ASSIGNMENT 1

Topic: IMMOBILIZED
MICROORGANISMS
AND THEIR APPLICATIONS

Submitted to Er Mukul Kumar

Submitted by Divyanshi
BTech Food
Technology
150713
INTRODUCTION:

Immobilization is as an action to stop or prevents something or someone from moving freely

or functioning normally as is it does by any mean possible

For an example, an action to stop a vehicle from moving is a work of immobilization. Thus
immobilization method can be regarded as a technique to stop an agent using a suitable
immobilizer.

In science, especially in organic chemistry, immobilization is an area of study, which is

focused on enzyme, cell, molecule and even atom immobilization utilizing various kind of old
and new immobilizer biomaterial.

The immobilization study also explored new technology, technique, preparation, application,
analysis and knowledge on the interaction between immobilizer and its immobilization agent.

However, in medical lingo, immobilization is something, which is related to a form of

movement disability.

Immobilization occurs naturally in microscopic life, after scientist discovers the phenomena
of immobilization in biology, we began to recognize its existence in nature and appreciate
its benefits to the diversity of life.

Naturally occurring immobilization can be observed in the form of microorganism that live
by adhering on the surface, known as biofilm.

Biofilm microorganisms were discovered and studied in the 1940s and twenty years later
its benefits are understood and its advantage began to be emulated. Biofilm can be
observed ubiquitously where microorganisms live: on the rock surface, on the skin and most
of other habitable surfaces for microorganism adhesion and growth.

Immobilization study begin as early as 1916 to 1940s, its development over the next each
decades were tremendous and described thoroughly as underdeveloped, developing,
developed, post-development and in the exciting phase now in the 21 century .

Nowadays, in the 21st century, latest, sophisticated and complex immobilization methods
have been explored.
The discovery of nanotechnology has accelerated research and development in
immobilization technology tremendously. The fact that smaller molecule can be arrested
and manipulated unlocked new ways to deal with it and solved an age-old problem in
immobilization previously staggered.

In biotechnology, immobilization is quite an important and greatly explored technology

because of its benefits and advantage in the related industry such as fermentation
system, medicine, pharmaceutical, diagnostics, food, beverage and energy production,
supplement, biosensor, environmental and water treatment. These are some of the
applications of immobilization, which has been proposed and studied so far. The list is
growing, as other application of immobilization will be discovered soon as more research
and development progress.

Immobilization of microbial cells in biological processes can occur either as a natural

phenomenon or through artificial process.

First immobilization technology : amino acylases by aspergillus oryzae for the production of
L amino acids in Japan.

Two main advantages of enzyme immobilization: Increased functional efficiency and

enhanced reproductivity.

NEED FOR IMMOBILIZATION

Accelerates the chemical reaction.

Cost effectiveNot difficult to separate
Attachment to polymers/matrix
Causes re-use.

TYPES OF IMMOBILIZATION

Immobilization involves the interaction of two entities known as biomaterial as an

immobilizer and an immobilization agent.

The immobilization preparation method depends on the suitability of both entities to react
and forms a stable and suitable immobilized phase. Typically the biomaterials used as an
immobilizer are an insoluble matrix, which is inert with the surrounding and also its
immobilized agent.

An immobilized system is prepared to achieve its specific objective, for example to obtain
a sustaining immobilization, to increase agent recyclability, agent purification, process
control or as temporal immobilization to increase release rate or for prolonged release or
as micro-devices to deliver agent at a targeted site.

However any objective it is, the determining factors to achieve the objective are the
suitability of immobilization preparation method, biomaterial, immobilization agent and
importantly the application purpose of the immobilization.

Physical and chemical immobilization categorization is based on its fundamental preparation

method, however nowadays immobilization can be prepared by combining multiple variations
of immobilizations.

1. PHYSICAL IMMOBILIZATION METHODS

Immobilization by physical method is the earliest form of immobilization, prepared mainly

for the purpose of protein isolation.

This method involves of physical interaction only, where both entities; either immobilizer
and immobilization agent are not changed or linked in order to immobilize them. Several
examples of physical immobilization methods preparation are by way of encapsulation,
entrapment or confinement, adsorption and also non-covalent interaction.

(A) Entrapment by encapsulation (B) Entrapment by lattice

(C) Adsorption

An encapsulation method can be described as a way to surround the immobilization agent

inside a vesicle of a nano-capsule structure made of suitable vesicle forming immobilizer
biomaterial.
On the other hand, in entrapment or confinement method immobilization agents are either
trapped or suspended in the nano-sphere particle or nano-crystals lattice, which are inert
to the agent itself.

Then, as for the adsorption immobilization, it is the simplest form of physical

immobilization. Adsorption occurs consequential from the interaction of immobilization agent
on the immobilizer biomaterial surface, layering it. The attachment interaction of the
agent was assisted by non-covalent interaction.

Non-covalent interactions are the opposite of covalent bonding, and it is formed by

temporal electromagnetic pull between immobilizer and immobilization agent. The
interactions in non-covalent are classified as electrostatic, Pi-interaction, van der Waals
force or hydrophobic effects.

Therefore, physical immobilization method presents several advantages over chemical

immobilization method. First of all, it preserves the purity and function of the
immobilization agent, which means that the agents are highly active and did not interact
with the immobilizer prior to action.

The releasing of agent are more rapid, natural and gentle due to the fact that no strong
bonding interaction existed and were required to be disassociated. Finally its preparation
are also much easier than the other methods, generally no special chemical is required to
form the immobilization steps. This method is natural and very organic in development and
application.

However the advantage of physical immobilization is also its drawbacks. Undisruptive

interaction of biomaterial and immobilized agent provides only weak adsorption force, thus
it is easily detached.

Weak non-covalent interaction can easily dissociated by harsh environment and

interferences. Consequently, agent leakage and loss may occur in between preparation and
application. Additionally the non covalent interaction is also non-specific to the target
agent and may adsorb other unwanted agent and competing agent.

