INTRODUCTION Fermentation is a biochemical reaction which relates to generation of energy by catabolism of the organic compounds.

Industrially fermentation is described as any process for the production of product by the mass culture of microorganisms.Fermentation is used in industry for the production of commercially important product. This project aims at optimization of hydrogen peroxide,as oxygen source for the production of protease enzyme by Aspergillus tamarii and to provide basic information on various parameters affecting the fermentation process,so as to take it to a large scale production and it also aims at maximizing the production of protease.Proteases is used instead of chemicals for dehairing the cattle hide in leather industries. PROTEASE: Protea ses occur naturally in all organisms. The se enzymes are involved in a multitude of physiological reactions from simple digestion of food proteins to highly regulated cascades (e.g., the blood-clotting cascade, the complement system, apoptosis pathways, and the invertebrate prophenoloxidase-activating cascade). Protease s can either break specific peptide bonds (limited proteolysis), depending on the amino acid se quence of a prote in, or break down a complete peptide to a mino acids (unlimited proteolysis). The activity can be a destructive change, abolishing a protein's function or digesting it to its principal components; it can be an activation of a function, or it can be a signal in a signaling pathway. Bacteria also secrete proteases to hydrolyse (dige st) the peptide bonds in prote ins and therefore break the proteins down into their constituent monomers. Bacterial and fungal proteases are particularly important to the global carbon and nitrogen cycles in the recycling of proteins, and such activity tends to be regulated in by nutritional signals in these organisms. [ 4 ] The net impact of nutritional regulation of protease activity among the thousands of species present in soil can be observed at the ove rall microbial

community le vel a s proteins are broken down in response to carbon, nitrogen, or sulfur limitation. [ 5 ] A secrete d bacterial protease may also ac t as an exotoxin, and be an example of a virulence factor in bacterial pathogenesis. Bacterial exotoxic proteases destroy extra cellular structures. Protea se enzyme s are a lso used extensively in the bread industry in bread improver. Protea ses, also known as proteinase s or proteolytic enz ymes, are a large group of enzyme s. Proteases belong to the class of enzymes known as hydrola ses, which catalyse the reaction of hydrolysis of various bonds with the participation of a water molecule. Protea ses are involved in digesting long protein chains into short fragments, splitting the peptide bonds that link amino acid residues. Some of them can detach the terminal a mino acids from the protein chain (exopeptidases, such as aminopeptidases, carboxypeptidase A); the others attack internal peptide bonds of a protein (endopeptidases, such a s trypsin, chymotrypsin, pepsin, papain, elasta se). Protea ses are divided into four major groups according to the character of their catalytic active site and conditions of action: se rine proteinases, cysteine (thiol) proteinases, aspartic proteinases, and metalloproteinases. Attachment of a protease to a certain group depends on the structure of catalytic site and the amino acid (as one of the c onstituents) essential for its activity. Proteases are used throughout an organism for various metabolic processes. Acid proteases secreted into the stomach (such as pepsin) and serine proteases present in duodenum (trypsin and chymotrypsin) enable us to digest the protein in food; proteases present in blood serum (thrombin, plasmin, Hage man factor, etc.) play important role in blood-clotting, as well as lysis of the clots, and the correc t action of the immune system. Other protease s are present in leukocytes (elastase, cathepsin G) and pla y se veral different roles in metabolic control. Proteases determine the lifetime of other proteins playing important physiological role like

