Bioreaction and separation in preparative batch chromatographic columns : the hydrology of lactose to yield glucose,galactose and oligosaccharides

The initial aim of this project was to improve the performance of a chromatographic bioreactor-separator (CBRS). In such a system, a dilute enzyme solution is pumped continuously through a preparative chromatographic column, while pulses of substrate are periodically injected on to the column. Enzymic reaction and separation are therefore performed in a single unit operation. The chromatographic columns used were jacketed glass columns ranging from 1 to 2 metres long with an internal diameter of 1.5 cm. Linking these columns allowed 1, 2, 3 and 4 metre long CBRS systems to be constructed. The hydrolysis of lactose in the presence of β~galactosidase was the reaction of study. From previous work at Aston University, there appeared to be no difficulties in achieving complete lactose hydrolysis in a CBRS. There did, however, appear to be scope for improving the separative performance, so this was adopted as an initial goal. Reducing the particle size of the stationary phase was identified as a way of achieving this improvement. A cation exchange resin was selected which had an average particle size of around half that previously used when studying this reaction. A CBRS system was developed which overcame the operational problems (such as high pressure drop development) associated with use of such a particle size. A significant improvement in separative power was achieved. This was shown by an increase in the number of theoretical plates (N) from about 500 to about 3000 for a 2 metre long CBRS, coupled with higher resolution. A simple experiment with the 1 metre column showed that combined bioreaction and separation was achievable in this system. Having improved the separative performance of the system, the factors affecting enzymic reaction in a CBRS were investigated; including pulse volume and the degree of mixing between enzyme and substrate. The progress of reaction in a CBRS was then studied. This information was related to the interaction of reaction and separation over the reaction zone. The effect of injecting a pulse over a length of time as in CBRS operation was simulated by fed batch experiments. These experiments were performed in parallel with normal batch experiments where the substrate is mixed almost instantly with the enzyme. The batch experiments enabled samples to be taken every minute and revealed that reaction is very rapid. The hydrodynamic characteristics of the two injector configurations used in CBRS construction were studied using Magnetic Resonance Imaging, combined with hydrodynamic calculations. During the optimisation studies, galactooligosaccharides (GOS) were detected as intermediates in the hydrolysis process. GOS are valuable products with potential and existing applications in food manufacture (as nutraceuticals), medicine and drug targeting. The focus of the research was therefore turned to GOS production. A means of controlling reaction to arrest break down of GOS was required. Raising temperature was identified as a possible means of achieving this within a CBRS. Studies were undertaken to optimise the yield of oligosaccharides, culminating in the design, construction and evaluation of a Dithermal Chromatographic Bioreactor-separator.

Increasing cell biomass in Saccharomyces cerevisiae increases recombinant protein yield:the use of a respiratory strain as a microbial cell factory

BACKGROUND: Recombinant protein production is universally employed as a solution to obtain the milligram to gram quantities of a given protein required for applications as diverse as structural genomics and biopharmaceutical manufacture. Yeast is a well-established recombinant host cell for these purposes. In this study we wanted to investigate whether our respiratory Saccharomyces cerevisiae strain, TM6*, could be used to enhance the productivity of recombinant proteins over that obtained from corresponding wild type, respiro-fermentative strains when cultured under the same laboratory conditions. RESULTS: Here we demonstrate at least a doubling in productivity over wild-type strains for three recombinant membrane proteins and one recombinant soluble protein produced in TM6* cells. In all cases, this was attributed to the improved biomass properties of the strain. The yield profile across the growth curve was also more stable than in a wild-type strain, and was not further improved by lowering culture temperatures. This has the added benefit that improved yields can be attained rapidly at the yeast's optimal growth conditions. Importantly, improved productivity could not be reproduced in wild-type strains by culturing them under glucose fed-batch conditions: despite having achieved very similar biomass yields to those achieved by TM6* cultures, the total volumetric yields were not concomitantly increased. Furthermore, the productivity of TM6* was unaffected by growing cultures in the presence of ethanol. These findings support the unique properties of TM6* as a microbial cell factory. CONCLUSIONS: The accumulation of biomass in yeast cell factories is not necessarily correlated with a proportional increase in the functional yield of the recombinant protein being produced. The respiratory S. cerevisiae strain reported here is therefore a useful addition to the matrix of production hosts currently available as its improved biomass properties do lead to increased volumetric yields without the need to resort to complex control or cultivation schemes. This is anticipated to be of particular value in the production of challenging targets such as membrane proteins.

