Critical and sustainable fluxes: theory, experiments and applications

Over the last ten years, numerous membrane filtration data have been viewed in the light of the concept of critical flux. This concept, used in a number of different ways often without explicit redefinition, is here clarified both from a theoretical and from an experimental viewpoint. Also, a link is make with the sustainable fluxes. Also covered are the various methods of measurement and the influence of membrane and suspension properties on the critical flux. Over the same period of time, models have been developed to explain the observed behaviour. Those for stable colloidal suspensions are based on the existence of repulsive interactions between soft matter constituents. The assumptions and usefulness of various models are discussed. The concept of a critical concentration for phase transition is introduced into the theoretical discussion. For theoreticians and experimentalist, this and the clarified concept of a small set of critical fluxes will continue to provide a valuable framework. For membrane users dealing with most industrial process streams (mixtures and complex fluid) the concept of a sustainable flux (shown as being derived from critical flux) is of a great utility; above a certain key flux (dependent on hydrodynamics, feed conditions and process time) the rate of fouling is economically and environmentally unsustainable. For many, knowledge of the point below which no major irreversible fouling occurs (the critical flux) in a membrane separation will always be of greatest utility

Development of a novel bioreactor and systems for suspension cell culture in biopharmaceutical production

Mammalian cells offer superior cellular machinery for the production of complex biological products. These cells provide proper post-translational processing machinery for recombinant protein expression to acquire the desired folding for optimal activity. With this advantage, mammalian cells have become the preferred choice for the production of biological products. These cells may grow either attached to a solid surface (adherent cells) or, where adapted, as suspension cultures. In order to grow these cells efficiently in suspension, a bioreactor is therefore required. Bioreactors play a key role in the production of biologicals. Due to the continuous advancement of medicine and the healthcare industry, the demand for biological drugs has increased in the last three decades. This has placed a significant pressure on the biopharmaceutical industry to meet this increasing demand and has become a key driving force behind the need to develop better, safer and more economical bioreactor designs and culture processes. Continuous stirred tank bioreactor is the norm for production of many bioproducts. However, these bioreactors exert high shear forces to cells due to the impeller speed, bubble disruption, and foam formation. In addition, at a large scale, improper mass transfer impairs the performance of cell lines and achieving high cell densities and prolonged viability with correct glycosylation of a secreted proteins is still a challenge during scale-up. Many cell lines, for example Vero cells, which are widely used to produce human vaccines are difficult to adapt into suspension culture. Fixed-bed bioreactors and the use of microcarriers provide an alternative platform for their growth to produce biologicals. However, a high surface area is required to achieve the high cell density which leading to an elevated cost of production (mainly from microcarriers) and ensuing a costly and technically challenging scaling-up of these systems. Other designs such as single-use bioreactors and novel bioreactors based on different operating principles have been explored, but their utilisation is limited from laboratory to pilot scale. Hence, a comprehensive bioreactor design which would be suitable for a large variety of cell lines to produce high-yielding products in suspension culture with the lowest cost and risk in the shortest span of time is still sought. In the current research, two approaches were investigated to address these challenges. Firstly, a horizontal tubular bioreactor (HTB) with a spiral impeller was designed and fabricated for the propagation of suspended mammalian cells with a focus to achieve middle to high cell density by improving mass transfer whilst reducing hydrodynamic shear and energy requirements through surface aeration. The second approach is to test the adaptation of adherent Vero cells into single-cell suspension culture in serum-free media by treating them with an anti-cancer drug, Puromycin amino nucleoside (PAN). The absence of a supporting surface for cell growth (e.g. microcarriers) and serum-free conditions are expected to reduce the cost of manufacturing and to achieve higher productivity of biological production per unit volume of bioreactor. In the first approach, the horizontal tubular vessel was designed to achieve the final volume of approximately 5.0 L. Design of the impeller is a key component that dictates the mixing patterns and mass transfer efficiency. Different geometric configurations were used to design the spiral impeller by considering various parameters such as impeller diameter, the pitch of the blade, pitch angle, height of the blade, the thickness of the blade, clearance efficiency and the position of the heating element. Another important aspect of the prototype design was incorporating an external magnetically-coupled motor drive which assisted in not only in aseptic handling but also reduction in mechanical stress and generation of fewer particles for cleanroom operations. The side plate was designed with the appropriate number of addition ports to allow execution of batches