The main variances among the product of physical immobilization methods are exhibited by
the novelty of the biomaterial used and its preparation methods, which will turn it into a
nano-capsule, nano-sphere, nano-crystals, nano-tubes, nanowires, nano-film or hydrogel
products.
CHEMICAL IMMOBILIZATION METHODS

Whereas in chemical immobilization, chemical reactions are involved in the preparation to

forms either strong bond between both entities. As a result of such chemical reaction,
immobilizer biomaterials are either covalently linked or cross linked to its immobilization
agents molecule depends on the type of bond formed. When specific covalent bonds
between both entities were established and electron sharing involved, the immobilization is
formed as a result of covalent bonding. In cross-linking, an intermediary links both
entities by covalent or ionic bonds, thus it is much stronger than covalent bonds which
provides more stability and longer immobilization.

Chemical immobilization is very favorable in the situation where high recovery and
recyclability of immobilized agent and biomaterial are necessary. This is because this
method provides a very strong immobilization and protection to the entities and from loss
and leaks.

However, for application of temporal immobilization of agent, releasing of agent would be

rather difficult and tough without a suitable competing or releasing agent. Moreover, care
should be taken in performing immobilization as to avoid potential agent activity loss due to
the destruction of its active site by chemical immobilization. Furthermore, its preparation
is also rather expensive, tedious and complex. For example, various factors needs be taken
into consideration when designing a chemical immobilization method, especially the
functional groups, which will involve in the covalent binding formation. Besides, a specific
pH ranges also influences covalent bonding, thus, biological pH environment should be
carefully evaluated for effective immobilization.
IMMOBILIZATION AND IMMOBILIZED PREPARATIONS

Among the procedures used to immobilize microorganisms are aggregation or flocculation,

adhesion or adsorption, covalent linkage of cell to cell or cell to support, entrapment or

encapsulation in gels or other polymers, and physical retention within membranous
structures or hollow fibers.

The object of immobilization is to restrict cell migration with minimal inhibition of catalytic
capacity.

Any immobilized microbial preparation destined for serious application must, among other
qualities, be stable in operation. The most commonly used method of immobilization has
been entrapment in gels and polymers, both natural and synthetic. Hydrocolloid gels,
especially alginates, continue to be the favorite support although with a range of variations
from the original procedure.
Alginic acid is a polymer , Solutions of sodium alginate are viscous. By contrast, calcium
alginates are gels, alginates are among the more favoured supports because they are
nontoxic and because the ease with which gel formation takes place allows mild conditions
to prevail during immobilization. In essence immobilization is effected by suspending cells in
a solution of sodium alginate which is then dropped, extruded or sprayed into a solution
containing calcium. The gel beads that form are usually allowed to harden for a period in
which calcium cross-linking is maximized. While it is possible to use a range of other
divalent ions for gel formation, calcium is generally considered to be the most appropriate.

The entrapped cells have an element of freedom within the gel structure and can multiply.
However, growth at or near the surface may result in loss of cells, with consequent
deterioration of catalytic ability and contamination of the substrate/product stream in
use.

Denaturation of the bead structure can occur as a result of competition with other ions
for binding sites and if calcium is removed by precipitation with phosphate

Apart from alginates, the most commonly favoured hydrogel for cell immobilization is
carrageenan. Its structure is similar to that of agar. It is galactopyranosyl-based with a
significant percentage of substituted residues. It is comprised of a number of major
fractions of which the K, the most frequently used, and t fractions form thermoreversible
gels.

Gel structures are achieved by formation of interchain helices. On warming to 50C, K-

carrageenan exists as a solution in which the polymer chains are present as random coils.
To maximize gel performance, the carrageenan can be washed with 3% (w/v) sucrose to
remove calcium ions.

The carrageenan solution is then mixed with an equal or less than equal volume of cells and
the mixture is dropped into a cold solution of either KCI or CaC12. The temperature of
the carrageenan solution used is critical in that it determines the efficiency of bead
formation and may have an adverse affect on cell viability. Nevertheless, the structural
integrity of a gel so formed is not as sensitive as those formed from alginates to the
presence of anions and cations in the macro environment .

Prepolymerized synthetic materials have also been used as entrapping or encapsulating

media as have a range of miscellaneous natural gels including agar, collagen, egg white
gelatin, locust bean gum various mixed gel systems and materials that can be
copolymerized by radiation Celite has been included in some entrapped biocatalyst systems
to improve on gel permeability and robustness. A more pragmatic approach to
immobilization of biocatalysts is to encourage the growth of cells in voids in porous
materials. The success of such systems derives from the natural tendency of microbial
cells to adhere to surfaces (adsorption) or to one another (flocculation).

In a typical example yeast cells have been immobilized in 1 cm 3 reticulated polyurethane

cubes by growing the cells in contact. The cubes can then be packed in columns or used in
tower fermenter systems. Such catalysts do not suffer the disadvantage of alginate beads
in being sensitive to the presence of anions and cations that might disrupt their structural
integrity nor are the cells subjected to possible heat shock denaturation as could be the
case in the preparation of carrageenan systems. They do not, however, afford the same
degree of control over contamination of product streams. Various cloths, foams, sponges
and metallic meshes have been used as immobilization supports.

Moreover, the processes of immobilization are generally quite simple.

In general, immobilized cell systems depend not only on physical entrapment factors
resulting from colony growth but also on a variety of adhesion forces. The latter may be
enhanced in porous or non-porous support matrices by any one of a variety of possible
modifications to the cells or to the supports.

One such modification involves the adsorption of aluminium ions onto the surface of
Saccharomyces cerevisiae. The aluminium ions neutralize repulsive charges and so facilitate
adhesion of the cells to glass plates. Aggregation and flocculation, a form of cell
immobilization, is frequently used to exploit the catalytic abilities of certain
microorganisms in established tower fermenters. Flocculation may be stimulated or
enhanced by the use of polyelectrolytes, various gas treatment techniques or by covalent
bonding.

One example is the cross-linking of permeabilized yeast cells using albumin and
glutaraldehyde. A disadvantage of this system and those like it is that the bifunctional
reagent used in cross-linking, by reacting with essential cellular components, may effect
significant loss of catalytic activity. Various membrane systems not only provide a means
of immobilizing cells they also provide the reactor vessel. Such systems may however
experience problems as a result of excessive cell growth and biomass build-up A typical
example of the use to which such systems may be put is the immobilization of yeast cells
within ultrafiltration hollow fibres . The fibres used had a nominal molecular weight cut-
off value of 10000 and to prevent the build-up of biomass a nitrogen-deficient medium
was used.
CO-IMMOBILIZATION

Though immobilization technology is mainly confined to potential monocultures, it is prudent

to examine the benefits of mixed cultures under immobilized state to accelerate the
fermentation processes. There are several reports of co-immobilization of two or more
cultures to derive the benefit of both cultures under monoculturing state.

