Lecture 15 FERMENTATION INDUSTRIES. Fermentation s Pros and Cons HISTORY OF FERMENTATION. Classic Fermentation Products I 2/22/13

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1 2/22/13 Lecture 15 FERMENTATION INDUSTRIES HISTORY OF FERMENTATION Age old applications: 1. Wine/ Beer/ Spirits 2. Cheese and Yoghurt Louis Pasteur: Hypothesised that bacteria spoils milk Suggested that milk be heated to kill bacteria Hence: pasteurization of milk Fermentation s Pros and Cons Extended shelf life of food (ex. Cheese) Eases Digestion (ex. Wild rice) New [better] flavours (ex. Chocolate) Can be unpredictable (i.e. bad bacteria win the battle) New [worse] flavours (ex. Mouldy bread tastes terrible) Classic Fermentation Products I Ethanol industrial solvent, beverage, fuel Saccharomyces cerevisiae Glycerol food and pharmaceutical use Lactobacillus delbrukki, bulgaricus Acetone-Butanol SOLVENT CLOSTRIDIUM ACETOBUTYLICUM 2,3-Butanediol synthetic rubber Bacillus polymyxa, Acetobacter aerogenes 1

2 Classic Fermentation Products II Organic Acids Acetic Acid Saccharomyces sp., Acetobacter Lactic Acid Lactobacillus delbruckii Citric Acid Aspergillus niger Itaconic Acid Aspergillus itaconicus Ethanol C in US Industrial Act denatured product was legalized in the US WWII: demands for industrial product increased use for synthetic rubber and smokeless gunpowder Whole grains, starches, sulfite liquors or saccharine materials are used as feed stocks Saccharomyces cerevesiae cannot ferment starch directly amylases must first break down starch to sugars Organic Acids Vinegar C2 French name vin + aigre Condiment and preservative Feedstock: sugary or starchy Slow Process: Orleans or French method - - mother of vinegar Generator Process: fast process, maximum air exposure Cider (apples), wine (grapes), malt (barley), sugar, glucose, spirit (grain) used for biomass Organic Acids Lactic Acid C by Scheele from milk Present in sour milk, sauerkraut, bread, muscle tissue, principal organic soil acid 1881 Commercial production by Chas. Avery, Littleton, Mass as substitute for cream of tartar Dextrose, maltose, lactose, sucrose, whey Starch, grapefruit, potatoes, molasses, beet juice PURAC for applications Dimerizes to lactide upon heating 2

3 Glycerol C3 Principal source is saponification of fats and oils Diverse use in explosives, foods, beverages, cosmetics, plastics, paints, coatings First identified by Pasteur WWI demand exceeded supply, esp. in Germany became leader in fermentation At least one integrated plant took directly to nitroglycerine Acetone- Butanol C3 and C4 True, anaerobic fermentation by Clostridium Major development during WWI: used for synthetic rubber via butadiene; critical commodity for cordite WWII production was solely by fermentation 1861 Pasteur first observed formation; 1905 Schardinger 1916 Chaim Weizmann procedure first industrial use in Canada, Terre Haute for WWI production 1926 Demand for lacquers: Peoria 96 fermentors in use, cap. 50,000 gallons each 2,3- Butanediol C4 Major interest in WWII by US and Canada Northern Regional Research Laboratory of USDA in Peoria Uses as antifreeze, butadiene synthesis 1936, Julius Nieuwland of Notre Dame with DuPont s Wallace Carothers- - DuPrene (neoprene) from it and later from petroleum sources Fermentation sources never commercialized Organic Acids Itaconic Acid C5 Resin and detergent industries Polymerizable alkene Competition with methacrylate Also produced by pyrolysis of citric acid Commercial production since 1940s Surface culture method shallow pans Submerged culture method vats Corn steep liquor: mixture of aa and sugars 3

4 Organic Acids Citric Acid C6 Made today by mold fermentation 1893: Carl Wehmer discovery 1917: Currie surface fermentation method 1945 Commercial, Landenburg Germany Molasses, cane blackstrap molasses, sugar Remarkable increase in production over past 60 years huge sales to China Originally produced directly from citrus fruit THE PROCESS Aerobic respiration release of energy from glucose or another organic substrate in the presence of Oxygen CO2, H2O, an energy produced Anaerobic respiration release of energy from glucose or another organic substrate in the absence of Oxygen Products: CO2, energy, and alcohol or various organic acids Fermentation Definition: Anaerobic respiration of food by micro organisms Types of fermentation: 1. Bacterial fermentation 2. Yeast fermentation 3. Mold and Enzyme fermentation 4

