PhD, EngD and MSc by research theses (Cranfield Health)

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Open Access
The biochemistry and physiology of poly-beta-hydroxybutyrate metabolism in methylosinus trichosporium OB3b
(Cranfield University, 1988-09) Williams, A. M.; Best, D. J.
The obligate methanotroph, Methylosinus trichosporium 0B3b synthesised up to 20 % of its dry weight as poly-beta-hydroxybutyrate when grown in batch culture or nitrogen limited continuous culture on a methanol/ammonium salts medium. Four soluble enzymes associated with polymer metabolism, D(-)-3-hydroxybutyrate dehydrogenase (3-HBD), acetoacetyl-CoA synthetase, beta-ketothiolase and acetoacetyl-CoA reductase have been purified from the crude extract of this organism. Detailed kinetic studies were conducted on each enzyme and their role in the regulation of polymer metabolism was assessed. Cont/d.
Open Access
Gelation and melting of gelatin.
(Cranfield University, 1990-03) Clegg, Stuart Mark; Morris, E. R.
Chiroptical, rheological and thermodynamic studies have been undertaken to investigate temperature-induced changes in the ý molecular organisation of gelatin. From the results obtained, a unified model for gelation and melting has been developed, and tested using Monte Carlo computer simulation. The temperature at which gelatin gels are formed has a major influence on the properties of the resulting network, with higher curing temperatures conferring increased thermal stability. In particular, gels formed by sequential curing at two different temperatures show biphasic melting. This is explained in terms of a temperature-dependence of helix length within the junction zones of the gel, and quantified by considering end-effects in the thermodynamics of helix stability. Measurements of 'initial slope' kinetics, performed over a broad concentration range, showed first-order kinetics at low gelatin concentrations, while at higher concentrations a second-order process was also evident. The results are interpreted as triple-helix nucleation at metastable 'hairpin turns' in one chain (bringing two chain segments into close proximity) together with a third strand from either the same chain (first order) or a different chain (second order). From simple geometric considerations, the maximum length of intermolecular helices ( which contribute to the gel network) is greater than that of twasted 9 intramolecular structures, giving a qualitative explanation of the increased strength of gels formed by precuring at higher temperatures (where only long helices are stable) over those quenched directly to low temperature. Monte Carlo simulation incorporating an initial assumption that helix propagation is rapid and proceeds to geometric limits gave unrealistic helix lengths and simulated melting profiles, and was replaced by the assumption that cis-trans isomerisation of peptide bonds is the controlling factor in helix propagation. Using the latter assumption, most aspects of the observed behaviour were successfully reproduced using program variables set within realistic ranges or, where possible, fixed at experimentally-determined values. In particularg the co-operativity of the simulated melting process was critically dependent on the value of a parameter x (the number of triplet units within each helix incapable of participating in bonding, due to end-effects), with a value of x=1 giving the best fits with experiment (consistent with accepted bonding patterns for the collagen triple helix). Other key parameters were the midpoint temperature for melting of the parent collagen, which gave best agreement when set at 37-38"C, and t6e proportion of cis peptide residues present in disordered gelatin chains, with an optimum lower limit of 0.15. Using these values, the simulation reproduced, with excellent precision, the helix fraction and melting profile of gels formed over a wide range of quench temperatures, and gave an acceptable approximation to the form of reaction progress curves obtained for helix formation. The biphasic melting of samples held at intermediate temperature before final quenching was also modelled realistically.
Open Access
Production of a lipase from a pseudomonad species
(Cranfield University, 1991-03) Smith, Catherine J.; Brown, D. E.
A lipase-producing micro-organism was found to be a strain of Pseudomonas aeruginosa. When it was grown batchwise in a stirred 2 dM3 fermenter in a simple defined minimal salts medium containing yeast extract and glucose, it produced lipase at a level of 0.173 LU/cm3 without the need for a lipid substrate. Batch cultures of cells were fed glucose at constant rates. An optimum rate was found to be 0.50 g/dm3/h and this produced a maximum lipase activity of 0.80 LU/cm3. Medium composition was changed until it was possible to produce 25 g/dm3 dry weight of cells. Evidence of linear growth due to nutient, limitations was obtained. A microcomputer controlled protocol was used successfully to feed glucose solutions at exponentially increasing rates. Problems were encountered with the production of lipase from the interaction with pyocyanin production and from possible trace nutrient limitations.
Open Access
Hydrophobicity in polysaccharide gelation
(Cranfield University, 1992-11) Jones, Amanda Kay; Morris, E. R.
