Biochemistry: the Molecules of Life

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Aeration, Agitation & its Kinetics

The majority of fermentation processes is aerobic and, therefore, requires the provision of oxygen. a microbial culture must be supplied with oxygen during growth at a rate sufficient to satisfy the organisms’ demand. The oxygen demand of an industrial fermentation process is normally satisfied by aerating and agitating the fermentation broth. However, the productivity of many fermentations is limited by oxygen availability and, therefore, it is important to consider the factors which affect a fermenter’s efficiency in supplying microbial cells with oxygen.

OXYGEN SUPPLY

Oxygen is normally supplied to microbial cultures in the form of air. The method varies with the scale of the process:

1. Laboratory-scale microbial cultures may be aerated by means of the shake-flask technique where the culture is grown in a conical flask shaken on a platform contained in a controlled environment chamber. Animal cell cultures are frequently grown in “T-flasks” which provide a large surface area for oxygen diffusion—the “T” referring to the total surface area available for cell growth; thus, a T-25 flask has a 25 cm2 growth area.

2. Microbial pilot- and industrial-scale fermentations are normally carried out in stirred, aerated vessels, termed fermenters. Laboratory scale experiments using culture volumes upward of approximately 500 cm3 are also performed in stirred, aerated fermenters as this enables the cultural conditions to be better monitored and controlled, and facilitates the addition of supplements and the removal of samples. Some fermenters are so designed that adequate oxygen transfer is obtained without agitation for example bubble columns and air-lift fermenters. Large-scale animal cell cultures have been grown in a range of fermenter types, including stirred aerated vessels based on microbial systems.

The rate of oxygen transfer from air bubble to the liquid phase may be described by the equation:

(1)

where C_L is the concentration of dissolved oxygen in the fermentation broth (mmoles dm^–3)

 t is time (hours)

dC_L/dt is the change in oxygen concentration over a time period, that is, the oxygen-transfer rate (mmoles O2 dm^–3 h^–1)

K_L is the mass transfer coefficient (cm h^–1)

a is the specific gas/liquid interface area per liquid volume (cm² cm^–3)

C* is the saturated dissolved oxygen concentration (mmoles dm^–3).

K_L may be considered as the sum of the reciprocals of the resistances to the transfer of oxygen from gas to liquid, and the difference between the saturated dissolved oxygen concentration and the actual concentration in the fermentation broth (C*–C_L) may be considered as the “driving force” across the resistances. It is extremely difficult to measure both K_L and “a,” the gas–liquid interface area, in a fermentation and, therefore, the two terms are generally combined in the term K_La, the volumetric mass-transfer coefficient, the units of which are reciprocal time (h^–1). The volumetric mass-transfer coefficient is used as a measure of the aeration capacity of a fermenter. The larger the K_La, the higher the aeration capacity of the system.

DETERMINATION OF K_La VALUES

The determination of the K_La of a fermenter is essential in order to establish its aeration efficiency and to quantify the effects of operating variables on the provision of oxygen. Dissolved oxygen is usually monitored using a dissolved oxygen electrode which records dissolved oxygen activity or dissolved oxygen tension (DOT) while the equations describing oxygen transfer are based on dissolved oxygen concentration.The solubility of oxygen is affected by dissolved solutes so that pure water and a fermentation medium saturated with oxygen would have different dissolved oxygen concentrations yet have the same DOT, that is, an oxygen electrode would record 100% for both. The dissolved oxygen concentration, for all practical purposes, will be zero and the K_La may then be calculated from the equation: (where OTR is the oxygen transfer rate.)

                                                           

  (2)

OXYGEN-BALANCE TECHNIQUE

The K_La of a fermenter may be measured during fermentation by the oxygen balance technique which determines, directly, the amount of oxygen transferred into solution in a set time interval. The procedure involves measuring the following parameters:

1. The volume of the broth contained in the vessel, VL (dm³).

2. The volumetric air flow rates measured at the air inlet and outlet, Q_i and Q_o, respectively (dm³ min^–1).

3. The total pressure measured at the fermenter air inlet and outlet, P_i and P_o, respectively (atm. absolute).

4. The temperature of the gases at the inlet and outlet, T_i and T_o, respectively (K).

5. The mole fraction of oxygen measured at the inlet and outlet, y_i and y_o, respectively.

The oxygen transfer rate may then be determined from the following equation:

(3)

Where 7.32 × 10⁵ is the conversion factor equaling (60 min h^–1) [mole/22.4 dm³ (STP)] (273 K/l atm).

