Fermentation (Biology) PDF

Definition ========== - **Fermentation** is a metabolic process that produces chemical changes in organic substrates through the action of enzymes. - The chemical breakdown of a substance by bacteria, yeasts, or other microorganisms, typically involving effervescence and the giving off...

Definition ========== - **Fermentation** is a metabolic process that produces chemical changes in organic substrates through the action of enzymes. - The chemical breakdown of a substance by bacteria, yeasts, or other microorganisms, typically involving effervescence and the giving off of heat. - The study of fermentation and its practical uses is named **zymology.** - If no oxygen is available, cells can obtain energy through the process of **anaerobic respiration.** - A common **anaerobic process is fermentation.** - Fermentation is not an efficient process and results in the formation of far fewer ATP molecules than aerobic respiration. Anaerobic Fermentation ====================== During fermentation, an organic electron acceptor (such as pyruvate or acetaldehyde) reacts with NADH to form NAD+ The main function of fermentation is to convert NADH back into the coenzyme NAD+ so that it can be used again for glycolysis. Fermentation yields net 2 ATP per glucose molecule (through glycolysis) The two fermentations most commonly used by humans to produce commercial foods are: a) Ethanol fermentation (used in beer and bread) B) Lactic acid fermentation (used to flavor and preserve dairy and vegetables). A) Ethanol fermentation:- ========================= - Ethanol fermentation is typically performed by yeast, which is a unicellular fungus. - Glucose → 2 ethanol + 2 carbon dioxide + 2ATP\ **C~6~H~12~O~6~ → 2 C~2~H~5~OH + 2 CO~2~** A diagram of a cycle Description automatically generated B) Lactic Acid Fermentation:- ============================= - This type of fermentation also occurs in muscle cells to produce ATP when the oxygen supply has been depleted during strenuous exercise and aerobic respiration is not possible. - Lactic acid that builds up in the tissue causes a burning, painful sensation. - glucose → 2 lactic acid\ **C~6~H~12~O~6~ → 2 C~3~H~6~O~3~**  Aerobic fermentation ==================== - Fermentation conducted in the presence of fresh air, as in crock, vat, tank is called aerobic fermentation. - Aerobic conditions are necessary **for yeast to reproduce rapidly.** - Bioreactors used for aerobic fermentation provide adequate supply of sterile air into the medium. - In addition, these fermenters may have a mechanism for stirring and mixing of the medium and cells. - **These fermenters may be either of** - i**) stirred tank type in which mechanical motor driven stirrers are provided** - ii\) **of air lift type in which no mechanical stirrers are used and the agitation is achieved by the air bubbles generated by the air supply**. Pharmaceutical production of antibiotics ======================================== **MANUFACTURING OF ANTIBIOTICS:** Antibiotics may be manufactured by fermentation process(most antibiotics) or by fermentation followed by chemical synthesis(ampicillin) or by synthesis (chloramphenicol). Fermentation process involves growing the organism in a nutrient broth under optimum conditions, followed by extraction of the antibiotic or its precursor from the growth medium. Originally the mold was cultivated on the surface of a liquid medium in numerous small flasks. Nowadays, antibiotics are made by deep (submerged) culture in which mold is encouraged to grow in a large tank of medium. **Choice of organism:** The organism should give high yields of antibiotic from economic sources of nutrients and should not produce excessive amounts of substances chemically related to the antibiotic as separation may be difficult. High yielding natural strain are often genetically unstable and also unable to resist attack by biological agents such as bacteriophages. More reliable strains are obtained by subjecting cultures to mutagens such as x-rays, UV light. **Choice of medium:** The basis of most fermentation media is corn liquor, the fluid obtained from the maize prior to starch extraction. It provides a variety of nutrients and has good buffering capacity For most fermentations, additional sugar (lactose or glucose) is needed as a source of energy for mycelium production. When nitrogen demand is high. Soya bean or peanut meal may be added. Buffering salts may be required when the antibiotic is pH sensitive. **Cultivation of the organism:** A slope is inoculated with spores from the master culture. After incubation the growth is checked for freedom from contamination and variation A spore suspension is transferred to culture flasks, which are incubated to obtain a large crop of spores The flasks are shaken during incubation to ensure that enough air is available to aerobic mould. The suspension in the flask is used to inoculate a 2000 dm3 tank, known as seed vessel. After further incubation, sterile compressed air is used to blow the contents through sterile pipes into huge fermentation tanks (fermenters) The growth of mold is allowed to continue until a satisfactory level of antibiotic has been reached. **Aeration:** The molds used in antibiotic production are aerobic They will not grow well in a tank of medium unless