Biotechnology Lecture Notes on Fermentation (PDF)

Biotechnology PM 504 Fermentation ROLE OF MEDIA IN FERMENTATION Media are required in several stages of most industrial fermentation processes. They may include inoculum propagation (which is known as starter culture), pilot-scale fermentations and the main production fermentation. The tech...

Biotechnology PM 504 Fermentation ROLE OF MEDIA IN FERMENTATION Media are required in several stages of most industrial fermentation processes. They may include inoculum propagation (which is known as starter culture), 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. Where biomass or primary metabolites are the target product, a production medium is designed to allow optimal growth of the microorganism for maximum biomass production. For secondary metabolite production, such as antibiotics, biosynthesis is not growth related. Consequently, for this purpose, media are designed to provide an initial period of cell growth, followed by conditions optimized for secondary metabolite production. In many cases, the supply of one or more nutrients (carbon, phosphorus or nitrogen source) may be limited and rapid growth ceases. ROLE OF MEDIA IN FERMENTATION Some considerations made when formulating media for fermentation: An optimally balanced culture medium containing all critical elements is mandatory for maximal production. The composition of culture media must be adapted to the fermentation process. In addition to product yield, product recovery must be examined in trial fermentations. If catabolite repression or phosphate repression cannot be eliminated by optimization of the nutrient medium or suitable fermentation management, deregulated mutants must be used as production strains. Besides material cost and product yield, it must be considered whether materials used are readily available in sufficient supply without high transportation costs. It should also be considered whether impurities will hinder product recovery or increase the cost of product recovery.  Media Preparation Medium formulation is a very important step in fermentation process. Most fermentation processes require liquid media. However, many solid-substrate fermentations are also known, such as enzyme fermentations. It is essential that the fermentation media satisfies all the nutritional requirements of the microorganisms and fulfill the technical objectives of the industrial process. certain criteria for an ideal medium: It should produce the maximum yield of product or biomass per gm of substrate used. It should produce maximum concentration of product or biomass. It should permit the maximum rate of product formation. There should be the minimum yield of undesired products. It should be of a consistent quality and be readily available throughout the year. It should cause minimal problems during media preparation and sterilization. It should cause minimal problems in other aspects of the production process particularly aeration and agitation, extraction, purification and waste treatment.  Media Preparation In order to successfully formulate a fermentation medium, it is of utmost importance that one should have a sound knowledge of the overall process including the stoichiometry for growth and product formation. consideration of the input of the carbon and nitrogen sources, minerals and oxygen and their conversion to cell biomass, metabolic products, carbon-dioxide, water and heat is very important as such information can help one to calculate the minimum quantities of each element required to produce a certain quantity of biomass or metabolite. knowledge of the complete elemental composition of the specific industrial microorganism allows further medium refinement. This ensures that no element is limiting, unless this is desired for a specific purpose.  Media Preparation An aerobic fermentation process is generally represented as: Carbon and energy source + Nitrogen source + O2 + other requirements → Biomass + products + CO2 + H2O + heat Based on this information, the minimum quantities of each element including carbon source, energy source, nitrogen source, aeration etc. required for producing a particular quantity of biomass and metabolic end product may be calculated. Once the amounts of nutrients to be incorporated have been detected, then suitable nutrient sources can be added into the media. Another consideration during media formulation is the scale of the fermentation. It is desirable that for small scale laboratory fermentations pure chemicals be used in well defined media. Pure chemicals are also desirable when fermentation is carried out for many purified end products of high value since crude media components can cause a problem in recovery of the product.  Media Preparation This is not possible for most industrial-scale fermentation processes simply due to cost, as media components may account for up to 60-80% of process expenditure. Many industrial scale fermentation processes use cost effective complex substrates, where many carbon and nitrogen sources are almost indefinable. Most are derived from natural plant and animal materials, often by-products of other industries, with varied and variable composition. Preparing a medium for culturing yeast for animal consumption, vegetable waste matter can be used as a starting material, although, such a waste matter is not suitable for direct feeding of animals. Corn-cob and/or stalks, reeds, sunflower stalks, sewage, wheat and paddy straw are other crude substrates of interest.  