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monika kaurav

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Fermentation technology
Fermentation • Fermentation is a metabolic process that produces chemical changes in organic substances through the action of enzymes. In biochemistry, it is narrowly defined as the extraction of energy from carbohydrates in the absence of oxygen. In food production, it may more broadly refer to any process in which the activity of microorganisms brings about a desirable change to a foodstuff or beverage. • Fermentation technology is the use of organisms to produce food, pharmaceuticals and alcoholic beverages on a large scale industrial basis. • The basic principle involved in industrial fermentation technology is that organisms are grown under suitable conditions, by providing raw materials meeting all the necessary requirements such as carbon, nitrogen, salts, trace elements and vitamins.
Fermentation • The end products formed as a result of their metabolism during their life span are released into the media, which are extracted for use by human being and that have a high commercial value. The major products of fermentation technology produced economically on a large scale industrial basis are wine, beer, cider, vinegar, ethanol, cheese, hormones, antibiotics, complete proteins, enzymes and other useful products.
Fermentation • Types of Fermentation Processes: • There are three different process of fermentation viz.: • (1) Batch fermentation • (2) Fed-batch fermentation and • (3) Continuous culture.
Fermentation: Methods Batch fermentation: • Batch fermentation is a process where all the substrate and nutrients are added at zero time or soon after inoculation takes place, and the vessel is allowed under a controlled environment to proceed until maximum end product concentration is achieved(i.e. the metabolite or target protein). In batch fermentation, six phases of the microbial growth are seen. • Lag phase  Acceleration phase  Log phase  Deceleration Phase  Stationary phase:  Death phase:
Fermentation: Methods • (a) Lag phase: • Immediately after inoculation, there is no increase in the numbers of the microbial cells for some time and this period is called lag phase. This is in order that the organisms adjust to the new environment they are inoculated into. • (b) Acceleration phase: • The period when the cells just start increasing in numbers is known as acceleration phase. • (c) Log phase: • This is the time period when the cell numbers steadily increase.
Fermentation: Methods • (d) Deceleration phase: • The duration when the steady growth declines. • (e) Stationary phase: • The period where there is no change in the microbial cell number is the stationary phase. This phase is attained due to depletion of carbon source or accumulation of the end products. • (f) Death phase: • The period in which the cell numbers decrease steadily is the death phase. This is due to death of the cells because of cessation of metabolic activity and depletion of energy resources. Depending upon the product required the different phases of the cell growth are maintained. For microbial mass the log phase is preferred. For production of secondary metabolites i.e. antibiotics, the stationary phase is preferred.
Fermentation: Methods • Fed-batch fermentation: • In this type of fermentation, freshly prepared culture media is added at regular intervals without removing the culture fluid. • This increases the volume of the fermentation culture. This type of fermentation is used for production of proteins from recombinant microorganisms.
Fermentation: Methods • Continuous fermentation: • In this type of fermentation the products are removed continuously along with the cells and the same is replenished with the developed cells and addition of fresh culture media. • This results in a steady or constant volume of the contents of the fermentor. This type of fermentation is used for the production of single cell protein (S.S.P), antibiotics and organic solvents.
Fermentation: Methods • Procedure of Fermentation: • (a) Depending upon the type of product required, a particular bioreactor is selected. • (b) A suitable substrate in liquid media is added at a specific temperature, pH and then diluted. • (c) The organism (microbe, animal/plant cell, sub-cellular organelle or enzyme) is added to it. • (d) Then it is incubated at a specific temperature for the specified time.
Fermentation: Methods • (e) The incubation may either be aerobic or anaerobic. • i. Aerobic conditions are created by bubbling oxygen through the medium. • ii. Anaerobic conditions are created by using closed vessels, wherein oxygen cannot diffuse into the media and the oxygen present just above is replaced by carbon dioxide released. • (f) After the specified time interval, the products are removed, as some of the products are toxic to the growing cell or at least inhibitory to their growth. The organisms are re-circulated. The process of removal of the products is called downstream processing
Fermentation: Methods • Types of fermenter • Available in various sizes • According to the sizes classified as • Small lab and research fermenter :1-50L • Pilot plant fermenter: 50-1000 L • Large size industrial production scale fermenter: more than 1000 L
Fermentation: Methods • Broadly fermenters are also classified as • I. surface fermenters • Tray fermenter • Packed bed column fermenter • II. Submerged fermenters • Simple fermenters (batch and continuous) • Fed batch fermenter • Air-lift • Bubble fermenter • Cyclone column fermenter • Tower fermenter • Other more advanced systems, etc
Fermentation: Types • Surface fermenters • Microbial cells cultured on surface layer of the nutrient medium (solid/liquid) held in dish or tray • Used for production of citric acid from Aspergillus niger and nicotinic acid from Aspergillus terrus • Microbial films can be developed on the surfaces of suitable packing medium, may be in the form of fixed bed, stones or plastic sheets.
Fermentation: Types • TRAY FERMENTER • One of the simplest and widely used fermenters. • Its basic part is a wooden, metal, or plastic tray, often with a perforated or wire mesh bottom to improve air circulation. • A shallow layer of less than 0.15 m deep, pretreated substrate is placed on the tray for fermentation. Solid as well as liquid medium are used
Fermentation: Types • TRAY FERMENTER • Temperature and humidity-controlled chambers are used for keeping the individual trays or stacks. • A spacing of at least one tray height is usually allowed between stacked trays. • Cheesecloth may be used to cover the trays to reduce contamination. • Inoculation and occasional mixing are done manually, often by hand. • If liquid medium, cells are allowed to float easily and to make a process continuous • If solid medium is used the micro-organisms are allowed grow on moist solid materials, process is called Solid State Fermentation
Fermentation: Types • Solid State Fermentation (SSF) • SSF defined as the growth of the micro-organisms on (moist) solid material in the absence or near-absence of free water • Used for production of antibiotics, enzymes, alkaloids, organic acids bio-pharmaceutical products • Advantages : • Produce higher yields than submerged liquid fermentation • Possibilities of contamination by bacteria and yeast is very less • All natural habitats of fungi are easily maintained in SSF • culture media very simple , provides all nutrients for growth of micro-organisms
Fermentation: Types • SSF Disadvantages: • Causes problems in monitoring of the process parameters such as pH, moisture content, and oxygen concentration • Despite some automation, tray fermenters are labor intensive • Difficulties with processing hundreds of trays limit their scalability • Aeration may be difficult due to high level of solid content • Substrates require pre treatment such as size reduction, chemical or enzymatic hydrolyses
Fermentation: Types • Packed bed fermenters • This is type of surface culture bioreactor • A bed of solid particles, with biocatalysts on or within the matrix of solids, packed in a column • The solids used may be porous or non- porous gels, and they may be compressible or rigid in nature.
Fermentation: Types • Packed bed fermenters • A nutrient broth flows continuously over the immobilised biocatalyst. The products obtained in the packed bed bioreactor are released into the fluid and removed. • The concentration of the nutrients can be increased by increasing the flow rate of the nutrient broth. • Because of poor mixing, difficult to control the pH of packed bed bioreactors by the addition of acid or alkali.
Fermentation: Types • Submerged fermenters • The microorganisms are dispersed in liquid nutrient medium at maintained environmental conditions. on the mechanism of agitation Submerged fermenters grouped as follows: • I. Mechanically stirred fermenter • batch operate fermenter • continuous stirred tank fermenter • II. Pneumatic fermenter • Fluidized bed reactor
Fermentation: Types • Submerged fermenters • III. Forced convection fermenters • Air –lift fermenter • Bubble column • Sparged tank fermenter • These are equipped with a mechanical agitator so as to maintain homogencity and rapid dispersion and mixing of materials • Examples includes stirred tank fermenter (batch or continuous operated) , multistage fermenter, paddle wheel reactor, and stirred loop reactor Mechanically stirred fermenter
Fermentation: Types • Stirred tank fermenter (STF • batch operated fermenter • agitators consists of one or more impellers mounted on the shaft • It is rotates with the help of electric motor • Advantage of this fermenter flexibility in design • Used in the range of 1- 100 ton capacity sizes Stirred tank fermenter
Fermentation: Types • Continuous stirred tank fermenter • A continuous stirred tank fermenter consists of a cylindrical vessel with motor driven central shaft that supports one or more agitators (impellers). • The shaft is fitted at the top of the bioreactor. The number of impellers is variable and depends on the size of the fermenter Continuous stirred tank fermenter (CSTF) Continuous stirred tank fermenter
Fermentation: Types • Continuous stirred tank fermenter • In this fresh medium is added continuously in the fermenter vessel • On the other end the medium is withdrawn for the recovery of fermentation products • As it is a continuous fermenter the Steady state conditions can be achieved by either Chemostatic or Turbidostatic principles.
Fermentation: Types • Different types of continuous fermenter are • a. Single stage: single fermenter is inoculated and kept in continuous operation by balancing the input and output culture media • b. Recycle continuous fermentation: a portion of the withdrawn culture or residual unused substrate plus the withdrawn culture is recycled • c. Multistage continuous operation: involves two or more stages with the fermenter being operated in sequence multistage
Fermentation: Types • STF Advantages of batch operated • Less risk of contamination because of short growth period • Process is more economical and simple • Raw material conversion level is high • Disadvantages: • Low productivity due to time required for the sterilizing, filling, cooling, emptying and cleaning • More expenses are required for subcultures for inoculation, labor and process control
Fermentation: Types • Advantages of continuous operated • Less labor expenses due to automation of fermentation process • Less toxicity risk to operator by toxins producing microorganisms • High yield and good quality product due invariable operating parameters and automation of the process • Less stress on the fermenter as sterilization is not frequent • Disadvantages: Higher investment costs in control and automation equipment • More risk of contamination and cell mutation
Fermentation: Types • Bubble column fermenters • In the bubble column bioreactor, the air or gas is introduced at the base of the column through perforated pipes or plates, or metal micro porous spargers (ref fig). • The flow rate of the air/gas influences the performance factors —O2 transfer, mixing. • May be fitted with perforated plates to improve performance. The vessel used for bubble column bioreactors is usually cylindrical with an aspect ratio of 4- 6 (i.e., height to diameter ratio). ..
Fermentation: Types • Air lift fermenter • Airlift fermenter (ALF) is generally classified as forced convection fermenters without any mechanical stirring arrangements for mixing. • The turbulence caused by the fluid (air/gas) flow ensures adequate mixing of the liquid. The baffle or draft tube is provided in the reactor. • A baffle or draft tube divides the fluid volume of the vessel into 2 inter- connected zones. • Only one of the 2 zones is sparged with air or other gas. • The sparged zone is known as " riser", the zone that receives no gas is "downcomer“.
Fermentation: Types • Air lift fermenter • Mainly 2 types • Internal-loop airlift bioreactor (ref Fig) has a single container with a central draft tube that creates interior liquid circulation channels. These bioreactors are simple in design, with volume and circulation at a fixed rate for fermentation. • External loop airlift bioreactor (ref fig) possesses an external loop so that the liquid circulates through separate independent channels. These reactors can be suitably modified to suit the requirements of different fermentations. Internal loop External loop
Fermentation: General requirements • Inoculum development • Inoculum production is a critical stage in an Industrial fermentation process. • Obviously, one loop of cell line requires a prolonged period if it is directly introduced in to fermentation. • Thus, inoculum is prepared as a stepwise sequence employing increasing volumes of media. • Constituent of Inoculum media :- • Chemical composition :- The Inoculum media must have a suitable Chemical composition. Generally, the medium should contain a source of carbon, a source of Nitrogen, growth factors and Mineral salts. • Buffering Capacity :- Maintenance of the PH in the optimum range is necessary for making the process successful. In order the control the PH of the medium, buffers (e.g. CaCo ) Should be added to the medium.
Fermentation: General requirements • Avoidance of foaming :- • 1.Foaming is a serious problem in a fermentation Industry. • 2. Hence, defoamers (e.g. oil mixed with Octadecanol for penicillin fermentations) should be used for controlling foam. • Consistency :- 1. Proper aeration and agitation.
Fermentation: General requirements
Fermentation: Media • NUTRIENTS • Most fermentations require liquid media, often referred to as broth; although some solid substrate fermentations (SSF) are operated. • Fermentation media must satisfy all the nutritional requirements of the microorganism and fulfil the technical objectives of the process. • All microorganisms require water, sources of energy, carbon, nitrogen, mineral elements and possibly vitamins plus oxygen if aerobic. • The nutrients should be formulated to promote the synthesis of the target product, either cell biomass or a specific metabolite.
Fermentation: Media • In most industrial fermentation processes there are several stages where media are required. They may include several inoculum (starter culture) propagation steps, pilot scale fermentations and the main production fermentation. • The technical objectives of inoculum propagation and the main fermentation are often very different, which may be reflected in differences in their media formulations.
Fermentation: Media • Medium formulation • Medium formulation is essential stage in manufacturing process Carbon & Nitrogen other Energy + sources + O2 + nutrients Biomass + products + CO2 +H2O +heat • Elemental composition of microorganisms may be taken as guide
Fermentation: Media • CARBON SOURCE • A carbon source is required for all biosynthesis leading to reproduction, product formation and cell maintenance. In most fermentations it also serves as the energy source. • Molasses • malted barley • Starch and Dextrins • Sulphite Waste Liquor
Fermentation: Media • Alkanes and Alcohols n-Alkanes • Oils and fats • Factors influencing the carbon source - Cost of the product - rate at which it is metabolized - geographical locations - government regulations - cellular yield coefficient
Fermentation: Media • Nitrogen Sources • Most industrial microbes can utilize both inorganic and organic nitrogen sources. • Inorganic nitrogen may be supplied as ammonium salts, often ammonium sulphate and diammonium hydrogen phosphate, or ammonia. Ammonia can also be used to adjust pH of the fermentation. • Organic nitrogen sources include amino acids, proteins and urea.:Corn Steep Liquor, Yeast Extracts, Peptones, Soya Bean Meal
Fermentation: Media • Minerals • All microorganisms require certain mineral elements for growth and metabolism. In many media, magnesium, phosphorous, potassium, sulphur, calcium and chlorine are essential components and must be added. • Others such as cobalt, copper, iron, manganese, molybdenum and zinc are present in sufficient quantities in the water supplies and as impurities in other media ingredients.
Fermentation: Media • Chelators • Many media cannot be prepared without precipitation during autoclaving. Hence some chelating agents are added to form complexes with metal ions which are gradually utilised by microorganism • Examples of chelators: EDTA, citric acid, polyphosphates etc., • It is important to check the concentration of chelators otherwise it may inhibit the growth. • In many media these are added separately after autoclaving Or yeast extract, peptone complex with these metal ions
Fermentation: Media • Vitamins and Growth Factors • Many bacteria can synthesize all necessary vitamins from basic elements. For other bacteria, filamentous fungi and yeasts, they must be added as supplements to the fermentation medium. • Most natural carbon and nitrogen sources also contain at least some of the required vitamins as minor contaminants
Fermentation: Media • Precursors • Precursors are defined as “substances added prior to or simultaneously with the fermentation which are incorporated without any major change into the molecule of the fermentation product and which generally serve to increase the yield or improve the quality of the product”. • They are required in certain industrial fermentations and are provided through crude nutritive constituents, e.g., corn steep liquor or by direct addition of more pure compounds.
Fermentation: Media • Inducers and Elicitors • If product formation is dependent upon the presence of a specific inducer compound or a structural analogue, it must be incorporated into the culture medium or added at a specific point during the fermentation. • The majority of enzymes of industrial interest are inducible. Inducers are often substrates such as starches or dextrins for amylase. • In plant cell culture the production of secondary metabolites, such as flavanoids and terpenoids can be triggered by adding elicitors.
Fermentation: Media • Inhibitors • Inhibitors are used to redirect metabolism towards the target product and reduce formation of other metabolic intermediates • others halt a pathway at a certain point to prevent further metabolism of the target product. • An example of an inhibitor specifically employed to redirect metabolism is sodium bisulphite
Fermentation: Media • WATER • All fermentation processes, except SSF, require vast quantities of water. Not only is water a major component of all media, but it is important for ancillary services like heating, cooling, cleaning and rinsing. • A reliable source of large quantities of clean water, of consistent composition, is therefore essential. • Assessing suitability of water - pH - dissolved salts - effluent contamination Reuse of water is important - It reduces water cost by 50% - Effluent treatment cost by 10 fold
Fermentation: Media • Oxygen • Depending on the amount of oxygen required by the organism, it may be supplied in the form of air containing about 21% (v/v) oxygen or occasionally as pure oxygen when requirements are particularly high. • The organism’s oxygen requirements may vary widely depending upon the carbon source. For most fermentations the air or oxygen supply is filter sterilized prior to being injected into the fermenter.
Fermentation: Media • Antifoams • Antifoams are necessary to reduce foam formation during fermentation. • Foaming is largely due to media proteins that become attached to the air-broth interface where they denature to form a stable foam “skin” that is not easily disrupted • An ideal antifoam should have the following properties • Disperse readily and have fast action • Active at low concentrations • Long acting in preventing new foam
Fermentation: Media • Should not be metabolized • Should not be toxic to m.o, humans etc • Cheap, should not cause problem in fermentation
Fermentation: sterilization • Sterilization is defined as the complete destruction or elimination of all viable organisms (in or on an object being sterilized). • There are no degrees of sterilization: an object is either sterile or not. • Sterilization procedures involve the use of heat, radiation, chemicals or physical removal of cells. • Media for industrial fermentations are usually sterilized. • In some cases the economics of the fermentation makes it unrealistic to sterilize. • The fermentations can proceed, however, these fermentations employ low contamination inhibitors (lactic acid) to hold in check the numbers of microorganisms. pH and other contaminating
Fermentation: sterilization • In other cases, sterilization is not required as the media components are poorly utilized by contaminating microorganisms. • Fermentation media are sterilized by the use of: filtration, radiation, ultrasonic treatment, chemical treatment or heat (boiling or passing live steam through the medium, or by subjecting the medium to steam under pressure - autoclaving). • Steam is used almost universally for the sterilization of fermentation media. The major exception is the use of filtration for the sterilization of animal cell culture.
Fermentation: sterilization • Heat: Heat is the most important and widely used method. For sterilization, the type of heat, time of application and temperature required to ensure destruction of all microorganisms must always be considered. Endospores of bacteria are the most thermo- resistant of all cells so their destruction usually guarantees sterility. • Incineration: In this process, organisms are burned and physically destroyed. It is widely used for needles, inoculating wires, glassware, tubes etc. and objects that cannot be destroyed in the incineration process. • Boiling: Boiling is done at >100˚C for 20-30 min. It kills everything except for some endospores. Tokill endospores and therefore perfectly sterilize the solution, very long or intermittent boiling is required.
