Recently significant attention has been paid globally to renewable energy as an alternative to fossil fuels, which are responsible for climate change and economic crises. Although various sources of clean energy exist, the amount generated from these sources has not reached levels capable of meeting the world's energy needs. Biogas produced from the digestion of organic material in the absence of oxygen is one of the most promising possible alternatives for use in generating electrical and heat energy or fuel for vehicles. In addition, it is considered a cheap alternative compared with biofuels produced from other biological processes. However, anaerobic digestion (means of producing biogas) faces many challenges, including low production levels and operational problems. The current research is a serious attempt to address these problems through the exploitation of possible ways to increase production of methane and reduce the operation and maintenance costs. The study suggests the use of a sparging system in anaerobic digesters to convert unspontaneous reactions to spontaneous reactions by reducing the partial pressure of intermediate gases; thus, the reactions become thermodynamically favourable and provide impetus for a higher level of production. In order to increase the momentum, mass and heat transfer rates, microbubbles generated by fluidic oscillation was used in a gaslift bioreactor. The application of microbbubles in airlift bioreactors has been experimented with studying the kinetics of carbon dioxide transfer mechanisms. The efficiency of CO2 removal from the liquid as a step toward upgrading biogas has been increased up to 29% by microbubble sparging (550μm) compared with fine bubble sparging (1300μm). The design and simulation of a gaslift bioreactor with microbubbles generated by fluidic oscillation is discussed in the present research. The simulation study was carried out by using computational fluidic dynamics. The effect of bubble size, location of the diffuser, and dead zone on the efficiency of the bioreactor has also been investigated. The simulation results show that the use of microbubbles not only increased the surface area to volume ratio, but also increased mixing efficiency through increasing the velocity of the circulation liquid around the draft tube. Moreover, the simulation data demonstrated that when the diameter of the microbubbles exceeded 200 μm, the 'downcomer' region, which is equivalent to more than half of the overall volume of the airlift bioreactor, is free from gas bubbles. The designed gaslift bioreactor was also used for processing the digested sludge from acidic gases (carbon dioxide and hydrogen sulphide), which have a pejorative effect on the environment when they are eventually released, as well as causing operational difficulties through creating cavitation phenomena and the accompanying pump load. The results obtained from the experiments show that the removal of acidic gases in an airlift digester is significantly greater with microbubble technology than with a conventional digester. Application of a sparging system in anaerobic digestion to break down the organic matter under mesophilic conditions was investigated in the present thesis. The study involved the use of different gases: nitrogen, nitrogen with carbon dioxide, undiluted biogas, diluted biogas by carbon dioxide, and finally pure carbon dioxide. The experiments were carried out with and without fluidic oscillation in three identical lab-scale gaslift anaerobic digesters fed by kitchen waste under different operational conditions. The results show that using pure nitrogen as a sparged gas in anaerobic digestion, with or without microbubble technology, leads to a reduction in methane production. The study concluded that the nitrogen sparging system causes the stripping of all intermediate gases, such as carbon dioxide and hydrogen, which are necessary for other bacteria to produce acetate and methane. Although the carbon dioxide was compensated for the second part of experiments, this action did not lead to an increase in methane production. Enhancement in methane production was observed when the undiluted and diluted biogas was recycled in the anaerobic digestion. The results show that 10-14% more methane was produced from the gaslift digester than was observed in the conventional anaerobic digester. In the final part the gaslift digester was sparged with pure carbon dioxide. The results show that using pure carbon dioxide led to the production of double the amount of methane than was produced by the conventional digester. The sparging system used in the study also incorporated a new heating system for anaerobic digestion that used direct-contact evaporation process. The study used Central Composite Rotatable Design to create an equation based on three parameters (liquid level, flow rate and heating time). This equation was obtained by taking the five values for the individual parameters in each experiment while the percentage of evaporation, water temperature and humidity were measured as objective variables. The results show that liquid level has a significant effect on the liquid temperature due to the existence of competition between the latent heat transferred and the sensible heat in the direct contact system. The conversion of heat supplied to sensible heat was observed with an increase in liquid level, which can be converted to latent heat when the liquid level is decreased. Finally, and according to the above results, the present thesis proposes the integration of anaerobic digestion unit, combined heat and power (CHP unit), removal of acid gases unit and upgrading of biogas unit. This integration, along with microbubbles generated by a fluidic oscillator, represents an effective way to increase the efficiency of biogas production and reduce the operational difficulties.