The fractionation of dextran polymer by ultrafiltration to yield clinical products

A review of ultrafiltration (UF) theory and equipment has been made. Dextran is fractionated industrially by ethanol precipitation, which is a high energy intensive process. The aims of this work were to investigate the fractionation of dextran using UF and to compare the efficiency and costs of UF fractionation with ethanol fractionation. This work is the continuation of research conducted at Aston, which was concerned with the fractionation of dextran using gel permeation chromatography (GPC) and hollow fibre UF membranes supplied by Amicon Ltd. Initial laboratory work centred on determining the most efficient make and configuration of membrane. UF membranes of the Millipore cassette configuration, and the DDS flat-sheet configuration, were examined for the fracationation of low molecular weight (MW) dextran. When compared to Amicon membranes, these membranes were found to be inferior. DDS membranes of 25 000 and 50 000 MW cut-offs were shown to be capable of fractionating high MW dextran with the same efficiency as GPC. The Amicon membranes had an efficiency comparable to that of ethanol fractionation. To increase this efficiency a theoretical UF membrane cascade was adopted to utilize favourable characteristics encountered in batch mode membrane experiments. The four stage cascade used recycled permeates in a counter- current direction to retentate flow, and was operated 24 hours per day controlled by a computer. Using 5 000 MW cut-off membranes the cascade improved the batch efficiency by at least 10% for a fractionation at 6 000 MW. Economic comparisons of ethanol fractionation, combined GPC and UF fractionation, and UF fractionation of dextran were undertaken. On an economic basis GPC was the best method for high MW dextran fractionation. When compared with a plant producing 100 tonnes pa of clinical dextran, by ethanol fractionation, a combined GPC and UF cascade fractionation could produce savings on operating costs and an increased dextran yield of 5%.

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.

Acoustic emission and yield in sand

An experimental investigation into the Acoustic Emission (AE) response of sand has been undertaken, and the use of AE as a method of yield point identification has been assessed. Dense, saturated samples of sand were tested in conventional triaxial apparatus. The measurements of stresses and strains were carried out according to current research practice. The AE monitoring system was integrated with the soil mechanics equipment in such a way that sample disturbance was minimised. During monotonically loaded, constant cell pressure tests the total number of events recorded was found to increase at an increasing rate in a manner which may be approximated by a power law. The AE response of the sand was found to be both stress level and stress path dependent. Undrained constant cell pressure tests showed that, unlike drained tests, the AE event rate increased at an increasing rate; this was shown to correlate with the mean effective stress variation. The stress path dependence was most noticeable in extension tests, where the number of events recorded was an order of magnitude less than that recorded in comparable compression tests. This stress path dependence was shown to be due to the differences in the work done by the external stresses. In constant cell pressure tests containing unload/reload cycles it was found that yield could be identified from a discontinuity in the event rate/time curve which occurred during reloading. Further tests involving complex stress paths showed that AE was a useful method of yield point identification. Some tests involving large stress reversals were carried out, and AE identified the inverse yield points more distinctly than conventional methods of yield point identification.

Genetic and environmentally derived variation in the cell wall composition of miscanthus and implications for thermo-chemical conversion