Fermentation: sterilization • Autoclaving: Autoclaving is the process of using steam under pressure in an autoclave or pressure cooker. It involves heating at 121˚C for 15-20 min under 15 psi pressure and can be used to sterilize almost anything. However heat labile substances will be denatured or destroyed. Sterilization of nutrient media is usually done using this process. • Dry Heat (Hot Air Oven): The process involves heating at 160˚C for 2 hours or at 170˚C for 1 hour. It is used for glassware, metal and objects that will not melt. • Sterilization in industry-scale fermenters (or bioreactors) is more complex. Steam is used to sterilize fermentation media. The medium can be sterilized in situ within the bioreactor. However, if the medium is sterilized in a separate vessel, the bioreactor needs to be sterilized before the sterile medium is added to it.
Fermentation: sterilization • Bioreactors are sterilized by passing steam through spargers. Spargers are devices that distribute gas bubbles (usually sterile air or steam) in a liquid phase. They have particular design criteria, e.g., providing small sized bubbles (the sparger breaks the incoming air into small bubbles). • Various designs can be used such as porous materials made of glass or metal. However, the most commonly used type of sparger used in modern bioreactors is the sparge ring. A sparge ring consists of a hollow tube in which small holes have been drilled and is easier to clean than porous materials and is also less likely to block during fermentation. During sparging, steam pressure is held at 15 psi in the vessel for 20 min.
Fermentation: Aeration • The purpose of aeration in fermentation is to supply oxygen to and, at the same time, to remove carbon dioxide from microbial cells suspended in the culture broth. The rate of aeration often controls the rates of cell growth and product formation. • Mixing in the gas and liquid phases affects the aeration characteristics of a fermenter. • Various types of aerobic fermenter could be classified into three major types: • (1) sparged mechanically stirred fermenter • (2) bubble column fermenter, and • (3) loop fermenter
Fermentation: Aeration • Transfer of gases in fermentation involves three phases, i.e., gas, culture medium, and microbial cells suspended in the medium. Oxygen absorbed from the gas-liquid interface diffuses through the culture medium to the cell surface and is consumed by the microbes. • Transfer of CO2 takes place in the reverse direction. Theoretically, resistances to gas transfer should exist in the gas film, the liquid film at the gas-liquid interface, the bulk of liquid, and the liquid film surrounding cells. • In case microbes form mycelial pellets, the diffusion resistance within the pellets could be significant. In some cases, a surfactant or antifoam agent may accumulate at the gas-liquid interface, giving an additional resistance to gas transfer.
Fermentation: Aeration • Aeration is to provide microorganism in submerged culture with sufficient oxygen for metabolic requirements. • Agitation ensures that a uniform suspension of microbial cells is achieved in a homogenous nutrient medium. • Aeration and agitation depends on fermentation.
AERATION SYSTEM  Syn : sparger  Adevice that introduce air intomedium  Has a pipe with minute holes (1/64 - 1/32 inch or large)  Hole – allows air under Pto escape intomedium  For mycelial growth – ¼ inch holes  Impeller blades disperses air released through sparger into medium
SPARGER TYPES  Porous  Orifice  Nozzle POROUS SPARGER:  Made of sintered glass, ceramics or metal  Used mainly on a large scale fermenters  Bubble size produced – 10-100times larger than pores  Throughput of air is low – Pdrop across it  Clogging of pores ORIFICE SPARGER  Those with drilled air holes on their under surface of the tubes making up ring or cross effluent  Without agitation used to a limited extend in yeast manufacture & treatment NOZZLE SPARGER  Modern mechanically stirred fermentors use them  Single open or partially closed pipes  Ideally, positioned centrally below impeller  Causes lower Pdrops  no clogging of pore
Fermentation: Stirring • Fine bubble aerator without agitation: • Advantage of lower equipment and power costs, • Agitation may be dispensed with only when aeration provides sufficient agitation. • E.g. in processes when broth of low viscosity and low total solids. • Mechanical agitation is required for fungal and actinomycetes fermentation.
Fermentation: Stirring • Agitation: Importance • 1. To increase the rate of oxygen transfer from the air bubble to the liquid medium. • 2. To increase the rate of oxygen and nutrients transfer from the medium to cells. • 3. To prevent formation of clumps of cells, aggregates of mycelium. • 4. To increase the rate of transfer of product of metabolism from cell to medium. • 5.To increase the rate or efficiency of heat transfer between the medium and the cooling surfaces of the fermenters.
Fermentation: Stirring • Effect of agitation on aeration • 1. by dispersing the air in smaller bubble. • 2.by causing the bubbles to follow a more tortuous path and dalaying their escape from the culture. • 3. by preventing the coalescence of bubbles. • 4. by decreasing the rate-limiting thickness of the liquid film at the gas/liquid interface.
FERMENTOR’S STRUCTURAL COMPONENTS IN AERA TION &AGIT A TION SYSTEM: The agitator Stirrer glands & bearings Baffles The aeration system
AGITATOR  Synonym : impeller  Mounted to a shaft through a bearing in the lid  Driven by an external power source or direct drive  Direct drive - action varied by using different impeller blades  Recent designs – driven by magnetic coupling to a motor mounted beneath the fermenter  High speed of rotation marked vortex occurs  Spinning of medium in circular direction  MIXING OBJECTIVES ITACHIEVE  Bulk fluid & gas  Heattransfer phase mixing  Air dispersion  Suspension of solid particles  O2 transfer  Maintenance of uniform environment throughout the vessel
CLASSIFICATION  Disc turbine  V anned disc  V ariable pitch open turbine  Marine propellers DISC TURBINE:  Adisc with series of rectangular vanes set in a vertical plane around the circumference.  Break up a fast air stream without itself becoming flooded in air bubbles
V ANED DISC  Aseries of rectangular vanesattached vertically to the underside  Air from sparger hits it’sunderside & the air gets displaced towards the vanes  Results in destruction of air bubbles VARIALBLE PITCH OPENTURBINE:  Vanesare attached directly to a boss on the agitator shaft  Air bubbles hit any surface by its action  Flood when super fial velocity exceed 21m/h
MARINE PROPELLER  Blades are attached directly to a boss on the agitator shaft  Air bubbles hit surface  Asingle low shear impeller  Mainly used in animla cell culture vessel  Flood when superfial velocity exceed 21m/h
MODERNAGITATORS  Rushton disc turbine  Scaba 6SRGT  Prochem max flowT  LighteningA315  Ekato intermig
BAFFLES  Metal strips  1/10th of the vesseldiameter  Attached radially to wall  4 baffles (normal)  Wider baffles -high agitation effect  Narrower baffles – low agitation effect  Can be attached with cooling coils  Not found in lab scale fermentors.  Verticalbaffles – increased aeration
Fermentation: Aeration and Stirring • Oxygen supply affected by following: • Type of agitation: • The shape, number and arrangement of impellers and baffles. • Either 2 or 3 impellers for large fermenters at suitable level on the stirrer shaft or 3 or 4 baffles on the wall of the vessel. • Speed of agitation: • 1000 or more for lab. Fermenters. • But this is not possible for large vessels. For penicillin fermentation requires 50rpm needs high input of energy and uneconomical. • Depth of liquid in the fermenters: • Bubble remain longer in the medium of a tall, deep fermenter. Greater hydrostatic pressure at the sparger improves solution of oxygen. • Height : diameter ratio of 3:1 or 4:1 is common.
Large scale production fermenter design and its various controls.
IDEAL FERMENTORPROPERTIES  Supports maximum growth of the organism  Aseptical operation  Adequate aeration and agitation  Low power consuming  Tempurature control system  pH control system  Sampling facilities  Minimum evaporation loss  Minimum use of labour  Range of processes  Smooth internal surfaces  Similar in geometry to both smaller & larger vessels in pilot plant
 Cheapest material usuage  Adequate service provisions  Provision for control of contaminants  Provision for intermittent addition of antifoams  Inoculum introduction facility  Mechanism for biomass/ product removal  Setting for rapid incorporation of sterile air  Withstands pressure  Ease of manipulation
BASIC DESIGN OF AFERMENTOR
Various components of an ideal process are fermenter for batch
Monitoring and controlling parts of are fermenter
SHAPE OF FERMENTER: FermentationAreAvailable In Different Shapes Like Conical Fermenter Cylindrical fermenter Spherical fermenter Pear In Shape Fermenter SIZES OFFERMENTER : The sizes of the fermenter are divided into the following groups. 1. The microbial cell (mm cube) 2. Shake flask (100-1000ml) 3. Laboratory fermenter (1-50 L) 4. Pilot scale (0.3 -10m cube) 5. Industrial scale (2-500m cube)
MATERIAL OF CONSTRUCTION Laboratory scale bioreactor: • In fermentation with strict aseptic requirements it is important to select materials that can withstand repeated sterilization cycles. On a small scale, it is possible to use glass and/or stainless steel. • Glass is useful because it gives smooth surfaces, is non-toxic, corrosion proof and it is usually easy to examine the interior of vessel. The glass should be 100% borosilicate, e.g. Pyrex® and Kimax®. • The following variants of the laboratory bioreactor can be made: 1. Glass bioreactor (without the jacket) with an upper stainless steel lid. 2. Glass bioreactor (with the jacket) with an upper stainless steel lid. 3. Glass bioreactor (without the jacket) with the upper and lower stainless steel lids. 4. Two-part bioreactor - glass/stainless steel. The stainless steel part has a jacket and ports for electrodes installation. 5. Stainless steel bioreactorwith peepholes. Vessels with two stainless steel plates cost approximately 50% more than those with just a top plate
Pilot scale and large scale bioreactors: • When all bioreactors are sterilized in situ, any materials use will have to assess on their ability to withstand pressure sterilization and corrosion and their potential toxicity and cost. • Pilot scale and large scale vessels are normally constructed of stainless steel or at least have a stainless steel cladding to limit corrosion. • The American Iron and Steel Institute (AISI) states that steels containing less than 4% chromium are classified as steel alloys and those containing more than 4% are classified as stainless steel. • Mild steel coated with glass or phenolic epoxy materials has occasionally been used. Wood, concrete and plastic have been used when contamination was not a problem in a process. • V essel shape: - • Typical tanks are vertical cylinders with specialized top plates and bottom plates. In some cases, vessel design eliminates the need for a stirrer system especially in air lift fermenter. A tall, thin vessel is the best shape with aspect ratio (height to diameter ratio) around 10:1. Sometimes a conical section is used in the top part of the vessel to give the widest possible area for gas exchange.
• Stainless steel top plates. • The top plates are of an elliptical or spherical dish shape. The top plates can be either removable or welded. Aremovable top plate provides best accessibility, but adds to cost and complexity. • Various ports and standard nozzles are provided on the stainless plate for actuators and probes. These include pH, thermocouple, and dissolved oxygen probes ports, defoaming, acid and base ports, inoculum port, pipe for sparging process air, agitator shaft and spare ports. • Bottom plates: • Tank bottom plates are also customized for specific applications. Almost most of the large vessels have a dish bottom, while the smaller vessels are often conical in shape or may have a smaller, sump type chamber located at the base of the main tank. These alternate bottom shapes aid in fluid management when the volume in the tank is low. One report states that a dish bottom requires less power than a flat one. • In all cases, it is imperative that tank should be fully drainable to recover product and to aid in cleaning of the vessel. Often this is accomplished by using a tank bottom valve positioned to eliminate any “dead section” that could arises from drain lines and to assure that all content will be removed from the tank upon draining.
If the bioreactor has a lower cover, then the following ports and elements should be placed and fastened there: 1. Discharge valve; 2. Sampling device; 3. Sparger; 4. Mixer's lower drive; 5. Heaters. Height-to-diameterratio (Aspect ratio). • The height-to-diameter ratio is also a critical factor in vessel design. Although a symmetrical vessel maximizes the volume per material used and results in a height-to-diameter ratio of one, most vessels are designed with higher ratio. The range of 2-3:1 is more appropriate and in some situation, where stratification of the tank content is not an issue or a mixer is used, will allow still higher ratio to be used in design. • The vessels for microbiological work should have an aspect ratio of 2.5- 3:1, while vessels for animal cell culture tend to have an aspect ratio closer to 1. The basic configuration of stirred tank bioreactors for mammalian cell culture is similar to that of microbial fermenter but the major difference is there in aspect ratio, which is usually smaller in mammalian cell culture bioreactor.
Common Measurement And Control Systems
Introduction : • Antibiotics are antimicrobial agents produced naturally by other microbes (usually fungi or bacteria) • The first antibiotic was discovered in 1896 by Ernest Duchesne and in 1928 "rediscovered" by Alexander Fleming from the filamentous fungus Penicilium notatum. • The antibiotic substance, named penicillin, was not purified until the 1940s (by Florey and Chain), just in time to be used at the end of the second world war. • Penicillin was the first important commercial product produced by an aerobic, submerged fermentation Cont..,
Cont.., • Penicillin is produced by the fungus Penicilium chrysogenum which requires lactose, other sugars, and a source of nitrogen (in this case a yeast extract) in the medium to grow well. • Like all antibiotics, penicillin is a secondary metabolite, so is only produced in the stationary phase.
• It exhibits the properties of a typical secondary metabolites. • It active against certain Gram- positive bacteria in presence of blood, pus and body fluids. • It is soluble in water. It is very soluble in acetone, ethyl alcohol and ether and it is less soluble in benzene, chloroform, ect.. • Aqueous solution of penicillin are unstable and must be stored under refrigeration. • Penicillin is most stable in the pᴴ range of 6.0 to 6.5 and reasonably stable over the pᴴ range of 5.5 to 7.5 . Properties of penicillin :
Types of penicillin : • Penicillin are compound of the general formula C₁₆H₁₈N₂O₅S –R, in which R represents the radical or group that is different for each day. The structural formula of the most common type ( F,G, X and k ) are given. • Penicillin F, G, X and K are produced by strain of the penicillin notatum – chrysogenum group of molds ; flavicidin ( flavicin) by Aspergilus flavus ; and dihydro F penicillin (gigantic acid ) by Aspergilus gigantic. Basic structure of penicillin : The basic structure of the penicillin's is 6- aminopenicillenic acid (6- APA), composed of a thiozolidine ring fused with a β- lactam ring whose 6- amino position carries a variety of acyl substituent's.
⦁ Also known as Penicillium notatum. ⦁ It is common in temperate and subtropical regions and can be found on salted food products, but it is mostly found in indoor environments, especially in damp or water-damaged buildings. ⦁ It is the source of several β-lactam antibiotics, most significantly penicillin which inhibits the biosynthesis of bacterial cell walls affecting lysis of the cell.
⦁ Kingdom: Fungi ⦁ Division:Ascomycota ⦁ Class: Eurotiomycetes ⦁ Order: Eurotiales ⦁ Family: Trichocomaceae ⦁ Genus: Penicillium ⦁ Species: Chrysogenum
⦁ Penicillium chrysogenum exhibits typical eukaryotic cell structure; it has a tubulin cytoskeleton which is used for motility. TAM structure of P . Chrysogenum Structure of P . Chrysogenum
⦁ This image displays the typical filamentous hyphae that contain many conidia. ⦁ The oblong structures in the image are conidia, the asexual spores of the fungus. ⦁ In P .chrysogenum, the conidia are blue to blue-green. ⦁ These conidia are the cause of pathogenicity in humans as in the cases of allergy and endophthalmitis. ⦁ The conidia originate from complexes known as conidiophores. ⦁ The growth of conidiophores begins when a stalk sprouts out of a foot cell. ⦁ The stalk swells at the end and forms a vesicle. Sterigmata form from the vesicle which give way to long chains of conidia.
⦁ It produces the hydrophobic β-lactam compound penicillin. ⦁ Penicillium chrysogenum remains the primary producer of Penicilian G and Penicilian V ⦁ P .chrysogenum has been used industrially to produce Penicilian G and Penicilian V and Xanthocillin X, and to produce the enzymes polyamine oxidase, phosphogluconate dehydrogenase, and glucose oxidase. ⦁ Penicillium chrysogenum can be used to assist crops to fight off other pathogenic species.
⦁ P.chrysogenum is high yielding strain and therefore most widely used as production strain. ⦁ Inoculum Preparation: ⦁Purpose is to develop a pure inoculum in an adequate amount. To do so various sequential steps are necessary like: 1) Astarter culture is needed for inoculation. 2) After getting growth on solid media, one or two growth stages should allowed in shaken flask cultures to create a suspension, which can be transferred to seed tanks for further growth. 3) After about 24-28 hours, the content of the seed tanks is transferred to the primary fermentation tanks.
4) •All the bio parameters like temperature, pH, aeration, agitation etc. should be properly maintained. • Bio parameters  pH: near 6.5  Temperature: 26°C to 28°C  Aeration: a continuous stream of sterilized air is pumped into it.  Agitation: have baffles which allow constant agitation (200rpm).
⦁ Fermentation broth contains all the necessary elements required for the proliferation of the microorganisms. ⦁ Generally, it contains a carbon source, nitrogen source, mineral source, precrsors and antifoam agents. Carbon Source ⦁ Lactose in a concentration of 6%. ⦁ Other carbohydrates like glucose & sucrose. ⦁ Complex as well as cheap sources like molasses, or soya meal can also be used which are made up of lactose and glucose sugars.
Nitrogen Source ⦁ Ammonium salts such as ammonium sulphate, ammonium acetate, ammonium lactate or ammonia gas are used for this reason. ⦁ Sometime corn steep liquor may be used. Mineral Source ⦁ These elements include phosphorus, sulphur, magnesium, zinc, iron, and copper which generally added in the form of water soluble salts. Precursors ⦁ Various types of precursors are added into production medium to produce specific type of penicillin.
⦁ For example, if phenyl acetic acid is provided then only penicillin-G will be produced but if hydroxy phenyl acetic acid is provided then penicillin-X will be produced. ⦁ Phenoxy acetic acid is provided as precursor for penicillin-Vproduction. ⦁ When corn steep liquor is provided as nitrogen source, it also provides phenyl acetic acid derivatives; therefore it is widely used in the production of penicillin-G.
Anti-foam agents ⦁ Anti-foaming agents such as lard oil, octadecanol and silicones are used to prevent foaming during fermentation. Recovery ⦁ The recovery of penicillin is carried out in three successive stages: 1. Removal of mycelium 2. Counter current solvent extraction of penicillin 3. Treatment of crude extracts
⦁ At harvest the fermentation broth is filtered on a rotatory vacuum filter to remove the mycelium and other solids. ⦁ Phosphoric or sulfuric acids are added to lower the pH (2 to 2.5) in order to transform the penicillin to the anionic form. ⦁ Then the broth is directly extracted in a Podbielniak Counter Current Solvent Extractor with an organic solvent such as methyl isobutyl ketone, amyl acetate or butyl acetate. ⦁ Penicillin is then again extracted into water from the organic solvent by adding an adequate amount of potassium or sodium hydroxide to form a salt of the penicillin. ⦁ The resulting aqueous solution is again acidified & re- extracted with methyl isobutyl ketone.
⦁ This shifts between water and solvent help in purification of the penicillin. ⦁ The solvent extract is carefully back extracted with NaOH and from this aqueous solution; various procedures are utilized to cause the penicillin to crystalize as sodium or potassium penicillinate. ⦁ The resulting crystalline penicillin salts are then washed and dried. ⦁ Sometimes the crude extract of penicillin is passed out from charcoal treatment to eliminate pyrogens; even sterilization can also be done.
