Production of Biofuels

CHAPTER-1INTRODUNCTION1.1 Introduction to membranes:Amembraneis a selective barrier. At times, it is also an outer covering of cell or cell organelle that allows the passage of certain constituents and retains other constituents found in the liquid. The influent of a membrane is known as the feed-stream, the liquid that passes through the membrane is known as the permeate and the liquid containing the retained constituents is the retentate or concentrate.

Many simple filtration processes use a dead-end technique the flow of liquid to be filtered is directed perpendicular to the filter surface. This is effective whenever the concentration of particles to be removed is low or the packing tendency of the filtered material does not produce a large pressure drop across the filter medium. Some common examples of dead-end filtration are home water filters, vacuum cleaners and oil filters in automobiles. Typical industrial uses include the sterile filtration of water, beer, and wine. In contrast, there are many process streams that have high concentrations of particles or macromolecules such as cells, proteins and precipitates that will rapidly compact on the filter surface when operated in a dead-end mode. Consequently, the filtration rate drops quickly to an unacceptable level. In these instances, a crossflow membrane system provides the means to maintain stable filtration rates. The key to the design of a crossflow system is selecting a membrane geometry that suits the physical characteristics of the process fluid. Crossflow membranes can be provided in tubular, flat sheet, spiral wound, and hollow fiber configurations, each of which provides certain advantages for specific process needs. Virtually any membrane design can be applied on water-like liquids with low concentrations of suspended solids, but viscous streams and fluids with large amounts of solids can only be handled with membranes specifically designed for this purpose. In general, the more difficult a stream is to process, the higher the cost of a membrane system and the higher the operating costs. Thus, an optimization study is an important component of any potential crossflow installation.

1.2 The Evolution of Membrane Technology:Modern crossflow technology has primarily evolved during the last forty years largely in step with the advancement of polymer chemistry. Durable, chemical-resistant polymers have made crossflow technology cost-effective. Today, 98 percent of crossflow installations utilize polymer-based membranes; inorganic materials such as ceramic are only selected in specific instances were pH, temperature, or cleaning chemistry prohibit the use of polymers. The theoretical principles of crossflow filtration are derived from Ficks law of diffusion, which addresses the migration of suspended solids/macromolecules in a flowing stream towards a filtration surface, and the potential back-diffusion into the bulk stream. This premise forms the basis for crossflow designthat the concentration of macromolecules at the membrane surface can be controlled as a function of the velocity of fluid flowing parallel to that surface. The design of a successful crossflow system relies on choosing a membrane geometry that can be installed and operated economically, provides consistent predictable results, and can be effectively cleaned using chemicals compatible with the membrane.The goal of membrane technologists is to use appropriate polymer material, module configuration, system design and operating conditions to achieve the most economical process possible. At the heart of this is the selection of a membrane with the optimum separation characteristics.1.3 How is a Specific Membrane Process Defined:Crossflow membranes are manufactured in a range of porosities tailored to address various applications. These span the range of salt removal from water to large particulate filtration in viscous fluids. Filtration ranges have been defined that correlate to physical aspects of the membrane process and the relative size exclusion involved. In addition to the choices of polymer and membrane geometry, pore size selection is an integral part of process optimization.The membrane regime with the smallest pores is reverse osmosis (RO), which involves appropriately enough reversal of the osmotic pressure of a solution in order to drive water away from dissolved molecules. Strictly speaking, RO is not a size exclusion process based on pore size; it depends on ionic diffusion to effect the separation. A common application of reverse osmosis is seawater desalination in which pure water is produced from a highly saline feed stream. In applications such as this, reverse osmosis serves a similar purpose to evaporation, yet provides better economics.RO is also used in many industrial processes including fruit juice concentration, ice-making, and car wash reclamation, and wastewater volume reduction. In each of these examples, the goal is either to produce a pure filtrate (typically water) or retain entirely the components of the feed stream as the product. Because the osmotic pressure (a measure of the dissolved ion concentration) of many process streams is quite high, RO membranes must be designed to operate at pressures of 400-1200 psi (29-83 bars) which restricts the available membrane geometries.

Figure 1.1: Relative size exclusionA natural extension of reverse osmosis is Nanofiltration (NF), the most recent development on the crossflow frontier. NF functions similarly to reverse osmosis, but is generally targeted to remove only divalent and larger ions, hence the nickname for NF-selective reverse osmosis. Monovalent ions such as sodium and chloride will pass through a Nanofiltration membrane, thus many of the uses of NF involve de-salting of the process stream. An example is the production of lactose from cheese whey; the NF process is designed to concentrate the lactose molecules while passing salts a procedure that purifies and concentrates the lactose stream. In water treatment, NF membranes are used for hardness removal (in place of water softeners), pesticide elimination and color reduction. Nanofiltration can also be used to reclaim spent NaOH solutions. In this case, the permeate (filtrate) stream is purified NaOH, allowing reuse many times over.Ultrafiltration (UF) is the next process on the pore size continuum. UF is not an osmotic process the pores of UF membranes are larger and the method of rejection is primarily physical size exclusion. While RO/NF membranes are generally categorized by the degree of salt rejection under standard conditions, UF membranes are specified by a molecular weight cut-off rating (MWCO). The range of MWCOs for UF is generally considered to be 1,000-1,000,000 Daltons which can be loosely correlated to pore size (roughly equivalent to 0.005-0.1 microns).1.4 Classification of Membrane:a) Classification based on Nature