The K_La may be determined, provided that CL and C* are known, from Eq. 1

(4)

EFFECT OF AIRFLOW RATE ON K_La

Non Mechanically agitated reactors Bubble columns and air-lift reactors are not mechanically agitated and, therefore, rely on the passage of air to both mix and aerate.

1. Bubble columns

The flow pattern of bubbles through a bubble column reactor is dependent on the gas superficial velocity. At gas velocities of below 1–4 cm s^–1 the bubbles will rise uniformly through the medium and the only mixing will be that created in the bubble wake. This type of flow is referred to as homogeneous. At higher gas velocities bubbles are produced unevenly at the base of the vessel and bubbles coalesce resulting in local differences in fluid density. The differences in fluid density create circulatory currents and flow under these conditions is described as heterogeneous 

The K_La in a bubble column is essentially dependent on the superficial gas velocity. for most non viscous situations the equation is:

(5)

Where V_s^c is the superficial air velocity corrected for local pressure. However, viscosity has an overwhelming influence on K_La in a bubble column that can be expressed as:

(6)

Where π is the liquid dynamic viscosity (N s m^–2).

2. Air-lift reactors

The difference between a bubble column and an air-lift reactor is that liquid circulation is achieved in the air-lift in addition to that caused by the bubble flow. The reactor consists of a vertical loop of two connected compartments, the riser and downcomer. Air is introduced into the base of the riser and escapes at the top. The degassed liquid is denser than the gassed liquid in the riser and flows down the downcomer. Thus, a circulatory pattern is established in the vessel—gassed liquid going up in the riser and degassed liquid coming down the downcomer. Circulation in an air-lift results in the bubbles being in contact with the liquid for a shorter time than in a corresponding bubble column. Thus, the K_La obtained in an air-lift will be less than that obtained in a bubble column at the same superficial air velocity, that is, less than 0.32 (V _s ^c) ^0.7 . The advantage of the air-lift lies in the circulation achieved, but this is at the cost of a lower K_La value.

Excerpts : Principles of fermentation technology, Stanbury & Whitaker

Scintillation Counting

Liquid Scintillation Counting (LS Counting) is a lab based strategy that utilizes a Liquid Scintillation Counter (LSC) to check the radioactive emanations from a liquid sample. It is regularly utilized in the organic sciences to quantify the take-up of radioactive isotopes into biological materials. Radioactive isotopes interact with matter in two different ways, ionization and excitation.

Excitation drives an energized molecule or compound (known as a fluor) to emanate photons of light. The procedure is known as scintillation. when the light is recognized by a photomultiplier, it frames the premise of scintillation counting. Basically, a photomultiplier changes over the energy of radiation into an electrical sign, and the  strength of the electrical signal that outcomes is legitimately corresponding to the energy of the first radioactive occasion. This implies two, or significantly more, isotopes can  be independently identified and estimated in a similar example, if they have  adequately different energy emitting spectra.

Types of scintillation counting

Solid scintillation counting

In solid scintillation counting the sample is placed adjacent to a solid fluor (e.g. sodium iodide). Solid scintillation counting is particularly useful for gamma emitting isotopes. This is because they can penetrate the fluor. The counters can be small handheld devices with the fluor attached to the photomultiplier tube or larger bench-top machines with a well-shaped fluor designed to automatically count many samples.

Liquid Scintillation Counting

In liquid scintillation counting, the sample is mixed with a scintillation fluid containing a solvent and one or more dissolved fluors. This method is particularly useful in quantifying weak b-emitters such as 3 H, 14C and 35S, which are frequently used in biological work. Scintillation fluids are called ‘cocktails’ because there are different formulations, made up of a solvent (such as Toulene) & fluors such as 2,5-diphenyloxazole (PPO), 1,4-bis(5- phenyloxazol-2-yl)benzene (nicknamed POPOP, or 2-(40 -t-butylphenyl)-5-(400-bi-phenyl)-1,3,4-oxydiazole (butyl-PBD).