For example, direct alcoholic fermentation of soluble starch and dextrin uses amylotic
yeasts Saccharomyces diastaticus, Schwanniomyces castellii, and Endomycopsis fibuligera.
Reilly and Scott reviewed several cases of mixed-culture fermentations to derive various
biochemicals. Co-immobilized mixed cultures of plasmid-free and plasmid-containing E. coli
HB101 in alginate matrix to study the stability of plasmid for long-time cultivation. This
study has indicated that the stability of the plasmid-recombinant cells was enhanced in
co-immobilized state. The co-immobilization of mixed cultures of algae and aerobic
bacteria by encapsulation has solved the problems of oxygen limitation under high cell
density conditions in the hydrogels.

Wikstrom have co-immobilized Chlorella vulgaris with Providencia sp. in agarose, and
employed them in a photoreactor for the production of a -keto-isocaproic acid from l-
leucine. It has been proved that the algae in the gel matrix acted as an in situ oxygen
generator. In a similar study, Cephalosporium acrymonium, an oxygen-consuming fungus,
and Chlorella pyrenoidosa, an oxygen-generating alga, were co-immobilized for the
cephalosporin C production. It was noticed that the co-immobilized microbial system
enhanced the production of cephalosporin C by 370% . Similar co-immobilization protocols
were worked out for in situ enzyme regeneration, complementation, multistep
biotransformation, exploitation of oxygen gradient in the polymer gel and photoproduction
of hydrogen.
APPLICATIONS

Current applications of immobilized microorganisms on an industrial-scale are, with few

exceptions, restricted to the production of antibiotics, amino acids and organic acids.
Nevertheless, research in many different countries shows that the potential of these
preparations for other applications is enormous.

Thus, apart from the above, they may find use in the production of ethanol and other
fuels, in the exploitation of biomass, in organic syntheses, in wastewater treatment and in
the food, dairy and drinks industries.

For example, immobilization of an organism with a view to producing cellulase could be

included under protein synthesis or biomass exploitation. Similarly, biomethanation of
organic nutrients in aqueous effluents could be listed under wastewater treatment or fuel
production.

ORGANIC ACIDS

Organic acids are important microbial products used in a variety of applications such as
food and medicines. Among the various organic acids, citric acid occupies predominant
position as a commercial biochemical.

Aspergillus niger is the widely used microorganism for the synthesis of citric acid. In
conventional practices, batch fermentation of A.niger is employed. The fungal
fermentations have serious disadvantage of rising viscosity during growth, leading to poor
oxygen supply to the cells. Therefore, it becomes necessary to aerate the cultures with
large volumes of sterile air. In case of immobilized cells, since growth is restricted, it is
possible to operate the fermentor without affecting the viscosity, thereby facilitating
good oxygen transfer rates with minimal cause. There are several investigations on citric
acid production using immobilized cells of A.niger

The methods most widely used for immobilization of A.niger cells are the entrapment in
alginate gels, agarose, and polyacrylamide.In addition, adsorption on various supports, such
as polyurethane foam, and entrapment in hollow fibres have also been attempted. Porous
cellulose carrier as support matrix for A.niger and observed that the immobilized cells
enhanced the productivity substantially.

A few reports are available on the use of yeast, Yarrowia lipolitica, in immobilized phase
for the production of citric acid1. Working with this yeast, evaluated several carriers such
as alginate, k -carrageenan, polyurethane gel, nylon web, and polyurethane foam for active
and passive immobilization of the cells. Amongst these various carriers, entrapped cells in
alginate exhibited highest citric acid productivity, 15. It has been observed that the
immobilized cells of A. niger required a lower initial sucrose concentration than the free-
cells for maximum productivity. High sucrose concentrations led to reduced yields and
increased polyol formation.

The use of immobilized cell technology to improve lactic acid fermentation processes has
been tried by several research workers1. Lactobacillus helveticus, Streptococcus salivarius,
L. delbrueckii Rhizopus oryzae, and Pedioccus halophilus are the important microorganisms
used for the production of lactic acid by immobilized cells. Similar to other microbial
fermentations, entrapment in calcium alginate or k -carrageenan is the method of
immobilization widely followed. Since many workers have reported shrinkage and decreased
mechanical strength of alginate beads during lactic acid fermentation, there was a
suggestion of a cell entrapment process using k -carrageenan and locust bean gum, which
significantly modified the mechanical properties of the gel. Using the above mixed gel,
several studies were carried out with various lactic acid-producing microorganisms130134.
The k -carrageenan locust-bean-gum-mixed gel matrix showed significant stability for 3
months in continuous fermentation in a stirred-tank reactor.

In another study, compared the performance of alginate-immobilized L.casei cells in

stirred-tank and packed-bed reactors. The total productivity of 1.6 g/l/h was achieved in
stirred-tank reactor with total utilization of glucose.

A novel method of extractive fermentation of lactic acid

with immobilized cells has been proposed . Since the product inhibition could be eliminated
by adsorption of lactic acid by selective resin, this method offers higher productivity in
bioparticle-continuous fermentation.

Fungal fermentation for lactic acid production has also been studied. Using R. oryzae cells,
Tamada et al.124 tried immobilization of the cells with polymer supports prepared from
polyethylene glycol and dimethyl- acrylate as monomers by induced polymerization. The
specific rate of lactic acid production from immobilized cells reached a value of 1.8 times
higher than the free-cells..

Several groups have investigated the production of acetic acid by the cells of Acetobactor
sp. immobilized in hollow fibres, ceramic supports, and carrageenan 1. It has been reported
that the addition of a -alumina (2%) to the aqueous solution of sodium alginate (4%) for
immobilization of viable cells of Acetobacter sp. allows the production of denser particles
than the ones obtained with sodium alginate alone. These particles allow for greater
oxygen transfer resulting in enhanced volumetric production rate of acetic acid, since
oxygen is the limiting factor of acetic acid production.
AMINO ACIDS

The production of amino acids, is one of the most successful applications of immobilized
microbial cells. Procedures for the formation of amino acids generally from ketoacids and
for the specific production of alanine, arginine, aspartate, glutamate, N-acetyl
methionine, phenylalanine, tryptophan and tyrosine

Chibata and co-workers at the Tanabe Seiyaku Co. in Osaka have developed a process
whereby asparate and alanine can be produced from ammonium fumarate by sequential
exploitation of the aspartase activity of immobilized E. coli and the aspartate
decarboxylase activity of immobilized P dacunhae.

