1.

Introduction
Fermentation: The term fermentation is derived from the Latin word fevere,to boil thus describing the appearance of the action of yeast on fruit extract or malted grain the boiling appearance is due to the production of CO2 bubbles caused by the anaerobic catabolism of the sugar present in the extract. However fermentation has come to have different meaning to biochemists and to industrial microbiologist. Its biochemical meaning relates to the generation of energy by catabolism of organic compound, whereas its meaning in industrial microbiology tends to be much broader. Antibiotics: In common usage, an antibiotic (from the Ancient Greek: anti-"against", and bios- "life") is a substance or compound that kills bacteria or inhibit their growth. Antibiotics belong to the broader group of antimicrobial compounds, used to treat infections caused by microorganisms, including fungi and protozoa. The term "antibiotic" was coined by Selman Waksman in 1942 to describe any substance produced by a microorganism that is antagonistic to the growth of other microorganisms in high dilution. This original definition excluded naturally occurring substances that kill bacteria but are not produced by microorganisms (such as gastric juice and hydrogen peroxide) and also excluded synthetic antibacterial compounds such as the sulfonamides. Many antibiotics are relatively small molecules with a molecular weight less than 2000 Da. Antibiotics are commonly classified based on their mechanism of action, chemical structure, or spectrum of activity. Most antibiotics target bacterial functions or growth processes. Antibiotics that target the bacterial cell wall (penicillin, cephalosporin), or cell membrane (polymixins), or interfere with essential bacterial enzymes (quinolones, sulfonamides) are usually bactericidal in nature. Those that target protein synthesis, such as the aminoglycosides, macrolides, and tetracyclines, are usually bacteriostatic. Further categorization is based on their target specificity: "Narrow-spectrum" antibiotics target particular types of bacteria, such as Gram-negative or Gram-positive bacteria, whereas broad-spectrum antibiotics affect a wide range of bacteria. In the last few years, three new classes of antibiotics have been brought into clinical use. 1

Beta-lactam antibiotics: The beta-lactam group of antibiotics is by far the largest group of antibacterial agents used in clinical medicine. It includes the first true antibiotic to be discovered and brought into clinical practice, and all the natural and semisynthetic derivatives with their wide-range of properties and clinical applications. With a few notable exceptions, they remain a cornerstone of antibiotic therapy for the spectrum of serious systemic infection. Structurally, all are based upon the four member nitrogen containing beta-lactam ring that gives these agents their antibacterial activity. They can be divided into four groups - penicillins, cephalosporins, carbapenems and monobactams - on the basis of the molecular structures surrounding and supporting this active site, A fifth group in clinical use are the beta-lactamase inhibitors that do not have intrinsic antibacterial activity. Penicillin: The penicillin may be divided into four main groups on the basis of their spectra of antibacterial activity. All the developments and manipulations of the penicillin nucleus have had the aim of extending the spectrum of the agents against gram-negative bacteria or increasing their resistance to beta-lactamase enzymes, or both. All of this "improved" penicillin retains activity against bacteria such as hemolytic streptococci and clostridia, which remain sensitive to benzyl penicillin. However, it must be remembered that all the alterations to the basic penicillin structure necessary to achieve these particular advantages have been at the expense of intrinsic activity against sensitive organisms. Early penicillin, Benzyl penicillin (penicillin G), the original member of the group, remains the most active antibacterial agent against sensitive bacteria. It is the drug of choice for serious infections with beta-hemolytic streptococci, alpha-hemolytic streptococci (combined with an aminoglycoside in sub-acute bacterial endocarditis), the pneumococcus, the meningococcus and clostridia other than Clostridium. Phenoxymethyl penicillin (penicillin V) remains useful when oral treatment of streptococcal infections is adequate.