                                                Liquid Scintillation Counting

There are a number of physical processes that may disrupt LSC. These include:

Process	Explanation	Examples	Reduce Problem By
Chemiluminescence	Generation of light due to chemical processes.	Bleaching agents, dioxane-based scintillators	Equilibrate sample for a period of time in the LSC
Photoluminescence	Emission of photons from excited molecular species.	Vials, caps, other materials in the LSC. Some samples such as proteinaceous materials when dissolved in alkaline solubilisers such as hyamine.	Acidify samples; avoid exposure to sunlight or fluorescent lighting. Dark adapt samples for several hours before counting.
Quenching	Reduction in the scintillation count rate.	Photon quenching, chemical impurity quenching, colour quenching (see diagram below).	Use Internal Standards to account for quenching. A standard with a known CPM/DPM (Counts per minute/Disintegrations per minute) is added and measured and the reduction due to quenching adjusted for in the measured samples.

Examples of the use of LS Counting

1. Viral Proteins: Proteins produced by viruses when they infect a cell are produced in very small amounts and are difficult to detect and purify. If virus-infected cells are fed a radioactive amino acid, then each time that amino acid is linked to form the growing protein a radioactive ‘label’ is attached to the protein. This radioactive ‘label’ is then used to monitor the identification and purification of the viral protein. Amino acids containing ³H, ¹⁴C and ³⁵S are often used to label proteins. ³⁵S is particularly useful as sulphur is only found in two amino acids – methionine and cysteine.
2. Environmental Monitoring: Checking for ³H spills in the laboratory. Tritium is such a weak emitter that its presence cannot be detected by a Geiger-Mueller counter. Wipe testing is usually used. This is where suspect surfaces are wiped with a piece of tissue. The tissue is placed in LS Cocktail in a LS vial and counted in the LS Counter.

 References:

1. Principles & Techniques of Biochemistry, 5th Edition. Keith Wilson & john Walker

2.http://simulab.ltt.com.au/5/Laboratory/StudyNotes/snLSCounting.htm

COMPLEMENT SYSTEM

Complement System

The complement system refers to a series of >20 proteins, circulating in the blood and tissue fluids. Most of the proteins are normally inactive, but in response to the recognition of molecular components of microorganisms they become sequentially activated in an enzyme cascade – the activation of one protein cleaves and activates the next protein in the cascade enzymatically. Complement can be activated through three different pathways,  each of which can cause the activation of C3, cleaving it into a large fragment, C3b, that acts as an opsonin, and a small fragment C3a (anaphylatoxin) that promotes inflammation. Activated C3 can trigger the lytic pathway, which can damage the plasma membranes of cells and some bacteria. C5a, produced by this process, attracts macrophages and neutrophils and also activates mast cells. Complement was discovered as a heat-labile component of normal plasma that causes the opsonisation and killing of bacteria.

Classical Pathway

This pathway involves complement components C1, C2 and C4. The pathway is triggered by antibody-antigen complexes binding to C1, which itself has three subcomponents C1q, C1r and C1s. The pathway forms a C3 convertase, C4b2a, which splits C3 into two fragments; the large fragment, C3b, can covalently attach to the surface of microbial pathogens and opsonise them; the small fragment, C3a, activates mast cells, causing the release of vasoactive mediators such as histamine.

Alternative Pathway

This pathway involves various factors, B, D, H & I, which interact with each other, and with C3b, to form a C3 convertase, C3bBb, that can activate more C3, hence the pathway is sometimes called ‘the amplification loop’. Activation of the loop is promoted in the presence of bacterial and fungal cell walls, but is inhibited by molecules on the surface of normal mammalian cells.

Mannose-binding Lectin Pathway

This pathway is activated by the binding of mannose-binding lectin (MBL) to mannose residues on the pathogen surface. This in turn activates the MBL-associated serine proteases, MASP-1 and MASP-2, which activate C4 and C2, to form the C3 convertase, C4b2a.

Role of Complement in Disease

The complement system plays a critical role in inflammation and defence against some bacterial infections. Complement may also be activated during reactions against incompatible blood transfusions, and during the damaging immune responses that accompany autoimmune disease. Deficiencies of individual complement components or inhibitors of the system can lead to a variety of diseases (Tabulated), which gives some indication of their role in protection against disease.

