10 General Microbiology BIO306 Assist. Prof. Betl AKCESME

10 General Microbiology BIO306 Assist. Prof. Betl AKCESME 1 Putting Microorganisms to Work Industrial Products and the Microorganisms That Make Them Industrial microbiologu vs biotechnology ??? Big amounts vs small amnounts!!!

Natural enhancment vs genetic engineering!! Production and Scale I. Industrial Products and the Microorganisms That Make Them Industrial microbiology Uses microorganisms, typically grown on a large scale, to produce products or carry out chemical transformation processes such as production of pharmaceuticals, food additives, enzymes, and chemicals were developed Major organisms used are fungi and genus Streptomyces Classical genetic methods are used to select for high-yielding microbial

variants goal is to increase the yield of the product to the point of being economically profitable. Industrial Products and the Microorganisms That Make Them 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 ( environment and human) Amenable to genetic manipulation Industrial Products and the Microorganisms

That Make Them Microbial products of industrial interest include Microbial cells Enzymes Antibiotics, steroids, alkaloids Food additives Commodity chemicals Inexpensive chemicals produced in bulk Include ethanol, citric acid, and many others Major Products of industrial microbiology Production and Scale Primary metabolite Produced during exponential growth

Example: alcohol Ethanol is a product of the fermentative metabolism of yeast or certain bacteria can grow only if they produce energy, ethanol grows in parallel with growth Production and Scale Secondary metabolites Produced during end of the growth or stationary phase Not essential for growth Formation depends on growth conditions Produced as a group of related compounds Often significantly overproduced by spore-forming microbes during sporulation, production is linked to the sporulation process itself.

Antibiotics: Virtually all antibiotics, for example, are produced by either fungi or spore-forming prokaryotes . Primary metabolite Cells Alcohol Sugar Time Penicillin, sugar, or cell number

Alcohol, sugar, or cell number Formation of alcohol by yeastan example of a primary metabolite. Penicillin production by the mold Penicillium chrysogenum an example of a secondary metabolite. Note that penicillin is not made until after the exponential phase. Secondary metabolite

Sugar Cells Penicillin Time Contrast between production of primary and secondary Production and Scale Secondary metabolites are often large organic molecules that require a large number of specific enzymatic steps for production Synthesis of tetracycline requires at least 72 separate enzymatic steps

Starting materials arise from major biosynthetic pathways Production and Scale Fermentor is where the microbiology process takes place. Any large-scale reaction is referred to as a fermentation!! whether or not it is, biochemically speaking, a fermentation. Most are aerobic processes Fermentors vary in size from 5 to 500,000 liters Aerobic and anaerobic fermentors Large-scale fermentors are almost always stainless steel Impellers and spargers supply oxygen Fermentor sizes for various industrial

fermantations Large-scale fermenters cylinder, closed at the top and bottom, into which various pipes and valves have been fitted Sterilization of the culture medium and removal of heat are vital for successful operation A critical part of the fermentor is the aeration system a high density of microbial cells, there is a tremendous oxygen demand by

the culture an aerator, called a sparger, and a stirring device, called an impeller monitored in real time for Motor pH Steam Sterile seal pH controller Acidbase

reservoir and pump Viewing port Filter Exhaust Impeller (mixing) External cooling water out

Cooling jacket Culture broth External cooling water in Sparger (highpressure air for aeration) Steam in Sterile air Valve

Harvest WHY necessary to alter the conditions in the fermentor?? It is often necessary to alter the conditions in the fermentor as the fermentation progresses. Computers are used to process environmental data as the fermentation proceeds and are programmed to respond by signaling for nutrient additions, increases in the rate of cooling water, impeller speed or sparger pressure, or changes in pH or other parameters, at just the right time to maintain high product yield. 15

The inside of an industrial fermentor, showing the impeller and internal heating and cooling coils. Production and Scale Industrial Fermentors Closely monitored during production run Growth and product formation must be measured Environmental factors must be controlled and altered as needed Including temperature, pH, cell mass, nutrients, and

product concentration Data on the process must be obtained in real time Production and Scale Scale-up from laboratory to commercial fermentor The transfer of a process from a small laboratory scale to large-scale commercial equipment Major task of the biochemical engineer Requires knowledge of the biology of producing organism and the physics of fermentor design and operation Many challenges in scale-up arise from aeration and mixing high cell densities, and this leads to high oxygen