Fig: 1 Basic 3D structure of Penicillin G

Fig: 2 Penicillin G structure As shown in the diagram, penicillin is not a single compound but a group of closely related compounds, all with the same basic ring-like structure (a lactam) derived from two amino acids (valine and cysteine) via a tripeptide intermediate. The third amino acid of this tripeptide is replaced by an acyl group (R in the diagram below) and the nature of this acyl group produces specific properties on different types of penicillin. 3

History of penicillin: Penicillin was the first naturally occurring antibiotic discovered. There are now more than 60 antibiotics, which are substances that are produced by microbes and that fight bacteria, fungi and other microbes harmful to humans the word means against (anti) life (bio). Penicillin is obtained in a number of forms from Penicillium moulds. In 1928, while working in St. Marys Hospital in London, bacteriologist Alexander Fleming was conducting research on the flu. He had been searching for antibacterial agents, influenced by his wartime experience. He had witnessed the deaths of many soldiers that died, not from the wounds they received during combat, but from secondary infections of those wounds. While he was on holidays, a bit of blue-green mould had fallen into a discarded culture plate containing Staphylococcus aureus, forming a clear patch in the surrounding area. From this he could conclude that the mould was producing an antibiotic substance. He named the antibiotic penicillin, after the Penicillium notatum mould that produced it and 1929; he published the results of his investigations, noting that his discovery might have therapeutic value if it could be produced in quantity. Unfortunately it couldnt, and it would be 10 years before another significant leap forward for penicillin would occur. In 1938 Dr. Howard W. Florey came across Flemings paper on penicillin (which oddly had languished in obscurity). While Flemings lab was poorly equipped with no staff support, Floreys lab was well equipped and staffed with a team of scientists at Oxford University that included Dr. Ernst B. Chain. It was Chain who began extracting penicillin into a purified and powerful antibiotic. At first penicillin was made using old dairy equipment and after a great deal of effort, enough was extracted for experimentation to begin. Eight white mice were inoculated with deadly Streptococcus germs, followed by injection of penicillin in half the mice. All of the untreated mice died the next day while the treated mice all recovered. Now it was time for the first human test. Albert Alexander, a 48-year-old London policeman had developed septicaemia as a result of a small cut on his face. When treated with penicillin Alexander began to recover within the day. However Floreys team didnt have enough of the drug to see the patient through to a full recovery and he later re-lapsed and died. However by 1941, it was acknowledged that penicillin was indeed a worthwhile drug and could save thousands of lives. In the same year Florey travelled to the United States (which at the time was still neutral) to continue his work with penicillin. 4

Because the United States intended to enter into World War II in another few months the penicillin project, which became declared a war project, was given top priority (and funding). Florey and his team were able to use beerbrewing technology to produce the huge amounts of the mouldy liquor needed for penicillin production. This underwent a slow purification process to produce the large amounts of clinically usable penicillin that became available for military use in early 1940s. In Peoria, Illinois a blue-green mould was found growing on a mouldy cantaloupe in a market. This mould was identified as Penicillium chrysogenum and produced approximately 200times 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. By late 1943, mass production of the drug had commenced and by the end of the war, many companies were manufacturing the drug, including the Merck, Squibb and Pfizer. In 1945 Fleming was awarded the Nobel Prize in Physiology and Medicine along with Florey and Chain. In the coming years, strains and techniques were improved upon and Penicillin saved tens of thousands of lives. However, it all began with a bit of blue-green mould falling onto a discarded culture plate.

2. Penicillin production and Recovery

Penicillin is produced in large scale by fermentation. The success of the production depends upon proper selection of strain that we are going to use, raw material selection, optimizing process parameters, monitoring and maintaining the parameters, product recovery, recovery of solvents and also effective effluent treatment. Sequential steps in Penicillin G production in terms of department: Microbiology department Quality assurance Pilot scale plant Fermentation plant (Production) Quality control Product recovery plant

2.1 Microbiology department:

In microbiology department two main processes are carried over: Strain/colony selection Shake flask analysis

2.1.1 Strain/colony selection:

In strain/colony selection the colony of organism which has a high productivity and genetic stability is selected mainly by analyzing zone of inhibition assays. The next step after isolation of microorganisms is their screening. A set of highly selective procedures, which allows the detection and isolation of microorganisms producing the desired metabolite, constitutes primary screening. Ideally, primary screening should be rapid, inexpensive, predictive, specific but effective for a broad range of compounds and applicable on a large scale.