hormones, antibodies, or other enzymesthis is one of the fa stest "switching on" and "switching off" regulatory mechanisms in the physiology of an organism. By complex cooperative action the proteases may proceed as cascade reactions, which re sult in rapid and efficient amplification of a n organism's response to a physiological signal. Bacterial Protease: Most commercial proteases, mainly neutral and alkaline, are produced by organisms belonging to the genus Bacillus. Bacterial neutral proteases are active in a narrow pH range (pH 5 to 8) and have relatively low thermotolerance. The bacterial neutral proteases are characterized by their high affinity for hydrophobic amino acid pairs. Their low thermotolerance is advantageous for controlling their reactivity during the production of food hydrolysates with a low degree of hydrolysis. Some of the neutral proteases belong to the metalloprotease type and require divalent metal ions for their activity, while others are serine proteinases, which are not affected by chelating agents. Bacterial alkaline proteases are characterized by their high activity at alkaline pH, e.g., pH 10, and their broad substrate specificity. Their optimal temperature is around 60C. These properties of bacterial alkaline proteases make them suitable for use in the detergent industry. FUNGAL PROTEASE: Fungi elaborate a wider variety of enzymes than do bacteria. The fungal proteases are active over a wide pH range (pH 4 to 11) and exhibit broad substrate specificity. However, they have a lower reaction rate and worse heat tolerance than do the bacterial enzymes. Fungal enzymes can be conveniently produced in a solid-state fermentation process. Fungal acid proteases have an optimal pH between 4 and 4.5 and are stable between pH 2.5 and 6.0. They are particularly useful in the cheesemaking industry due to their narrow pH and temperature specificities. Fungal neutral proteases are metalloproteases that are active at pH 7.0 and are inhibited by chelating agents. In view of the accompanying peptidase activity and their specific function in hydrolyzing hydrophobic amino acid bonds, fungal neutral proteases supplement the action of plant, animal, and bacterial proteases in reducing the bitterness of food

protein hydrolysates. Fungal alkaline proteases are also used in food protein modification. ADVANTAGES OF FUNGAL PROTEASE: y Wide pH range(4 to 11) y Broad substrate specificity Sources of Protease: Produced by fungi, bacteria, plants, and in the digestive system of humans and animals, protease enzymes play an important role in digestion. Proteins are made up of long chains of amino acids linked by peptide bonds. These peptide bonds require a specific protease enzyme to cleave the bond creating free amino acids, which can then be used throughout the body. These foods contain natural proteases that can promote digestion and the breakdown of proteins. y Papaya Papaya is a rich source of papain, a protease found in the fruit and leaves of the plant. The richest source of papain comes from green or unripe papaya. Ripe papaya also contains papain but in smaller amounts. Papain has been found to reduce pain from ulcers and heartburn and improve overall digestion. Papain supplements are widely available as digestive aids. y Pineapple Pineapple contains bromelain, a protease found in the stem and fruit of the plant. Bromelain is known for its anti-inflammatory and digestive benefits and has been used in the treatment of heart disease, arthritis, and other infections. This enzymes works by breaking down proteins in the stomach as well as in swollen tissues around the body. Add bromelain to the diet by mixing pineapple chunks into smoothies or adding pineapple salsa on fish. Bromelain is also available in tablet form. y Cheese Cheese is not known as a digestive aid; however, it is a source of protease enzymes. Cheese is made using rennet, a group of enzymes that work to coagulate the milk then separate it into curds and whey. Rennet contains many protease enzymes including chymosin, trypsin and pepsin, all of which are produced in the stomach and pancreas of the animal providing

the milk. Many digestive aid supplements are produced using proteases from the pancreas and stomach of pigs and oxen. TYPES: Based on functional group at active site:
y y y y y y

Serine proteases Threonine proteases Cysteine proteases Aspartate proteases Metalloproteases Glutamic acid proteases

Based on optimal pH:

y y

Acid proteases Neutral proteases involved in type 1 hypersensitivity. Here, it is released by mast cells and causes activation of complement and kinins.[3] This group includes the calpains. Basic proteases (or alkaline proteases)