SCALE UP Flask laboratory fermentor(1-10 liter) pilot plant(300-3000 l) commercial fermentor(10000-500000 l) In all stages of scale-up, aeration is the key variable that is closely monitored; as scale-up proceeds, oxygen dynamics are carefully measured to determine how increases in volume affect oxygen demand in the fermentation. 19 A bank of small research fermentors used in process

development. The fermentors are the glass vessels with the stainless steel tops. The small plastic bottles collect overflow. (b) A large bank of outdoor industrial-scale fermentors (each 240 m3) used in commercial production of alcohol in Japan. Drugs, Other Chemicals, and Enzymes Antibiotics: Isolation, Yield, and Purification Industrial Production of Penicillins and Tetracyclines Vitamins and Amino Acids Enzymes as Industrial Products

Antibiotics: Isolation, Yield, and Purification Antibiotics Compounds that kill or inhibit the growth of other microbes Typically secondary metabolites Most antibiotics in clinical use are produced by filamentous fungi or actinobacteria Modern drug discovery relies heavily on computer modeling of drugtarget interactions - Before discovered by laboratory screening Microbes are obtained from nature in pure culture Assayed for products that inhibit growth of test bacteria Some antibiotics produced commercially

b) EFB, endospore-forming bacterium; F, fungus; A, actinomycete. Isolation of antibiotic producers. (a) Isolation using media selective for Streptomyces and identification of antibiotic producers by screening using an indicator organism. Photo: Most of the colonies are Streptomyces species, and some are producing antibiotics as shown by zones of growth inhibition of the indicator organism (Staphylococcus aureus).

24 Antibiotics: Isolation, Yield, and Purification Cross-streak method: Method of testing an organism for its antibiotic spectrum of activity. Used to test new microbial isolates for antibiotic production Most isolates produce known antibiotics Most antibiotics fail toxicity and therapeutic tests in animals Time and cost of developing a new antibiotic is approximately 15 years and $1 billion Involves clinical trials and U.S. FDA approval Antibiotic purification and extraction often involves elaborate methods

Method of testing an organism for its antibiotic spectrum of activity. (SECREENING) The producer was streaked across one-third of the plate and the plate incubated. After good growth was obtained, the five species of test bacteria were streaked perpendicular to the producing organism, and the plate was further incubated.

The failure of several species to grow near the producing organism indicates that it produced an antibiotic active against these bacteria. Photo: Test organisms streaked vertically (left to right) include Escherichia coli, Bacillus subtilis, S. aureus, Klebsiella pneumoniae, Mycobacterium smegmatis. 26

Once a new antibiotic has been characterized and proven medically effective and nontoxic in tests on experimental animals, it is ready for clinical trials on humans. If the new drug proves clinically effective and passes toxicity and other tests, it is given FDA approval and is ready to be produced commercially. 27 Yield and purification One of the major tasks of the industrial microbiologist is to isolate high-yielding strains mutagenizing the wild-type organism to obtain mutant derivatives that are so altered that they overproduce the

antibiotic of interest The next challenge is to purify the antibiotic specifically and efficiently, and elaborate methods for extraction and purification of the antibiotic are often necessary 28 Industrial Production of Penicillins and Tetracyclines Penicillins are -lactam antibiotics are produced by fungi of the genera Penicillium and Aspergillus and by certain prokaryotes. Commercial penicillin is produced in the United States using high-yielding strains of

the mold Penicillium chrysogenum. Natural and biosynthetic penicillins Semisynthetic penicillins Broad spectrum of activity Industrial Production of Penicillins and Tetracyclines Penicillin G is produced in fermantors of 40.000-200.000 liters. Highly aerobic process. Typical secondary metabolite, very little is produced during the growth phase. Can be extended several days by additions (carbon, nitrogen) High levels of glucose repress penicillin production but high levels of lactose do not, so lactose is added At the end of the production phase, the cells are removed by filtration and the

pH is made acidic. The penicillin can then be extracted and concentrated into an organic solvent and, finally, crystallized. 30 Add precursor I Penicillin fermentation Biosynthetic penicillin I