Criteria for the choice of organism: 1. Nutritional characteristics of the organism. 2. Optimum temperature of the organism. 3. Suitability of the organism to the type of process to be used. 4. Stability of the microorganism. 5. Productivity of the organism. 6. Ease of product recovery.

Fig: 3 Zone of inhibition assay 2.1.2 Shake flask analysis: Culture flasks (usually Erlenmeyer) of 250 or 500 ml or larger are used for growing microorganisms; these are shaken, generally, by a gyratory shaker at 200-250 R.P.M. Shake cultures are usually applied to aerobic processes. At this stage, the spores will begin to revive and form vegetative cells. Temperature is normally maintained at 23-28C and pH at ~6.5, although there may be some changes made to facilitate optimum growth. In general, filamentous microorganisms are grown for the production of secondary metabolites, which begins 1-3 days after inoculation and continues 3-4 days thereafter. In all such cases, the shake cultures are used for: Strain improvement Determination of the optimum conditions for the fermentation process. In many industrial processes, they are also used for the initial stages of inoculum development. 7

Shake cultures are a convenient method of growing microorganisms in submerged cultures under aerobic conditions created by shaking; it is a small scale equivalent of stirred tank bioreactor. and, Both the devices are extensively used with filamentous micro-organisms often, with other types of micro-organisms as well.

Usually, complex media are used for shake flask cultures, but the objective is to devise a synthetic medium for the fermentation process. Studies on inoculum size, temperature, agitation, nutrition are initially done using these cultures to monitor their influences on growth and product formation.

2.2 Quality assurance:

Quality assurance, or QA for short, refers to a program for the systematic monitoring and evaluation of the various aspects of a project, service, or facility to ensure that standards of quality are being met. Quality assurance, in its broadest sense, is any action taken to prevent quality problems from occurring. In practice, this means devising systems for carrying out tasks which directly affect product quality. Two key principles characterize QA: "fit for purpose" (the product should be suitable for the intended purpose) and "right first time" (mistakes should be eliminated). QA includes regulation of the quality of raw materials, assemblies, products and components; services related to production; and management, production and inspection processes. Even goods with low prices can be considered quality items if they meet a market need. QA is more than just testing the quality of aspects of a product, service or facility, it analyzes the quality to make sure it conforms to specific requirements and comply with established plans. SOPs are formulated and maintained for maintaining the quality. Steps for Quality Assurance Process Test previous article Plan to improve Design to include improvements and requirements Manufacture with improvements Review new item and improvements Test new item

Raw materials : Quality of the raw materials used in the fermentation is the deciding factor of the productivity; hence the quality analysis of each and every raw material should be carried over. When performing any kind of fermentation, the selection of media is of critical importance to the overall performance of the fermentation. The aim of the media is to provide all the elements required for the synthesis of cell materials and the formation of the desired product. At the same time, the media must provide a favourable environment for the culture in question (an example of this would be control of pH by addition of calcium carbonate or inorganic phosphates). As well as this it must remain cost effective. Typically, all microorganisms require Carbon, Hydrogen, Oxygen, Sulphur and Nitrogen for cell growth and cell maintenance. In many cases, microorganisms require small amounts of trace elements such as Cu, Mn and Co (this will frequently depend on the water source as most water sources contain small amounts of the elements) or growth factors such as vitamins or amino acids as well. Each and every raw material is given a reference number which is called M.I.N (Material inward number). Raw materials used in the fermentation of Penicillin-G are: Acetic acid, Activated carbon, Ammonium sulphate, n-butyl acetate, n-butyl alcohol, Calcium carbonate, Corn steep liquor, Citric acid monohydrate, Copper sulphate pentahydrate, Corrugated box, Demulsifier, Dextrose monohydrate, Dextrose syrup, Ferrous sulphate heptahydrate, Filter aid, Formaldehyde, Maize oil, Magnesium sulphate heptahydrate, Monopotassium phosphate, Pen G first crystal label, Maganese sulphate, Phenyl acetic acid, Polypropylene glycol, Polythene bag, Potassium carbonate, Sodium phenyl acetate solution, Sodium hydroxide solution, Sodium sulphate, Sucrose, Sulphuric acid, Zinc sulphate, Ferrous sulphate and High maltose corn syrup.