Citric Acid Production
CONTENT  Introduction  History  Microbes in Citric acid Production  Citric acid fermentation techniques  Factors affecting Citric acid production  Industrial production of citric acid  Applications/Uses of citric acid  Side effects  Largest producers in the world
Overview • Citric acid is a usually occuring acid found primarily in Several Varieties of fruits and vegetables with citrus fruits such as lemons and limes containing the highest amounts of citric acid. • This Organic acid has many uses, including as a food additive /Preservative, ingredient in Cosmetic products and as a powerful cleaving agent.
Introduction: • Citric acid (2-hydroxy-1,2,3- propane tri carboxylic acid) is the most important commercial product, which is found in almost all plant & animal tissues. • Citric acid is the most important organic acid produced in tonnage and is extensively used in food and pharmaceutical industries. • Citric acid is a weak organic acid found in citrus fruits(lemon). • It is good ,natural preservative and is also used to add an acidic taste to food and soft drinks. • More than million tonnes are produced every year by fermentation.
HISTORY: • The discovery of citric acid has been credited to the 8th century Muslim alchemist Jabir Ibn Hayyan (Geber). • Citric acid was first isolated in 1784 by the Swedish chemist carl Wilhelm Scheele, who crystallize it from lemon juice. • Industrial scale citric acid production began in 1890 based on the Italian citrus fruit industry. • In 1893, C. Wehmer discovered penicillin mold could produce citric acid from sugar. However, microbial production of citric acid did not become industrially important until world war I disrupted Italian citrus exports.
Structure of citric acid
Micro-organisms used for citric acid production: • Large number of micro-organisms including bacteria, fungi and yeasts have been employed to produce citric acid. • The main advantages of using this micro-organisms are: • Its easy of handling • Its ability to ferment a variety of cheap raw • materials • High yields
Micro organisms: • Fungi: • Aspergillus nagger • A. aculeatus • A. awamori • A. carbonarius • A. wentii • A. foetidus • Penicillium janthinelum • Bacteria: • Bacillus licheniformis Arthrobacter paraffinens Corynebacterium species • Yeasts: • Saccahromicopsis lipolytica Candida tropicalis • C. oleophila • C. guilliermondii • C. parapsilosis • C. citroformans Hansenula anamosa
Citric acid production: • Fermentation is the most economical and widely used ay for synthesis citric acid production. • The industrial citric acid production can be carried in three different ways: • surface fermentation • submerged fermentation • solid state fermentation
Surface Fermentation:  Surface fermentation using Aspergillus niger may be done on rice bran as is the case in Japan, or in liquid solution in flat aluminium or stainless steel pans.  Special strains of Aspergillus niger which can produce citric acid despite the high content of trace metals in rice bran are used.
SUBMERGED FERMENTATION: • In this case , the strains are inoculated of about 15cm depth in fermentation tank. • The culture is enhanced by giving aeration using air bubbles. • And its allowed to grow for about 5 to 14 days at 27 to 33 degree Celsius. • The citric acid produced in the fermentation tank and it is purified.
Solid state fermentation: • It is simplest method for citric acid production. • Solid state fermentation is also known as koji process, was first developed in Japan. • Citric acid production reached a maximum(88g/kg dry matter)when fermentation as carried out with cassava having initial moisture of 62% at 26degree Celsius for 120 hours.
Separation: • The biomass is separated by filtration. • The liquid is transferred to recovery process • Separation of citric acid from the liquid precipitation. • Calcium hydroxide is added to obtain calcium citrate.
Tetra hydrate Wash the precipitate Dissolve it with dilute sulfuric acid, yield citric acid and calcium sulfate precipitate Bleach and crystallization Anhydrous or mono hydrate citric acid Separation process:
PURIFICATION: • Purification is a simple form of getting a pure citric acid followed by two simple techniques. • Precipitation • Filtration React citric acid with calcium carbonate Filter precipitate React precipitate with sulfuric acid Filter precipitate Purified citricAcid
Citric acid production A. niger CA16 and 79/20 Substrate cut , dried and powdered Grown in PDAagar slant Mixed with water at different concentration A. Niger spores 7days old culture Filtration @ sterilization inoculation with 1 10 spores/25mLf FILTERATION Filtrate for citric acid CELLBIOMASS
Factors affecting citric acid production: • Nitrogen source • pH • Aeration • Trace elements • Temperature
Industrial production of citric acid: • 99% of world production: microbial processes surface or submerged culture. • 70% of total production of 1.5 million tons per year is used in food and beverage industry as on acidifier or antioxidant to preserve or enhance the flavors and aromas of fruit juices, ice cream and marmalades. • 20% used: pharmaceutical industry as anti oxidant to preserve vitamins, effervescent, pH corrector, blood preservative, or in the form of iron citrate.
Tablets, ointments and cosmetic preparations • Chemical industry remaining 10% softening and treatment of textile. • Also used in the detergent industry as a Phosphate substitute, because of less entropic effect hardening of cement
Applications/uses of citric acid: • Food & drink: • Preservative and flavoring agent Emulsifying agent in ice-cream. • Household cleaner: • Kitchen Bathroom sprays. • Cosmetics: • Shampoos Body wash • WASH CLEANERS: • Nail polish • Hand soap and other cosmetic products
• To cure kidney disorders: • Sodium citrate, acetic acid is used to prevent kidney stones. • Side effects: • Taking excess of citric acetate in combination with sodium citrate may lead to kidney failure. • Taking citric acid with empty stomach may lead to stomach or intestinal side-effects. • It may also lead to muscle twisting or cramps. • It can also cause weight gain, swelling, fast heart rate, slow o rapid .
Vitamin B12
 Avitamin is an organic compound and a vital nutrient that an organism requires in limited amounts.  They are of great value in the growth and metabolism of the living cells.  Vitamins are obtained with food, but a few are obtained by other means ; humans can produce some vitamins from precursors they consume while certain microorganism produce vitamins too.  Thirteen vitamins are universally recognized at present, vitamins are classified by their biological and chemical activity.  Vitamins can be classified as “Fat soluble vitamins” and “Water soluble vitamins”
FAT SOLUBLE VITAMINS W ATERSOLUBLE VITAMINS  VitaminA(Retinol)  Vitamin D 1. Vitamin D2 (Egrocalciferol) 2. Vitamin D3 (Cholecalciferol)  Vitamin E (Tocopherol)  Vitamin K (Phylloquinone)  Vitamin B Complex 1. Vitamin B1 (Thiamine) 2. Vitamin B2 (Riboflavin) 3. Vitamin B3 (Niacin) 4. Vitamin B5 (Pantothenic acid) 5. Vitamin B6 (Pyridoxine) 6. Vitamin B7 (Biotin) 7. Vitamin B9 (FolicAcid) 8. Vitamin B12 (Cobalamin)  Vitamin C (Ascorbic acid)
 Vitamin B12, also called Cobalamin, is a water- soluble vitamin that has a key role in the normal functioning of the brain and nervous system, and the formation of red blood cells.  It is involved in the metabolism of every cell of the human body, especially affecting DNAsynthesis, fatty acid and amino acid metabolism.  It is synthesized only by microorganisms and not by animals (including humans) and plants.  People with B12 deficiency may eventually develop Pernicious anemia.  It is the largest and most structurally complicated vitamin and can be produced
 Cyanocobalamin, is the industrially produced stable Cobalamin form which is not found in nature.  Vitamin B12 is entirely produced on a commercial basis by the fermentation. It is usually manufactured by submerged culture process. Such a fermentation process is completed in 3-5 days.  Most of the B12 fermentation processes use glucose as a carbon source.  The microorganisms that maybe employed in the industrial production process are : i. Streptomyces griseus ii. Streptomyces olivaceus iii. Bacillus megaterium iv. Bacillus coagulans v. Pseudomonas denitrificans vi. Propionibacterium freudenreichii vii. Propinibacteriun shermanii
Step 1 • Formulation of the medium Step 2 • Sterilization of the medium Step 3 • Making starter culture Step 4 • Anaerobic fermentation Step 5 •Aerobic fermentation Step 6 •Recovery
 Production by Streptomyces olivaceus yields about 3.3mg / L of vitamin B12.  Process : A. Preparation Of Inoculum : Pure slant culture of S. olivaceus is inoculated in 100-250ml of inoculum medium, contained in Erlenmeyer flask. Seeded flask is incubated on platform of a mechanical shaker to aerate the system. This flask culture is then subsequently used to inoculate larger inoculum tanks. (2 or 3 successive transfers are made to obtain required amount of inoculum cultures.)
 Media used in preparation of inoculum is Bennett’s agar. Component Amount (g/L) Yeast extract 1.0 Beef extract 1.0 N-Z-AmineA (Enzymatic hydrolase of casein) 2.0 Glucose 10.0 Agar 15.0 D/W 1000 mL pH 7.3
B. Production Medium :  Consist of carbohydrate, proteinaceous material, and source of cobalt and other salts.  It is necessary to add cobalt to the medium for maximum yield of cobalamin.  Cyanide is added for conversion of other cobalamins to vitamin B12. Component Amount (%) Distiller’s Solubles 4.0 Dextrose 0.5 - 1 CaCO3 0.5 COCL2.6H2O 1.5 – 10 ppm pH 7
C. Sterilization of the medium :  Sterilization can be done batchwise of continuously.  Batch – medium heated at 250°F for 1 hour.  Continuous – 330°F for 13 min by mixing with live steam. D. Temperature , pH ,Aeration andAgitation : • Temperature : A temperature of 80°F in production tank is satisfactory during fermentation. • pH : At starting of process pH falls due to rapid consumption of sugar, then rises after 2 to 4 due to lysis of mycelium. pH 5 is maintained with H2SO4 and reducing agent Na2SO4. • Aeration and Agitation : Optimum rate of aeration is 0.5 volume air/volume medium/min.
E.Antifoam agent , Prevention of contamination :  Antifoam agent : Defoaming agents like soya bean oil , corn oil, lard oil and silicones can be used.  Prevention of contamination : Essential to maintain sterility, contamination results in reduced yields, equipments must be sterile and all transfers are carried out under aseptic conditions. F . Recovery :  During fermentation, most of cobalamin is associated with the mycelium; boiling mixture at pH 5 liberates the cobalamin quantitatively from mycelium.  Broth containing cobalamin is subjected to further process to obtain crystalline B12.
Filtration of broth to remove mycelium. Filtered broth is treated with cyanide to bring conversion of cobalamin to cyanocobalamin. Adsorption of cyanocobalamin from the solution is done by passing it through adsorbing agents packed in a column. Cyanocobalamin is then eluted from the adsorbent by the use of an aqueous solution of organic bases or solutions of Na-Cyanide and Na- thiocyanate. Extraction is carried out by countercurrent distribution between cresol, amylphenol, or benzyl alcohol and water or a single extraction into an organic solvent (e.g. Phenol) is carried out. Chromatography on alumina and final crystallization completes the process.
 Production by Propionibacterium freudenreichii yields about 20mg/Lof vitamin B12. A. Production media : glucose , corn- steep , betaine , & cobalt.  Betaine -0.5 %  Cobalt – 5µg./ml (excess cause reduced cobalamin formation) B. pH -7.5 C. Temperature – 30°C
D. Fermentation : It involves to cycles; anaerobic fermentation cycle of 70 hours andAerobic fermentation cycle of 50 hours.  Anaerobic fermentation :  Formation of cobinamide occurs.  The pH falls from 7.5 to 6.5 and then rises upto 8.5.  Necessary to add 0.1% of 5, 6 – dimethyl benziminazole to the production medium.  Aerobic fermentation :  Nucleotide formation takes place.  This nucleotide then links with cobinamide to give cobalamin.
Species Medium Aeration Temp. (°C) Time (hour) Yield (mg/L) B.megaterium Molasses , mineral salts, cobalt Aerobic 30 18 0.45 P . shermanii Glucose , corn- steep, ammonia , cobalt pH 7.0 Anaerobic (3 days), aerobic (4 days) 30 150 23 B. coagulants Citric acid , triethanolam ine , corn – steep , cobalt. Aerobic 55 18 6.0
GLUTAMIC ACID FERMENTATION
Introduction Amino acids have always played an important role in biology of life, in biochemistry and in (industrial) chemistry. Amino acids are the building blocks of proteins and they play an essential role in the metabolism regulation of living organisms. Large scale chemical and microbial production processes have been commercialised for a number of essential amino acids. Current interest in developing peptide-derived chemo- therapeutics has heightened the importance of rare and non- proteinogenic pure amino acids.
Amino acids are versatile chiral (optically active) building blocks for a whole range of fine chemicals. Amino acids are, therefore, important as nutrients (food), seasoning, flavourings and starting material for pharmaceuticals, cosmetics and other chemicals.  Amino acid can be produced by :  Chemical synthesis  Isolation from natural materials  Fermentation  Chemo-enzyme methods Importance of Amino acids
Glutamic acid Glutamic acid is an α-amino acid that used in biosynthesis of proteins. It contains an α-amino group which is in the protonated −NH3+. An α-carboxylic acid group which is in the deprotonated −COO.  And a side chain carboxylic acid. Polar negatively charged (at physiological pH), aliphatic amino acid. It is non-essential in humans, meaning the body can synthesize it.
Glutamic Acid  Food Production:  As flavor enhancer, to improve flavor.  As nutritional supplement.  Beverage  As flavor enhancer: in soft drink and wine. Cosmetics  As Hair restorer: in treatment of Hair Loss.  As Wrinkle: in preventing aging. Agriculture/Animal Feed  As nutritional supplement: in feed additive to enhance nutrition. Other Industries  As intermediate: in manufacturing of various organic chemicals.
Biosynthesis of Glutamic acid Reactants Products Enzymes Glutamine + H2O → Glu + NH3 GLS, GLS2 NAcGlu + H2O → Glu + Acetate (unknown) α-ketoglutarate + NADPH + NH4 + → Glu + NADP+ + H2O GLUD1, GLUD2 α-ketoglutarate + α-amino acid → Glu + α-oxo acid transaminase 1-pyrroline-5-carboxylate + NAD+ + H2O → Glu + NADH ALDH4A1 N-formimino-L-glutamate + FH4 ⇌ Glu + 5-formimino-FH4 FTCD  An amino acid precursor is converted to the target amino acid using 1 or 2 enzymes.  Allows the conversion to a specific amino acid without microbial growth, thus eliminating the long process from glucose.  Raw materials for the enzymatic step are supplied by chemical synthesis.  The enzyme itself is either in isolated or whole cell form which is prepared by microbial fermentation.
Industrial Production and use of Microorganisms  Industrial microbiology  Microorganisms, typically grown on a large scale, to produce products or carry out chemical transformations.  The glutamic acid is produced through the fermentation process  Major organism used is Corynebacterium glutamicum .  Classic methods are used to select for high-yielding microbial variants.  Properties of a useful industrial microbe include  Produces spores or can be easily inoculated.  Grows rapidly on a large scale in inexpensive medium.  Produces desired product quickly.  Should not be pathogenic.  Amenable to genetic manipulation. Corynebacterium glutamicum
The manufacturing process of glutamic acid by fermentation comprises :- a. fermentation, b. crude isolation, c. purification processes.  There are 4 types of fermentation are used:  (1) Batch Fermentation.  (2) Fed-batch Fermentation.  (3) Continuous Fermentation. Industrial production of glutamic acid
(1)Batch Fermentation  Widely use in the production of most of amino acids. Fermentation is a closed culture system which contains an initial, limited amount of nutrient. A short adaptation time is usually necessary (lag phase) before cells enter the logarithmic growth phase (exponential phase). Nutrients soon become limited and they enter the stationary phase in which growth has (almost) ceased. In glutamic acid fermentations, production of the acid normally starts in the early logarithmic phase and continues through the stationary phase.
For economical reasons the fermentation time should be as short as possible with a high yield of the amino acid at the end. A second reason not to continue the fermentation in the late stationary phase is the appearance of contaminant- products. The lag phase can be shortened by using a higher concentration of seed inoculum. The seed is produced by growing the production strain in flasks and smaller fermenters.
(2) Fed-batch fermentation Batch fermentations which are fed continuously, or intermittently, with medium without the removal of fluid.  In this way the volume of the culture increases with time. The residual substrate concentration may be maintained at a very low level. This may result in a removal of catabolite repressive effects and avoidance of toxic effects of medium components.  Oxygen balance. The feed rate of the carbon source (mostly glucose) can be used to regulate cell growth rate and oxygen limitation,especially when oxygen demand is high in the exponential growth phase.
(3) Continuous fermentation  In continuous fermentation, an open system is set up.  Sterile nutrient solution is added to the bioreactor continuously.  And an equivalent amount of converted nutrient solution with microorganisms is simultaneously removed from the system.
 Natural product such as sugar cane is used.  Then, the sugar cane is squeezed to make molasses. The heat sterilize raw material and other nutrient are put in the tank of the fermenter. The microorganism (Corynebacterium glutamicum) producing glutamic acid is added to the fermentation broth. The microorganism reacts with sugar to produce glutamic acid. Then, the fermentation broth is acidified and the glutamic acid is crystallized. Industrial production of glutamic acid
Separation and purification After the fermentation process, specific method is require to separate and purify the amino acid produced from its contaminant products, which include:  Centrifugation.  Filtration.  Crystallisation.  Ion exchange.  Electrodialysis.  Solvent extraction.  Decolorisation.  Evaporation.
The glutamic acid crystal is added to the sodium hydroxide solution and converted into monosodium glutamate (MSG). MSG is more soluble in water, less likely absorb moisture and has strong umami taste. The MSG is cleaned by using active carbon, which has many micro holes on their surface. The clean MSG solution is concentrated by heating and the monosodium glutamate crystal is formed. The crystal produce are dried with a hot air in a closed system. Then, the crystal is packed in the packaging and ready to be sold. Separation and purification of Glutamic acid
PREPARATION OF GRISEOFULVIN BY FERMENTATION
GRISOFULVIN- INTRODUCTION  Griseofulvin is an antifungal antibiotic first isolated from a Penicillium species in 1939. It is a secondary metabolite produce by thefungus Penicillium griseofulvum.  The compound is insoluble in water,and slightly soluble in ethanol, methanol, acetone, benzene, CHCl3, ethyl acetate, and acetic acid.
MolecularFormula: C17H17ClO6
MODE OF ACTION  Griseofulvin inhibit fungal cell mitosis and nuclear acid synthesis. It also binds to and interferes with the function of spindle and cytoplasmic microtubules by binding to alpha and beta tubulin.  It binds to keratin in human cells, and then once it reaches the fungal site of action, it binds to fungal microtubules thus altering the fungal process of mitosis.
USES It is used in the treatment of ⚫ Ringworm of the Beard ⚫ Ringworm of Scalp ⚫ Fungal Disease of the Nails ⚫ Ringworm of Groin Area ⚫ Athlete's Foot ⚫ Ringworm of the Body.