b) Classification based on Morphology

Synthetic membrane can be fabricated from a large number of different materials. It can be made from organic or inorganic materials including solids such asmetal or ceramic,homogeneousfilms (polymers),heterogeneoussolids (polymeric mixes, mixed glasses), and liquids.Ceramic membranes are produced from inorganic materials such asaluminumoxides,silicon carbide, andzirconiumoxide. Ceramic membranes are very resistant to the action of aggressive media (acids, strong solvents). They are very stable chemically, thermally, and mechanically, and biologicallyinert. Even though ceramic membranes have a high weight and substantial production costs, they are ecologically friendly and have long working life. Ceramic membranes are generally made as monolithic shapes of tubularcapillaries.1.5 Application areas of membranes: Membrane separation processes operate without heating and therefore use less energy than conventional thermal separation processes such asdistillation,sublimationorcrystallization. The separation process is purely physical and both fractions (permeateandretentate) can be used. Cold separation using membrane technology is widely used in the food technology biotechnologyandpharmaceuticalindustries. Furthermore, using membranes enables separations to take place that would be impossible using thermal separation methods. Depending on the type of membrane, the selective separation of certain individual substances or substance mixtures is possible. Important technical applications include the production of drinking water byreverse osmosis(worldwide approximately 7 million cubic meters annually), filtrations in thefood industry, the recovery of organic vapours such as petro-chemical vapour recovery and theelectrolysisfor chlorine production. Inwaste watertreatment, membrane technology is becoming increasingly important. With the help of UF and MF (Ultra/Microfiltration) it is possible to remove particles, colloids and macromolecules, so that waste-water can be disinfected in this way. This is needed if waste-water is discharged into sensitive waters especially those designated for contact water-sports and recreation. About half of the market is in medical applications such as use in artificial kidneys to remove toxic substances byhemodialysis and asartificial lungfor bubble-free supply of oxygen in theblood. The importance of membrane technology is growing in the field of environmental protection. Even in modern energy recovery techniques membranes are increasingly used, for example infuel cellsand inosmotic power plants1.6 Different membrane processes:According to driving force of the operation it is possible to distinguish: Pressure driven operations Microfiltration Ultrafiltration Nano filtration Reverse osmosis Concentration driven operations Dialysis Pervaporation Forward osmosis Gas separation Operations in electric potential gradient Electrodialysis Membrane electrolysis Electrodeionization Electrofiltration Operations in temperature gradient Membrane distillation Microfiltration: -When particles of diameter greater than 100 nm have to be retained, it is possible to use an open membrane structure. The hydrodynamic resistance is low and small driving forces are sufficient to obtain high fluxes. It is commonly abbreviated to MF. It is a type of physical filtration process where a contaminated fluid is passed through a special pore-sized membrane to separate microorganisms and suspended particles from process liquid. It is commonly used in conjunction with various other separation processes such as ultrafiltration and reverse osmosis to provide a product stream which is free of undesired contaminants. Microfiltration usually serves as a pre-treatment for other separation processes such as ultrafiltration, and a post-treatment for granular media filtration. The typical particle size used for microfiltration ranges from about 0.1 to 10 m. In terms of approximate molecular weight these membranes can separate macromolecules generally less than 100,000 g/mol. The filters used in the microfiltration process are specially designed to prevent particles such as, sediment, algae, protozoa or large bacteria from passing through a specially designed filter. More microscopic, atomic or ionic materials such as water (H2O), monovalent species such as Sodium (Na+) or Chloride (Cl-) ions, dissolved or natural organic matter, and small colloids and viruses will still be able to pass through the filter. The suspended liquid is passed though at a relatively high velocity of around 13m/s and at low to moderate pressures (around 100-400 kPa) parallel or tangential to the semi-permeable membrane in a sheet or tubular form. A pump is commonly fitted onto the processing equipment to allow the liquid to pass through the membrane filter. There are also two pump configurations, either pressure driven or vacuum. A differential or regular pressure gauge is commonly attached to measure the pressure drop between the outlet and inlet streams. The most abundant use of microfiltration membranes are in the water, beverage and bio-processing industries. The exit process stream after treatment using a micro-filter has a recovery rate which generally ranges to about 90-98%. Ultrafiltration: To separate macromolecules from an aqueous solution, the membrane structure must be denser and hence its hydrodynamic resistance also increases. The applied pressure is now greater than in microfiltration. It is commonly abbreviated to UF. It is a variety of membrane filtration in which forces like pressure or concentration gradients leads to a separation through a semipermeable membrane. Suspended solids and solutes of high molecular weight are retained in the so-called retentate, while water and low molecular weight solutes pass through the membrane in the permeate. This separation process is used in industry and research for purifying and concentrating macromolecular (103 - 106 Da) solutions, especially protein solutions. Ultrafiltration is not fundamentally different from microfiltration. Both of these separate based on size exclusion or particle capture. It is fundamentally different from membrane gas separation, which separate based on different amounts of absorption and different rates of diffusion. Ultrafiltration membranes are defined by the Molecular Weight Cut Off (MWCO) of the membrane used. Ultrafiltration is applied in cross-flow or dead-end mode. Industries such as chemical and pharmaceutical manufacturing, food and beverage processing, and waste water treatment, employ ultrafiltration in order to recycle flow or add value to later products. But also blood dialysis belongs to ultrafiltration. Nanofiltration: - Nanofiltration membranes have pore size of 1-10 Angstrom, smaller than that used in microfiltration and ultrafiltration. But the hydrodynamic resistance also increases consequently the higher driving forces are needed. It is abbreviated to NF. It is a relatively recent membrane filtration process used most often with low total dissolved solids water such as surface water and fresh groundwater, with the purpose of softening (polyvalent cation removal) and removal of disinfection by-product precursors such as natural organic matter and synthetic organic matter. Nanofiltration is also becoming more widely used in food processing applications such as dairy, for simultaneous concentration and partial (monovalent ion) demineralisation. Nanofiltration is a membrane filtration based method that uses nanometer sized cylindrical through-pores that pass through the membrane at a 90. Nanofiltration membranes have pore sizes from 1-10 Angstrom, smaller than that used in microfiltration and ultrafiltration, but just larger than that in reverse osmosis. Membranes used are predominantly created from polymer thin films. Materials that are commonly used include polyethylene terephthalate or metals such as aluminium. Pore dimensions are controlled by pH, temperature and time during development with pore densities ranging from 1 to 106 pores per cm2. Reverse Osmosis: In this process it is possible to separate low molecular weight components of approximately equal size from each other. So a very dense membrane is used, resulting in a very high hydrodynamic resistance. It is abbreviated to RO. It is a water purification technology that uses a semipermeable membrane. This membrane technology is not properly a filtration method. In reverse osmosis, an applied pressure is used to overcome osmotic pressure, a colligative property that is driven by chemical potential, a thermodynamic parameter. Reverse osmosis can remove many types of molecules and ions from solutions, and is used in both industrial processes and the production of potable water. The result is that the solute is retained on the pressurized side of the membrane and the pure solvent is allowed to pass to the other side. To be "selective", this membrane should not allow large molecules or ions through the pores (holes), but should allow smaller components of the solution (such as the solvent) to pass freely. In the normal osmosis process, the solvent naturally moves from an area of low solute concentration (high water potential), through a membrane, to an area of high solute concentration (low water potential). The movement of a pure solvent is driven to reduce the free energy of the system by equalizing solute concentrations on each side of a membrane, generating osmotic pressure. Applying an external pressure to reverse the natural flow of pure solvent, thus, is reverse osmosis. The process is similar to other membrane technology applications. However, key differences are found between reverse osmosis and filtration. The predominant removal mechanism in membrane filtration is straining, or size exclusion, so the process can theoretically achieve perfect exclusion of particles regardless of operational parameters such as influent pressure and concentration. Moreover, reverse osmosis involves a diffusive mechanism, so that separation efficiency is dependent on solute concentration, pressure, and water flux rate.[1] Reverse osmosis is most commonly known for its use in drinking water purification from seawater, removing the salt and other effluent materials from the water molecules. Dialysis: - It works on the principles of the diffusion of solutes and ultrafiltration of fluid across a semi-permeable membrane. The two main types of dialysis, hemodialysis and peritoneal dialysis, removes waste and excess water from the blood in different ways. Pervaporation: - In pervaporation there are two process- permeation through the membrane by the permeate then its evaporation into the vapour phase. The heat of vaporization has to be supplied. It is mainly used to dehydrate organic mixtures. Forward Osmosis: - It is an osmotic process which uses a semi-permeable membrane to effect separation of water from dissolved solutes. The driving force for this separation is an osmotic pressure gradient. Gas separation: - In gas separation two completely different types of membranes can be used a dense membrane where transport takes place via diffusion and a porous membrane where Knudsen flow occurs. Electrodialysis and Membrane Electrolysis: - It is used to transport ions from one solution through ion-exchange membranes to another solution under the influence of an applied electric potential difference. This is done in a configuration called an electrodialysis cell. Elctrodeionization: - Electrodeionizationis awater treatmenttechnology that utilizes anelectrodetoionizewatermoleculesand separate dissolvedions(impurities) from water. It differs from other water purification technologies in that it is done without the use of chemical treatments and is usually a tertiary treatment toreverse osmosis Electofiltration: - Electrofiltration is highly innovative technique for separation, respectively concentration of colloidal substances- for instancebiopolymers. The principle of electrofiltration is based on overlayingelectric fieldon a standarddead-end filtration. Membrane Distillation: - It is a thermally driven separational process in which separation is due to phase change. A hydrophobic membrane displays a barrier for the liquid phase letting the vapour phase pass through the membrane pores.