Synthesis of Polyhydroxyalkanoates (PHAs) by Pseudomonas mendocina, using different vegetable oils such as, coconut oil, groundnut oil, corn oil and olive oil, as the sole carbon source was investigated for the first time. The PHA yield obtained was compared with that obtained during the production of PHAs using sodium octanoate as the sole carbon source. The fermentation profiles at shaken flask and bioreactor levels revealed that vegetable oils supported the growth of Pseudomonas mendocina and PHA accumulation in this organism. Moreover, when vegetable oil (coconut oil) was used as the sole carbon source, fermentation profiles showed better growth and polymer production as compared to conditions when sodium octanoate was used as the carbon source. In addition, comparison of PHA accumulation at shaken flask and fermenter level confirmed the higher PHA yield at shaken flask level production. The highest cell mass found using sodium octanoate was 1.8 g/L, whereas cell mass as high as 5.1 g/L was observed when coconut oil was used as the feedstock at flask level production. Moreover, the maximum PHA yield of 60.5% dry cell weight (dcw) was achieved at shaken flask level using coconut oil as compared to the PHA yield of 35.1% dcw obtained using sodium octanoate as the sole carbon source. Characterisations of the chemical, physical, mechanical, surface and biocompatibility properties of the polymers produced have been carried out by performing different analyses as described in the second chapter of this study. Chemical analysis using GC and FTIR investigations showed medium chain length (MCL) PHA production in all conditions. GC-MS analysis revealed a unique terpolymer production, containing 3-hydroxyoctanoic acid, 3-hydroxydecanoic acid and 3-hydroxydodecanoic acid when coconut oil, groundnut oil, olive oil, and corn oil were used as the carbon source. Whereas production of the homopolymer containing 3-hydroxyoctanoic acid was observed when sodium octanoate was used as the carbon source. MCL-PHAs produced in this study using sodium octanoate, coconut oil, and olive oil exhibited melting transitions, indicating that each of the PHA was crystalline or semi-crystalline polymer. In contrast, the thermal properties of PHAs produced from groundnut and corn oils showed no melting transition, indicating that they were completely amorphous or semi-crystalline, which was also confirmed by the X-Ray Diffraction (XRD) results obtained in this study. Mechanical analysis of the polymers produced showed higher stiffness of the polymer produced from coconut oil than the polymer from sodium octanoate. Surface characterisation of the polymers using Scanning Electron Microscopy (SEM) revealed a rough surface topography and surface contact angle measurement revealed their hydrophobic nature. Moreover, to investigate the potential applicability of the produced polymers as the scaffold materials for dental pulp regeneration, multipotent human Mesenchymal stem cells (hMSCs) were cultured onto the polymer films. Results indicated that these polymers are not cytotoxic towards the hMSCs and could support their attachment and proliferation. Highest cell growth was observed on the polymer samples produced from corn oil, followed by the polymer produced using coconut oil. In conclusion, this work established, for the first time, that vegetable oils are a good economical source of carbon for production of MCL-PHA copolymers effectively by Pseudomonas mendocina. Moreover, biocompatibility studies suggest that the produced polymers may have potential for dental tissue engineering application.