The 1984 production rates of alanine and aspartate were 10 tonnes/month and 100
tonnes/month, respectively.

The operational stability of the cellular aspartase , by contrast with the instability of the
free enzyme, and the use of mutants of E. coli lacking fumarase activity (thereby
eliminating the conversion of fumarate to malate) contributed significantly to these
impressive production. The interfering fumarase (E. colt) and alanine racemase (P..
dacunhae) activities could also be selectively eliminated by appropriate treatment of the
cells prior to immobilization .

Note than L-phenylalanine is important not only as an essential amino acid in human
nutrition but like asparate is also a raw material for synthesis of the synthetic sweetener,
aspartame. Thus, demand for the amino acid is rising. One may also note that the
feedstock containing a- acetamidocinnamate, lactate and NH4OH is cheap and that
reducing equivalents are recycled by the sequential operation of lactate and phenylalanine
dehydrogenases.

ANTIBIOTICS

The use of cell-free immobilized microorganisms has had considerable impact on the
industrial production of antibiotics especially in those reactions involving only a single step .

Antibiotic production

Production of antibiotics is one of the key areas in the field of applied microbiology. The
conventional method of production is in stirred tank batch reactors. Since it is a non
growth associated process, it is difficult to produce the antibiotic in continuous
fermentations with free-cells. But it is a suitable case for cell immobilization, since
growth and metabolic production can be uncoupled without affecting metabolite yields.
Therefore, several attempts have been made to immobilize various microbial species on
different support matrices for antibiotic production.

The most widely studied system is the production of penicillin G using immobilized cells of
Penicillium chrysogenum. Gaucher group entrapped the conidia of the fungi in k -
carrageenan and used for batch and continuous production of penicillin, and compared it
with fungi adsorbed on celite. It has been reported that the adsorption on celite was five
times more productive than entrapment in k -carrageenan.

The adsorbed cells exhibited maximum specific reaction rates compared to the free-cells.
This may be due to better oxygen availability to the cells. In another study, entrapment
of P. chrysogenum in calcium alginate and used in bubble column reactors with limited
success. It demonstrated the use of immobilized cells of Streptomyces rimosus for the
continuous production of oxytetracycline. Among the various methods of immobilization,
adsorption on hydrophilic urethane prepolymer was found to be superior to the other
methods..

Many of the reports on antibiotic production are confined to shake-flask level, however
there are a few reports on attempts at using different types of bioreactors. Air-lift
bioreactor is used to improve the oxygenation of the culture. In an another study it was
demonstrated that a three-phase fluidized bed reactor, using immobilized P. urdicae for
the production of patulin, could operate continuously for 360 h with 35% higher antibiotic
production85.

As antibiotic fermentations are not growth associated, the requirements of nutrients are
different from growth and metabolite synthesis. In principle, proliferation of immobilized
cells is prevented. Hence, it is possible to use a very dilute medium for the synthesis of
the metabolite. The decoupling of growth with the product synthesis can be achieved by
manipulating key nutrients for growth. Reports are available on using glucose-free medium
for oxytetracycline production70 and phosphate limitation for hybrid antibiotic production.
Such limitations of nutrients were reported to be effective in case of bacitracin, patulin,
cephalosporin C, and cephamycin.

Though entrapment of microbial cells in polymer gels is the choice for many, it might not
be the best. Since most of the antibiotic producers are highly aerobic, the limitation of
oxygen supply in gel matrix adversely affect their performance.

PRODUCTION OF ALCOHOLS

Ethanol fermentation using immobilized cells of yeast is one of the widely studied systems.
In fact, almost all the methods of immobilization, namely, gel entrapment, adsorption on
the surfaces of the various carriers, crosslinking were tried for alcohol fermentation.
There are specialized reviews on ethanol fermentation with immobilized Saccharomyces
cerevisiae. There are also several reports on Zymomonas mobilis for alcohol production.
Many reactor systems are being operated on continuous basis with encouraging results.
However, except for one or two attempts on large scale, the remaining investigations are
confined to laboratory scale.

One of the problems usually encountered in alcohol fermentation is the insitu generation of
CO2 in large quantities, resulting in severe operational difficulties in continuous
fermentation. Several studies have been carried out to overcome the problems associated
with gas generation such as bed compaction, resulting higher pressure drop, breakage of
the gel particles, and improper CO2 ventilation. Giletal have used ceramic-like matrix
material constructed of aluminum silicate composition as a carrier for yeast immobilization.
It has been reported that the continuous process was operated over a period of 2 years
producing 98 g/l of alcohol.

Similarly, another research group used polyurethane foam to entrap Z. mobilis to produce
high concentrations of ethanol.

The first time a large-scale continuous alcohol fermentation system by immobilized living
cells of yeast. The yeast cells were mixed with photo-crosslinkable resin, and were
polymerized by light sources. A pilot-plant-unit, producing 250 litres of alcohol/day, was
operated for 8000 h continuously operating a pilot plant of 4.0 kl capacity, using alginate-
entrapped cells of yeast for alcohol fermentation for a period of 4000 h with a constant
alcohol production rate of 8.5 to 9.0% by volume. Using Ca-alginate-entrapped cells of S.
cereviceae, Jamuna and Ramakrishna reported rapid fermentation of high concentration
sugar solution, thereby obtaining 20% (w/v) alcohol in 30 h.
BIOMASS conversion Biomass may be defined as everything excluding fossil fuels that has
been derived as a result of photosynthesis. Whatever the definition used, biomass,
whether primary (trees account for 90%) or secondary (via wastes or residues of forest,
agricultural, domestic or industrial origin), is potentially a vast and renewable reservoir of
fuel and chemical feedstocks. Realization of this potential requires that the major
components of biomass, the polysaccharides cellulose and hemicellulose, be degraded to
their monomeric forms and sequentially or simultaneously fermented to desired end
products such as ethanol. The major bottleneck in achieving these goals is the fact that
cellulose, an insoluble crystalline material, is not very amenable to hydrolysis. Thus, large
amounts of cellulase are required for its conversion. To this end various groups of
investigators are actively searching for improved cellulase producing organisms or are
attempting by mutation or recombinant DNA techniques to improve on the cellulase-
producing abilities of known organisms or to confer on non-cellulolytic species the ability to
utilise this substrate. In the present survey we note that procedures for immobilization of
Trichoderna reesei, one of the most powerful cellulase-producing fungi, and other
organisms to produce cellulase when immobilize. The ability of immobilized cellulolytic
organisms to saccharify cellulosic substrates including sugar cane bagasse , sawdust and
cereal chaff.Anaerobic digestion of 'wastes' such as pig slurry by immobilized consortia
capable of producing methane may also be cited as examples of the use of immobilized
microorganisms in exploitation of the potential of biomass.

