Developing a scalable model of recombinant protein yield from Pichia pastoris:the influence of culture conditions, biomass and induction regime

Background The optimisation and scale-up of process conditions leading to high yields of recombinant proteins is an enduring bottleneck in the post-genomic sciences. Typical experiments rely on varying selected parameters through repeated rounds of trial-and-error optimisation. To rationalise this, several groups have recently adopted the 'design of experiments' (DoE) approach frequently used in industry. Studies have focused on parameters such as medium composition, nutrient feed rates and induction of expression in shake flasks or bioreactors, as well as oxygen transfer rates in micro-well plates. In this study we wanted to generate a predictive model that described small-scale screens and to test its scalability to bioreactors. Results Here we demonstrate how the use of a DoE approach in a multi-well mini-bioreactor permitted the rapid establishment of high yielding production phase conditions that could be transferred to a 7 L bioreactor. Using green fluorescent protein secreted from Pichia pastoris, we derived a predictive model of protein yield as a function of the three most commonly-varied process parameters: temperature, pH and the percentage of dissolved oxygen in the culture medium. Importantly, when yield was normalised to culture volume and density, the model was scalable from mL to L working volumes. By increasing pre-induction biomass accumulation, model-predicted yields were further improved. Yield improvement was most significant, however, on varying the fed-batch induction regime to minimise methanol accumulation so that the productivity of the culture increased throughout the whole induction period. These findings suggest the importance of matching the rate of protein production with the host metabolism. Conclusion We demonstrate how a rational, stepwise approach to recombinant protein production screens can reduce process development time.

Liquid-liquid extraction in a pilot scale rotating disc contactor

A study of the hydrodynamics and mass transfer characteristics of a liquid-liquid extraction process in a 450 mm diameter, 4.30 m high Rotating Disc Contactor (R.D.C.) has been undertaken. The literature relating to this type of extractor and the relevant phenomena, such as droplet break-up and coalescence, drop mass transfer and axial mixing has been revjewed. Experiments were performed using the system C1airsol-350-acetone-water and the effects of drop size, drop size-distribution and dispersed phase hold-up on the performance of the R.D.C. established. The results obtained for the two-phase system C1airso1-water have been compared with published correlations: since most of these correlations are based on data obtained from laboratory scale R.D.C.'s, a wide divergence was found. The hydrodynamics data from this study have therefore been correlated to predict the drop size and the dispersed phase hold-up and agreement has been obtained with the experimental data to within +8% for the drop size and +9% for the dispersed phase hold-up. The correlations obtained were modified to include terms involving column dimensions and the data have been correlated with the results obtained from this study together with published data; agreement was generally within +17% for drop size and within +14% for the dispersed phase hold-up. The experimental drop size distributions obtained were in excellent agreement with the upper limit log-normal distributions which should therefore be used in preference to other distribution functions. In the calculation of the overall experimental mass transfer coefficient the mean driving force was determined from the concentration profile along the column using Simpson's Rule and a novel method was developed to calculate the overall theoretical mass transfer coefficient Kca1, involving the drop size distribution diagram to determine the volume percentage of stagnant, circulating and oscillating drops in the sample population. Individual mass transfer coefficients were determined for the corresponding droplet state using different single drop mass transfer models. Kca1 was then calculated as the fractional sum of these individual coefficients and their proportions in the drop sample population. Very good agreement was found between the experimental and theoretical overall mass transfer coefficients. Drop sizes under mass transfer conditions were strongly dependant upon the direction of mass transfer. Drop Sizes in the absence of mass transfer were generally larger than those with solute transfer from the continuous to the dispersed phase, but smaller than those with solute transfer in the opposite direction at corresponding phase flowrates and rotor speed. Under similar operating conditions hold-up was also affected by mass transfer; it was higher when solute transfered from the continuous to the dispersed phase and lower when direction was reversed compared with non-mass transfer operation.