The role of hydrophobic substituents on the gelation mechanism of highly esterified pectin and the cellulose derivatives methylcellulose and hydroxypropylmethylcellulose (HPMC) has been explored by monitoring the behaviour of the amphiphilic polysaccharides in varying combinations of an ethylene glycoVwater solvent. The gelling ability (mechanical spectroscopy, visual observation) of very highly esterified (- 100%) pectin in high concentrations of ethylene glycol (>60%) is greatly reduced, however, the polymer still undergoes conformational ordering (CD, OR). A model for gel formation involving a two stage process has been proposed, comprising adoption of the ordered structure stabilised by hydrogen bonding between OH groups of contiguous polysaccharide chains, followed by (or coincident with) aggregation of the ordered sequences by 'hydrophobic' clustering of the fundamental structural subunits to form the three dimensional gel network. It has been found that ethylene glycol promotes the fIrst stage (ordering) but is antagonistic to the second (aggregation). The reversibility (mechanical spectroscopy) of the thermo-gelling cellulose derivatives can be largely abolished in the presence of ethylene glycol (40% for methylcellulose, 10% for HPMC) , attributed to solubilisation of the proposed ordered 'bundle' structure at low temperatures removing the enthalpic advantage (DSC) of gel melting. The increased sensitivity of HPMC to modification of the solvent environment is due to the presence of the polar hydroxypropyl substituent causing an inceptive destabilisation of the 'bundle' structure. It is suggested that gelation is driven by the entropic advantage of melting-out 'cages' of structured water surrounding the hydrophobic groups giving rise to intermolecular 'hydrophobic' aggregation.
Open Access
Operational characteristics of rotor/stator mixers
(Cranfield University, 1993-08) Sparks, T.; Brown, D. E.
Fast chemical reactors require rapid mixing, and literature shows that rotor/stator mixers can provide this. Results have been published, at small scale, showing rotor/stator mixers to be promising as chemical reactors. An industrial size rotor/stator mixer has been procured and fitted into a test loop. Instrumentation was used to measure shaft torque, shaft speed, differential pressure and differential temperature. Measurements were used to produce an energy balance for the mixer that could be used to assess its mixing performance, based upon general descriptions of mixing. Residence time distribution (RTD) curves were obtained and it was shown that the mixer exhibited substantial backmixing. Modifications to the mixer are proposed that may produce a RTD that better approximates plug flow. It is concluded that if the mixer could be made to better approach plug flow, as mixing rates would be very high, use of such a device could improve the product quality from a competitive chemical reaction.
Open Access
Electrochemistry of metal complexes and their use in amperometric sensors
(Cranfield University, 1994-02) Somasundrum, Mithran; Bannister, J. V.
This thesis concerns the utilization of metal complexes in amperometric sensors. Chapter One provides a general introduction to the area. The electrochemical theories relating to the development and use of amperometric sensors, are described, and applications for such sensors are outlined. These include trace element analysis for environmental and clinical use and the determination of NADH for the detection of clinical analytes. In Chapter Two, the electrochemical changes occurring in a ligand upon complexation, are examined as a possible method of selective metal ion detection. Screen-printing is used to produce disposable, single-use electrodes modified with the ligand bis- cyclohexanone oxaldihydrazone. At +250 mV vs SCE, the electrodes give a linear response to copper(II) across the range 30-300 pM (r = 0.983, n= 13). The effect on the electrode response of variations in pH, temperature, ligand content and storage time are outlined; as well as the effect of competing cations. In Chapter Three, pre-formed metal complexes are used as electron-transfer mediators. Part I considers homogeneous mediation from the enzyme NADH oxidase, using the Ru(lII/IV) redox couple. A scheme for enzyme amplification of the NADH response is outlined using alcohol dehydrogenase (ADH) and NADH oxidase. Additionally, ethanol determination is performed using an ADH/NADH oxidase bilayer. In Part 11, mediator immobilisation is examined using a novel ion-exchange/hydrogel composite (Nf/PVA). The structure of the composite is investigated by following the diffusional characteristics of both hydrophobic and hydrophilic mediators, incorporated within the film. An analytical application of the Nf(PVA layer is illustrated following the co-immobilisation of a mediator with glucose oxidase. The effect of protein adsorption onto the composite is also examined. Part HI of Chapter Three considers a possible alternative to mediated electrocatalysis, by using an electro- deposited film of poly(indole-5-carboxylic acid) (PICA). The overpotential. for the oxidation of ascorbate and NADH is lowered, apparently without the action of a redox mediating species. Strategies for the development of a PICA-based biosensor are outlined. Chapter Four provides an overview and general discussion of the experimental results and suggests areas for further work. These include further improvements to the design of the screen-printed electrodes in Chapter 2; the preferable choice of mediator for the immobilisation matrix in Chapter 3 Part 11, as well as possible methods of improving the biocompatability of the matrix; and a possible route to the immobilisation of NAD,, for the PICA-based system described in Chapter 3 Part III.
Open Access
Tertiary nitrification of wastewater in trickling filters
(Cranfield University, 1994-10) Biddle, Jeremy R.; Wheatley, A. D.; Brown, D. E.
