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.

The production of the enzyme dextransucrase by batch and continuous fermentation techniques

A review of the literature of work carried out on dextransucrase production, purification, immobilization and reactions has been carried out. A brief review has also been made of the literature concerning general enzyme biotechnology and fermentation technology. Fed-batch fermentation of the bacteria Leuconostoc mesenteroides NRRL B512 (F) to produce dextransucrase has formed the major part of this research. Aerobic and anaerobic fermentations have been studied using a 16 litre New Brunswick fermenter which has a 3-12 litre working volume. The initial volume of broth used in the studies was 6 litres. The results of the fed-batch fermentations showed for the first time that yields of dextransucrase are much higher under the anaerobic conditions than during the aerobic fermentations. Dextransucrase containing 300-350 DSU/cm3 of enzyme activity has been obtained during the aerobic fermentations, while in the anaerobic fermentations, enzyme yields containing 450-500 DSU/cm3 have been obtained routinely. The type of yeast extract used in the fermentation medium has been found to have significant effects on enzyme yield. Of the different types studied, the Gistex Standard was found to be the type that favoured the highest enzyme production. Studies have also been carried out on the effect of agitation rate and antifoam on the enzyme production during the anaerobic experiments. Agitation rates of up to 600 rpm were found not to affect the enzyme yield, however, the presence of antifoam in the medium led to a significant reduction in enzyme activity (less than 300 DSU/cm3). Scale-up of the anaerobic fermentations has been performed at up to the 1000 litre level with enzyme yields containing more than 400 DSU/cm3 of activity being produced. Some of the enzyme produced at this scale was used for the first time to produce dextran on an industrial scale via the enzyme route, with up to 99% conversion of sucrose to dextran being obtained. An attempt has been made at continuous dextransucrase production. Cell washout was observed to occur at dilution rates of greater than 0.4 h-1. Dextransucrase containing up to 25 DSU/cm3/h has been produced continuously.

Batch and continuous fermentation methods for the production of dextransucrase

The available literature concerning dextransucrase and dextran production and purification has been reviewed along with the reaction mechanisms of the enzyme. A discussion of basic fermentation theory is included, together with a brief description of bioreactor hydrodynamics and general biotechnology. The various fermenters used in this research work are described in detail, along with the various experimental techniques employed. The micro-organism Leuconostoc mesenteroides NRRL B512 (F) secretes dextransucrase in the presence of an inducer, sucrose, this being the only known inducer of the enzyme. Dextransucrase is a growth related product and a series of fed-batch fermentations have been carried out to extend the exponential growth phase of the organism. These experiments were carried out in a number of different sized vessels, ranging in size from 2.5 to 1,000 litres. Using a 16 litre vessel, dextransucrase activities in excess of 450 DSU/cm3 (21.67 U/cm3) have been obtained under non-aerated conditions. It has also been possible to achieve 442 DSU/cm3 (21.28 U/cm3) using the 1,000 litre vessel, although this has not been done consistently. A 1 litre and a 2.5 litre vessel were used for the continuous fermentations of dextransucrase. The 2.5 litre vessel was a very sophisticated MBR MiniBioreactor and was used for the majority of continuous fermentations carried out. An enzyme activity of approximately 108 DSU/cm3 (5.20 U/cm3) was achieved at a dilution rate of 0.50 h-1, which corresponds to the maximum growth rate of the cells under the process conditions. A number of continuous fermentations were operated for prolonged periods of time, with experimental run-times of up to 389 h being recorded without any incidence of contamination. The phenomenon of enzyme enhancement on hold-up of up to 100% was also noted during these fermentations, with dextransucrase of activity 89.7 DSU/cm3 (4.32 U/cm3) being boosted to 155.7 DSU/cm3 (7.50 U/cm3) following 24 hours of hold-up. These findings support the recommendation of a second reactor being placed in series with the existing vessel.

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.