Drying of portland cement raw material slurries:predicting the performance of a counter-current spray drier

An extensive review of literature has been carried out concerning the drying of single drops, sprays of droplets and the prediction of spray drier performances. The experimental investigation has been divided into two broad parts mainly: (1) Single Drop Experiments, and (2) Spray Drying and Residence Time Distribution Experiments. The thermal conductivity of slurry cakes from five different sources have been experimentally determined using a modified Lee's Disc Apparatus and the data collected was correlated by the polynominal... Good agreement was observed between the experimental thermal conductivity values and the predicted ones. The fit gave a variance ... for the various samples experimented on. A mathematical model for estimating crust mass transfer coefficient at high drying temperatures was derived.

Studies of the drying of particulate slurries

The literature relating to the drying characteristics of pure liquid drops and particulate slurry drops has been reviewed. The experimental investigation was, therefore, divided into three parts: Pure water drops, Aqueous sodium sulphate decahydrate drops, and, Slurry drops from nine detergent formulations. The value of the constant,'Ψ, reported by Ranz and Marshall, was found to be temperature dependent. In the temperature range o 26.5≤T≤118.5°C,Ψ , for pure water drops, varied between 0.38 and 0.47. A revised correlation of the mass transfer coefficients is therefore proposed.  A mathematical model for estimating the variation of crust thickness, for aqueous sodium sulphate drops, with time is proposed: β = R _ {R3 - ( 1.5G/πCo ) ( ΔHD - ΔHU) Δ} 1/3 Experimental crust thickness evaluated from stereoscan micrographs showed good agreement with theoretical prediction. It has been shown that drying characteristics of detergent drops can be evaluated from the porosity:thickness ratio, {ε/\β}. Formulations having large {ε/β I-ratios dry better than those with smaller values. The agreement between the experimental and theoretical mass transfer coefficients shows, in addition to the above correlation, that the overall mass transfer coefficient can be predicted from the expression1/K = 1/K + β/DMε 1.5 The crust is the controlling resistance to transfer in particulate slurry drops. For aqueous sodium sulphate drops, the crust provides 64.2% of the total resistance while for detergents with thicker, but less porous crusts, the value is 97.5%.

Liquid-phase adsorption of n-paraffins on type 5A molecular sieves

As a basis for the commercial separation of normal paraffins a detailed study has been made of factors affecting the adsorption of binary liquid mixtures of high molecular weight normal paraffins (C12, C16, and C20) from isooctane on type 5A molecular sieves. The literature relating to molecular sieve properties and applications, and to liquid-phase adsorption of high molecular weight normal paraffin compounds by zeolites, was reviewed. Equilibrium isotherms were determined experimentally for the normal paraffins under investigation at temperatures of 303oK, 323oK and 343oK and showed a non-linear, favourable- type of isotherm. A higher equilibrium amount was adsorbed with lower molecular weight normal paraffins. An increase in adsorption temperature resulted in a decrease in the adsorption value. Kinetics of adsorption were investigated for the three normal paraffins at different temperatures. The effective diffusivity and the rate of adsorption of each normal paraffin increased with an increase in temperature in the range 303 to 343oK. The value of activation energy was between 2 and 4 kcal/mole. The dynamic properties of the three systems were investigated over a range of operating conditions (i.e. temperature, flow rate, feed concentration, and molecular sieve size in the range 0.032 x 10-3 to 2 x 10-3m) with a packed column. The heights of adsorption zones calculated by two independent equations (one based on a constant width, constant velocity and adsorption zone and the second on a solute material balance within the adsorption zone) agreed within 3% which confirmed the validity of using the mass transfer zone concept to provide a simple design procedure for the systems under study. The dynamic capacity of type 5A sieves for n-eicosane was lower than for n-hexadecane and n-dodecane corresponding to a lower equilibrium loading capacity and lower overall mass transfer coefficient. The values of individual external, internal, theoretical and experimental overall mass transfer coefficient were determined. The internal resistance was in all cases rate-controlling. A mathematical model for the prediction of dynamic breakthrough curves was developed analytically and solved from the equilibrium isotherm and the mass transfer rate equation. The experimental breakthrough curves were tested against both the proposed model and a graphical method developed by Treybal. The model produced the best fit with mean relative percent deviations of 26, 22, and 13% for the n-dodecane, n-hexadecane, and n-eicosane systems respectively.