Documentation :
Every analysis result should be recorded and a clear documentation should be prepared. All analysis should be performed as per the S.O.P prepared and they should be recorded having a reference number for future reference.

2.3 Pilot scale plant:

Pilot scale reactor will be similar in design to the bench-top reactor except it will have a size of about 100-1000 litres. The aim here is to examine the effect of scale-up on the culture. At this stage, hopefully growth will continue as before, however, there are often sudden changes or loss in performance. This can be due to changes in the morphology of the culture (remember Penicillium chrysogenum is a filamentous fungi and hence pseudoplastic) that may or may not be correctable. If the pilot-plant stage is successful then work can begin on an industrial scale operation. This is now very much an engineering problem. The reactor must be capable of running aseptically and the design must reflect safety and contamination requirements. At this stage the medium being added to the reactor will change. Now we wish to emphasis penicillin production over growth while maintaining a constant volume. Carbon and nitrogen will be added sparingly alongside precursor molecules for penicillin fed-batch style. Another note is that the presence of penicillin in the reactor is itself inhibitory to the production of penicillin. Therefore, we must have an efficient method for the removal of this product and to maintain constant volume in the reactor. Other systems, such as cooling water supply, must also be considered. In spic pharmaceuticals there are two 70litre fermenters, two1000 and one 1500litre fermenters for pilot scale. Here pilot scale studies are carried over. Optimization of media constituents, their flow rates, optimum temperature, pH and various other additions are estimated. Pilot plant is equipped with latest control system SCADA (Supervisory control and data acquisition, Eurotherm suite). Pilot plants are used to reduce the risk associated with construction of large process plants. They provide valuable data for design of the full-scale plant. Scientific data about reactions, material properties, corrosiveness, for instance, may be available, but it is difficult to predict the behavior of a process of any complexity. Engineering data from other process may be available, but this data cannot always be clearly applied to the process of interest. Designers use data from the pilot plant to refine their design of the production scale facility. If a system is well defined and the engineering parameters are known, pilot plants are not used. For instance, a business that wants to expand production capacity by building a new plant that does the same thing as an existing plant may choose to not use a pilot plant. 10

Fig: 3 Pilot scale fermeter (70 liter) 11

2.4 Fermentation plant:

Penicillin G production is done by two stage fed batch fermentation. In the first stage, primary metabolism will be emphasized. Media for this stage will typically be focused on achieving maximum growth and biomass production. In next stage (production stage) we desire to produce PenG a secondary metabolite, hence the flow rates of the feed is adjusted to serve for only maintenance energy and not for growth, biomass production in this stage is undesired. In SPIC pharmaceutical there about eleven 100m 3 fermenters and six 60m3 seed fermenters are there for production.

