DOWNSTREAM PROCESS

1. AQUEOUS 2 PHASE EXTRACTION

2. MEMBRANE FILTRATION

Downstream processing refers to the recovery and purification of biosynthetic products, 1. 2. 3. 4. Removal of insolubles: Product Isolation:Product Purification:Product Polishing:-

Downstream processing is an integral part of any product development, and the final cost of the product depends largely on the cost incurred during extraction and purification techniques.

The conventional techniques used for product recovery, for example precipitation and column chromatography, are not only expensive but also result in lower yields.

 Furthermore solid liquid separation by filtration or centrifugation results in some technical difficulties, Example filter fouling and viscous slurries. Therefore, there is an ongoing need for new, fast, cost-effective, ecofriendly simple separation techniques. Thus, for separation of biomolecules, AQUEOUS TWO PHASE SYSTEMS (ATPS) offer an attractive alternative that meets the above-mentioned requirements as well as the criteria for industrially compatible procedures The advantage of using this technique is that it substantially reduces the number of initial downstream steps & clarification, concentration, & partial purification can be integrated in one unit.

 Furthermore, scale-up processes based on aqueous two phase systems are simple & a continuous steady state is possible. Aqueous two-phase system was developed in Sweden during mid1950s for the separation of macromolecules, cells & organelles. These systems were initially applied to the separation of organelles & viruses. During the last two decades, lot of work has been done to develop feasible separation processes using aqueous two phase systems for various biological materials, and proteins and recombinant proteins.

 Two-phase aqueous systems have been known since the late nineteenth century, and a large variety of natural and synthetic hydrophilic polymers are used today to create two (or more) aqueous phases. Phase separation occurs when hydrophilic polymers are added to an aqueous solution, and when the concentrations exceed a certain value two immiscible aqueous phases are formed. Settling time for the two phases can be prolonged, depending on the components used and vessel geometry. Phase separation can be improved by using centrifugal separators & other novel techniques

Many systems are available:

 Since these phase components are inert towards biological materials, these can therefore be employed for partitioning of biomolecules, cell organelles and whole cells also. The basis of partitioning depends upon surface properties of the particles and molecules, which include size, charge, and hydrophobicity. More-over, the most characteristic feature of the two-phase system is that the water content in it is as high as 8599%, which when complemented with suitable buffers and salts results in providing a suitable protection for biological materials, as well as in an easy scale-up possibilities.

In addition, the low surface tension between the two phases results in partitioning of proteins possible without any loss in their activity.

 Partitioning of the two phases is a complex phenomenon, taking into account the interaction between the partitioned substance and the component of each phase. A number of different chemical and physical interactions are involved, for example hydrogen bond, charge interaction, van der Waals forces, hydrophobic interaction and steric effects. Moreover the distribution of molecules between the two phases depends upon the molecular weight and chemical properties of the polymers and the partitioned molecules of both the phases. Thus, the distribution of molecules between the two phases is characterized by the partition coefficient, Kpart, defined as the ratio of the concentrate in the top (Ctop) and bottom (Cbottom) phase, respectively.

 The ability of a certain substance to partition in such a phase system maybe described as the sum of different factors which can be expressed as:

where Kelectrical, Khydrophobic, Khydrophilc,& Kconformation denote partition factors due to electrical, hydrophobic, hydrophilic and conformation effects respectively.

Thus, different factors of the system can be manipulated in order to achieve the desired effect.

 Aqueous two phase systems meet all the characteristics of a ideal extraction technology, specially for proteins, since it is less time consuming and has the potential to give high yield and high purity, involving low investment, less energy, and lower labour costs. However, it is not being commercially exploited, since most of the phase-recycling processes have not been defined. To adopt this technique for large-scale processing involves developing recycling operations to make it economically feasible. This would also save cost of effluent treatment and diminish environmental load.

