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Production of the biocontrol agent Pantoea agglomerans PBC-1 in a stirred tank reactor by batch and fed-batch cultures

Abstract

Concerns about food safety as well as the development of resistance to many fungicides by major postharvest pathogens have increased recently. Biological control, using microorganisms antagonistic to the fungal plant pathogens, appears to be promising as an alternative to fungicides. The microbial biocontrol agent has to be produced on an industrial scale, maintaining its biocontrol efficacy. The purpose of the current study was to optimize the conditions for microbial biomass production of the biocontrol agent Pantoea agglomerans PBC-1 in a 2-l mechanically stirred reactor (STR), defining mixing and mass transfer technological parameters and the growth kinetics for different saccharides. In the batch mode, different impellers and spargers were tested. Despite the oxygen mass transfer improvement achieved with marine propeller combined with porous sparger, the biomass did not increase, if compared with the use of a Rushton turbine and L-sparger, pointing out the relevance of a radial flux for better broth homogenization. Different carbon sources were used: sucrose, glucose and fructose; each of which led to viable populations 3.9 × 109, 1.4 × 109, 3.9 × 109 c.f.u/ml, respectively, after 20 h of incubation. Fed-batch technology allows the maintenance of high cell viability for longer periods of time in the stationary growth phase, which can be crucial for the scale-up of biocontrol agent production process that is achieved together with a reduction of 85% on the incidence caused by the pathogens, brought about by fresh microbial biomass preparation on artificially wounded apples or oranges, stored for 7 days at 25°C against Penicillium expansum and Penicillium digitatum.

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References

  1. Abadias M, Teixido N, Usall J, Vinas I (2003) Optimization of growth conditions of the postharvest biocontrol agent Candida sake CPA-1 in a lab-scale fermenter. J Appl Microbiol 95:301–309. doi:10.1046/j.1365-2672.2003.01976.x

  2. Belo I, Pinheiro R, Mota M (2003) Fed-batch cultivation of Saccharomyces cerevisiae in a hyperbaric bioreactor. Biotechnol Progr 19:665–671. doi:10.1021/bp0257067

  3. Bonaterra A, Mari M, Casalini L, Montesinos E (2003) Biological control of Monilinia laxa and Rhizopus stolonifer in postharvest of stone fruit by Pantoea agglomerans EPS125 and putative mechanisms of antagonism. Int J Food Microbiol 84:93–104. doi:10.1016/S0168-1605(02)00403-8

  4. Cascaval D, Galaction AI, Folescu E, Turnea M (2006) Comparative study on the effects of n-dodecane addition on oxygen transfer in stirred bioreactors for simulated, bacterial and yeasts broths. Biochem Eng J 3:56–66. doi:10.1016/j.bej.2006.05.019

  5. Chalutz E, Wilson CL (1990) Postharvest biocontrol of green and blue mold and sour rot of citrus fruit by Debaryomyces hansenii. Plant Dis 74:134–137

  6. Costa E, Teixidó N, Usall J, Atarés E, Viñas I (2002) Production of the biocontrol agent Pantoea agglomerans strain CPA-2 using commecial products and by-products. Appl Microbiol Biotechnol 56:367–371. doi:10.1007/s002530100666

  7. de Mare L, Velut S, Ledung E, Cimander C, Norrman B, Karlsson EN, Holst O, Hagander P (2005) A cultivation technique for E. coli fed-batch cultivations operating close to the maximum oxygen transfer capacity of the reactor. Biotechnol Lett 27:983–990. doi:10.1007/s10529-005-7844-6

  8. Doran PM (1995) Fluid flow and mixing. In: Doran PM (ed) Bioprocess engineering principles. Academic Press, London, pp 129–163

  9. El-Enshasy H, Abul-Hamd A, El-Sehrawi M, Sidkey N, Azazzy A (2007) Improvement of cell mass production of thermotolerant Baker’s yeast Saccharomyces cerevisiae using sugar cane molasses based medium in stirred tank bioreactor in batch and fed-batch cultures. Deut Lebensm-Rundsch 103:273–278

  10. Fredlund E, Blank LM, Schnurer J, Sauer U, Passoth V (2004) Oxygen- and glucose-dependent regulation of central carbon metabolism in Pichia anomala. Appl Environ Microbiol 70:5905–5911. doi:10.1128/AEM.70.10.5905-5911.2004

  11. Fujasová M, Linek V, Moucha T (2007) Mass transfer correlations for multiple-impeller gas-liquid contactors. Analysis of the effect of axial dispersion in gas and liquid phases on “local” k(L)a values measured by the dynamic pressure method in individual stages of the vessel. Chem Eng Sci 62:1650–1669. doi:10.1016/j.ces.2006.12.003