1.7 Selection of Membrane Separation Process Membrane separation processes have very important role in separation industry. Nevertheless, they were not considered technically important until mid-1970. Membrane separation processes differ based on separation mechanisms and size of the separated particles. Microfiltration and ultrafiltration is widely used in food and beverage processing (beer microfiltration, apple juice ultrafiltration), biotechnological applications andpharmaceutical industry(antibiotic production, protein purification), water purification andwastewater treatment, microelectronics industry, and others. Nanofiltration and reverse osmosis membranes are mainly used for water purification purposes. Dense membranes are utilized for gas separations (removal of CO2from natural gas, separating N2from air, organic vapor removal from air or nitrogen stream) and sometimes in membrane distillation. The later process helps in separating of azeotropic compositions reducing the costs of distillation processes.

CHAPTER-2 PROTEIN SEPERATION USING MICROFILTRATION AND ULTRAFILTRATION UNDER ELECTRIC FIELDProtein separations have been carried out using a variety of techniques like microfiltration, ultrafiltration,membrane chromatography, high performance tangential flow filtration.Membrane based methods have been the basis for all types of separation and purification of proteins and other biotechnologically important products. Here we have designed a unique method for the separation of proteins under the influence of an electric field. We hypothesize that the application of an electric field will utilize the charges present on the proteins thus separating them which in turn reduce the time required for their separation and the flux resistance. With the use of electric field, the separation of proteins enhances. Towards this aim we have designed a unique separation apparatus which will separate proteins in a continuous manner, varying the electric field. For this investigation, polyvinylidene fluoride (PVDF) membrane with 0.2 and 0.5 micrometer pore size diameters will be used and proteins will be separated. This apparatus can be used for separation of proteins from whey by microfiltration under influence of electrical field. Effect of various parameters such as voltage, membrane pore size, feed composition and flow rate was observed with and without electric field. It was observed that the concentration of proteins was increased in the presence of electric field and it increased with the increase in voltage.A newly developed type of electric force an electro microfiltration has been used in this study for the separation of protein from whey. Effect of parameters such as voltage, membrane pore size, feed flow rate and composition of feed has been studied. Microfiltration was chosen to increase the permeate flux. Whey Protein is a mixture of globular proteins isolated from whey, the liquid material created as a by-product of cheese production. Whey protein is commonly marketed and ingested as a dietary supplement, and various health claims have been attributed to it in the alternative medical community. Whey is leftover when milk is coagulated during the process of cheese production, and contains everything that is soluble from milk after pH is dropped to 4.6 during the coagulation process. Whey can be denatured by heat . High heat (such as sustained temperatures above 72 associated with the pasteurization process) denatures whey protein. Denaturing the whey protein triggers hydrophobic interactions with other proteins and the formation of a protein gel. The protein in cows milk is 20% whey protein and 80% casein protein whereas the protein in human milk is 60% casein protein and 40%whey protein. The protein fractions in whey constitute approximately 10% of the total dry solids in whey. This protein is typically a mixture of beta-lactoglobulin (65%), alpha-lactalbulin (25%), bovine serum albumin (8%), and rest immunoglobulins. Laboratory experiments have suggested that whey protein and its components might reduce the risk of cancer in animals, suggesting an avenue for future medical research. The use of whey proteins as a source of amino acids and its effect on reducing the risk of diseases such as heart disease, cancer and diabetes has been the focus of ongoing research. Whey is an abundant source of branched chain amino acids (BCAAs) which are used to stimulate protein synthesis.UF is used extensively in the dairy industry; particularly in the processing of cheese whey to obtain whey protein concentrate (WPC) and lactose-rich permeate. In a single stage, a UF process is able to concentrate the whey 10-30 times the feed the original alternative to membrane filtration of whey was using steam heating followed by drum drying or sprays drying. The product of these methods had limited applications due to its granulated texture and insolubility. Existing methods also had inconsistent product composition, high capital and operating costs and due to the excessive heat used in drying would often denature some of the proteins. Compared to traditional methods, UF processes used for this application. Are more energy efficient Have consistent product quality, 35-80% protein product depending on operating conditions Do not denature proteins as they use moderate operating conditionsThe potential for fouling is widely discussed, being identified as a significant contributor to decline in productivity. Cheese whey contains high concentrations of calcium phosphate which can potentially lead to scale deposits on the membrane surface. As a result substantial pretreatment must be implemented to balance pH and temperature of the feed to maintain solubility of calcium salts.The purification of proteins was first studied in membrane process under the influence of an electric field by Young G.Park (2005). An example is presented of the membrane process showing how filtration time was reduced by the use International Journal of Engineering Research & Technology (IJERT) of electric field. Trans membrane pressure was reduced by 20% as electric field was increased. The concentration of proteins in the membrane process in the presence of electric field was reduced by over 300% in comparison with the membrane process without electric field. Hydraulic electro filtration provided another substitute to cross flow filtration for the purification proteins. Electrically enhanced cross filtration for the separation of lactoferrin from whey protein mixture was done by Guillaume Brisson, Michel Britten, Yves Pouliot (2007). The effect of applying an external electric field during lactoferrin(LF) and whey protein solution microfiltration was studied. The impact of electric field strength and polarity on the permeation flux and protein separation were investigated. The influence of LF iron saturation was also assessed. The electrically enhanced microfiltration (EMF) were performed on a purpose built flat sheet module and operated in a full recirculation mode at low trans membrane pressure(0.7105Pa) and feed velocity (0.05m/s). The results showed than application of an electric field had an important impact on protein transmission.

