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The use of microfiltration as an alternative to pasteurisation to reduce the microbial load of raw skimmed milk is a well established technology. However, its application in reducing bacteria from highly viscous dairy based solutions has not due to issues of low flux and high fouling tendency. This study involves the application of microfiltration to remove spores from high solids content Milk Protein Isolate (MPI) solutions. MPI feeds were inoculated with Bacillus mycoides spores a safer alternative to Bacillus cereus, a psychrotrophic spore forming bacteria found in dairy feeds. Suitable protocols for MPI resolubilisation, Bacillus mycoides cell and spore preparations were established and the membranes, MPI and spores were fully characterised by scanning electron microscopy (SEM), particle size distribution, rheology and pure water flux (PWF) measurements. Feed and permeate samples collected during experiments were analysed for solids content by oven drying, protein content using the Bradford assay and spore content using PetrifilmTM Aerobic count plates. To try and determine an optimum protocol for MPI filtration, a variety of filtration rig set-ups, modules and membranes were tested. Experiments were carried out at different MPI concentrations (4 – 16 wt%), cross flow velocities (CFV’s) (0.7 – 2.0 m s-1) and transmembrane pressures (TMP’s ) (1 and 2 bar). The filtration of 15 wt% MPI proved challenging. The best set of results were obtained using the 12.0 μm membrane at 1.4 m s-1, producing a 27 LMH flux, 96.5% protein transmission and a 2.1 log spore reduction. These results indicate that large pore ceramic microfiltration may be a suitable technology to replace or augment pasteurisation for high solids content dairy feeds. The effect of backwashing using different durations and frequencies was investigated. Backwashing parameters of 10 seconds every 5 minutes at 1 bar were found to be the most effective. The optimum cleaning regime found for MPI fouled ceramic membranes involved a long rinsing backflush at 1 bar, acid and alkali steps without backwashing, which produced a 99.6% flux recovery.

The major drawback of crossflow membrane filtration is that permeate flux declines with time as a result of the increase in total membrane resistance. Pulsatile flow is well known to reduce the resistance and enhance permeate flux. This study applied pulsatile flow induced by the oscillation of a collapsible tube to microfiltration and ultrafiltration, to improve filtration performance expressed as permeate flux enhancement and backflushable resistance reduction. Three membranes (ceramic tubular microfiltration, PVDF spiral-wound microfiltration and PS hollow-fibre ultrafiltration) and two media (bentonite suspension and whey solution) were used. In bentonite pulsatile microfiltration with the tubular membrane, up to 300% of permeate flux enhancement and 90% of backflushable resistance reduction were achieved. In bentonite and whey pulsatile microfiltration with the spiral-wound membrane, moderate improvements were gained: for bentonite, the highest increase in permeate flux was 51% and decrease in backflushable resistance was 45%; for whey, the highest permeate flux enhancement and backflushable resistance reduction were 36% and 38% respectively. In ultrafiltration of both media, no significant performance improvement was found. This is thought due in the one case to the relatively minute membrane pore size, and in the other to the large irreversible resistance created by whey solution. Transmural pressure at the collapsible tube downstream end indicates the tube compression and influences the pulsation vigour. Increasing the transmural pressure was an effective way to improve filtration performance. In bentonite microfiltration with the tubular membrane, increasing crossflow velocity was also effective, but increasing transmembrane pressure was not. Analysis of pulsatility parameters showed that the pulsatile flow always resulted in enhanced wall shear, and induced pore backflush always in the tubular membrane and sometimes in the HF membrane. These mechanistic findings helped to understand the filtration performance improvements. The analysis of energy consumption in bentonite microfiltration with the tubular membrane clearly demonstrated the benefit of applying the collapsible-tube-induced pulsatile flow in energy utilisation. The system specific energy could be reduced more than 70 % relative to the equivalent steady microfiltration permeate flux. For a given specific energy, the permeate flux could be increased by a factor of nearly four.

