DIALYSIS

Dialysis is a separation technique that facilitates the removal of small, unwanted compounds from
macromolecules in solution by selective and passive diffusion through a semi-permeable membrane. In
dialysis separation of small molecules and protein based upon their size. A sample and a buffer solution
(dialysate) are placed on opposite sides of the membrane. Sample molecules that are larger than the
membrane-pores are retained on the sample side of the membrane, but small molecules and buffer salts
pass freely through the membrane, reducing the concentration of those molecules in the sample. Changing
the dialysate buffer removes the small molecules that are no longer in the sample and allows more
contaminants to diffuse into the dialysate. In this way, the concentration of small contaminants within the
sample can be decreased to acceptable or negligible levels.

Mainly used membranes are such as a cellulose membrane with pores.

Dialysis works by diffusion, a process that results from the thermal, random movement of molecules in
solution and leads to the net movement from areas of higher to lower concentration (until an equilibrium
is reached). The bag is filled with a concentrated solution containing proteins. Molecules that are small
enough to pass through the pores of the membrane diffuse out of the bag into the buffer solution, or
dialysate. Dialysis is sometimes used to change buffers. The molecules go from an area of high
concentration to low concentration. When the level of concentration is equal between the bag and the
buffer, there is no more net movement of molecules. The bag is taken out and inserted into another buffer,
causing the concentration to be higher in the bag relative to the buffer. This causes more diffusion of
molecules. This process is repeated several times to ensure that all or most of the unwanted small
molecules are removed (usually done overnight). In general, dialysis is not a means of separating proteins,
but is a method used to remove small molecules such as salts. At equilibrium, larger molecules that are
unable to pass through the membrane remain inside the dialysis bag while much of the small molecules
have diffused out.

ION EXCHANGE CHROMATOGRAPHY

Ion exchange chromatography is one of the most efficient methods for the separation of charged particles.

Ion exchange chromatography is most often performed in the form of column chromatography. However,
there are also thin-layer chromatographic methods that work basically based on the principle of ion
exchange. In the following, we will exclusively deal with column chromatographic applications.
Column materials used for ion exchange chromatography contain charged groups covalently linked to the
surface of an insoluble matrix. When suspended in an aqueous solution, the charged groups of the matrix
will be surrounded by ions of the opposite charge. In this “ion cloud”, ions can be reversibly exchanged
without changing the nature and the properties of the matrix.

The charged groups of the matrix can be positively or negatively charged. A positively charged matrix
will bind negatively charged ions from the solution. Therefore, it is called an anion exchanger. Cation
exchanger matrices have negative charges.

Based on the structure of the ion exchange matrix, we distinguish ion exchangers with hydrophobic and
hydrophilic matrices. Ion exchangers with a hydrophobic matrix are most often highly substituted
polystyrene resins. These are suitable for the binding of inorganic ions, e.g. in water softening
applications. However, they tend to denature proteins due to the high hydrophobicity of their matrix and
their high surface charge density.

Ion exchangers with hydrophilic matrices were first produced from modified cellulose. However,
cellulose has disadvantageous mechanical properties: cellulose fibres are prone to break, making it
difficult to create a well-utilisable column. This disadvantage has been partially remedied in Sephadex
(dextran-based) ion exchange matrices.

MEMBRANE REACTOR

A membrane reactor (MR) is a device for simultaneously performing a reaction and a membrane-based
separation in the same physical device. A membrane reactor is really just a plug-flow reactor that contains
an additional cylinder of some porous material within it. The main requirement for a membrane reactor
(MR) is a semi permeable membrane which allows the free passage of the product molecules but contains
the enzyme molecules. The membrane is a barrier that only allows certain components to pass through it.
The selectivity of the membrane is controlled by its pore diameter.

The usual choice for a membrane reactor is a hollow-fibre reactor consisting of a preformed module
containing hundreds of thin tubular fibres each having a diameter of about 200 mm and a membrane
thickness of about 50 mm. Membrane reactors may be used in either batch or continuous mode and allow
the easy separation of the enzyme from the product. They are normally used with soluble enzymes,
avoiding the costs and problems associated with other methods of immobilisation and some of the
diffusion limitations of immobilised enzymes.

