton JH, Costich EW, Everett HJ (1950b) Power characteristics of mixing impellers. Part II. Chem Eng Progr 46:467–479Google Scholar
  24. Sable SB, Cheruvu M, Nandakumar S, Sharma S, Bandyopadhyay K, Kellar KL, Posey JE, Plikaytis BB, Amara RR, Shinnick TM (2011) Cellular immune responses to nine Mycobacterium tuberculosis vaccine candidates following intranasal vaccination. PLoS ONE 6:e22718CrossRefGoogle Scholar
  25. Seletzky JM, Noak U, Fricke J, Welk E, Eberhard W, Knocke C, Buchs J (2007) Scale-up from shake flasks to fermenters in batch and continuous mode with Corynebacterium glutamicum on lactic acid based on oxygen transfer and pH. Biotechnol Bioeng 98:800–811CrossRefGoogle Scholar
  26. Silberbach M, Maier B, Zimmermann M, Buchs J (2003) Glucose oxidation by Gluconobacter oxydans: characterization in shaking-flasks, scale-up and optimization of the pH profile. Appl Microbiol Biotechnol 62:92–98CrossRefGoogle Scholar
  27. Tough AJ, Prosser JI (1996) Experimental verification of a mathematical model for pelleted growth of Streptomyces coelicolor A3(2) in submerged batch culture. Microbiol 142(Pt 3):639–648Google Scholar
  28. Tough AJ, Pulham J, Prosser JI (1995) A mathematical model for the growth of mycelial pellet populations. Biotechnol Bioeng 46:561–572CrossRefGoogle Scholar
  29. Vallin C, Ramos A, Pimienta E, Rodriguez C, Hernandez T, Hernandez I, Del SR, Rosabal G, Van ML, Anne J (2006) Streptomyces as host for recombinant production of Mycobacterium tuberculosis proteins. Tuberculosis (Edinb.) 86:198–202CrossRefGoogle Scholar
  30. Yun SI, Yahya AR, Malten M, Cossar D, Anderson WA, Scharer JM, Moo-Young M (2001) Peptidases affecting recombinant protein production by Streptomyces lividans. Can J Microbiol 47:1137–1140Google Scholar

Copyright information

© Springer Science+Business Media Dordrecht 2013

Authors and Affiliations

  • Ramsés A. Gamboa-Suasnavart
    • 1
    • 2
  • Luz D. Marín-Palacio
    • 1
    • 2
  • José A. Martínez-Sotelo
    • 3
  • Clara Espitia
    • 3
  • Luis Servín-González
    • 2
  • Norma A. Valdez-Cruz
    • 2
  • Mauricio A. Trujillo-Roldán
    • 1
    • 2
  1. 1.Unidad de Bioprocesos, Instituto de Investigaciones BiomédicasUniversidad Nacional Autónoma de MéxicoMexicoMexico
  2. 2.Departamento de Biología Molecular y Biotecnología, Instituto de Investigaciones BiomédicasUniversidad Nacional Autónoma de MéxicoMexicoMexico
  3. 3.Departamento de Inmunología, Instituto de Investigaciones BiomédicasUniversidad Nacional Autónoma de MéxicoMexicoMexico

Personalised recommendations