Properties of Media Due to the inconsistency of the raw substrate, small-scale trials are usually performed with each new batch of substrate, particularly to examine the impact on product yield and product recovery. The main factors that affect the final choice of individual raw materials are as follows: Cost and availability: ideally, materials should be inexpensive, of consistent quality and should have year-round availability. Ease of handling in solid or liquid forms, along with associated transport and storage costs. Sterilization requirements and any potential denaturation problems. Formulation, mixing, complexing and viscosity characteristics that may influence agitation, aeration and foaming during fermentation and downstream processing stages. The concentration of target product attained, its rate of formation and yield per gram of substrate utilized. The levels and range of impurities, and the potential for generating further undesired products during the process. Overall health and safety implications.  Properties of Media A medium that is easily sterilized with minimum thermal damage is vitally important. Thermal damage not only reduces the level of specific ingredients but can also produce potentially inhibitory by-products that may also interfere with downstream processing. Medium Components 1. Carbon sources A carbon source is required for all biosynthesis leading to multiplication of cells, cell maintenance and product formation. it serves as the energy source. Carbohydrates are traditional carbon and energy sources for microbial fermentation, although other sources are increasingly gathering importance, such as alcohols, alkanes and organic acids. In addition to main carbon source, animal fats and plant oils may be incorporated into some media as supplements  Properties of Media Simple sugars such as pure glucose or sucrose can seldom be used as the sole carbon source. Since these substrates are relatively expensive, so they are used primarily to produce high cost, low volume products. Molasses, a byproduct of sugar production, is one of the cheapest sources of carbohydrates. Besides a large amount of sugar, molasses contains nitrogenous substances, vitamins, and trace elements. The composition of molasses varies depending on the raw material used for sugar production. It requires removal of certain metal ions which can reduce the yield of the fermentation product. Malt extract, an aqueous extract of malted barley, is an excellent substrate for many fungi, yeasts, and actinomycetes. Dry malt extract consists of about 90-92% carbohydrates, and is composed of hexoses (glucose, fructose), disaccharides (maltose, sucrose), trisaccharides (maltotriose), and dextrins.  Properties of Media In addition to carbohydrates, malt extracts serve as a rich source of nitrogen. Nitrogenous substances present in malt extract include proteins, peptides, amino acid, purines, pyrimidines, and vitamins. The amino acids composition of different malt extracts varies according to the grain used, but proline always makes up about 50% of the total amino acids present. sterilization of medium containing malt extract needs special consideration and should be done carefully. This is because when overheating occurs, the Maillard reaction results, due to the low pH value and the high proportion of reducing sugar. In this conversion, the amino groups of amines, amino acid (especially lysine), or proteins react with the carbonyl groups of reducing sugars, aldehydes or ketones, which results in the formation of brown condensation products. These reaction products are not suitable substrates for microorganisms and may inhibit their growth. The Maillard reaction is one of the main causes of damage to culture media during heat sterilization, resulting in considerably reduced yields.  Properties of Media Starch and dextrin can be directly metabolized as carbon sources by microorganisms that are capable of producing amylase. In addition to glucose syrup, which is frequently used as a fermentation substrate, starch is finding more importance as a substrate for ethanol fermentation. Sulfite waste liquors are sugar-containing waste products of the paper industry. Sulphite waste liquors have a dry weight of 9-13% and are primarily used in the cultivation of yeasts. Sulfite liquors from coniferous trees have a total sugar content of 2-3%, and 80% of the sugars are hexoses (glucose, mannose, galactose), the others being pentoses (xylose, marabinose). Sulfite liquors from deciduous trees contain mainly pentose sugars.  Properties of Media Cellulose is being extensively studied as a substrate for conversion to sugar or alcohol due to its wide availability and low cost. It is usually not possible to use cellulose directly as a carbon source, so it must first be hydrolyzed chemically or enzymatically. The sugar syrup formed from cellulose hydrolysis has been used for ethanol fermentation, and the fermentative production of butanol, acetone, and isopropanol is also being considered. Whey a byproduct of the dairy industry, is produced annually on a world-wide basis to the amount of 74 million tons which might contain 1.2 million tons of lactose and 0.2 million tons of milk protein. Only about 56% of this product is used for human or animal feed. The lactose is used primarily to produce ethanol or single-cell protein but is also used in the production of vitamin B12, 2,3-butandiol, lactic acid, and gibberellic acid. because of storage and transportation costs, whey is often not economical as a substrate.  