A study of liquid-liquid extraction in a sieve plate column

The literature relating to sieve plate liquid extraction columns and relevant hydrodynamic phenomena have been surveyed. Mass transfer characteristics during drop formation, rise and coalescence, and related models were also reviewed. Important design parameters i.e. flooding, dispersed phase hold-up, drop size distribution, mean drop size, coalescence/flocculation zone height beneath a plate and jetting phenomena were investigated under non-mass transfer and mass transfer conditions in a 0.45m diameter, 2.3m high sieve plate column. This column had provision for four different plate designs, and variable plate spacing and downcomer heights, and the system used was Clairsol `350' (dispersed) - acetone - deionised water (continuous) with either direction of mass transfer. Drop size distributions were best described by the functions proposed by Gal-or, and then Mugele-Evans. Using data from this study and the literature, correlations were developed for dispersed phase hold-up, mean drop size in the preferred jetting regime and in the non-jetting regime, and coalescence zone height. A method to calculate the theoretical overall mass transfer coefficient allowing for the range of drop sizes encountered in the column gave the best fit to experimental data. This applied the drop size distribution diagram to estimate the volume percentage of stagnant, circulating and oscillating drops in the drop population. The overall coefficient Kcal was then calculated as the fractional sum of the predicted individual single drop coefficients and their proportion in the drop population. In a comparison between the experimental and calculated overall mass transfer coefficients for cases in which all the drops were in the oscillating regime (i.e. 6.35mm hole size plate), and for transfer from the dispersed(d) to continuous(c) phase, the film coefficient kd predicted from the Rose-Kintner correlation together with kc from that of Garner-Tayeban gave the best representation. Droplets from the 3.175mm hole size plate, were of a size to be mainly circulating and oscillating; a combination of kd from the Kronig-Brink (circulating) and Rose-Kintner (oscillating) correlations with the respective kc gave the best agreement. The optimum operating conditions for the SPC were identified and a procedure proposed for design from basic single drop data.

Behaviour of concrete frame structures under localised fire scenarios

This thesis encompasses an investigation of the behaviour of concrete frame structure under localised fire scenarios by implementing a constitutive model using finite-element computer program. The investigation phase included properties of material at elevated temperature, description of computer program, thermal and structural analyses. Transient thermal properties of material have been employed in this study to achieve reasonable results. The finite-element computer package of ANSYS is utilized in the present analyses to examine the effect of fire on the concrete frame under five various fire scenarios. In addition, a report of full-scale BRE Cardington concrete building designed to Eurocode2 and BS8110 subjected to realistic compartment fire is also presented. The transient analyses of present model included additional specific heat to the base value of dry concrete at temperature 100°C and 200°C. The combined convective-radiation heat transfer coefficient and transient thermal expansion have also been considered in the analyses. For the analyses with the transient strains included, the constitutive model based on empirical formula in a full thermal strain-stress model proposed by Li and Purkiss (2005) is employed. Comparisons between the models with and without transient strains included are also discussed. Results of present study indicate that the behaviour of complete structure is significantly different from the behaviour of individual isolated members based on current design methods. Although the current tabulated design procedures are conservative when the entire building performance is considered, it should be noted that the beneficial and detrimental effects of thermal expansion in complete structures should be taken into account. Therefore, developing new fire engineering methods from the study of complete structures rather than from individual isolated member behaviour is essential.