SIDE EFFECTS The most common side-effects are ⚫ Nausea ⚫ Vomiting ⚫ Diarrhoea ⚫ Heartburn ⚫ Flatulence, cracking at the side of the mouth ⚫ Soreness and/or blackening of the tongue and thirst ⚫ Headache
CULTIVATION
PREPARATION OF MEDIA Medium ⚫ Czapek Dox Medium Chemicals ⚫ Glucose ⚫ Sodium Nitrate ⚫ Potassium Hydrogen Phosphate ⚫ Magnesium Sulphate 7H20 5% 0.2% 0.1% 0.05%
Industrial preparation of griseofulvin by submerged fermentation
STEPS INVOLVED IN THE MANUFACTURING PROCESS  Fermentation  Pre treatment of fermentation broth  Filtration  Extraction  Decolorization  Isolation and separation  Precipitation and purification
FERMENTATION  The pH of Czapek-Dox medium was adjusted between 6.0-7.2. The medium was dispensed in the fermenter .  The fresh sample of mycelial suspension of fungus Peccillium griseofulvum from the fresh slope on raper steep agar (Czapek-Dox medium + corn steep+ agar) was obtained.  The solution was autoclaved for 200 minutes at 120°C at 15lbs pressure and fermented for 14 days at 24°C.
PRE TREATMENT OF FERMENTATION BROTH  The broth is heated above 60°C for 20- 30minutes.  After heating, sufficient coagulation of material occurs to produce a valuable improvement in separation characteristics of the broth.  The period of heating may be short, 5-10 minutes at 80°C having been found to provide a satisfactory increase in filtration rate.
FILTRATION  Drum covered with diatomaceous earth matter and allowed to rotate under vacuum with half immersed in the slurry tank. Small amount of coagulation agent added to broth and pumped into the slurry tank. As drum rotates in the slurry tank under vacuum thin layer of coagulated particles adhere to drum.  The layer thickens to from cake. As the cake portion in the drum comes to the upper region which is not immersed in the liquid it is washed with water and dewatered immediately by blowing air over it.  Then before the dried portion is again immersed into the liquid it is cut off from drum by knife.
EXTRACTION  Griseofulvin is extracted in the cold acetone when it is used as an extraction agent.  The extractions with the cold acetone may be carried out with the efficiencies between 75-96% or even upto 99.5%. the quantity of the solvent used in the extraction at large scale production should be kept minimum.  The volume of acetone should be 3-5 times of the mycelial felt.
DECOLORIZATION  The color of the extract can be improved by the addition of calcium hydroxide usually 2.5-50 g/liter preferably 5- 30 g/liter. The pH of the extract should be above 10. It can be neutralize by the removal of lime or by using mineral acid.
ISOLATION AND SEPARATION  The impurities or waxy substances are removed by washing the extract with a solvent in which extract is immiscible and also griseofulvin is insoluble.  Hydrocarbon solvents, generally aliphatic hydrocarbons such as hexane or petroleum containing a high portion of hexane are in general suitable for this step.
PRECIPITATION AND PURIFICATION  Griseofulvin can be precipitated from the solvent extract in various ways. One of the method is using the liquid solvent in which griseofulvin is substantially insoluble. Griseofulvin non-solvent is preferably water.  The alkaline water is more effective for the removal of colored impurities present in the crystals of the griseofulvin.  Water is made alkaline with ammonia or an alkali metal carbonate or alkali metal hydroxide. The suitable pH is about 8.5.  The purity of the precipitate is generally improved by washing with a solvent for the small quantities of impurities remaining. The suitable washing media are dry or wet acetone, a lower alkanol for example methanol or butanol. Marked purification is obtained with the use of methanol for this step.
Blood Products: Collection, Processing and Storage
Blood • The importance of blood in health and disease has been appreciated since ancient times but blood transfusion was not practised on a large scale until early this century. • Previous attempts were often frustrated by clotting and ignorance of the existence of blood groups; therefore, for centuries blood letting rather than transfusion remained a pillar of medical therapy.
Blood Products • COLLECTION • The blood is collected aseptically from the median cubital vein, in front of the elbow, into a sterile container containing an anticoagulant solution. During collection the bottle is gently shaken to ensure that blood and anticoagulant are well mixed, thus preventing the formation of small fibrin clots. • Not more than 420 ml is taken at one attendance. Immediately afterwards the container is sealed and cooled to 4-6°C. • The equipment used for taking blood is made from plastics, and is disposable.. The container is most often the Medical Research Council blood bottle but plastic bags have been used in America
Blood • “Whole Human’ Blood • This is human blood that has been mixed with a suitable anticoagulant. Any person in good health is accepted as a donor provided that he or she • 1. Is not suffering from any disease that can be transmitted by transfusion. This includes syphilis, malaria, and serum jaundice. • 2. Is not anaemic. The haemoglobin content of the blood should not be less than 12.5 and 13.3 per cent for female and male donors respectively.
Blood • COLLECTION • The blood is collected aseptically from the median cubital vein, in front of the elbow, into a sterile container containing an anticoagulant solution. During collection the bottle is gently shaken to ensure that blood and anticoagulant are well mixed, thus preventing the formation of small fibrin clots. • Not more than 420 ml is taken at one attendance. Immediately afterwards the container is sealed and cooled to 4-6°C. • The equipment used for taking blood is made from plastics, and is disposable.. The container is most often the Medical Research Council blood bottle but plastic bags have been used in America for some years and are likely to be the containers of the future (see Gunn and Carter, 1965).
Blood • BLOOD CLOTTING • According to classical theory blood clotting takes place in two phases • In response to injury, the tissues and blood platelets free substances that activate the clot promoting enzyme thromboplastin. This, with the assistance of ionised calcium and other factors, converts prothrombin into the active clotting enzyme thrombin which acts on fibrinogen, converting it into insoluble fibrin, the matrix of the clot.
Blood • 1. Citrates • The solution most often used as a blood anti coagulant is known as Acid-citrate-dextrose (ACD) and has the composition • Sodium acid citrate 2:0 to 25.G • Dextrose : 30G • Water for Injections to 120ml • The citrate prevents clotting by binding ‘the calcium ions as unionised calcium citrate.
Blood Products • “At one time the normal (trisodium) citrate was used but it has a very alkaline pH in solution which causes considerable caramelisation (darkening) of the dextrose during sterilisation and the two solutions have to be autoclaved separately. The acid citrate produces a pH of about 5 and causes little or no caramelization. In addition, it is less likely to induce flaking of the glass of the container. The higher concentration (2:5 G/120 ml) is often preferred because it’ more effectively reduces the formation of small clots. • The dextrose delays haemolysis of the erythrocytes in vitro and prolongs their life after transfusion. Its function may be connected with the synthesis of compounds, such as adenosine tri-phosphate (ATP)
Blood Products • 2. Heparin • This is a naturally occurring anticoagulant made by the mast ceils of the connective tissue surrounding blood vessels. It inhibits clotting in the circulatory system. Occasionally it is used in blood for transfusion when large volumes must be given to one patient and the corresponding amounts of citrate would be harmful, e.g. in cardiac surgery. • It quickly loses activity in blood in vitro and normal quantities are effective for about a day. ADC, on the other hand, prolongs the storage life to three weeks. Heparin is expensive and may continue its action after transfusion, necessitating the administration of neutralising substances such as protamine sulphate.
Blood Products • Disodium Edetate • This is a chelating agent which, because it has a strong affinity for divalent metals, will bind calcium firmly. It is sometimes preferred when preservation of blood platelets is essential, although the stability of these seems to depend much more on preventing contact with the glass surfaces.
Blood Products • Testing • At the time that blood is taken, two small additional amounts are collected. • One, which is often obtained by draining the collecting tube, is put into a small 5ml bottle and is firmly attached to the main container. This is for testing compatibility with the blood of the recipient before administration; using a separate specimen avoids the dangerous procedure of attempting to remove a sample from the main bottle without causing bacterial contamination. • With a plastic bag it is possible to leave the blood-filled collecting tube attached to the bag and to seal it at several points with a special tool; then a section can be separated for testing without contaminating the bulk.
Blood Products • Testing • The second, somewhat larger sample is used as soon as possible: • (a) for a serological test to confirm the absence of syphilis, and • (b) to determine the ABO grouping of the cells and plasma and the Rh grouping of the cells. • Blood groups . • Fundamentally, the aim of blood grouping is to prevent ‘an antigen-antibody reaction. Red cells carry an antigen that reacts with the corresponding antibody in the plasma of individuals of certain other groups. If the cells are transfused-into an individual with the equivalent antibody in his plasma they are rapidly destroyed, with serious consequences.
Blood Products • ABO System. • The first sign of the haemolytic antigen-antibody reaction is agglutination and, therefore, red-cell antigens and plasma antibodies are called agelutinogens and agglutinins respectively. • The agglutinated cells haemolyse, freeing haemoglobin and other constituents and causing jaundice and kidney damage. if the latter is extreme the patient may die. • Fortunately, most transfusion reactions are mild.
Blood Products • The red cells of some individuals carry an antigen that, because it is also found in the rhesus monkey is Known as the rhesus (Rh) factor. If Rh+ bloodis transfused info an Rh negative recipient production of antibodies to Rh + cells may be stimulated. • If this occurs, subsequent transfusion of Rh + blood to the same patient will cause a haemolytic reaction.
Blood Products • STORAGE • Apart from short periods for transport and examination, which must not exceed thirty minutes, blood must be kept at 4 to 6°C until required for use. • Even at this temperature deleterious changes take place. The leucocytes disintegrate in a few hours and the platelets in a few days. The red cells. show a fall in ATP and other organic - phosphates, a reduction in oxygen-carrying capacity and, due partly to loss of lipid from .their membranes, increased fragility. • Storage at room temperature, even for only a day, seriously reduces post-transfusion survival of the erythrocytes.
Blood Products • The fitness of blood for transfusion is based on its appearance. On standing, the cells sediment, leaving a layer of yellow supernatant plasma. • If the blood has been taken shortly after a heavy fatty meal the plasma may be turbid and show a white layer of fat on its surface. On top of the red cells there may be a complete or partial greyish layer of leucocytes. • The most important feature, however, is the line of demarcation between cells and plasma, which must be sharp; if it is obscured by a diffuse red colouration, indicating haemolysis, the blood is unfit for use.
Blood Products • The fitness of blood for transfusion is based on its appearance. On standing, the cells sediment, leaving a layer of yellow supernatant plasma. • If the blood has been taken shortly after a heavy fatty meal the plasma may be turbid and show a white layer of fat on its surface. On top of the red cells there may be a complete or partial greyish layer of leucocytes. • The most important feature, however, is the line of demarcation between cells and plasma, which must be sharp; if it is obscured by a diffuse red colouration, indicating haemolysis, the blood is unfit for use.
Blood Products • The volume of the blood in the body can be reduced to a dangerously low level by haemorrhage,, shock, burns, and uncontrollable diarrhoea and vomiting. • Haemorrhage and certain diseases may result in deficiency or absence of vital blood constituents such as red cells, platelets, or clotting factors. The transfusion of whole blood can be of great value in all these circumstances but often, because of the risk of transfusion reactions, it is not used where the need is: • solely to make up blood volume but is restricted to haemorrhage and • certain diseases where there is deficiency of the vital oxygen-carrying erythrocytes.
Blood Products • Normally whole blood is not administered unless the ABO and Rh groups of donor and recipient are known and a sample of the donor’s blood has been tested for compatibility with that of the recipient. • In an emergency, group O, Rh negative blood may be given while the above precautions are being taken.
Dried human plasma Whole blood has several serious disadvantages— 1. It has poor keeping properties necessitating use within three weeks. 2. It requires refrigerated storage. 3. It must be compatible with the blood of the recipient. Dried plasma, on the other hand, has the following advantages— 1. Properly stored it keeps well for at least five years. . 2. If protected from light it can be stored at room temperature provided this is below 20°C. 3. It can be given to patients of any blood group. Consequently, in suitable circumstances, dried plasma is used as a substitute for whole blood
Dried human plasma Whole blood has several serious disadvantages— 1. It has poor keeping properties necessitating use within three weeks. 2. It requires refrigerated storage. 3. It must be compatible with the blood of the recipient. Dried plasma, on the other hand, has the following advantages— 1. Properly stored it keeps well for at least five years. . 2. If protected from light it can be stored at room temperature provided this is below 20°C. 3. It can be given to patients of any blood group. Consequently, in suitable circumstances, dried plasma is used as a substitute for whole blood
Dried human plasma It consist about 55 % of blood. This is yellow colour fluid. It is used for plasma transfusion where medically needed. It was first developed in British 1930. • Collection • Take blood from blood donor which want eligible for donating blood. to donate blood and • Separate the blood plasma from blood by the process of centrifugation
Procedure • T ake 400 ml blood plasma which are separated from blood. • Packed in to MRC bottle (medical research council bottle • The bottle is sealed with • Bacteriological efficient. • Freezing- • The bottle is then centrifuged at 18°c
Primary drying- • The bottle are mounted horizontally in the drying chamber at 50°c • This process takes place about 2 weeks Secondary drying- • This is done in another chamber by vaccume desiccation over phosphorus pentaoxide • It takes place about a day • Atleast 0.5% moisture should be left
Storage- • It is kept below 28°c and protected from moisture, sunlight and remains usable for 5 year • Uses- • Blood clotting • Autoimmune disorders • hemophilia • Other medical emergencies
Plasma Substitutes • The limited supplies of plasma, the cost of ‘producing the dried form and the risk of transmitting serum hepatitis stimulated attempts to find substitutes of non-human origin that could be used to restore the blood volume temporarily while the recipient replaced the lost protein.
Plasma Substitutes • PROPERTIES OF AN IDEAL PLASMA SUBSTITUTE • 1. The same colloidal osmotic pressure as whole blood. • 2. A viscosity similar to that of plasma. • 3. A molecular weight such that the molecules do not easily diffuse through the capillary walls. • 4. A fairly low rate of excretion or destruction by the body. • 5, Eventual and complete elimination from the body.
Plasma Substitutes • PROPERTIES OF AN IDEAL PLASMA SUBSTITUTE • 6. Freedom from toxicity, e.g. no impairment of renal function. • 7. Freedom from antigenicity, pyrogenicity, and confusing effects on important tests such as blood grouping and the erythrocyte sedimentation rate. • 8. Isotonicity, in solution, equal to that of blood plasma. • 9. High stability in liquid form at normal and sterilising temperatures and during transport and storage. • 10. Ease of preparation, ready availability and low cost.
Plasma Substitutes • GUM SALINE • Synonym for Injection of Sodium Chloride and Acacia having 6% acacia in 0.9% Sodium Chloride solution. • DISADVANTAGES: • Signs of liver dysfunction as gum was not metabolized but stored in various organs. • Polyvinylpyrrolidone – a synthetic colloid. Disadvantage – suspected carcinogenicity.
Plasma Substitutes • Dextran • To date this is the most satisfactory plasma substitute. It is a polysaccharide produced when the bacterium Leuconostoc mesenteroides is grown in a sucrose- containing medium. In the sugar industry it occurs as a slime that clogs pipes and filters and interferes with crystallisation. • The organism secretes.an enzyme that converts sucrose to dextran according to the following reaction
Plasma Substitutes • Different strains produce dextrans of two main groups • 1. Long, practically unbranched chains of glucose units joined by 1:6 glucosidic linkages. • 2. Highly branched polymers consisting of short chains of 1:6 units joined by 1:4 and 1:3 linkages to branches. • Branched chains are more likely to give rise to allergic reactions when injected, and in dextrans used for plasma substitutes the linkages should be almost entirely of the 1:6 type. This is achieved by choosing a suitable specially developed strain of the organism that produces dextran in which about 95 per cent of the linkages are 1:6.
• Production • Production involves laboratory culture followed by growth in and then in 4500 cubic dm fermenters. seed tanks in the factory • PRECAUTIONS – need to prevent the hydrolysis of sucrose to glucose and fructose during sterilization of the culture media. Prevention measures include the adjustment of the media to neutral pH before sterilization, and the avoidance of overheating. • When maximum conversion to dextran has been obtained it is precipitated by adding a suitable organiac solvent. Plasma Substitutes
• Natural dextran consists of chains of approximately 200,000 weights up to about 50 million. glucose units with molecular • Very large molecules i.e., those with a molecular weight above about 250,000 have serious drawbacks: – They yield very viscous solutions that are difficult to administer. – They may cause renal damage and allergic reactions. – They interfere with blood matching and sedimentation tests by causing rouleaux formation. Rouleaux are aggregates of red cells that resemble piles of plates. – They produce colloidal osmotic pressures that are lower than those of small molecules. Plasma Substitutes
• Therefore to produce a material suitable for medical use it is necessary to reduce the size of the natural molecules. This can be accomplished in several ways: • Acid hydrolysis (most widely used). • Thermal degradation. • Ultrasonic disintegration. • Seeding the fermenter. Plasma Substitutes
• The very small molecules, i.e. those of below a molecular weight of about 60,000 also have disadvantages: • They are rapidly excreted in urine. • They pass into the tissue fluids causing an adverse osmotic pressure. Plasma Substitutes
• The selected fraction still requires considerable purification to remove- • Reducing sugars: by further solvent precipitation. The main contaminant is fructose, the by-product of fermentation. • Fractionation solvents: by evaporation under reduced pressure. • Inorganic salts: by demineralization in a mixed bed ion exchanger. • Colour: by adsorption on to activated charcoal. • Pyrogrens: by adsorption on to asbestos, or cellulose derivatives. Plasma Substitutes
• Micro organisms: by filtration. • The solution is diluted to a concentration of 5% in either 5% dextrose injection or sodium chloride injection, packed in sulphur treated soda- lime bottles and closed with lacquered rubber plugs. • Finally, it is sterilized, usually by heating in an autoclave. Plasma Substitutes
Thank you

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UNIT V.pptx

  • 2. Fermentation • Fermentation is a metabolic process that produces chemical changes in organic substances through the action of enzymes. In biochemistry, it is narrowly defined as the extraction of energy from carbohydrates in the absence of oxygen. In food production, it may more broadly refer to any process in which the activity of microorganisms brings about a desirable change to a foodstuff or beverage. • Fermentation technology is the use of organisms to produce food, pharmaceuticals and alcoholic beverages on a large scale industrial basis. • The basic principle involved in industrial fermentation technology is that organisms are grown under suitable conditions, by providing raw materials meeting all the necessary requirements such as carbon, nitrogen, salts, trace elements and vitamins.
  • 3. Fermentation • The end products formed as a result of their metabolism during their life span are released into the media, which are extracted for use by human being and that have a high commercial value. The major products of fermentation technology produced economically on a large scale industrial basis are wine, beer, cider, vinegar, ethanol, cheese, hormones, antibiotics, complete proteins, enzymes and other useful products.
  • 4. Fermentation • Types of Fermentation Processes: • There are three different process of fermentation viz.: • (1) Batch fermentation • (2) Fed-batch fermentation and • (3) Continuous culture.