This thesis investigates the fouling of a microfiltration membrane by biological solutions. Membrane fouling is recognized as a major drawback for the application of microfiltration in the purification of biotechnology products. Membrane fouling was analyzed and compared for filtrations performed with a hollow fiber microfiltration module operated at constant permeate flux using bovine serum albumin (BSA) solutions or Chinese hamster ovary (CHO) cell culture broths as feed solutions. A mechanistic model was developed to represent the fouling of a cross-flow microfiltration membrane operated at constant permeate flux. Fouling was observed as an increase in the transmembrane pressure (TMP) and assumed to occur first by pore blockage followed by cake formation over the blocked pores. The effect of the cross-flow action was described by the removal of deposits from the membrane surface thereby reducing the pore blockage and the mass of the cake. The model was fitted to the TMP profiles obtained during the filtration of BSA solutions and Chinese hamster ovary (CHO) cell culture broths with a 0.45 µm polysulfone hollow fiber membrane. According to the fitted model, pores blocked faster and more cake was formed with increasing BSA concentration. In the case of CHO cell culture broth, increasing the wall shear rate (proportional to cross-flow velocity) seemed to lead to the formation of a less pronounced cake but more pore blockage. The cross-flow mechanistic model was applied to the microfiltration of CHO cell culture supernatants harvested at different days of the fermentation process. The filtrations were performed at two different shear rates and with two different membrane pore sizes. The cell culture supernatant caused membrane fouling observed as an increase in both the TMP and the membrane hydraulic resistance estimated from water flux measurements at the end of the filtrations. The highest TMP increase was observed for the filtrations with the smaller membrane pore size (0.2 µm) and the higher shear rate (8000 s-1). The hydraulic resistance estimates of the fouled membrane also revealed a higher irreversible fouling for the smaller (0.2 µm) membrane pore size while the model analysis indicates that more fouling occurred at the entrance of the membrane pores. The shear rate was found to strongly influence the contribution of the reversible fouling to the total hydraulic resistance of the membrane. The cross-flow mechanistic model indicates a higher pore blockage for the most severe membrane fouling observed experimentally for the smaller membrane pore size and the higher shear rate. At the same time, a smaller cake deposit was predicted for the higher shear rate. The different cell culture harvest time investigated in this study did not reveal any differences in membrane fouling. BSA solutions were used to evaluate the contribution of BSA aggregates, fresh cell culture medium and the non-ionic surfactant Pluronic F-68 to membrane fouling. A simple empirical model was developed to represent the TMP increase and to derive the initial fouling rate. The initial fouling rate, the normalized TMP and the irreversible membrane resistance at the end of the filtration were analyzed to determine the effect of BSA solution characteristics on membrane fouling. The initial fouling rate increased with increasing BSA aggregate content consistent with a two-step fouling mechanism that was proposed for membrane fouling by BSA. Increased BSA concentration and the use of fresh cell culture medium compared to potassium phosphate buffer resulted in an increase in initial fouling rate, TMP and irreversible membrane fouling. The addition of the non-ionic surfactant Pluronic-F68 to the BSA solutions decreased the long-term fouling and the irreversible fouling but did not affect the initial fouling rate.