APPLICATIONS: y Digestion of natural proteins y Removing blood stain y Dehairing of skin Food Industry: Certain proteases have been used in food processing for centuries and any record of the discovery of their activity has been lost in the mists of time. Rennet (mainly chymosin), obtained from the fourth stomach (abomasum) of unweaned calves has been used traditionally in the production of cheese. Similarly, papain from the leaves and unripe fruit of the pawpaw (Carica papaya) has been used to tenderise meats. These ancient discoveries have led to the development of various food applications for a wide range of available

proteases from many sources, usually microbial. Proteases may be used at various pH values, and they may be highly specific in their choice of cleavable peptide links or quite non-specific. Proteolysis generally increases the solubility of proteins at their isoelectric points. Medical and related uses: Proteases (also sometimes referred to as proteolytic enzymes or peptidases) are in use, or have been proposed or tried, for a number of purposes related to medicine or surgery. Some preparations involving protease have undergone successful clinical trials and have regulatory authorization;[1] and some further ones have shown apparently useful effects in experimental medical studies.[2] Proteases have also been used by proponents of alternative therapies, or identified in materials of traditional or folk medicine.[3] Some of these uses rely directly on the proteolytic activity: others rely on observations of anti-inflammatory activity. For at least one significant use, the mechanism of action is unclear.[4] 1. Medical and surgical applications based on proteolytic effect
o o o

1.1 Treatment of blood clots in ischemic stroke 1.2 Wound debridement 1.3 Applications of proteases auxiliary to antibiotic therapy

2 .Applications of protease based on anti-inflammatory activity Leather Industry: The use of proteases as alternatives to hazardous chemicals such as sodium sulphide has proved successful in improving the leather quality and in reducing the environmental pollution.Currently,microbial alkaline protease are used to ensure the faster absorption of water and to reduce time required for soaking.Alkaline protease is used extensively to removal of hair hides.The bating following the dehairing process involves the degradation of elastin and keratin,the removal of hair residues and the deswelling of collagen,which produces good,soft leather useful making leather clothes and goods. Functions of Proteases:

1.Extracellular acid proteases are implied in a breakage of cell wall polypeptide linkages during germination of Dictyostelium discoideum spores. 2.The microbial enzymes and mammalian extracellular enzymes such as those secreted by pancreas are primarily involved in keeping the cells alive by providing them with the necessary amino acid pool as nutrition. 3.Recently,a new physiological function has been attributed to the ATPdependent proteases conserved between the bacteria and eukaryotes.It is implied that they act as chaperons and mediate not only proteolysis but also insertion of protein into membranes. Molecular and Biotechnological Aspects of Microbial Proteases: Proteases represent the class of enzymes which occupy a pivotal position with respect to their physiological roles as well as their commercial applications. They perform both degradative and synthetic functions. Since they are physiologically necessary for living organisms, proteases occur ubiquitously in a wide diversity of sources such as plants, animals, and microorganisms. Microbes are an attractive source of proteases owing to the limited space required for their cultivation and their ready susceptibility to genetic manipulation. Proteases are divided into exo- and endopeptidases based on their action at or away from the termini, respectively. They are also classified as serine proteases, aspartic proteases, cysteine proteases, and metalloproteases depending on the nature of the functional group at the active site. Proteases play a critical role in many physiological and pathophysiological processes. Based on their classification, four different types of catalytic mechanisms are operative. Proteases find extensive applications in the food and dairy industries. Alkaline proteases hold a great potential for application in the detergent and leather industries due to the increasing trend to develop environmentally friendly technologies. There is a renaissance of interest in using proteolytic enzymes as targets for developing therapeutic agents. Protease genes from several bacteria, fungi, and viruses have been cloned and sequenced with the prime aims of (i) overproduction of the enzyme by gene amplification, (ii) delineation of the role of the enzyme in pathogenecity, and (iii) alteration in enzyme properties to suit its commercial application. Protein engineering techniques have been exploited to obtain proteases which show unique specificity and/or enhanced stability at high temperature or pH or in the presence of detergents and to understand the structure-function relationships of