Polyhydroxyalkanoates (PHAs) are a family of polyhydroxyesters of 3-, 4-, 5- and 6- hydroxyalkanoic acids produced by bacterial fermentation in a nutrient limiting conditions with excess carbon. They can be produced easily using renewable carbon sources. They are biodegradable and biocompatible in nature. Their physical properties are highly tailorable and a range of desired properties can be achieved based on the type of application. Owing to these properties, there has been a considerable interest in the commercial exploitation of PHAs, particularly for biomedical applications. The main aim of this research project was to produce MCL-PHAs from Pseudomonas mendocina and use them for biomedical applications. In this study, an economical production of MCL-PHAs using renewable and cheap carbon sources such as sugarcane molasses, biodiesel waste and pure glycerol was carried out. Maximum PHA yield of 43.2% dcw was obtained in the media containing biodiesel waste. The results demonstrated the successful utilisation of these cheap carbon sources by P. mendocina for the economical production of MCL-PHAs. One of the main objectives of this project was to utilize the PHAs produced for biomedical applications. Multifunctional novel 2D P(3HO)/bacterial cellulose composite films were developed for their potential use in tissue engineering applications. Chemically modified bacterial cellulose microcrystals were used as the reinforcing agent to improve the properties of P(3HO). Mechanical properties such as the Young’s modulus and tensile strength values of the P(3HO)/bacterial cellulose composite films were significantly higher in comparison to the neat P(3HO) film. Also, the composite film had a rougher and more hydrophilic surface compared to the neat P(3HO) film. It is known from literature that surface roughness and hydrophilicity affects protein adsorption on the surface of the biomaterial. Protein adsorption, in turn, plays an important role in determining the biocompatibility of a material being used for medical applications (Das et al., 2007). In this study, protein adsorption was higher in the P(3HO)/25% bacterial cellulose composite film compared to the neat P(3HO) film. In vitro biocompatibility studies using Human microvascular endothelial cells (HMEC-1) was carried out. Both neat and composite films were able to support the proliferation of HMEC-1 cells. However, the biocompatibility of the P(3HO)/25% bacterial cellulose composite films had increased. The cell proliferation significantly higher on the P(3HO)/25% bacterial cellulose composite film as compared to the neat P(3HO) film on day 7. In addition, multifunctional 2D P(3HO)/P(3HB) blend films with varying percentages of P(3HO) and P(3HB) were developed and assessed for their suitability in the development of biodegradable stents. Mechanical, thermal and microstructural properties of the P(3HO)/P(3HB) blends were characterised. The results highlighted the role of P(3HB) in enhancing the mechanical properties and thermal stability of the blend films compared to the neat P(3HO) films. However, the results suggested that the mechanical properties of the P(3HO)/P(3HB) had to be further improved to meet the desired values required for the development of a biodegradable stent. The overall protein adsorption and % cell viability was significantly higher in the blend films compared to the neat P(3HO) film. Hydrolytic degradation was faster in the blend films and the degradation rate could potentially be tailored to achieve the optimum rate required for a particular medical application. From the literature, it is known that the surface topography determines the compatibility of a biomaterial by governing important processes such as wettability, protein adsorption, cell adhesion and proliferation (Duncan et al., 2007). In this part of the study, P(3HO)/P(3HB) 50:50 blend films were micropatterned using the laser micropatterning technique to improve their biocompatibility. The results demonstrated an increase in hydrophilicity and protein adsorption on the micropatterned blend films compared to the plain P(3HO)/P(3HB) 50:50 blend films. Cell attachment, proliferation and alignment was significantly higher on the micropatterned blend films compared to the P(3HO)/P(3HB) 50:50 blend films which was a desirable outcome. Furthermore, an investigation of the P(3HO)/P(3HB) 50:50 2D films as the base material for the development of a drug eluting biodegradable stent was carried out by incorporating aspirin within the film. The percentage viability of the HMEC-1 cells was higher in the blend films with aspirin compared to the blend films without aspirin indicating an increased biocompatibility of the P(3HO)/P(3HB) 50:50 blend film containing aspirin. Controlled release of aspirin was observed without any burst release and 96.6% release was achieved within 25 days, ideal for the development of biodegradable drug eluting stents. Finally, a drug delivery system for the controlled delivery of aspirin was successfully developed. In this part of the study, 2D solvent cast films and microspheres (average size=30 μm) were developed using P(3HB). Drug release pattern from P(3HB) films as well as P(3HB) microspheres were monitored. The results demonstrated that the P(3HB) films with aspirin were suitable for sustained long term drug release whereas P(3HB) microspheres with aspirin were more suitable for fast release. In conclusion, this project has led to the successful production of PHAs, and their utilisation in the development of a range of composites, blends and drug elution structures with promising potential medical applications.

