Development of novel approaches for industrial fermentation of menaquinone-7
Ranmadugala, D. N. A. (2018). Development of novel approaches for industrial fermentation of menaquinone-7 (Thesis, Doctor of Philosophy (PhD)). The University of Waikato, Hamilton, New Zealand. Retrieved from https://hdl.handle.net/10289/12246
Permanent Research Commons link: https://hdl.handle.net/10289/12246
Vitamin K, an anti-haemorrhagic factor discovered by the Danish nutritional biochemist Henrik Carl Peter Dam in 1929, is capable of correcting dietary-induced bleeding disorders in chickens. It is essential for blood coagulation, and therefore must be supplied in the diet. Menaquinones (MK) together with phylloquinone (PK) fall under the common name ‘vitamin K’. Recent studies show that dietary intake of menaquinones results in more health benefits than phylloquinone. Menaquinone-7 (MK-7) plays an important role in maintaining human health in the liver, bone and arterial vessels with potential to prevent osteoporosis and cardiovascular diseases as well as cancer. Therefore, MK-7 can be taken as the obvious choice for the prevention of these health complications. MK-7 is produced industrially by a fermentation process of Bacillus subtilis at low concentrations. To date, there have been several attempts to improve MK-7 yield via optimising the fermentation media, genetic mutation of Bacillus subtilis and reducing downstream processing steps. Despite this progress, industrial production of MK-7 has been difficult, mainly due to biofilm formation, low MK-7 production by Bacillus subtilis and the presence of so many tedious unit operations in the process. As a result, the price of MK-7 is currently US$5 million/kg. It appears worthwhile to develop novel strategies to address the current MK-7 production challenges. The main goal of the current research was, therefore, to develop a novel cost-effective fermentation process by eliminating biofilm formation while enhancing MK-7 production and reducing the fermentation process steps. In combating biofilm formation in Bacillus subtilis fermentation, a major challenge was to adopt strategies which would maintain the viability of the bacterial cells as required by the fermentation process. In this regard, as a first step, a nanobiotechnological approach was taken in which superparamagnetic iron oxide nanoparticles (Fe3O4) were used to decorate Bacillus subtilis cells. In comparison to naked iron oxide nanoparticles, iron oxide nanoparticles coated with 3-aminopropyltriethoxysilane (IONs@APTES) significantly reduced the adherence and pellicle biofilm biomass of Bacillus subtilis in a concentration-dependent manner without affecting the growth and viability of Bacillus subtilis. Further, the research embodied in this thesis also addresses the low MK-7 production by immobilising Bacillus subtilis cells using IONs@APTES. Immobilisation of Bacillus subtilis cells with IONs@APTES significantly enhanced the MK-7 production, implying that binding of IONs@APTES to Bacillus subtilis cells might have changed the state or composition of the cell membranes resulting in enhanced production and secretion of MK-7 to the fermentation medium. In addition to a strategy that minimised bacterial cell attachment and subsequent biofilm formation, the effect of a biofilm detachment strategy was also investigated. As nutrient conditions in the medium can greatly influence biofilm detachment, nutrient components which would interfere with the structural integrity of biofilms and bring about biofilm detachment were investigated by supplementing the medium with salts and urea. In this study the optimum conditions to minimise biofilm formation while maximising MK-7 production was determined by response surface optimisation. These investigations finally led to an optimum fermentation medium for minimum biofilm formation and maximum MK-7 production which consists of 5% (w/v) yeast extract, 18.9% (w/v) soy peptone, 5% (w/v) glycerol, 0.05% (w/v) K₂HPO₄, 0.32% (w/v) CaCl₂, 0.10% (w/v) urea and 200 µg/mL IONs@APTES. A ~ 47% reduction in biofilm biomass and ~ 16% increase in MK-7 production was observed after 60 hours of fermentation when the medium was supplemented with 0.32% (w/v) CaCl₂, 0.10% (w/v) urea and 200 µg/mL IONs@APTES in comparison to the medium without added CaCl₂, urea and IONs@APTES. This was found useful as a non-antibiotic antifouling strategy which would permit the growth and viability of bacterial cells in the fermentation process while minimising biofilm formation and maximising MK-7 production. Using this medium, the feasibility of designing a milking process for enhancing MK-7 productivity was assessed. Initial studies involved selecting biocompatible organic solvent/s for milking MK-7. Milking MK-7 with n-hexane proved to be an effective strategy to enhance the total MK-7 production by ~ 1.7-fold in subtilis fermentation without compromising the bacterial cell viability in comparison to non-milking conditions. Not only did the total MK-7 increase by ~ 1.7-fold, but the maximum total MK-7 production also reached earlier i.e. within 72 hoursin comparison to previously recorded liquid state fermentation studies for MK-7 production, which is important from a commercial point of view. In addition, a novel, accurate, reliable and high throughput MK-7 analysis protocol is also presented in the thesis. Hence, by examining and integrating a range of strategies, this thesis provides a feasible approach for industrial production and analysis of MK-7.
The University of Waikato
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