Biotechnology and Bioprocess Engineering

, Volume 4, Issue 4, pp 287–293 | Cite as

The effect of inoculum size on the growth of cell and root cultures ofHyoscyamus muticus: Implications for reactor inoculation

  • Edgard B. Carvalho
  • Wayne R. CurtisEmail author


Cell suspensions inoculated at low cell concentrations displayed a typical growth reduction, whereas root cultures displayed an improvement in growth. Specific growth rate ofHyoscyamus muticus cell suspensions decreased from 0.25 to 0.12 d−1 as inoculum concentration was reduced from 4.0 to 0.02 g fresh weight per liter. In contrast, roots show an increase in growth rate from 0.24 to 0.43 d−1. These contrasting growth patterns can be explained as the result of: a) the high specific surface area of cells as compared to roots and, b) the differentiated structure of roots. The dispersed nature of cell suspensions makes them more prone to leakage of key growth factors/cellular contents to medium. The results of this work indicate that cell cultures require substantially higher inoculum concentrations. In contrast, roots can be inoculated at very low concentrations. These facts imply that whereas seed vessels must be employed by cell suspensions, their use for root cultures is a compromise between an easier handling of an entwined root mass and the reduction of the contamination risk of large medium volumes.

Key words

minimum inoculum density hairy roots bioreactors medium conditioning stress 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. [1]
    Syono, K., and T. Furuya (1968) Studies on plant tissue cultures I. Relationship between inocula size and growth of calluses in liquid cultures.Plant Cell Physiol. 9: 103–114.Google Scholar
  2. [2]
    Costa, E. L., J. M. Novais, M. S. Pais, and J. M. S. Cabral (1988) Effect of aeration onCynara cardunculus plant cell cultures. p. 333–351 In: M. S. S. Pais, F. Mavituna, and J. M. Novais (eds.)Plant Cell Biotechnology. Vol. H14. Springer-Verlag, Berlin Germany.Google Scholar
  3. [3]
    Street, H. E. (1977) Cell (suspension) cultures-techniques, p. 61–102 In: H. E. Street (ed.)Plant Tissue and Cell Culture, University of California Press, Berkeley, U.S.A.Google Scholar
  4. [4]
    Stuart, R., and H. E. Street (1971) Studies on the growth in cultures of plant cells. X. Further studies on the conditioning of culture media by suspensions ofAcer pseudoplatanus.J. Exp. Bot 22(10): 96–106.CrossRefGoogle Scholar
  5. [5]
    Wijnsma, R., R. Verpoorte, P. A. A. Harkes, F. van Iren, and H. J. G. ten Hoopen (1988) Conditioning of media: an elaborate method of optimizing initial growth hormone concentration. p. 297–303 In: M. S. S. Pais, F. Mavituna, and J. M. Novais (eds.)Plant Cell Biotechnology. Vol. H14. Springer-Verlag. Berlin, Hermany.Google Scholar
  6. [6]
    van Gulik, W. M., A. M. Nuutila, K. L. Vinke, H. J. G. Hoopen, and J. J. Heijnen (1994) Effect of carbon dioxide, air flow rate, and inoculation density on the batch growth ofCatharanthus roseus cell suspension in stirred fermenters.Biotechnol. Prog. 10: 335–339.CrossRefGoogle Scholar
  7. [7]
    Stuart, R. and H. E. Street (1969) Studies on the growth in culture of plant cells. IV. The initiation of division in suspensions of stationary phase cells ofAcer pseudoplatanus L..J. Exp. Bot. 20(64): 556–571.CrossRefGoogle Scholar
  8. [8]
    Dix, P. H. (1986) Cell line selection. p. 143–201. In: M. M. Yeoman (ed.)Plant Cell Culture Technology. Blackwell Scientific Publications, Boston, U.S.A.Google Scholar
  9. [9]
    Singh, G. and W. R. Curtis (1994) Reactor design for plant cell culture. p. 151–183 In: P. D. Shargod, and T. T. Ngo (eds.)Biotechnological applications of Plant Cultures. CRC Press, Boca Raton, U.S.A.Google Scholar
  10. [10]
    Bhadra, R. and J. V. Shanks (1995) Statistical design of the effect of inoculum conditions on growth of hairy root cultures ofCatharanthus roseus.Biotechnol. Tech. 9: 681–686.CrossRefGoogle Scholar
  11. [11]
    Falk, L. R. and P. M. Doran (1996) Influence of inoculum morphology on growth ofAtropa belladonna hairy roots and production on tropane alkaloids.Biotechnol. Lett. 18(9): 1099–1104.CrossRefGoogle Scholar
  12. [12]
    Flint-Wandel, J. and M. Hjortso (1993) A flow cell for the study of growth kinetics of single hairy roots.Biotechnol. Tech. 7(6): 447–452.CrossRefGoogle Scholar
  13. [13]
    Kanokwan, K. and P. M. Doran (1997) Effect of inoculum size on growth ofAtropa belladonna hairy roots in shake flasks.J. Ferment. Bioeng. 84(4): 378–381.CrossRefGoogle Scholar
  14. [14]
    Hjortso, M. A. (1997) Mathematical modelling of hairy root growth. p. 169–178 In: P. Doran (ed.)Hairy Roots: Culture and Applications. Harwood Academics Publishing. The Netherlands.Google Scholar
  15. [15]
    Gamborg, O. L., R. A. Miller, and K. Ojima (1968) Nutrient requirements of suspensions of soybean root cells.Exp. Cell Res. 50: 148–151.CrossRefGoogle Scholar
  16. [16]
