Bioprocess and Biosystems Engineering

, Volume 42, Issue 2, pp 223–232 | Cite as

Effects of medium components in a glycerol-based medium on vitamin K (menaquinone-7) production by Bacillus subtilis natto in biofilm reactors

  • Ehsan Mahdinia
  • Ali DemirciEmail author
  • Aydin Berenjian
Research Paper


Menaquinone-7 (MK-7) as the most important form of Vitamin K has been reported to have miraculous benefits such as preventing cardiovascular diseases and osteoporosis along with antitumor effects. Therefore, there have been numerous studies in the past decades to improve MK-7 production via microbial fermentation. Unfortunately, both solid and liquid state fermentation strategies that are utilized for MK-7 production, face fundamental operational and scale-up issues as well as intense heat and mass transfer problems during fermentation. In this regard, biofilm reactors seem to be a practical solution to overcome these issues and enhance the production in agitated liquid fermentation. Therefore, this study was undertaken to utilize biofilm reactors in investigating and optimizing different media components in a glycerol-based medium. Using response surface methodology, the effects of glycerol, yeast extract, and soytone were studied in the fermentation medium on MK-7 production in biofilm reactor. With a composition of 48.2 g/L of glycerol, 8.1 g/L of yeast extracts, 13.6 g/L of soytone and 0.06 g/L of K2HPO4, MK-7 concentrations could reach 14.7 ± 1.4 mg/L in biofilm reactors, which was 57% higher compared to the MK-7 concentration achieved in suspended-cell reactors under similar conditions, while glycerol was depleted by the end of the fifth day in biofilm reactors, but glycerol was never depleted in suspended-cell reactors. Evidently, biofilm reactors present a reliable strategy to address the operational issues that occur during MK-7 biosynthesis on an industrial scale production.


MK-7 Menaquinone-7 Vitamin K Biofilm reactor Bacillus subtilis RSM optimization 



This work was supported by the USDA National Institute of Food and Agriculture Federal Appropriations under Project PEN04561 and Accession number 1002249. The authors thank the Statistical Consulting Center at The Pennsylvania State University for their support in providing useful consultation for data processing.