In the search of alternative new materials for biodegradable plastics, biopolymers provide attractive solutions with their vast range of applications. A challenge in industrial production of biopolymers is their high cost, and one approach to minimise the cost is expanding the number of valuable products obtained from a single batch. The aim of this thesis was the dual production of biopolymers, P(3HB) and γ-PGA from cheap substrates with the view to lay grounds for a feasible, innovative, low cost production process. A common denominator between the two biopolymers focused in this thesis, (P(3HB) and γ-PGA), was that they both could be produced by Bacillus sp. One out of five strains screened, Bacillus subtilis OK2, was selected and the structures of both biopolymers produced were confirmed. Subsequently, optimisation of the production medium via statistical optimisation tools, and scaling-up of the process from shaken flasks to fermenters were carried out. Statistical design tool Placket Burman (PB), (Design Expert 6.0), was used to determine the effect of medium components on γ-PGA and P(3HB) production and to identify the crucial medium components in production media. The outcome of PB analysis of dual polymer production did not match the PB analysis of single polymer production. Considering the complexity of the dual polymer production mechanism, central composite design was applied after the number of parameters was reduced from five to three. A medium composed of 20 g/L glucose, 1.5 g/L yeast extract, 2.4 g/L citric acid, 32 g/L glutamic acid and 12 g/L ammonium sulphate was identified as the dual polymer production medium. Using an inoculum medium different from the production medium proved to have a positive effect on the production. Consequently, 1 g/L P(3HB) and 0.4 g/L γ-PGA in shaken flasks and 0.6 g/L P(3HB) and 0.2 g/L γ-PGA in single batch fermenters were produced with the strain Bacillus subtilis OK2. Selection of biowaste for the dual production was conducted using four biowastes; rapeseed cake, wheat bran, Spirulina powder and orange peel; using four pre-treatment methods, acid treatment, alkaline treatment, water infusion, and microwave exposure. γ-PGA production could not be detected when any of the waste materials was used as a sole medium component. Orange peel using water infusion pre-treatment was found to be the most suitable biowaste for the production of P(3HB). Bioreactor experiments showed that 1.24 g/L P(3HB) could be produced using orange peel as carbon source supplemented with yeast extract and citric acid. Dual polymer production using orange peel as carbon source proved to be more challenging as some of the ingredients in orange peel interfered with the dual production and inhibited production of both polymers. Although the different sugars in orange peel had a positive effect on production, pH control coupled with DOT control proved to be essential to overcome inhibition and 0.2 g/L of each polymer were produced in 79 h. For the separation of the two polymers from the culture broth, magnetic field, floatation, and sedimentation methods were investigated. Exposure to magnetic field was found to be inhibitory for P(3HB) production. The use of floatation and sedimentation for the online separation of cells with and without polymer to facilitate a recycle strategy exhibited negative results. This was found to be due to cells undergoing cell lyses at the early stages of the fermentation releasing P(3HB) granules into the fermentation medium. The size distribution of these granules was identified. The results elicit the possibility of using cell auto-lysis behaviour for the separation of the two polymers from the culture broth leading to a reduction of costs.

Adherent cell culture is a key experimental method for biological investigations in diverse areas such as developmental biology, drug discovery and biotechnology. Light microscopy-based methods, for example phase contrast microscopy (PCM), are routinely used for visual inspection of adherent cells cultured in transparent polymeric vessels. However, the outcome of such inspections is qualitative and highly subjective. Analytical methods that produce quantitative results can be used but often at the expense of culture integrity or viability. In this work, an imaging-based strategy to adherent cell cultures monitoring was investigated. Automated image processing and analysis of PCM images enabled quantitative measurements of key cell culture characteristics. Two types of segmentation algorithms for the detection of cellular objects on PCM images were evaluated. The first one, based on contrast filters and dynamic programming was quick (<1s per 1280×960 image) and performed well for different cell lines, over a wide range of imaging conditions. The second approach, termed ‘trainable segmentation’, was based on machine learning using a variety of image features such as local structures and symmetries. It accommodated complex segmentation tasks while maintaining low processing times (<5s per 1280×960 image). Based on the output from these segmentation algorithms, imaging-based monitoring of a large palette of cell responses was demonstrated, including proliferation, growth arrest, differentiation, and cell death. This approach is non-invasive and applicable to any transparent culture vessel, including microfabricated culture devices where a lack of suitable analytical methods often limits their applicability. This work was a significant contribution towards the establishment of robust, standardised, and affordable monitoring methods for adherent cell cultures. Finally, automated image processing was combined with computer-controlled cultures in small-scale devices. This provided a first demonstration of how adaptive culture protocols could be established; i.e. culture protocols which are based on cellular response instead of arbitrary time points.