Seed stage:
At this stage, sugar will usually be the primary source of carbohydrate for the culture. The seed media contains sugar, MSP, AMS, CSM, CaCO3, CSL, and PPG. Before inoculation Steam is used to keep the reactor running aseptically. This is achieved because the reactor is designed as a pressure vessel and steam is sent through at a minimum temperature/pressure of 121C/15 psi for 15-30min. The media thus prepared is maintained under positive pressure by giving sterile air to avoid contamination. About 10% of volume of the reactor is inoculated in the vessel, the Penicillium chrysogenum starts to adapt itself in the environment. Sugar is given as carbohydrate source because it is easily used for energy metabolism and thus gives the best yields in terms of growth. At this stage substrate corn steep liquor is frequently added that will give a readily usable source of Nitrogen. Both these substrates allow maximal growth of the culture at the expense of product (antibiotic) formation. This is because growth and antibiotic production are mutually interrelated and inversely proportionate. Readily available carbon and nitrogen sources tend to inhibit antibiotic production (this is known as Catabolite Repression, where a secondary metabolite is inhibited by the presence of a more readily usable substrate). The parameters to be maintained in seed stage are: Temperature : 25C Pressure : 0.5Kg/Cm2 Airflow : 1.0vvm Agitation : 2.3m/s 12

The seed maturation criteria: Age : 60-64 hrs PMV : above 20% pH : increasing trend

Production stage:
In this stage the Penicillin G is produced. The constituents used for this production are sugar (45%), MSP, AMS, CSM, CSL, CaCO3, CSL, and PPG. Once the broth is seeded with the seed broth from a matured seed fermeter the time is noted as log0. At the log0 the fermenter is aerated and agitated to maintain DO2, temperature is set at 25C; pH 6.5 is maintained with AMH. After 5hrs of feeding, precursor PAA is added as NaPA and care should be taken that its concentration should be in the range of 0.3-0.8g/l if concentration exceeds it is toxic to organism. Once feeding is started Pen-G production will start. The concentration of PenG or PAA, Nitrogen, and PMV are monitored at every 8 hr intervals. Depending on residual PAA value PAA addition is given. Initially the growth of the microorganism will go on up to 50hrs and the production will be increasing slowly (log phase). From 50-120hrs the production will be maximum (production phase), after 170 hrs the production will decrease and come to an end due to lysis of organism. The parameters to be maintained in seed stage are: Temperature : 25C Pressure : 0.6Kg/Cm2 Airflow : 1.0vvm DO : 60% PAA : 0.3-0.8g/l

13

Fig: 4 100m3 Fermenter used for production The parameters for the production are monitored by a centralized control system called DCS (Distributed Control System). By this system all the parameters can be monitored and can be controlled by using various control valves.

Harvesting of fermenter:
Once the production is stopped, 0.5% of formaldehyde is added to the broth to kill the microorganism. This is sent to recovery plant to recover Pen-G.

2.5 Quality control:

Quality control is a process by which entities review the quality of all factors involved in production. This approach places an emphasis on three aspects: Total Quality Control is the most important inspection control of all in cases where, despite statistical quality control techniques or quality improvements implemented, sales decrease. If the original specification does not reflect the correct quality requirements, quality cannot be inspected or manufactured into the product. 14

For instance, the parameters for a pressure vessel should include not only the material and dimensions, but also operating, environmental, safety, reliability and maintainability requirements. Elements such as controls, job management, defined and well managed processes, performance and integrity criteria, and identification of records competence, such as knowledge, skills, experience, and qualifications soft elements, such as personnel integrity, confidence, organizational culture, motivation, team spirit, and quality relationships. The quality of the outputs is at risk if any of these three aspects is deficient in any way.

Instruments used:
1. KF titrator (Karl Fischer) Principle: The determination of total moisture by Karl Fisher titration is a calculation based on the concentration of iodine in the KF titrating reagent (i.e. titer) and the amount of KF re-agent consumed in the titration. The end-point of the titration is determined by the dead-stop end-point method. Reagents Procedure Standardization: New bottles containing Composite 5 or Titrant 5 must be standardized against sodium tartrate dihydrate. 230.10g sodium tartrate dehydrate corresponds to 36.4g H2O. Using approx 0.1g of sodium tartrate dihydrate as sample. Fresh solvent is used between each standardization. The standardization is accepted when two determinations agree within 0.5% relative. Karl Fisher reagent Methanol (moisture free) Sodium sulphate (Na2SO4), moisture free Sodium tartrate dihydrate (Na2C4H4O6 2 H2O)