This thesis investigates the fouling of a microfiltration membrane by biological solutions. Membrane fouling is recognized as a major drawback for the application of microfiltration in the purification of biotechnology products. Membrane fouling was analyzed and compared for filtrations performed with a hollow fiber microfiltration module operated at constant permeate flux using bovine serum albumin (BSA) solutions or Chinese hamster ovary (CHO) cell culture broths as feed solutions. A mechanistic model was developed to represent the fouling of a cross-flow microfiltration membrane operated at constant permeate flux. Fouling was observed as an increase in the transmembrane pressure (TMP) and assumed to occur first by pore blockage followed by cake formation over the blocked pores. The effect of the cross-flow action was described by the removal of deposits from the membrane surface thereby reducing the pore blockage and the mass of the cake. The model was fitted to the TMP profiles obtained during the filtration of BSA solutions and Chinese hamster ovary (CHO) cell culture broths with a 0.45 µm polysulfone hollow fiber membrane. According to the fitted model, pores blocked faster and more cake was formed with increasing BSA concentration. In the case of CHO cell culture broth, increasing the wall shear rate (proportional to cross-flow velocity) seemed to lead to the formation of a less pronounced cake but more pore blockage. The cross-flow mechanistic model was applied to the microfiltration of CHO cell culture supernatants harvested at different days of the fermentation process. The filtrations were performed at two different shear rates and with two different membrane pore sizes. The cell culture supernatant caused membrane fouling observed as an increase in both the TMP and the membrane hydraulic resistance estimated from water flux measurements at the end of the filtrations. The highest TMP increase was observed for the filtrations with the smaller membrane pore size (0.2 µm) and the higher shear rate (8000 s-1). The hydraulic resistance estimates of the fouled membrane also revealed a higher irreversible fouling for the smaller (0.2 µm) membrane pore size while the model analysis indicates that more fouling occurred at the entrance of the membrane pores. The shear rate was found to strongly influence the contribution of the reversible fouling to the total hydraulic resistance of the membrane. The cross-flow mechanistic model indicates a higher pore blockage for the most severe membrane fouling observed experimentally for the smaller membrane pore size and the higher shear rate. At the same time, a smaller cake deposit was predicted for the higher shear rate. The different cell culture harvest time investigated in this study did not reveal any differences in membrane fouling. BSA solutions were used to evaluate the contribution of BSA aggregates, fresh cell culture medium and the non-ionic surfactant Pluronic F-68 to membrane fouling. A simple empirical model was developed to represent the TMP increase and to derive the initial fouling rate. The initial fouling rate, the normalized TMP and the irreversible membrane resistance at the end of the filtration were analyzed to determine the effect of BSA solution characteristics on membrane fouling. The initial fouling rate increased with increasing BSA aggregate content consistent with a two-step fouling mechanism that was proposed for membrane fouling by BSA. Increased BSA concentration and the use of fresh cell culture medium compared to potassium phosphate buffer resulted in an increase in initial fouling rate, TMP and irreversible membrane fouling. The addition of the non-ionic surfactant Pluronic-F68 to the BSA solutions decreased the long-term fouling and the irreversible fouling but did not affect the initial fouling rate.

Nanoparticles (NPs) have attracted considerable attention in the field of separation science, especially in filtration studies for direct membrane integrity tests, investigating pore-size distribution, and their potential to be used as surrogates for various types of viruses encountered in water treatment and bioprocessing applications. Although the effect of adding surfactants to stabilize NP suspension have been explored for a number of different applications, there is significant variation in the amounts and types of surfactants used in filtration studies. This study used three different sizes (59, 188, and 490 nm) of fluorescent polystyrene nanoparticles (PNPs) to mimic the length, width, and aggregates of Rhabdovirus Maraba, a bullet-shape envelope virus. The PNPs were suspended in solutions with varying concentrations of the nonionic surfactant Tween 20 (0.0005% to 0.1% (v/v) in the carbonate buffer feed solution) and were tested in constant-flux filtration studies using two commercial microfiltration (MF) membranes (Durapore PVDF and MiniSart PES) with 0.22 micron pore size ratings. Results clearly demonstrate that adding a nonionic surfactant to a PNP solution will cause a shift from full retention to complete transmission during the dead-end MF of PNPs that are smaller than the pore size of an MF membrane. In a separate study, in order to have a better resemblance of virus particles in terms of surface properties, 188 nm PNPs were coated with different (lysozyme, α -lactalbumin and bovine serum albumin) proteins in order to gain similar surface properties to actual virus particles. Filtration results with one type of commercial MF membranes (Durapore PVDF) 0.22 μm pore size, clearly indicate that the transmission behavior of PNPs strongly depends on their surface properties. PNPs fully covered with BSA and α–lactalbumin could completely pass through the membranes while uncovered or partially covered PNPs resulted in no transmission or partial transmission. / Thesis / Master of Applied Science (MASc) / Nanoparticles (NPs) has been employed enormously in various applications for a variety of purposes. One of the areas that have been greatly influenced by NPs, is the field of separation science. In the pharmaceutical industry, purification of therapeutics involves a sequence of filtration and in this step, therapeutic virus filtration, sterile filtration, in particular, have been poorly studied. There is also a growing interest in the use of engineered viruses for cancer treatment due to its magnificent implication on human health. However, there are significant challenges in running filtration experiments with pathogenic substances. Therefore it has been determined that a detailed and comprehensive study of sterile filtration of virus-size NPs can benefit this area. In this work, fluorescently-labeled NPs has been used as surrogates of oncolytic viruses to extract fundamental aspects affecting the transmission of virus-sized particles through commercial microfiltration sterilizing grade membranes.