Fifteen Miscanthus genotypes grown in five locations across Europe were analysed to investigate the influence of genetic and environmental factors on cell wall composition. Chemometric techniques combining near infrared reflectance spectroscopy and conventional chemical analyses were used to construct calibration models for determination of acid detergent lignin, acid detergent fibre, and neutral detergent fibre from sample spectra. The developed equations were shown to predict cell wall components with a good degree of accuracy and significant genetic and environmental variation was identified. The influence of nitrogen and potassium fertiliser on the dry matter yield and cell wall composition of M. x giganteus was investigated. A detrimental affect on feedstock quality was observed to result from application of these inputs which resulted in an overall reduction in concentrations of cell wall components and increased accumulation of ash within the biomass. Pyrolysis-gas chromatography-mass spectrometry and thermo-gravimetric analysis indicates that genotypes other than the commercially cultivated M. x giganteus have potential for use in energy conversion processes and in the bio-refining. The yields and quality parameters of the pyrolysis liquids produced from Miscanthus compared favourably with that produced from SRC willow and produced a more stable pyrolysis liquid with a higher lower heating value. Overall, genotype had a more significant effect on cell wall composition than environment. This indicates good potential for dissection of this trait by QTL analysis and also for plant breeding to produce new genotypes with improved feedstock characteristics for energy conversion.

Antifoam addition to shake flask cultures of recombinant Pichia pastoris increases yield

Pichia pastoris is a widely-used host for recombinant protein production. Initial screening for both suitable clones and optimum culture conditions is typically carried out in multi-well plates. This is followed by up-scaling either to shake-flasks or continuously stirred tank bioreactors. A particular problem in these formats is foaming, which is commonly prevented by the addition of chemical antifoaming agents. Intriguingly, antifoams are often added without prior consideration of their effect on the yeast cells, the protein product or the influence on downstream processes such as protein purification. In this study we characterised, for the first time, the effects of five commonly-used antifoaming agents on the total amount of recombinant green fluorescent protein (GFP) secreted from shake-flask cultures of this industrially-relevant yeast.

The intermediate pyrolysis and catalytic steam reforming of brewers spent grain

Brewers spent grain (BSG) is a widely available feedstock representing approximately 85% of the total by-products generated in the brewing industry. This is currently either disposed of to landfill or used as cattle feed due to its high protein content. BSG has received little or no attention as a potential energy resource, but increasing disposal costs and environmental constraints are now prompting the consideration of this. One possibility for the utilisation of BSG for energy is via intermediate pyrolysis to produce gases, vapours and chars. Intermediate pyrolysis is characterised by indirect heating in the absence of oxygen for short solids residence times of a few minutes, at temperatures of 350-450 °C. In the present work BSG has been characterised by chemical, proximate, ultimate and thermo-gravimetric analysis. Intermediate pyrolysis of BSG at 450 °C was carried out using a twin coaxial screw reactor known as Pyroformer to give yields of char 29%, 51% of bio-oil and 19% of permanent gases. The bio-oil liquid was found to separate in to an aqueous phase and organic phase. The organic phase contained viscous compounds that could age over time leading to solid tars that can present problems in CHP application. The quality of the pyrolysis vapour products before quenching can be upgraded to achieve much improved suitability as a fuel by downstream catalytic reforming. A Bench Scale batch pyrolysis reactor has then been used to pyrolyse small samples of BSG under a range of conditions of heating rate and temperature simulating the Pyroformer. A small catalytic reformer has been added downstream of the reactor in which the pyrolysis vapours can be further cracked and reformed. A commercial reforming nickel catalyst was used at 500, 750 and 850 °C at a space velocity about 10,000 L/h with and without the addition of steam. Results are presented for the properties of BSG, and the products of the pyrolysis process both with and without catalytic post-processing. Results indicate that catalytic reforming produced a significant increase in permanent gases mainly (H2 and CO) with H2 content exceeding 50 vol% at higher reforming temperatures. Bio-oil yield decreased significantly as reforming temperature increased with char remaining the same as pyrolysis condition remained unchanged. The process shows an increase in heating value for the product gas ranging between 10.8-25.2 MJ/m as reforming temperature increased. © 2012 Elsevier B.V. All rights reserved.

Econometrics of yield spreads in the money market:a note

The literature on bond markets and interest rates has focused largely on the term structure of interest rates, specifically, on the so-called expectations hypothesis. At the same time, little is known about the nature of the spread of the interest rates in the money market beyond the fact that such spreads are generally unstable. However, with the evolution of complex financial instruments, it has become imperative to identify the time series process that can help one accurately forecast such spreads into the future. This article explores the nature of the time series process underlying the spread between three-month and one-year US rates, and concludes that the movements in this spread over time is best captured by a GARCH(1,1) process. It also suggests the use of a relatively long term measure of interest rate volatility as an explanatory variable. This exercise has gained added importance in view of the revelation that GARCH based estimates of option prices consistently outperform the corresponding estimates based on the stylized Black-Scholes algorithm.