Properties of Media Animal fats such as lard, animal and plant oils are readily utilized by some microorganisms but are generally added as supplemental substrates rather than as the sole fermentable carbon source. Oils contain approximately 2.4 times the energy of glucose on per weight basis and occupy less volume, thereby, giving a better fermenter working volume. Methanol is the cheapest fermentation substrate with respect to its carbon content, but it can be metabolized by only a few bacteria and yeasts. Methanol has commonly been used as a substrate for single cell protein production. production of glutamic acid, serine, and vitamin B12 using methanol as the sole carbon source or as a co-substrate.  Properties of Media Ethanol is available in ample supply from the fermentation of either saccharified starch or cellulose and can be metabolized by many microorganisms as the sole carbon source or as a co-substrate. Acetic acid is presently made by the oxidation of ethanol. at the present time, the cost of ethanol is too high to make it utilizable as a general industrial carbon source. Hydrocarbons such as n-alkanes with a chain length of C12 to C18 are readily metabolized by many microorganisms. The use of alkanes as an alternative to carbohydrates depends on the price of petroleum. On a dry weight basis, n-alkanes have approximately twice the carbon and thrice the energy content of the same weight of sugar. Hydrocarbons and their derivatives might be used as feedstocks in fermentation of high value products such as pharmaceuticals, fine chemicals and agricultural chemicals  Properties of Media 2. Nitrogen Sources Most of the industrially important microorganisms can utilize both inorganic and organic nitrogen sources. Inorganic nitrogen supplied as ammonium salts, often ammonium sulfate and diammonium hydrogen phosphate, or ammonia. These are readily available to the microorganisms. large-scale processes utilize ammonium salts, urea, or gaseous ammonia as nitrogen source. However, ammonium salts such as ammonium sulfate usually lead to decrease in pH of the fermentation broth when ammonium ions are utilized, thereby generating free acid. Ammonium nitrate leads to alkaline conditions. Initially, a drop in pH is observed as ammonium ions are utilized and acid is produced. Once the ammonium ions are exhausted, nitrate is used as an alternative source.  Properties of Media Organic nitrogen sources include amino acids, proteins and urea. In many fermentations, nitrogen is supplied in crude forms that are essentially by-products of other industries, such as corn steep liquor, yeast extracts, peptones and soya meal. Growth rate is increased with a supply of organic nitrogen. A few microorganisms have an absolute requirement of amino acids. Purified amino acids act as precursors for specific products and are thus added accordingly. A nitrogen source, which is efficiently metabolized, is corn steep liquor, which is formed during starch production from corn. A nitrogen source, which is efficiently metabolized, is corn steep liquor, which is formed during starch production from corn. The concentrated extract (about 4% nitrogen) contains numerous amino acids, such as alanine, arginine, glutamic acid, isoleucine, threonine, valine, phenylalanine, methionine and cystine.  Properties of Media The sugar present in corn steep liquor is largely converted to lactic acid (9-20%) by lactic acid bacteria. Corn steep Liquor, in addition to acting as a carbon and nitrogen source, also acts as a precursor in penicillin production. Complex nitrogen sources when used in antibiotic fermentation help in creating favorable physiological conditions in exponential phase of growth (trophophase) that lead to antibiotic production in stationary phase  Properties of Media Yeast extracts are other excellent substrates for many microorganisms. They are produced from baker‘s yeast through autolysis/plasmolysis in the presence of high concentrations of NaCl. Yeast extract contains amino acids and peptides, water-soluble vitamins and carbohydrates. The glycogen and trehalose of yeast cells are hydrolyzed to glucose during yeast extract production. Peptones are protein hydrolysates which can be utilized by many microorganisms, but they are relatively expensive for industrial application. Sources of peptones include meat, casein, gelatin, keratin, peanut seeds, soy meal, cotton seeds, and sunflower seeds. peptone from gelatin is rich in proline and hydroxyproline but has almost no sulfur-containing amino acids peptone from keratin has a large proportion of proline and cysteine but lacks lysine. Peptones of plant origin such as soy peptone and cottonseed peptone have large proportions of carbohydrates. The end product is also influenced by the type of hydrolysis, whether acid or enzymatic, especially regarding its tryptophan content. Best nitrogen sources for some secondary metabolites  Properties of Media Soy meal, the residue from soybeans after the extraction of soybean oil, is a complex substrate which contains a protein content of 50%, a carbohydrate content of 30% (sucrose, stachyose, raffinose, arabinoglucan, arabinan, and acidic polysaccharides), 1% residual fat, and 1.8% lecithin. Soy meal is frequently used in, antibiotic fermentations; catabolite regulation does not occur because of the slow catabolism of this complex mixture. 3. Water Most fermentation, except solid substrate fermentation, require large quantities of water in which medium is formulated. Water is required in heating, cooling, cleaning and rinsing. Many early industrial establishments were located close to a river or stream which provided ample, clean water for use. It also provides trace mineral elements.  