CHAPTER-3LITERATURE SURVEYLR-1, S.M.Chavan,2014,protein separation using microfiltration under electric fieldIntroduction:Protein separations have been carried out using a variety of techniques like microfiltration, ultrafiltration,membrane chromatography, high performance tangential flow filtration.Membrane based methods have been the basis for all types of separation and purification of proteins and other biotechnologically important products. Here we have designed a unique method for the separation of proteins under the influence of an electric field. We hypothesize that the application of an electric field will utilize the charges present on the proteins thus separating them which in turn reduce the time required for their separation and the flux resistance. With the use of electric field, the separation of proteins enhances. Towards this aim we have designed a unique separation apparatus which will separate proteins in a continuous manner, varying the electric field. For this investigation, polyvinylidene fluoride (PVDF) membrane with 0.2 and 0.5 micrometer pore size diameters will be used and proteins will be separated. This apparatus can be used for separation of proteins from whey by microfiltration under influence of electrical field. Effect of various parameters such as voltage, membrane pore size, feed composition and flow rate was observed with and without electric field. It was observed that the concentration of proteins was increased in the presence of electric field and it increased with the increase in voltage.

Result Discussion:A. Effect of VoltageThe experiments were performed by varying voltage applied as 0 V, 10 V, 20 V and keeping membrane size, flow rate and composition of feed constant. The quantitative analysis of fractions collected on either side of equipment was carried out and plotted in terms of concentration on Y- axis vs voltage values occupying X-axis entries. As mentioned elsewhere, two electrodes were used for separation. As expected Proteins were more attracted towards anode predominantly. Our experiment also supports the same. Presence of electric field facilitates the separation of proteins, which has been observed in our experimental studies. As the applied voltage increases, the concentration of proteins in anode side fractions i.e., Right hand side product increases.

B. Effect of Flow RateThe experiments were performed by varying flow rate as 0.1354 l/s and 0.054 l/s and keeping membrane size, composition of feed and voltage applied constant. The quantitative analysis of fractions collected on either side of equipment was carried out and plotted in terms of concentration on Y- axis vs flow rate occupying X-axis entries. Flow rate determines residence time of feed material inside the equipment, during which electric field is applied. As the flow rate decreases, residence time of feed solution inside the equipment increases and thus the time of exposure of feed solution to the electric field increases which results in increase in the concentration of proteins in anode side fractions i.e., Right hand side product increases. And adverse effect has been observed for higher values of flow rate.

C. Effect of Membrane SizeThe experiments were performed by varying membrane size as 0.2 m and 0.5 m and keeping flow rate, composition of flow and voltage applied constant. The quantitative analysis of fractions collected on either side of equipment was carried out and plotted in terms of concentration on Y- axis vs membrane size occupying X-axis entries.As the membrane pore size increases, more amounts of proteins are observed in both the fractions.

D. Effect of Composition of FeedThe experiments were performed by varying composition of feed as 1:2 and 1:4 (whey:water) and keeping membrane size, flow rate and voltage applied constant. The quantitative analysis of fractions collected on either side of equipment was carried out and plotted in terms of concentration on Y-axis vs. composition of feed occupying X-axis entries. On diluting the feed solution, the concentration of proteins decreases.

Conclusion:The separation of proteins increases by the application of electric field as compared to without electric field. It is directly proportional to the applied electric field. The introduction of electric field indicates more concentration of proteins in the Right Hand Side (RHS) of the apparatus ascompared to the Left Hand Side (LHS). This can be attributed to the fact that the electrode present on the RHS of the apparatus is having positive charge (anode) and since most of the proteins are negatively charged, they get attracted towards it and hence we observe large concentration. The concentration of Proteins increases on increasing the pore size of the membrane and less dilution of feed.

LR-2, Young G.Park,2005,Effect of an electric field during purification of protein using microfiltrationIntroduction:The purification of protein was investigated in a membrane process under the influence of an electric field. An example is presented of the membrane process showing how filtration time was reduced by the use of an electric field. Transmembrane pressure was reduced by 20% as the electric field increased. The concentration of protein in the membrane process in the presence of an electric field was reduced by over 300% in comparison with the membrane process without an electric field. Hydraulic electrofiltration provided another substitute to cross flow filtration for the purification of protein.The purification of protein has been widely used in electro dialysis, ultra filtration and microfiltration when comparing studies on the use of conventional membranes for different protein separation. However, they were not used for the purpose of enhancing permeability. The newly developed techniques superimpose additional forces such as pressured hydraulic force. Recently dynamic filtration has represented a further possibility for reducing the surface layer on the membrane of rotating disc filtration.However, the principle disadvantage of this technique is that it cannot be separated in high concentrations of protein. Another superimposed force, that is, an electric field, was induced as a force on charged protein in order to reduce the surface layer of a membrane. A newly developed type of electric force has been invented an electro-microfiltration. Membrane. The current studies were done to test the performance of the new type of electro microfiltration membrane at low hydraulic pressure that induces large amounts of permeates. As such, membrane filtration was investigated based on the membrane properties measured experimentally using cake filtration theory. The microfiltration (MF) technique was chosen to reduce transmembrane pressure (TMP) and to increase permeate flux. The study shows that this membrane system produced with an electro-MF technique offers significant advantages.