Table . Diseases associated with complement deficiencies

Complement Deficiency	Disease
C3 and Factor B	Severe bacterial infections
C3b-INA, C6 and C8	Severe Neisseria infections
Deficiencies of early C components C1, C4, C2.	Systemic lupus erythematosus (SLE)

REFERENCES:
Zaahira Gani, Cambridge, UK
https://www.immunology.org/public-information/bitesized-immunology/systems-and-processes/complement-system
https://www.youtube.com/watch?v=mfCeCvkQbuI&t=63s

FED BATCH KINETICS

FED BATCH KINETICS


Fed Batch as the name indicates means batch cultures which need to be fed continuously, with medium without removing culture fluid. The culture broth is harvested usually only at the end of the operational period, either fully or partially (the remainder serving as the inoculum for the next repeated run). This process may be repeated (repeated fed-batch) a number of times if the cells are fully viable and productive. Thus, there are one or more feed streams but no effluent during the course of operation. Sources of carbon, nitrogen, phosphates, nutrients, precursors, or inducers are fed either intermittently or continuously into the culture by manipulating the feed rates during the run. The products are harvested only at the end of the run. Therefore, the culture volume increases during the course of operation until the volume is full. Thereafter, a batch mode of operation is used to attain the final results. Thus, the fed-batch culture is a dynamic operation. By manipulating the feed rates, the concentrations of limiting nutrients in the culture can be manipulated either to remain at a constant level or to follow a predetermined optimal profile until the culture volume reaches the maximum, and then a batch mode is used to provide a final touch. In so doing, the concentration of the desired product or the yield of product at the end of the run is maximized. This type of operation was first called a fed-batch culture or fed-batch fermentation. 
The fed-batch culture has been practiced since the early 1900s, when it was recognized in yeast production from malt wort that the malt concentration in the medium had to be kept low enough to suppress alcohol formation and maximize the yield of yeast cells. High malt concentration would accelerate the cell growth, which in turn would cause anaerobic conditions that favored ethanol formation and lowered the yield of yeast cells. Additional wort was added at a rate that was always less than the rate at which the yeast cells could use it. Intermittent or incremental feeding of nutrients to an initially dilute medium was introduced thereafter in large-scale yeast production to improve the yeast yields while obviating the production of ethanol.4 However, there is some speculation that a small amount of ethanol may be necessary to ensure the quality of the baker’s yeasts.
The oldest and first well-known industrial application of a fed-batch operation was introduced after the end of World War I. It was the yeast cell production in which sugar (glucose) was added incrementally during the course of fermentation to maintain a low sugar concentration to suppress alcohol formation. The manufacture of yeast by fed-batch culture has gone through a series of improvements and is an industrially important fed-batch process. This process was historically followed by penicillin fermentation, in which the energy source (e.g., glucose) and precursors (e.g., phenyl acetic acid) were added incrementally during the course of fermentation8 to improve penicillin production. Prior to this practice, a slowly metabolized but more expensive substrate, lactose, was used in place of glucose in a batch culture. Oversupply of a carbon source resulted in more mycelial growth and low penicillin formation, while undersupply resulted in slower mycelial growth and, eventually, slower penicillin formation.
The primary candidates in the list of compounds that may be fed during the course of the operation include the limiting substrate, inducers, precursors, a carbon source, a nitrogen source, a phosphate source, inducers, and other nutrient sources. The feeding patterns are open loop or feedback controlled to maintain some key variables at constant optimum values such as the specific growth rate, respiratory quotient, pH, partial pressure of carbon dioxide, dissolved oxygen, substrate concentration, and some metabolite concentrations. The optimum feed rates sometimes require keeping these parameters to follow certain optimum profiles rather than keeping them at constant values. To maximize the cell formation rate for the case of constant cell mass yield, it is obvious that the substrate concentration should be maintained at the value that maximizes the specific growth rate, Sm, until the reactor is full. Therefore, it is also obvious that the initial substrate concentration should be Sm, that is, S(0) = Sm, and that the substrate concentration should be maintained at Sm throughout the course of fermentation. This will lead to the maximum cell concentration at the end of the run. To achieve this, the feed rate must be regulated properly to hold the substrate concentration constant at Sm. If it is not possible to set the initial substrate concentration to Sm for one reason or another, the substrate concentration should be brought to this value as soon as possible by applying at the beginning the maximum substrate feed rate (S(0) < Sm) or a batch period (S(0) > Sm) and then regulating thereafter to maintain the substrate concentration to remain at Sm until the fermentor is full. Once the fermentor is full, it is run in a batch mode to reduce the substrate concentration to a desired level, and the cells containing the product are harvested. This is the basis for the simplest case of a fed-batch culture.


 Initial establishment of fed batch is in BATCH mode and then the feeding is done by applying one of several strategies. 

lets see the strategies.... Quite interesting..uhmm

1.Use the same medium which you have used to establish the batch culture and add it. what will happen? increase in volume.. right???

2.Take the solution of limiting substrate; keeping concentration same as it was in initial medium & add it... again . tell me the result.... increase in volume... yes ! exactly..

