Protein microfiltration

In the current work, we employed a modified approach, with predeposition of a secondary membrane of yeast (SMY) before starting the filtration of protein. Backflushing was employed periodically to remove the deposited secondary membrane to recover the flux, and a new secondary membrane was deposited subsequently with the start of each new cycle, prior to restarting the filtration of protein. Microfiltration experiments were performed with yeast as the secondary membrane and BSA-only solutions and yeast-BSA mixtures as the feed. Ultrafiltration experiments were performed with yeast as the secondary membrane deposition medium and cellulase enzyme solutions, used in the conversion of biomass into ethanol, as the feed. In this article, we also present direct visual observation images (19) of the formation of the secondary membrane and its subsequent removal. 

Ouammou. M. et al.. Elux decay in protein microfiltration through charged membranes as a function of pH. Colloids Surf. A, 298, 267, 2007. 

Bowen, W.R., Calvo, J.I., Hernandez, A. (1995), Steps of membrane blocking in flux decline during protein microfiltration. Journal of Membrane Science, 101, 153-165. 

Laboratory Microfiltration membranes have countless laboratory uses, such as recovering biomass, measuring particulates in water, clarifying and sterilizing protein solutions, and so on. There are countless examples for both general chemistry and biology, especially for analytical proc ures. Most of these apphcations are run in dead-end flow, with the membrane replacing a more conventional medium such as filter paper. 

Many process mixtures, notably fermentations, require sample preconcentration, microdialysis, microfiltration, or ultrafiltration prior to analysis. A capillary mixer has been used as a sample preparation and enrichment technique in microchromatography of polycyclic aromatic hydrocarbons in water.8 Microdialysis to remove protein has been coupled to reversed phase chromatography to follow the pharmacokinetics of the metabolism of acetaminophen into acetaminophen-4-O-sulfate and acetaminophen-4-O-glucu-ronide.9 On-line ultrafiltration was used in a process monitor for Aspergillus niger fermentation.10... 

Upon completion of the homogenization step, cellular debris and any remaining intact cells can be removed by centrifugation or by microfiltration. As mentioned previously, these techniques are also used to remove whole cells from the medium during the initial stages of extracellular protein purification. 

As discussed previously, the technique of microfiltration is effectively utilized to remove whole cells or cell debris from solution. Membrane filters employed in the microfiltration process generally have pore diameters ranging from 0.1 to 10 pm. Such pores, while retaining whole cells and large particulate matter, fail to retain most macromolecular components, such as proteins. In the case of ultrafiltration membranes, pore diameters normally range from 1 to 20 nm. These pores are sufficiently small to retain proteins of low molecular mass. Ultrafiltration membranes with molecular mass cut-off points ranging from 1 to 300 kDa are commercially available. Membranes with molecular mass cut-off points of 3,10, 30, 50, and 100 kDa are most commonly used. 

Generally, a distinction can be made between membrane bioreactors based on cells performing a desired conversion and processes based on enzymes. In ceU-based processes, bacteria, plant and mammalian cells are used for the production of (fine) chemicals, pharmaceuticals and food additives or for the treatment of waste streams. Enzyme-based membrane bioreactors are typically used for the degradation of natural polymeric materials Hke starch, cellulose or proteins or for the resolution of optically active components in the pharmaceutical, agrochemical, food and chemical industry [50, 51]. In general, only ultrafiltration (UF) or microfiltration (MF)-based processes have been reported and little is known on the application of reverse osmosis (RO) or nanofiltration (NF) in membrane bioreactors. Additionally, membrane contactor systems have been developed, based on micro-porous polyolefin or teflon membranes [52-55]. 

The range of application of the three pressure-driven membrane water separation processes—reverse osmosis, ultrafiltration and microfiltration—is illustrated in Figure 1.2. Ultrafiltration (Chapter 6) and microfiltration (Chapter 7) are basically similar in that the mode of separation is molecular sieving through increasingly fine pores. Microfiltration membranes filter colloidal particles and bacteria from 0.1 to 10 pm in diameter. Ultrafiltration membranes can be used to filter dissolved macromolecules, such as proteins, from solutions. The mechanism of separation by reverse osmosis membranes is quite different. In reverse osmosis membranes (Chapter 5), the membrane pores are so small, from 3 to 5 A in diameter, that they are within the range of thermal motion of the polymer... 

In contrast to hemodialysis that uses ultrafiltration membranes, plasma separation (also called plasmapheresis) requires microfiltration membranes with a pore size from 0.2 to 0.6 pm, in order to transmit all proteins and lipids, including LDL cholesterol (2000kDa) and retain completely platelets (2 pm diameter), red blood cells (8 pm diameter) and white blood cells. Thus, membrane plasmapheresis can yield high-quality platelet-free plasma and red cells can be either continuously returned to the donor or saved in another bag for blood transfusion. But it is important, in the case of plasma collection from donors, to minimize the membrane area, in order to reduce the cost of disposable hollow-fiber filters and to avoid the risk of hemolysis (free hemoglobin release) due to RBC damage by contact at the membrane if the pressure difference across the membrane is too high. 

Marcinkowsky et al. (16) were the first to use dynamic secondary membranes in reverse osmosis for rejection of salts. Giiell et al. (17) later investigated protein transmission and permeate fluxes in microfiltration of protein mixtures using yeast to form a predeposited secondary membrane, and they observed higher flux and protein transmission in the presence of the secondary layer. Kuberkar and Davis (18) also observed higher flux and transmission of BSA in the presence of a cake layer of yeast,... 

Fig. 6. Normalized microfiltration permeate flux vs time for different concentrations of yeast in secondary feed reservoir ( ) 1.36 g/L ( ) 0.68 g/L ( ) 0.34 g/L. The cycle conditions were tf = 280 s, = 10 s, and th = 10 s, with Pf=Ph = 7.5 psi. The primary feed contained a mixture of yeast (with the same concentration of yeast as in the secondary feed) and 2.0 g/L of BSA. The dashed line represents the normalized permeate flux during filtration of protein alone without deposition of SMY and without backflushing. Error bars represent SD for two to three repeats.

The goal of ultrafiltration, in contrast to microfiltration, is to retain protein molecules by the membrane while passing smaller solutes through the membrane with the permeate. Ultrafiltration experiments were performed with polysulfone membranes (30,000-Dalton mol wt cutoff). Figure 9 shows a comparison of the permeate flux vs time obtained during ultrafiltration of cellulase in the presence and absence of SMY that was periodically removed by backflushing and then replaced with a new SMY. 

Fig. 8. Protein transmission during microfiltration of 2.0 g/L BSA only (O) and 2.0 g/L of BSA in presence of SMY and backflushing (A), and permeate flux vs time for...

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