15

Fig 5: KF Titrator 2. Kjeldahl Method for Determining Nitrogen The Kjeldahl method was developed over 100 years ago for determining the nitrogen contents in organic and inorganic substances. Although the technique and apparatus have been modified over the years, the basic principles introduced by Johan Kjeldahl still endure today. Kjeldahl nitrogen determinations are performed on a variety of substances such as meat, feed, grain, waste water, soil, and many other samples. Various scientific associations approve and have refined the Kjeldahl method, including the AOAC International (formerly the Association of Official Analytical Chemists), Association of American Cereal Chemists, American Oil Chemists Society, Environmental Protection Agency, International Standards Organization, and United States Department of Agriculture.The Kjeldahl method has three main steps: digestion, distillation, and titration. Digestion Digestion is accomplished by boiling a homogeneous sample in concentrated sulfuric acid. The end result is an ammonium sulfate solution. The general equation for the digestion of an organic sample is shown below: Organic N + H2SO4 (NH4)SO4 + H2O + CO4 + other sample matrix byproducts 16

Distillation: Excess base is added to the digestion product to convert NH4 to NH3 as indicated in the following equation. The NH3 is recovered by distilling the reaction product. (NH4)2SO4 + 2NaOH 2NH3 + Na2SO4 + 2H2O Titration: Titration quantifies the amount of ammonia in the receiving solution. The amount of nitrogen in a sample can be calculated from the quantified amount of ammonia ion in the receiving solution. There are two types of titrationback titration and direct titration. Both methods indicate the ammonia present in the distillate with a color change. In back titration (commonly used in macro Kjeldahl), the ammonia is captured by a carefully measured excess of a standardized acid solution in the receiving flask. The excess of acid in the receiving solution keeps the pH low, and the indicator does not change until the solution is "back titrated" with base. 2NH3 + 2H2SO4 (NH4)2SO4 + H2SO4 + (NH4)2SO4 + H2SO4

2NaOH Na2SO4 + (NH4)2SO4 + 2H2O

In direct titration, if boric acid is used as the receiving solution instead of a standardized mineral acid, the chemical reaction is: NH3 + H3BO3 NH4 + H2BO-3 + H3BO3

The boric acid captures the ammonia gas, forming an ammonium-borate complex. As the ammonia is collected the color of the receiving solution changes. 2NH4 + H2BO3 H2SO4 (NH4)2SO4 + 2H3BO3

The boric acid method has the advantages that only one standard solution is necessary for the determination and that the solution has a long shelf life. 17

Applications The Kjeldahl method's precision and reproducibility have made it the internationally-recognized method for estimating the protein content in foods and it is the standard method against which all other methods are judged.

Fig 6: Kjeldahl Method for Determining Nitrogen

3. HPLC
High-performance liquid chromatography (or high-pressure liquid chromatography, HPLC) is a form of column chromatography used frequently in biochemistry and analytical chemistry to separate, identify, and quantify compounds based on their idiosyncratic polarities and interactions with the column's stationary phase. HPLC utilizes different types of stationary phase (typically, hydrophobic saturated carbon chains), a pump that moves the mobile phase(s) and analyte through the column, and a detector that provides a characteristic retention time for the analyte. The detector may also provide other characteristic information (i.e. UV/Vis spectroscopic data for analyte if so equipped). Analyte retention time varies depending on the strength of its interactions with the stationary phase, the ratio/composition of solvent(s) used, and the flow rate of the mobile phase. 18

In QC we use HPLC mainly for determining PAA and PenicillinG concentration and activity. We use reverse phase chromatography, where the stationary phase is non-polar and mobile phase is polar. The mobile phase we commonly used for PenG activity estimation is Acetonitryl in 0.1%PBS and the stationary phase is ODS (Octo Dodecyl Silane).