Add precursor II Chemical or enzymatic Add precursor treatment III of penicillin G Biosynthetic penicillin II Biosynthetic penicillin III

Natural penicillins (for example, penicillin G) 6-Aminopenicillanic acid Add side chains chemically Semisynthetic penicillin (for example, ampicillin, amoxycillin, methicillin) Industrial production of penicillins. The -lactam ring is circled in red. The

normal fermentation leads to the natural penicillins. If specific precursors are added during the fermentation, various biosynthetic penicillins are formed. Semisynthetic penicillins are produced by chemically adding a specific side chain to the 6-aminopenicillanic acid nucleus on the R group shown in purple. Semisynthetic penicillins are the most widely prescribed of all the penicillins today, primarily because of their broad spectrum of activity and ability to be taken orally. penicillin fermentation with Penicillium chrysogenum. Kinetics of the penicillin fermentation with Penicillium chrysogenum. Note that penicillin is produced as cells are entering the stationary

phase, when most of the carbon and nitrogen has been exhausted. Nutrient feedings keep penicillin production high over several days. Biomass (g/liter), carbohydrate, ammonia, penicillin (g/liter 10)) Figure :Kinetics of the Glucose feeding 10)0)

Nitrogen feeding 90) 80) Penicillin 70) 60) 50) 40) 30)

Cells 20) Lactose 10) Ammonia 0) 20) 40) 60) 80) 10)0)120)140) Fermentation time (h)

Industrial Production of Penicillins and Tetracyclines Biosynthesis of tetracycline has a large number of enzymatic steps More than 72 intermediates More than 300 genes involved! (studies of Streptomyces aureofaciens) Complex biosynthetic regulation Glucose and phosphate repress the synthesis, need for low phosphate concentration. Inoculum (spores on agar slant or in sterile soil)

Agar plates Spores as inoculum some key regulatory signals are known and are accounted for in the production scheme

Shake flask 24 h Prefermentor Medium 2% Meat extract; 0).0)5% asparagine; 1% glucose; 0).5% K2HPO4; 1.3% agar Growth in optimal medium 2% Corn steep liquor; 3% sucrose; 0).5% CaCO3 Medium mimics production

medium Same as for shake culture Production scheme for chlortetracycl ine using Streptomyces aureofaciens. 1924 h pH 5.26.2 1% Sucrose; 1% corn steep liquor; 0).2% (NH4)2HPO4; Production 0).1% CaCO3;

medium, no 60)65 h glucose, low pH 5.86.0) 0).0)25% MgSO4 phosphate 0).0)0)5% ZnSO4 0).0)0)0)33% and each of CuSO4, MnCl2 Antibiotic purification from broth after cell removal Fermentor Chlortetracycline

Glucose is used to grow the inoculum, but not for commercial production. Vitamins and Amino Acids Production of vitamins is second only to antibiotics in terms of total pharmaceutical sales Most of them are made commercially by chemical synthesis. Vitamin B12 produced exclusively by microorganisms 10,000 tons per year.

Deficiency results in pernicious anemia low production of red blood cells and nervous system disorders Cobalt is present in B12 vitamin are greatly increased by addition of small amounts of cobalt to the culture medium B12 Vitamins produced by microorganisms on an industrial sc Vitamins and Amino Acids Amino acids Used as food additives in the food industry Used as nutritional supplements in nutraceutical

industry Used as starting materials in the chemical industry Examples include Glutamic acid (monosodium glutamate, MSG) Over one million tons of this amino acid are produced annually by the gram-positive bacterium Amino acids used in the food industry Enzymes as Industrial Products Exoenzymes Enzymes that are excreted into the medium instead of being held within the cell; they are extracellular Can digest insoluble polymers such as cellulose,

protein, and starch Enzymes are useful as industrial catalysts Produce only one stereoisomer High substrate specificity Microbial enzymes and their applications Enzymes as Industrial Products Enzymes are produced from fungi and bacteria Bacterial proteases are used in laundry detergents (can also contain amylases, lipases, and reductases) Isolated from alkaliphilic bacteria Usually acttive pH between 9-10 (alkaline pH of laundary detergant)

