Enzymatic production of oligosaccharides in centrifugal fields

The aims of this work have been to identify an enzymatic reaction system suitable to investigate and develop the high-speed centrifuge as a novel reaction system for performing such reactions. The production of galacto-oligosaccharides by the trans-galactosyl activity of the enzyme β-galactosidase on lactose monohydrate was identified as a model enzymatic system to elucidate the principles of this type of process. Galacto-oligosaccharides have attracted considerable commercial interest as food additives which have been shown to be beneficial to the health of the human gastrointestinal tract. The development of a single unit operation capable of controlling the biosynthesis of galacto-oligosaccharides whilst simultaneously separating the enzyme from the reaction products would reduce downstream processing costs. This thesis shows for the first time that by using a combination of (a) immobilised or insolubilised β-galactosidase , (b) a rate-zonal centrifugation technique, and (c) various applied centrifugal fields, that a high-speed centrifuge could be used to control the formation of galacto-oligosaccharides whilst removing the enzyme from the reaction products. By layering a suspension of insolubilised β-galactosidase on top of a lactose monohydrate density gradient and centrifuging, the applied centrifugal fields generated produced sedimentation of the enzyme particles through the substrate. The higher sedimentation rate of the enzyme compared to those of the reaction products allowed for separation to take place. Complete sedimentation, or pelleting of the enzyme permits the possible recovery and re-use. Insolubilisation of the enzyme allowed it to be sedimented through the substrate gradient using much lower applied centrifugal fields than that required to sediment free soluble enzyme and this allowed for less expensive centrifugation equipment to be used. Using free soluble and insolubilised β-galactosidase stirred-batch reactions were performed to investigate the kinetics of lactose monohydrate hydrolysis and galacto-oligosaccharide formation. Based on these results a preliminary mathematical model based on Michaelis-Menten kinetics was produced. It was found that the enzyme insolubilisation process using a chemical cross-linking agent did not affect the process of galacto-oligosaccharide formation. Centrifugation experiments were performed and it was found that by varying the applied centrifugal fields that the yield of galacto-oligosaccharides could be controlled. The higher the applied centrifugal fields the lower the yield of galacto-oligosaccharides. By increasing the applied centrifugal fields the 'contact time' between the sedimenting enzyme and the substrate was reduced, which produced lower yields. A novel technique involving pulsing the insolubilised enzyme through the substrate gradient was developed and this was found to produce higher yields of galacto-oligosaccharide compared to using a single enzyme loading equivalent to the total combined activity of the pulses. Comparison of the galacto-oligosaccharide yields between stirred-batch and centrifugation reactions showed that the applied centrifugal fields did not adversely affect the transgalactosyl activity of the insolubilised enzyme.

Development of a sediment yield model for South East Ghana in the regions around Lake Volta

Soil erosion is one of the most pressing issues facing developing countries. The need for soil erosion assessment is paramount as a successful and productive agricultural base is necessary for economic growth and stability. In Ghana, a country with an expanding population and high potential for economic growth, agriculture is an important resource; however, most of the crop production is restricted to low technology shifting cultivation agriculture. The high intensity seasonal rainfall coincides with the early growing period of many of the crops meaning that plots are very susceptible to erosion, especially on steep sided valleys in the region south of Lake Volta. This research investigated the processes of soil erosion by rainfall with the aim of producing a sediment yield model for a small semi-agricultural catchment in rural Ghana. Various types of modelling techniques were considered to discover those most applicable to the sub-tropical environment of Southern Ghana. Once an appropriate model had been developed and calibrated, the aim was to look at how to enable the scaling up of the model using sub-catchments to calculate sedimentation rates of Lake Volta. An experimental catchment was located in Ghana, south west of Lake Volta, where data on rainstorms and the associated streamflow, sediment loads and soil data (moisture content, classification and particle size distribution) was collected to calibrate the model. Additional data was obtained from the Soil Research Institute in Ghana to explore calibration of the Universal Soil Loss Equation (USLE, Wischmeier and Smith, 1978) for Ghanaian soils and environment. It was shown that the USLE could be successfully converted to provide meaningful soil loss estimates in the Ghanaian environment. However, due to experimental difficulties, the proposed theory and methodology of the sediment yield model could only be tested in principle. Future work may include validation of the model and subsequent scaling up to estimate sedimentation rates in Lake Volta.