5 1. Bacterial Fermentation (4 types) 2. Yeast Fermentation a) Lactic Acid Bacteria (pickles, sauerkraut) b) Acetic Acid Bacteria (vinegar) c) Carbon Dioxide Bacteria (Edam, Gouda, Swiss) d) Proteolytic Bacteria (cocoa, chocolate) bacteria Gluà Ethyl alcohol + CO2 Best temperature: 27 degree C (warm) Other sugars will ferment (mal, suc, fru) Too much salt ruins the process When baking: follow the recipe 3. Mold and Enzyme Fermentation Enzymes in Mold can be useful: - Break down cellulose thus grains easier to chew - Add flavour and texture to cheeses (ex- blue) WINE Dates back to Middle east 3000 bc Fermentation of grapes Scientific process yet so many variables Growing years affect vintages 5

6 2/22/13 Coffee TEA Coffee beans fermented by bacteria and enzymes (2 methods): Wet Method: soaked for hours and dried Dry Method: washed then dried for 2-3 weeks CHOCOLATE 1.5 million tons cocoa produced each year Supply: W. Africa Produced: S. America Enzyme fermentation in the sun via proteolytic bacteria Bitter beans become sweeter and brown 3000 AD (at the latest)- Cultivated in China Rolled leaves begin to ferment Lets stand at 27 degree C for 2-3 hrs Types: Green, Oolong, Black FERMENTATION AROUND THE WORLD Food, drink, sauces, et cetera 6

7 INDUSTRIAL FERMENTATION Industrial fermentation uses microorganisms, typically grown on a large scale, to produce valuable commercial products or to carry out important chemical transformations. This process is commonly referred to as FERMENTATION RANGES OF FERMENTATION PROCESS Microbial cell (Biomass) Yeast Microbial enzymes Glucose isomerase Microbial metabolites Penicillin Food products Cheese, yoghurt, vinegar Vitamins B12, riboflavin Transformation reactions Steroid biotransformation FERMENTATION AEROBIC FERMENTATION Adequate aeration Aerobic Anaerobic Bioreactors- adequate supply of sterile air In addition, these fermenters may have a mechanism for stirring and mixing of the medium and cells Antibiotics, enzymes, vitamins. 7

8 ANAEROBIC FERMENTATION In anaerobic fermentation, a provision for aeration is usually not needed. Lactic acid, ethanol, wine INDUSTRIAL FERMENTORS View looking down into a 125m 3 stainless steel fermentor 8

9 INDUSTRIAL FERMENTORS INDUSTRIAL FERMENTORS m 3 Conditions in the fermenter are carefully monitored to regulate cell growth. Fermenter and all pipe work must be sterile before fermentation begins This is usually achieved by flushing the whole system with superheated steam before the production begins. Process if frequently aerobic so fermentor has to be well aerated. The aeration will be sufficient to mix many cultures If the culture is thick or sticky, additional stirring is required by a motor driven paddle called an impeller. INDUSTRIAL FERMENTORS While initially the culture may need warming to start of the process once it has started a cooling system is vital. Cooling is achieved by either a water jacket or cooling coils inside the fermenter. 9

10 FERMENTATION Fermentation could be: Batch mode Fed batch mode (continuous) BATCH FERMENTATION Most fermentations are batch processes Nutrients and the inoculum are added to the sterile fermenter and left to get on with it! Anti- foaming agent may be added. Once the desired amount of product is present in the fermenter the contents are drained off and the product is extracted. After emptying, the tank is cleaned & prepared for a new batch. CONTINUOUS FERMENTATION Some products are made by a continuous culture system. Sterile medium is added to the fermentation with a balancing withdrawal of broth for product extraction. MICROBIAL GROWTH KINETICS Microbial Growth Kinetics describe how the microbe grows in the fermenter. This information is important to determine optimal batch times. The growth of microbes in a fermenter can be broken down into four stages: Lag Phase Exponential Phase Stationary Phase Death Phase 10