  12. Gogate PR, Beenackers A, Pandit A (2000) Multiple-impeller systems with a special emphasis on bioreactors: a critical review. Biochem Eng J 6:109–144. doi:10.1016/S1369-703X(00)00081-4

  13. Hadjiev D, Sabiri NE, Zanati A (2006) Mixing time in bioreactors under aerated conditions. Biochem Eng J 27:323–330. doi:10.1016/j.bej.2005.08.009

  14. He L, Xu Y, Zhang X (2008) Medium factor optimization and fermentation kinetics for phenazine-1-carboxilic acid production by Pseudomonas sp. M18G. Biotechnol Bioeng 100:250–259. doi:10.1002/bit.21767

  15. Irvine TS (1990) Laboratory fermenters. In: McNeil B, Harvey LM (eds) Fermentation a practical approach. IRL, Oxford, pp 17–38

  16. Kao PM, Chen CI, Huang SC, Chang YC, Tsai P, Liu YC (2007) Effects of shear stress and mass transfer on chitinase production by Paenibacillus sp CHE-N1. Biochem Eng J 34:172–178. doi:10.1016/j.bej.2006.11.028

  17. Kurtzman CP, Droby S (2001) Metschnikowia fructicola, a new ascosporic yeast with potential for biocontrol of postharvest fruit rots system. Appl Microbiol 24:395–399. doi:10.1078/0723-2020-00045

  18. Leckie F, Scragg AH, Cliffe KR (1991a) Effect of impeller design and speed on the large-scale cultivation of suspension cultures of Catharanthus roseus. Enzyme Microb Techn 13:801–810. doi:.1016/0141-0229(91)90063-G

  19. Leckie F, Scragg AH, Cliffe KR (1991b) An investigation into the role of initial K L a on the growth and alkaloid accumulation by cultures of Catharanthus roseus. Biotechnol Bioeng 37:364–370. doi:10.1002/bit.260370410

  20. Lee IY, Kim JH, Seo WT, Jung JK, Lee HW, Park YH (1991) Influence of agitation speed on production of curdlan by Agrobacterium species. Bioprocess Eng 20:283–287. doi:10.1007/PL00009049

  21. Littlejohns JV, Daugulis AJ (2007) Oxygen transfer in a gas-liquid system containing solids of varying oxygen affinity. Chem Eng J 129:67–74. doi:10.1016/j.cej.2006.11.002

  22. Mounir R, Durieux A, Bodo E, Allard C, Simon JP, Achbani EH, El-Jaafari S, Douira A, Jijalki MH (2007) Production, formulation and antagonistic activity of the biocontrol like-yeast Aureobasidium pullulans against Penicillium expansum. Biotechnol Lett 29:553–559. doi:10.1007/s10529-006-9269-2

  23. Nunes C, Usall J, Teixidó N, Viñas I (2001) Biological control of postharvest pear disease using bacterium Pantoea agglomerans CPA-2. Int J Food Microbiol 70:53–61. doi:10.1016/S0168-1605(01)00523-2

  24. Nunes C, Usall J, Teixidó N, Fons E, Viñas I (2002) Post-harvest biological control of Pantoea agglomerans (CPA-2) on Golden Delicious apples. J Appl Microbiol 92:247–255. doi:10.1046/j.1365-2672.2002.01524.x

  25. Nunes C, Usall J, Teixidó N, Abadias M, Asensio A, Viñas I (2007) Biocontrol of postharvest decay using a new strain of Pseudomonas syringae CPA-5 in different cultivars of pome fruits. Agr Food Sci 16:56–65. doi:10.2137/145960607781635877

  26. Nunes C, Manso T, Lima-Costa ME (2009) Postharvest biological control of citrus fruit. Tree Forestry Sci Biotechnol 3:116–126

  27. Patiño-Vera M, Jimenez B, Balderas K, Ortiz M, Allende R, Carrillo A, Galindo E (2005) Pilot-scale production and liquid formulation of Rhodotorula minuta, a potential biocontrol agent of mango anthracnose. J Appl Microbiol 99:540–550. doi:10.1111/j.1365-2672.2005.02646.x

  28. Pollard DJ, Kirschner TF, Hunt GR, Tong IT, Stieber R, Salmons PM (2007) Scale up of a viscous fungal fermentation: application of scale-up criteria with regime analysis and operating boundary conditions. Biotechnol Bioeng 96:307–317. doi:10.1002/bit.21112

  29. Potumarthi R, Ch S, Jetty A (2007) Alkaline protease production by submerged fermentation in stirred tank reactor using Bacillus licheniformis NCIM-2042: effect of aeration and agitation regimes. Biochem Eng J 34:185–192. doi:10.1016/j.bej.2006.12.003

  30. Pusey PL, Wilson CL (1984) Postharvest biological control of stone fruit brown rot by Bacillus subtilis. Plant Dis 68:753–756