Limbal epithelial stem cells (LESCs) are adult stem cells in the eye responsible for maintenance and repair of the corneal surface. Cultured LESC therapy aims to deliver stem cells to patients with a deficiency of these LESCs due to injury or disease and has been shown to be to be effective for restoring the corneal surface. The aim of this thesis was to characterise the culture of LESCs and improve the current method used to produce these LESCs for therapeutic use. Since human tissue for research is in very short supply, models of human LESC culture and the human LESC niche are required to identify mechanisms involved in LESC regulation. In this study rabbit limbal tissue and cultured cells were evaluated for this purpose. At present the 3T3 co-culture system seems to be optimal for the expansion of LESCs, and is approved for clinical use. NIBSC 3T3 J2s have recently (2006) been banked under GMP and this study showed that the GMP scale-up process had not affected the ability of these feeder cells to support the expansion of LESCs. As human limbal epithelial cells typically only survive for a few passages in vitro before they senesce, conditions in the in vivo stem cell niche were mimicked in an attempt to improve the in vitro culture of LESCs. Sub-atmospheric oxygen and ascorbic acid supplementation were investigated and found to be beneficial for maintenance and expansion of LESC progenitors in vitro. Microfluidic technology was investigated as a possible method for sorting LESCs, and was found to show promise as a tool for the identification of LESCs when used in conjunction with methods such as photoluminescence and fourier transform infrared spectroscopy.

This study reveals the development of new 3-D support for ethanol production by Saccharomyces cerevisiae W303. The production of bioethanol by immobilising this organism had been demonstrated to have greater advantages over a suspended culture of free cells of S.cerevisiae W303. The production of ethanol is proportional to the growth of yeast, so preparing suitable supports with unlimited spaces that permit the cell proliferation are crucial for a long term continuous operation. The 3-D scaffold prepared from high internal phase emulsion (HIPE) offers good mechanical strength, and has a high surface area for allowing monolayer cell proliferation which was necessary in order to avoid additional stresses for the nutrient and oxygen transfer in the micro-environment. The enhanced porosity of this 3-D scaffold that was characterised by highly interconnected pores, not only promoted the dynamic condition in the monolith, but also facilitated easy flushing of dead cells and metabolic product. This study reveals that sulphonated polyHIPEs, a highly hydrophilic polymer which had pore and interconnect sizes of 45 μm and 16 μm respectively, had shown good bio-compatibility with the model organism, subsequently allowing its growth and glucose conversion. The ethanol productivity in the microreactor was greatly enhanced to 4.72 gL-1h-1, being over 12 times higher than that observed in the suspended shake flask culture (0.41 gL-1h-1) by free cell S.cerevisiae W303, despite only 60.1% of glucose being consumed. Since the remaining sugar must be kept low, the glucose utilization was further enhanced by introducing the two-stage reactor in series. The consumption of glucose was enhanced by 20.1% (compared to single stage reactor), where nearly 72.2 % of the supplied glucose was converted per pass during the pseudo steady state condition. This, on the other hand, increased the ethanol productivity to 5.84 gL-1h-1, which was 14 fold higher than the productivity obtained in the shake flask culture. The increment might be associated with the altered metabolical functions in the immobilised cells. This alteration is attributed to the reduction of the diffusion path of the growth nutrient (e.g: carbon, nitrogen and oxygen) that enhanced the availability and promoted the growth of yeast in the microreactor, thus enhancing the catalytic conversion of glucose to ethanol.

The enzymatic degradation of polysaccharides is a major goal of the biotechnology industry, most notably for both first (starch) and second (cellulose/chitin) generation biofuels. The classical model for enzymatic polysaccharide breakdown involves the action of endo- and exo- acting glycoside hydrolases which act in synergy; endo-acting enzymes generating free chain-ends for the processive exo-glycosidases. In 2010 a new enzyme class was discovered which overturned the classical hydrolase model. These enzymes, now known as Lytic Polysaccharide Monooxygenases (LPMOs), are understood to be copper-dependent oxygenase enzymes that cause chain cleavage within polysaccharides, facilitating their degradation by classical hydrolases. This research showcases the findings for an LPMO of the “Auxiliary Activity” class AA10 from the bacterium Bacillus amyloliquefaciens (BaAA10). Methods of gene expression and protein production are described, including the incorporation of a SUMO-tag which resulted in increased yields of BaAA10. The nature of the copper binding was assessed using Isothermal Titration Calorimetry and Differential Scanning Fluorimetry, which demonstrated the extremely tight binding of copper to the enzyme and its increased stability when copper is bound. Additionally, the detection of lactone and aldonic acid products of oxidative degradation, in MALDI-TOF Mass Spectrometry results, determined for the first time that BaAA10 was active on both alpha and beta forms of chitin. Finally, a Cu(II) structure of BaAA10 was able to be obtained following the use of a spiral data collection technique as a method of preventing photoreduction of Cu(II) to Cu(I) in the X-ray beam. This structure can now provide us with details regarding the copper active site and, together with potential spectroscopic studies in the future, can help us to determine the mechanism of action of these LPMO enzymes, so that we can better exploit their use within the biofuel industry.