  • 5. Fermentation: Methods Batch fermentation: • Batch fermentation is a process where all the substrate and nutrients are added at zero time or soon after inoculation takes place, and the vessel is allowed under a controlled environment to proceed until maximum end product concentration is achieved(i.e. the metabolite or target protein). In batch fermentation, six phases of the microbial growth are seen. • Lag phase  Acceleration phase  Log phase  Deceleration Phase  Stationary phase:  Death phase:
  • 6. Fermentation: Methods • (a) Lag phase: • Immediately after inoculation, there is no increase in the numbers of the microbial cells for some time and this period is called lag phase. This is in order that the organisms adjust to the new environment they are inoculated into. • (b) Acceleration phase: • The period when the cells just start increasing in numbers is known as acceleration phase. • (c) Log phase: • This is the time period when the cell numbers steadily increase.
  • 7. Fermentation: Methods • (d) Deceleration phase: • The duration when the steady growth declines. • (e) Stationary phase: • The period where there is no change in the microbial cell number is the stationary phase. This phase is attained due to depletion of carbon source or accumulation of the end products. • (f) Death phase: • The period in which the cell numbers decrease steadily is the death phase. This is due to death of the cells because of cessation of metabolic activity and depletion of energy resources. Depending upon the product required the different phases of the cell growth are maintained. For microbial mass the log phase is preferred. For production of secondary metabolites i.e. antibiotics, the stationary phase is preferred.
  • 8. Fermentation: Methods • Fed-batch fermentation: • In this type of fermentation, freshly prepared culture media is added at regular intervals without removing the culture fluid. • This increases the volume of the fermentation culture. This type of fermentation is used for production of proteins from recombinant microorganisms.
  • 9. Fermentation: Methods • Continuous fermentation: • In this type of fermentation the products are removed continuously along with the cells and the same is replenished with the developed cells and addition of fresh culture media. • This results in a steady or constant volume of the contents of the fermentor. This type of fermentation is used for the production of single cell protein (S.S.P), antibiotics and organic solvents.
  • 10. Fermentation: Methods • Procedure of Fermentation: • (a) Depending upon the type of product required, a particular bioreactor is selected. • (b) A suitable substrate in liquid media is added at a specific temperature, pH and then diluted. • (c) The organism (microbe, animal/plant cell, sub-cellular organelle or enzyme) is added to it. • (d) Then it is incubated at a specific temperature for the specified time.
  • 11. Fermentation: Methods • (e) The incubation may either be aerobic or anaerobic. • i. Aerobic conditions are created by bubbling oxygen through the medium. • ii. Anaerobic conditions are created by using closed vessels, wherein oxygen cannot diffuse into the media and the oxygen present just above is replaced by carbon dioxide released. • (f) After the specified time interval, the products are removed, as some of the products are toxic to the growing cell or at least inhibitory to their growth. The organisms are re-circulated. The process of removal of the products is called downstream processing
  • 12. Fermentation: Methods • Types of fermenter • Available in various sizes • According to the sizes classified as • Small lab and research fermenter :1-50L • Pilot plant fermenter: 50-1000 L • Large size industrial production scale fermenter: more than 1000 L
  • 13. Fermentation: Methods • Broadly fermenters are also classified as • I. surface fermenters • Tray fermenter • Packed bed column fermenter • II. Submerged fermenters • Simple fermenters (batch and continuous) • Fed batch fermenter • Air-lift • Bubble fermenter • Cyclone column fermenter • Tower fermenter • Other more advanced systems, etc
  • 14. Fermentation: Types • Surface fermenters • Microbial cells cultured on surface layer of the nutrient medium (solid/liquid) held in dish or tray • Used for production of citric acid from Aspergillus niger and nicotinic acid from Aspergillus terrus • Microbial films can be developed on the surfaces of suitable packing medium, may be in the form of fixed bed, stones or plastic sheets.
  • 15. Fermentation: Types • TRAY FERMENTER • One of the simplest and widely used fermenters. • Its basic part is a wooden, metal, or plastic tray, often with a perforated or wire mesh bottom to improve air circulation. • A shallow layer of less than 0.15 m deep, pretreated substrate is placed on the tray for fermentation. Solid as well as liquid medium are used
  • 16. Fermentation: Types • TRAY FERMENTER • Temperature and humidity-controlled chambers are used for keeping the individual trays or stacks. • A spacing of at least one tray height is usually allowed between stacked trays. • Cheesecloth may be used to cover the trays to reduce contamination. • Inoculation and occasional mixing are done manually, often by hand. • If liquid medium, cells are allowed to float easily and to make a process continuous • If solid medium is used the micro-organisms are allowed grow on moist solid materials, process is called Solid State Fermentation
  • 17. Fermentation: Types • Solid State Fermentation (SSF) • SSF defined as the growth of the micro-organisms on (moist) solid material in the absence or near-absence of free water • Used for production of antibiotics, enzymes, alkaloids, organic acids bio-pharmaceutical products • Advantages : • Produce higher yields than submerged liquid fermentation • Possibilities of contamination by bacteria and yeast is very less • All natural habitats of fungi are easily maintained in SSF • culture media very simple , provides all nutrients for growth of micro-organisms
  • 18. Fermentation: Types • SSF Disadvantages: • Causes problems in monitoring of the process parameters such as pH, moisture content, and oxygen concentration • Despite some automation, tray fermenters are labor intensive • Difficulties with processing hundreds of trays limit their scalability • Aeration may be difficult due to high level of solid content • Substrates require pre treatment such as size reduction, chemical or enzymatic hydrolyses
  • 19. Fermentation: Types • Packed bed fermenters • This is type of surface culture bioreactor • A bed of solid particles, with biocatalysts on or within the matrix of solids, packed in a column • The solids used may be porous or non- porous gels, and they may be compressible or rigid in nature.
  • 20. Fermentation: Types • Packed bed fermenters • A nutrient broth flows continuously over the immobilised biocatalyst. The products obtained in the packed bed bioreactor are released into the fluid and removed. • The concentration of the nutrients can be increased by increasing the flow rate of the nutrient broth. • Because of poor mixing, difficult to control the pH of packed bed bioreactors by the addition of acid or alkali.
  • 21. Fermentation: Types • Submerged fermenters • The microorganisms are dispersed in liquid nutrient medium at maintained environmental conditions. on the mechanism of agitation Submerged fermenters grouped as follows: • I. Mechanically stirred fermenter • batch operate fermenter • continuous stirred tank fermenter • II. Pneumatic fermenter • Fluidized bed reactor
  • 22. Fermentation: Types • Submerged fermenters • III. Forced convection fermenters • Air –lift fermenter • Bubble column • Sparged tank fermenter • These are equipped with a mechanical agitator so as to maintain homogencity and rapid dispersion and mixing of materials • Examples includes stirred tank fermenter (batch or continuous operated) , multistage fermenter, paddle wheel reactor, and stirred loop reactor Mechanically stirred fermenter
  • 23. Fermentation: Types • Stirred tank fermenter (STF • batch operated fermenter • agitators consists of one or more impellers mounted on the shaft • It is rotates with the help of electric motor • Advantage of this fermenter flexibility in design • Used in the range of 1- 100 ton capacity sizes Stirred tank fermenter
  • 24. Fermentation: Types • Continuous stirred tank fermenter • A continuous stirred tank fermenter consists of a cylindrical vessel with motor driven central shaft that supports one or more agitators (impellers). • The shaft is fitted at the top of the bioreactor. The number of impellers is variable and depends on the size of the fermenter Continuous stirred tank fermenter (CSTF) Continuous stirred tank fermenter
  • 25. Fermentation: Types • Continuous stirred tank fermenter • In this fresh medium is added continuously in the fermenter vessel • On the other end the medium is withdrawn for the recovery of fermentation products • As it is a continuous fermenter the Steady state conditions can be achieved by either Chemostatic or Turbidostatic principles.
  • 26. Fermentation: Types • Different types of continuous fermenter are • a. Single stage: single fermenter is inoculated and kept in continuous operation by balancing the input and output culture media • b. Recycle continuous fermentation: a portion of the withdrawn culture or residual unused substrate plus the withdrawn culture is recycled • c. Multistage continuous operation: involves two or more stages with the fermenter being operated in sequence multistage
  • 27. Fermentation: Types • STF Advantages of batch operated • Less risk of contamination because of short growth period • Process is more economical and simple • Raw material conversion level is high • Disadvantages: • Low productivity due to time required for the sterilizing, filling, cooling, emptying and cleaning • More expenses are required for subcultures for inoculation, labor and process control
  • 28. Fermentation: Types • Advantages of continuous operated • Less labor expenses due to automation of fermentation process • Less toxicity risk to operator by toxins producing microorganisms • High yield and good quality product due invariable operating parameters and automation of the process • Less stress on the fermenter as sterilization is not frequent • Disadvantages: Higher investment costs in control and automation equipment • More risk of contamination and cell mutation
  • 29. Fermentation: Types • Bubble column fermenters • In the bubble column bioreactor, the air or gas is introduced at the base of the column through perforated pipes or plates, or metal micro porous spargers (ref fig). • The flow rate of the air/gas influences the performance factors —O2 transfer, mixing. • May be fitted with perforated plates to improve performance. The vessel used for bubble column bioreactors is usually cylindrical with an aspect ratio of 4- 6 (i.e., height to diameter ratio). ..
  • 30. Fermentation: Types • Air lift fermenter • Airlift fermenter (ALF) is generally classified as forced convection fermenters without any mechanical stirring arrangements for mixing. • The turbulence caused by the fluid (air/gas) flow ensures adequate mixing of the liquid. The baffle or draft tube is provided in the reactor. • A baffle or draft tube divides the fluid volume of the vessel into 2 inter- connected zones. • Only one of the 2 zones is sparged with air or other gas. • The sparged zone is known as " riser", the zone that receives no gas is "downcomer“.
  • 31. Fermentation: Types • Air lift fermenter • Mainly 2 types • Internal-loop airlift bioreactor (ref Fig) has a single container with a central draft tube that creates interior liquid circulation channels. These bioreactors are simple in design, with volume and circulation at a fixed rate for fermentation. • External loop airlift bioreactor (ref fig) possesses an external loop so that the liquid circulates through separate independent channels. These reactors can be suitably modified to suit the requirements of different fermentations. Internal loop External loop
  • 32. Fermentation: General requirements • Inoculum development • Inoculum production is a critical stage in an Industrial fermentation process. • Obviously, one loop of cell line requires a prolonged period if it is directly introduced in to fermentation. • Thus, inoculum is prepared as a stepwise sequence employing increasing volumes of media. • Constituent of Inoculum media :- • Chemical composition :- The Inoculum media must have a suitable Chemical composition. Generally, the medium should contain a source of carbon, a source of Nitrogen, growth factors and Mineral salts. • Buffering Capacity :- Maintenance of the PH in the optimum range is necessary for making the process successful. In order the control the PH of the medium, buffers (e.g. CaCo ) Should be added to the medium.
  • 33. Fermentation: General requirements • Avoidance of foaming :- • 1.Foaming is a serious problem in a fermentation Industry. • 2. Hence, defoamers (e.g. oil mixed with Octadecanol for penicillin fermentations) should be used for controlling foam. • Consistency :- 1. Proper aeration and agitation.
  • 35. Fermentation: Media • NUTRIENTS • Most fermentations require liquid media, often referred to as broth; although some solid substrate fermentations (SSF) are operated. • Fermentation media must satisfy all the nutritional requirements of the microorganism and fulfil the technical objectives of the process. • All microorganisms require water, sources of energy, carbon, nitrogen, mineral elements and possibly vitamins plus oxygen if aerobic. • The nutrients should be formulated to promote the synthesis of the target product, either cell biomass or a specific metabolite.
  • 36. Fermentation: Media • In most industrial fermentation processes there are several stages where media are required. They may include several inoculum (starter culture) propagation steps, pilot scale fermentations and the main production fermentation. • The technical objectives of inoculum propagation and the main fermentation are often very different, which may be reflected in differences in their media formulations.
  • 37. Fermentation: Media • Medium formulation • Medium formulation is essential stage in manufacturing process Carbon & Nitrogen other Energy + sources + O2 + nutrients Biomass + products + CO2 +H2O +heat • Elemental composition of microorganisms may be taken as guide
  • 38. Fermentation: Media • CARBON SOURCE • A carbon source is required for all biosynthesis leading to reproduction, product formation and cell maintenance. In most fermentations it also serves as the energy source. • Molasses • malted barley • Starch and Dextrins • Sulphite Waste Liquor
  • 39. Fermentation: Media • Alkanes and Alcohols n-Alkanes • Oils and fats • Factors influencing the carbon source - Cost of the product - rate at which it is metabolized - geographical locations - government regulations - cellular yield coefficient
  • 40. Fermentation: Media • Nitrogen Sources • Most industrial microbes can utilize both inorganic and organic nitrogen sources. • Inorganic nitrogen may be supplied as ammonium salts, often ammonium sulphate and diammonium hydrogen phosphate, or ammonia. Ammonia can also be used to adjust pH of the fermentation. • Organic nitrogen sources include amino acids, proteins and urea.:Corn Steep Liquor, Yeast Extracts, Peptones, Soya Bean Meal
  • 41. Fermentation: Media • Minerals • All microorganisms require certain mineral elements for growth and metabolism. In many media, magnesium, phosphorous, potassium, sulphur, calcium and chlorine are essential components and must be added. • Others such as cobalt, copper, iron, manganese, molybdenum and zinc are present in sufficient quantities in the water supplies and as impurities in other media ingredients.
  • 42. Fermentation: Media • Chelators • Many media cannot be prepared without precipitation during autoclaving. Hence some chelating agents are added to form complexes with metal ions which are gradually utilised by microorganism • Examples of chelators: EDTA, citric acid, polyphosphates etc., • It is important to check the concentration of chelators otherwise it may inhibit the growth. • In many media these are added separately after autoclaving Or yeast extract, peptone complex with these metal ions
  • 43. Fermentation: Media • Vitamins and Growth Factors • Many bacteria can synthesize all necessary vitamins from basic elements. For other bacteria, filamentous fungi and yeasts, they must be added as supplements to the fermentation medium. • Most natural carbon and nitrogen sources also contain at least some of the required vitamins as minor contaminants
  • 44. Fermentation: Media • Precursors • Precursors are defined as “substances added prior to or simultaneously with the fermentation which are incorporated without any major change into the molecule of the fermentation product and which generally serve to increase the yield or improve the quality of the product”. • They are required in certain industrial fermentations and are provided through crude nutritive constituents, e.g., corn steep liquor or by direct addition of more pure compounds.
  • 45. Fermentation: Media • Inducers and Elicitors • If product formation is dependent upon the presence of a specific inducer compound or a structural analogue, it must be incorporated into the culture medium or added at a specific point during the fermentation. • The majority of enzymes of industrial interest are inducible. Inducers are often substrates such as starches or dextrins for amylase. • In plant cell culture the production of secondary metabolites, such as flavanoids and terpenoids can be triggered by adding elicitors.
  • 46. Fermentation: Media • Inhibitors • Inhibitors are used to redirect metabolism towards the target product and reduce formation of other metabolic intermediates • others halt a pathway at a certain point to prevent further metabolism of the target product. • An example of an inhibitor specifically employed to redirect metabolism is sodium bisulphite
  • 47. Fermentation: Media • WATER • All fermentation processes, except SSF, require vast quantities of water. Not only is water a major component of all media, but it is important for ancillary services like heating, cooling, cleaning and rinsing. • A reliable source of large quantities of clean water, of consistent composition, is therefore essential. • Assessing suitability of water - pH - dissolved salts - effluent contamination Reuse of water is important - It reduces water cost by 50% - Effluent treatment cost by 10 fold
  • 48. Fermentation: Media • Oxygen • Depending on the amount of oxygen required by the organism, it may be supplied in the form of air containing about 21% (v/v) oxygen or occasionally as pure oxygen when requirements are particularly high. • The organism’s oxygen requirements may vary widely depending upon the carbon source. For most fermentations the air or oxygen supply is filter sterilized prior to being injected into the fermenter.
  • 49. Fermentation: Media • Antifoams • Antifoams are necessary to reduce foam formation during fermentation. • Foaming is largely due to media proteins that become attached to the air-broth interface where they denature to form a stable foam “skin” that is not easily disrupted • An ideal antifoam should have the following properties • Disperse readily and have fast action • Active at low concentrations • Long acting in preventing new foam
  • 50. Fermentation: Media • Should not be metabolized • Should not be toxic to m.o, humans etc • Cheap, should not cause problem in fermentation
  • 51. Fermentation: sterilization • Sterilization is defined as the complete destruction or elimination of all viable organisms (in or on an object being sterilized). • There are no degrees of sterilization: an object is either sterile or not. • Sterilization procedures involve the use of heat, radiation, chemicals or physical removal of cells. • Media for industrial fermentations are usually sterilized. • In some cases the economics of the fermentation makes it unrealistic to sterilize. • The fermentations can proceed, however, these fermentations employ low contamination inhibitors (lactic acid) to hold in check the numbers of microorganisms. pH and other contaminating
  • 52. Fermentation: sterilization • In other cases, sterilization is not required as the media components are poorly utilized by contaminating microorganisms. • Fermentation media are sterilized by the use of: filtration, radiation, ultrasonic treatment, chemical treatment or heat (boiling or passing live steam through the medium, or by subjecting the medium to steam under pressure - autoclaving). • Steam is used almost universally for the sterilization of fermentation media. The major exception is the use of filtration for the sterilization of animal cell culture.
  • 53. Fermentation: sterilization • Heat: Heat is the most important and widely used method. For sterilization, the type of heat, time of application and temperature required to ensure destruction of all microorganisms must always be considered. Endospores of bacteria are the most thermo- resistant of all cells so their destruction usually guarantees sterility. • Incineration: In this process, organisms are burned and physically destroyed. It is widely used for needles, inoculating wires, glassware, tubes etc. and objects that cannot be destroyed in the incineration process. • Boiling: Boiling is done at >100˚C for 20-30 min. It kills everything except for some endospores. Tokill endospores and therefore perfectly sterilize the solution, very long or intermittent boiling is required.
  • 54. Fermentation: sterilization • Autoclaving: Autoclaving is the process of using steam under pressure in an autoclave or pressure cooker. It involves heating at 121˚C for 15-20 min under 15 psi pressure and can be used to sterilize almost anything. However heat labile substances will be denatured or destroyed. Sterilization of nutrient media is usually done using this process. • Dry Heat (Hot Air Oven): The process involves heating at 160˚C for 2 hours or at 170˚C for 1 hour. It is used for glassware, metal and objects that will not melt. • Sterilization in industry-scale fermenters (or bioreactors) is more complex. Steam is used to sterilize fermentation media. The medium can be sterilized in situ within the bioreactor. However, if the medium is sterilized in a separate vessel, the bioreactor needs to be sterilized before the sterile medium is added to it.