with minimum cross-contamination and for the ease of operation. Thereafter, the engineering characterisation of the HTB was carried out. The performance of the HTB was evaluated for (i) oxygen mass transfer (kLa) through the dynamic gassing-in method, (ii) mixing time and fluid flow by tracer and phenolphthalein method, (iii) minimum stirring speed (Njs) through alginate beads mimicking cell loading and modelling through modifying Zwietering equation, (iv) power consumption through heat calorimetry (temperature method) and (v) shear stress by determining specific death constant (kd) at different impeller speeds. The general characterisation profile of HTB has shown that at high agitation speed, homogeneity and mass transfer efficiency improved while power consumption increases with an increase in agitation speed. The bioreactor operated well at 2 L and 3 L capacity when the impeller is 40 - 90 % immersed in the liquid. The maximum mass transfer coefficient (kLa) of 16 h-1 was measured with a 3 L volume with an impeller speed of 500 rpm. These results are comparable with the other culture systems of the same scale. The HTB was also tested for suitability to grow mammalian cells. Three batches were carried out, of which one was with the Chinese hamster ovary (CHO) cells expressing the somatic angiotensinconverting enzyme (sACE) and the two with plain CHO cells without expressing any recombinant protein. The maximum cell density achieved was of 5.48 x 106 cells mL-1 with plain CHO cells and 4.14 x 106 cells mL-1 with CHO cells expressing sACE with a maximum protein productivity of 465 mg mL-1 . The specific death rate constant of 0.025 (h-1 ) was obtained when impeller speed was increase from 150 rpm (normal) to 300 rpm (induced shear) for 72 h. In this study, CHO cells have been successfully adapted to suspension in serum-free conditions using the slow weaning of serum method and propagated in the HTB whereas Vero cells have been adapted successfully to serum-free media in adherent conditions. Attempt to suspend Vero cells based on literature using the weaning method remains timeous. Therefore, an alternative approach was explored using an anti-cancer drug (PAN) which is known to suppress the expression of integrin (cell adhesion receptors). The expectations from this approach were that the suppression of integrin would allow cells to detach and grow as a suspended culture (Krishnamurti et al., 2001). The results indicated that the anti-cancerous drug may have modulated the structure and function of the integrin which resulted in dislodging of the cells from the surface and form clumps which were viable for a week in suspension culture without increase in cell density. The viability of the cell clumps and few suspended cells were tested by re-seeding of these cells back to tissue culture (TC) flasks in serum-containing media without the presence of PAN. The culture in the TC flask regained confluency in the 2-3 day which confirms the viability of the cells and the likeliness of integrin re-modulating itself in the absence of PAN. As the suspended Vero cells did not grow, they were not tested for growth in the HTB. To investigate the biological activity of these Vero cells, Isothermal microcalorimetry was used to evaluate the heat generation profile of the Vero cells quantitatively before and after drug treatment. The heat flow data (metabolic heat) from the treated and normal cells showed a distinct decrease in the heat generation profile which indicated that the treated cells were viable but not as active as the normal (non-treated) cells. It was evident from the heat flow data obtained for the PAN-treated Vero cells (-0.13 µW) from that of non-treated cells (13.12 µW) and thereafter when PAN-treated Vero cells regrown in serum-containing media, they regain their metabolic activities which were indicated by their heat flow values as positive control (9.30 µW), 100 µg mL-1 (10.12 µW), 200 µg mL-1 (10.18 µW), and 250 µg mL1 (9.15 µW). It is recommended that dielectric spectroscopy and total DNA in the culture from the lysed cells could also be used to measure the bioactivity of the pre and post treated cells and data can be compared with IMC for more insight into the behaviour of the cells It has been concluded that the horizontal tubular bioreactor (HTB) can sustain the middle to high cell density by imparting desired mixing and mass and heat transfer requirements whilst exerting minimum hydrodynamic shear. For the improvement of the design, it is recommended that more batches at different agitation speeds in combination with different airflow rate would further unravel the suitability of HTB to grow mammalian cells and stringently decode the optimum process conditions to achieve high cell densities with extended longevity. Additionally, changes in the pitch of the impeller blades could result in the improved fluid flow profile, mixing and mass transfer while drawing low power input. Subsequently, different modes of operation, e.g. fed-batch or continuous operation are suggested to investigate the suitability of the HTB for integrity, sterility, and possible higher productivities of products. In suspending Vero cells, it has been concluded that the presence of serum-containing media reversibly stimulates the re-modulation of the integrin which poses hurdles in suspending Vero cells by reattaching the cells to the TC flasks. Therefore, it is recommended that a thorough investigation of the drug-treated cell integrin profile is examined through fluorescence-activated cell sorting (FACS) which would give details of the inhibition of the different integrin subunits. This information could form the basis of adapting cell-lines into suspension in a single step, which is otherwise difficult to adapt