Fed batch using above two strategies will be described as Variable Volume Fed Batch Culture

3.In this strategy concentrated solution of limiting substrate is to be added but at a rate less than no.1 & 2 strategy with the result is increase in volume.

Fed Batch employing above strategy is called as Intermediate Fed Batch Culture

4. This time a very concentrated solution of limiting substrate is added but at a rate less than strategy 1,2&3, resulting in overall increase in specific volume.

Fed Batch utilising above strategy is called as Fixed Volume Fed Batch Culture

So now we have an idea that what the heck is this fed batch strategy ..lets move on to the devilish part that is kinetics!!!! Start with Variable Volume Fed Batch culture

Variable Volume Fed Batch Culture

Pirt the king of kinetics ( That's what i call him......) , Dunn & Mor has developed this culture. I'm discussing Pirt's kinetics only. He said consider a batch culture in which growth is limited by concentration of one substrate; so whats the equation if we want to describe the biomass at any point of time::::

Equation for fed batch kinetics (variable volume)

Thus from above equation it may be concluded that input of substrate is compensated by substrate consumption. Thus ds/dt =0

Total biomass (X) concentration increases with time but cell concentration (x) remains constant, i.e. dx/dt = 0 and we can now write 

μ= D

This situation is quasi steady state

(P.S. ::: please go back to basics of kinetics if you have trouble following above equation.. )

The difference between steady state in chemostat and quasi steady statein fed batch is that μ is constant in chemostat and it decreases in fed batch.

Fixed volume in next blog ....till then understand it and let me know if youy face any issues

CREDITS::: Whitaker, Pirt, Cambridge.

Phagocytosis

Phagocyosis is the process of engulfment and digestion of microorganisms performed by macrophages & microphages.

The macrophages:

These cells originate from bone marrow promonocytes; after circulating in blood for few hours they migrate to tissues and constitute the mononuclear phagocyte system. They have been given specific names in accordance with their residing tissues; in lungs ; alveolar macrophages, in  liver ; kupffer cells, microglial cells in brain, mesangial cells in kidney and in bone as osteoclasts.

Macrophages are capable of ingesting and digesting exogeneous antigens such a s whole microorganisms and insoluble particles and endogeneous matter such as injured or dead cells. Macrophages act by process of chemotaxis in which they get attract towards variety of substances. In the next step antigen adheres to the cell membrane of macrophage. Adherence induces the protrusions called Pseudopodia to extend around the attached antigen. Fusion of pseudopodia encloses the antigen with in membrane bound structure called Phagosome which then enters the phagocytic processing pathway. Here Phagosome fuses with the lysosome and makes phagolyosome. Lysome has hydrolytic enzymes which digest the ingested material. The digested contents are then released through the process of exocytosis.

It has been found that phagocytosis of antigen can be enhances many times in the presence of specific antibody; so antibody functions as opsonin; a molecule that binds to antigen and macrophage and enhance the process of engulfment. This whole process whereby antigens are rendered more susceptible to phagocytosis is called as opsonisation.

for further reads:::

kuby's immunology

RNA-laced bandages could treat the worst wounds on the genetic level

couldn't help sharing this!!!
A breakthrough in RNA interference.
check it out!


http://io9.com/rna-laced-bandages-could-treat-the-worst-wounds-on-the-509956299

Proteases in leather Industry

Proteases in leather Industry

Proteases belong to a group of proteolytic enzymes. They are obtained by microbial fermentations and are meant to use in leather industry for Dehairing, bating and soaking purposes.   Their major use is in detergent industry too where they are used for breaking proteinaceous matter caused by body secretions, food stuffs and blood stains. These enzymes are obtained from plants, animals and microbial sources. Animal and Microbial Proteases from Fungi and bacteria re used in the pretanning processes of leather manufacture.

Animal Proteases are mixture of Trypsin, Chymotrypsin and various Peptidases which may contain amylase or lipase as secondary enzymes.

Proteases differ in their pH range as follows:

Acid Proteases:              pH 2.5 – 6.0 derived from A. satoi

Alkaline Proteases:        belong to group of serine proteases. More heat sensitive and heat                              

  deactivate at 60⁰ C

Neutral Proteases:        Obtained from Aspergillus and Penicillium spp.

Enzymes in Dehairing

Dehairing involves the digestion of basal cells of hair bulb and cells of malphigian layer. This is followed by loosening of hair with an attack on outermost sheath and breakdown of inner root sheath and parts of hair that are not keratinized.