11 MICROBIAL GROWTH KINETICS Lag Phase This is the first phase in the fermentation process The cells have just been injected into a new environment and they need time to adjust accordingly Cell growth is minimal in this phase. MICROBIAL GROWTH KINETICS Exponential Phase The second phase in the fermentation process The cells have adjusted to their environment and rapid growth takes place Cell growth rate is highest in this phase MICROBIAL GROWTH KINETICS Exponential Phase (Continued) At some point the cell growth rate will level off and become constant The most likely cause of this leveling off is substrate limited inhibition Substrate limited inhibition means that the microbes do not have enough nutrients in the medium to continue multiplying. MICROBIAL GROWTH KINETICS Stationary phase This is the third phase in the fermentation process The cell growth rate has leveled off and become constant The number of cells multiplying equals the number of cells dying 11

12 MICROBIAL GROWTH KINETICS Death phase The fourth phase in the fermentation process The number of cells dying is greater than the number of cells multiplying The cause of the death phase is usually that the cells have consumed most of the nutrients in the medium and there is not enough left for sustainability MEDIA FOR INDUSTRIAL FERMENTATIONS The media is the feed solution It must contain the essential nutrients needed for the microbe to grow Factors of consideration when choosing media - Quality consistence and availability - Ensure there are no problems with Media Prep or other aspects of production process Ex. Cane molasses, beet molasses, cereal grains STERILIZATION Sterilizing the feed solution is essential because the media cannot contain foreign microbes because this could severely hinder the growth of the production microbe Most popular method is heat sterilization of the feed solution THE DEVELOPMENT OF INOCULA FOR INDUSTRIAL FERMENTATIONS The inoculum is the starter culture that is injected into the fermenter It must be of sufficient size for optimal growth kinetics Since the production fermenter in industrial fermentations is so large, the inoculum volume has to be quite large - A seed fermenter is usually required to produce the inoculum volume - The seed fermenter s purpose is not to produce product but to prepare inoculum 12

13 DESIGN OF A FERMENTER Factors to consider when designing a fermenter Aseptic and regulatory capability, long- term reliability Adequate aeration and agitation Low power consumption Temperature and ph controls Sampling facilities 14 L fermenter shown is a copyright of New Brunswick Scientific INSTRUMENTATION AND CONTROL The success of a fermentation process is highly dependent on environmental factors The fermenter needs to be able to control such factors as temperature, ph, and dissolved oxygen levels AERATION AND AGITATION Most industrial fermentations are aerobic processes meaning that the production microbe requires oxygen to grow The oxygen demand is met by sparging air through the fermentation vessel and using an agitator increase the amount of dissolved oxygen INDUSTRIAL ETHANOL PRODUCTION The Philippines Biofuels Act 2006 requires oil companies to use biofuels in all "liquid fuels for motors and engines sold in the Philippines." All gasoline sold in the country must contain at least 5 percent ethanol by February 2009, and by 2011, the mandated blend can go up to 10 percent. 13

14 Ethanol can enter the environment as emissions from its manufacture, use as a solvent and chemical intermediate, and release in fermentation and alcoholic beverage preparation. It naturally occurs as a plant volatile, microbial degradation product of animal wastes, and in natural fermentation of carbohydrates. Produced naturally from a wide range of microbiological processes (by fungi, bacteria, etc), and possibly some plants. When spilled on land it is apt to volatilize, biodegrade, and leach into the ground water, but no data on the rates of these processes could be found. Its fate in ground water is unknown. ethanol is a clean- burning, high- octane fuel that is produced from renewable sources. at its most basic, ethanol is grain alcohol, produced from crops such as corn. 14

15 STEPS TO MAKING ETHANOL In 2005, 97 ethanol plants in 21 states produced a record billion gallons of ethanol A bushel of corn weighs 56 pounds and will produce at least 2.8 gallons of ethanol, 17 pounds of distillers grain & 18 Pounds of CO 2 Ethanol is produced using the following process: Wheat or corn kernels are ground in a hammer mill to expose the starch. The ground grain is mixed with water, cooked briefly and enzymes are added to convert the starch to sugar using a chemical reaction called hydrolysis. Yeast is added to ferment the sugars to ethanol. The ethanol is separated from the mixture by distillation and the water is removed from the mixture using dehydration. MAKING ETHANOL ETHANOL PRODUCTION FLOW CHART The unprocessed product, in fact, is a lot like beer: 8 percent alcohol and 92 percent water. Not something that's going to burn in a car engine. To make a usable fuel, all but 0.5 percent of the water must be removed. This is done by a series of distillation and chemical extractions that use even more energy than was used to grow the corn. And that doesn't count the diesel fuel needed to ship corn to the ethanol plant or ethanol to the pump. In theory, all of these energy costs should make ethanol uneconomical to produce. 15