A novel mechanistic model for nitrogen removal in algal-bacterial photo sequencing batch reactors

© 2018 Elsevier Ltd A comprehensive mathematical model was constructed to evaluate the complex substrate and microbial interaction in algal-bacterial photo sequencing batch reactors (PSBR). The kinetics of metabolite, growth and endogenous respiration of ammonia oxidizing bacteria, nitrite oxidizing bacteria and heterotrophic bacteria were coupled to those of microalgae and then embedded into widely-used activated sludge model series. The impact of light intensity was considered for microalgae growth, while the effect of inorganic carbon was considered for each microorganism. The integrated model framework was assessed using experimental data from algal-bacterial consortia performing sidestream nitritation/denitritation. The validity of the model was further evaluated based on dataset from PSBR performing mainstream nitrification. The developed model could satisfactorily capture the dynamics of microbial populations and substrates under different operational conditions (i.e. feeding, carbon dosing and illuminating mode, light intensity, influent ammonium concentration), which might serve as a powerful tool for optimizing the novel algal-bacterial nitrogen removal processes

Design of the aerobic hail reactor - towards improved energy efficiency

This dissertation presents the results of an investigation into the design of a novel low aspect ratio reactor, dubbed the HAIL (horizontal air-injected loop) reactor. Current industrial high cell density aerobic reactors for cultivation of bacteria and yeast are typically either stirred tank reactors (STR's), bubble column reactors (BCR's) or airlift reactors (ALR's). These systems can attain high mass transfer rates and short mixing times; however, their energy efficiency remains a concern. Many studies have attempted to further optimise these reactors, but they are ultimately limited by their high aspect ratios. These lead to large pressure heads that the air compressor needs to overcome on sparging, contributing significantly to energy costs. Low aspect ratio (LAR) reactors, such as the wave bag, orbital shaker and raceway reactors offer an alternative to these systems, providing superior energy efficiency for both mixing and aeration. However, each has core issues preventing their usage in high cell density aerobic culture. Their maximum mass transfer coefficient is typically too low to support high cell density cultures. Additionally, these reactors tend to have poor scalability, making them unfeasible for large scale industrial usage. To overcome these challenges, the HAIL reactor makes use of a tubular loop design. The anticipated benefit of the loop design was that it forces the air to travel the length of the reactor before leaving the system, enabling significant surface aeration and residence time in the reactor. These both impact the mass transfer coefficient. Additionally, the loops can be stacked upon one another, overcoming the scalability issue. The reactor would also be energy efficient based on its LAR. To establish target performance ranges, a literature review on the gas-liquid mass transfer coefficient, mixing time and efficiency of current low and high aspect ratio (HAR) reactors was conducted. This was supplemented with experimental results (including mass transfer coefficients, cell density and viscosity) from the fed-batch STR cultivation of Saccharomyces cerevisiae, an easy to work with highly aerobic yeast. A fed-batch feeding profile was developed for this. To better compare reactor performance, a term was introduced called the mass transfer energy efficiency, with units m3 ∙h -1 ∙W-1 , obtained via the quotient of the kLa and the power input per unit volume. The literature mass transfer energy efficiency ranges for the STR, BCR and ALR were found to be 0.022-0.236 m3 ∙h -1 ∙W-1 , 0.084-0.317 m3 ∙h -1 ∙W-1 and 0.142-0.493 m3 ∙h -1 ∙W-1 respectively, with maximum kLa values ranging up to 1000 h-1 depending on the power input. Mixing times for these systems differ depending on scale and configuration, ranging from below a minute up to 20 minutes. Experimental fed-batch and sterile water systems had efficiency ranges of 0.044-0.245 m3 ∙h -1 ∙W-1 and 0.059-0.285 m3 ∙h -1 ∙W-1 respectively, with a maximum kLa of 240 h-1 and 226 h-1 . Based on cellular growth results, the theoretical minimum kLa required was calculated as 372 h-1 . The most notable literature efficiencies for LAR reactors were held by the travelling loop, raceway, and wave reactors with ranges of 0.286- 0.295 m3 ∙h -1 ∙W-1 , 0.034-0.867 m3 ∙h -1 ∙W-1 , and 0.112-0.742 m3 ∙h -1 ∙W-1 . For the wave and travelling loop reactors, mixing times below a minute were attainable. A 6.2 L proof-of-concept and 31.4 L laboratory-scale prototype of the HAIL reactor were developed. In the proof-of-concept prototype, preliminary studies were carried out on the impact of sparger depth and angle on circulation time. Using the laboratory-scale system a range of sparger designs, including different angled jets, outlet areas and a circular sparger design, were investigated. The circular sparger design was found to be the ideal sparger type. A mixing time of 7-19 minutes depending on the power input was found for the 31.4 L configuration. The power efficiency range determined was 0.120- 0.281 m3 ∙h -1 ∙W-1 ; however, the calculation used to determine this is an underapproximation. The maximum kLa of 13.84 h-1 is an order of magnitude (between 10 and 100) lower than the values that can be obtained in HAR reactors for industrial aerobic culture. It was found that HAIL reactor performance did not change substantially with an increase in viscosity from 1 to 1.4 cP. The HAIL reactor did not compete with existing low and high aspect ratio reactors in its current configuration in terms of mass transfer. Additional research on the design is recommended to enhance gas - liquid contacting and associated mass transfer. These ongoing studies will enable the potential relevance and application of the novel reactor to be determined