Properties of Media The mineral content of water is important in breweries and decides the type of beer produced. For instance, hard water containing CaSO4 is good for producing English bitter beer while water rich in carbonate content is better for stout beer. Removal of suspended solids, colloids and microorganisms and removal of hardness is usually required. In order to minimize water costs, recycle/ reusage of water is practiced, which also reduces the volume requiring wastewater treatment. 4. Minerals Usually sufficient quantities of cobalt, copper, iron, manganese, molybdenum and zinc are also present in water supplies. certain trace elements also occur as impurities in other media ingredients. For example, corn steep liquor satisfies the requirements of minor and trace mineral needs. Specific salts of calcium, magnesium, phosphorus, potassium, sulfur and chloride ions have to be added in the medium as inorganic salts fulfill the requirements  Properties of Media 5. Vitamins and Growth Factors Some bacteria, filamentous fungi and yeasts cannot synthesize all necessary vitamins from basic elements; 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. Many vitamins such as B-complex vitamins are rapidly destroyed at elevated temperatures. When a single vitamin is required by a culture, it is better to use it in pure form as it will be more economical than using a larger amount of cheaper source. Other necessary growth factors, amino acids, nucleotides, fatty acids and sterols are added either in pure form, or as plant and animal extracts  Properties of Media 6. Precursors Precursors are the chemicals which when added to the fermentation media are directly incorporated into the desired product They are often added in controlled quantities and in a relatively pure form. Use of precursors was an increase in the yield of penicillin from 20 units cm-3 to 100 units cm-3 on addition of corn steep liquor in the medium which can be attributed to the presence of phenylethylamine. 7. Inducers and Elicitors Majority of enzymes which are of industrial use, being inducible, require a specific inducer or a structural analogue, which must be incorporated into the culture medium or added at some specific stage. Inducers are often necessary in fermentation by genetically modified microorganisms  Properties of Media Common examples include use of substrates such as starch for amylase production, pectin for pectinase production and cellulase for cellulose production. Fatty acids induce the production of lipase, however their inclusion in fermentation medium might not be financially feasible. During streptomycin fermentation both streptomycin and mannosidostreptomycin are produced. Mannosidostreptomycin has only about 20% of the biological efficiency of streptomycin, making it an undesirable product. Addition of yeast mannan induces the production of β- mannosidase by Streptomyces griseus which converts mannosidostreptomycin into streptomycin.  Properties of Media 8. Inhibitors When certain inhibitors are added to fermentation medium, a specific product may be produced or a metabolic intermediate which is normally metabolized is accumulated. For example: glycerol production sodium bisulphite is used as an inhibitor. For glycerol production to takes place ethanol fermentation is modified by removing acetaldehyde. When sodium bisulphite is added to the medium, formation of acetaldehyde bisulphite takes place thereby making acetaldehyde unavailable for reoxidation of NADH2. As a result, dihydroacetone phosphate acts as a hydrogen acceptor producing glycerol 3- phosphate which is then converted into glycerol.  Properties of Media Other inhibitors include bromide for tetracycline production from Streptomyces auriofaciens penicillin for glutamic acid production using Micrococcus glutamicus alkali metal for citric acid production by Aspergillus niger. 9. Buffers Buffers are chemical compound which specifically serve to maintain the pH of the medium. Maintenance of pH of medium is of utmost importance for the optimal productivity in a fermentation process. Media are buffered at pH 7.0 by incorporation of calcium carbonate. Apart from this, sodium and potassium phosphates can be used as important buffering compounds. At small scale sulfuric acid or sodium hydroxide can be used for maintaining the pH of the medium. The balanced use of carbon and nitrogen sources also aids in pH control, as buffering capacity can be provided by proteins, peptides and amino acids  Properties of Media 10. Cell Permeability Modifiers These compounds generally increase the cell permeability by modifying cell walls and/or membranes, promoting the release of intracellular products into the fermentation medium. Penicillins and surfactants are frequently added to amino acids fermentations, including processes for producing L-glutamic acid using the members of genera Corynebacterium and Brevibacterium. 11. Anti Foams protein components of the medium, such as corn-steep-liquor, peanut meal, soybean meal, yeast extract, meat extract and malt extract may lead to foam production in the fermentation broth. Foaming can be a major problem in fermentation industry as it might lead to removal of cells from the medium as well as autolysis of the cells leading to the release of more microbial proteins thereby increasing stability of foam.  