they are supplied with plenty of oxygen This is achieved by pumping in large volumes of air through an inlet (sparger) at the bottom of fermenter. Air is introduced as streams of bubbles that are further broken down and distributed by a high powered stirrer (agitator). Because of their protein content, culture media froth readily. This can be reduced by using antifoaming agents eg silicone fluids, vegetable oils or octadecanol. **Sterilization:** The culture medium, vessels, pipe lines and air must be sterile to prevent contamination by extraneous organisms that might reduce yields and produce unwanted metabolism In case of penicillin, penicillinase-producing organism would rapidly destroy the antibiotic. The fermenter, and pipe lines are usually sterilized by steam under pressure Sterilization of the medium may be carried out in the fermenter or in a separate vessel In fermenter, high pressure steam is passed through heating coils inside the tank Contamination during the process is prevented by careful design A continuous method of air sterilization is appropriate A compressor of high capacity and capable of high compression is often used The air is passed through a sterilization filter immediately before entry to the tank **Temperature Control:** Part of energy produced by carbohydrate break down during the metabolism of mold is liberated as heat This must be controlled because high temperatures inhibit the mold and destroy the antibiotic An optimum temperature (about 24°c for penicillin) is maintained by running cold water over the surface of fermenter **Isolation of product:** After fermentation, the mycelium is removed by a rotary filter The antibiotic is obtained from the filtrate followed by removal of unwanted substances and extraction of the active material with solvent system. The crude product of extraction is purified in variety of ways eg, recrystallization. Final drying is done under vacuum and may involve freeze drying. **Production of antibiotics**: Antibiotics can be classified in several ways. The major points of antibiotics on microorganism include: Inhibition of cell wall synthesis, (penicillins, cephalosporins) Damage to the cytoplasmic membrane Inhibition of protein synthesis (chloramphenicol, aminoglycoside, tetracyclines). Inhibition of nucleic acid synthesis (rifamycin) Inhibition of specific enzyme systems Penicillins =========== Penicillin is produced by Penicillium notatum, Pencillium chrysogenum and other species of moulds. It was isolated by Fleming in 1929 as contaminant on culture plate. Penicillins are a class of beta-lactam antibiotics of related structure with slightly different properties and activities These are potassium, sodium or other metallic salts of derivatives of 6-amino penicillanic acid (6-apa). All penicillins have a fused beta-lactam-thiazolidine ring with different side chains They are produced by fermentation followed by chemical synthesis In pure crystalline forms the penicillins are colorless. 6-apa is obtained by one of the following methods and required penicillin is synthesized from it by acylation of the 6-apa 1\. Fermentation and synthesis :- direct method, indirect method. 2\. Fermentation alone A diagram of a structure Description automatically generated  2. Cephalosporins ================= Cephalosporins are group of antibiotics produced by a species of marine fungus, Cephalosporium acremonium. The nucleus of cephalosporins resemble that of penicillin as is called 7- aminocephalosporanic acid. It contains a beta lactam ring having an acylated amino group. Semisynthetic cephalosporins have been manufactured commercially for therapeutic use. These have a broader antibacterial spectrum, are longer lasting in the body, and are resistant to many beta-lactamases (cephalosporinases) They are effective against gram positive and gram negative bacteria. They are effective therapeutically and have a low toxicity. The mode of action is that of inhibition of the cross linking transpeptidase. Cephalosporins inhibit cell wall synthesis in same way as penicillins. They are more widely used than any other b- lactam antibiotics. They are bactericidal to growing cells.  The first -generation cephalosporins have a relatively narrow spectrum of activity, primarily against gram-positive bacteria and certain gram negative rods. These are variably absorbed from the intestines. Example cephalothin and cephalexin. Second generation cephalosporins have a more extended gram-negative spectrum. Examples are the intravenous drug cefamandole and the oral drug cefaclor. The third generation drugs, such as ceftazidime, cefotaxime and ceftriaxone, are the most active against gram-negative bacteria, including some pseudomonads, but most must be injected. An oral third-generation cephalosporin is cefixime. The fourth -generation drugs, such as cefepime, require injections but have the most extended spectrum of activity. These have improved activity against the gram-negative bacteria involved with urinary tract infections. 