ASSIGNMENT REPORT ON INDUSTRIAL PRODUCTION OF PENICILLIN

BIOPROCESS TECHNOLOGY SUBMITED TO

SUJEET SINGH

SUBMITTED BYSHRISHTI SINGH SENGAR B.TECH(BIOTECH) VII SEM SECTION-C ROLL NO-50 ENROLLMENT-A7104108176

ANTIBIOTICS
The word antibiotic comes from the Greek anti meaning 'against' and bios meaning 'life' (a bacterium is a life form).' Antibiotics are also known as antibacterial, and they are drugs used to treat infections caused by bacteria. Bacteria are tiny organisms that can sometimes cause illness to humans and animals. The singular word for bacteria is bacterium. Such illnesses as tuberculosis, salmonella, syphilis and some forms of meningitis are caused by bacteria. Some bacteria are not harmful, while others are good for us. Before bacteria can multiply and cause symptoms our immune system can usually destroy them. We have special white blood cells that attack harmful bacteria. Even if symptoms do occur, our immune system can usually cope and fight off the infection. There are occasions, however, when it is all too much and our bodies need some help - from antibiotics. The first antibiotic was penicillin. Such penicillin-related antibiotics as ampicillin, amoxicillin and benzylpenicilllin are widely used today to treat a variety of infections - these antibiotics have been around for a long time. There are several different types of modern antibiotics and they are only available with a doctor's prescription in industrialized countries. n antibiotic is given for the treatment of an infection caused by bacteria. Antibiotics target microorganisms such as bacteria, fungi and parasites. However, they are not effective against viruses. If you have an infection it is important to know whether it is caused by bacteria or a virus. Most upper respiratory tract infections, such as the common cold and sore throats are generally caused by viruses - antibiotics do not work against these viruses. If antibiotics are overused or used incorrectly there is a chance that the bacteria will become resistant - the antibiotic becomes less effective against that type of bacterium. A broad-spectrum antibiotic can be used to treat a wide range of infections. A narrow-spectrum antibiotic is only effective against a few types of bacteria. There are antibiotics that attack aerobic bacteria, while others work against anaerobic bacteria. Aerobic bacteria need oxygen, while anaerobic bacteria don't.

MODE OF ACTION OF ANTIBIOTICSAntimicrobial agents or drugs are either bactericidal (they kill microbes directly) or bacteriostatic (they prevent microbes from growing). In bacteriostatic, the host own defense such as phagosytosis and antibody production usually destroy the microorganism. The major modes of actions are given below: Cell Wall Synthesis Inhibitors: Bacteria contain murein or peptidoglycan that is highly essential in maintaining the cell wall structure. Cell wall synthesis inhibitors such as beta-lactams, cephalosporin and glycopeptides block the ability of microorganisms to synthesize their cell wall by inhibiting the synthesis of peptidoglycan. Penicillin and other antibiotics prevent the synthesis of intact peptidoglycan. Since penicillin targets the synthesis process, only actively growing cells are affected by these antibiotics. Interfering with Protein Synthesis: These classes of antibiotics inhibit the protein synthesis machinery in the cell. Some examples include: Tetracycline ( react with the 30s portion of the 70s prokaryotic ribosome), Chloramphenicol (reacts with the 50s portion of the 70s prokaryotic ribosome and inhibit the formation of peptide bond in the growing polypeptide chain.) Amino glycosides (interfere with the initial steps of protein synthesis by changing the shape of the 30s portion of the 70s prokaryotic ribosome)

Cell Membrane Inhibitors: Antibiotics such as polymyxins disrupt the integrity and structure of cell membranes, thereby killing them. These set of antibiotics are mostly effective on gram negative bacteria because these are the bacteria that contain a definite cell membrane. Effect on Nucleic Acids: Polymyxin B causes disruption of the plasma membrane by attaching to the phospholipids of the membrane. Amphotericin B is effective against a considerable range of fungal diseases.

Inhibition of nucleic acid synthesis: Antibiotics such as rifampin and the quinolones interfere with the process of DNA replication and transcription in microorganisms. They interfere with the mammalian DNA and RNA as well.

PENICILLIN- Penicillin: C16H18N2O5S

Penicillium fungi.[1] They include penicillin G, procaine penicillin, benzathine penicillin, and penicillin V. Penicillin antibiotics are historically significant because they are the first drugs that were effective against many previously serious diseases such as syphilis and infections caused by staphylococci and streptococci. Penicillin are still widely used today, though many types of bacteria are now resistant. All penicillin are beta-lactam antibiotics and are used in the treatment of bacterial infections caused by susceptible, usually Gram Penicillin (sometimes abbreviated PCN or pen) is a group of antibiotics derived from -positive, organisms.