Results and discussion:Effect of pressure on microfiltrationThe fouling on the surface mainly consisted of organic aggregates and protein. As discussed previously and in accordance with other works, the protein in the suspension took a determinant part in cake formation. For example, initial fouling rates were significantly decreased while volumetric flow rate increased. When the pump delivered a uniform volume of influent into the membrane, the MF rate remained constant while filtering incompressible material. However, to achieve this constant rate, the pressure delivered had to be increased by 3.5 kgf/cm2 to overcomethe increasing resistance to filtration caused by cake deposition. A constant rate filtration was easily observed on the plot of the filtrate volume using the protein solution relative to time. Experimental results are shown in a graph where the plot of the volume filtrate collected against the filtration time produced a linear line, and all the filtration displayed some form of compressibility in practice; increasing the filtration pressure resulted in an increased filtration time.A plot of the filtration volume against the filtrate time increased linearly. The slopes in the figure for MF, depending upon hydraulic pressures of 2, 2.5, 2.8 and 3.1 kgf/cm2, had values of 0.61, 1.27, 1.28 and 1.61, respectively, which permeated more than 200% in comparison with the highest hydraulic pressure of 3.1 kgf/cm2. The most economical way of removing liquid from an incompressible filter cake is to filter at the lowest pressure required to overcome the fluid drag within the cake, but the filter cake contributed to the decrease of concentration of the protein. The overall removal efficiencies during MF of pore sizes of 0.22 m and 0.45 m had 63.5% and 59.6% in the case of hemoglobin but the removal efficiencies were significantly different, depending upon the pore sizes of the membranes. When the TMP increased, hemoglobin and some aggregated organic matter were deposited on the membrane front face, but this deposit remained insufficient to lead to full coverage of the surface. The membrane experiment shows that the permeate rate was difficult to penetrate in the membrane with a pore size of 0.22 m. The rejection efficiencies in the cross-flow membrane decreased more with the increase of TMP, and these efficiencies appeared very differently depending upon the pore size of membrane. As TMP increased from 1.8 kgf/cm2 to 3.5 kgf/cm2, the rejection efficiencies were decreased by 25.0% in the 0.22 m pore size membrane and 27.1% in the 0.45 m pore size one. For example,the rejection efficiencies in the 0.22 m membrane were 0.7 at 1.8 kgf/cm2 and 0.525 at 3.5 kgf/cm2, but those in the 0.45 m membrane were 0.576 at 1.8 kgf/cm2 and 0.425 at 3.5 kgf/cm2.

Effects of membrane pore size and protein concentration during electro-microfiltrationIf hydraulic pressure is increased, the highly compressible protein layers are compressed. This leads to the effect that an electric field acts along the cake layer. As the electric field is greater, the proteins migration increases. Thus, the electric field and therewith the electrophoretic velocity of the constituents close to the cathode-side membrane are reduced at smaller total concentrations than in electro filtrations with higher pressure. As an electric field increased, the permeate rate was also increased depending upon the size of the electric field. As a result, the permeate velocities were increased by 118.7% and 126.1% in the membrane with a 0.45 m pore size and by 99.0% and 118.7% in the membrane with a 0.22 m pore size as the electric field was increased at 9 V and 15 V, respectively, as shown This indicates that the permeate velocity increased to the direction of an electric field and the amount of permeate also increased. Therefore, the rejection efficiencies significantly decreased as the electric field increased. The TMP was significantly decreased as the electric field increased. For example, the differences of TMP were approximately 20% in the pore sizes between 0.22 m and 0.45 m, respectively. Also, the TMP was significantly varied depending on protein concentration. As the protein concentration increased four times from 0.05 g/L to 0.2 g/L, the TMP was approximately decreased by 20%. When the volume of the permeate as a function of time was measured through the MF membrane at a pressure of 2 kgf/cm2, the amounts of the permeation of 0.05 g/L protein concentration were increased by 334% as the pore sizes of membrane were increased from 0.22 m to 2.5 m. But when the applied electric field was 15 V, the amounts of the permeation of 0.05 g/L protein concentration were increased by 246% when the pore sizes of the membranes were from 0.22 m to 2.5 m. As the protein concentration increased to 0.2 g/L, the amount of permeation in the electro-MF membrane was also about five times greater than that with the MF membrane. This indicates that the electric field induces the enhancement of permeation rate in the membrane, even though the protein concentration increased. By the same previous procedure, the amounts of the permeation for 0.05 g/L and 0.2 g/L hemoglobin concentration in the presence of an electric field were increased by 762%, 544%, 555% and 160%, 435%, 449% in comparison with the absence of an electric field as the pore sizes of membrane were increased 0.22 m, 0.45 m and 2.5 m. This indicates that the permeation rate by an electric field was greatly increased in more porous membranes. Based on Table 2, Rm was calculated from the slope of the extrapolated line in the initial time for the filtration data. The membrane resistance in the electro-MF membrane at 15 V was significantly reduced by 455% to 1.997109 m!1 in comparison with Rm = 9.902109 m!1 during MF whereas the membrane resistances in the electro-MF at 15 V were reduced by 529% and 506% incomparison with the MF membranes with a poremembrane size of 0.22 m and 0.45 m, respectively.This indicates that the membrane resistances in the presence of an electric field were significantly reduced in less porous membranes as shown.The amounts of permeate in the presence of an electric field can be varied depending upon electric fields. When hemoglobin almost penetrated into the membrane, the concentrations of hemoglobin maintained equal amounts during the electro-mF, but hemoglobin was significantly reduced by 45% during MF in the absence of an electric field. Therefore, it is important for the electro-MF membrane to play a significant role in the transport of the protein in the membrane process.