Fig 7: HPLC With HPLC, a pump (rather than gravity) provides the higher pressure required to propel the mobile phase and analyte through the densely packed column. The increased density arises from smaller particle sizes. This allows for a better separation on columns of shorter length when compared to ordinary column chromatography. 4. Gas chromatography (GC) It is a common type of chromatography used in analytic chemistry for separating and analyzing compounds that can be vaporized without decomposition. Typical uses of GC include testing the purity of a particular substance, or separating the different components of a mixture (the relative amounts of such components can also be determined). In some situations, GC may help in identifying a compound. In preparative chromatography, GC can be used to prepare pure compounds from a mixture. In gas chromatography, the moving phase (or "mobile phase") is a carrier gas, usually an inert gas such as helium or an unreactive gas such as nitrogen. The stationary phase is a microscopic layer of liquid or polymer on an inert 19

solid support, inside a piece of glass or metal tubing called a column. The instrument used to perform gas chromatography is called a gas chromatograph. The gaseous compounds being analyzed interact with the walls of the column, which is coated with different stationary phases. This causes each compound to elute at a different time, known as the retention time of the compound. The comparison of retention times is what gives GC its analytical usefulness. Gas chromatography is in principle similar to column chromatography (as well as other forms of chromatography, such as HPLC, TLC), but has several notable difference. Firstly, the process of separating the compounds in a mixture is carried out between a liquid stationary phase and a gas moving phase, whereas in column chromatography the stationary phase is a solid and the moving phase is a liquid. (Hence the full name of the procedure is "Gas-liquid chromatography", referring to the mobile and stationary phases, respectively.). Secondly, the column through which the gas phase passes is located in an oven where the temperature of the gas can be controlled, whereas column chromatography (typically) has no such temperature control. Thirdly, the concentration of a compound in the gas phase is solely a function of the vapor pressure of the gas. Gas chromatography is also similar to fractional distillation, since both processes separate the components of a mixture primarily based on boiling point (or vapor pressure) differences.

Fig 8: Gas chromatography 20

5. Polari-meter:
A polarimeter is a scientific instrument used to measure the angle of rotation caused by passing polarized light through an optically active substance. Some chemical substances are optically active, and polarized light will rotate either to the left (counter-clockwise) or right (clockwise) when passed through these substances. The amount by which the light is rotated is known as the angle of rotation. Polarimeters measure this by passing monochromatic light through the first of two polarizing plates, creating a polarized beam. This first plate is known as the polarizer. This beam is then rotated as it passes through the sample. The sample is usually prepared as a tube where the optically active substance is dissolved in an optically inactive chemical such as distilled water, ethanol and methanol. Some polarimeters can be fitted with tubes that allow for sample to flow through continuously. After passing through the sample, a second polarizer, known as the analyzer, rotates either via manual rotation or automatic detection of the angle. When the analyzer is rotated to the proper angle, the maximum amount of light will pass through and shine onto a detector.Semi-automatic polarimeter requires visual detection but use push-buttons to rotate the analyzer and offer digital displays. The most modern polarimeters are fully automatic, and simply require the user to press a button and wait for a digital readout. The angle of rotation of an optically active substance can be affected by: Concentration of the sample Wavelength of light passing through the sample (generally, angle of rotation and wavelength tend to be inversely proportional) Temperature of the sample (generally the two are directly proportional) Length of the sample cell (input by the user into most automatic polarimeters to ensure better accuracy) Polarimeters can be calibrated or at least verified by measuring a quartz plate, which is constructed to always read at a certain angle of rotation (usually +34, but +17 and +8.5 are also popular depending on the sample). Quartz plates are preferred by many users because a solid sample are much less affected by variations in temperature, and does not need to be mixed on-demand like sucrose solutions. Applications Because many optically active chemicals are stereoisomers, a polarimeter can be used to identify which isomer is present

21

in a sample if it rotates polarized light to the left, it it a levo-isomer, and to the right, a dextro-isomer. If the specific rotation of a sample is already known, then the concentration and/or purity of a solution containing it can be calculated. Most automatic polari-meters make this calculation automatically, given input on variables from the user. Concentration and purity measurements are especially important to determine product or ingredient quality in the food & beverage and pharmaceutical industries. Samples that display specific rotations that can be calculated for purity with a polarimeter include: Antibiotics Vitamins Amino Acids Starches Sugars

Polarimeters are used for determining quality of sugar. Often it is used a modified polarimeter with a flow cell called a saccharimeter. These instruments use the International Sugar Scale (as defined by ICUMSA).