  • 55. Fermentation: sterilization • Bioreactors are sterilized by passing steam through spargers. Spargers are devices that distribute gas bubbles (usually sterile air or steam) in a liquid phase. They have particular design criteria, e.g., providing small sized bubbles (the sparger breaks the incoming air into small bubbles). • Various designs can be used such as porous materials made of glass or metal. However, the most commonly used type of sparger used in modern bioreactors is the sparge ring. A sparge ring consists of a hollow tube in which small holes have been drilled and is easier to clean than porous materials and is also less likely to block during fermentation. During sparging, steam pressure is held at 15 psi in the vessel for 20 min.
  • 56. Fermentation: Aeration • The purpose of aeration in fermentation is to supply oxygen to and, at the same time, to remove carbon dioxide from microbial cells suspended in the culture broth. The rate of aeration often controls the rates of cell growth and product formation. • Mixing in the gas and liquid phases affects the aeration characteristics of a fermenter. • Various types of aerobic fermenter could be classified into three major types: • (1) sparged mechanically stirred fermenter • (2) bubble column fermenter, and • (3) loop fermenter
  • 57. Fermentation: Aeration • Transfer of gases in fermentation involves three phases, i.e., gas, culture medium, and microbial cells suspended in the medium. Oxygen absorbed from the gas-liquid interface diffuses through the culture medium to the cell surface and is consumed by the microbes. • Transfer of CO2 takes place in the reverse direction. Theoretically, resistances to gas transfer should exist in the gas film, the liquid film at the gas-liquid interface, the bulk of liquid, and the liquid film surrounding cells. • In case microbes form mycelial pellets, the diffusion resistance within the pellets could be significant. In some cases, a surfactant or antifoam agent may accumulate at the gas-liquid interface, giving an additional resistance to gas transfer.
  • 58. Fermentation: Aeration • Aeration is to provide microorganism in submerged culture with sufficient oxygen for metabolic requirements. • Agitation ensures that a uniform suspension of microbial cells is achieved in a homogenous nutrient medium. • Aeration and agitation depends on fermentation.
  • 59. AERATION SYSTEM  Syn : sparger  Adevice that introduce air intomedium  Has a pipe with minute holes (1/64 - 1/32 inch or large)  Hole – allows air under Pto escape intomedium  For mycelial growth – ¼ inch holes  Impeller blades disperses air released through sparger into medium
  • 60. SPARGER TYPES  Porous  Orifice  Nozzle POROUS SPARGER:  Made of sintered glass, ceramics or metal  Used mainly on a large scale fermenters  Bubble size produced – 10-100times larger than pores  Throughput of air is low – Pdrop across it  Clogging of pores ORIFICE SPARGER  Those with drilled air holes on their under surface of the tubes making up ring or cross effluent  Without agitation used to a limited extend in yeast manufacture & treatment NOZZLE SPARGER  Modern mechanically stirred fermentors use them  Single open or partially closed pipes  Ideally, positioned centrally below impeller  Causes lower Pdrops  no clogging of pore
  • 61. Fermentation: Stirring • Fine bubble aerator without agitation: • Advantage of lower equipment and power costs, • Agitation may be dispensed with only when aeration provides sufficient agitation. • E.g. in processes when broth of low viscosity and low total solids. • Mechanical agitation is required for fungal and actinomycetes fermentation.
  • 62. Fermentation: Stirring • Agitation: Importance • 1. To increase the rate of oxygen transfer from the air bubble to the liquid medium. • 2. To increase the rate of oxygen and nutrients transfer from the medium to cells. • 3. To prevent formation of clumps of cells, aggregates of mycelium. • 4. To increase the rate of transfer of product of metabolism from cell to medium. • 5.To increase the rate or efficiency of heat transfer between the medium and the cooling surfaces of the fermenters.
  • 63. Fermentation: Stirring • Effect of agitation on aeration • 1. by dispersing the air in smaller bubble. • 2.by causing the bubbles to follow a more tortuous path and dalaying their escape from the culture. • 3. by preventing the coalescence of bubbles. • 4. by decreasing the rate-limiting thickness of the liquid film at the gas/liquid interface.
  • 64. FERMENTOR’S STRUCTURAL COMPONENTS IN AERA TION &AGIT A TION SYSTEM: The agitator Stirrer glands & bearings Baffles The aeration system
  • 65. AGITATOR  Synonym : impeller  Mounted to a shaft through a bearing in the lid  Driven by an external power source or direct drive  Direct drive - action varied by using different impeller blades  Recent designs – driven by magnetic coupling to a motor mounted beneath the fermenter  High speed of rotation marked vortex occurs  Spinning of medium in circular direction  MIXING OBJECTIVES ITACHIEVE  Bulk fluid & gas  Heattransfer phase mixing  Air dispersion  Suspension of solid particles  O2 transfer  Maintenance of uniform environment throughout the vessel
  • 66. CLASSIFICATION  Disc turbine  V anned disc  V ariable pitch open turbine  Marine propellers DISC TURBINE:  Adisc with series of rectangular vanes set in a vertical plane around the circumference.  Break up a fast air stream without itself becoming flooded in air bubbles
  • 67. V ANED DISC  Aseries of rectangular vanesattached vertically to the underside  Air from sparger hits it’sunderside & the air gets displaced towards the vanes  Results in destruction of air bubbles VARIALBLE PITCH OPENTURBINE:  Vanesare attached directly to a boss on the agitator shaft  Air bubbles hit any surface by its action  Flood when super fial velocity exceed 21m/h
  • 68.
  • 69. MARINE PROPELLER  Blades are attached directly to a boss on the agitator shaft  Air bubbles hit surface  Asingle low shear impeller  Mainly used in animla cell culture vessel  Flood when superfial velocity exceed 21m/h
  • 70. MODERNAGITATORS  Rushton disc turbine  Scaba 6SRGT  Prochem max flowT  LighteningA315  Ekato intermig
  • 71. BAFFLES  Metal strips  1/10th of the vesseldiameter  Attached radially to wall  4 baffles (normal)  Wider baffles -high agitation effect  Narrower baffles – low agitation effect  Can be attached with cooling coils  Not found in lab scale fermentors.  Verticalbaffles – increased aeration
  • 72. Fermentation: Aeration and Stirring • Oxygen supply affected by following: • Type of agitation: • The shape, number and arrangement of impellers and baffles. • Either 2 or 3 impellers for large fermenters at suitable level on the stirrer shaft or 3 or 4 baffles on the wall of the vessel. • Speed of agitation: • 1000 or more for lab. Fermenters. • But this is not possible for large vessels. For penicillin fermentation requires 50rpm needs high input of energy and uneconomical. • Depth of liquid in the fermenters: • Bubble remain longer in the medium of a tall, deep fermenter. Greater hydrostatic pressure at the sparger improves solution of oxygen. • Height : diameter ratio of 3:1 or 4:1 is common.
  • 73. Large scale production fermenter design and its various controls.
  • 74. IDEAL FERMENTORPROPERTIES  Supports maximum growth of the organism  Aseptical operation  Adequate aeration and agitation  Low power consuming  Tempurature control system  pH control system  Sampling facilities  Minimum evaporation loss  Minimum use of labour  Range of processes  Smooth internal surfaces  Similar in geometry to both smaller & larger vessels in pilot plant
  • 75.  Cheapest material usuage  Adequate service provisions  Provision for control of contaminants  Provision for intermittent addition of antifoams  Inoculum introduction facility  Mechanism for biomass/ product removal  Setting for rapid incorporation of sterile air  Withstands pressure  Ease of manipulation
  • 76. BASIC DESIGN OF AFERMENTOR
  • 77. Various components of an ideal process are fermenter for batch
  • 78. Monitoring and controlling parts of are fermenter
  • 79. SHAPE OF FERMENTER: FermentationAreAvailable In Different Shapes Like Conical Fermenter Cylindrical fermenter Spherical fermenter Pear In Shape Fermenter SIZES OFFERMENTER : The sizes of the fermenter are divided into the following groups. 1. The microbial cell (mm cube) 2. Shake flask (100-1000ml) 3. Laboratory fermenter (1-50 L) 4. Pilot scale (0.3 -10m cube) 5. Industrial scale (2-500m cube)
  • 80. MATERIAL OF CONSTRUCTION Laboratory scale bioreactor: • In fermentation with strict aseptic requirements it is important to select materials that can withstand repeated sterilization cycles. On a small scale, it is possible to use glass and/or stainless steel. • Glass is useful because it gives smooth surfaces, is non-toxic, corrosion proof and it is usually easy to examine the interior of vessel. The glass should be 100% borosilicate, e.g. Pyrex® and Kimax®. • The following variants of the laboratory bioreactor can be made: 1. Glass bioreactor (without the jacket) with an upper stainless steel lid. 2. Glass bioreactor (with the jacket) with an upper stainless steel lid. 3. Glass bioreactor (without the jacket) with the upper and lower stainless steel lids. 4. Two-part bioreactor - glass/stainless steel. The stainless steel part has a jacket and ports for electrodes installation. 5. Stainless steel bioreactorwith peepholes. Vessels with two stainless steel plates cost approximately 50% more than those with just a top plate
  • 81. Pilot scale and large scale bioreactors: • When all bioreactors are sterilized in situ, any materials use will have to assess on their ability to withstand pressure sterilization and corrosion and their potential toxicity and cost. • Pilot scale and large scale vessels are normally constructed of stainless steel or at least have a stainless steel cladding to limit corrosion. • The American Iron and Steel Institute (AISI) states that steels containing less than 4% chromium are classified as steel alloys and those containing more than 4% are classified as stainless steel. • Mild steel coated with glass or phenolic epoxy materials has occasionally been used. Wood, concrete and plastic have been used when contamination was not a problem in a process. • V essel shape: - • Typical tanks are vertical cylinders with specialized top plates and bottom plates. In some cases, vessel design eliminates the need for a stirrer system especially in air lift fermenter. A tall, thin vessel is the best shape with aspect ratio (height to diameter ratio) around 10:1. Sometimes a conical section is used in the top part of the vessel to give the widest possible area for gas exchange.
  • 82. • Stainless steel top plates. • The top plates are of an elliptical or spherical dish shape. The top plates can be either removable or welded. Aremovable top plate provides best accessibility, but adds to cost and complexity. • Various ports and standard nozzles are provided on the stainless plate for actuators and probes. These include pH, thermocouple, and dissolved oxygen probes ports, defoaming, acid and base ports, inoculum port, pipe for sparging process air, agitator shaft and spare ports. • Bottom plates: • Tank bottom plates are also customized for specific applications. Almost most of the large vessels have a dish bottom, while the smaller vessels are often conical in shape or may have a smaller, sump type chamber located at the base of the main tank. These alternate bottom shapes aid in fluid management when the volume in the tank is low. One report states that a dish bottom requires less power than a flat one. • In all cases, it is imperative that tank should be fully drainable to recover product and to aid in cleaning of the vessel. Often this is accomplished by using a tank bottom valve positioned to eliminate any “dead section” that could arises from drain lines and to assure that all content will be removed from the tank upon draining.
  • 83. If the bioreactor has a lower cover, then the following ports and elements should be placed and fastened there: 1. Discharge valve; 2. Sampling device; 3. Sparger; 4. Mixer's lower drive; 5. Heaters. Height-to-diameterratio (Aspect ratio). • The height-to-diameter ratio is also a critical factor in vessel design. Although a symmetrical vessel maximizes the volume per material used and results in a height-to-diameter ratio of one, most vessels are designed with higher ratio. The range of 2-3:1 is more appropriate and in some situation, where stratification of the tank content is not an issue or a mixer is used, will allow still higher ratio to be used in design. • The vessels for microbiological work should have an aspect ratio of 2.5- 3:1, while vessels for animal cell culture tend to have an aspect ratio closer to 1. The basic configuration of stirred tank bioreactors for mammalian cell culture is similar to that of microbial fermenter but the major difference is there in aspect ratio, which is usually smaller in mammalian cell culture bioreactor.
  • 84. Common Measurement And Control Systems
  • 85. Introduction : • Antibiotics are antimicrobial agents produced naturally by other microbes (usually fungi or bacteria) • The first antibiotic was discovered in 1896 by Ernest Duchesne and in 1928 "rediscovered" by Alexander Fleming from the filamentous fungus Penicilium notatum. • The antibiotic substance, named penicillin, was not purified until the 1940s (by Florey and Chain), just in time to be used at the end of the second world war. • Penicillin was the first important commercial product produced by an aerobic, submerged fermentation Cont..,
  • 86. Cont.., • Penicillin is produced by the fungus Penicilium chrysogenum which requires lactose, other sugars, and a source of nitrogen (in this case a yeast extract) in the medium to grow well. • Like all antibiotics, penicillin is a secondary metabolite, so is only produced in the stationary phase.
  • 87. • It exhibits the properties of a typical secondary metabolites. • It active against certain Gram- positive bacteria in presence of blood, pus and body fluids. • It is soluble in water. It is very soluble in acetone, ethyl alcohol and ether and it is less soluble in benzene, chloroform, ect.. • Aqueous solution of penicillin are unstable and must be stored under refrigeration. • Penicillin is most stable in the pᴴ range of 6.0 to 6.5 and reasonably stable over the pᴴ range of 5.5 to 7.5 . Properties of penicillin :
  • 88. Types of penicillin : • Penicillin are compound of the general formula C₁₆H₁₈N₂O₅S –R, in which R represents the radical or group that is different for each day. The structural formula of the most common type ( F,G, X and k ) are given. • Penicillin F, G, X and K are produced by strain of the penicillin notatum – chrysogenum group of molds ; flavicidin ( flavicin) by Aspergilus flavus ; and dihydro F penicillin (gigantic acid ) by Aspergilus gigantic. Basic structure of penicillin : The basic structure of the penicillin's is 6- aminopenicillenic acid (6- APA), composed of a thiozolidine ring fused with a β- lactam ring whose 6- amino position carries a variety of acyl substituent's.
  • 89.
  • 90. ⦁ Also known as Penicillium notatum. ⦁ It is common in temperate and subtropical regions and can be found on salted food products, but it is mostly found in indoor environments, especially in damp or water-damaged buildings. ⦁ It is the source of several β-lactam antibiotics, most significantly penicillin which inhibits the biosynthesis of bacterial cell walls affecting lysis of the cell.
  • 91. ⦁ Kingdom: Fungi ⦁ Division:Ascomycota ⦁ Class: Eurotiomycetes ⦁ Order: Eurotiales ⦁ Family: Trichocomaceae ⦁ Genus: Penicillium ⦁ Species: Chrysogenum
  • 92. ⦁ Penicillium chrysogenum exhibits typical eukaryotic cell structure; it has a tubulin cytoskeleton which is used for motility. TAM structure of P . Chrysogenum Structure of P . Chrysogenum
  • 93. ⦁ This image displays the typical filamentous hyphae that contain many conidia. ⦁ The oblong structures in the image are conidia, the asexual spores of the fungus. ⦁ In P .chrysogenum, the conidia are blue to blue-green. ⦁ These conidia are the cause of pathogenicity in humans as in the cases of allergy and endophthalmitis. ⦁ The conidia originate from complexes known as conidiophores. ⦁ The growth of conidiophores begins when a stalk sprouts out of a foot cell. ⦁ The stalk swells at the end and forms a vesicle. Sterigmata form from the vesicle which give way to long chains of conidia.
  • 94. ⦁ It produces the hydrophobic β-lactam compound penicillin. ⦁ Penicillium chrysogenum remains the primary producer of Penicilian G and Penicilian V ⦁ P .chrysogenum has been used industrially to produce Penicilian G and Penicilian V and Xanthocillin X, and to produce the enzymes polyamine oxidase, phosphogluconate dehydrogenase, and glucose oxidase. ⦁ Penicillium chrysogenum can be used to assist crops to fight off other pathogenic species.
  • 95. ⦁ P.chrysogenum is high yielding strain and therefore most widely used as production strain. ⦁ Inoculum Preparation: ⦁Purpose is to develop a pure inoculum in an adequate amount. To do so various sequential steps are necessary like: 1) Astarter culture is needed for inoculation. 2) After getting growth on solid media, one or two growth stages should allowed in shaken flask cultures to create a suspension, which can be transferred to seed tanks for further growth. 3) After about 24-28 hours, the content of the seed tanks is transferred to the primary fermentation tanks.
  • 96. 4) •All the bio parameters like temperature, pH, aeration, agitation etc. should be properly maintained. • Bio parameters  pH: near 6.5  Temperature: 26°C to 28°C  Aeration: a continuous stream of sterilized air is pumped into it.  Agitation: have baffles which allow constant agitation (200rpm).
  • 97. ⦁ Fermentation broth contains all the necessary elements required for the proliferation of the microorganisms. ⦁ Generally, it contains a carbon source, nitrogen source, mineral source, precrsors and antifoam agents. Carbon Source ⦁ Lactose in a concentration of 6%. ⦁ Other carbohydrates like glucose & sucrose. ⦁ Complex as well as cheap sources like molasses, or soya meal can also be used which are made up of lactose and glucose sugars.
  • 98. Nitrogen Source ⦁ Ammonium salts such as ammonium sulphate, ammonium acetate, ammonium lactate or ammonia gas are used for this reason. ⦁ Sometime corn steep liquor may be used. Mineral Source ⦁ These elements include phosphorus, sulphur, magnesium, zinc, iron, and copper which generally added in the form of water soluble salts. Precursors ⦁ Various types of precursors are added into production medium to produce specific type of penicillin.
  • 99. ⦁ For example, if phenyl acetic acid is provided then only penicillin-G will be produced but if hydroxy phenyl acetic acid is provided then penicillin-X will be produced. ⦁ Phenoxy acetic acid is provided as precursor for penicillin-Vproduction. ⦁ When corn steep liquor is provided as nitrogen source, it also provides phenyl acetic acid derivatives; therefore it is widely used in the production of penicillin-G.
  • 100. Anti-foam agents ⦁ Anti-foaming agents such as lard oil, octadecanol and silicones are used to prevent foaming during fermentation. Recovery ⦁ The recovery of penicillin is carried out in three successive stages: 1. Removal of mycelium 2. Counter current solvent extraction of penicillin 3. Treatment of crude extracts
  • 101. ⦁ At harvest the fermentation broth is filtered on a rotatory vacuum filter to remove the mycelium and other solids. ⦁ Phosphoric or sulfuric acids are added to lower the pH (2 to 2.5) in order to transform the penicillin to the anionic form. ⦁ Then the broth is directly extracted in a Podbielniak Counter Current Solvent Extractor with an organic solvent such as methyl isobutyl ketone, amyl acetate or butyl acetate. ⦁ Penicillin is then again extracted into water from the organic solvent by adding an adequate amount of potassium or sodium hydroxide to form a salt of the penicillin. ⦁ The resulting aqueous solution is again acidified & re- extracted with methyl isobutyl ketone.
  • 102. ⦁ This shifts between water and solvent help in purification of the penicillin. ⦁ The solvent extract is carefully back extracted with NaOH and from this aqueous solution; various procedures are utilized to cause the penicillin to crystalize as sodium or potassium penicillinate. ⦁ The resulting crystalline penicillin salts are then washed and dried. ⦁ Sometimes the crude extract of penicillin is passed out from charcoal treatment to eliminate pyrogens; even sterilization can also be done.