Longitudinal dispersion in spiral wound RO modules and its effect on the performance of batch mode RO operations

Operation of reverse osmosis (RO) in cyclic batch mode can in principle provide both high energy efficiency and high recovery. However, one factor that causes the performance to be less than ideal is longitudinal dispersion in the RO module. At the end of the batch pressurisation phase it is necessary to purge and then refill the module. During the purge and refill phases, dispersion causes undesirable mixing of concentrated brine with less concentrated feed water, therefore increasing the salt concentration and energy usage in the subsequent pressurisation phase of the cycle. In this study, we quantify the significance of dispersion through theory and experiment. We provide an analysis that relates the energy efficiency of the batch operation to the amount of dispersion. With the help of a model based on the analysis by Taylor, dispersion is quantified according to flow rate. The model is confirmed by experiments with two types of proprietary spiral wound RO modules, using sodium chloride (NaCl) solutions of concentration 1000 to 20,000 ppm. In practice the typical energy usage increases by 4% to 5.5% compared to the ideal case of zero dispersion.

Desalination of brackish water by a batch reverse osmosis desalink system for use with solar thermal energy

For remote, semi-arid areas, brackish groundwater (BW) desalination powered by solar energy may serve as the most technically and economically viable means to alleviate the water stresses. For such systems, high recovery ratio is desired because of the technical and economical difficulties of concentrate management. It has been demonstrated that the current, conventional solar reverse osmosis (RO) desalination can be improved by 40–200 times by eliminating unnecessary energy losses. In this work, a batch-RO system that can be powered by a thermal Rankine cycle has been developed. By directly recycling high pressure concentrates and by using a linkage connection to provide increasing feed pressures, the batch-RO has been shown to achieve a 70% saving in energy consumption compared to a continuous single-stage RO system. Theoretical investigations on the mass transfer phenomena, including dispersion and concentration polarization, have been carried out to complement and to guide experimental efforts. The performance evaluation of the batch-RO system, named DesaLink, has been based on extensive experimental tests performed upon it. Operating DesaLink using compressed air as power supply under laboratory conditions, a freshwater production of approximately 300 litres per day was recorded with a concentration of around 350 ppm, whilst the feed water had a concentration range of 2500–4500 ppm; the corresponding linkage efficiency was around 40%. In the computational aspect, simulation models have been developed and validated for each of the subsystems of DesaLink, upon which an integrated model has been realised for the whole system. The models, both the subsystem ones and the integrated one, have been demonstrated to predict accurately the system performance under specific operational conditions. A simulation case study has been performed using the developed model. Simulation results indicate that the system can be expected to achieve a water production of 200 m3 per year by using a widely available evacuated tube solar collector having an area of only 2 m2. This freshwater production would satisfy the drinking water needs of 163 habitants in the Rajasthan region, the area for which the case study was performed.

Concentration polarization model of spiral-wound membrane modules with application to batch-mode RO desalination of brackish water

Batch-mode reverse osmosis (batch-RO) operation is considered a promising desalination method due to its low energy requirement compared to other RO system arrangements. To improve and predict batch-RO performance, studies on concentration polarization (CP) are carried out. The Kimura-Sourirajan mass-transfer model is applied and validated by experimentation with two different spiral-wound RO elements. Explicit analytical Sherwood correlations are derived based on experimental results. For batch-RO operation, a new genetic algorithm method is developed to estimate the Sherwood correlation parameters, taking into account the effects of variation in operating parameters. Analytical procedures are presented, then the mass transfer coefficient models are developed for different operation processes, i.e., batch-RO and continuous RO. The CP related energy loss in batch-RO operation is quantified based on the resulting relationship between feed flow rates and mass transfer coefficients. It is found that CP increases energy consumption in batch-RO by about 25% compared to the ideal case in which CP is absent. For continuous RO process, the derived Sherwood correlation predicted CP accurately. In addition, we determined the optimum feed flow rate of our batch-RO system.