16 THE MAJOR STEPS IN THE DRY MILL PROCESS ARE: Ethanol can be made by a dry mill process or a wet mill process. Most of the ethanol in the U.S. is made using the dry mill method. In the dry mill process, the starch portion of the corn is fermented into sugar then to alcohol. 1. Milling. The feedstock passes through a hammer mill which grinds it into a fine powder called meal. 2. Liquefaction. The meal is mixed with water and alpha- amylase, then passed through cookers where the starch is liquefied. Heat is applied at this stage to enable liquefaction. Cookers with a high temperature stage ( degrees Celsius) and a lower temperature holding period (95 degrees Celsius) are used. High temperatures reduce bacteria levels in the mash. 3. Saccharification. The mash from the cookers is cooled and the secondary enzyme (gluco-amylase) is added to convert the liquefied starch to fermentable sugars (dextrose). 4. Fermentation. Yeast is added to the mash to ferment the sugars to ethanol and carbon dioxide. Using a continuous process, the fermenting mash is allowed to flow through several fermenters until it is fully fermented and leaves the final tank. In a batch process, the mash stays in one fermenter for about 48 hours before the distillation process is started. 16

17 5. Distillation. The fermented mash, now called beer, contains about 10% alcohol plus all the non-fermentable solids from the corn and yeast cells. The mash is pumped to the continuous flow, multi-column distillation system where the alcohol is removed from the solids and the water. The alcohol leaves the top of the final column at about 96% strength, and the residue mash, called stillage, is transferred from the base of the column to the co-product processing area. 6. Dehydration. The alcohol from the top of the column passes through a dehydration system where the remaining water will be removed. Most ethanol plants use a molecular sieve to capture the last bit of water in the ethanol. The alcohol product at this stage is called anhydrous ethanol (pure, without water) and is approximately 200 proof. 7. Denaturing. Ethanol that will be used for fuel must be denatured, or made unfit for human consumption, with a small amount of gasoline (2-5%). This is done at the ethanol plant. 8. Co-Products. There are two main co-products created in the production of ethanol: distillers grain and carbon dioxide. Distillers grain, used wet or dry, is a highly nutritious livestock feed. Carbon dioxide is given off in great quantities during fermentation and many ethanol plants collect, compress, and sell it for use in other industries. 17

18 Distillers grain can be fed to livestock wet or dry. Dried distillers grain (DDG) is the most common variety. Drying the distillers grain increases its shelf life and improves its ability to be transported over longer distances. If a consistent nearby market can be secured, ethanol producers can supply the feed as wet distillers grain (WDG). The wet form is not as easily transportable, but the cost of drying the product is removed. The personal care products industry is one of the largest users of industrial ethanol, or ethyl alcohol. Check the labels hairspray, mouthwash, aftershave, cologne, and perfume all contain large amounts of alcohol by volume. Ethanol is also used in many deodorants, lotions, hand sanitizers, soaps, and shampoos. Ethanol melts at C, boils at 78.5 C, and has a density of g/ml at 20 C. Pure, 100% ethanol is not generally used as a motor fuel; instead, a percentage of ethanol is combined with unleaded gasoline. This is beneficial because the ethanol: Its low freezing point has made it useful as the fluid in thermometers for temperatures below 40 C, the freezing point of mercury, and for other low- temperature purposes. decreases the fuel's cost increases the fuel's octane rating decreases gasoline's harmful emissions 18

19 BLENDING WITH GASOLINE E10-10% ethanol and 90% unleaded gasoline Any amount of ethanol can be combined with gasoline, but the most common blends are: E10 is approved for use in any make or model of vehicle sold in the U.S. Many automakers recommend its use because of its high performance, clean- burning characteristics. In 2004, about one- third of America's gasoline was blended with ethanol, most in this 10% variety. E85-85% ethanol and 15% unleaded gasoline E85 is an alternative fuel for use in flexible fuel vehicles (FFVs). There are currently more than 4 million FFVs on America's roads today, and automakers are rolling out more each year. In conjunction with more flexible fuel vehicles, more E85 pumps are being installed across the country. When E85 is not availible, these FFVs can operate on straight gasoline or any ethanol blend up to 85%. ETHANOL Physical properties: Colorless liquid. Pleasant alcoholic odor detectable at 49 to 716 ppm. Miscible with water and most organic solvents. Melting Point ( C): Boiling Point ( C): 78.3 Specific Gravity: Vapor Density:

20 COMMERCIAL PRODUCTION OF BEER Essential Ingredients of Beer Malted Barley Hops Yeast Water Not required, but frequently found ingredient Starch adjuncts Corn and rice starches MAKING BEER: A THREE STEP PROCESS Malting Brewing Fermentation MALTING Takes place in malt houses Occasionally in a brewery (Coors) Controlled germination of barley Moisture Temperature Carbon dioxide Goal Produce enzymes useful for brewing 20

21 Malting Soaking the grain Allow for controlled germination Maximum enzyme production Minimum enzymatic activity and plant growth Kiln drying Stop germination Stabilize malted barley Impart color and flavor Brewing Functions: Enzymatic conversion of starch to maltose, proteins to amino acids Extraction of hop flavors and aromatic compounds Sterilize maltose/aa/hop flavor solution Brewing Milling of malted barley Careful cracking of malted barley Shatter endosperm Keep husk in large pieces Adding water Controlled temperature for enzymatic action Mash Tun The mash tun is a vessel in which the milled malted barley is mixed with water And the enzymes are allowed to degrade the starches and proteins into Substrates that the yeast can utilize during fermentation 21

22 These photos show the milled Malted barley being mixed with Warm water. The enzymes Convert the starch to maltose and The proteins to amino acids creating What is known as sweet wort. Mash Lautering (filtering) The sweet wort Is separted from The spent barley By a filtration step Known as Lautering. The Barley husks serve As the primary Filtering material. Here, the remaining Spent grains are Being removed from The sweet wort With this screen. Mash Tun with used Mash Scraping out the used mash These are the spent malt that acted as a filtering bed for the sweet wort. 22

23 Used mash heading towards feedlot Sweet Wort Kettle Sweet Wort Bring to boil Add hops Extract flavors (bitter acids) and aromatic compounds Sterilizes hopped wort Fermentation Tanks After the yeast is added to the hopped wort, fermentation of the maltose to Ethanol occurs in these tanks. 23

24 Adding yeast to the fermenter Blow- off hoses on fermentation tanks Fermentation produces both ethanol and carbon dioxide. The carbon Dioxide is allowed to vent out through these blow-off hoses whose ends Are immersed in a tank of water, producing an air-lock and preventing Oxygen from entering the fermentation tanks. Cleaning fermentation tanks Cleanliness is critical in producing Quality beer. Microbial contamination Can result in off flavors and aromas. Grape Wine Next 24

25 Grape Wine Introduction Grapes are cultivated in many countries of the world. India produces only about 2.77 per cent of the total world production. However, in productivity India stood first with tonnes /Ha. Grape is one of the most perishable fruits and during the process of distribution and marketing, substantial losses are incurred which ranges from a slight loss of quality to total spoilage. Processing is an alternative method of preservation of this fruit for long durations. Fermentation of juices for the preparation of alcoholic beverages is being practiced for the last many centuries. Wine is a fermented beverages produced End from grape Previous and has a large Next acceptability across End Grape Wine Alcoholic Beverages These are the beverages which are prepared after alcoholic fermentation of sugars by yeast, contain varying amounts of ethyl alcohol (5-42%), and are consumed directly or after dilution in water. Wine Product made by alcoholic fermentation of grapes or grape juice unless otherwise specified, by yeast (Saccharomyces cerevisiae and a subsequent ageing process. Alcohol content is %, but may be as low as 7 %. Previous Next Grape Wine Grape Wine Definitions Fortified wines Contain added alcohol/ distillate of wine (brandy). Alcohol content of fortified wines is 19-21% Table Wines Low alcohol content and little or no sugar Source: Sharma (2010) Dessert Wines End Previous These are fortified sweet wines. Next End Previous Next 25