Mechanism of action
Bacteria constantly remodel their peptidoglycan cell walls, simultaneously building and breaking down portions of the cell wall as they grow and divide. -Lactam antibiotics inhibit the formation of peptidoglycan cross-links in the bacterial cell wall, but have no direct effect on cell wall degradation. The -lactam moiety (functional group) of penicillin binds to the enzyme (DDtranspeptidase) that links the peptidoglycan molecules in bacteria. The enzymes that hydrolyze the peptidoglycan cross-links continue to function, which weakens the cell wall of the bacterium (in other words, the antibiotic causes cytolysis or death due to osmotic pressure). In addition, the build-up of peptidoglycan precursors triggers the activation of bacterial cell wall hydrolyses and autolysins, which further digest the bacteria's existing peptidoglycan. This imbalance between cell wall production and degradation is responsible for the rapid cell-killing action of this class of drugs, even in the absence of cell division. In addition, the relatively small size of the penicillin molecule allows it to penetrate deeply into the cell wall, affecting its entire depth. This is in contrast to the other major class of cell wall synthesis inhibiting antibiotics, the glycopeptides antibiotics (which include vancomycin and teicoplanin). Penicillin and other -lactam antibiotics act by inhibiting penicillin-binding proteins, which normally catalyze cross-linking of bacterial cell walls.

STRUCTURE OF PENICILLIN-

molecular weight 313[6] to 334[7][8] g/mol

ACTION OF PENICILLIINASE-

NATURAL PENICILLINExamples:Penicillin G (Benzyl penicillin) Procaine Penicillin G Penicillin V (phenoxymethyl penicillin) Benzathine The -lactam ring, especially the bond between N and CO reacts with the enzymes involved in the synthesis of the bacterial cell wall and produces the inhibitory effect. Natural penicillin has some disadvantages. Many staphylococci and certain other species develop resistance to Penicillin G. This is due to the fact that they produce the enzyme penicillinase (lactamase) which hydrolyses the lactam bond and converts penicillin to penicilloic acid (which lacks the antibacterial activity).

SEMI-SYNTHETIC PENICILLINThese compounds consist of the basic Penicillin structure, but have been purposefully modified chemically by removing the acyl group to leave 6-aminopenicillanic acid and then adding acyl groups that produce new properties. These modern semi-synthetic penicillin have various specific properties such as resistance to stomach acids so that they can be taken orally, a degree of resistance to penicillinase (or -lactamase) (a penicillin-destroying enzyme produced by some Bacteria) and an extended range of activity against some Gram-negative bacteria.

Semi-synthetic Penicillin [Methicillin; Flucloxacillin]

Prepared by adding different side chains to the penicillin nucleus (A-B) Penicillinase-resistant - effective against penicillin resistant bacteria

Used in severe infections due to penicillinase-producing staphylococci e.g. septicemia,

meningitis, pneumonia

Broad spectrum semi-synthetic Penicillin [Ampicillin; Amoxicillin; Carbenicillin; Ticarcillin; Piperacillin]

Are semi synthetic penicillin with a wider antibacterial spectrum Effective against both gram positive & gram negative bacteria Ampicillin & Amoxicillin are not effective against penicillinase producing bacteria Carbenicillin and Ticarcillin are uniquely effective against pseudomonas Piperacillin has a wider spectrum of activity

INDUSTRIAL PRODUCTION OF PENICILLIN

In Peoria, Illinois a blue-green mould was found growing on a moldy cantaloupe in a Market. This mould was identified as Penicillium chrysogenum and produced approximately 200 times as much penicillin than what Floreys team was working with (Penicillium notatum). Scientists began to try to increase the amount of penicillin produced by P. chrysogenum, by irradiating it with X-rays and UV rays in order to induce mutations of this species. They succeeded and developed a mutant that produced 1000 times the amount of penicillin than Flemings original culture. At the same time scientists began to grow the mould in the first deep tank fermenters.

INOCULUM DEVELOPMENTInoculum development begins on solid media and subsequently liquid media are used. Inoculate 100 ml medium in 500 ml flask with spores of Penicillium chrysogenum strains and incubate at 25oC by keeping on a rotator shaker. After 4 days transfer the content of the flask to another flask containing the 2 liters medium and incubate for 2 days. The inoculum is prepared usually in the form of spore suspension, which is transferred into the fermentor by placing it in a metal vessel that is attached to the fermentor.