Mechanisms of chromium deposition and dissolution under direct and pulse reverse plating conditions

Baths containing sulphuric acid as catalyst and others with selected secondary catalysts (methane sulphonic acid - MSA, SeO2, a KBrO3/KIO3 mixture, indium, uranium and commercial high speed catalysts (HEEF-25 and HEEF-405)) were studied. The secondary catalysts influenced CCE, brightness and cracking. Chromium deposition mechanisms were studied in Part II using potentiostatic and potentiodynamic electroanalytical techniques under stationary and hydrodynamic conditions. Sulphuric acid as a primary catalyst and MSA, HEEF-25, HEEF-405 and sulphosalycilic acid as co-catalysts were explored for different rotation, speeds and scan rates. Maximum current was resolved into diffusion and kinetically limited components, and a contribution towards understanding the electrochemical mechanism is proposed. Reaction kinetics were further studied for H2SO4, MSA and methane disulphonic acid catalysed systems and their influence on reaction mechanisms elaborated. Charge transfer coefficient and electrochemical reaction rate orders for the first stage of the electrodeposition process were determined. A contribution was made toward understanding of H2SO4 and MSA influence on the evolution rate of hydrogen. Anodic dissolution of chromium in the chromic acid solution was studied with a number of techniques. An electrochemical dissolution mechanism is proposed, based on the results of rotating gold ring disc experiments and scanning electron microscopy. Finally, significant increases in chromium electrodeposition rates under non-stationary conditions (PRC mode) were studied and a deposition mechanisms is elaborated based on experimental data and theoretical considerations.

Modelling of wastewater treatment plants:how far shall we go with sophisticated modelling tools?

Several levels of complexity are available for modelling of wastewater treatment plants. Modelling local effects rely on computational fluid dynamics (CFD) approaches whereas activated sludge models (ASM) represent the global methodology. By applying both modelling approaches to pilot plant and full scale systems, this paper evaluates the value of each method and especially their potential combination. Model structure identification for ASM is discussed based on a full-scale closed loop oxidation ditch modelling. It is illustrated how and for what circumstances information obtained via CFD (computational fluid dynamics) analysis, residence time distribution (RTD) and other experimental means can be used. Furthermore, CFD analysis of the multiphase flow mechanisms is employed to obtain a correct description of the oxygenation capacity of the system studied, including an easy implementation of this information in the classical ASM modelling (e.g. oxygen transfer). The combination of CFD and activated sludge modelling of wastewater treatment processes is applied to three reactor configurations, a perfectly mixed reactor, a pilot scale activated sludge basin (ASB) and a real scale ASB. The application of the biological models to the CFD model is validated against experimentation for the pilot scale ASB and against a classical global ASM model response. A first step in the evaluation of the potential of the combined CFD-ASM model is performed using a full scale oxidation ditch system as testing scenario.

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.

Estudo da absorção das espécies pesadas do gás natural em octanol: efeitos da temperatura e vazão

In the oil industry, natural gas is a vital component of the world energy supply and an important source of hydrocarbons. It is one of the cleanest, safest and most relevant of all energy sources, and helps to meet the world's growing demand for clean energy in the future. With the growing share of natural gas in the Brazil energy matrix, the main purpose of its use has been the supply of electricity by thermal power generation. In the current production process, as in a Natural Gas Processing Unit (NGPU), natural gas undergoes various separation units aimed at producing liquefied natural gas and fuel gas. The latter should be specified to meet the thermal machines specifications. In the case of remote wells, the process of absorption of heavy components aims the match of fuel gas application and thereby is an alternative to increase the energy matrix. Currently, due to the high demand for this raw gas, research and development techniques aimed at adjusting natural gas are studied. Conventional methods employed today, such as physical absorption, show good results. The objective of this dissertation is to evaluate the removal of heavy components of natural gas by absorption. In this research it was used as the absorbent octyl alcohol (1-octanol). The influence of temperature (5 and 40 °C) and flowrate (25 and 50 ml/min) on the absorption process was studied. Absorption capacity expressed by the amount absorbed and kinetic parameters, expressed by the mass transfer coefficient, were evaluated. As expected from the literature, it was observed that the absorption of heavy hydrocarbon fraction is favored by lowering the temperature. Moreover, both temperature and flowrate favors mass transfer (kinetic effect). The absorption kinetics for removal of heavy components was monitored by chromatographic analysis and the experimental results demonstrated a high percentage of recovery of heavy components. Furthermore, it was observed that the use of octyl alcohol as absorbent was feasible for the requested separation process.