Resistance of filter mediumDarcys law is related to the hydraulic total resistance; thus, the total resistance (Rt) of a porous medium is the sum of the resistance of the membrane (Rm) and that of the cake (Rc) asfollows:

(Rt = Rm + Rc = P/J)

where J is the permeate flux. The membrane resistance in the electro-MF membrane wassignificantly reduced by 485% to 1.997109 m!1 in comparison with that in the membrane at 9,902109 m!1 whereas the total resistance in the electro-MF membrane was reduced by 16.35% in comparison with the MF membrane. The cake resistance was slightly increased in the MF membrane rather than electro-MF because the accumulation rate of the cake was similar between the membranes for electro-MF and MF, and the accumulation of the cake layer is dominated by the hydraulic pressure rather than an electric field. Therefore, it is important for the electro-MF membrane to play a significant role in the transport of the solute in the process.

Effects of pressure electro-microfiltrationYukawa et al. modified Darcys law to account for the electrophoretic and the electro osmotic effect: where PE is the electro osmotic pressure, E is the electric field strength and Ecr is the critical electric field strength. Fig. 5 shows that the permeate rate in the electro-MF membrane can be enhanced by hydraulic pressure on the membrane since the electric field increased from 3 V to 24 V (3 V, 9V, 15 V and 12 V). When the hydraulic pressure was 1.3 kgf/cm2, the permeate rates were not different even though the electric field varied. This indicates that an electric field did not exclusively play a role to enhance the permeability. But as the hydraulic pressure increased with applied electric field, the permeate rates significantly increased. For example, the permeate rates were 269%, 274%, 295% and 327% as an electric field increased from 3 V, 9 V, 15 V and 24 V, respectively, when the hydraulic pressure is 2.1 kgf/cm2, in comparison with an hydraulic pressure of 1.3 kgf/cm2. This indicates that the permeate rate in the electro-MF membrane is enhanced by the hydraulic pressure with an electric field. In cases of hydraulic pressures of 2.8 kgf/cm2 and 3.5 kgf/cm2, the permeate rates were 362%, 476%, 490%, 500% and 394%, 530%, 554% and 568% as the electric field increased from 3 V, 9 V, 15 V and 24 V, respectively, in comparison with the hydraulic pressure of 1.3 kgf/cm2. Therefore, the permeate rate in the pressure electro-MF followed Equation that the permeation rate linearly increased with hydraulic pressure and electric field.

ConclusionsThe permeability of protein was proportional to hydraulic pressure with an electric field, indicating that the protein quickly is oriented in the field direction through the membrane pores.The experimental results can be described as follows. First, TMP was lowered in the electro- MF membrane by 20% in comparison with the conventional MF membrane. Second, the permeate rate of the electro-MF membrane was over 200% higher than the conventional MF membrane. Third, membrane resistance (Rm) in the pore-filled membrane was significantly reduced by 500% in comparison with the conventional MF membrane and resistance in the cake layer was reduced by 16%. Fourth, TMP was approximately decreased by 20% as protein concentration increased four times from 0.05 g/L to 0.2 g/L. Fifth, the amount of hemoglobin was significantly reduced by 45% in MF in the absence of an electric field. The concentrations of protein maintained equal amounts during electro-MF. The study experimentally demonstrated that the permeability inside a membrane can be controlled using an electric field.

LR-3,Arunima Saxena,2005,Membrane based techniques for separation and purification of proteins.Introduction:Membrane processes are increasingly reported for various applications in both upstream and downstream technology, such as microfiltration, ultrafiltration, emerging processes as membrane chromatography, high performance tangential flow filtration and electrophoretic membrane contactor. Membrane-based processes are playing critical role in the field of separation/purification of biotechnological products. Membranes became an integral part of biotechnology and improvements in membrane technology are now focused on high resolution of bioproduct. In bioseparation, applications of membrane technologies include protein production/ purification, proteinvirus separation. This manuscript provides an overview of recent developments and published literature in membrane technology, focusing on special characteristics of the membranes and membrane-based processes that are now used for the production and purification of proteins.

Result Discussion:

Electrically enhanced membrane filtration (EMF) is an advanced technique, which consists in superimposing an electrical field to conventional membrane filtration unit. In EMF, the electrical field acts as an additional driving force to the transmembrane pressure. Accordingly, differences in protein electrophoretic mobility are coupled to the membrane sieving effect to enhance the selectivity of membrane fractionation in EMF. It has been mainly used as a strategy to improve protein solutions permeation flux by preventing concentration polarization and membrane fouling. Furthermore, selectivity enhancement for biomolecules separation (amino acids and peptides) was recently obtained using EMF. However, only few studies reported the effect of EMF on complex protein solutions separation selectivity. The purification and separation of protein has been widely used in electrodialysis, UF and MF when comparing studies on the use of conventional membranes for different protein separation. The newly developed techniques superimposed additional forces such as pressured hydraulic force. Recently dynamic filtration has represented a further possibility for reducing the surface layer on the membrane of rotating disc filtration. However, the principle disadvantage of this technique is that it cannot be used in high concentrations of protein. Another superimposed force, that is, an electric field, was induced as a force on charged protein in order to reduce the surface layer of a membrane. Park et al. studied the purification of protein through membrane process under the influence of an electric field and explained how filtration time was reduced by the use of an electric field. In this case, concentration of protein in the membrane process in the presence of an electric field was reduced by over 300% in comparison with the membrane process without an electric field. For this investigation, polyvinylidene fluoride (PVDF) membrane and hemoglobin as a protein was used. It was observed that an electric field is a superimposed force which, induced as a force on charged protein to reduce the surface layer formation at the membrane interface and that led the development of EMF. Pouliot et al. studied the effect of applying an external electrical field during lactoferrin (LF) and whey protein solutions by MF under influence of electrical field strength (03333 V m1) and polarity on the permeation flux and protein transmission through microfilter membrane with flat-sheet module. In this case, electrical field had an important impact on protein transmission. Selectivity enhancements were obtained, particularly when the cathode was on the retentate side. In that configuration (3333 V m1), the separation factors obtained between LF and the two main whey proteins -LG and -LAwere, respectively, 3.0 and 9.1. PVDF membrane with 0.5 m of pore size diameters was used for this study.