This study was designed to investigate, at laboratory and pilot scale, the effects of various loading and climatic factors on the nitrification perfon-nance of four media, and to undertake a comparative assessment of the media. The media used were blast furnace slag and three random plastic media: Flocor RS, Etapak 160 and a new medium, Etapak 210. Laboratory experiments using pure cultures of Nitrosomonas europaea have determined the effect on nitrification of temperature, pH and substrate, BOD and inhibitor concentrations. Optimal values have been resolved for the temperature and pH and half- saturation constants for the substrate and inhibition are calculated for pure culture conditions. The presence of glucose and glutamic acid has been shown to have a beneficial effect on nitrification, although this observation could not be adequately explained. Pilot scale research, conducted over a two year period, has provided an accurate representation of a nitrifying trickling filter by using effluent from Cranfield STW in filters exposed to the full climadc variation. Data obtained from these filters have indicated the superior performance of the blast furnace slag media due mostly to its ability to maintain a large active bacterial Population without excessive accumulation. Results have been used to support or challenge previous publications, and to generate a set of desion curves. Conclusions from this research have been used in the design of a full scale nitrifying trickling filter which has been constructed at Cranfield STW, early data from which are presented.
Open Access
Quinoprotein dehydrogenase and pyrroloquinoline quinone modified enzyme electrodes
(Cranfield University, 1995-05) Loughran, Michael Gerard; Turner, Anthony P. F.; Hall, J. M.
This thesis concerns the use of the coenzyme PQQ and quinoprotein dehydrogenase enzymes for the development of enzyme electrodes. A general introduction to the area which describes the properties of quinoprotein dehydrogenases is given. The reactivity and redox properties of the quinone cofactor, PQQ, are also outlined. Cont/d.
Open Access
Fluid mixing in rotor/stator mixers
(Cranfield University, 1996-12) Sparks, T.; Brown, D. E.
An industrial rotor-stator mixer was fitted into a flow loop to carry out overall power balance, flow visualisation and residence time distribution experiments. These were performed on various rotor-stator geometries and a half-scale unit. The overall power measurements showed that a large amount of power was given to the fluid by the rotor and estimates of the local turbulent energy dissipation rate per unit mass, e, were made using these data. It was found that the pumping efficiency of rotor- stator mixers is - 10 to 20% and an expression for the motor power (when the flow rate is controlled) was found. The flow pattern was characterised by a high tangential velocity in the rotor followed by an abrupt transformation to radial flow through the stator. It was suggested that the kinetic energy of the fluid in the rotor is transformed to pumping, ftiction and turbulence in the stator and that this is the region of greatest importance for mixing. The residence time distribution is characterised by a region of plug flow in series with a region of mixed flow. The flow through the volute has a dominant effect on the overall residence time distribution and the RTD is insensitive to operating condition (flow rate, rotor speed) or geometry. The knowledge gained from the above experiments was used to design diazo-coupling experiments (a mixing-sensitive competitive chemical reaction with well known kinetics) such that they gave qualitative information (e. g. best feed position) and quantitative information (e. g. turbulent energy dissipation rate) on the performance of a rotor-stator mixer. e was found to be proportional to the power given the fluid by the rotor and estimates for e of order 500 W kg-' were made using a micro-mixing model.
Open Access
Fluorescence-based optical biosensors for clinical and environmental applications
(Cranfield University, 1996-12) Psoma, Sotiria D.; Turner, Anthony P. F.
The aim of this thesis was to investigate the feasibility of simultaneous utilisation of pH and oxygen-dependent fluorescent indicators for the development of a novel fibre-optical fluorescence-based bio sensor. This approach would be used to measure simultaneously changes in the two indicator species generated by a single enzyme-catalysed reaction in response to one analyte where both the indicators and the enzyme are immobilised in the same sol-gel matrix, and to offer more accurate and reliable results using this portable optical biosensor in the clinical and environmental fields. HPTS (1-hydroxypyrene-3,6,8-trisulfonic acid) and tris(2,2'-bipyridyl)ruthenium(II) chloride hexahydrate, respectively, were used as the target fluorescent indicators; these two indicators had no cross sensitivity separate or in the same solution and well-separated emission bands at 510 nm and 610 mn, respectively. The catalytic oxidation of glucose by the enzyme glucose oxidase was initially investigated using the two indicators, and subsequently the same principle was applied in other biocatalysed oxidations such as of lactate, xanthine and phenol. Substrate concentration was assessed by simultaneously measuring two parameters: oxygen consumption, through the reduction of the fluorescence intensity of tris(2,2'-bipyridyl) ruthenium(II) chloride hexahydrate; and the production of acid, through pH changes affecting the fluorescence intensity of HPTS.A thorough spectroscopic study of the enzymatic oxidation of glucose was performed using glucose oxidase in solution in a cuvette, in the presence of both indicators. A number of combinations of wavelengths of the indicators for excitation and fluorescence were utilised in order to establish calibration curves with the optimum performance for glucose detection in the diabetic range. Similarly results were taken from the kinetic studies of lactate oxidase, xanthine oxidase and polyphenol oxidase for the detection of lactate and xanthine in blood and phenol in water at ppb-levels, using the above principle. The application and characterisation of immobilisation techniques for the fluorescence-based blood-glucose b iosenor were carried out. The advantages of the microcapsulation sol-gel method over conventional immobilisation techniques for application in an optical biosensor, were elucidated and this immobilisation technique was implemented for glucose and phenol detection. Finally, additional solution studies were conducted and used to evaluate the implementation and performance of the above method when used for the detection and measurement of glucose concentration in biological samples such as human serum.
