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Continuous 2-keto-l-gulonic acid fermentation from l-sorbose by Ketogulonigenium vulgare DSM 4025

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A single-stage continuous fermentation process for the production of 2-keto-l-gulonic acid (2KGA) from l-sorbose using Ketogulonigenium vulgare DSM 4025 was developed. The chemostat culture with the dilution rate that was calculated based on the relationship between the 2KGA production rate and the 2KGA concentration was feasible for production with high concentration of 2KGA. In this system, 112.2 g/L of 2KGA on the average was continuously produced from 114 g/L of l-sorbose. A steady state of the fermentation was maintained for the duration of more than 110 h. The dilution rate was kept in the range of 0.035 and 0.043 h−1, and the 2KGA productivity was 3.90 to 4.80 g/L/h. The average molar conversion yield of 2KGA from l-sorbose was 91.3%. Under the optimal conditions, l-sorbose concentration was kept at 0 g/L. Meanwhile, the dissolved oxygen level was changing in response to the dilution rate and 2KGA concentration. In the dissolved oxygen (DO) range of 16% to 58%, it was revealed that the relationship between DO and D possessed high degree of positive correlation under the l-sorbose limiting condition (complete consumption of l-sorbose). Increasing D closer to the critical value for washing out point of the continuous fermentation, DO value tended to be gradually increased up to 58%. In conclusion, an efficient and reproducible continuous fermentation process for 2KGA production by K. vulgare DSM 4025 could be developed using a medium containing baker’s yeast without using a second helper microorganism.

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  1. Aiba S, Humphrey AE, Millis NF (1984) Biochemical engineering second edition. University of Tokyo Press, Tokyo, pp 139–172

  2. Carlsen M, Jochumsen KV, Emborg C, Nielsen J (1997) Modeling the growth and proteinase A production in continuous cultures of recombinant Saccharomyces cerevisiae. Biotechnol Bioeng 55:447–454

  3. Hoshino T, Ojima S, Sugisawa T (1990a) Fermentation process to produce 2-Keto-L-gulonic acid. United States Patent 4960695

  4. Hoshino T, Sugisawa T, Tazoe M, Shinjoh M, Fujiwara A (1990b) Metabolic pathway for 2-keto-L-gulonic acid formation from L-sorbose and D-sorbitol by Gluconobacter melanogenes IFO 3293. Agric Biol Chem 54:1211–1218

  5. Hoshino T, Ojima S, Sugisawa T (1994) Process for producing 2-Keto-L-gulonic acid. United States Patent 5312741

  6. Hoshino T, Sugisawa T, Takagi Y (2004) Production of 2-KGA. International publication number WO2004/029265 A2

  7. Jeude M, Dittrich B, Niederschulte H, Anderlei T, Knocke C, Klee D, Büchs J (2006) Fed-batch mode in shake flasks by slow-release technique. Biotechnol Bioeng 95:433–445

  8. Krieg P, Ettlinger L (1970) Kinetic studies on sorbose fermentation. Pathol Microbiol 36:343

  9. Leduc S, de Troostembegh J-C, Lebeault J-M (2004) Folate requirements of the 2-keto-L-gulonic acid-producing strain Ketogulonigenium vulgare LMP P-20356 in L-sorbose/CSL. Appl Microbiol Biotechnol 65:163–167

  10. Martin CKA, Perlman D (1976) Conversion of L-sorbose to 2-keto-L-gulonic acid by mixtures of immobilized cells of Gluconobacter melanogenes IFO3293 and Pseudomonas species. Eur J Appl Microbiol 3:91–95

  11. Okazaki H, Kanzaki T, Doi M, Nara K, Motizuki K (1968) 2-Keto-L-gulonic acid fermentation Part II. Identification of metabolic products from sorbitol. Agric Biol Chem 32:1250–1255

  12. Reichstein T, Gruessner A, Oppenauer R (1934) Eine ergiebige synthese der L-ascorbinsaeure (C-vitamin). Helv Chim Acta 17:311–328