the enzyme. Protein sequences of acidic, alkaline, and neutral proteases from diverse origins have been analyzed with the aim of studying their evolutionary relationships. Despite the extensive research on several aspects of proteases, there is a paucity of knowledge about the roles that govern the diverse specificity of these enzymes. Deciphering these secrets would enable us to exploit proteases for their applications in biotechnology. Fermentation: The fermentation unit in industrial microbiology is analogous to a chemical plant in a chemical industry.A fermentation process is a biological process and therefore has requirements of sterility and use of cellular enzymatic reactions instead of chemical reactions aided by inanimate catalysts,sometimes operated at elevated temperature and pressure. Types of Fermentation: 1.Solid substrate fermentation. 2.Submerged fermentation. Solid substrate fermentation: Solid-state fermentation has emerged as a potential technology for the production of microbial products such as feed, fuel, food, industrial chemicals and pharmaceutical products. Its application in bioprocesses such as bioleaching, biobeneficiation, bioremediation, biopulping, etc. has offered several advantages. Utilisation of agro-industrial residues as substrates in SSF processes provides an alternative avenue and value-addition to these otherwise under- or non-utilised residues. Today with better understanding of biochemical engineering aspects, particularly on mathematical modelling and design of bioreactors (fermenters), it is possible to scale up SSF processes and some designs have been developed for commercialisation. It is hoped that with continuity in current trends, SSF technology would be well developed at par with submerged fermentation technology in times to come. Submerged fermentation: Submerged liquid culturing is usually preferred for large scale fermentations and has been used extensively for industrial production of not only enzymes,but also antibiotics,amino acids,ethanol and organic acids.The

enzyme protease is produced due to the fermentation process,taking place in the flasks.The production of protease by Aspergillus tamarii is carried out bby submerged fermentation,wherein usually the substrate either gets dissolved in the aqueous medium or remains suspended. Reaction calorimeter: The first operative calorimeter ever described in literature was more devoted to study of biological heat generation than heat production by chemical reaction. The Bio-RC1 is a heat bench scale calorimeter by MettlerToledoAG(Scwerzenbach, Switzerland),initially developed to investigate chemical reactions.It is essentially a standard 2-1 jacketed glass reactor,which may operate in isothermal,adiabatic or isoperiboloic models.In order to increase the stability and sensitivity of the system,a plexiglass housing for thermostating the reactor was built up. In the isothermal mode temperature of the reactant medium(Tr) is maintained constant by a proper control of the jacket temperature(Tj) by circulating at high rate(21 per sec) low viscosity silicone oil through the reactor jacket.The jacket temperature is carefully controlled metering valve. Thus a process dissipating or taking up heat will result, respectively in a decrease or increase in Tj leading to temperature gradient across the reactor wall which is directly proportional to the thermal flux liberated by or absorbed by the process (Qr) according to: Qr=UA(Tr-Tj) Where, U - overall heat transfer coefficient(W/m2K) A- Heat transfer area(m2) (Tr-Tj) - temperature difference between the reactor content and jacket oil. Applications of Bio-Calorimetry: The main objective of calorimetry in biotechnology includes process development work in monitoring biological activity as a function of process

condition.Bioprocess and bioreactor control are used for heat dissipation measurement at production scale as on-line probe for indirect determination of biomass concentration, product formation, state of culture and in research, induction-repression kinetics in continous culture, growth energetics and Biothermodynamics.

Scope and Objectives: y To increase the protease production by submerged fermentation in shaker level and Bio-calorimeter. y To study the production of protease by Aspergillus tamarii in submerged culture. y Optimization of Hydrogen peroxide as a source of oxygen. y Growth kinetics of Aspergillus tamarii.

