Efficient bioconversion of waste bread into 2-keto-d-gluconic acid by Pseudomonas reptilivora NRRL B-6


Development of bioprocess routes for production of value-added materials from food waste is very attractive in terms of bioprocess economics and environmental protection. In this study, production of 2-keto-d-gluconic acid, a significant platform chemical, from food waste was performed by Pseudomonas reptilivora NRRL B-6 for the first time. The nitrogen source preference of the strain for 2-keto-d-gluconic acid production under different glucose concentrations in synthetic medium has been elucidated. At higher glucose concentrations, utilization of organic nitrogen source (yeast extract) led to higher product formation while at lower glucose concentration, utilization of inorganic nitrogen source (ammonium chloride) was also very feasible. The results indicated that favorable nitrogen source for 2-keto-d-gluconic acid production can change depending on the glucose concentration. The waste bread hydrolysate (glucose 181.43 g/L, protein 1.21% (w/v)) prepared by sequential application of α-amylase, amyloglucosidase, and protease was utilized in medium formulation at different glucose concentrations. Bread hydrolysate without additional supplements provided higher product formation than the optimum medium prepared with the hydrolysate. Utilization of 50 g/L CaCO3 led the maximum product formation. The maximum 2-keto-d-gluconic acid production from waste bread was 142.81 g/L with the productivity of 3.02 g/L/h and molar yield of 0.95. The proposed approach in this study confirms that waste bread as a sole source of nutrients can be valorized for microbial production of various industrially relevant platform chemicals.

This is a preview of subscription content, access via your institution.

We’re sorry, something doesn't seem to be working properly.

Please try refreshing the page. If that doesn't work, please contact support so we can address the problem.

Fig. 1
Fig. 2
Fig. 3


  1. 1.

    Sun L, Sun W, Wang D, Cui F, Qi X, Xu Z (2018) A novel 2-keto-D-gluconic acid high-producing strain Arthrobacter globiformis JUIM02. Appl Biochem Biotechnol 185:947–957

    Article  Google Scholar 

  2. 2.

    Stottmeister U, Aurich A, Wilde H, Andersch J, Schmidt S, Sicker D (2005) White biotechnology for green chemistry: fermentative 2-oxocarboxylic acids as novel building blocks for subsequent chemical syntheses. J Ind Microbiol Biotechnol 32(11−12):651–664

    Article  Google Scholar 

  3. 3.

    Andersch J, Sicker D (1999) Reductive cyclization of carbohydrate 2-nitrophenyl-hydrazones to chiral functionalized 1,2,4-benzotriazines and benzimidazoles. J Heterocycl Chem 36:589–594

    Article  Google Scholar 

  4. 4.

    Francis AJ (1990) Microbial dissolution and stabilization of toxic metals and radionuclides in mixed wastes. Experientia 46:840–851

    Article  Google Scholar 

  5. 5.

    Raghuvanshi R, Chaudhari A, Kumar GN (2017) 2-Ketogluconic acid and pyrroloquinoline quinone secreting probiotic Escherichia coli Nissle 1917 as a dietary strategy against heavy metal induced damage in rats. J Funct Foods 37:541–552

    Article  Google Scholar 

  6. 6.

    Chia M, Van Nguyen TB, Choi WJ (2008) DO-stat fed-batch production of 2-keto-D-gluconic acid from cassava using immobilized Pseudomonas aeruginosa. Appl Microbiol Biotechnol 78:759–765

    Article  Google Scholar 

  7. 7.

    Zeng W, Cai W, Liu L, Du G, Chen J, Zhou J (2019) Efficient biosynthesis of 2-keto-D-gluconic acid by fed-batch culture of metabolically engineered Gluconobacter japonicus. Synth Syst Biotechnol 4:134–141

    Article  Google Scholar 

  8. 8.

    Sun W, Alexander T, Man Z, Xiao F, Cui F, Qi X (2018) Enhancing 2-ketogluconate production of Pseudomonas plecoglossicida JUIM01 by maintaining the carbon catabolite repression of 2-ketogluconate metabolism. Molecules 23:2629. https://doi.org/10.3390/molecules23102629

    Article  Google Scholar 

  9. 9.

    Shi YY, Li KF, Lin JP, Yang SL, Wei DZ (2015) Engineered expression vectors significantly enhanced the production of 2-keto-D-gluconic acid by Gluconobacter oxidans. J Agric Food Chem 63(22):5492–5498

    Article  Google Scholar 

  10. 10.

    Sun WJ, Zhou YZ, Zhou Q, Cui FJ, Yu SL, Sun L (2012) Semi-continuous production of 2-keto-gluconic acid by Pseudomonas fluorescens AR4 from rice starch hydrolysate. Bioresour Technol 110:546–551

    Article  Google Scholar 

  11. 11.

