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.

Bioreaction and separation in batch chromatographic columns

The objective of this work has been to study the behaviour and performance of a batch chromatographic column under simultaneous bioreaction and separation conditions for several carbohydrate feedstocks. Four bioreactions were chosen, namely the hydrolysis of sucrose to glucose and fructose using the enzyme invertase, the hydrolysis of inulin to fructose and glucose using inulinase, the hydrolysis of lactose to glucose and galactose using lactase and the isomerization of glucose to fructose using glucose isomerase. The chromatographic columns employed were jacketed glass columns ranging from 1 m to 2 m long and the internal diameter ranging from 0.97 cm to 1.97 cm. The stationary phase used was a cation exchange resin (PUROLITE PCR-833) in the Ca2+ form for the hydrolysis and the Mg2+ form for the isomerization reactions. The mobile phase used was a diluted enzyme solution which was continuously pumped through the chromatographic bed. The substrate was injected at the top of the bed as a pulse. The effect of the parameters pulse size, the amount of substrate solution introduced into the system corresponding to a percentage of the total empty column volume (% TECV), pulse concentration, eluent flowrate and the enzyme activity of the eluent were investigated. For the system sucrose-invertase complete conversions of substrate were achieved for pulse sizes and pulse concentrations of up to 20% TECV and 60% w/v, respectively. Products with purity above 90% were obtained. The enzyme consumption was 45% of the amount theoretically required to produce the same amount of product as in a conventional batch reactor. A value of 27 kg sucrose/m3 resin/h for the throughput of the system was achieved. The systematic investigation of the factors affecting the performance of the batch chromatographic bioreactor-separator was carried out by employing a factorial experimental procedure. The main factors affecting the performance of the system were the flowrate and enzyme activity. For the system inulin-inulinase total conversions were also obtained for pulses sizes of up to 20 % TECV and a pulse concentration of 10 % w/v. Fructose rich fractions with 100 % purity and representing up to 99.4 % of the total fructose generated were obtained with an enzyme consumption of 32 % of the amount theoretically required to produce the same amount of product in a conventional batch reactor. The hydrolysis of lactose by lactase was studied in the glass columns and also in an SCCR-S unit adapted for batch operation, in co-operation with Dr. Shieh, a fellow researcher in the Chemical Engineering and Applied Chemistry Department at Aston University. By operating at up to 30 % w/v lactose feed concentrations complete conversions were obtained and the purities of the products generated were above 90%. An enzyme consumption of 48 % of the amount theoretically required to produce the same amount of product in a conventional batch reactor was achieved. On working with the system glucose-glucose isomerase, which is a reversible reaction, the separation obtained with the stationary phase conditioned in the magnesium form was very poor although the conversion obtained was compatible with those for conventional batch reactors. By working with a mixed pulse of enzyme and substrate, up to 82.5 % of the fructose generated with a purity of 100 % was obtained. The mathematical modelling and computer simulation of the batch chromatographic bioreaction-separation has been performed on a personal computer. A finite difference method was used to solve the partial differential equations and the simulation results showed good agreement with the experimental results.

Mechanics of rolling continuously cast stock

A review of published literature was made to establish the fundamental aspects of rolling and allow an experimental programme to be planned. Simulated hot rolling tests, using pure lead as a model material, were performed on a laboratory mill to obtain data on load and torque when rolling square section stock. Billet metallurgy and consolidation of representative defects was studied when modelling the rolling of continuously cast square stock with a view to determining optimal reduction schedules that would result in a product having properties to the high level found in fully wrought billets manufactured from large ingots. It is difficult to characterize sufficiently the complexity of the porous central region in a continuously cast billet for accurate modelling. However, holes drilled into a lead billet prior to rolling was found to be a good means of assessing central void consolidation in the laboratory. A rolling schedule of 30% (1.429:1) per pass to a total of 60% (2.5:1) will give a homogeneous, fully recrystallized product. To achieve central consolidation, a total reduction of approximately 70% (3.333:1) is necessary. At the reduction necessary to achieve consolidation, full recrystallization is assured. A theoretical analysis using a simplified variational principle with experimentally derived spread data has been developed for a homogeneous material. An upper bound analysis of a single, centrally situated void has been shown to give good predictions of void closure with reduction and the reduction required for void closure for initial void area fractions 0.45%. A limited number of tests in the works has indicated compliance with the results for void closure obtained in the laboratory.

Shaken not stirred:mixing semantics into XPDL

Ubiquitous computing requires lightweight approaches to coordinating tasks distributed across smart devices. We are currently developing a semantic workflow modelling approach that blends the proven robustness of XPDL with semantics to support proactive behaviour. We illustrate the potential of the model through an example based on mixing a dry martini.