Ethanol and other solvents or fuels even a cursory perusal of the pertinent literature
would show that the production of ethanol is the most actively researched area of
application of immobilized microorganisms.

As further examples of the potential of biomass as a source of fuels one notes that the
possibility of using immobilized microorganisms to produce ethanol from hydrolysates of
wood , bagasse and other cellulosic raw materials has been demonstrated as has that from
cellobiose , sucrose, apple juice, beet juice , cane juice, molasse, sweet sorghum juice [4,
banana fruit pulp sugar, fructose, starches sugars, such as xylose , derived directly or
indirectly from hemicellulose and from whey permeate or lactose .
Food, dairy and drinks INDUSTRIES

The fermentative properties of microorganisms have long been used in the making of those
staples of life, bread, cheese, beer and wine. It is not surprising therefore to find that
much effort has been expended on examination of the possibility of improving on the
efficiency of some of these applications by using immobilized microbial cells.

Examples of specific applications of immobilized microrganisms include the reduction of the

limonin and nomilin contents of citrus juices so as to prevent the development of
bitterness. However, the greatest application of these preparations is in the production of
high fructose syrups.

Isomaltulose, a natural component of honey, is a sugar of reduced cariogeneity used in

foodstuffs and pharmaceuticals. Its production using immobilized microorganisms has
recently been investigated .Process for isomaltulose production using immobilized Erwinia
rhapontici . Sorbose, much of which is used in the synthesis of vitamin C, produced by the
oxidation of sorbitol using immobilized preparations of Gluconobacter suboxydans or
Acetobacter melanogenum .

Soy sauce, long produced by traditional fermentation procedures, may also be prepared
using immobilized cell technology , using column type reactors containing various immobilized
microorganisms reduced the time taken for soy sauce production from 6 months to 2
weeks. In the dairy industry immobilized microbial cells are also finding increasing use.
Examples include a glucose oxidase/E, coli coimmobilisate for the preservation of milk ,
lactic acid production/prefermentation of milk for cheese making, the production of
yoghurt , Emmental and Roquefort flavours and protein concentrates.

Immobilized microorganisms appear to have great promise in brewing, i.e. in speeding up

the time taken to make beer or in improving the quality of the finished product .

Other applications include mead production wine-making, e.g. the production of sparkling
wines and malolactic fermentation.

CHEESE PRODUCTION involves the conversion of the milk protein, K-casein, to para-
casein by a defined, limited hydrolysis catalysed by chymosin (rennin) Since chymosin -
only be extracted from calves killed before they are weaned (pepsin is produced instead of
chymosin after weaning) - the enzyme is in short supply - also ethical issues, there has
been a large-scale search for an acceptable substitute Proteases from animals (pepsin),
plants (ficain and papain) and over a thousand micro-organisms have been tried, either on
their own or mixed with calf chymosin.

THE CLARIFICATION OF CIDER, WINES AND FRUIT JUICES (e.g. apple) is usually
achieved by treatment with fungal pectinases. Pectinases are a group including
polygalacturonases, which break the main chains of pectins, and pectinesterases, which
hydrolyse methyl esters. Their action releases the trapped particles and allows them to
flocculate (pectins of fruit and vegetables play an important role in jam-making and other
processes by bringing about gel formation)

PROTEIN PRODUCTION

Most large-scale protein production processes involve either traditional liquid cultivation or
solid-state fermentation.
Steroid transformation of steroids has in the past been carried out by conventional
fermentation procedures because of the difficulties involved in chemical transformation.
The possibility of using immobilized cell-free enzymes is impractical since the more
important steroid-transforming enzymes, dehydrogenases and hydroxylases are highly
labile and difficult to obtain in quantity.

For these reasons steroid transformation using immobilized microorganism is becoming the
state of the art. Indeed, the first such transformation was carried out in 1970.

ENVIRONMENTAL DECONTAMINATION

Environmental decontamination is one area in which immobilized microorganisms already

have application. Indeed, one aspect of this topic, i.e., wastewater treatment, is
currently the major large-scale application of surface-bound microbial cells . For example,
the Norton Co. in Oak Ridge, TN, have since 1976 operated a large- scale wastewater
treatment unit involving microorganisms adsorbed on anthracite in a tapered up-flow
anaerobic bioreactor. Advances in the aerobic and anaerobic treatment of wastewaters,
effluents and condensates using immobilized whole cells have been reviewed by several
investigators .Processes for removal/recovery of heavy metals , as have those for removal
of phenols , chlorinated phenols, benzoates and hydrocarbons.

Dehydroabietic acid, a component of many pulp mill effluents, is toxic to fish. However, it
can be converted to harmless products using mycelia of Mortierella isabellina free or
entrapped in alginate. Entrapment stabilized the hydroxylase activity involved in
detoxication. A procedure, based on the use of immobilized Pseudomonas indigofera, has
been developed for the degradation of anabasine, an insecticide con- taminating soils

NITROGEN FIXATION The possibility of using alginate beads as inoculant carriers for the
slow release of bacteria that affect plant growth is under investigation.
 IMMOBILIZATION TECHNOLOGIES IN PROBIOTIC FOOD PRODUCTION

Various supports and immobilization techniques have been proposed and tested for
application in functional food production. In the present review, the use of probiotic
microorganisms for the production of novel foods is discussed, while the benefits and
criteria of using probiotic cultures are analyzed.