    Sun WJ, Yun QQ, Zhou YZ, Cui FJ, Yu SL, Zhou Q, Sun L (2013) Continuous 2-keto-gluconic acid (2KGA) production from corn starch hydrolysate by Pseudomonas fluorescens AR4. Biochem Eng J 77:97–102

    Article  Google Scholar 

  12. 12.

    Sun W, Xiao F, Wei Z, Cui F, Yu L, Yu S, Zhou Q (2015) Non-sterile and buffer-free bioconversion of glucose to 2-keto-gluconic acid by using Pseudomonas fluorescens AR4 free resting cells. Process Biochem 50:493–499

    Article  Google Scholar 

  13. 13.

    Alexandri M, Papapostolou H, Stragier L, Verstraete W, Papanikolaou S, Koutinas AA (2017) Succinic acid production by immobilized cultures using spent sulphite liquor as fermentation medium. Bioresour Technol 238:214–222

    Article  Google Scholar 

  14. 14.

    Samray MN, Masatcioglu TM, Koksel H (2019) Bread crumbs extrudates: a new approach for reducing bread waste. J Cereal Sci 85:130–136

    Article  Google Scholar 

  15. 15.

    Haroon S, Vinthan A, Negron L, Das S, Berenjian A (2016) Biotechnological approaches for production of high value compounds from bread waste. Am J Biochem Biotechnol 12(2):102–109

    Article  Google Scholar 

  16. 16.

    Guevarra ED, Tabuchi T (1990) Accumulation of itaconic, 2-hydroxyparaconic, itatartaric and malic acids by strains of the genus Ustilago. Agric Biol Chem 54(9):2353–2358

    Google Scholar 

  17. 17.

    Anonymous (2004) ICC standard methods: methods Nr. 104/1. International association for cereal science and technology, Vienna

  18. 18.

    Rojo F (2010) Carbon catabolite repression in Pseudomonas: optimizing metabolic versatility and interactions with the environment. FEMS Microbiol Rev 34:658–684

    Article  Google Scholar 

  19. 19.

    Sun WJ, Liu CF, Yu L, Cui FJ, Zhou Q, Yu SL, Sun L (2012) A novel bacteriophage KSL-1 of 2-Keto-gluconic acid producer Pseudomonas fluorescens K1005: isolation, characterization and its remedial action. BMC Microbiol 12(127):2–8 http://www.biomedcentral.com/1471-2180/12/127

    Google Scholar 

  20. 20.

    Yi XN, Li TM, Wang BZ, Liu JL, Du HY, Feng HY (2014) Production of 2-keto-D-gluconic acid by metabolically engineered Gluconobacter suboxydans. China Biotechnol 34:97–106 (in Chinese)

    Google Scholar 

  21. 21.

    Svitel J, Sturdik E (1995) 2-ketogluconic acid production by Acetobacter pasteurianus. Appl Biochem Biotech 53:53–63

    Article  Google Scholar 

  22. 22.

    Tian Y, Fan Y, Liu J, Zhao X, Chen W (2016) Effect of nitrogen, carbon sources and agitation speed on acetoin production of Bacillus subtilis SF4-3. Electron J Biotechnol 19:41–49

    Article  Google Scholar 

  23. 23.

    Canete-Rodriguez AM, Santos-Duenas IM, Jimenez-Hornero JE, Torija-Martinez MJ, Mas A, Garcia-Garcia I (2016) Revalorization of strawberry surpluses by bio-transforming its glucose content into gluconic acid. Food Bioprod Process 99:188–196

    Article  Google Scholar 

  24. 24.

    Wei D, Xu J, Sun J, Shi J, Hao J (2013) 2-Ketogluconic acid production by Klebsiella pneumoniae CGMCC 1.6366. J Ind Microbiol Biotechnol 40:561–570

    Article  Google Scholar 

Download references


The postdoctoral fellowship from “The Scientific and Technological Research Council of Turkey (TUBITAK)” has been gratefully acknowledged (S. Yegin).

Author information



Corresponding author

Correspondence to Sirma Yegin.

Ethics declarations

Conflict of interest

The authors declare that they have no conflict of interest.

Additional information

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Mention of trade names or commercial products in this article is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the US Department of Agriculture. USDA is an equal opportunity provider and employer.

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Yegin, S., Saha, B.C., Kennedy, G.J. et al. Efficient bioconversion of waste bread into 2-keto-d-gluconic acid by Pseudomonas reptilivora NRRL B-6. Biomass Conv. Bioref. 10, 545–553 (2020). https://doi.org/10.1007/s13399-020-00656-7

Download citation


  • 2-Keto-d-gluconic acid
  • l-Ascorbic acid
  • Pseudomonas reptilivora
  • Bread waste
  • Valorization