Parameter Identification of a Fed-Batch Cultivation of S. Cerevisiae using Genetic Algorithms

Fermentation processes as objects of modelling and high-quality control are characterized with interdependence and time-varying of process variables that lead to non-linear models with a very complex structure. This is why the conventional optimization methods cannot lead to a satisfied solution. As an alternative, genetic algorithms, like the stochastic global optimization method, can be applied to overcome these limitations. The application of genetic algorithms is a precondition for robustness and reaching of a global minimum that makes them eligible and more workable for parameter identification of fermentation models. Different types of genetic algorithms, namely simple, modified and multi-population ones, have been applied and compared for estimation of nonlinear dynamic model parameters of fed-batch cultivation of S. cerevisiae.

Element characteristic tolerance for semi-batch fixed bed biomass pyrolysis

Biomass pyrolysis to bio-oil is one of the promising sustainable fuels. In this work, relation between biomass feedstock element characteristic and crude bio-oil production yield and lower heating value was explored. The element characteristics considered in this study include moisture, ash, fix carbon, volatile matter, C, H, N, O, S, cellulose, hemicellulose, and lignin content. A semi-batch fixed bed reactor was used for biomass pyrolysis with heating rate of 30 °C/min from room temperature to 600 °C and the reactor was held at 600 °C for 1 h before cooling down. Constant nitrogen flow (1bar) was provided for anaerobic condition. Sago and Napier glass were used in the study to create different element characteristic of feedstock by altering mixing ratio. Comparison between each element characteristic to crude bio-oil yield and low heating value was conducted. The result suggested potential key element characteristic for pyrolysis and provide a platform to access the feedstock element acceptance range.

Recovery of glass fibre and carbon fibres from reinforced thermosets by batch pyrolysis and investigation of fibre re-using as reinforcement in LDPE matrix

A semi-batch pyrolysis process was used to recover samples carbon fibre and glass fibre from their respective wastes. The mechanical properties of the recovered fibres were tested and compared to those of virgin fibres, showing good retention of the fibre properties. The recovered fibres were then used to prepare new LDPE composite materials with commercial and laboratory-synthesized compatibilizers. Mild oxidation of the post-pyrolysis recovered fibres and the use of different compatibilizers gave significant improvements in the mechanical properties of the LDPE composites; however some of the manufactured composites made from recovered fibres had properties similar to those made from virgin fibres.

Algorithms for scheduling parallel batch processing machines with non-identical job ready times

This research is motivated by a practical application observed at a printed circuit board (PCB) manufacturing facility. After assembly, the PCBs (or jobs) are tested in environmental stress screening (ESS) chambers (or batch processing machines) to detect early failures. Several PCBs can be simultaneously tested as long as the total size of all the PCBs in the batch does not violate the chamber capacity. PCBs from different production lines arrive dynamically to a queue in front of a set of identical ESS chambers, where they are grouped into batches for testing. Each line delivers PCBs that vary in size and require different testing (or processing) times. Once a batch is formed, its processing time is the longest processing time among the PCBs in the batch, and its ready time is given by the PCB arriving last to the batch. ESS chambers are expensive and a bottleneck. Consequently, its makespan has to be minimized. ^ A mixed-integer formulation is proposed for the problem under study and compared to a formulation recently published. The proposed formulation is better in terms of the number of decision variables, linear constraints and run time. A procedure to compute the lower bound is proposed. For sparse problems (i.e. when job ready times are dispersed widely), the lower bounds are close to optimum. ^ The problem under study is NP-hard. Consequently, five heuristics, two metaheuristics (i.e. simulated annealing (SA) and greedy randomized adaptive search procedure (GRASP)), and a decomposition approach (i.e. column generation) are proposed—especially to solve problem instances which require prohibitively long run times when a commercial solver is used. Extensive experimental study was conducted to evaluate the different solution approaches based on the solution quality and run time. ^ The decomposition approach improved the lower bounds (or linear relaxation solution) of the mixed-integer formulation. At least one of the proposed heuristic outperforms the Modified Delay heuristic from the literature. For sparse problems, almost all the heuristics report a solution close to optimum. GRASP outperforms SA at a higher computational cost. The proposed approaches are viable to implement as the run time is very short. ^