3. Gentamycin ============= In 1963, Weinstein isolated gentamicin, in mixture of water soluble basic antibiotic from the species of Micromonospora ie, from Micromonospora purpurea and M.echinospora. It forms salts with mineral and strong organic acids, the sulphate (official) being used clinically. Gentamicin, an aminoglycoside, is administered for serious infections of the urinary tract caused by gram-negative bacteria. It is especially useful against Pseudomonas infections. Pseudomonads are a major problem for persons suffering from cystic fibrosis It is active against aerobacter, Escherichia, klebsiella, salmonella and some proteus species. It is not absorbed orally and must be given by intramuscular injection as sulphate. 4. Erythromycin =============== The best-known macrolide in clinical use is erythromycin. It was discovered in 1952 and is prepared by the fermentation of soil organism Streptomyces erythreus. Its mode of action is the inhibition of protein synthesis. Erythromycin is not able to penetrate the cell walls of most gram-negative bacilli. Its spectrum of activity is therefore similar to that of penicillin G, and it is a frequent alternative drug to penicillin. Because it can be administered orally it is recommended for use against gram-positive bacteria in patients with penicillin allergy. **Applications of Erythromycin** It is an antibiotic of low toxicity with an antibacterial spectrum similar to benzyl penicillin It is active against many gram positive and some gram negative bacteria It is administered orally 5. Tetracyclines ================ Tetracyclines structure is based on four benzenoid rings. These include chlortetracycline, oxytetracycline, tetracycline, doxycycline and minocycline. They are produced by the submerged culture of Streptomyces viridifaciens (tetracycline), Streptomyces aureofaciens (chlortetracycline) and Streptomyces rimosus (oxytetracycline) Demethylchlortetracycline is the most stable, gives the highest blood levels and is the most active against Pseudomonas aeruginosa and Proteus vulgaris. These are broad spectrum antibiotics, acting on a wide range of organisms including gram negative bacteria, spirochaetes, mycoplasma, rickettsia etc. They are usually given orally but in emergencies may be administered by slow I/v infusion or by I/m injection A diagram of a chemical structure Description automatically generated **5. Tetracyclines**: A side effect of prolonged treatment with tetracyclines is over growth of yeasts and molds in the alimentary tract Because the natural bacterial flora of the gut is depressed by the antibiotic, the fungi find conditions more favorable for their growth causing gastrointestinal upsets and often leading to superinfections, particularly by the fungus Candida albicans They also interfere with the growth of teeth and should not be given to pregnant women or children under 12, unless no other drug will control the infection 6. Rifamycins ============= These drugs are structurally related to the macrolides and inhibit the synthesis of mRNA. The best-known derivative of the rifamycin family of antibiotics is rifampin. In 1959, rifampicin B was isolated from a culture of Streptomyces mediterranei 7. Griseofulvin =============== It was isolated in 1939 from mycelium of Penicillium griseofulvum It is now prepared commercially by deep fermentation of Penicillium patulum It is an antifungal antibiotic Its activity is restricted to fungi with chitinous cell walls. It accumulates in the keratinous layers of dermis on which fungi exists and exerts fungistatic action Griseofulvin interferes with mitosis by binding to microtubules. There is little evidence of acute toxicity. It is very active against fungal infections of the skin, hair, and nails, such as ringworm and athlete's foot. Griseofulvin BP 125mg **Processing** The manufacturing technology for antibiotics can be divided into:- i. Upstream processes ii. Down stream processes **Upstream process** is defined as the microbial growth required to produce antibiotic or other biomolecules and involves a series of events including the selection of cell line, culture media, growth parameters, and process optimization to achieve optimal conditions for cell growth and antibiotic production.The main goal of the upstream process is the transformation of substrates into the desired metabolic products. This requires well-controlled conditions and involves the use of large-scale bioreactors. Several factors should be considered such as the type of process (batch, fed-batch, continuous, etc.) temperature, pH, and oxygen supply control, sterilization of materials and equipment employed, and maintenance of the environment to ensure it is free of contaminating microorganisms. **Downstream process: Isolation and purification of antibiotics** The aim of downstream process should be to deliver the highest yield of the purest product at the shortest time/cost. Downstream processing includes all steps required to purify a product from cell culture broth to final purified product. It involves multiple steps to capture the target biomolecule and to remove host cell related impurities, process related impurities (e.g., buffers, antifoam, etc.) and product related impurities (e.g., aggregates, fragments, clipped species, etc.). Downstream processing usually encompasses three main stages, namely: i. initial recovery (extraction or isolation) ii. (purification (removal of most contaminants), and iii. polishing (removal of specified contaminants and unwanted forms of the target biomolecule that may have formed during isolation and purification).