LITERATURE REVIEW:

Aspergillus is a genus consisting of several hundred mold species found in various climates worldwide. Aspergillus was first catalogued in 1729 by the Italian priest and biologist Pier Antonio Micheli. Viewing the fungi under a microscope, Micheli was reminded of the shape of an aspergillum (holy water sprinkler), from Latin spargere (to sprinkle), and named the genus accordingly. Aspergillus species are highly aerobic and are found in almost all oxygen-rich environments, where they commonly grow as molds on the surface of a substrate, as a result of the high oxygen tension. Commonly, fungi grow on carbon-rich substrates such as monosaccharides (such as glucose) and polysaccharides (such as amylose). Aspergillus species are common

contaminants of starchy foods (such as bread and potatoes), and grow in or on many plants and trees. In addition to growth on carbon sources, many species of Aspergillus demonstrate oligotrophy where they are capable of growing in nutrient-depleted environments, or environments in which there is a complete lack of key nutrients. More than 60 Aspergillus microbial species are medically relevant pathogens.They are used for commercial fermentations.Alcoholic beverages like the sake,citric acid and other enzymes are produced using this genus species.Aspergillus tamarii is one important species under this genus of fungal organism. Aspergilli have varying morphological and growth response to different nutrients so it is important to standardize conditions. Species identification depends upon pure cultures grown on known media. The early taxonomic micrographs used a defined medium often called 'Czapek-Dox medium' which contains sucrose as the carbon source and nitrate as the nitrogen source. Strain variation is quite extensive within species and a variety of subtle effects such as air exchange, light and volume of the medium can affect morphology. Contemporary taxonomists usually grow strains on several media, at several temperatures, to identify species. Aspergillus tamarii is a non pathogenic, aerobic strain which comes under Aspergillus genus. It is considered as GRAS. It is used in the production of protease enzyme .The growth is observed after three days of inoculation in the media. Morphology and physical appearance: Colony Characteristics: Colonies (CzA) growing rapidly, dark brown.

Microscopy: Conidial heads compact and spherical or loosely radiate, 500-600 micrometer diam. Conidiophore stipes usually 1-2mm in length, hyaline, usually roughened. Vesicles spherical, 10-50 micrometer diam. Conidiogenous cells uniseriate and biseriate. Metulae or phialides covering the entire surface of the vesicle. Conidia echinulate to tuberculate, sub spherical, 5-8 micrometer diameter. Colonies on malt extract agar at room temperature attained diameters of 6.0 to 7.0 cm in 10 days, producing abundant conidial heads in dull yellowish green shades becoming metallic bronze at maturity .The conidiophore stipe was hyaline and rough walled; the conidialheads were radiate; the vesicles were globose to subglobose, 25 to 50 um in diameter. The phialides were borne directly on the vesicle or on metulae (mostly on large heads). The conidia were globose to subglobose, 5 to 6.5 um in diameter, and brownish yellow. However, in contrast with those of typical wild A. tamarii isolates some conidia of this isolate were not ornamented with tubercules and warts but were smooth walled and hyaline. The isolate grew well at37C but was unable to grow at 42C on malt extract agar medium. Colonies sporulated heavily, and the colors of the colonies were old gold to olive lake while the colony color of A. tamarii isolates was olive brown Pathogenicity: The species was implicated in a case of eyelid infection. The species had grown in the eye leading to ulcer. It was found that the anterior chamber showed a moderate number of cells. When the ulcer was cleaned and the species was identified and it was found to be Aspergillus tamarii.This was the only reported case of pathogenicity of Aspergillus tamarii. Uses: This common mould is involved in many industrial processes including enzymes (e.g. amylases), commodity chemicals (e.g. citric acid) and food stuffs

(e.g. soy sauce).Aspergillus and other moulds play an important role in these consortia because they are adept at recycling starches, hemicelluloses, celluloses, pectins and other sugar polymers. Some aspergilli are capable of degrading more refractory compounds such as fats, oils, chitin, and keratin. Maximum decomposition occurs when there is sufficient nitrogen, phosphorus and other essential inorganic nutrients. Fungi also provide food for many soil organisms. . Both paper and textiles (cotton, jute, and linen) are particularly vulnerable to Aspergillus degradation. Aspergiillus tamarii is known for production of hydrolytic enzymes which can degrade various biomolecules.It is known to produce the protease enzyme, which is the product required in our project.Apart from protease enzyme ,it produces six other enzymes they are