26 Red table wine Grape Wine Grape Wine Nutritional value of red table wine Nutritional value per 100 g (3.5 oz) Energy 355 kj (85 kcal) Carbohydrates 2.6 g - Sugars 0.6 g Fat 0.0 g Protein 0.1 g Alcohol 10.6 g Process for preparation of wine 10.6 g alcohol is 13%vol., 100 g wine is approximately 100 ml (3.4 fl oz.), Sugar and alcohol content can vary. Source: USDA Nutrient Database End Previous Next End Previous Next Grape Wine Grape Wine Steps in wine making Determination of alcohol content? Record starting specific gravity of must (S1), Record finished specific gravity (S2) Calculate by using the formula S1 S2 Alcohol (%) = 7.36 Source: winetutorial4.html End Previous Next End Previous Next 26

27 Grape Wine What happens during fermentation? Yeast Saccharomyces cerevisae, which causes fermentation, is a single cell organism that converts the sugar in the fruit to alcohol and carbon dioxide. The carbon dioxide escapes into the air and what is left is wine. Wine Making Problems Grape Wine The major cause of wine failures is a lack of proper sterilization procedures and practices. Important Problems encountered are: 1. Corkiness Symptoms: An unpleasant flavor in wine Possible Causes: a. Bottling with a defective cork b. Not a complete seal and the outside air allowed to enter into the bottle c. Inferior cork End Previous Next 2. Soapiness Symptoms: End Previous Soapy taste in your favorite wine Next Grape Wine Grape Wine Wine Making Problems 3. Woody Symptoms: Aroma of wood in your wine. Possible Causes: a. Over-soaking of corks b. Over-aging with oak chips 4. Flowers of Wine Symptoms: A white film or skin that forms on the surface of wines Possible Causes: a. Undue exposure to air Wine Making Problems 5. Stuck Fermentation Symptoms: Wine has stopped fermenting before reaching a specific gravity of Possible Causes: a. cold temperatures / too hot b. Bad yeast, using a yeast that's reached its limit of alcohol tolerance c. Too much sugar d. Insufficient nutrients or acids e. Insufficient oxygen f. Too much carbon dioxide End Previous Next End Remedy: Add a high powered yeast Previous Next 27

28 Grape Wine Let us sum up Wine is a product made by alcoholic fermentation of grapes or grape juice unless otherwise specified, by yeast (Saccharomyces cerevisiae and subsequent ageing process. Alcohol content is %, but may be as low as 7 %. Fortified wines contain added alcohol or distillate of wine while sweet wines consist of unfermented sugar. Ethyl alcohol and CO 2 are produced during fermentation Alcohol content is calculated by dividing the difference between initial and final specific gravity by End Previous Next Corkiness, soapiness and flower of wines are problems of wine making. PRODUCTION OF ANTIBIOTICS ANTIBIOTICS SOME ANTIBIOTICS PRODUCED BY MICROORGANISMS Of all the microbial products manufactured commercially, antibiotics are the most important. Antibiotics are chemical substances produced by microorganisms to kill other microorganisms. They are used in the treatment of infectious diseases. Antibiotic Cephalosporin Chloramphenicol Erythromycin Griseofulvin Penicillin Streptomycin Tetracycline Gentamicin Producing microorganism Cephalosporium acrimonium Streptomyces venezuelae Streptomyces erythreus Penicillium griseofulvin Penicillium chrysogenum Streptomyces griseus Streptomyces aureofaciens Micromonospora purpurea 28

29 Thanks to work by Alexander Fleming ( ), Howard Florey ( ) and Ernst Chain ( ), penicillin was first produced on a large scale for human use in At this time, the development of a pill that could reliably kill bacteria was a remarkable development and many lives were saved during World War II because this medication was available. PRODUCTION OF PENICILLIN During world war II- importance realized, as penicillin had been used to treat many wounded soldiers. A. Fleming E. Chain H. Florey A tale by A. Fleming In 1928, Sir Alexander Fleming, a Scottish biologist, observed that Penicillium notatum, a common mold, had destroyed staphylococcus bacteria in culture. A tale by A. Fleming He took a sample of the mold from the contaminated plate. He found that it was from the Penicillium family, later specified as Penicillium notatum. Fleming presented his findings in 1929, but they raised little interest. He published a report on penicillin and its potential uses in the British Journal of Experimental Pathology. 29