Open Access
Bioremediation of the pesticides Dieldrin, Simazine, Trifluralin using tropical and temperate white-rot fungi
(Cranfield University, 1997-08) Elyassi, Ali; Magan, Naresh
The natural breakdown of three pesticides on the UK Red Lis~ dieldrin~ simazine and trifluralin in water and soil varied with environmental conditions. In both sterile and unsterile water trifluralin was degraded to some extent at 20 and 30°C. In contras~ dieldrin and simazine were stable over the 42 days incubation period. A gradient HPLC method was developed for the simultaneous quantification of the three pesticides in soil. In field capacity soil mixtures of the three pesticides (5 and 10 ppm) showed a similar stability with limited degradation at 20°C but increased rates of degradation at 30°C. At the higher concentration the pesticides naturally degraded at a slower rate. Simazine and trifluralin degradation was significantly enhanced with increasing temperature from 20 to 30°C. Water potential (field capacity~ -0.065 MPa~ and - 0.28 MPa) had little effect on the natural breakdown rate of dieldrin. Simazine showed a greater breakdown in the mid-wetness soil~ while trifluralin was degraded rapidly in the field capacity soil, but not at all in the driest treatment over the 70 day experimental period. In vitro studies on solid agar media overlayed with cellophane showed that of four fungi examined~ Trametes cingulata, Trametes socotrana (tropical species) and Phanerochaete chrysosporium and Polystictus versicolor (temperate species) all except P.chrysosporium were able to grow in the presence of 5 ppm of any of the three pesticides at 20 and 30°C, with the latter only growing at 30°C. At 10 ppm concentration P. chrysosporium did not grow, regardless of temperature or time of incubation (up to 56 days). HPLC was used to quantify the temporal rates of degradation in the solid agar media and this showed that P. versicolor and T. socotrana were very effective at breaking down the three pesticides, at 20 and 30°C. The chosen fungi were grown on chopped straw as a carrier and incorporated into soil microcosms in the ratio of 1:10 containing mixtures of the three pesticides (5, 10 ppm) at 20 and 30°C, and subsequently under different water potential regimes at 20°C only, over periods of 70 days. P. versicolor alone significantly increased breakdown of 5 ppm dieldrin by 26% over untreated controls, while simazine breakdown was increased by 16%. However, for simazine at 30°C there was no difference between temporal rates of natural breakdown and those containing fungal inocula, regardless of concentration. With 5 ppm trifluralin, a maximum breakdown in untreated soil was 67% after 70 days. By contras~ this pesticide was undetectable after 28 days in the presence of the inoculant P . versicolor. This increased to 42 days where a mixture of the two fungi were used. Generally the mixture of fungi used in this study were not as effective in bioremediation of these pesticides as a single species. Field capacity soil appeared to be the best condition for P. versicolor to degrade dieldrin and trifluralin added at 10 ppm. However, for simazine this occurred in the driest water potential (-0.28 MPa) used.
Open Access
Studies on production and ecophysiology of fungal antagonists for improved biological control of Fusarium oxysporum f.sp. Iycopersici and Monilinia laxa.