  31. Puthli MS, Rathod VK, Pandit AB (2005) Gas-liquid mass transfer studies with triple impeller system on a laboratory scale bioreactor. Biochem Eng J 23:25–30. doi:10.1016/j.bej.2004.10.006

  32. Puthli MS, Rathod VK, Pandit AB (2006) Optimization of lipase production in a triple impeller bioreactor. Biochem Eng J 27:287–329. doi:10.1016/j.bej.2005.08.016

  33. Raposo S, Lima-Costa ME (2006) Rheology and shear stress of Centaurea calcitrapa cell suspension cultures grown in bioreactor. Biotechnol Lett 28:431–438. doi:10.1007/s10529-005-6181-0

  34. Raposo S, Lima-Costa ME (2008) Mixing and mass transfer on aspartic proteinases production by Centaurea calcitrapa cell suspension. (in press)

  35. Schena L, Nigro F, Pentimone I, Ligorio A, Ippolito A (2003) Control of postharvest rots of sweet cherries and table grapes with endophytic isolates of Aureobasidium pullulans. Postharvest Biol Tec 30:209–220. doi:10.1016/S0925-5214(03)00111-X

  36. Smilanick JL, Denis-Arrue R (1992) Control of green mold of lemons with Pseudomonas species. Plant Dis 76:481–485

  37. Spadaro D, Gullino ML (2004) State of the art and future prospects of biological control of postharvest fruit diseases. Int J Food Microbiol 91:185–194. doi:10.1016/S0168-1605(03)00380-5

  38. Stanbury PF, Whitaker A, Hall SJ (1995) Media for industrial fermentations. In: Stanbury PF, Whitaker A, Hall SJ (eds) Principles of fermentation technology, 2nd edn. Pergamon Press, Oxford, pp 93–121

  39. Teixidó N, Usall J, Palou L, Asensio A, Nunes C, Viñas I (2001) Improving control of green and blue molds of oranges by combining Pantoea agglomerans (CPA-2) and sodium bicarbonate. Eur J Plant Pathol 107:685–694. doi:10.1023/A:1011962121067

  40. Torres R, Teixidó N, Usall J, Abadias M, Viñas I (2005) Postharvest control of Penicillium expansum on pome fruits by the bacterium Pantoea ananatis CPA-3. J Hortic Sci Biotech 80:75–81

  41. Ungerman AJ, Heindel T (2007) Carbon monoxide mass transfer for syngas fermentation in a stirred tank reactor with dual impeller configurations. Biotechnol Progr 23:613–620. doi:10.1021/bp060311z

  42. Verma M, Brar SK, Tyagi RD, Surampalli RY, Valero JR (2006) Dissolved oxygen as principal parameter for conidia production of biocontrol fungi Trichoderma viride in non-Newtonian wastewater. J Ind Microbiol Biotechnol 33:941–952. doi:10.1007/s10295-006-0164-6

  43. Viñas I, Usall J, Teixidó N, Sanchis V (1998) Biological control of major postharvest pathogens on apple with Candida sake. Int J Food Microbiol 40:9–16. doi:10.1016/S0168-1605(98)00009-9

  44. Visnovsky GA, Smalley DJ, O’Callaghan M, Jackson TA (2008) Influence of culture medium composition, dissolved oxygen concentration and harvesting time on the production of Serratia entomophila, a microbial control agent of the New Zealand grass grub. Biocontrol Sci Technol 18:87–100. doi:10.1080/09583150701760513

  45. Xie L, Ye X, Liu D, Ying Y (2009) Quantification of glucose, fructose and sucrose in bayberry juice by NIR and PLS. Food Chem 114:1135–1140. doi:10.1016/j.foodchem.2008.10.076

  46. Zhang ZY, Jin B, Kelly JM (2007) Production of lactic acid and byproducts from waste potato starch by Rhizopus arrhizus: role of nitrogen sources. World J Microb Biotechnol 23:229–236. doi:10.1007/s11274-006-9218-1

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Acknowledgments

The present work was conducted in the frame of a doctorate scholarship awarded to Teresa Manso (SFHR/BD/21922/2005) and supported by the project POCTI/AGR/45098/2002, by the Fundação para a Ciência e a Tecnologia, Ministério da Ciência e Superior.

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Correspondence to Maria Emília Lima-Costa.

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Manso, T., Nunes, C., Raposo, S. et al. Production of the biocontrol agent Pantoea agglomerans PBC-1 in a stirred tank reactor by batch and fed-batch cultures. World J Microbiol Biotechnol 26, 725–735 (2010). https://doi.org/10.1007/s11274-009-0229-6

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Keywords

  • Batch culture
  • Fed-batch culture
  • Mixing
  • Mass transfer
  • Cell growth
  • Stirred bioreactor
  • Biocontrol agent
  • Pantoea agglomerans