Protein separation by UF:Protein fractionation is rapidly becoming more selective through improvements in membrane and module design. Compared to chromatographic methods, membrane separation techniques offer advantages of lower cost and ease to scale-up for commercial production. However, the lack of membrane selectivity and its fouling due to protein absorption during filtration has severely restricted UF applications. Now, UF has been widely used as preferred method for protein concentration and buffer exchange, and replaced size exclusion chromatography in these applications. UF membranes, based on variety of synthetic polymers, have high thermal stability, chemical resistivity, and restricted the use of fairly harsh cleaning chemicals [4,63]. The choice of membrane was usually guided by its molecular weight cut-off (MWCO), which is defined asthe equivalent molecular weight of the smallest protein that would exhibit above 90% rejection. Although this choice is arbitrary, but it has been adopted by most of the UF membrane user community. However, the experimental conditions and systems used to evaluate 90%MWCO have not been standardized [63]. Hollow fiber, flat-sheet cassettes, spiral-wound cartridges, tubular modules, and enhanced masstransfer devices have been developed for UF. These modules provide physical separation of the retentate and filtrate streams, mechanical support for the membrane (if needed), high membrane packing densities (membrane area per device volume), easy access for cleaning and replacement, and good mass-transfer characteristics. Spiralwound modules are sensitive for plugging/fouling, and are more difficult to clean. However, they have more limited range of scalability than hollow-fiber modules or flat-sheet cassettes. Rotating and Dean Vortex systems have been also developed for UF. These devices showed high mass-transfer coefficients but lower packing densities. Additionally, their scale-up (or down) is difficult due to changes in hydrodynamic conditions. PES is widely used UF membrane material, because of its high rigidity, creep resistance, good thermal and dimensional stabilities. Ghosh et al. studied purification of lysozyme from chicken egg white using hollow-fiber PES UF membrane (30 kDa MWCO). UF of fermented cheese whey broth was also studied using a lab scale cross-flowmembrane system with PES membranes (5, 20 kDaMWCO). Separation of -LG from whey protein was achieved by its fractionation using two-stage UF with PES membrane (30 and 10 kDa MWCO) in stirred rotating disk module followed by ion-exchange membrane chromatography. Other types of polymeric UF membranes such as polyacrylonitrile membrane, regenerated cellulose membrane,cellulose acetate membrane and ceramic membranes etc., were extensively studied for the separation of proteins. A schematic diagram of UF membrane set-up used for protein separation/purifications is shown in fig. Fractionation of dairy wastewater into lactose-enriched and protein-enriched streams using UF membrane technique was also studied. Three regenerated cellulose membranes of 3, 5 and 10 kDaMWCOwere used to determine the efficiency of the process. The performance was determined under various processing conditions that include the operating temperature and transmembrane pressure across and the concentration of lactose in the feed solution.

Conclusion:

Membranes have been traditionally used to separate species of different size such as proteins from cells, fermentation broths, cell debris and separation of low molecular weight components from proteins. Since long it has been an integral part of biotechnology processes, the well known examples are MF and UF, which have become routine methods for protein separation/fractionation. The development of membrane chromatography, HPTFF and electrophoretic membrane contactor enable for the complete purification/separation of proteins using membrane systems. Although not implemented in any commercial processes, small-scale studies using this process show comparable yield, purification, and product quality with a conventional process. Continued efforts to develop improved membrane materials, modules, and process designs should enable membrane systems to play an important role in the next generationof biotechnology processes. New applications of membrane processes continue to emerge, such as membrane biosensors and molecularly imprinted polymeric membranes for separation of molecules. Their industrial success will depend on their advantages over the existing technologies. Thus, deep understanding of physical and chemical phenomena across the membrane interfaces under the operating conditions will help to improve their performance in the biotechnology-based industries. Future trends of membranes in biotechnology will be driven by higher selectivity, lower cost of production, and enhanced membrane throughput. There is also trend towards increased use of disposable systems (bioreactors, ultrafilter membranes, and buffer bags), which are attractive for production scale manufacturing, eliminating the need for the development and validation of cleaning cycles. Future developments will determine whether such a membrane-based process can provide the required product quality, purity, yield and throughput with low cost for biotechnology industry.

LR-4, Prafulla. G. Bansod, V.S.Sapkal,2002, Protein separation from fermentation broth (Ecoli) using polyethersulphone MFand UF membranes.

Introduction:

In this paper, the intracellular Ecoli fermentation broth was developed in fermentor. The broths were separated using 0.2m microfiltration polyethersulfone membranes. The Cells of Ecoli was retained in microfiltration membrane, were broken in high pressure homogenization. The cells debris and protein were separated using polyethersulfone 0.2m PES Microfiltration membranes. In microfiltration, cells of Ecoli were rejected and proteins collected in permeate sides. After microfiltration, 30KD Ultra filtration membrane was used to separate proteins. About 91.01 % of the proteins were separated by the ultra filtration polyethersulfone membrane.

Result Discussions:

Polyethersulfone microfiltration 0.2m membrane was used for concentrating and separating cells of E-coli -fermented broth. Seven liters E-coli -fermented broth [7L] were used as a feed for microfiltration membrane, where cells of E-coli were concentrated (635ml) and 6.325 L as permeated. The concentrated (0.635 L) cells were passed in high pressure Homogenizer, where intracellular proteins given out from the cells. After Homogenization, cell debris and proteins were separated in 0.2m microfiltration membranes.2.185L protein were collected in permeate sides and 0.410L cells debris collected in retained sides of microfiltration membranes. The permeate which were collected in microfiltration were passed to 30KD PES ultra filtration membranes. The total proteins concentration in permeate were estimated by using Folin-Lowry method of protein assay .The proteins concentration of feed to 30KD UF membranes were found845g/ml and in permeated sides 75g/ml.The total proteins rejected was found out to be 91.01 percentages in 30 KD polyethersulfone ultra filtration membranes. The fig. shows that, when transmembrane pressure increased concentrate flow rate decreased, whereas in Fig 2, showed that when transmembrane pressure increased permeate flow rate also increased continuously.After some times, it was found that permeate flow rate decreased with increase in transmembranes pressure. The resistance creates due to fouling and plugging problems of the membrane and permeates flux decreased

Conclusion:Ecoli fermentation broth was developed and separated using 0.2m polyethersulfone microfiltration membranes. The concentrated cells were passed to high pressure homogenizer where intracellular proteins separated out. The cell debris separated using 0.2m PES microfiltration membranes as retained and proteins collected as permeate side. The proteins were separated in 30KD PES ultra filtration membrane. The total 90.01% proteins separated using polyethersulfones UF membranes. Ultra filtration can be successfully used to separate protein and bacteria cells from Ecoli fermentation broth. Cells and proteins were retained by the ultra filtration membrane with MWCO of 30,000 Daltons. Increased transmembrane pressures caused higher permeate flux.