  13. Saito Y, Ishii Y, Hayashi H, Imao Y, Akashi T, Yoshikawa K, Noguchi Y, Soeda S, Yoshida M, Niwa M, Hosoda J, Shimomura K (1997) Cloning of genes coding for L-sorbose and L-sorbosone dehydrogenases from Gluconobacter oxydans and microbial production of 2-keto-L-gulonate, a precursor of L-ascorbic acid, in a recombinant G. oxydans strain. Appl Environ Microbiol 63:454–460

  14. Shin HW, Shin BS, Shin CS (1997) Continuous L-sorbose production in immobilized cell reactors. Sanop Misaengmul Hakhoei 25:68–74

  15. Shinjoh M, Setoguchi Y, Hoshino T, Fujiwara A (1990) L-Sorbosone dissimilation in 2-keto-L-gulonic acid-producing mutant UV10 derived from Gluconobacter melanogenes IFO 3293. Agric Biol Chem 54:2257–2263

  16. Shinjoh M, Tomiyama N, Asakura A, Hoshino T (1995) Cloning and nucleotide sequencing of the membrane-bound L-sorbosone dehydrogenase gene of Acetobacter liquefaciens IFO12258 and its expression in Gluconobacter oxydans. Appl Environ Microbiol 61:413–420

  17. Sonoyama T, Tani H, Matsuda K, Kageyama B, Tanimoto M, Kobayashi K, Yagi S, Kyotani H, Mitsushima K (1982) Production of 2-keto-L-gulonic acid from D-glucose by two-stage fermentation. Appl Environ Microbiol 43:1064–1069

  18. Spassov G, Christov P, Pramatarova V (1995) Conversion of D-sorbitol to L-sorbose by cells of Acetobacter suboxydans immobilized in sintered grass. Appl Microbiol Biotechnol 43:35–37

  19. Stanbury PF, Whitaker A (1987) Principles of fermentation technology. Pergamon, New York, pp 12–16

  20. Stoddard SF, Liaw HJ, Eddington J, Yang Y (1998) Bacterial strains and use thereof in fermentation process for 2-keto-L-gulonic acid production. United States Patent 5834231

  21. Sugisawa T, Hoshino T, Masuda S, Nomura S, Setoguchi Y, Tazoe M, Shinjoh M, Someha S, Fujiwara A (1990) Microbial production of 2-keto-L-gulonic acid from L-sorbose and D-sorbitol by Gluconobacter melanogenes. Agric Biol Chem 54:1201–1209

  22. Sugisawa T, Hoshino T, Fujiwara A (1991a) Isolation and characterization of NAD(P)-dependent L-sorbosone from Gluconobacter melanogenes UV 10. Agric Biol Chem 55:665–670

  23. Sugisawa T, Hoshino T, Nomura S, Fujiwara A (1991b) Membrane-bound L-sorbose dehydrogenase from Gluconobacter melanogenes UV 10. Agric Biol Chem 55:363–370

  24. Urbance JW, Bratina BJ, Stoddard SF, Schmidt TM (2001) Taxonomic characterization of Ketogulonigenium vulgare gen. nov., sp. nov. and Ketogulonigenium robustum sp. nov., which oxidize L-sorbose to 2-keto-L-gulonic acid. Int J Syst Evol Microbiol 51:1059–1070

  25. Yanmada S, Wada M, Chibata I (1979) Studies on aerobic fermentation. III. Oxygen transfer as a parameter of automatic control of the continuous cultivation for the conversion of sorbitol to sorbose by Acetobacter suboxydans. J Ferment Technol 57:210–214

  26. Yin G, Tao Z, Yan Z, Ning W, Wang C, Wang S (1990) Fermentation process. United States Patent 4935359

  27. Yuan Z, Wei D, Yin G, Yuan W (1992) Co immobilization of Gluconobacter oxydans and Bacillus cereus for bioconversion of 2-keto-L-gulonic acid. Ann NY Acad Sci 672:628–633

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Correspondence to Yoshinori Takagi.

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Takagi, Y., Sugisawa, T. & Hoshino, T. Continuous 2-keto-l-gulonic acid fermentation from l-sorbose by Ketogulonigenium vulgare DSM 4025. Appl Microbiol Biotechnol 82, 1049–1056 (2009). https://doi.org/10.1007/s00253-008-1822-6

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  • 2-Keto-l-gulonic acid
  • l-Sorbose
  • Continuous fermentation
  • Ketogulonicigenium vulgare