Subsequently, immobilization applications in the food industry aiming at the prolongation of

cell viability are described together with an evaluation of their potential future impact,
which is also highlighted and assessed.

INTRODUCTION

Probiotics have rapidly gained interest in the area of self-care and complementary
medicine under the general term functional foods. Modern consumers are increasingly
interested in their personal health and particularly in foods which are capable of
preventing and/or curing illness. Microbes have been used for years in food and alcoholic
fermentations but only recently have undergone scientific scrutiny to examine their
possible health benefits.

The word probiotic comes from the Greek words pro and biotic, meaning for the
life. The concept of probiotics appeared a long time ago.

The Nobel laureate Elie Metchnikoff was the first microbiologist in the beginning of the
20th century who suggested that the longevity of Bulgarian peasants could be related to
their large consumption of sour milk containing Lactobacillus bulgaricus. The most commonly
used definition for probiotics comes from Fuller in 1989 defining that probiotics are live
microbial food supplements, which beneficially affect the host animal by improving its
intestinal microbial balance. Salminen altered the term to probiotics are microbial cell
preparations or components of microbial cells that have a beneficial effect on the health
and well-being of the host.

According to this definition, probiotics are not necessary to be viable, as nonviable forms
have also been proved to provide health effects. Today, the term probiotic refers to
live microorganisms which, administered in adequate amounts, confer a beneficial
physiological effect on the host, according to the Food and Agriculture Organization and
World Health Organization .

A variety of microorganisms have been studied for potential probiotic effects. Most
microbial strains with probiotic activity belong primarily to Lactobacillus and
Bifidobacterium genera. However, the potential probiotic roles of other microbes are also
under investigation
Immobilization of probiotics is an exciting field of food technology that has emerged and
developed rapidly in the past decade. The most excellent application of probiotic
immobilization technology is the controlled and continuous delivery of cells in the gut.

The potential benefit of this therapeutic strategy is to maintain greater cell viability
despite the acidity of the stomach. In their viable state, probiotics exert a health benefit
on the host.

There is growing scientific evidence to support the concept that the maintenance of
healthy gut microbiota may provide protection against gastrointestinal disorders, such as
gastrointestinal infections and inflammatory bowel diseases .

The use of probiotic bacterial cultures may stimulate the growth of preferred
microorganisms, crowd out potentially harmful bacteria, and reinforce the body's natural
defence mechanisms . Beneficial effects of probiotic consumption also include enhancement
of bioavailable nutrients, reduction of symptoms of lactose intolerance, decrease of the
occurrence of allergic symptoms in susceptible individuals, and reduced risk of certain
cancers . However, the mechanisms by which probiotics exert their effects are largely
unknown and may involve modification of gut pH, antagonism of pathogens through
production of antimicrobial and antibacterial compounds, competition for pathogen receptor
sites and for available nutrients and growth factors, stimulation of immunomodulatory
cells, and production of lactase.

CRITERIA OF A CULTURE TO BE USED AS PROBIOTIC

Several aspects, including safety, functional and technological characteristics, have to be

taken into consideration in the selection process of probiotic microorganisms. Many
microorganisms could be considered as potential probiotics, but only a few are able to
satisfy the necessary criteria. Safety aspects include specifications such as origin (healthy
human gastrointestinal tract), nonpathogenicity, nondigestive upsets, and nonantibiotic
resistance characteristics.

Functional aspects include viability and persistence in the gastro-intestinal (GI) tract,
surviving the digestive stresses , immunomodulation, and antagonistic and antimutagenic
properties.Careful screening of probiotic strains for their technological suitability can also
allow selection of strains with the best manufacturing and food technology characteristics.
Moreover, they should not produce off-flavours.

Safety Criteria

It is essential that probiotics must be considered as generally recognized as safe (GRAS)

organisms for human use according to the US Food and Drug Administration [13]. The
congruent safety aspects include mainly human origin of strains in order to exclude
negative characteristics as harmful effects, pathogenecity, digestive upsets, and antibiotic
resistance. In particular:

1. It is highly recommended that strains used for products addressed to humans should be of
human origin. Additionally, a probiotic strain is expected to function better in a similar
environment from where it was originally isolated (e.g., human GI-tract). Generally,
probiotics should be isolated from healthy human GI tract. It is also considered that the
safety criteria depend on our experience in food fermentations
2. There should be no association with disease. Most intestinal microorganisms are not
considered pathogenic in healthy individuals, but some intestinal bacteria are potentially
pathogenic. Their growth and metabolism are influenced by the normal immune system in the
digestive tract. The pathogenic microbes can potentially cause an infection even in a healthy
host
3. Metabolic activity in the food matrix and in the intestine following consumption is an
important safety criterion. For example, although tolerance of bile salts is an essential
criterion for the selection of potential probiotic strains, microbial bile salts hydrolase
activity has been mooted to be potentially detrimental to the human host, and thus it is yet
not completely clear whether it is in fact a desirable trait in a probiotic bacterium
4. The selected strains should not carry transmissible antibiotic resistance genes.

FUNCTIONAL CRITERIA

For the selection of a probiotic strain, several criteria of functionality have to be considered. The
functional criteria of probiotics should be established based on both in vitro and in vivo assays, and
the results should be also reflected in controlled human studies. To deliver the health benefits,
probiotics should be able to survive the acidic conditions of the upper GI tract and proliferate in
the intestine, a requirement that is not always fulfilled. Feeding trials of Wistar rats with
fermented milk containing free or immobilized L. casei ATCC 393 showed that the levels of the
probiotic strain at the faeces and in the intestinal tissue dropped sharply and were undetected
48h after discontinuation of administration [15, 16]. Apparently, daily consumption of probiotic
products is a prerequisite for retaining cell levels at an effective concentration, information that
could be valuable in the food industry. The continued existence of the probiotic strain in the
human GI-tract has therefore been considered an essential trait.

The survival of different probiotic strains in different parts of the GI-tract may greatly
vary. Some strains are rapidly killed in the stomach, while others are able to pass through
the whole gut in high numbers. Bifidobacteria differ significantly in their survival in gastric
juices and bile salts.