  1. 1.
    Dam H (1935) The antihaemorrhagic vitamin of the chick: occurrence and chemical nature. Nature 135:652–653CrossRefGoogle Scholar
  2. 2.
    Widhalm JR, Ducluzeau A-L, Buller NE, Elowsky CG, Olsen LJ, Basset GJC (2012) Phylloquinone (vitamin K1) biosynthesis in plants: two peroxisomal thioesterases of Lactobacillales origin hydrolyze 1,4-dihydroxy-2-naphthoyl-CoA. Plant J 71:205–215. CrossRefGoogle Scholar
  3. 3.
    Booth SL (2012) Vitamin K: food composition and dietary intakes. Food Nutr Res 56(1):5505. CrossRefGoogle Scholar
  4. 4.
    Binkley SB, Maccorquodale DW, Thayer A, Doisy EA (1939) The isolation of vitamin K1. J Biol Chem 130:219–234Google Scholar
  5. 5.
    Mahdinia E, Demirci A, Berenjian A (2017) Production and application of menaquinone-7 (vitamin K2): a new perspective. World J Microbiol Biotechnol 33:2. CrossRefGoogle Scholar
  6. 6.
    Schurgers LJ, Teunissen KJF, Hamulyák K, Knapen MHJ, Vik H, Vermeer C (2007) Vitamin K-containing dietary supplements: comparison of synthetic vitamin K1 and natto-derived menaquinone-7. Blood 109:3279–3283. CrossRefGoogle Scholar
  7. 7.
    Howard LM, Payne AG (2006) Health benefits of vitamin K2: a revolutionary natural treatment for heart disease and bone loss, 1st edn. Basic Health Publications, Inc., Laguna BeachGoogle Scholar
  8. 8.
    Gast GCM, de Roos NM, Sluijs I, Bots ML, Beulens JWJ, Geleijnse JM, Witteman JC, Grobbee DE, Peeters PHM, van der Schouw YT (2009) A high menaquinone intake reduces the incidence of coronary heart disease. Nutr Metab Cardiovasc Dis 19:504–510. CrossRefGoogle Scholar
  9. 9.
    Geleijnse JM, Vermeer C, Grobbee DE, Schurgers LJ, Knapen MHJ, van der Meer IM, Hofman A, Witteman JCM (2004) Dietary intake of menaquinone is associated with a reduced risk of coronary heart disease: the Rotterdam study. J Nutr 134:3100–3105CrossRefGoogle Scholar
  10. 10.
    Yamaguchi M (2006) Regulatory mechanism of food factors in bone metabolism and prevention of osteoporosis. Yakugaku Zasshi 126:1117–1137. CrossRefGoogle Scholar
  11. 11.
    Davidson RT, Foley AL, Engelke JA, Suttie JW (1998) Conversion of dietary phylloquinone to tissue menaquinone-4 in rats is not dependent on gut bacteria. J Nutr 128:220–223CrossRefGoogle Scholar
  12. 12.
    Walther B, Chollet M (2017) Menaquinones, bacteria, and foods: vitamin K2 in the diet. In: Vitamin K2—vital for health and wellbeing. IntechOpen, London, UK, pp 63–82.
  13. 13.
    Berenjian A, Mahanama R, Kavanagh J, Dehghani F (2015) Vitamin K series: current status and future prospects. Crit Rev Biotechnol 35:199–208. CrossRefGoogle Scholar
  14. 14.
    Berenjian A, Mahanama R, Talbot A, Biffin R, Regtop H, Kavanagh J (2011) The effect of amino-acids and glycerol addition on MK-7 production. In: Proc. world congr. eng. comput. sci. II, pp 19–21Google Scholar
  15. 15.
    Goodman SR, Marrs BL, Narconis RJ, Olson RE (1976) Isolation and description of a menaquinone mutant from Bacillus licheniformis. J Bacteriol 125:282–289Google Scholar
  16. 16.
    Wu W-J, Ahn B-Y (2011) Improved menaquinone (vitamin K2) production in cheonggukjang by optimization of the fermentation conditions. Food Sci Biotechnol 20:1585–1591. CrossRefGoogle Scholar
  17. 17.
    Singh R, Puri A, Panda BP (2015) Development of menaquinone-7 enriched nutraceutical: inside into medium engineering and process modeling. J Food Sci Technol 52:5212–5219. CrossRefGoogle Scholar
  18. 18.
    Pandey A (2003) Solid-state fermentation. Biochem Eng J 13:81–84. CrossRefGoogle Scholar
  19. 19.
    Ikeda H, Doi Y (1990) A vitamin-K2-binding factor secreted from Bacillus subtilis. Eur J Biochem 192:219–224. CrossRefGoogle Scholar
  20. 20.
    Kuchma SL, O’Toole GA (2000) Surface-induced and biofilm-induced changes in gene expression. Curr Opin Biotechnol 11:429–433. CrossRefGoogle Scholar
  21. 21.
    Mahdinia E, Demirci A, Berenjian A (2017) Strain and plastic composite support (PCS) selection for vitamin K (menaquinone-7) production in biofilm reactors. Bioprocess Biosyst Eng 40:1507–1517. CrossRefGoogle Scholar
  22. 22.
    Demirci A, Pongtharangkul T, Pometto IIIAL (2007) Applications of biofilm reactors for production of value-added products by microbial fermentation. Blackwell Publishing and The Institude of Food Technologists, IowaGoogle Scholar
  23. 23.