Concentration polarisation and fouling reduce performance as well as increase costs. In order to mitigate these effects, understanding the cause and effects of these phenomena is crucial. It has been hypothesized, and to a certain extent shown, that amelioration of fouling can be achieved through the use of time varying shear for example use of air-sparging or sharp changes in crossflow velocity. Nevertheless the effect of shear on membrane fouling, in particular its effect on the foulant deposition and the transmission of small molecules in microfiltration, is not well understood. The goal of this project was to achieve an understanding of various foulant be- haviour. The work was divided into two parts. Firstly, the observations of fouling by freshwater algae, Chlorella Sorokiniana, were carried out at Nanyang Technolo- gical University, Singapore. Observations using macroscopic parameters were ex- amined with an optical non-invasive observation technique called Direct Observa- tion Through Membrane (DOTM). The result yielded a novel relationship between operating flux, crossflow velocity and transmission of extracellular polysaccharide. Interestingly shear was shown to have positive as well as negative effects on fouling of microfiltration membrane. The analysis of permeate has clearly shown that a maximisation of shear rate was not ideal. The second part was concerned with observations of the effect of shear patterns on membrane fouling using newly fabricated special membrane filtration cell, Direct Shear Stress Test Cell (DSSTC), designed to fit an Anton Paar rheometer and op- erate at constant flux. Unlike the-constant-shear-filtration cells, one could impose a very wide variety of shear regimes including intermittent sharp changes of direction and sinusoidal oscillations in the DSSTC. The effect of shear patterns on transmis- sion and fouling of a model polysaccharide (Dextran Blue) through microfiltration membranes was carried out at various conditions. Again, the results showed that the maximisation of shear rate was not ideal. The effect of shear patterns and EPS on fouling by yeast suspension was also studied using the DSSTC. The benefit of oscillatory shear is foulant dependent. For example, square wave oscillatory shear led to lower relative fouling for yeast EPS, but it resulted in higher relative fouling for unwashed yeast.

Membrane filtrations are widely used in process industries but are almost always limited by fouling, a highly studied and significant problem. This is defined as unwanted material deposited on a membrane surface or within its pores, which can significantly impair performance and/or reduce operating life. The strategies to mitigate fouling include cleaning in place, modified membranes, and optimisation of operating conditions. In order to correctly select or target improvements to any such measures a detailed mechanistic understanding of the fouling process is important, which requires more than just performance data from unit operations. One key mechanism is that of cake fouling, which describes the build-up of particle layers on the surface of the membrane. Its growth and physical properties are difficult to assess. In this project the technique of fluid dynamic gauging (FDG) has been explored as a means to study cake fouling. This simple, yet robust method allows for estimation of thickness and strength of cake fouling at high concentrations and opacity, without any prerequisite knowledge of feed properties. Studies described herein focused on microfiltrations through cellulosic membranes. FDG was used to track cake growth during filtrations of polydisperse yeast suspensions (which contained large agglomerates), demonstrating its capability to work with non-ideal, food-like substances. Later studies used more predictable suspensions of hollow glass spheres, which were used to assess various filtration models. The most effective was found to be an interpretation of the critical flux laws, which were used to successfully identify pore fouling during filtrations of Kraft lignin, an observation supported by FDG measurements. Another novel achievement of this project was the development of an automated apparatus for performing FDG in cross-flow membrane filtration. This allowed for much faster acquisition of results, and demonstrated the potential for its development into an autonomous system capable of making thickness measurements on the fly during filtrations. The most reliable protocol for determining cake growth rates was by repeated filtrations in which destructive thickness testing was performed at selected time points. This was because continuous or even repeated thickness measurements during a single filtration were found to cause too much disturbance to the fouling layer. Computational fluid dynamics was used to simulate shear stress profiles on the fouling layer, while also providing a more accurate means to calibrate the automated apparatus. Erosion caused by FDG readings, when viewed under a microscope, was found to conform to the shear stress profiles predicted by simulations.