(Cranfield University, 1998-02) Lopez, Susana Pascual; Magan, Naresh; Melgarejo, P. (supervisor)
This work investigated the production of the biocontrol agents: Penicillium oxalicum to control Fusarium oxysporum f.sp. Iycopersici in the rhizosphere; and Epicoccum nigrum and Penicillium frequentans to control Monilinia laxa in the phyllosphere. Ecophysiological studies were carried out to categorise different inoculum types, and to test their biocontrol efficacy. A method was developed for the induction of submerged conidiation of P.oxalicum for the first time. This was achieved by transferring 24 h cultures to a nitrogen free medium, and further stimulated by a high C:N ratio with 20 mM calcium. Optimum submerged conidial concentrations obtained were 35x10 6 spores mr1. The hydrophobicity of submerged and aerial conidia of P.oxalicum was similar. However, different results were obtained depending on the method used. Appearance of both spore types under the SEM was similar in size and shape. However, submerged spores were covered by a filamentous matrix, absent in aerial ones. Viability of aerial and submerged P.oxalicum spores was higher than 80% after 27 weeks, when stored fresh at either 4 or 25°C, but aerial spores survived slightly better. Freeze-drying severely affected viability, especially of submerged spores. Aerial spores effectively colonised sterile soil in tomato seedbeds with water potentials in the range 1-7 (-MPa), and this was further favoured by the addition of nutrients. Aerial conidia of P.oxalicum applied to seedbeds were able to significantly (P<0.05) reduce Fusarium wilt of tomato at concentrations as low as 6x10 4 spores mr 1 substratum, indicating that the amount of the antagonist needed is not a limiting factor for the practical application of this antagoniSt. Aerial spores were slightly more effective than submerged ones in the control of the disease. However, mycelium was ineffective. Coating of tomato seeds with formulations of aerial spores of P.oxalicum in alginate or methyl cellulose significantly (P<0.05) enhanced the growth promotion effect of the antagonist in vitro, which may be related to the ability of the fungus to control the disease. E.nigrum spores were produced by solid fermentation on wheat grains at different water activities (aw). Maximum levels of sporulation (7-11x10 6 spores g-1 grain) were obtained at high aw (0.996) or reduced aw (0.98) adjusted with a mixture glycerol/water. E.nigrum and P.frequentans were both produced in culture medium at reduced aw, to improve their ecological competence in the phyllosphere and therefore their biocontrol ability. E.nigrum produced at reduced aw showed improved germ tube extension and in some cases colony growth rate when placed on medium at reduced aw, showing water stress tolerance of such modified inocula. Furthermore, such inocula showed an enhanced ability to compete with the pathogen, M.laxa, at reduced aw, shown by a higher Niche Overlap Index (the proportion of the carbon compounds utilised by M.laxa that were also utilised by E.nigrum). E.nigrum spores produced at reduced aw had improved survival when stored fresh at 4 or 25°C. Freeze-drying severely affected the viability of both spore types (produced at high or reduced aw). E.nigrum and P.frequentans produced at reduced aw accumulated low molecular weight polyols as compatible solutes. Improvement of biocontrol of peach twig blight was obtained in the case of E.nigrum, which is more sensitive to conditions of low water availability than the xerotolerant P.frequentans. However, both fungi accumulated glycerol as the main compatible solute, indicating that different accumulation mechanisms may be responsible for the different tolerance to low water availability. Glycerol was also the main compatible solute in E.nigrum spores produced by solid fermentation at reduced aw. E.nigrum produced at high or reduced aw was also able to control brown rot of cherries, under optimum conditions for the development of the disease. The results presented in this work show that the conditions during the production of biocontrol agents are critical in determining their efficacy. Therefore, when developing mass-production systems it is necessary to aim not only for high propagule numbers but also for inoculum quality, defined by parameters such as ecological competence or survival during storage.
Open Access
Molecular Engineering of the Biosensor Interface.
(Cranfield University, 1999-10) Weston , D. G.; Cullen, David C.
The research described in this thesis concerns the investigation of technologies for the molecular engineering of the biosensor interface. Two avenues of investigation have been explored: the use of polymer matrices to modify the properties and functions of optical biosensor interfaces and the conjugation of photochromic dyes to protein systems to achieve photomodulation of protein function for biosensor applications. A comparison of the industry standard polymer, carboxymethyl dextran (CMD) was made against carboxymethyl cellulose and mixed systems, including a novel synthetic polymer, carboxylated polynoxylin. While CMD was found to provide the highest surface loading of protein, mixed polymers demonstrated the ability to allow prediction of surface loading, and showed features such as improved resistance to biological degradation. A novel method of depositing interfaces was investigated, allied to a study of liquid handling methods. A system was developed that allowed a printed heterogeneous array to be produced which showed preferential binding of specific analytes to defined areas of the sensor, whilst the other printed arrays retaining a high degree of non-specific interaction. The use of photochromic dyes to modulate protein function was applied to glucose oxidase and horseradish peroxidase. From the initial results, a hypothesis regarding the mechanism of photomodulation and its effect concerning the molecular weight of the conjugated protein was proposed. This was examined by the photomodulation of members of the peroxidase super family, antibodies and Fab fragments. From these results, the hypothesis was proved to be correct but incomplete, and was modified to include the disruption of the hydration shell around the protein caused by photochromic switching. Further research directly related to these experiments, and in novel fields of investigation have been proposed.