Makespan minimization in a job shop with a batch processing machine

A job shop with one batch processing and several discrete machines is analyzed. Given a set of jobs, their process routes, processing requirements, and size, the objective is to schedule the jobs such that the makespan is minimized. The batch processing machine can process a batch of jobs as long as the machine capacity is not violated. The batch processing time is equal to the longest processing job in the batch. The problem under study can be represented as Jm:batch:Cmax. If no batches were formed, the scheduling problem under study reduces to the classical job shop scheduling problem (i.e. Jm:: Cmax), which is known to be NP-hard. This research extends the scheduling literature by combining Jm::Cmax with batch processing. The primary contributions are the mathematical formulation, a new network representation and several solution approaches. The problem under study is observed widely in metal working and other industries, but received limited or no attention due to its complexity. A novel network representation of the problem using disjunctive and conjunctive arcs, and a mathematical formulation are proposed to minimize the makespan. Besides that, several algorithms, like batch forming heuristics, dispatching rules, Modified Shifting Bottleneck, Tabu Search (TS) and Simulated Annealing (SA), were developed and implemented. An experimental study was conducted to evaluate the proposed heuristics, and the results were compared to those from a commercial solver (i.e., CPLEX). TS and SA, with the combination of MWKR-FF as the initial solution, gave the best solutions among all the heuristics proposed. Their results were close to CPLEX; and for some larger instances, with total operations greater than 225, they were competitive in terms of solution quality and runtime. For some larger problem instances, CPLEX was unable to report a feasible solution even after running for several hours. Between SA and the experimental study indicated that SA produced a better average Cmax for all instances. The solution approaches proposed will benefit practitioners to schedule a job shop (with both discrete and batch processing machines) more efficiently. The proposed solution approaches are easier to implement and requires short run times to solve large problem instances.

Minimizing makespan for hybrid flowshops with batch, discrete processing machines and arbitrary job sizes

This research aims at a study of the hybrid flow shop problem which has parallel batch-processing machines in one stage and discrete-processing machines in other stages to process jobs of arbitrary sizes. The objective is to minimize the makespan for a set of jobs. The problem is denoted as: FF: batch1,sj:Cmax. The problem is formulated as a mixed-integer linear program. The commercial solver, AMPL/CPLEX, is used to solve problem instances to their optimality. Experimental results show that AMPL/CPLEX requires considerable time to find the optimal solution for even a small size problem, i.e., a 6-job instance requires 2 hours in average. A bottleneck-first-decomposition heuristic (BFD) is proposed in this study to overcome the computational (time) problem encountered while using the commercial solver. The proposed BFD heuristic is inspired by the shifting bottleneck heuristic. It decomposes the entire problem into three sub-problems, and schedules the sub-problems one by one. The proposed BFD heuristic consists of four major steps: formulating sub-problems, prioritizing sub-problems, solving sub-problems and re-scheduling. For solving the sub-problems, two heuristic algorithms are proposed; one for scheduling a hybrid flow shop with discrete processing machines, and the other for scheduling parallel batching machines (single stage). Both consider job arrival and delivery times. An experiment design is conducted to evaluate the effectiveness of the proposed BFD, which is further evaluated against a set of common heuristics including a randomized greedy heuristic and five dispatching rules. The results show that the proposed BFD heuristic outperforms all these algorithms. To evaluate the quality of the heuristic solution, a procedure is developed to calculate a lower bound of makespan for the problem under study. The lower bound obtained is tighter than other bounds developed for related problems in literature. A meta-search approach based on the Genetic Algorithm concept is developed to evaluate the significance of further improving the solution obtained from the proposed BFD heuristic. The experiment indicates that it reduces the makespan by 1.93 % in average within a negligible time when problem size is less than 50 jobs.