    Ercan D, Demirci A (2013) Production of human lysozyme in biofilm reactor and optimization of growth parameters of Kluyveromyces lactis K7. Appl Microbiol Biotechnol 97:6211–6221. CrossRefGoogle Scholar
  24. 24.
    Izmirlioglu G, Demirci A (2016) Ethanol production in biofilm reactors from potato waste hydrolysate and optimization of growth parameters for Saccharomyces cerevisiae. Fuel 181:643–651. CrossRefGoogle Scholar
  25. 25.
    Ho KG, Pometto ALI, Hinz PN, Dickson JS, Demirci A (1997) Ingredient selection for plastic composite supports for l-(1)-lactic acid biofilm fermentation by Lactobacillus casei subsp. rhamnosus. Appl Environ Microbiol 63:2516–2523Google Scholar
  26. 26.
    Khiyami MA, Pometto AL, Kennedy WJ (2006) Ligninolytic enzyme production by Phanerochaete chrysosporium in plastic composite support biofilm stirred tank bioreactors. J Agric Food Chem 54:1693–1698. CrossRefGoogle Scholar
  27. 27.
    Mahdinia E, Demirci A, Berenjian A (2018) Optimization of Bacillus subtilis natto growth parameters in glycerol-based medium for vitamin K (menaquinone-7) production in biofilm reactors. Bioprocess Biosyst Eng 41:195–204. CrossRefGoogle Scholar
  28. 28.
    Berenjian A, Mahanama R, Talbot A, Biffin R, Regtop H, Valtchev P, Kavanagh J, Dehghani F (2011) Efficient media for high menaquinone-7 production: response surface methodology approach. N Biotechnol 28:665–672. CrossRefGoogle Scholar
  29. 29.
    Sato T, Yamada Y, Ohtani Y, Mitsui N, Murasawa H, Araki S (2001) Production of menaquinone (vitamin K2)-7 by Bacillus subtilis. J Biosci Bioeng 91:16–20. CrossRefGoogle Scholar
  30. 30.
    Berenjian A, Mahanama R, Talbot A, Regtop H, Kavanagh J, Dehghani F (2012) Advances in menaquinone-7 production by Bacillus subtilis natto: fed-batch glycerol addition. Am J Biochem Biotechnol 8:105–110. CrossRefGoogle Scholar
  31. 31.
    Berenjian A, Chan NL-C, Mahanama R, Talbot A, Regtop H, Kavanagh J, Dehghani F (2013) Effect of biofilm formation by Bacillus subtilis natto on menaquinone-7 biosynthesis. Mol Biotechnol 54:371–378. CrossRefGoogle Scholar
  32. 32.
    Ercan D, Demirci A (2014) Enhanced human lysozyme production in biofilm reactor by Kluyveromyces lactis K7. Biochem Eng J 92:2–8. CrossRefGoogle Scholar
  33. 33.
    Mahdinia E, Demirci A, Berenjian A (2018) Utilization of glucose-based medium and optimization of Bacillus subtilis natto growth parameters for vitamin K (menaquinone-7) production in bio fi lm reactors. Biocatal Agric Biotechnol 13:219–224. CrossRefGoogle Scholar
  34. 34.
    Shahami M, Ransom R, Shantz DF (2017) Synthesis and characterization of tin, tin/aluminum, and tin/boron containing MFI zeolites containing MFI zeolites. Microporous Misoporous Mater 251:165–172. CrossRefGoogle Scholar
  35. 35.
    Rahimi M, Schoener Z, Zhu X, Zhang F, Gorski CA, Logan BE (2017) Removal of copper from water using a thermally regenerative electrodeposition battery. J Hazard Mater 322:551–556. CrossRefGoogle Scholar
  36. 36.
    Rahimi M, Angelo AD, Gorski CA, Scialdone O, Logan BE (2017) Electrical power production from low-grade waste heat using a thermally regenerative ethylenediamine battery. J Power Sources 351:45–50. CrossRefGoogle Scholar
  37. 37.
    Fisher SH (1999) MicroReview regulation of nitrogen metabolism in Bacillus subtilis. Mol Microbiol 32:223–232. CrossRefGoogle Scholar
  38. 38.
    Ashby RD (2005) Synthesis of short-/medium-chain-length poly(hydroxyalkanoate) blends by mixed culture fermentation of glycerol. Biomacromol 6:2106–2112. CrossRefGoogle Scholar
  39. 39.
    Nishikawa M, Ogawa K (2006) Inhibition of epsilon-poly-l-lysine biosynthesis in Streptomycetaceae bacteria by short-chain polyols. Appl Environ Microbiol 72:2306–2312. CrossRefGoogle Scholar
  40. 40.
    Qureshi N, Annous BA, Ezeji TC, Karcher P, Maddox IS (2005) Biofilm reactors for industrial bioconversion processes: employing potential of enhanced reaction rates. Microb Cell Fact 4(1):24. CrossRefGoogle Scholar
  41. 41.
    Mahdinia E, Demirci A, Berenjian A (2018) Implementation of fed-batch strategies for vitamin K (menaquinone-7) production by Bacillus subtilis natto in biofilm reactors. Appl Microbiol Biotechnol 102(21):9147–9157. CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Department of Agricultural and Biological EngineeringThe Pennsylvania State UniversityUniversity ParkUSA
  2. 2.Faculty of Science and EngineeringThe University of WaikatoHamiltonNew Zealand

Personalised recommendations