Open Access
The development of a solid-phase fermentation system for the production of pharmaceutically useful secondary metabolites in fungi
(Cranfield University, 2000-05) Aldred, David; Magan, Naresh; Penn, J. (external supervisor); Martin, S. (external supervisor)
A Phoma species, producing a squalestatin (Si) was grown on agar media derived from wheat, oats, oil seed rape and malt extract over a range of water availability values corresponding to water activity (aa) levels of. 0.998,0.995,0.990,0.980 and 0.960. Growth of the organism was not significantly affected by aw, except at the lowest value, but production of SI was significantly enhanced at intermediate aW in the range 0.990 - 0.980. For example, at 25°C and 0.98 aw wheat extract produced a l Ox increase in titre compared to 0.998 aw at the same temperature. Wheat extract was shown to be the best substrate for SI production. For example, at 25°C and 0.98 aW, this substrate produced 2x, 5x and 8x increases in titres compared to oat, malt and oil seed rape extracts respectively. A range of raw and processed agricultural products, including those used as extracts with the Phoma species, as well as maize, rice, soya, wheat flakes, bulgar wheat, couscous and "shredded wheat", were selected as candidate materials for solid substrate fermentation (SSF). Moisture sorption isotherms were prepared for each of these so that aW could be accurately set in experimental work. Small scale fermentations (40 cm3 wet substrate volume) were carried out with these materials and the fungi Epicoccum nigrum, Sarophorum palmicola, Drechslera dematioidea and Corynespora cassiicola over the aw range 0.998 - 0.970. Studies with E. nigrum in particular produced a range of unique metabolites at low aw, and other metabolites where titres were increased by as much as 20x compared to high aW conditions. The optimum aw level for metabolite production in this fungus appeared to be in the range 0.990-0.980. Ultimately, E. nigrum was chosen as the model fungus and bulgar wheat as the model substrate, with 3 key target metabolites being followed (metabolites 1,2 and 3). A series of scale-up studies (40 cm3-3 litres wet volume) were carried out utilising the model system. These studies typically produced reasonable levels of metabolites, but were subject to problematic water and heat accumulation, and bacterial contamination. These were identified as critical parameters. A system was ultimately developed around a Bioengineering AG submerged liquid fermenter, modified for use with solid substrates, and incorporating forced aeration and mechanical agitation. This apparatus gave encouraging levels of metabolites, producing most of these rapidly and uniformly, and showed good critical parameter control. The overall scale-up achieved in the final fermenter studies was 75x, in terms of wet substrate volume. Increased titres were achieved for all three target metabolites compared to small-scale studies with the same substrate. These increases were approx. 17x for metabolite 1, approx. 3x for metabolite 3, while metabolite 2 was absent from small scale studies at the relevant aW level.
Unknown
Effect of cosolutes on polysaccharides gelation.
(Cranfield University, 2001-06) Tsoga, Areti K.; Kasapis, S.; Morris, E. R.
In the first stage of the investigation, the effect of high levels of sugars (mixture of 50% sucrose with 351/'o glucose syrup) on agarose (0.7 w-L °'o) was characterized by low amplitude oscillatory measurements of storage modulus (G'), loss modulus (G") and loss tangent (tan 6" as well as large deformation techniques. Samples were prepared at 90°:, and measured immediately, or after Storage at 5°C. The combined Williams-T andel-Ferry W F; /free volume theory was used to derive the glass transition temperature, the fractional free volume, and the thermal expansion coefficierit of the glass. Solution of high concentrations of sucrose Crystallizes, but addition of the polymer encourages Bitei- olecular interactions, which transfoý the mixture into a high v iscosi Ly glass. The mechanical properties of glucose syrup follow ýýý. iý behavior in the glass transition region and revert to an Arrhenius type prediction in the CrIa-SSy State. feasurements on sugar Samples and aýarose-Sugar ii 1x üres were resolved into a bas iiriCuuu Gi temperature alone and a basic function of equency (time) alone. T ie former traces the energetic cost of vitrification, which increases sharply with decreasing tempera ure. The layer, at long time scales, is goes erned by the mtinite molecular weight of the äcräiGSZ ne ork. In the region of S1 O1 tImes, tie effect of Ze vole ie is active rega dless of In a continuation of, investigating the significance of polymer-cosolute interactions, the effect Gi sucrose, glucose, fructose, sorbitol, hylitol, glycerol and ethan-1,2-dial on gelation of high methoxy pectin was studied under different experimental conditions. The main changes in procedure in comparison with the work on agarose were: (i) the polymer concentration was increased from 0.7 to 1.0 wt °'o, (ii) the mixtures prepared at pH AT and subsequently acidified to pH 3, rather than beinte prepared at neutral pH, (i11) the cosolute concentration was varying from 50 to 65 w IL °'o and (vi) the mixtures were studied through rheology, calorimetry and optical rotation. The samples were prepared at 95°C and changes in storage modulus (G') and loss modulus (G") during cooling to 5°C, heating to 900 and re-cooling to 5°C, at 1°C/min, were measured at 1 rad s"1 and 0.5% strain. In all cases, the onset temperature for gelation during cooling and the moduli recorded at 5'C increased With increasing concentration of cosolute. However, both values were substantially lower for the liquid cosolutes than for mixtures with solid cosolutes at the same concentrations. The difference is attributed to inhibition of pectin-pectin interactions by pectin-cosolute interactions, which in turn are inhibited by cosolute-cosolute interactions. On heating there was an initial reduction in modulus, with the same temperature-course as the increase on cooling; for the solids, this was followed by an increase attributable to hydrophobic association of methyl ester substituents. No SUCH increase was seen with the liquid cosolutes, but DSC studies showed two reversible I. thermal ti ansitions in all cases, one over the temperature-range of the initial gelation process ý nfý r 'A f+ .