Pectinase,Asparaginase,Amylase, Glucoamylase, Cellulase, Lipase, Catalase. Aspergillus tamarii consumes the glucose present in the media first for increasing the biomass and its growth. Once the glucose is drained then the strain depends on the nitrogen source for it growth. When the organism depends on nitrogen source the strain starts producing protease enzyme during metabolism. Hence the protease is produced only in the stationery phase.Therefore it is not a growth dependent process. As the Aspergillus tamarii grows the viscosity of the medium starts increasing. Even when aeration is provided the oxygen transfer rate is reduced greatly, because of the increased viscosity of the medium. So to make oxygen available to the organisms, hydrogen peroxide is added to the medium as a source of oxygen. Aspergillus tamarii produces an enzyme catalase i.e. peroxidases, which degrade the hydrogen peroxide to water and oxygen, which is readily

taken up the organism. Hydrogen peroxide (H2O2) is the simplest peroxide. Hydrogen peroxide is a clear liquid, slightly more viscous than water. In dilute solution, it appears colorless. With its oxidizing properties, hydrogen peroxide is often used as a bleach or cleaning agent. Hydrogen peroxide is also naturally produced in organisms as a by-product of metabolism. It has a density of 1.450 g/cm3 (20 C, pure) .Pure hydrogen peroxide has a pH of 6.2; thus it is considered to be a weak acid. Hydrogen peroxide decomposes (disproportionate)

exothermically into water and oxygen gas spontaneously: 2 H2O2 2 H2O + O2

Hydrogen peroxide is generally recognized as safe (GRAS) as an agent. It acts as disinfectant above particular concentration. Hence it is important to optimize the amount of hydrogen peroxide being added. Catalases: Catalase (E.C1.11.1.6) is a common enzyme found in nearly all living organisms that are exposed to oxygen, where it catalyzes the decomposition of hydrogen peroxide to water and oxygen.[1] Catalase has one of the highest turnover numbers of all enzymes; one catalase molecule can convert millions of molecules of hydrogen peroxide to water and oxygen each second. Catalase is a tetramer of four polypeptide chains, each over 500 amino acids long. It contains four porphyrin heme (iron) groups that allow the enzyme to react with the hydrogen peroxide. The optimum pH for human catalase is approximately 7, and has a fairly broad maximum (the rate of reaction does not change appreciably at pHs between 6.8 and 7.5) The pH optimum for other

catalases varies between 4 and 11 depending on the species. The optimum temperature also varies by species. Catalase was first noticed as a substance in 1818 when Louis Jacques Thnard, who discovered H2O2 (hydrogen peroxide), suggested that its breakdown is caused by a substance. In 1900, Oscar Loew was the first to give it the name catalase, and found its presence in many plants and animals. The reaction of catalase in the decomposition of hydrogen peroxide is: 2 H2O2 2 H2O + O2

Hydrogen peroxide is a harmful by-product of many normal metabolic processes: to prevent damage, it must be quickly converted into other, less dangerous substances. To this end, catalase is frequently used by cells to rapidly catalyze the decomposition of hydrogen peroxide into less reactive gaseous oxygen and water molecules. Catalase performs its rapid destruction of hydrogen peroxide in two steps. First, a molecule of hydrogen peroxide binds and is broken apart. One oxygen atom is extracted and attached to the iron atom, and the rest is released as harmless water. Then, a second hydrogen peroxide molecule binds. It is also broken apart and the pieces are combined with the iron-bound oxygen atom, releasing water and oxygen gas. Catalase is found in the middle of this two-step reaction. The oxygen atom is bound to the iron, ready for the second hydrogen peroxide molecule to bind. The histidine and asparagine amino acids assist with the reaction. Bacterial and Fungal Protease: Most commercial proteases, mainly neutral and alkaline, are produced by organisms belonging to the genus Bacillus. Bacterial neutral