Moreover, because viable and biologically active microorganisms are usually required at the
target site in the host, it is essential that probiotics are able to withstand the host's
natural barriers against ingested bacteria. Several studies have shown that many strains
of Bifidobacterium sp. intrinsically lack the ability to survive the harsh conditions of
acidity and bile concentration commonly encountered in the GI-tract of humans.
The reduction of viable cell levels might not always constitute a major issue, as a high
number of studies reporting that nonviable probiotics could also have beneficial effects on
human health or even be more efficient than alive cells are available. For example,
lyophilized heat-killed Lactobacillus acidophilus was more effective than living lactobacilli in
the treatment of chronic diarrhea. Likewise, in the case of lactose tolerance by lactase-
deficient subjects, viable and non-viable cultured milks show similar effects. Similarly, in
the treatment of acute gastroenteritis, some probiotics showed clinical efficacy in
shortening the duration of diarrhoea both in viable and non-viable forms.

Saccharomyces boulardii was required in a viable form for the treatment of candidiasis, in
contrast to lactic acid bacteria which showed efficacy both in the viable and non-viable
forms, stimulation of the human immune system by oral administration of fermented milks
or probiotic cultures has been observed with viable bacteria only, effects in faecal
bacterial enzyme activities are observed following the consumption of viable bacteria only ,
and so forth. Hence, the association of viable cell levels with the clinical outcome is still
dubious and seems to depend on the microbial species and on the disorder. Future work
should focus on controlled blinded studies to further clarify issues concerning viability of
probiotics during product manufacture and in the host, as well as to set the essential
dosage for each case.

The health benefits of potential probiotic strains should be also assessed. Potential
benefits may vary from maintenance of normal intestinal flora to anticancer effects .
However, the positive activity might be strain specific and may be affected by the food
matrix. Long-term clinical studies employing both animal models and humans are thus
required to get fully proven health effects, especially to healthy populations.

TECHNOLOGICAL CRITERIA

Even though a probiotic strain fulfills the necessary safety and functional criteria, its
selection should also satisfy technological criteria, as aspects related to probiotic food
production and processing are also very important.

Viability of bacteria is often reduced during the food manufacture, distribution, and
storage. Non-viable cultured products usually have longer shelf-life and easier storage
which favour the adoption of the technology by the industrial sector, but it has been
claimed that only probiotic products with viable microorganisms have beneficial health
effects.

As it is strongly suggested that probiotic products should contain an adequate amount of

live bacteria, the food industry has adopted the recommended level of probiotic cells at
the time of consumption. Thus, a daily intake of at least 108109 viable cells, which could
be achieved with a daily consumption of at least 100g of probiotic food, has been
suggested as the minimum intake to provide a probiotic effect. Apart from high survival
rates, the probiotic cultures should also not have a detrimental effect on sensory
characteristics, for example, provide unpleasant flavours or textures.

Many surveys have shown large fluctuations and poor viability of probiotic bacteria, and
especially bifidobacteria, in food products, such as yoghurt preparations . Several factors
have been claimed to affect the viability of Bifidobacterium cultures in fermented milk
products, including acidity, pH, concentration of lactic and acetic acids, hydrogen
peroxide,and dissolved oxygen content. The sensitivity of bifidobacteria to low pH and
hydrogen peroxide combined with low viability in dairy products during storage at
refrigeration temperature remains a problem in most fermented products.

Consequently, industrial demand for technologies ensuring bifidobacteria stability in foods

is still strong, since high cell survival is important for both economical (a lower cell
addition in the product is necessary when stability is high) and health effects, as well as
business ethics issues (industry should not mislead the consumer by mentioning only the
presence of probiotics without clarifying the amount of live bacteria at the time of
consumption). Bifidobacteria are also very sensitive to environmental parameters and
require expensive media for propagation and the addition of growth-promoting factors, due
to their stringent growth requirements. They are mainly marketed through fermented
dairy foods, which are well suited to promote the health image of probiotics for several
reasons. Firstly, fermented foods, and dairy products in particular, already have a
positive health image, and consumers are familiar with the fact that these products
contain living microorganisms . Secondly, the image of yoghurt-like products as healthy
foods facilitates the recommendation of daily consumption of bifidobacteria. Moreover,
bifidobacteria have no adverse effects on the taste or aroma of dairy products and do not
enhance acidification during product-shelf life . Lastly, bifidobacteria are protected by
milk proteins during digestion, which allows better delivery to the host.

As with all fermented dairy products containing living bacteria, products containing
bifidobacteria must be cooled during storage, which is necessary both to guarantee high
survival rates and to ensure product stability.

APPLICATION OF IMMOBILIZATION TECHNOLOGY IN PROBIOTIC FOOD

PRODUCTION

Foods used for dissemination of probiotics are usually fermented foods even if probiotics
could also be present in infant formulas, fruit drinks, whey drinks, and sweet milk.
Fermented milk and cheese are the most common foods containing probiotics , while
drinking and frozen yoghurts, ice creams , and fermented soya products are well
established in the market . In fermented dairy products, most commonly lactobacilli such
as Lactobacillus acidophilus and bifidobacteria, often referred to as bifidus, are used as
probiotics. Although strains of Bifidobacterium and Lactobacillus are currently the most
widely used probiotics for human consumption, other microorganisms including Enterococcus,
Streptococcus, and Propionibacterium, as well as some yeasts might also promote human
intestinal health.

In Europe, probiotic applications are restricted to fermented milk products. In the United
States, however, probiotics are found most frequently in the supplements sector and in
yoghurts, while in Japan and Korea the use of probiotics is also widespread in food
products that claim to assist the digestion process. On the other hand, research is also
currently oriented to nondairy foods, like fermented meat and bakery products.

Among the numerous immobilization supports, only a few are considered suitable for food
production. For example, inorganic materials are usually excluded because they are
characterized as unsuitable for human or animal nutrition. Instead, biopolymers and
natural supports of food-grade purity are preferable. It is also very interesting to exploit
materials with nondigestible carbohydrates and to investigate their application in probiotic
food production.