Estudo da absorção das espécies pesadas do gás natural em octanol: efeitos da temperatura e vazão

In the oil industry, natural gas is a vital component of the world energy supply and an important source of hydrocarbons. It is one of the cleanest, safest and most relevant of all energy sources, and helps to meet the world's growing demand for clean energy in the future. With the growing share of natural gas in the Brazil energy matrix, the main purpose of its use has been the supply of electricity by thermal power generation. In the current production process, as in a Natural Gas Processing Unit (NGPU), natural gas undergoes various separation units aimed at producing liquefied natural gas and fuel gas. The latter should be specified to meet the thermal machines specifications. In the case of remote wells, the process of absorption of heavy components aims the match of fuel gas application and thereby is an alternative to increase the energy matrix. Currently, due to the high demand for this raw gas, research and development techniques aimed at adjusting natural gas are studied. Conventional methods employed today, such as physical absorption, show good results. The objective of this dissertation is to evaluate the removal of heavy components of natural gas by absorption. In this research it was used as the absorbent octyl alcohol (1-octanol). The influence of temperature (5 and 40 °C) and flowrate (25 and 50 ml/min) on the absorption process was studied. Absorption capacity expressed by the amount absorbed and kinetic parameters, expressed by the mass transfer coefficient, were evaluated. As expected from the literature, it was observed that the absorption of heavy hydrocarbon fraction is favored by lowering the temperature. Moreover, both temperature and flowrate favors mass transfer (kinetic effect). The absorption kinetics for removal of heavy components was monitored by chromatographic analysis and the experimental results demonstrated a high percentage of recovery of heavy components. Furthermore, it was observed that the use of octyl alcohol as absorbent was feasible for the requested separation process.

Advanced thermal analysis of microelectronics using spreading resistance models

Thermal analysis of electronic devices is one of the most important steps for designing of modern devices. Precise thermal analysis is essential for designing an effective thermal management system of modern electronic devices such as batteries, LEDs, microelectronics, ICs, circuit boards, semiconductors and heat spreaders. For having a precise thermal analysis, the temperature profile and thermal spreading resistance of the device should be calculated by considering the geometry, property and boundary conditions. Thermal spreading resistance occurs when heat enters through a portion of a surface and flows by conduction. It is the primary source of thermal resistance when heat flows from a tiny heat source to a thin and wide heat spreader. In this thesis, analytical models for modeling the temperature behavior and thermal resistance in some common geometries of microelectronic devices such as heat channels and heat tubes are investigated. Different boundary conditions for the system are considered. Along the source plane, a combination of discretely specified heat flux, specified temperatures and adiabatic condition are studied. Along the walls of the system, adiabatic or convective cooling boundary conditions are assumed. Along the sink plane, convective cooling with constant or variable heat transfer coefficient are considered. Also, the effect of orthotropic properties is discussed. This thesis contains nine chapters. Chapter one is the introduction and shows the concepts of thermal spreading resistance besides the originality and importance of the work. Chapter two reviews the literatures on the thermal spreading resistance in the past fifty years with a focus on the recent advances. In chapters three and four, thermal resistance of a twodimensional flux channel with non-uniform convection coefficient in the heat sink plane is studied. The non-uniform convection is modeled by using two functions than can simulate a wide variety of different heat sink configurations. In chapter five, a non-symmetrical flux channel with different heat transfer coefficient along the right and left edges and sink plane is analytically modeled. Due to the edge cooling and non-symmetry, the eigenvalues of the system are defined using the heat transfer coefficient on both edges and for satisfying the orthogonality condition, a normalized function is calculated. In chapter six, thermal behavior of two-dimensional rectangular flux channel with arbitrary boundary conditions on the source plane is presented. The boundary condition along the source plane can be a combination of the first kind boundary condition (Dirichlet or prescribed temperature) and the second kind boundary condition (Neumann or prescribed heat flux). The proposed solution can be used for modeling the flux channels with numerous different source plane boundary conditions without any limitations in the number and position of heat sources. In chapter seven, temperature profile of a circular flux tube with discretely specified boundary conditions along the source plane is presented. Also, the effect of orthotropic properties are discussed. In chapter 8, a three-dimensional rectangular flux channel with a non-uniform heat convection along the heat sink plane is analytically modeled. In chapter nine, a summary of the achievements is presented and some systems are proposed for the future studies. It is worth mentioning that all the models and case studies in the thesis are compared with the Finite Element Method (FEM).