Applications
Apart from the large-scale purification of extra cellular proteins, the aqueous two phase systems can be applied to the following as well: (i) Separation of membrane proteins, for example cholesterol oxidase and bacteriorhodopsin (ii) For structural analysis of the biological materials such as thylakoid membranes (iii) For the concentration and purification of viruses (iv) Bioremediation.

Membrane Filtration:Basic Principles of Membrane Filtration:A membrane filtration process is defined by two basic criteria: 1. The filtration system must be a pressure- or vacuum-driven process and remove particulate matter larger than 1 mm using an engineered barrier, primarily via a size exclusion mechanism. 2. The process must have a measurable removal efficiency of a target organism that can be verified through the application of a direct integrity test.

 The common membrane classifications are.., Micro filtration (MF), Ultra filtration (UF), Nano-filtration (NF), and Reverse-osmosis (RO) as well as Membrane cartridge filtration (MCF) device The most common membrane module configurations are.., 1. 2. Hollow-fiber (consisting of hollow-fiber membrane), Spiral wound (consisting of flat sheet membrane wrapped around a central collection tube), and 3. Cartridges (consisting of flat sheet membrane, that is often pleated to increase the surface area).

Membrane Materials & Systems: There are a number of different types of membrane materials, modules, and associated systems that are utilized by the various classes of membrane filtration. In general, MF and UF use hollow-fiber membranes NF and RO use spiral-wound membranes. MCF systems use flat sheet material configured into a cartridge filtration device. The terms hollow-fiber, spiral-wound, tubular and ceramic refer to the module in which the membrane media is made.

 There are three types of construction that are commonly used in production of membranes: symmetric, asymmetric and composite. Symmetric membranes are constructed of a single (i.e.,homogeneous) material, while composite membranes use different (i.e., heterogeneous) materials.

 Asymmetric membranes may be either homogeneous or heterogeneous. In symmetric membrane, the membrane is uniform in density or pore structure throughout the cross-section, while in an asymmetric membrane there is a change in density of the membrane material across the cross sectional area.

Membrane Materials: The membrane material refers to the substance from which the membrane itself is made. Normally,the membrane material is manufactured from a synthetic polymer, although other forms, including ceramic & metallic "membranes, may be available. The material properties of the membrane may significantly impact the design and operation of the filtration system.

 Mechanical strength is another consideration, since a membrane with greater strength can withstand larger transmembrane pressure(TMP) levels allowing for greater operational flexibility and the use of higher pressures with pressure-based direct integrity testing Material properties influence the exclusion characteristic of a membrane as well. A membrane with a particular surface charge may achieve enhanced removal of particulate or microbial contaminants of the opposite surface charge due to electrostatic attraction. In addition, a membrane can be characterized as being hydrophilic (i.e., water attracting) or hydrophobic (i.e., water repelling). Each of these materials have different properties with respect to surface charge,degree of hydrophobicity, pH & oxidant tolerance, strength, and flexibility.

 MF and UF membranes may be constructed from a wide variety of materials: Cellulose acetate (CA), Polyvinylidene fluoride (PVDF), Polyacrylonitrile (PAN), Polypropylene (PP), Polysulfone (PS), Polyethersulfone (PES), or other polymers. NF and RO membranes are generally manufactured from cellulose acetate or polyamide materials (and their respective derivatives), and there are various advantages and disadvantages associated with each. While cellulose membranes are susceptible to biodegradation & must be operated within relatively narrow pH range of about 4 to 8, they do have some resistance to continuous low

Micro filtration (MF): Micro Filtration is a pressure-driven process that separates micron or submicron particles from the liquid or gaseous feed stream with the help of a membrane MF membranes are generally considered to have a pore size range of 0.1 0.2 m (nominally 0.1 m), although there are exceptions, with pores sizes of up to 10 m exist. The operating conditions are 550 psi (0.33.3 bar) pressure and 36 m/s cross flow velocities. MF is carried as a multistage (stages-in-series) operation in industrial scale & can efficiently removes starch, bacteria, molds, yeast, and emulsified oils.