LR-5,G.Akay,1997,Seperation of products from fermentation broth using microfiltration and ultrafiltration.Introduction:Microorganisms are sources of valuable enzymes, proteins and other bio-products. They produce two basic types of biological molecules: extracellular, which are excreted into a growth medium, and intracellular, which are retained inside the cytoplasm of the cells. A variety of host microorganisms have been studied. The most often used organisms are E. coli, S. cerevisiae and Bacillus subtilis. Several other microbial strains have been used for production of microbial enzymes, such as Aspergillus niger and Kluyveromyces fragilis (for production of catalase), Saccharomyces lactis and Kluyveromyces lactis (b-galactosidase), Bacillus coagulans and Streptomyces sp. (glucose isomerase) and Penicilium notatum (glucose oxidase). A large proportion of potentially useful microbial products is retained within the cells of the microorganisms. The isolation of intracellular molecules requires either the cell to be genetically engineered (so that intracellular molecules can be excreted into the growth medium) or the cells must be disintegrated by physical, chemical or enzymatic means to release their cytoplasmic content. A major application of MF in biotechnologies is the recovery of intracellular molecules produced from fermentation broth [66]. In typical processes, fermentation is followed by cell harvesting, where the cells are concentrated into a paste by centrifugation or MF. The paste is then diluted in a buffer and homogenized by shear impingement or milling to rupture the cell walls and release the product. The overexpressed molecule needs to be separated from the cell debris in the resulting lysate before it is introduced to the down-stream purification process. Changes in fermentation media affect not only the performance of the fermentation itself (with regard to the kinetics of biomass and product formation and the yields obtained) but also the initial product recovery operations downstream of the fermentor. In the case of crossflow MF, this may occur due to a reduction in permeate flux or reduced transmission of the target molecule through the membrane. In addition, several studies have observed the effects of individual media components on MF operations, for exampleantifoams and oils.ProteinsCross-flow MF may be used to recover soluble proteins from the cellular debris and other insoluble components. As reviewed by Belfort et al. however, a rapid flux decline occurs initially due to cake formation, followed by a relatively slow decline due to protein fouling of the cake and membrane. Moreover, the proteins are also retained by the microporous membrane and the overlying cake layer of rejected solids, so that the protein transmission coefficient is typically less than unity. For example, Parnham and Davis investigated MF of bacterial cell debris and obtained low long-term permeate fluxes in the range 10_410_3 cm s_1, with an average total protein transmission of only 60%. Low protein transmission values are undesirable, as they represent a significant reduction in yield of valuable product. Backflushing is one approach to the problem in which the permeate is periodically forced back through the membrane in the reverse direction to normal permeate flow in order to flush out the accumulated fouling material from the membrane pores and the membrane surface. Proteins recovered by MF from fermentation broth include heterogeneous IgG from transgenic goat milk and naturally glycosylated therapeutic proteins produced from animal cell cultures. The work of Baruah and Belfort on the optimization of monoclonal antibodies recovery from transgenic goat milk by MF is an interesting example. The optimization involved varying pH, TMP, milk feed concentration, membrane module type and axial velocity. Operation in the pressure-dependent regime at low uniform TMPs using permeate circulation in co-flow, at the pI of the protein (9 in this case) was shown to increase IgG recovery from less than 1% to over 95%.Fig shows, for example, the effect of axial velocity evaluated with a short helical module at pH 9.0. A yield of 95% was obtained for velocities _0.95 m/s. This method was claimed to be generalizable to the recovery of target proteins found in other complex suspensions of biological origin.

AntibioticsOne of the main advantages of membrane systems for the initial recovery of antibiotics from a fermentation broth is the ability to obtain very high yield using a combined filtration and diafiltration process. Complete retention of the cells and particulate matter can be achieved using membranes with pores sizes up to 0.2 0.45 mm, and essentially complete passage of the antibiotics can be achieved as long as the nominal MWCO is greater than about 20 kDa. Davies et al. showed that a change of medium composition affected the production of an antibiotic, erythromycin, and its transmission and rate of flow through the membrane used to separate the biomass from whole fermentation broths. As a model system, the authors studied the growth of Saccharopolyspora erythraea (a spore forming, Gram-positive, hyphae-producing bacterium) on both a soluble complex medium and an oil-based process medium. These systems were then processed using a Minitan II (Millipore) cross-flow filtration module, fitted with a single 60 cm2 Durapore (Millipore) hydrophilic 0.2 mm membrane, operated in concentration mode. The media composition changed throughout the period of the fermentation as media components were utilized and biomass growth took place. Residual glucose and oil concentrations are shown in fig. The variation of permeate flux and erythromycin transmission as a function of fermentation time was also investigated.

Lactic acidLactic acid and lactate salts are widely used in the food industry as acidulants, preservatives or flavour enhancers. In the food industry, the natural grade of lactic acid of biological origin makes it more attractive as a food additive than that of chemical origin. However, the production of lactic acid from fermentation requires the use of an efficient and economic downstream process to recover lactic acid and isolate it from various impurities present in the fermentation broth .In some studies, UF membranes have been used for separation of microbial cells and protein molecules from lactic acid fermentation broth. However, it was observed that UF membrane in the first step of separation, presented serious fouling problem. The use of MF in the first step was shown to be more effective in subsequent downstream purification.PolysaccharidesPolysaccharides are increasingly used in many industrial applications, for instance as thickening or gelling agents (agar) in the food industry. Polysaccharides produced by cultures of microorganisms (also called biopolymers) constitute a promising alternative to petrochemical and vegetal polymers because they are produced from renewable resources and their characteristics can be adjusted by controlling cell culture conditions in fermentors. Some authors investigated the extraction of polysaccharides from fermentation broths of Sinorhizobium meliloti M5N1CS using crossflow filtration through ceramic membranes of various pore sizes from 0.1 to 0.8 mm.The most interesting results were obtained with dynamic filtration (0.2 mm nylon membrane using a rotating disc filter) because it allowed operation at highshear rates and low TMP. Sieving coefficients remained between 90 and 100%.

Other productsOther products recovered from fermentation broth include yeast alcohol dehydrogenease (ADH) from bakers yeast rBDNF inclusion bodies from E. coli cell suspensions by cross-flow MF and diafiltration and the separation of hyaluronic acid from fermentation broth by cross-flow MF and UF .