  • 104. CONTENT  Introduction  History  Microbes in Citric acid Production  Citric acid fermentation techniques  Factors affecting Citric acid production  Industrial production of citric acid  Applications/Uses of citric acid  Side effects  Largest producers in the world
  • 105. Overview • Citric acid is a usually occuring acid found primarily in Several Varieties of fruits and vegetables with citrus fruits such as lemons and limes containing the highest amounts of citric acid. • This Organic acid has many uses, including as a food additive /Preservative, ingredient in Cosmetic products and as a powerful cleaving agent.
  • 106. Introduction: • Citric acid (2-hydroxy-1,2,3- propane tri carboxylic acid) is the most important commercial product, which is found in almost all plant & animal tissues. • Citric acid is the most important organic acid produced in tonnage and is extensively used in food and pharmaceutical industries. • Citric acid is a weak organic acid found in citrus fruits(lemon). • It is good ,natural preservative and is also used to add an acidic taste to food and soft drinks. • More than million tonnes are produced every year by fermentation.
  • 107.
  • 108. HISTORY: • The discovery of citric acid has been credited to the 8th century Muslim alchemist Jabir Ibn Hayyan (Geber). • Citric acid was first isolated in 1784 by the Swedish chemist carl Wilhelm Scheele, who crystallize it from lemon juice. • Industrial scale citric acid production began in 1890 based on the Italian citrus fruit industry. • In 1893, C. Wehmer discovered penicillin mold could produce citric acid from sugar. However, microbial production of citric acid did not become industrially important until world war I disrupted Italian citrus exports.
  • 110. Micro-organisms used for citric acid production: • Large number of micro-organisms including bacteria, fungi and yeasts have been employed to produce citric acid. • The main advantages of using this micro-organisms are: • Its easy of handling • Its ability to ferment a variety of cheap raw • materials • High yields
  • 111. Micro organisms: • Fungi: • Aspergillus nagger • A. aculeatus • A. awamori • A. carbonarius • A. wentii • A. foetidus • Penicillium janthinelum • Bacteria: • Bacillus licheniformis Arthrobacter paraffinens Corynebacterium species • Yeasts: • Saccahromicopsis lipolytica Candida tropicalis • C. oleophila • C. guilliermondii • C. parapsilosis • C. citroformans Hansenula anamosa
  • 112. Citric acid production: • Fermentation is the most economical and widely used ay for synthesis citric acid production. • The industrial citric acid production can be carried in three different ways: • surface fermentation • submerged fermentation • solid state fermentation
  • 113. Surface Fermentation:  Surface fermentation using Aspergillus niger may be done on rice bran as is the case in Japan, or in liquid solution in flat aluminium or stainless steel pans.  Special strains of Aspergillus niger which can produce citric acid despite the high content of trace metals in rice bran are used.
  • 114. SUBMERGED FERMENTATION: • In this case , the strains are inoculated of about 15cm depth in fermentation tank. • The culture is enhanced by giving aeration using air bubbles. • And its allowed to grow for about 5 to 14 days at 27 to 33 degree Celsius. • The citric acid produced in the fermentation tank and it is purified.
  • 115. Solid state fermentation: • It is simplest method for citric acid production. • Solid state fermentation is also known as koji process, was first developed in Japan. • Citric acid production reached a maximum(88g/kg dry matter)when fermentation as carried out with cassava having initial moisture of 62% at 26degree Celsius for 120 hours.
  • 116. Separation: • The biomass is separated by filtration. • The liquid is transferred to recovery process • Separation of citric acid from the liquid precipitation. • Calcium hydroxide is added to obtain calcium citrate.
  • 117. Tetra hydrate Wash the precipitate Dissolve it with dilute sulfuric acid, yield citric acid and calcium sulfate precipitate Bleach and crystallization Anhydrous or mono hydrate citric acid Separation process:
  • 118. PURIFICATION: • Purification is a simple form of getting a pure citric acid followed by two simple techniques. • Precipitation • Filtration React citric acid with calcium carbonate Filter precipitate React precipitate with sulfuric acid Filter precipitate Purified citricAcid
  • 119. Citric acid production A. niger CA16 and 79/20 Substrate cut , dried and powdered Grown in PDAagar slant Mixed with water at different concentration A. Niger spores 7days old culture Filtration @ sterilization inoculation with 1 10 spores/25mLf FILTERATION Filtrate for citric acid CELLBIOMASS
  • 120. Factors affecting citric acid production: • Nitrogen source • pH • Aeration • Trace elements • Temperature
  • 121. Industrial production of citric acid: • 99% of world production: microbial processes surface or submerged culture. • 70% of total production of 1.5 million tons per year is used in food and beverage industry as on acidifier or antioxidant to preserve or enhance the flavors and aromas of fruit juices, ice cream and marmalades. • 20% used: pharmaceutical industry as anti oxidant to preserve vitamins, effervescent, pH corrector, blood preservative, or in the form of iron citrate.
  • 122. Tablets, ointments and cosmetic preparations • Chemical industry remaining 10% softening and treatment of textile. • Also used in the detergent industry as a Phosphate substitute, because of less entropic effect hardening of cement
  • 123. Applications/uses of citric acid: • Food & drink: • Preservative and flavoring agent Emulsifying agent in ice-cream. • Household cleaner: • Kitchen Bathroom sprays. • Cosmetics: • Shampoos Body wash • WASH CLEANERS: • Nail polish • Hand soap and other cosmetic products
  • 124. • To cure kidney disorders: • Sodium citrate, acetic acid is used to prevent kidney stones. • Side effects: • Taking excess of citric acetate in combination with sodium citrate may lead to kidney failure. • Taking citric acid with empty stomach may lead to stomach or intestinal side-effects. • It may also lead to muscle twisting or cramps. • It can also cause weight gain, swelling, fast heart rate, slow o rapid .
  • 126.  Avitamin is an organic compound and a vital nutrient that an organism requires in limited amounts.  They are of great value in the growth and metabolism of the living cells.  Vitamins are obtained with food, but a few are obtained by other means ; humans can produce some vitamins from precursors they consume while certain microorganism produce vitamins too.  Thirteen vitamins are universally recognized at present, vitamins are classified by their biological and chemical activity.  Vitamins can be classified as “Fat soluble vitamins” and “Water soluble vitamins”
  • 127. FAT SOLUBLE VITAMINS W ATERSOLUBLE VITAMINS  VitaminA(Retinol)  Vitamin D 1. Vitamin D2 (Egrocalciferol) 2. Vitamin D3 (Cholecalciferol)  Vitamin E (Tocopherol)  Vitamin K (Phylloquinone)  Vitamin B Complex 1. Vitamin B1 (Thiamine) 2. Vitamin B2 (Riboflavin) 3. Vitamin B3 (Niacin) 4. Vitamin B5 (Pantothenic acid) 5. Vitamin B6 (Pyridoxine) 6. Vitamin B7 (Biotin) 7. Vitamin B9 (FolicAcid) 8. Vitamin B12 (Cobalamin)  Vitamin C (Ascorbic acid)
  • 128.  Vitamin B12, also called Cobalamin, is a water- soluble vitamin that has a key role in the normal functioning of the brain and nervous system, and the formation of red blood cells.  It is involved in the metabolism of every cell of the human body, especially affecting DNAsynthesis, fatty acid and amino acid metabolism.  It is synthesized only by microorganisms and not by animals (including humans) and plants.  People with B12 deficiency may eventually develop Pernicious anemia.  It is the largest and most structurally complicated vitamin and can be produced
  • 129.
  • 130.  Cyanocobalamin, is the industrially produced stable Cobalamin form which is not found in nature.  Vitamin B12 is entirely produced on a commercial basis by the fermentation. It is usually manufactured by submerged culture process. Such a fermentation process is completed in 3-5 days.  Most of the B12 fermentation processes use glucose as a carbon source.  The microorganisms that maybe employed in the industrial production process are : i. Streptomyces griseus ii. Streptomyces olivaceus iii. Bacillus megaterium iv. Bacillus coagulans v. Pseudomonas denitrificans vi. Propionibacterium freudenreichii vii. Propinibacteriun shermanii
  • 131. Step 1 • Formulation of the medium Step 2 • Sterilization of the medium Step 3 • Making starter culture Step 4 • Anaerobic fermentation Step 5 •Aerobic fermentation Step 6 •Recovery
  • 132.  Production by Streptomyces olivaceus yields about 3.3mg / L of vitamin B12.  Process : A. Preparation Of Inoculum : Pure slant culture of S. olivaceus is inoculated in 100-250ml of inoculum medium, contained in Erlenmeyer flask. Seeded flask is incubated on platform of a mechanical shaker to aerate the system. This flask culture is then subsequently used to inoculate larger inoculum tanks. (2 or 3 successive transfers are made to obtain required amount of inoculum cultures.)
  • 133.
  • 134.  Media used in preparation of inoculum is Bennett’s agar. Component Amount (g/L) Yeast extract 1.0 Beef extract 1.0 N-Z-AmineA (Enzymatic hydrolase of casein) 2.0 Glucose 10.0 Agar 15.0 D/W 1000 mL pH 7.3
  • 135. B. Production Medium :  Consist of carbohydrate, proteinaceous material, and source of cobalt and other salts.  It is necessary to add cobalt to the medium for maximum yield of cobalamin.  Cyanide is added for conversion of other cobalamins to vitamin B12. Component Amount (%) Distiller’s Solubles 4.0 Dextrose 0.5 - 1 CaCO3 0.5 COCL2.6H2O 1.5 – 10 ppm pH 7
  • 136. C. Sterilization of the medium :  Sterilization can be done batchwise of continuously.  Batch – medium heated at 250°F for 1 hour.  Continuous – 330°F for 13 min by mixing with live steam. D. Temperature , pH ,Aeration andAgitation : • Temperature : A temperature of 80°F in production tank is satisfactory during fermentation. • pH : At starting of process pH falls due to rapid consumption of sugar, then rises after 2 to 4 due to lysis of mycelium. pH 5 is maintained with H2SO4 and reducing agent Na2SO4. • Aeration and Agitation : Optimum rate of aeration is 0.5 volume air/volume medium/min.
  • 137. E.Antifoam agent , Prevention of contamination :  Antifoam agent : Defoaming agents like soya bean oil , corn oil, lard oil and silicones can be used.  Prevention of contamination : Essential to maintain sterility, contamination results in reduced yields, equipments must be sterile and all transfers are carried out under aseptic conditions. F . Recovery :  During fermentation, most of cobalamin is associated with the mycelium; boiling mixture at pH 5 liberates the cobalamin quantitatively from mycelium.  Broth containing cobalamin is subjected to further process to obtain crystalline B12.
  • 138. Filtration of broth to remove mycelium. Filtered broth is treated with cyanide to bring conversion of cobalamin to cyanocobalamin. Adsorption of cyanocobalamin from the solution is done by passing it through adsorbing agents packed in a column. Cyanocobalamin is then eluted from the adsorbent by the use of an aqueous solution of organic bases or solutions of Na-Cyanide and Na- thiocyanate. Extraction is carried out by countercurrent distribution between cresol, amylphenol, or benzyl alcohol and water or a single extraction into an organic solvent (e.g. Phenol) is carried out. Chromatography on alumina and final crystallization completes the process.
  • 139.  Production by Propionibacterium freudenreichii yields about 20mg/Lof vitamin B12. A. Production media : glucose , corn- steep , betaine , & cobalt.  Betaine -0.5 %  Cobalt – 5µg./ml (excess cause reduced cobalamin formation) B. pH -7.5 C. Temperature – 30°C
  • 140. D. Fermentation : It involves to cycles; anaerobic fermentation cycle of 70 hours andAerobic fermentation cycle of 50 hours.  Anaerobic fermentation :  Formation of cobinamide occurs.  The pH falls from 7.5 to 6.5 and then rises upto 8.5.  Necessary to add 0.1% of 5, 6 – dimethyl benziminazole to the production medium.  Aerobic fermentation :  Nucleotide formation takes place.  This nucleotide then links with cobinamide to give cobalamin.
  • 141. Species Medium Aeration Temp. (°C) Time (hour) Yield (mg/L) B.megaterium Molasses , mineral salts, cobalt Aerobic 30 18 0.45 P . shermanii Glucose , corn- steep, ammonia , cobalt pH 7.0 Anaerobic (3 days), aerobic (4 days) 30 150 23 B. coagulants Citric acid , triethanolam ine , corn – steep , cobalt. Aerobic 55 18 6.0
  • 143. Introduction Amino acids have always played an important role in biology of life, in biochemistry and in (industrial) chemistry. Amino acids are the building blocks of proteins and they play an essential role in the metabolism regulation of living organisms. Large scale chemical and microbial production processes have been commercialised for a number of essential amino acids. Current interest in developing peptide-derived chemo- therapeutics has heightened the importance of rare and non- proteinogenic pure amino acids.
  • 144. Amino acids are versatile chiral (optically active) building blocks for a whole range of fine chemicals. Amino acids are, therefore, important as nutrients (food), seasoning, flavourings and starting material for pharmaceuticals, cosmetics and other chemicals.  Amino acid can be produced by :  Chemical synthesis  Isolation from natural materials  Fermentation  Chemo-enzyme methods Importance of Amino acids
  • 145. Glutamic acid Glutamic acid is an α-amino acid that used in biosynthesis of proteins. It contains an α-amino group which is in the protonated −NH3+. An α-carboxylic acid group which is in the deprotonated −COO.  And a side chain carboxylic acid. Polar negatively charged (at physiological pH), aliphatic amino acid. It is non-essential in humans, meaning the body can synthesize it.
  • 146. Glutamic Acid  Food Production:  As flavor enhancer, to improve flavor.  As nutritional supplement.  Beverage  As flavor enhancer: in soft drink and wine. Cosmetics  As Hair restorer: in treatment of Hair Loss.  As Wrinkle: in preventing aging. Agriculture/Animal Feed  As nutritional supplement: in feed additive to enhance nutrition. Other Industries  As intermediate: in manufacturing of various organic chemicals.
  • 147. Biosynthesis of Glutamic acid Reactants Products Enzymes Glutamine + H2O → Glu + NH3 GLS, GLS2 NAcGlu + H2O → Glu + Acetate (unknown) α-ketoglutarate + NADPH + NH4 + → Glu + NADP+ + H2O GLUD1, GLUD2 α-ketoglutarate + α-amino acid → Glu + α-oxo acid transaminase 1-pyrroline-5-carboxylate + NAD+ + H2O → Glu + NADH ALDH4A1 N-formimino-L-glutamate + FH4 ⇌ Glu + 5-formimino-FH4 FTCD  An amino acid precursor is converted to the target amino acid using 1 or 2 enzymes.  Allows the conversion to a specific amino acid without microbial growth, thus eliminating the long process from glucose.  Raw materials for the enzymatic step are supplied by chemical synthesis.  The enzyme itself is either in isolated or whole cell form which is prepared by microbial fermentation.
  • 148. Industrial Production and use of Microorganisms  Industrial microbiology  Microorganisms, typically grown on a large scale, to produce products or carry out chemical transformations.  The glutamic acid is produced through the fermentation process  Major organism used is Corynebacterium glutamicum .  Classic methods are used to select for high-yielding microbial variants.  Properties of a useful industrial microbe include  Produces spores or can be easily inoculated.  Grows rapidly on a large scale in inexpensive medium.  Produces desired product quickly.  Should not be pathogenic.  Amenable to genetic manipulation. Corynebacterium glutamicum
  • 149. The manufacturing process of glutamic acid by fermentation comprises :- a. fermentation, b. crude isolation, c. purification processes.  There are 4 types of fermentation are used:  (1) Batch Fermentation.  (2) Fed-batch Fermentation.  (3) Continuous Fermentation. Industrial production of glutamic acid
  • 150. (1)Batch Fermentation  Widely use in the production of most of amino acids. Fermentation is a closed culture system which contains an initial, limited amount of nutrient. A short adaptation time is usually necessary (lag phase) before cells enter the logarithmic growth phase (exponential phase). Nutrients soon become limited and they enter the stationary phase in which growth has (almost) ceased. In glutamic acid fermentations, production of the acid normally starts in the early logarithmic phase and continues through the stationary phase.
  • 151. For economical reasons the fermentation time should be as short as possible with a high yield of the amino acid at the end. A second reason not to continue the fermentation in the late stationary phase is the appearance of contaminant- products. The lag phase can be shortened by using a higher concentration of seed inoculum. The seed is produced by growing the production strain in flasks and smaller fermenters.
  • 152. (2) Fed-batch fermentation Batch fermentations which are fed continuously, or intermittently, with medium without the removal of fluid.  In this way the volume of the culture increases with time. The residual substrate concentration may be maintained at a very low level. This may result in a removal of catabolite repressive effects and avoidance of toxic effects of medium components.  Oxygen balance. The feed rate of the carbon source (mostly glucose) can be used to regulate cell growth rate and oxygen limitation,especially when oxygen demand is high in the exponential growth phase.
  • 153. (3) Continuous fermentation  In continuous fermentation, an open system is set up.  Sterile nutrient solution is added to the bioreactor continuously.  And an equivalent amount of converted nutrient solution with microorganisms is simultaneously removed from the system.
  • 154.  Natural product such as sugar cane is used.  Then, the sugar cane is squeezed to make molasses. The heat sterilize raw material and other nutrient are put in the tank of the fermenter. The microorganism (Corynebacterium glutamicum) producing glutamic acid is added to the fermentation broth. The microorganism reacts with sugar to produce glutamic acid. Then, the fermentation broth is acidified and the glutamic acid is crystallized. Industrial production of glutamic acid
  • 155. Separation and purification After the fermentation process, specific method is require to separate and purify the amino acid produced from its contaminant products, which include:  Centrifugation.  Filtration.  Crystallisation.  Ion exchange.  Electrodialysis.  Solvent extraction.  Decolorisation.  Evaporation.
  • 156. The glutamic acid crystal is added to the sodium hydroxide solution and converted into monosodium glutamate (MSG). MSG is more soluble in water, less likely absorb moisture and has strong umami taste. The MSG is cleaned by using active carbon, which has many micro holes on their surface. The clean MSG solution is concentrated by heating and the monosodium glutamate crystal is formed. The crystal produce are dried with a hot air in a closed system. Then, the crystal is packed in the packaging and ready to be sold. Separation and purification of Glutamic acid
  • 157. PREPARATION OF GRISEOFULVIN BY FERMENTATION
  • 158. GRISOFULVIN- INTRODUCTION  Griseofulvin is an antifungal antibiotic first isolated from a Penicillium species in 1939. It is a secondary metabolite produce by thefungus Penicillium griseofulvum.  The compound is insoluble in water,and slightly soluble in ethanol, methanol, acetone, benzene, CHCl3, ethyl acetate, and acetic acid.
  • 160.
