title: Development of novel approaches for industrial fermentation of menaquinone-7

authors: Ranmadugala, Dinali Niluka Achala

subjects: Menaquinone-7

abstract: 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.

text: Development of novel approaches for industrial fermentation of menaquinone-7 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.

year: 2018

title: Control-theoretical stress-testing for cross-platform compliance

authors: McCarthy, Harry George Andrew

subjects: LQR

abstract: Modern systems are required more and more to be able to provide performance reliability qualities for not just one system but cross platform as well. Stress testing applications allow for performance testing of a system, however, when applied across different platforms the performance of the application is not a one to one comparison. This thesis introduces the idea of applying control theory to provide consistent resource percentage utilisation when stress testing cross platform. Proportional Integral Derivative (PID) control, Linear Quadratic Regulator (LQR) control and a PID Assisted LQR control were utilised as three control system in this thesis. While the PID controller provided the best results across multiple platforms, its inability to control all the variables in the system along with multiple different state
objectives (CPU and memory) leads to the need for a system model approach. LQR controllers provide this model based approach but at the cost of being unable to adapt across different platforms and to unpredictability in the system. Finally, a combined approach PID Assisted LQR control. PID Assisted LQR control gave the best of both PID and LQR control approach and had improvements in the case of where the system was most unpredictable but lacked any significant improvements in areas where the model gave an accurate representation of the system.

text: Control-theoretical stress-testing for cross-platform compliance Modern systems are required more and more to be able to provide performance reliability qualities for not just one system but cross platform as well. Stress testing applications allow for performance testing of a system, however, when applied across different platforms the performance of the application is not a one to one comparison. This thesis introduces the idea of applying control theory to provide consistent resource percentage utilisation when stress testing cross platform. Proportional Integral Derivative (PID) control, Linear Quadratic Regulator (LQR) control and a PID Assisted LQR control were utilised as three control system in this thesis. While the PID controller provided the best results across multiple platforms, its inability to control all the variables in the system along with multiple different state
objectives (CPU and memory) leads to the need for a system model approach. LQR controllers provide this model based approach but at the cost of being unable to adapt across different platforms and to unpredictability in the system. Finally, a combined approach PID Assisted LQR control. PID Assisted LQR control gave the best of both PID and LQR control approach and had improvements in the case of where the system was most unpredictable but lacked any significant improvements in areas where the model gave an accurate representation of the system.

year: 2020

title: Caught in the cross-fire: the realities of being Māori at a bicultural law school

authors: Papuni-Ball, Makere

abstract: This thesis examines the first Waikato Māori Law Graduates’ experiences at law school and our employment choices. It has been written as a result of a research project which was conducted in 1994. The paper begins with establishing the importance of other Māori the guidelines on this project. In this discussion, my role as the researcher is explained together with how the original research project impacted upon this thesis.

Waikato School of Law was established in an era of socio-political changes in our society. Te Matahauariki Report supported its establishment based upon proven demand for law graduates. It also envisaged the School as being a meeting place for all who attended, an institution which might encourage relationships between Māori and Pakeha societies. The Council of Legal Education supported the need to have at least one law school which would not reflect the traditional legal education system and acknowledged the need to incorporate a Māori perspective. Tainui as tangata whenua hoped the school would provide opportunities for Māori students, including Māori in the curriculum and assist in promoting research ensuring the recognition of Māori lore/law.

Amongst this backdrop, the most important reason why we came to law school was because of our whānau expectations. It is the accountability and responsibility we have as Māori to our whānau and the wider Māori community that is a major theme throughout this thesis. As students we were expected to utilise our skills to benefit our wider Māori networks.

We had expected that the School would provide us with a legal education which would be enriched in Māori kaupapa. Our experiences tell another story. Instead of being educated in Māori tikanga and the true discourse of the law as a tool of oppression, we were exposed to continual suppression of our cultural heritage. In class we had become the teachers of the Māori content.

The School, although admiral in its aims, was not prepared in structure, staff or mental processes for biculturalism. The consequences for the Māori students who were exposed to this structure was devastating. Our experiences of being discriminated against and made to justify our Māori perspective against the backdrop of the dominant Pakeha legal culture have affected our perceptions of the law. The myths of one law for all and the neutrality of the justice system have resulted in our choosing to work in jobs that will continue to benefit Māori.

It is suggested that our legal education has influenced our choices to work in areas which focus on benefiting the Māori community. In those monocultural work areas which have few Māori practising, this is because such places repel those of us who know that such work places perpetuate the dominant Pakeha culture. Māori struggle against the oppressive presence of the dominant culture.

This thesis calls upon my own experiences and the experiences of my fellow Māori law graduates. It is a starting point for other Māori to write about their experiences and to reassert Māori rangatiratanga not only in law school, but in the wider community.

text: Caught in the cross-fire: the realities of being Māori at a bicultural law school This thesis examines the first Waikato Māori Law Graduates’ experiences at law school and our employment choices. It has been written as a result of a research project which was conducted in 1994. The paper begins with establishing the importance of other Māori the guidelines on this project. In this discussion, my role as the researcher is explained together with how the original research project impacted upon this thesis.

Waikato School of Law was established in an era of socio-political changes in our society. Te Matahauariki Report supported its establishment based upon proven demand for law graduates. It also envisaged the School as being a meeting place for all who attended, an institution which might encourage relationships between Māori and Pakeha societies. The Council of Legal Education supported the need to have at least one law school which would not reflect the traditional legal education system and acknowledged the need to incorporate a Māori perspective. Tainui as tangata whenua hoped the school would provide opportunities for Māori students, including Māori in the curriculum and assist in promoting research ensuring the recognition of Māori lore/law.

Amongst this backdrop, the most important reason why we came to law school was because of our whānau expectations. It is the accountability and responsibility we have as Māori to our whānau and the wider Māori community that is a major theme throughout this thesis. As students we were expected to utilise our skills to benefit our wider Māori networks.

We had expected that the School would provide us with a legal education which would be enriched in Māori kaupapa. Our experiences tell another story. Instead of being educated in Māori tikanga and the true discourse of the law as a tool of oppression, we were exposed to continual suppression of our cultural heritage. In class we had become the teachers of the Māori content.

The School, although admiral in its aims, was not prepared in structure, staff or mental processes for biculturalism. The consequences for the Māori students who were exposed to this structure was devastating. Our experiences of being discriminated against and made to justify our Māori perspective against the backdrop of the dominant Pakeha legal culture have affected our perceptions of the law. The myths of one law for all and the neutrality of the justice system have resulted in our choosing to work in jobs that will continue to benefit Māori.

It is suggested that our legal education has influenced our choices to work in areas which focus on benefiting the Māori community. In those monocultural work areas which have few Māori practising, this is because such places repel those of us who know that such work places perpetuate the dominant Pakeha culture. Māori struggle against the oppressive presence of the dominant culture.

This thesis calls upon my own experiences and the experiences of my fellow Māori law graduates. It is a starting point for other Māori to write about their experiences and to reassert Māori rangatiratanga not only in law school, but in the wider community.

year: 1996