Ultra filtration (UF): Ultra filtration is a low-pressure operation. UF membranes are used in numerous industries for concentration and clarification of large process streams,where in higher molecular weight compounds are retained while passing solvents and low molecular- weight compounds. For UF, pore sizes generally range from 0.01 0.05 m (nominally 0.01m) or less & in terms of pore size the lower cutoff for a UF membrane is approximately 0.005 m. Because some UF membranes have the ability to retain larger organic macromolecules, they have been characterized as molecular weight cutoff (MWCO) membranes.

 Because organic macromolecules are morphologically difficult to define and are typically found in solution rather than as suspended solids, it may be convenient in conceptual terms to use a MWCO rather than a particular pore size to define UF membranes. The concept of the MWCO (expressed in Daltons a unit of mass) is a measure of the removal characteristic of a membrane in terms of atomic weight (or mass) rather than size Typical MWCO level for UF membrane ranges from 10,000500,000 Daltons.

Because the flux through such a membrane is inversely proportional to its thickness, asymmetric membranes are used where the membrane(~0.3 m thick) is supported by a mesh around 0.3 mm thick.

 When considering the feasibility of ultra filtration it is important to remember that factors other than the molecular weight of the solute affect the passage of molecules through the membranes There may be concentration polarization caused by accumulation of solute at the membrane surface which can be reduced by increasing the shear forces at the membrane surface either by conventional agitation or by the use of a cross-flow system. Secondly a slurry of protein may accumulate on the membrane surface forming a gel layer which is not easily removed by agitation. Formation of the gel layer may be partially controlled by careful choice of conditions such as pH. Finally, equipment and energy costs may be considerable because of the high pressures necessary; this also limits the life of ultra filtration membranes

Nanofiltration (NF): The NF membrane rejects divalent ions(like Ca+2 or SO4 -2) while allowing a majority of monovalent ions (like Na+ or Cl-)to pass. Also, organic molecules in the 200300 molecular weight ranges are rejected.
The typical range of MWCO for NF is between 200-1000 Daltons.

NF is used in the municipal drinking water plants and dairy industry for cheese whey desalting. Since NF is more cost effective than reverse osmosis (RO) in certain applications, new growing markets include RO pretreatment, pharmaceutical concentration, kidney dialysis units.

Reverse Osmosis (RO): Reverse osmosis membranes concentrate low-molecular-weight organic materials and salts while allowing water and solvents to pass through. During the operation, water flows from the concentrated feed stream to the dilute permeate a direction that is just the reverse of what would occur naturally during osmosis. High pressures of the order of 35100 atm are maintained in order to overcome the high osmotic pressures across the membrane. RO membrane is widely used technology for desalinating water & the membranes also reject organic molecules (mol wt >150) such as sugars based on its size & shape.

 Reverse osmosis (Figure Next page), is the reversal of the natural osmotic process, accomplished by applying pressure in excess of the osmotic pressure to the more concentrated solution. This pressure forces the water through the membrane against the natural osmotic gradient, thereby increasingly concentrating the water on one side (i.e., the feed) of the membrane and increasing the volume of water with a lower concentration of dissolved solids on the opposite side (i.e., the filtrate or permeate). The typical range of MWCO level is less than 100 Daltons for RO. The required operating pressure depends on membrane properties and temperature, and can range from less than 100 psi for some NF applications to more than 1,000 psi for seawater desalting using RO.

Benefits of Membrane Filtration: MF offers an unique advantage that it is a process that can take place while temperatures are low. It is a process that does not require much energy and thus, energy costs are low. The process just requires energy to pump liquids through the membrane. This is far too low when compared to the total amount of energy required for a technique such as evaporation & the process can easily be expanded/scaled up. Process management of membrane filtration systems is simple. Membrane filtration systems can be managed in both dead-end flow as well as cross-flow.