INVESTIGATION OF LIPID AND BIODIESEL PRODUCTION FROM Chlorella vulgaris (UTEX 2714) CULTURED IN PHOTO-BIOREACTORS (Spine title: Lipid & Biodiesel production from C. vulgaris grown on dairy effluent)

The use of microalgae as feed stock for biodiesel production has been under consideration for a number of years. However the process design always had limitations which were associated with the extraction and the harvesting processes. Biodiesel production from microalgae is an important process component in the microalgal biofuel area. In this project, a method for the cultivation of Chlorella vulgaris (UTEX 2714) on anaerobically digested dairy farm effluent is proposed. The first part of the work provides a study on the feasibility of microalgae cultivation on pre-treated dairy farm effluent. The microalgae were cultivated using 2% (v/v) and 4% (v/v) CO2 and two different pre-treatment techniques were employed i.e. using aluminum sulphate and the other using ferric sulphate. The pre treatment method employing doses of aluminum sulphate and ferric sulphate which provided the best results were 11 g/L and 7 g/L respectively. Triglyceride extractions were carried out using a modified Bligh and Dyer method which yielded triglyceride content to be in the range 17.65 - 22.85 % by weight. The microalgae were capable of removing 80.3 - 83.2 % of the initial NO3\u27, 100 % of the initial NH/, 39.8 - 45.1 % of the initial phosphate and 52.8 - 65.4 % of the initial COD concentrations. The maximum dry weight recorded in these experiments was 2.05 g/L. The post harvesting and concentration resulted in a microalgal slurry with a final dry weight concentration of 15 g/L was obtained. The harvested microalgae cells were then freeze dried for solvent extraction and Insitu biodiesel production. In the second part of the project, the focus was drawn to performing triglyceride extractions and biodiesel production from microalgae using an acid catalyst. For the various microalgae samples iii cultivated in the tubular photo bioreactor setup, yield calculations and FAME profiles were developed. A biodiesel yield maximum of 26.67 % by wt. was determined for the microalgae cultivated on dairy farm effluent using a 7 g/L ferric sulphate salt solution as the pre-treatment method. The FAME profile obtained in majority of the microalgae cultivated was mainly composed of C16, C18:0, Cl8:1, C18:2 fatty acids. This composition obtained for the microalgae very closely resembled the triglyceride composition obtained for canola oil derived biodiesel