proteases are active in a narrow pH range (pH 5 to 8) and have relatively low thermotolerance. The bacterial neutral proteases are characterized by their high affinity for hydrophobic amino acid pairs. Their low thermotolerance is advantageous for controlling their reactivity during the production of food hydrolysates with a low degree of hydrolysis. Some of the neutral proteases belong to the metalloprotease type and require divalent metal ions for their activity, while others are serine proteinases, which are not affected by chelating agents. Bacterial alkaline proteases are characterized by their high activity at alkaline pH, e.g., pH 10, and their broad substrate specificity. Their optimal temperature is around 60C. These properties of bacterial alkaline proteases make them suitable for use in the detergent industry. Fungi elaborate a wider variety of enzymes than do bacteria. The fungal proteases are active over a wide pH range (pH 4 to 11) and exhibit broad substrate specificity. However, they have a lower reaction rate and worse heat tolerance than do the bacterial enzymes. Fungal enzymes can be conveniently produced in a solid-state fermentation process. Fungal acid proteases have an optimal pH between 4 and 4.5 and are stable between pH 2.5 and 6.0. They are particularly useful in the cheesemaking industry due to their narrow pH and temperature specificities. Fungal neutral proteases are metalloproteases that are active at pH 7.0 and are inhibited by chelating agents. In view of the accompanying peptidase activity and their specific function in hydrolyzing hydrophobic amino acid bonds, fungal neutral proteases supplement the action of plant, animal, and bacterial proteases in reducing the bitterness of food protein hydrolysates. Fungal alkaline proteases are also used in food protein modification.

EQUATION: d2Ca/dr2 +(2/r)(dCa/dr) - (OUR/Do) =0 Assuming, y=Ca x=r c=(OUR/Do) Substituting, d2y/dx2 + (2/x)(dy/dx) - c = 0 D=d/dx , D2=d2/dx2 [D2+(2/x)D]y= c Multiplying by x2, [x2D2+2xD]y = cx2 Put, X=ez, z=logx xD= , =d/dz x2D2 = ( -1) [ ( -1) +2 ]y = ce2z [
2

+2 ]y= ce2z

[ 2+ ]y=c.e2z m2+m=0 m(m+1)=0 m=0,m=-1 CF=Ae0z+Be-z PI= ce2z/f( )

=ce 2z /( 2+ ) =ce2z/(22+2) PI= ce2z/6 y=CF +PI y=A+Be-z+ce2z/6 y=A+B/x + cx2/6 thus, Ca=A+B/x+(OUR/Do)(x2/6) Ca=A+ B/r + (OUR/Do).(r2/6)-----eqn1 dCa/dr = -B/r2 + (OUR/Do).(r/3) Multiplying by r2 r2(dCa/dr) = -B +(OUR/Do).(r3/3) Applying boundary condition, When r=0,(dCa/dr) =0 Thus B=0 When r=R, Ca=Cao Cao=A+ (OUR/Do).(R2/6) A=Cao (OUR/Do).(R2/6) Subs values of A &B in eqn 1, Ca=Cao - (OUR/Do).(R2/6)[1-(r/R)2] When r=a, (dCa/dr)=0 0= -(B/a2) + (OUR/Do)(a/3) B/a2=(OUR/Do)(a/3) B=(OUR/Do)(a3/3)

When, r=R, Ca=Cao Cao = A+ (OUR/Do)(a3/3R) + (OUR/Do)(R2/6) A=Cao - (OUR/3Do)(a3/R + R2/2) Substituting A &B in eqn 1, Ca= Cao - (OUR/3Do)(a3/R + R2/2) + (OUR/Do)(a3/3r) + (OUR/Do)(r2/6) Ca = Cao -(OUR/3Do)[a3/R + R2/2 a3/r r2/2] Ca = Cao -(OUR/3Do)[a3(1/R-1/r) +(1/2)(R2-r2)