RESEARCH PAPER:

Topic: Immobilization of microbial cells: A promising tool for treatment of toxic pollutants
in industry

INTRODUCTION

Large-scale production of wastewater is an inevitable consequence of all contemporary

societies. Most wastewaters are usually hazardous to human populations and the
environment and must be treated prior to disposal into streams, lakes, seas, and land
surfaces. Although, the field of environmental biotechnology has been around for decades,
starting in the early 20th century, the introduction of new technologies has enabled
engineers and scientists to tackle the more contemporary environment problems such as
detoxification of hazardous wastes through the use of living organisms .Traditional
biological treatment processes can eliminate a large fraction of biodegradable organic
compounds existed in wastewater.

Moreover, the biological treatment cost is much lower than that of physical and
chemical wastewater methods. However, many hazardous compounds are poorly removed in
conventional biological processes due to their toxicity. Furthermore, they also have
adverse impact on the composition and activities of microorganism communities in activated
sludge flocs, thus reducing the overall performance of these facilities.
The removal of these compounds is a real challenge for waste treatment engineers and
scientists. Immobilization of microbial cells has received increasing interest in the field of
waste treatment. Compared with conventional suspension system, the immobilized
microorganism technology offer a multitude of advantages, such as high biomass, high
metabolic activity and strong resistance to toxic chemicals.

Moreover, immobilized microorganisms could be cost effective since they can be used
several times without significant loss of activity. Therefore, immobilized microorganism
technology has been explored as promising for wastewater treatment in the past few
decades and in the near future.

CELL IMMOBILIZATION

Immobilization is a general term describing a wide variety of the cell or the particle
attachment or entrapment . It can be applied to basically all types of biocatalysts
including enzymes, cellular organelles, animal and plant cells. Currently, different kinds of
immobilization have found wide applications not only in the field of biotechnology, but also
in pharmaceutical, environmental, food and biosensor industries. Immobilization of
microrganisms containing specific enzymes has further advantages such as elimination of
long and expensive procedures for separation and purification and it is vital to expand
their application by enabling easy separation and purification of products from reaction
mixtures and efficient recovery of catalyst. In the case of the immobilization of microbial
cells, their field of application spreads from industrial to environmental process.
Microorganisms retained on a carrier can be used in continuous and semi-continuous
production processes allowing for significant cost decrease, as the biocatalyst does not
need to be refilled.

Cell immobilization has been defined as the physical confinement or localization of viable
microbial cells to a certain defined region of space in such a way as to limit their free
migration and exhibit hydrodynamic characteristic which differ from those of the
surrounding environment while retaining their catalytic activities for repeated and
continuous use.

SUPPORT MATERIALS

The support selection is one of the crucial decisions to be made in the course of
preparation of the immobilization process. For treatment of wastewater, support materials
need to meet the following criteria: insoluble, non-biodegradable, non-toxic, nonpolluting,
light weight; flexibility in overall shape, high mechanical and chemical stability, high
diffusivity, simple immobilization procedure, high biomass retention, minimal attachment of
other organisms and preferably a low cost price.

Other criteria, such as physical characteristics (porosity, swelling, compression, material

and mean particle behavior), as well as, possibility for microbial growth and solubility, are
more application specific.

The carriers are classified as inorganic material (zeolite, clay, anthracite, porous glass,
activated charcoal, and ceramics) and organic polymers. Inorganic carriers were selected
to immobilize microorganisms because they can resist microbial degradation and are thermo
stable. The organic polymeric carriers are more abundant than inorganic carriers and can
be natural and synthetic polymeric carriers.

Several synthetic (acrylamide, polyurethane, polyvinyl, resins) and natural polymer

derivatives of algal polysaccharides (alginate, carrageenan, agar, agarose), and chitosan,
an amino polysaccharide derived from chitin, has been experimentally used. The most
commonly used polymers are the natural polymers alginate and carrageenan but these
natural polymers are less stable in wastewater than synthetic polymers.

Alginates (polymers made of different proportions and sequences of mannuronic and

guluronic acids extracted from brown algae) are easy to handle, nontoxic to humans, the
environment, and the entrapped microorganisms, legally safe for human use, available in
large quantities, and inexpensive. From a physiological perspective, a major advantage of
alginate is that immobilized cells do not suffer extreme changes in physicochemical
condition during the procedure of immobilization and the gel is transparent and permeable.
However, this substance cannot be maintained for a long period in aqueous solution because
the encapsulation immobilized microorganism can easily be broken during the operation.
Chitosan is inexpensive, non-toxic property and possesses potentially reactive amino
functional groups which can enhance the affinity of the carrier with the microorganisms.
However, the mechanical stability of the carrier would decrease because of the
biodegradability in the course of usage. Other natural gels, such as agar, collagen
andagarose, also can be used as microbial encapsulation carriers. Some natural polymers
are more vulnerable to environmental degradation by microbes.

However, diffusivity is higher in natural polymers and they are less hazardous to produce.
Synthetic polymeric supports are not easily biodegradable and have much better mechanical
performance compared with nature carrier.

Materials, such as polyacrylamide (PAM), polyvinyl alcohol (PVA), polyethyleneglycol (PEG)

and polycarbamoyl sulphonate (PCS) were synthesized as encapsulation carriers.
In order to improve the stability of gel carrier, various synthetic plastics, for example
polypropylene (PP), polyethylene (PE), polyvinylchloride (PVC), poly-urethane (PU) and
polyacrylonitrile (PAN) have been explored extensively as immobilized microorganism
carriers more recently.

Among the various extensively used plastics carriers, polyurethane (PU) is one kind of
outstanding carrier for entrapping microorganisms in piloted applications in practical
wastewater treatment reported potential of the Gramnegative bacterium Serratia
marcescens and the yeast Candida rugosa to immobilization on polyurethane foam.

CONCLUSIONS

Immobilized microbial systems currently offer various advantages over free systems. One
of the most promising areas of research is using this technology to reduce environmental
pollutions through biodegradation of many harmful compounds. The application of
immobilization technology to environmental area is in its preliminary stages, but the results
seen so far are promising. The immobilized cells will be useful to treat the waste to
convert the toxicant into nutrient, biomass and CO2 via biodegradation through their
intermediates. Better biodegradation rate was observed in immobilized cells due to absence
of internal and external mass transfer resistance. An immobilized cell is one of the
approaches for incorporating fungal biomass into an engineering process. The advantage of
the process based on immobilized biomass include enhancing microbial cell stability, allowing
continuous process operation and avoiding the biomass - liquid separation requirement.

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