Investigação experimental das propriedades termofísicas e da convecção forçada de nanofluido de grafeno

This work presents an experimental investigation of thermal hydraulic performance of the nanofluid composed by graphene nanoparticles dispersed in a mixture of water and ethylene glycol at a ratio of 70:30% by volume. The tests were carried out under forced convection inside a circular tube with uniform heat flux on the wall for the laminar-turbulent transition regime. The mass flow rate ranged from 40 to 70 g/s corresponding to Reynolds numbers between 3000 and 7500. The heat flux was maintained constant at values of 11, 16 and 21 kW/m², as well as the inlet temperature of 15, 20 and 25°C. Three samples were produced with the nanofluid volumetric concentration of 0.05%, 0.10% and 0.15%. Thermophysical properties were experimentaly measured for all samples that were critically compared and discussed with theoretical models most commonly used in the literature. Initially, experiments with distilled water confirmed the validity of the experimental equipment for the thermo-hydraulic tests. Therefore, nanofluid samples that showed the highest thermal conductivity, corresponding to the volumetric concentrations of 0.15% and 0.10%, were subjected to the tests. The thermal-hydraulic performance for both samples was unsatisfactory. The heat transfer coefficients for convection of nanofluids reduced 21% in average, for the sample with = 0.15% and 26% and for =0.10%. The pressure drop of the samples was higher than the base fluid. Finally, the pressure drop and heat transfer coefficient by convection of both samples were also compared to theoretical models. The models used for pressure drop showed an excellent agreement with experimental results, which is remarkable considering the transitional flow.

FLOW REGIME DRIVEN THERMAL ENHANCEMENT IN INTERNALLY-GROOVED TUBES

Internally-grooved refrigeration tubes maximize tube-side evaporative heat transfer rates and have been identified as a most promising technology for integration into compact cold plates. Unfortunately, the absence of phenomenological insights and physical models hinders the extrapolation of grooved-tube performance to new applications. The success of regime-based heat transfer correlations for smooth tubes has motivated the current effort to explore the relationship between flow regimes and enhanced heat transfer in internally-grooved tubes. In this thesis, a detailed analysis of smooth and internally-grooved tube data reveals that performance improvement in internally-grooved tubes at low-to-intermediate mass flux is a result of early flow regime transition. Based on this analysis, a new flow regime map and corresponding heat transfer coefficient correlation, which account for the increased wetted angle, turbulence, and Gregorig effects unique to internally-grooved tubes, were developed. A two-phase test facility was designed and fabricated to validate the newly-developed flow regime map and regime-based heat transfer coefficient correlation. As part of this setup, a non-intrusive optical technique was developed to study the dynamic nature of two-phase flows. It was found that different flow regimes result in unique temporally varying film thickness profiles. Using these profiles, quantitative flow regime identification measures were developed, including the ability to explain and quantify the more subtle transitions that exist between dominant flow regimes. Flow regime data, based on the newly-developed method, and heat transfer coefficient data, using infrared thermography, were collected for two-phase HFE-7100 flow in horizontal 2.62mm - 8.84mm diameter smooth and internally-grooved tubes with mass fluxes from 25-300 kg/m²s, heat fluxes from 4-56 kW/m², and vapor qualities approaching 1. In total, over 6500 combined data points for the adiabatic and diabatic smooth and internally-grooved tubes were acquired. Based on results from the experiments and a reinterpretation of data from independent researchers, it was established that heat transfer enhancement in internally-grooved tubes at low-to-intermediate mass flux is primarily due to early flow regime transition to Annular flow. The regime-based heat transfer coefficient outperformed empirical correlations from the literature, with mean and absolute deviations of 4.0% and 32% for the full range of data collected.