ýýn on cooling and t he oLlleICoincidenf t with the increase in modulus ulon hea ing in the presence o1- ý solid COSO1UteS. The absence of any detectable increase in modulus on heating with the liquid COSOtUteS is a trlbuted to accumulation of cosolute around tiie polymer chains promoting hydrophobic association between methyl ester groups on the same Chain; Or within Cl sters o chains, v with, therefore no contribution to network strucLarz. At high concentrations of the solid cosolutes, the increase in modulus on heating was followed by a decrease at higher ýempei atur e; this -was attributed to excessive aggregation, and was reflected in lower moduli on subsequent re-coolIný to 5'C, in coast to the enhancement in gel strength after heating aril cooling observed at lower concentration of the same COSül teS. in the presence of fructose as cosolute, calorimetric studies showed an intense endotherm follovýied immediately by an intense exotherm on hea ng. These transitions occurred over approximately the same teMperat'ure-rande as initial gelation on cooling and increased in mag liLude with increasing concentration of the sugar. The displacement of both transitions to progressively hid her temperature as the rate of heating was increased was much Greater than anticipated from a simple thermal lag, indicating that the undelying structural chancres are slow. The proposed interpretation is that fructose is capable of site-binding to pectin in both the ordered and disordered state
Unknown
Biodesulphurisation of coal
(Cranfield University, 2001-07) Prayuenyong, P.; Tothill, Ibtisam E.
The emission of sulphur oxides during the combustion of coal is one of the causes of an environmental problem known as acid rain. Biodesulphurisation technology applied as a method to remove sulphur before coal combustion was investigated in this work. The desulphurisation abilities of three specific bacterial strains including Rhodococcus erythropolis IGTS8, R. erythropolis X309 and Shewanella putrefaciens strain NCIMB 8768 have been evaluated. R. erythropolis IGTS8 and X309 were found to be able to remove both inorganic and organic sulphur from model compounds and coal samples. Their abilities to remove sulphur from benzothiophene were observed for the first time. A novel desulphurising bacterium, S. putrefaciens was also found to be able to remove inorganic and organic sulphur from coal samples. The bacterium, however, lost its ability to remove organic sulphur from model compounds during the investigation. R. erythropolis IGTS8 presented the greatest desulphurisation efficiency among the three bacterial strains. Nevertheless, the desulphurisation activity of R. erythropolis IGTS8 was too low for an economical coal biodesulphurisation process as it removed only 32.0% of total sulphur in bituminous coal, and 21.1% of total sulphur in anthracite coal. Alternatively, coal biodesulphurisation can be carried out in inexpensive conditions by using the bacteria inherent in the coal itself. The type of coal has an important effect on desulphurisation efficiency since the sulphur reduction in bituminous coal, which is in a lower rank than anthracite, was greater than the sulphur reduction in anthracite coal. This work also developed and evaluated the analytical methods used in the field. A HPLC method was developed to detect the desulphurisation metabolites of model compounds. The techniques for measuring sulphur in coal were improved.
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The study of molecular markers for the progression of Barrett's Oesophagus to adenocarcinoma to identify markers that can be used as diagnostic tools.
(Cranfield University, 2002-05) Cadd, Verity Anne; Warner, P.; Barr, H.; Shepherd, N. A.
Barrett's oesophagus is a complication of gastro-oesophageal reflux disease and is the single most important predisposing factor for the development of adenocarcinoma of the oesophagus. New molecular markers are needed for early diagnosis and to monitor disease progression. Telomerase is a ribonuclear protein with reverse transcriptase activity, which uses its own RNA component as a template for the addition of telomeric repeats to the end of chromosomes. Telomerase activity has been studied during the neoplastic progression of Barrett's oesophagus using a TRAP based ELISA technique, which found telomerase to be reactivated early during . disease progression. A non-isotopic method of in situ hybridisation for the detection of the RNA component of telomerase has also been successfully developed. Plasminogen activation is an inducible extracellular proteolytic system involved in the regulation of cellular interactions and invasion. The components of the urokinase-type Plasminogen Activator system have been fully investigated during the progression of Barrett's oesophagus to adenocarcinoma utilising immunohistochemistry and ELISA techniques. Changes in the expression of this invasive phenotype were found to occur late during disease progression in malignant tissues. Two-oesophageal cell-lines have been characterised using molecular biological techniques to detect a range of molecular markers to produce ex vivo models of oesophageal adenocarcinoma and oesophageal squamous cell carcinoma. In order to assess the effects of bile salts and acidity on oesophageal tissues these celllines were then utilised as ex vivo models. Exposure to acidic conditions both alone and with bile salts altered the morphological appearance of the cells and disrupted adhesion molecules in the cellular membrane. Investigations into both telomerase reactivation and the plasminogen activator system have provided new information concerning the nature and timing of molecular changes during the Barrett's metaplasia/dysplasia/ adenocarcinoma sequence.