FERMENTORWhen a reactor is being designed for a specific purpose there are a number of important parameters that will greatly affect the reactors process performance. 1. Reactor Size: How large does the reactor need to be in order to achieve optimum rates of production? 2. Reactor Configuration: How will the reactor be configured? For example should impellers be used to exert mechanical agitation (stirred tank) or will an air-jet system is used for mixing (bubble column)?

3. Mode of operation: How will substrate be added? Will it be batch fed or continuously fed? 4. Conditions inside the reactor: What temperature and pH should the reactor be maintained at? How will contamination be avoided? And how will these conditions are controlled?

Antibiotics are produced in stainless steel fermentor 200-500 m3 volume. Inoculums of strongly growing hyphae are added. Agitation is provided by the impeller batch or fed batch mode. The fermentor is stem sterilized and loaded with sterilized growth medium but air lift system is also used. Oxygen is a vital component for aerobic metabolism. The sparger delivers the oxygen. One problem is the formation of foam and the impellers break the foam. An external cooling jacket is there to control the temperature. The pH requires adjustment from time to time, to neutralize the ammonia produced by the fungi. Temperature is set at first to give the maximum growth rate and then altered to favour the protein synthesis. Steam is used to keep the reactor free from contamination. Aside from what is described above, there are a few specific characteristics of Penicillin that must be considered when attempting fermentation: Most penicillin form filamentous broths that are pseudo plastic (nonNewtonian) in nature. This means they can be difficult to mix due to their high (and not constant) viscosity. Also the increasing viscosity of the broth can hinder oxygen transfer. Which leads to the next point? Penicillin is an aerobic organism; therefore the rate of oxygen supply is critical to the fermentation. Thus, the reactor must have an efficient oxygen supply system. The optimum pH for penicillin growth is 6.5. Thus the reactor must maintain pH efficiently (this is frequently done by addition of NaOH Strain Stability problems do exist and careful strain maintenance is required. Biomass doubling is about 6h.

PRODUCTION MEDIUMThe production medium contains the following compound: Lactose 3-4% Glucose or Molasses- 10% Corn steep liquor- 4% CaCO3- 1% KH2PO4- 0.4% Phenyl acetic acid- 0.5 0.8% and Antifoam- 0.25-0.5%

PRODUCTION PROCESSProduction fermenters are agitated tank 200-250 m3 in volume made of stainless steel. Mechanical agitation is provided at the rate of 100-300 rpm. Temperature is controlled around 25-28oC by using cooling coils. Antifoam is added to reduce foam formation. Dissolved oxygen is controlled at >2mg/L and pH at 6.5. Three phases of growth can be differentiated during cultivation of Penicillium chrysogenum.

First phase- In this phase, growth of the mycelium occurs, yield of antibiotic is quite low.
Lactic acid present in corn steep liquor is utilized at the maximum rate by the microorganisms. Lactose is used slowly. Ammonia is liberated into the medium resulting into the rise in pH.

Second phase- There was intense synthesis of penicillin in this phase, due to rapid
consumption of lactose and ammonia nitrogen. The mycelia mass increases, the pH remain unchanged.

Third phase- The concentration of antibiotic decreases in the medium. The autolysis of
mycelium starts, liberation of ammonia and slight rise in pH.

RECOVERYOnce the formation is completed the broth is separated from fungal mycelium and processed by adsorption, precipitation and crystallization to yield the final product. Penicillin is recovered by solvent extraction at an acidic pH at temperature below 10oC. The solid can be removed by ultra filteration. Mycelium can be treated, dried and used as soil conditioner. The penicillin rich solvent can be treated with activated carbon, to remove pigments and other impurities and the penicillin recovered as the potassium or the sodium salt by adding potassium or sodium acetate to the solvent. Further impurities can be removed by washing the recovered salt with a dry solvent such as isopropanol or n-butanol.

PENICILLIN DISADVANTAGES Acid liability- most of these drugs are destroyed by gastric acid. Short duration of action- because of this short half life, the penicillin must be
administered at short interval, usually after every 4 hrs. Lack of activity against most Gram-negative organisms. Drug hypersensitivity- about 10% of population has allergy. Painful if given intramuscularly.

PENICILLIN ADVANTAGES Relatively nontoxic Have excellent tissue penetration. Efficacious in the treatment of infections. Bactericidal against sensitive strains. SIDE EFFECTS Allergic or hypersensitive reactions are thought to be the most frequent side effect. The most serious side effect is Anaphylaxis that can cause skin rashes, itching etc. Mild diarrhea, vomiting, headache, sore mouth or tongue, white patches on mouth or
tongue. On rare cases can cause severe abdominal stomach cramps, pain or bloody diarrhea.