If the substrate is able to diffuse through the membrane, it may be introduced to either side of the
membrane with respect to the enzyme, otherwise it must be within the same compartment as the enzyme,
a configuration that imposes a severe restriction on the flow rate through the reactor, if used in continuous
mode. Due to the ease with which membrane reactor systems may be established, they are often used for
production on a small scale (g to kg), especially where a multi-enzyme pathway or coenzyme
regeneration is needed. They allow the easy replacement of the enzyme in processes involving
particularly labile enzymes and can also be used for biphasic reactions. The major disadvantage of these
reactors concerns the cost of the membranes and their need to be replaced at regular intervals.

Membrane reactors are commonly used in dehydrogenation reactions (e.g., dehydrogenation of ethane),
where only one of the products (molecular hydrogen) is small enough to pass through the membrane. This
raises the conversion for the reaction, making the process more economical.

CONCEPT OF EFFECTIVENESS FACTOR

It is defined as the rate with diffusion limitation versus the rate without diffusion limitation. The rate of a
reaction catalysed by an immobilised enzyme (v) is normally lower than the rate due to the same amount
of free enzyme in solution (vfree).

This is due to the controlling necessity for the substrate to diffuse from the bulk phase to the catalytic
surface.

The substrate concentration within the microenvironment ([S]) is lower than that in the bulk ([S0]) due to
its depletion by the reaction.

The change in reaction rate can be expressed quantitatively by introducing the effectiveness factor (η),
where:
 η= v/vfree

The effectiveness factor generally lies between 0 and 1 and is dependent on the bulk substrate
concentration, amongst other factors. It may sometimes be greater than unity due to non-isothermal
operation, because of partition or inhibitory effects, or if the immobilised enzyme is stabilised relative to
the free enzyme over the time course of its assay.

Catalytic reactions take place on the exposed surface of a catalyst. Consequently, a higher surface area
available for the reaction yields a higher rate of reaction. It is therefore necessary to disperse an expensive
catalyst on a support of small volume and high surface area. However, use of such a supported catalyst in
the form of a pellet is not without its drawback. Reactants have to diffuse through the pores of the support
for the reaction to take place, and therefore, the actual rate can be limited by the rate at which the
diffusing reactants reach the catalyst. This actual rate can be determined in terms of intrinsic kinetics and
pertinent physical parameters of the diffusion rate process. Thiele was one of the first to use the concept
of an effectiveness factor. He defined the effectiveness factor as:

𝜂 = 𝑔𝑙𝑜𝑏𝑎𝑙 𝑟𝑎𝑡𝑒 / 𝑖𝑛𝑡𝑟𝑖𝑛𝑠𝑖𝑐 𝑟𝑎𝑡𝑒

By definition, the global rate is simply the intrinsic rate multiplied by the effectiveness factor. In order to
obtain an expression for the effectiveness factor, conservation equations for the diffusion and reaction
taking place in a pellet are normally solved. The effectiveness factor has been popularly used for
estimating the efficiency of catalytic particles when a catalytic reactor is designed. Wijngaarden et al. [4]
pointed out that there are three main aspects in which the conversion rate inside the porous catalyst
depends. These are: a) Micro properties of the catalyst pellet; the most important being pore size
distribution, pore tortuosity, diffusion rate of the reaction components in the gas phase, and diffusion rate
of the reacting components under Knudsen flow. b) Macro properties which include size and shape of the
pellet, and possible occurrence of anisotropy of the catalyst pellet. c) Reaction properties such as reaction
kinetics, number of reactions involved, and complexity of the reaction scheme under consideration. The
micro properties cannot be determined easily. Moreover, due to the complexity of diffusion of the
reactions in a solid matrix, the micro properties are usually accounted for by a lumped parameter, the so-
called effective diffusion co-efficient, De . For solid catalyst particles, this approach has proved to be very
useful, provided that the particles can be regarded as homogenous on a micro scale. Here it is assumed
that it is possible to use the concept of an effective diffusion co-efficient without too large error. Hence,
the effect of micro properties is not usually of much concern as it is assumed that the value De is known.
The discussion is restricted usually to the impact of the macro properties and reaction properties on the
effectiveness factor.