Properties of Media Uncontrolled foaming can reduce working volume of fermenter, lower the mass and heat transfer, interfere with sensory electrodes, oxygen availability and deposition of cells leading to clogging and wetting of filters microbial infection and siphoning leading to loss of product. In case of excessive foaming, antifoams should be used. Additionally, a well defined medium lacking protein components can be used. Mechanical foam breaker can be used. Antifoams are surface active agents which reduce the surface tension in the foams and destabilize protein films. Foam production can also be controlled in very severe cases by modification of medium composition. Natural antifoams include plant oils Soya, sunflower and rapeseed, decolorized fish oil, mineral oils and tallow. The synthetic antifoams are mostly silicon oils, poly alcohols and alkylated glycols  Properties of Media An ideal anti foam is active at low concentration, inert, cheap, heat sterilizable, nontoxic to microorganism, humans and animals and should not interfere with downstream processing of the product. High molecular weight alcohols, esters, fatty acids and their derivatives example cotton seed oil, olive oil, castor oil, cord liver oil and sunflower oil, silicones, sulphonates are also some good antifoaming agents. Anti foams have been observed to reduce the oxygen transfer rates by up to 50%. A severe decrease in oxygen transfer rates due to addition of anti foams can be remedified using mechanical foam breaker. 12. Oxygen Oxygen is a very important component of the medium. Depending on the amount of oxygen required by the organism, it may be supplied in the form of air or alternately as pure oxygen, when requirements are high. The medium may influence the oxygen availability due to rapid metabolism of sugar by the culture, oxygen deficiency might be encountered leading to high oxygen demand. increase in viscosity of the medium due to various components may influence the oxygen availability. Additionally, antifoams used in the medium may reduce oxygen transfer rate.  MEDIUM OPTIMIZATION Different combinations and sequences of process conditions must be investigated to determine growth conditions. Medium optimization can be carried out by the classical method, in which one independent variable is changed while keeping all others at a certain level. Many statistical designs are currently employed for lab scale, pilot scale as well as industrial scale fermentation processes. Strategies such as Plackett- Burman Design, Response Surface Methodology, Artificial Neural Network or any other method may help in selecting the best possible conditions for a novel fermentation process.  MEDIA STERILIZATION A pure or mixed culture of defined organism is responsible for the production of fermentation product. invasion by a foreign microorganism may cause contamination leading to various problems includes: 1. Loss of productivity because of utilization of medium by the contaminant. 2. Displacement of fermentation organism by rapidly growing contaminant. 3. Difficult downstream processing of fermentation product. 4. Degradation of fermentation product. For example: Degradation of β-lactam antibiotics such as penicillin by β-lactamase producing bacteria. 5. Lysis of bacterial culture by phage contaminants. In order to avoid such loss of productivity, the entire fermentation process needs to be carried out in aseptic conditions.  MEDIA STERILIZATION This involves sterilization of the medium used, sterilization of the fermentation vessel, sterilization of all the materials such as air, acid-alkali and anti foam. Certain fermentations known as protected fermentations do not require extensive sterilization as only selective microorganisms grow because of development of certain growth conditions. Example, during brewing of beer, resins are added which inhibit the growth of many microorganisms. Moreover, growth of yeast decreases the pH of the medium, thereby decreasing the chances of growth of contaminants. Brewing does not necessarily involve sterilization. Instead boiling and disinfection can suffice.  MEDIA STERILIZATION Media can be sterilized by heat treatment, chemical treatment, filtration, radiation or ultra sonic treatment. However, in most fermentation processes, steam is used for sterilization of all heat tolerant components of medium. For heat labile components filter sterilization is the method of choice. The destruction of microorganism by moist heat takes place by coagulation of proteins. However, thermal death time of different microorganisms is different. Bacillus stearothermophillus is used as an indicator microorganism in order to access the completion of sterilization since it is one of the most heat resistant microorganisms. The bioindicator strain proposed for validation of this sterilization process is spores of Bacillus stearothermophilus for which the D-value is 1.5-2 minutes at 121 °C, using about 106 spores per indicator. MEDIA STERILIZATION Fermentation medium is not an inert mixture and various reactions taking place during the sterilization process may lead to loss of nutritive quality. The method of sterilization is chosen; the procedure must be validated for each type of product or material in order to ensure and rule out any adverse change that might take place within the product.  MEDIA STERILIZATION It is necessary to correlate temperature measurements, made with sensory devices to demonstrate heat penetration and heat distribution, with the destruction of biological indicators. Loss of nutrient quality can be because of interaction between various components of the medium leading to Maillard type browning reaction which results in discoloration of the medium and loss of nutrients. It is advisable that sugar be separately sterilized and added into the medium only after cooling. Another reaction that might lead to loss of nutrients is degradation of heat labile components of the medium such as vitamins, amino acids and proteins.  