    Laemmli, U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4.Nature 227: 680–685.CrossRefGoogle Scholar
  17. [17]
    Larsen, W. A., J. T. Hsu, H. E. Flores, and A. F. Humphrey (1993) A study of nicotine release from tobacco hairy roots by transient technique.Biotechnol. Tech. 7(8): 557–562.CrossRefGoogle Scholar
  18. [18]
    Renaudin, J. P. (1981) C14 Tabernanthine, by cell suspension cultures ofCatharanthus reseus (L.) G. Don andAcer pseudoplatanus L.Plant Science Lett. 22: 59–69.CrossRefGoogle Scholar
  19. [19]
    Bordonaro, J. L. and W. R. Curtis (1997) Development of a fluorescent tracer technique to evaluate mixing in plant root culture.Biotechnol. Tech. 11(8): 597–600.Google Scholar
  20. [20]
    Yu, S., M. G. P. Mahagamasekera, G. R. C. Williams, K. Kanokwaree, and P. D. Doran (1997) Oxygen effects in hairy root culture. p. 139–150 In: P. Doran (ed.)Hairy Roots: Culture and Anplications. Harwood Academics Publishing. The Netherlands.Google Scholar
  21. [21]
    Ramakrishnan, D. and W. R. Curtis (1995) Elevated meristematic respiration in plant root cultures: implications to reactor design.J. Chem. Eng. Japan 28(4): 491–493.CrossRefGoogle Scholar
  22. [22]
    Carvalho, E. C., S. Holihan, B. Pearsal, and W. R. Curtis (1997) Effect of root morphology on reactor design and operation for production of chemicals. p. 151–167 In: P. Doran (ed.)Hairy Roots: Culture and Applications. Harwood Academics Publishing. The Netherlands.Google Scholar
  23. [23]
    Ramakrishnan, D. and W. R. Curtis (1994) Fluid dynamic studies on plant root cultures for application to bioreactor design. p. 281–305 In: S. Furusaki and D. D. Y. Ryu (eds.)Studies in Plant Science, 4: Advances in Plant Biotechnology. Elsevier, Amsterdam, The Netherlands.Google Scholar
  24. [24]
    Mukundan, U., E. B. Carvalho, and W. R. Curtis (1998) Growth and pigment production in hairy root cultures ofBeta vulgaris L. in a bubble column reactor.Biotechnol. Lett. 20(5): 469–474.CrossRefGoogle Scholar
  25. [25]
    Tescione, L. D., D. Ramakrishnan, and W. R. Curtis (1997) The role of liquid mixing and gasphase dispersion in a submerged, sparged root reactor.Enzyme Microb Technol. 20: 207–213.CrossRefGoogle Scholar
  26. [26]
    Beesch, S. C. (1952) Acetone-butanol fermentation of sugars.Ind. Eng. Chem. 44(7): 1677–1682.CrossRefGoogle Scholar
  27. [27]
    Kalk, J. P. and A. F. Langlykke (1986) Cost estimation for biotechnology projects. p. 363–385 In: A. L. Demain and N. A. Solomon (eds.)Manual of Industrial Microbiology and Biotechnology. American Society for Microbiology Washington, D.C.Google Scholar
  28. [28]
    Ramakrishnan, D., J. Salim, and W. R. Curtis (1994) Inoculation and tissue distribution in pilot-scale plant root bioreactors.Biotechnol. Tech. 8(9): 639–644.CrossRefGoogle Scholar
  29. [29]
    Wilson, P. D. G. (1997) The pilot-scale cultivation of transformed roots. p. 179–190 In: P. Doran (ed.)Hairy Roots: Culture and Applications. Harwood Academics Publishing. The Netherlands.Google Scholar
  30. [30]
    Carvalho, E. B. and W. R. Curtis (1998) Characterization of fluid-flow resistance in root cultures with a convective flow tubular reactor.Biotechnol. Bioeng. 60(30): 375–384.CrossRefGoogle Scholar
  31. [31]
    Hsiao, T. Y., F. T. Bacani, E. B. Carvalho, and W. R. Curtis (1999) Development of a low capital investment reactor system application for plant cell suspension culture.Biotechnol. Preg. 15: 114–122.CrossRefGoogle Scholar
  32. [32]
    Curtis, W. R. (1999) Achieving economic feasibility for moderate-value food and flavor adcitives. p. 225–236 In: T.-J. Fu, G. Singh, und W. R. Curtis (eds.)Plant Cell Culture for the Production of Food Ingredients. Kluwer Academic Plenum Publishers, New York, U.S.A.Google Scholar
  33. [33]
    Curtis, W. R., F. T. Bacani, T. Y. Hsiao, E. B. Carvalho, and C. M. Srubar (1999) Development and application of a low capital investment bioreactor. American Chemical Society National Meeting, Paper No. 157 (BIOT), March 21–25, Anaheim, CA, U.S.A.Google Scholar
  34. [34]
    Lin, S.-M., J. A. Swanzy, and P. T. Jacob (1999) Method of hydrogen peroxide plasma sterilization.U.S. Patent 5, 876, 666.Google Scholar
  35. [35]
    Panda, A. K., S. Mishra, and V. S. Bisaria (1992) Alkaloid production by plant cell suspension cultures ofHolarrhena antidysenterica: I. Effect of major nutrients.Biotechnol. Bioeng. 39: 1043–1051.CrossRefGoogle Scholar
  36. [36]
    Westphal, K. (1990) Large scale production of new biological active compounds in plant cell cultures. p. 601–608. In: H. J. J. Nijkamp, L. H. W. van der Plas, and J. van Aartrijk (eds.)Progress in Plant Cellular and Molecular Biology. Kluwer Academic Publishers. The Netherlands.Google Scholar

Copyright information

© The Korean Society for Biotechnology and Bioengineering 1999

Authors and Affiliations

  1. 1.Department of Chemical EngineeringThe Pennsyivania State UniversityState CollegeUSA

Personalised recommendations