  • 161. MODE OF ACTION  Griseofulvin inhibit fungal cell mitosis and nuclear acid synthesis. It also binds to and interferes with the function of spindle and cytoplasmic microtubules by binding to alpha and beta tubulin.  It binds to keratin in human cells, and then once it reaches the fungal site of action, it binds to fungal microtubules thus altering the fungal process of mitosis.
  • 162.
  • 163. USES It is used in the treatment of ⚫ Ringworm of the Beard ⚫ Ringworm of Scalp ⚫ Fungal Disease of the Nails ⚫ Ringworm of Groin Area ⚫ Athlete's Foot ⚫ Ringworm of the Body.
  • 164.
  • 165. SIDE EFFECTS The most common side-effects are ⚫ Nausea ⚫ Vomiting ⚫ Diarrhoea ⚫ Heartburn ⚫ Flatulence, cracking at the side of the mouth ⚫ Soreness and/or blackening of the tongue and thirst ⚫ Headache
  • 167. PREPARATION OF MEDIA Medium ⚫ Czapek Dox Medium Chemicals ⚫ Glucose ⚫ Sodium Nitrate ⚫ Potassium Hydrogen Phosphate ⚫ Magnesium Sulphate 7H20 5% 0.2% 0.1% 0.05%
  • 168.
  • 169. Industrial preparation of griseofulvin by submerged fermentation
  • 170. STEPS INVOLVED IN THE MANUFACTURING PROCESS  Fermentation  Pre treatment of fermentation broth  Filtration  Extraction  Decolorization  Isolation and separation  Precipitation and purification
  • 171.
  • 172. FERMENTATION  The pH of Czapek-Dox medium was adjusted between 6.0-7.2. The medium was dispensed in the fermenter .  The fresh sample of mycelial suspension of fungus Peccillium griseofulvum from the fresh slope on raper steep agar (Czapek-Dox medium + corn steep+ agar) was obtained.  The solution was autoclaved for 200 minutes at 120°C at 15lbs pressure and fermented for 14 days at 24°C.
  • 173.
  • 174.
  • 175. PRE TREATMENT OF FERMENTATION BROTH  The broth is heated above 60°C for 20- 30minutes.  After heating, sufficient coagulation of material occurs to produce a valuable improvement in separation characteristics of the broth.  The period of heating may be short, 5-10 minutes at 80°C having been found to provide a satisfactory increase in filtration rate.
  • 176. FILTRATION  Drum covered with diatomaceous earth matter and allowed to rotate under vacuum with half immersed in the slurry tank. Small amount of coagulation agent added to broth and pumped into the slurry tank. As drum rotates in the slurry tank under vacuum thin layer of coagulated particles adhere to drum.  The layer thickens to from cake. As the cake portion in the drum comes to the upper region which is not immersed in the liquid it is washed with water and dewatered immediately by blowing air over it.  Then before the dried portion is again immersed into the liquid it is cut off from drum by knife.
  • 177.
  • 178.
  • 179. EXTRACTION  Griseofulvin is extracted in the cold acetone when it is used as an extraction agent.  The extractions with the cold acetone may be carried out with the efficiencies between 75-96% or even upto 99.5%. the quantity of the solvent used in the extraction at large scale production should be kept minimum.  The volume of acetone should be 3-5 times of the mycelial felt.
  • 180.
  • 181. DECOLORIZATION  The color of the extract can be improved by the addition of calcium hydroxide usually 2.5-50 g/liter preferably 5- 30 g/liter. The pH of the extract should be above 10. It can be neutralize by the removal of lime or by using mineral acid.
  • 182. ISOLATION AND SEPARATION  The impurities or waxy substances are removed by washing the extract with a solvent in which extract is immiscible and also griseofulvin is insoluble.  Hydrocarbon solvents, generally aliphatic hydrocarbons such as hexane or petroleum containing a high portion of hexane are in general suitable for this step.
  • 183. PRECIPITATION AND PURIFICATION  Griseofulvin can be precipitated from the solvent extract in various ways. One of the method is using the liquid solvent in which griseofulvin is substantially insoluble. Griseofulvin non-solvent is preferably water.  The alkaline water is more effective for the removal of colored impurities present in the crystals of the griseofulvin.  Water is made alkaline with ammonia or an alkali metal carbonate or alkali metal hydroxide. The suitable pH is about 8.5.  The purity of the precipitate is generally improved by washing with a solvent for the small quantities of impurities remaining. The suitable washing media are dry or wet acetone, a lower alkanol for example methanol or butanol. Marked purification is obtained with the use of methanol for this step.
  • 184.
  • 185.
  • 186. Blood Products: Collection, Processing and Storage
  • 187. Blood • The importance of blood in health and disease has been appreciated since ancient times but blood transfusion was not practised on a large scale until early this century. • Previous attempts were often frustrated by clotting and ignorance of the existence of blood groups; therefore, for centuries blood letting rather than transfusion remained a pillar of medical therapy.
  • 188. Blood Products • COLLECTION • The blood is collected aseptically from the median cubital vein, in front of the elbow, into a sterile container containing an anticoagulant solution. During collection the bottle is gently shaken to ensure that blood and anticoagulant are well mixed, thus preventing the formation of small fibrin clots. • Not more than 420 ml is taken at one attendance. Immediately afterwards the container is sealed and cooled to 4-6°C. • The equipment used for taking blood is made from plastics, and is disposable.. The container is most often the Medical Research Council blood bottle but plastic bags have been used in America
  • 189. Blood • “Whole Human’ Blood • This is human blood that has been mixed with a suitable anticoagulant. Any person in good health is accepted as a donor provided that he or she • 1. Is not suffering from any disease that can be transmitted by transfusion. This includes syphilis, malaria, and serum jaundice. • 2. Is not anaemic. The haemoglobin content of the blood should not be less than 12.5 and 13.3 per cent for female and male donors respectively.
  • 190. Blood • COLLECTION • The blood is collected aseptically from the median cubital vein, in front of the elbow, into a sterile container containing an anticoagulant solution. During collection the bottle is gently shaken to ensure that blood and anticoagulant are well mixed, thus preventing the formation of small fibrin clots. • Not more than 420 ml is taken at one attendance. Immediately afterwards the container is sealed and cooled to 4-6°C. • The equipment used for taking blood is made from plastics, and is disposable.. The container is most often the Medical Research Council blood bottle but plastic bags have been used in America for some years and are likely to be the containers of the future (see Gunn and Carter, 1965).
  • 191. Blood • BLOOD CLOTTING • According to classical theory blood clotting takes place in two phases • In response to injury, the tissues and blood platelets free substances that activate the clot promoting enzyme thromboplastin. This, with the assistance of ionised calcium and other factors, converts prothrombin into the active clotting enzyme thrombin which acts on fibrinogen, converting it into insoluble fibrin, the matrix of the clot.
  • 192. Blood • 1. Citrates • The solution most often used as a blood anti coagulant is known as Acid-citrate-dextrose (ACD) and has the composition • Sodium acid citrate 2:0 to 25.G • Dextrose : 30G • Water for Injections to 120ml • The citrate prevents clotting by binding ‘the calcium ions as unionised calcium citrate.
  • 193. Blood Products • “At one time the normal (trisodium) citrate was used but it has a very alkaline pH in solution which causes considerable caramelisation (darkening) of the dextrose during sterilisation and the two solutions have to be autoclaved separately. The acid citrate produces a pH of about 5 and causes little or no caramelization. In addition, it is less likely to induce flaking of the glass of the container. The higher concentration (2:5 G/120 ml) is often preferred because it’ more effectively reduces the formation of small clots. • The dextrose delays haemolysis of the erythrocytes in vitro and prolongs their life after transfusion. Its function may be connected with the synthesis of compounds, such as adenosine tri-phosphate (ATP)
  • 194. Blood Products • 2. Heparin • This is a naturally occurring anticoagulant made by the mast ceils of the connective tissue surrounding blood vessels. It inhibits clotting in the circulatory system. Occasionally it is used in blood for transfusion when large volumes must be given to one patient and the corresponding amounts of citrate would be harmful, e.g. in cardiac surgery. • It quickly loses activity in blood in vitro and normal quantities are effective for about a day. ADC, on the other hand, prolongs the storage life to three weeks. Heparin is expensive and may continue its action after transfusion, necessitating the administration of neutralising substances such as protamine sulphate.
  • 195. Blood Products • Disodium Edetate • This is a chelating agent which, because it has a strong affinity for divalent metals, will bind calcium firmly. It is sometimes preferred when preservation of blood platelets is essential, although the stability of these seems to depend much more on preventing contact with the glass surfaces.
  • 196. Blood Products • Testing • At the time that blood is taken, two small additional amounts are collected. • One, which is often obtained by draining the collecting tube, is put into a small 5ml bottle and is firmly attached to the main container. This is for testing compatibility with the blood of the recipient before administration; using a separate specimen avoids the dangerous procedure of attempting to remove a sample from the main bottle without causing bacterial contamination. • With a plastic bag it is possible to leave the blood-filled collecting tube attached to the bag and to seal it at several points with a special tool; then a section can be separated for testing without contaminating the bulk.
  • 197. Blood Products • Testing • The second, somewhat larger sample is used as soon as possible: • (a) for a serological test to confirm the absence of syphilis, and • (b) to determine the ABO grouping of the cells and plasma and the Rh grouping of the cells. • Blood groups . • Fundamentally, the aim of blood grouping is to prevent ‘an antigen-antibody reaction. Red cells carry an antigen that reacts with the corresponding antibody in the plasma of individuals of certain other groups. If the cells are transfused-into an individual with the equivalent antibody in his plasma they are rapidly destroyed, with serious consequences.
  • 198. Blood Products • ABO System. • The first sign of the haemolytic antigen-antibody reaction is agglutination and, therefore, red-cell antigens and plasma antibodies are called agelutinogens and agglutinins respectively. • The agglutinated cells haemolyse, freeing haemoglobin and other constituents and causing jaundice and kidney damage. if the latter is extreme the patient may die. • Fortunately, most transfusion reactions are mild.
  • 199. Blood Products • The red cells of some individuals carry an antigen that, because it is also found in the rhesus monkey is Known as the rhesus (Rh) factor. If Rh+ bloodis transfused info an Rh negative recipient production of antibodies to Rh + cells may be stimulated. • If this occurs, subsequent transfusion of Rh + blood to the same patient will cause a haemolytic reaction.
  • 200. Blood Products • STORAGE • Apart from short periods for transport and examination, which must not exceed thirty minutes, blood must be kept at 4 to 6°C until required for use. • Even at this temperature deleterious changes take place. The leucocytes disintegrate in a few hours and the platelets in a few days. The red cells. show a fall in ATP and other organic - phosphates, a reduction in oxygen-carrying capacity and, due partly to loss of lipid from .their membranes, increased fragility. • Storage at room temperature, even for only a day, seriously reduces post-transfusion survival of the erythrocytes.
  • 201. Blood Products • The fitness of blood for transfusion is based on its appearance. On standing, the cells sediment, leaving a layer of yellow supernatant plasma. • If the blood has been taken shortly after a heavy fatty meal the plasma may be turbid and show a white layer of fat on its surface. On top of the red cells there may be a complete or partial greyish layer of leucocytes. • The most important feature, however, is the line of demarcation between cells and plasma, which must be sharp; if it is obscured by a diffuse red colouration, indicating haemolysis, the blood is unfit for use.
  • 202. Blood Products • The fitness of blood for transfusion is based on its appearance. On standing, the cells sediment, leaving a layer of yellow supernatant plasma. • If the blood has been taken shortly after a heavy fatty meal the plasma may be turbid and show a white layer of fat on its surface. On top of the red cells there may be a complete or partial greyish layer of leucocytes. • The most important feature, however, is the line of demarcation between cells and plasma, which must be sharp; if it is obscured by a diffuse red colouration, indicating haemolysis, the blood is unfit for use.
  • 203. Blood Products • The volume of the blood in the body can be reduced to a dangerously low level by haemorrhage,, shock, burns, and uncontrollable diarrhoea and vomiting. • Haemorrhage and certain diseases may result in deficiency or absence of vital blood constituents such as red cells, platelets, or clotting factors. The transfusion of whole blood can be of great value in all these circumstances but often, because of the risk of transfusion reactions, it is not used where the need is: • solely to make up blood volume but is restricted to haemorrhage and • certain diseases where there is deficiency of the vital oxygen-carrying erythrocytes.
  • 204. Blood Products • Normally whole blood is not administered unless the ABO and Rh groups of donor and recipient are known and a sample of the donor’s blood has been tested for compatibility with that of the recipient. • In an emergency, group O, Rh negative blood may be given while the above precautions are being taken.
  • 205. Dried human plasma Whole blood has several serious disadvantages— 1. It has poor keeping properties necessitating use within three weeks. 2. It requires refrigerated storage. 3. It must be compatible with the blood of the recipient. Dried plasma, on the other hand, has the following advantages— 1. Properly stored it keeps well for at least five years. . 2. If protected from light it can be stored at room temperature provided this is below 20°C. 3. It can be given to patients of any blood group. Consequently, in suitable circumstances, dried plasma is used as a substitute for whole blood
  • 206. Dried human plasma Whole blood has several serious disadvantages— 1. It has poor keeping properties necessitating use within three weeks. 2. It requires refrigerated storage. 3. It must be compatible with the blood of the recipient. Dried plasma, on the other hand, has the following advantages— 1. Properly stored it keeps well for at least five years. . 2. If protected from light it can be stored at room temperature provided this is below 20°C. 3. It can be given to patients of any blood group. Consequently, in suitable circumstances, dried plasma is used as a substitute for whole blood
  • 207. Dried human plasma It consist about 55 % of blood. This is yellow colour fluid. It is used for plasma transfusion where medically needed. It was first developed in British 1930. • Collection • Take blood from blood donor which want eligible for donating blood. to donate blood and • Separate the blood plasma from blood by the process of centrifugation
  • 208. Procedure • T ake 400 ml blood plasma which are separated from blood. • Packed in to MRC bottle (medical research council bottle • The bottle is sealed with • Bacteriological efficient. • Freezing- • The bottle is then centrifuged at 18°c
  • 209. Primary drying- • The bottle are mounted horizontally in the drying chamber at 50°c • This process takes place about 2 weeks Secondary drying- • This is done in another chamber by vaccume desiccation over phosphorus pentaoxide • It takes place about a day • Atleast 0.5% moisture should be left
  • 210. Storage- • It is kept below 28°c and protected from moisture, sunlight and remains usable for 5 year • Uses- • Blood clotting • Autoimmune disorders • hemophilia • Other medical emergencies
  • 211. Plasma Substitutes • The limited supplies of plasma, the cost of ‘producing the dried form and the risk of transmitting serum hepatitis stimulated attempts to find substitutes of non-human origin that could be used to restore the blood volume temporarily while the recipient replaced the lost protein.
  • 212. Plasma Substitutes • PROPERTIES OF AN IDEAL PLASMA SUBSTITUTE • 1. The same colloidal osmotic pressure as whole blood. • 2. A viscosity similar to that of plasma. • 3. A molecular weight such that the molecules do not easily diffuse through the capillary walls. • 4. A fairly low rate of excretion or destruction by the body. • 5, Eventual and complete elimination from the body.
  • 213. Plasma Substitutes • PROPERTIES OF AN IDEAL PLASMA SUBSTITUTE • 6. Freedom from toxicity, e.g. no impairment of renal function. • 7. Freedom from antigenicity, pyrogenicity, and confusing effects on important tests such as blood grouping and the erythrocyte sedimentation rate. • 8. Isotonicity, in solution, equal to that of blood plasma. • 9. High stability in liquid form at normal and sterilising temperatures and during transport and storage. • 10. Ease of preparation, ready availability and low cost.
  • 214. Plasma Substitutes • GUM SALINE • Synonym for Injection of Sodium Chloride and Acacia having 6% acacia in 0.9% Sodium Chloride solution. • DISADVANTAGES: • Signs of liver dysfunction as gum was not metabolized but stored in various organs. • Polyvinylpyrrolidone – a synthetic colloid. Disadvantage – suspected carcinogenicity.
  • 215. Plasma Substitutes • Dextran • To date this is the most satisfactory plasma substitute. It is a polysaccharide produced when the bacterium Leuconostoc mesenteroides is grown in a sucrose- containing medium. In the sugar industry it occurs as a slime that clogs pipes and filters and interferes with crystallisation. • The organism secretes.an enzyme that converts sucrose to dextran according to the following reaction
  • 216. Plasma Substitutes • Different strains produce dextrans of two main groups • 1. Long, practically unbranched chains of glucose units joined by 1:6 glucosidic linkages. • 2. Highly branched polymers consisting of short chains of 1:6 units joined by 1:4 and 1:3 linkages to branches. • Branched chains are more likely to give rise to allergic reactions when injected, and in dextrans used for plasma substitutes the linkages should be almost entirely of the 1:6 type. This is achieved by choosing a suitable specially developed strain of the organism that produces dextran in which about 95 per cent of the linkages are 1:6.
  • 217. • Production • Production involves laboratory culture followed by growth in and then in 4500 cubic dm fermenters. seed tanks in the factory • PRECAUTIONS – need to prevent the hydrolysis of sucrose to glucose and fructose during sterilization of the culture media. Prevention measures include the adjustment of the media to neutral pH before sterilization, and the avoidance of overheating. • When maximum conversion to dextran has been obtained it is precipitated by adding a suitable organiac solvent. Plasma Substitutes
  • 218. • Natural dextran consists of chains of approximately 200,000 weights up to about 50 million. glucose units with molecular • Very large molecules i.e., those with a molecular weight above about 250,000 have serious drawbacks: – They yield very viscous solutions that are difficult to administer. – They may cause renal damage and allergic reactions. – They interfere with blood matching and sedimentation tests by causing rouleaux formation. Rouleaux are aggregates of red cells that resemble piles of plates. – They produce colloidal osmotic pressures that are lower than those of small molecules. Plasma Substitutes
  • 219. • Therefore to produce a material suitable for medical use it is necessary to reduce the size of the natural molecules. This can be accomplished in several ways: • Acid hydrolysis (most widely used). • Thermal degradation. • Ultrasonic disintegration. • Seeding the fermenter. Plasma Substitutes
  • 220. • The very small molecules, i.e. those of below a molecular weight of about 60,000 also have disadvantages: • They are rapidly excreted in urine. • They pass into the tissue fluids causing an adverse osmotic pressure. Plasma Substitutes
  • 221. • The selected fraction still requires considerable purification to remove- • Reducing sugars: by further solvent precipitation. The main contaminant is fructose, the by-product of fermentation. • Fractionation solvents: by evaporation under reduced pressure. • Inorganic salts: by demineralization in a mixed bed ion exchanger. • Colour: by adsorption on to activated charcoal. • Pyrogrens: by adsorption on to asbestos, or cellulose derivatives. Plasma Substitutes
  • 222. • Micro organisms: by filtration. • The solution is diluted to a concentration of 5% in either 5% dextrose injection or sodium chloride injection, packed in sulphur treated soda- lime bottles and closed with lacquered rubber plugs. • Finally, it is sterilized, usually by heating in an autoclave. Plasma Substitutes