The study presented in this document is the result of three years of research into the complex world of Molecular Dynamics applied to biological cell membranes. The simulation of biological tissues involves not only an excellent knowledge of the numerical calculus and its related tools, but a profound comprehension of the biological and medical literature associated with the phenomenon. By the other hand, the use of high performance facilities is essential for the computation of the Molecular Dynamics models in order to obtain results in acceptable times, so the latest technological advances have played a decisive and important part in this eld of research. The presented obtained results about shock wave interaction with biological membranes, as well as the air ow through the alveolar surface, are part of a new line of research usually known as 'virtual experimental'. This name comes from the fact that any physical or chemical situation can be re-created into a computer system to calculate its propagation in time. The results of the interaction of shock waves with biological cell membranes have been particularly satisfactory and they have opened a new line of investigation into cancer research. A numerical proportional relation between the shock wave impulse and the value of lateral di usion (from 9.80 to 12.84 10 .7 cm2 s ), as well as the simulation of the transient provoked by the wave into a NPT ensemble are a successful achievement. Other computations of this type of interaction have been simulated into an NVE ensemble as well, however the obtained results for the lateral di usion, in the order of 10 .7 cm2 s , showed no trend regarding the shock wave and the transient e ect could not be simulated. On the other hand, the recreation of the air ow through the alveolar surface is an initial step into the solution of all the controversy surrounding this extremely complex system known as alveolar surface network. An alveolar membrane of around 7 nm has been successfully simulated in agreement with Scarpelli's experiments. This lipid-protein membrane model simulated can serve as a virtual experiment in order to solve the controversy about the alveolar surface. It points to the possibility of air ow through a stable two-layered DPPC phospholipid structure either from a numerical or physical and biological point of view and the existence of an alveolar membrane at the end of the bronchial tubes.

Researchinto enzyme catalysis has enormously enriched the success of biological organic synthesis; particularly in the last decade, remarkable progress in biological catalysis has taken place leading to the application of enzymes to a wider extent in industrial processes. The benefits of establishing enzymes as industrial catalysts are founded on the need to develop a ‘clean’ and ‘pure’ technology which carry out isomer and region selective reactions; generate or transform pure isomers compared with racemic mixtures and produce pure compounds in comparison with mixtures of by-products often arising from conventional chemical synthesis. With the onset of molecular biology and genetics, the manufacturing of high value proteins in recombinant host cells has become commonplace; nevertheless, there needs to be a better understanding of the optimum conditions for the production of every recombinant protein. In this study, the over expression of an industrially important amine oxidase – D-Amino Acid Oxidase (DAAO) – was attempted using two different expression systems: Escherichia coli and Pichia pastoris in order to establish the optimum conditions for industrial production. Amine oxidases exhibit stereoselectivity; ergo they can theoretically be used for the deracemisation of nonopticallypure mixtures of amines leading to optically pure amines of high value. Furthermore, DAAO is involved in the production of 7-aminocephalosporanic acid (7-ACA) from cephalosporin c (CPC), which can be used in the synthesis of novel cephalosporin antibiotic derivatives. In order to accelerate development of these new antibiotics DAAO must be readily available. Overexpression of DAAO in E. coli was studied under varying experimental conditions in order to ascertain the best conditions for enzymatic activity whilst simultaneously investigating the variations between both defined and complex media to understand suitability for scale-up. Although E. coli is still regarded as the ‘go to’ organisms for initial laboratory scale up the methylotrophic yeast P. pastoris is considered an industrial workhorse for the production of recombinant proteins and thus the expression of DAAO was also investigated to ascertain how enzyme activities are affected by this expression system. Furthermore, the supplementation of media with different nutrient sources of varying complexity was investigated, as was the implementation of co-feeding inorder to improve carbon flux during recombinant protein production. Finally, the oxygen availability was investigated in order to gain important understandings on its effects on microbial physiology and DAAO activity.