Methods of Sterilization Sterilization is necessary for the complete removal of all microorganisms (including spore-forming and non-spore-forming bacteria, viruses, fungi, and protozoa) that could contaminate fermentation products such as pharmaceuticals and constitute a health hazard. The efficacy of any sterilization process will depend on the nature of the product, the extent and type of any contamination, and the conditions under which the final product has been prepared. The requirements for Good Manufacturing Practice should be observed through all the stages of manufacture and sterilization. Classical sterilization techniques using saturated steam under pressure or hot air are the most reliable and should be used whenever possible.  Methods of Sterilization Other sterilization methods include filtration, ionizing radiation (gamma and electron-beam radiation), and gas (ethylene oxide, formaldehyde). Exposure of microorganisms to saturated steam under pressure achieves their destruction by the irreversible denaturation of enzymes and structural proteins. The temperature at which denaturation occurs varies inversely with the amount of water present Sterilization in saturated steam thus requires precise control of time, temperature, and pressure. Fats and oils may be sterilized at 121°C for 2 hours but, whenever possible, should be sterilized by filtration.  Methods of Sterilization In certain cases (e.g., thermolabile substances), sterilization may be carried out at temperatures below 121°C, provided that the chosen combination of time and temperature has been validated. Sterilization by filtration is employed mainly for thermolabile solutions. These may be sterilized by passage through sterile bacteria-retaining filters, plastic, porous ceramic, or suitable sintered glass filters, or combinations of these. Asbestos-containing filters should not be used. Appropriate measures should be taken to avoid loss of solute by adsorption onto the filter and to prevent the release of contaminants from the filter. Suitable filters will prevent the passage of microorganisms, but the filtration must be followed by an aseptic transfer of the sterilized solution to the final containers which are then immediately sealed with great care to exclude any recontamination. Membranes of not greater than 0.22 μm pore size should be used. The effectiveness of the filtration method must be validated if larger pore sizes are employed. Air is generally sterilized by passage through membrane filters.  Thermal Resistance of Microorganisms Bacterial spores are by far the most heat resistant forms of microorganisms. The thermal resistance of bacterial spores is inherently different among different species and even among strains of the same species. The most important factors in nutrient media sterilization include factors such as the pH of the nutrient medium during sterilization and the osmotic nature of the media. The presence of suspended solids also affects sterilization by physically insulating spores from heat exposure. Because bacterial spores are the most heat resistant forms of microorganisms, their germinated cells are the most frequent contaminants encountered in industrial fermentations due to improper sterilization.  Batch Sterilization The most common method of heat sterilizing nutrient media is the batch method. The medium ingredients are introduced directly into the fermenter. The fermenter and medium are sterilized by heat transferred across the jacket and/or coil surfaces from condensing steam. The medium is agitated and often steam is injected directly into the medium through the air sparger to speed up the sterilization. Injection of steam introduces some dilution to the medium, but this is taken into account during batch make-up prior to sterilization. Batch sterilization conditions are often specified as a holding period at a certain temperature. The heat effects involved during the time required to reach the desired sterilizing temperature and the time required in cooling down from this temperature are usually neglected. In large-scale equipment, the rising and falling temperature portions of the heating cycle are much longer than the constant temperature portion.  Continuous Sterilization Continuous sterilization process has become more popular over conventional batch sterilization owing to several advantages that it offers. In one type of continuous sterilization, preheated unsterile medium passes through an injection heater in which steam is introduced. This vigorous and instantaneous mixing leads to an increase in temperature immediately for effective sterilization. The temperature is maintained for the required amount of time in an insulated retention tube through which the hot medium flows.  Continuous Sterilization The hot medium passes through a heat exchanger where it is cooled to below its flash point at the same time preheating the unsterile medium. The medium cools down to the process temperature in the fermenter. Bacterial spore destruction requires high activation energy. It is, therefore, an advantage to use flash sterilization a high temperature-short exposure time sterilization operation whenever nutrient degradation occurs to the extent that it lowers the process yield. Continuous sterilization not only overcomes unfavorable nutrient destruction but has a number of operational advantages.