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Natural antifungal systems for prevention of mould spoilage in bakery products
(Cranfield University, 2003-04) Arroyo, Mariona; Magan, Naresh
Growth of spoilage fungi in bread and other bakery products is currently controlled with the addition of weak acid preservatives. Consumers demand more natural products and thus there is a need to reduce the amount of chemical preservatives added to foods, or to identify alternative, more 'natural' antifungal compounds with strong biological activity. This thesis reports on several areas of research undertaken in the project: evaluation of sub-optimal concentrations of existing preservatives, under different environmental conditions, on growth and ochratoxin A (OT A) production by six bread spoilage moulds; efficacy of new/natural antifungal compounds for possible use in bread preservation; evaluation of the impact of preservation hurdles on ecophysiology of the spoilage fungi, including niche overlap; and mechanisms of action of preservatives on hydrolytic enzymes. It was found that the use of currently applied levels of the existing preservatives potassium sorbate, calcium propionate and sodium benzoate were effective, under low pH environments (pH 4.5) at completely controlling growth of spoilage moulds (Aspergillus ochraceus, Eurotium repens, Cladosporium herbarum, Penicillium corylophilum and Penicillium verrucosum) on wheat flour-based substrates over a period of 30 days. At higher pH levels, the efficacy decreased being almost nil at pH 7.5. The use of sub-optimal concentrations of weak acid preservatives led in most cases to reductions in lag times and/or stimulation of mould growth and ochratoxin A production by P. verrucosum strains. Cont/d.
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Identification and carcinogenicity of polycyclic aromatic hydrocarbons in transformer oil.
(Cranfield University, 2003-05) Pillai, Ilona G.; Saini, S.; Setford, S.; Heyward, R.
Insulating oils are rich in polycyclic aromatic hydrocarbons (PAHs) which act as inhibitors of oil breakdown, but are believed to be the main source of oil mutagenicity when converted to their epoxide form by mammalian enzymes. The current industry- recognised measurement of oil risk, the IP 346 method, measures total aromatic content and therefore cannot be directly related to PAH risk. This thesis describes efforts made to establish the contribution of PAH species alone to total oil mutagenicity. Cont/d.
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Ecology and control of Fusarium species and mycotoxins in wheat grain
(Cranfield University, 2003-08) Hope, Russell; Magan, Naresh
This study investigated the effect of interacting environmental factors on the ecophysiology of Fusarium culmorum and F. graminearum and mycotoxin production, and potential for controlling growth and deoxynivalenol (DON) and nivalenol (NIV) production using antioxidants and essential oils on wheat grain. Two- dimensional temporal profiles of growth and DON/NIV production for F. culmorum and F. graminearum were constructed for the first time and showed that the water activity (aw, ) ranges required for growth and DON/NIV production were different in vitro and on wheat grain. Growth occurred over a wider aW range (0.9-0.99) while DON/NIV production occurred over a significantly narrower range (0.95 - 0.99 a,,, ) at both 15 and 25°C over a 40 day incubation period. Interactions between aW, temperature, and sub-optimal concentrations of fungicides had a significant impact on growth and competitiveness of F. culmorum in interspecific interactions with 5-6 other wheat grain fungi. This had an impact on both colonisation capacity in vitro and in situ and affected DON/NIV production. Interactions were complex changing with environmental or fungicide stress resulting in significant stimulation or reduction of both fungal growth and mycotoxin production. An Index of Dominance was developed to compare interspecific interactions between F. culmorum and other wheat spoilage fungi and showed that competitiveness of F. culmorum increased as temperature and a,, level approached the optimum conditions for growth and DON/NIV production. Niche overlap indices (NOI) and niche maps were developed for the first time and showed that interspecific competitiveness was closely related to the number of nutritional carbon sources shared between spoilage fungi. Hydrolytic enzyme profiles for seven different enzymes by F. culmorum, other Fusaria and spoilage fungi showed that the most competitive species produced the highest specific activity of N-acetyl-ß-D-glucosaminidase. Potential for control of Fusarium species post-harvest using 23 essential oils and 8 antioxidants was investigated. Cinnamon, clove, bay, butylated hydroxyanisole and propyl paraben (500 µg g"1) were able to significantly reduce (>90%) the colonisation of wheat grain by three Fusarium species and DON/NIV production. Bay essential oil was the most effective at controlling total fungal populations and DONNIV in naturally contaminated wheat grain.