Calculation of Effectiveness Factor: Calculations of the effectiveness factor normally involve

dimensionless numbers. Most common among these numbers are: Thiele modulus (Ø), Arrhenius
number, γ, and the heat of reaction parameter, β. Wijngaarden et al. [4] has however introduced two other
quantities called zeroth Aris number (An0) and first Aris number (An1). The earlier ones are presented
below.

Amperometric Biosensors:

These electrodes function by the production of a current when potential is applied between two

electrodes, the magnitude of current being proportional to the substrate concentration.

During amperometric measurement, a constant potential is maintained between a working

electrode and a reference electrode.

The current generated by oxidation or reduction of an electroactive species at the surface of the

working electrode is measured.

The signal generated is thus dependent on the mass transfer of the electroactive species to the

electrode surface, so that a continuous use may cause fowling of electrode surface, causing loss

of sensitivity.

Oxido- reductases are the best suited enzymes for these biosensors, since electron transfer is

involved in transduction.

The simplest amperometric biosensors make use of Clarks oxygen electrode, which determines

the quantity of oxygen (present in the analyte) reduced. These are described as first generation

amperemetric biosensors. However, the reaction depends on concentration of O2 in the solution

and to overcome this problem, mediators like ferrocenes (they transfer electrons to electrodes,

rather than reducing O2) have been used leading to the production of second generation

biosensors. However, the latest third generation amperometric biosensors remove the electrons

directly from the reduced enzymes (without the help of mediators) and the electrodes are coated

with electrically conducting organic salts.

Figure 2: Schematic diagram of a simple amperometric biosensor. A

potential is applied between the central platinum cathode and the

annular silver anode. This generates a current (I) which is carried

between the electrodes by means of a saturated solution of KCl. This

electrode compartment is separated from the biocatalyst (here shown

glucose oxidase) by a thin plastic membrane, permeable only to

oxygen. The analyte solution is separated from the biocatalyst by

another membrane, permeable to the substrate(s) and product(s).

This biosensor is normally about 1 cm in diameter but has been

scaled down to 0.25 mm diameter using a Pt wire cathode within a silver plated steel needle

anode and utilising dip-coated membranes.

Submerged Fermentation:

Batch Culture:

Batch culture is a closed culture system, which contains limited amount of nutrient medium. After

inoculation, the culture enters lag phase, during which there is increase in the size of the cells and not in

their number. The culture then enters lag phase or exponential growth phase during which cells divide at a

maximal rate and their generation time reaches minimum.

The increasing population of bacterial cells, after sometime, enters into a stationary-phase due to

depletion of the nutrients and the accumulation of inhibitory end products in the medium. Eventually, the

stationary, phase of bacterial population culminates into death-phase when the viable bacterial cells begin

to die.
If we collect data of the increase in cell number at various intervals of time and plot this data in two ways

(logarithm of number of bacteria and arithmetic number of bacteria versus time), we find a characteristic

growth curve (Fig. 14.9). This typical growth curve is only obtained in a batch culture.

Fed-Batch Culture:

When a butch culture is subsequently led with fresh nutrient medium without removing the growing

microbial culture, it is called fed-batch culture. Fed-batch culture allows one to supplement the medium

with such nutrients that are depleted or that may be needed for the terminal stages of the culture, e.g.,

production of secondary metabolites.

Therefore, the volume of a fed- batch culture increases with time. Fed-batch cultures achieve higher cell

densities than batch cultures. It is used when high substrate concentration causes growth inhibition. It

allows the substrate to be used at lower nontoxic levels, followed by subsequent feeding. It allows the

maximum production of cellular melabolities by the culture.