Amylases and glucoamylases are also commercially important Produce high-fructose syrup Production of glucose from starch then converted by a second enzyme, glucose isomerase, to fructose, which is a much sweeter sugar than glucose. Widely used in the food industry to sweeten soft drinks, juices, and many other products. Worldwide production of high-fructose syrups is over 10 billion kilograms per year. Enzymes as Industrial Products Extremozymes

Enzymes that function at some environmental extreme Produced by extremophiles This feature makes these enzymes of interest to a variety of biotechnical applications Taq polymerase Cold-tolerant Extremozymes Food processor cold-wash detergent Acid-tolerant Extremozymes Catalyses for the synthesis of compounds in acidic solution

additives for animal feed Alkali-tolerant Extremozymes Detergent (protease, lipase etc.) Dye Salt-tolerant Extremozymes Oil exploitation 43 Thermostability of the enzyme pullulanase from Pyrococcus woesei, a hyperthermophile whose growth temperature optimum is 100C. At 110C the enzyme denatures, but calcium improves the heat stability of this enzyme dramatically.

Percent enzyme activity remaining 10)0) 10) Pullulanase Starch oligosaccharides 90)C 10)0)C 110)C

110)C plus Ca2 1 1 2 Time (h) 3 4 An acid-tolerant enzyme mixture used as a feed supplement for poultry. The enzymes function in the birds stomach to digest fibrous materials in

the feed, thereby improving the nutritional value of the feed and promoting more rapid growth. Enzymes as Industrial Products Immobilized enzymes are attached to a solid surface makes it easier to carry out the enzymatic reaction under large-scale continuous flow conditions, also helps stabilize the enzyme to retard denaturation. Three ways to immobilize an enzyme Bonding of enzyme to a carrier Cross-linking of enzyme molecules Enzyme inclusion

Procedures for the immobilization of enzymes. Carrier-bound Cross-linked enzyme enzyme Enzyme inclusion in Enzyme inclusion fibrous polymers in microcapsules Biofuels A biofuel is a type of fuel whose energy is derived from biological carbon fixation.

Fermentation of recently grown plant materials rather than being of ancient origin (fossils). Biodisels made from vegatable oils. Algal fuels- from green algea. Gasohol-produced by adding ethanol to gasoline. 47 Biofuels Ethanol Biofuels Ethanol is a major industrial commodity chemical In USA most ethanol is obtained from by yeast fermentation of glucose obtained from cornstarch.

Various yeast have been used but most ethanol is produced by Saccharomyces. The increased demand for corn as a biofuel feedstock has driven up the price of human foods and livestock feeds. Sugar cane, whey, sugar beets, and even wood chips and waste paper are used as feedstocks for the fermentation Cellulosic materials, the cellulose must first be treated to release glucose, which is then fermented to alcohol. 49 Petroleum Biofuels

Production of butanol Synthesis of petroleum from green algae during growth the colonial green alga Botryococcus braunii excretes long-chain (C30C36) hydrocarbons that have the consistency of crude oil. 50 http://science.howstuffworks.com/environmental/greenscience/algae-biodiesel3.htm 51 (a)A bioethanol production plant in Nebraska (USA). In the plant, glucose obtained from corn starch is fermented by

Saccharomyces cerevisiae to ethanol plus CO2. The large tank in the left foreground is the ethanol storage tank, and the tanks and pipes in the background are for distilling ethanol from the fermentation broth. (b)Switchgrass, a promising feedstock for bioethanol production. The cellulose from this rapidly growing plant can be treated to yield glucose that can then be fermented to ethanol or butano 52 (c) The petroleum-producing colonial green alga, Botryococcus braunii. Note the excreted oil droplets that appear as bubbles along the margin of the cells. 54

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