Synthesis of hydrazine by the fixation process

A critical review of the literature concerning organic derivatives of hydrazine, the ammonia-chlorine reaction and the electrolytic formation of hydrazine has been carried out. Apparatus was constructed to study the electrolysis of liquid ammonia, the formation of chloramine and the fixation of chloramine with a ketone to form an isohydrazone. In the latter case the reaction was carried out in a 3" diameter stirred tank and also in a 1" diameter, 2' high column reactor where the liquid phase was continuously recirculated. Two methods of analysis of azines and isohydrazones in a ketone solution have been developed. One is a colorimetric technique using p-dimethylaminobenzaldehyde and the other involves the hydrolysis of the organic derivative to hydrazine sulphate. Hydrazine was detected in low concentration in some of the electrolytic experiments carried out but it was concluded that this method did not show sufficient promise to warrant further investigation. The gas phase formation of chloramine and acetone isohydrazone has also been studied but in this system difficulties were encountered with the chlorine jet blocking with ammonium chloride. The formation of isohydrazones in a stirred tank reactor has been investigated in some detail and the effect of several parameters was determined. The yield was found to be extremely sensitive to chlorine concentration and in order to obtain yields of more than 90 per cent, the molar concentration of chlorine in the gas phase had to be of the order of 5 per cent. An optimum temperature in the region of 0°C was also detected. These results disagree with those quoted in previous studies but extensive experimental work has confirmed the information presented in this thesis. It has also been shown that at high yields the chloramine formation reaction took place in the gas phase.

Modelling vortex formation in an unbaffled stirred tank reactors

Agitating liquids in unbaffled stirred tank leads to the formation of a vortex in the region of the impeller shaft when operating in the turbulent flow regime. A numerical model is presented here that captures such a vortex. The volume of fluid model, a multiphase flow model was employed in conjunction with a multiple reference frame model and the shear stress turbulence model. The dimensions of the tank considered here, were 0.585 m for the liquid depth and tank diameter with a 0.2925 m diameter impeller at a height of 0.2925 m. The impeller considered was an eight-bladed paddle type agitator that was rotating with an angular velocity of 7.54 rad s (72 rpm) giving a Reynolds number of 10 and Froude number of 0.043. Preliminary results of a second investigation into the effect of liquid phase properties on the vortex formed are also presented. © 2006 Elsevier B.V. All rights reserved.

Agitation conditions for the culture and detachment of hMSCs from microcarriers in multiple bioreactor platforms

In our recent work in different bioreactors up to 2.5L in scale, we have successfully cultured hMSCs using the minimum agitator speed required for complete microcarrier suspension, N JS. In addition, we also reported a scaleable protocol for the detachment from microcarriers in spinner flasks of hMSCs from two donors. The essence of the protocol is the use of a short period of intense agitation in the presence of enzymes such that the cells are detached; but once detachment is achieved, the cells are smaller than the Kolmogorov scale of turbulence and hence not damaged. Here, the same approach has been effective for culture at N JS and detachment in-situ in 15mL ambr™ bioreactors, 100mL spinner flasks and 250mL Dasgip bioreactors. In these experiments, cells from four different donors were used along with two types of microcarrier with and without surface coatings (two types), four different enzymes and three different growth media (with and without serum), a total of 22 different combinations. In all cases after detachment, the cells were shown to retain their desired quality attributes and were able to proliferate. This agitation strategy with respect to culture and harvest therefore offers a sound basis for a wide range of scales of operation.

Continuously measured 222Rn activity concentrations (daily means) in near surface air at Neumayer Station in the period 1995 through 2011

We report on continuously measured 222Rn activity concentrations in near-surface air at Neumayer Station in the period 1995-2011. This 17-year record showed no long-term trend and has overall mean ± standard deviation of (0.019 ± 0.012) Bq/m**3. A distinct and persistent seasonality could be distinguished with maximum values of (0.028 ± 0.013) Bq/m**3 from January to March and minimum values of (0.015 ± 0.009) Bq/m**3 from May to October. Elevated 222Rn activity concentrations were typically associated with air mass transport from the Antarctic Plateau. Our results do not support a relation between enhanced 222Rn activity concentrations at Neumayer and cyclonic activity or long-range transport from South America. The impact of oceanic 222Rn emissions could not be properly assessed but we tentatively identified regional sea ice extent (SIE) variability as a significant driver of the annual 222Rn cycle.