Continuous Culture:

Contrary to the batch culture where the exponential growth of microbial population is restricted only for a

few generations, it is often desirable to maintain prolonged exponential growth of microbial population in

industrial processes.
This condition is obtained by growing microbes in a continuous culture, a culture in which nutrients are

supplied and end products are continuously removed. A continuous culture, therefore, is that in which the

growth of bacterial population can be maintained in a steady state over a long period of time.

CHARACTERISTICS OF THE ENZYME.

 All enzymes are protein
 Enzymes are easily influenced by environmental changes
 Enzymes possess great catalytic power.
 Enzymes are highly specific. It shows varying degree of specificities.
 It is sensitive and labile to heat
 It is water soluble
 Like proteins enzymes can be coagulated by alcohol, heat, concentrated acids
and alkaline reagents.
 The rate of reaction involving an enzyme is high at optimum temperature and
pH.

DIFFERENCE BETWEEN THE TYPES OF ENZYME INHIBITORS.

COMPETITIVE NON COMPETITIVE UN COMPETITIVE

Bind specifically at the
Other than catalytic site Other than catalytic site
catalytic site.
Bind to E Binds to E or ES Only binds to ES
Cannot be reversed by Cannot reversed by
Reversible by substrate
substrate inhibition
Decreased by [1 + Decreased by [1 +
Vmax unchanged
([I0]+Ki)] ([I0]+Ki)]
Km increased by Decreased by [1 +
Unchanged
[1 + ([I0]+Ki)] ([I0]+Ki)]
Substrate & inhibitor
Different shape Different shape
same shape
Chemically similar Not similar Not similar
Doesnot change shape
Change shape Change shape
of active site
Eg: Succinate Eg: Inhibition of
Eg: Pyruvate kinase
dehydrogenase is arylsulphatase by
inhibited by alanine
inhibited by malonate hydrazine.
VARIOUS FACTORS AFFECTING ENZYME ACTIVITY.

1. Enzyme Concentration – concentration of enzyme increases the reaction rate also

increases.
2. Substrate Concentration – reaction rate increases with increase in substrate
concentration.
3. Inhibitors – molecules which reduce enzymatic activity. Different type of
inhibitors.
4. Temperature – at optimum temperature maximum number of collisions taking
place. If temperature increase beyond optimum Temp increased energy level of
molecules disrupts bonds in enzyme & between enzyme & substrate and
denaturation occurs. At colder temperature movement of molecule is slower and
reaction rate will be low.
5. pH - Enzymes are affected by changes in pH. The most favorable pH value - the
point where the enzyme is most active - is known as the optimum pH. Extremely
high or low pH values generally result in complete loss of activity for most
enzymes. pH is also a factor in the stability of enzymes.

FLUIDISED BED REACTOR

 In this type of reactor, a fluid (gas or liquid) is passed through a solid granular
material (usually a catalyst possibly shaped as tiny spheres) at high
enough velocities to suspend the solid and cause it to behave as though it were a
fluid. This process, known as fluidization.
 The solid substrate (the catalytic material upon which chemical species react)
material in the fluidized bed reactor is typically supported by a porous plate, known
as a distributor.
 The fluid is then forced through the distributor up through the solid material.
 At lower fluid velocities, the solids remain in place as the fluid passes through the
voids in the material. This is known as a packed bed reactor.
 As the fluid velocity is increased, the reactor will reach a stage where the force of the
fluid on the solids is enough to balance the weight of the solid material.
 Thi stage is known as incipient fluidization and occurs at this minimum fluidization
velocity.
 Once this minimum velocity is surpassed, the contents of the reactor bed begin to
expand and swirl around much like an agitated tank or boiling pot of water.
 The reactor is now a fluidized bed.
 Depending on the operating conditions and properties of solid phase various flow
regimes can be observed in this reactor.
Advantages
 Uniform Particle Mixing
 Uniform Temperature Gradients
 Ability to Operate Reactor in Continuous State

Disadvantages
 Increased Reactor Vessel Size
 Pumping Requirements and Pressure Drop
 Particle Entrainment
 Lack of Current Understanding
 Erosion of Internal Components
 Pressure Loss Scenarios