A shape-based decomposition of the yield adjustment term in the arbitrage-free Nelson and Siegel (AFNS) model of the yield curve

The appealing feature of the arbitrage-free Nelson-Siegel model of the yield curve is the ability to capture movements in the yield curve through readily interpretable shifts in its level, slope or curvature, all within a dynamic arbitrage-free framework. To ensure that the level, slope and curvature factors evolve so as not to admit arbitrage, the model introduces a yield-adjustment term. This paper shows how the yield-adjustment term can also be decomposed into the familiar level, slope and curvature elements plus some additional readily interpretable shape adjustments. This means that, even in an arbitrage-free setting, it continues to be possible to interpret movements in the yield curve in terms of level, slope and curvature influences. © 2014 © 2014 Taylor & Francis.

A generalized Drucker–Prager viscoplastic yield surface model for asphalt concrete

A generalized Drucker–Prager (GD–P) viscoplastic yield surface model was developed and validated for asphalt concrete. The GD–P model was formulated based on fabric tensor modified stresses to consider the material inherent anisotropy. A smooth and convex octahedral yield surface function was developed in the GD–P model to characterize the full range of the internal friction angles from 0° to 90°. In contrast, the existing Extended Drucker–Prager (ED–P) was demonstrated to be applicable only for a material that has an internal friction angle less than 22°. Laboratory tests were performed to evaluate the anisotropic effect and to validate the GD–P model. Results indicated that (1) the yield stresses of an isotropic yield surface model are greater in compression and less in extension than that of an anisotropic model, which can result in an under-prediction of the viscoplastic deformation; and (2) the yield stresses predicted by the GD–P model matched well with the experimental results of the octahedral shear strength tests at different normal and confining stresses. By contrast, the ED–P model over-predicted the octahedral yield stresses, which can lead to an under-prediction of the permanent deformation. In summary, the rutting depth of an asphalt pavement would be underestimated without considering anisotropy and convexity of the yield surface for asphalt concrete. The proposed GD–P model was demonstrated to be capable of overcoming these limitations of the existing yield surface models for the asphalt concrete.

Comparison of Multivariate and Univariate Models for Genetic Evaluation of Milk Yield based on Test Day Data

2000 Mathematics Subject Classification: 62H12, 62P99

Serum-free process development:improving the yield and consistency of human mesenchymal stromal cell production

Background aims: The cost-effective production of human mesenchymal stromal cells (hMSCs) for off-the-shelf and patient specific therapies will require an increasing focus on improving product yield and driving manufacturing consistency. Methods: Bone marrow-derived hMSCs (BM-hMSCs) from two donors were expanded for 36 days in monolayer with medium supplemented with either fetal bovine serum (FBS) or PRIME-XV serum-free medium (SFM). Cells were assessed throughout culture for proliferation, mean cell diameter, colony-forming potential, osteogenic potential, gene expression and metabolites. Results: Expansion of BM-hMSCs in PRIME-XV SFM resulted in a significantly higher growth rate (P < 0.001) and increased consistency between donors compared with FBS-based culture. FBS-based culture showed an inter-batch production range of 0.9 and 5 days per dose compared with 0.5 and 0.6 days in SFM for each BM-hMSC donor line. The consistency between donors was also improved by the use of PRIME-XV SFM, with a production range of 0.9 days compared with 19.4 days in FBS-based culture. Mean cell diameter has also been demonstrated as a process metric for BM-hMSC growth rate and senescence through a correlation (R² = 0.8705) across all conditions. PRIME-XV SFM has also shown increased consistency in BM-hMSC characteristics such as per cell metabolite utilization, in vitro colony-forming potential and osteogenic potential despite the higher number of population doublings. Conclusions: We have increased the yield and consistency of BM-hMSC expansion between donors, demonstrating a level of control over the product, which has the potential to increase the cost-effectiveness and reduce the risk in these manufacturing processes.