ABSTRACT PERFORMANCE OF A WASTEWATER TREATMENT POND SYSTEM WITH MICROFILTRATION Eric Martin The Woodlands Wastewater Treatment Plant (WWWTP) treats wastewater from the Woodlands housing community near Nipomo, California. The treated effluent is recycled for irrigation of a golf course within the community. The treatment facility consists of three facultative ponds in series followed by a microfiltration system and chlorine disinfection. Microfiltration of wastewater pond effluent is a fairly new, and potentially challenging, application of microfiltration. This thesis describes the operating conditions and behavior of the WWWTP pond system followed by a microfiltration system for the purpose of producing recyclable water fit for reuse under the regulations of Title 22. Water quality data were compiled in two ways. Weekly influent and effluent water quality and flow measurements conducted by the WWWTP operators over the course of three years were studied to show the treatment trends of the treatment plant as a whole. In addition, weekly water quality tests were performed on samples of wastewater influent, effluent, and intermediate stages of treatment for 20 weeks and studied to show treatment performance of each individual pond and the microfiltration system. Pond treatment performance was analyzed based on removal of biochemical oxygen demand (BOD) and total suspended solids (TSS) and accumulation of sludge within the pond system. Microfiltration performance was analyzed in terms of meeting the TSS discharge requirement and the membrane fouling rate. The power consumption of the pond system components and the microfiltration system were estimated. The data show that the WWWTP is producing very high quality discharge. Without microfiltration, five-day carbonaceous soluble BOD (csBOD5) averaged 3.0 mg/L and TSS averaged 42.5 mg/L. BOD5 and TSS removal efficiencies were greater than 90%. Microfilter effluent BOD5 and TSS concentrations averaged 3.0 mg/L and 1.6 mg/L, respectively. Total ammonia nitrogen was reduced to 1.61 mg/L. pH remained between 6.5 and 8.5 with few temporary exceptions. The sludge accumulation was at the high end of the range of typical accumulation rates. However, the measured rate is for the first three years of operation and so likely over-estimates the long-term accumulation rate. Although the treatment performance of the WWWTP is excellent, the power consumed was high.

Efficient separation of target protein from impurities is crucial in bioseparation for large scale production and purity of the target protein. Two separation process approaches were considered in this study. The first approach focused on identifying major impurity and optimization of solution properties for target protein purification. The second approach consisted of designing an adsorbent that interacted specifically with the target molecule. The first study included modification of protein solution properties (pH, ionic strength, buffer ions) in order to maximize lysozyme purification by a strong cation exchange resin. The interaction of phytic acid, a major impurity, present in transgenic rice extracts, that contributes to decreased lysozyme adsorption capacity on SP Sepharose was evaluated. The target protein was lysozyme, which is used in a purified form as a baby formula additive to reduce gastrointestinal tract infections. At constant ionic strength, lysozyme in pH 4.5 acetate buffer had a higher binding capacity and stronger binding strength than at pH 6.0. Lysozyme in sodium phosphate buffer of pH 6.0 exhibited lower adsorption capacity than in pH 6 Tris buffer. Binding capacity and strength were significantly affected by phytic acid in all studies buffers. The second study consisted of surface modification of microfiltration membranes for protein purification and separation and reduces fouling. This study describes adsorption and fouling of chemically modified microfiltration membranes with bovine serum albumin (BSA) and immunoglobulin G (IgG). Least fouling resulted with polyethylene glycol (PEG) membranes when BSA protein was used. Amine-functionalized membranes showed specific interaction with BSA. There was multi-layer deposition of IgG on amine-functionalized membrane. G3 membrane synthesized to selectively bind IgG seemed a noble option to separate IgG from a protein mixture.