Various Enzymes used in dehairing process are:

CLARIZYME: has been developed especially for dehairing of skins and hides. It is an alkaline protease derived from A. Flavus. It grows rapidly on wheat bran and produce large amount of protease.

Enzymes from bacteria have gained much importance because of commercial interest , easy production, high yield and easy recovery of enzyme. Examples are, Bacillus and Streptomyces spp.

Keratinases: hydrolyse Keratins are obtained from S. fradiae.

Enzymes in Soaking:

Soaking is the first operation where in hides and skins are cleaned and softened with water. Soaking is necessary for solubilisation and elimination of salts and globulin proteins contained within fibrous structures of hides and skins. It is carried out at alkaline conditions at temperature of 10-20⁰C.

Advantages of soaking are production of leather with less wrinkled skin.

Alkaline Proteases of Bacterial & Fungal origin has been used for soaking which reduces the need of chemicals.

Enzymes in Bating:

Bating is a process in which hides are softened by treating them in warm infusion of animal dung and product used for such purposes is called as BATE. Main objective of bating is to remove proteinaceous materials like albumin, globulins, mucoids from hides and skin and allow splitting up of collagen fibres to facilitate penetration of tanning materials and other chemicals, thereby giving finished leather with desired properties like feel, softness and pliability etc. Principal materials which a bate contains are proteolytic enzyme, a carrier for enzyme, wood flour and deliming agent like Ammonium chloride.

Enzymes from Aspergillus spp. are used in bating.

A combination of both mold and pancreatic enzymes are ideal bates.

Enzymes in Degreasing:

Degreasing is an essential step for production of glove and clothing leather. This process involves removal of excess natural fats from greasy skin. This grease results in defects like uneven dyeing, finishing and staining. Degreasing helps to obtain soft and pliable;e leather for garment manufacture.

It is carried out using aqueous emulsification with detergents or by solvent extraction since solvents are hazardous. So Lipase is used as an alternative for various solvents.

Acid Lipase from Rhizopus has been very effective in degreasing of sheep skins.

Fungal Lipase from A. Niger is also a good degreasing agent.

Alkaline Bacterial Lipase at pH of 9-9.4 is good for degreasing pig skin.

Enzyme (Cellulase ) Production

CELLULASE PRODUCTION

Cellulase term is referred to all enzymes which cleave beta, 1-4 glycosidic linkages in cellulose. Many bacteria and fungi are celluloytic but preparations marketed for industrial applications are derived from Aspegillus niger, Neurospora and Trichoderma viridae. Aspergillus enzyme exerts good activity on CMC (carboxy methyl cellulose) but fails to attack on solid cellulose because it lacks a important component i.e. C₁ Cellulase. Cellulase is multienzyme complex with 2 important components called C₁ and C_x. Both have important functions.

C₁ can attack upon native cellulose of higher crystallinity while C_x cannot attack such kind of cellulose but can split in turn the cellulose fragments which have been derived by action of C_1.

Trichoderma produces an enzyme complex with high levels of C₁ cellulase which extensively degrades insoluble cellulose.

Cultivation & Purification

In large scale fermentation A. niger is mostly cultured by wheat bran tray method. This process yields high levels of enzyme. Extraction of cellulose from solid substrate cultures is performed by percolation of dried mold bran with 0.02 to 0.1 M lactic acid.

Neurospora and Trichoderma are grown by submerged culture. For continuous culture it is advantageous that T. Viride produces a suspension of short mycelia threads, rarely forming pellets. For submerged cultivations bran and wheat straw pre-treated with alkali can be used as  a source of cellulose. Ammonium ions can be used a suitable nitrogen source. A correct pH profile is necessary to give optimum enzyme yields in batch culture concentration and purification of enzyme is carried out by precipitation, adsorption and gel filtration techniques.

Ammonium sulphate is used for precipitation followed by centrifugation at 10,000 g for 10 minutes. After separating the sediment supernatant is subjected to re centrifugation for 10,000 g for 10 minutes. Precipitates are suspended in 0.5 M acetate buffer with pH 5 and crystallisation procedures are followed.

Industrial Uses

Ø  Cellulase is currently used to improve texture and palatability of poor quality vegetables.

Ø  It also accelerates drying of vegetables.

Ø  A potential use of cellulose is conversion of cellulosic material to glucose and other sugars which in turn can be used as microbial substrates in variety of fermentations e.g. Alcohol.