30 MOA OF PENICILLIN All penicillin like antibiotics inhibit synthesis of peptidoglycan, an essential part of the cell wall. They do not interfere with the synthesis of other intracellular components. These antibiotics do not affect human cells because human cells do not have cell walls. Spectrum of Activity Penicillins are active against Gram positive bacteria Some members (e.g. amoxicillin) are also effective against Gram negative bacteria but not Pseudomonas aeruginosa PRODUCTION OF PENICILLIN Penicillin was the first important commercial product produced by an aerobic, submerged fermentation First antibiotic to have been manufacture in bulk. Used as input material for some semi synthetic antibiotics. When penicillin was first made at the end of the second world war using the fungus Penicillium notatum, the process made 1 mg dm - 3. Today, using a different species (P. chrysogenum) and a better extraction procedures the yield is 50 g dm - 3. There is a constant search to improve the 30

31 The yield of penicillin can be increased by: Improvement in composition of the medium Isolation of better penicillin producing mold sp. Penicillium chrysogenum which grow better in huge deep fermentation tank Development of submerged culture technique for cultivation of mold in large Primary and Secondary Metabolites Primary metabolites are produced during active cell growth, and secondary metabolites are produced near the onset of stationary phase. Commercial Production Of Penicillin Like all antibiotics, penicillin is a secondary metabolite, so is only produced in the stationary phase. INDUSTRIAL PRODUCTION OF ANTIBIOTIC- PENICILLIN The industrial production of penicillin was broadly classified in to two processes namely, Upstream processing Downstream processing 31

32 UPSTREAM PROCESSING Upstream processing encompasses any technology that leads to the synthesis of a product. Upstream includes the exploration, development and production. DOWNSTREAM PROCESSING The extraction and purification of a biotechnological product from fermentation is referred to as downstream processing. UPSTREAM PROCESSING INOCULUM PREPARATION The medium is designed to provide the organism with all the nutrients that it requires. Inoculation method- submerged technique RAW MATERIALS CARBON SOURCES: Lactose acts as a very satisfactory carbon compound, provided that is used in a concentration of 6%. Others such as glucose & sucrose may be used. NITROGEN SOURCES: Corn steep liquor (CSL) Ammonium sulphate and ammonium acetate can be used as nitrogenous sources. MINERAL SOURCES: Elements namely potassium, phosphorus, magnesium, sulphur, zinc and copper are essential for penicillin production. Some of these are applied by corn steep liquor. Calcium can be added in the form of chalk to counter the natural acidity of CSL PAA- precursor 32

33 FERMENTATION PROCESS The medium is inoculated with a suspension of conidia of Penicillium chrysogenum. The medium is constantly aerated and agitated, and the mould grows throughout as pellets. After about seven days, growth is complete, the ph rises to 8.0 or above, STAGES IN DOWNSTREAM PROCESSING Removal of cells The first step in product recovery is the separation of whole cells and other insoluble ingredients from the culture broth by technique such as filtration and centrifugation. ISOLATION OF BENZYL PENICILLIN The PH is adjusted to with the help of phosphoric or sulphuric acids. In aqueous solution at low PH values there is a partition coefficient in favor of certain organic solvents such as butyl acetate. This step has to be carried out quickly for penicillin is very unstable at low PH values. Antibiotic is then extracted back into an aqueous buffer at a PH of 7.5, the partition coefficient now being strongly in favor of the aqueous phase. The resulting aqueous solution is again acidified & re- extracted with an organic solvent. These shifts between the water and solvent help in the The treatment of the crude penicillin extract varies according to the objective, but involves the formation of an appropriate penicillin salt. The solvent extract recovered in the previous stage is carefully extracted back with aqueous sodium hydroxide. This is followed by charcoal treatment to eliminate pyrogens and by sterilization. Pure metal salts of penicillin can be safely sterilized by dry heat, if desired. Thereafter, the aqueous solution of penicillin is subjected to crystallization. 33

34 FURTHER PROCESSING The main stages of Penicillin production are: For parental use, the antibiotic is packed in sterile vials as a powder or suspension. For oral use, it is tabletted usually now with a film coating. Searching tests (ex: for purity, potency) are performed on the appreciable number of random samples of the finished product. It must satisfy fully all the strict government standards before being marketed 34

35 PRODUCTION OF PENICILLIN V Phenoxy methyl penicillin Addition of different Acyl groups to the medium. Phenoxyacetic acid as precursor instead of phenyl acetic acid. 35