Fig: 9 Polarimeter 22

6. LOD analyzer:
The classic laboratory method of measuring high level moisture in solid or semi-solid materials is loss on drying (LOD). In this technique a sample of material is weighed, heated in an oven for an appropriate period, cooled in the dry atmosphere of a desiccators, and then reweighed. If the volatile content of the solid is primarily water, the LOD technique gives a good measure of moisture content. Because the manual laboratory method is relatively slow, automated moisture analyzers have been developed that can reduce the time necessary for a test from a couple hours to just a few minutes. These analyzers incorporate an electronic balance with a sample tray and surrounding heating element. Under microprocessor control the sample can be heated rapidly and a result computed prior to the completion of the process, based on the moisture loss rate, known as a drying curve.

Fig: 10 LOD analyzer 7.Bulk density apparatus: The bulk and tapped density of pharmaceutical powders are often measured for processability. The tapped density is measured for two primary purposes: (i) the tapped value is more reproducibly measured than the bulk value, and (ii) the "flowability" of a powder is inferred from the ratio of these two measured densities. The density ratio method is compared to flowability measurements on a Johanson Flow Indicizer. These methods are evaluated for monodisperse glass beads as well as for common pharmaceutical excipients and blends. The "tapped" density of a pharmaceutical powder is determined using a tapped 23

density tester, which is set to tap the powder at a fixed impact force and frequency. The methods for measurement in the U.S. pharmaceutical industry are specified in the U.S. Pharmacopeia (USP). Tapped density by the USP method is determined by a linear progression of the number of taps. In light of experiments performed over the past decade, exhibiting a slow relaxation approach to the final packing state (logarithmic with the number of taps), the tapped density measured with the USP method is further explored. Additionally, the dependence of the tapped density on the conditions specified in the USP method, e.g., sample size and time separation between taps, is examined.

Fig:11 Bulk density analyzer

2.6 Product recovery plant:

Penicillin G recovery is carried over by 8 steps: 1. Extraction with n-Butyl acetate 2. Removal of color(carbon treatment) 3. Extraction of PenG in aqueous phase 4. Crystallization using butanol 5. Crystal washing 6. Drying 24

1st Extraction unit

2nd Extraction unit

3rd Extraction unit

Dryer

Crystal washing

Crystallization

Fig: 12 Schematic representation recovery processes

1 st Extraction unit:
In 1st stage the fermentation broth is collected in a two 70m 3 reactor and one more reactor is kept for receiving partial discharge. The broth is first treated with H2SO4 and the pH is brought down to 2. This is done because in this pH Pen G soluble in organic phase which is provided by adding n-Butyl acetate. The broth can form emulsion and the extraction will become difficult, to avoid this situation we are adding a demulsifying agent (Quaternary ammonium salt). With these additions Pen G is extracted in n-Butyl acetate as Rich BAC and this is then sent to color removal unit. The color removal is done by using Activated carbon. Our penicillin rich solution is then treated with 0.25-5% activated carbon to remove pigments and impurities. Activated carbon is an amorphous solid, and absorbs molecules from the liquid phase through is highly developed internal pore structure. It is obtained in powered, pelleted or granular form and is produced from coal, wood and coconut shells.

2nd Extraction unit:

In this unit PenG is converted into Potassium salt so that it can be extracted in aqueous phase. This is achieved by adding 13-15% K 2CO3 solution. In this unit PenG salt is recovered in aqueous phase, then this phase along with some BAC. Then the rich aqueous phase is sent to 3rd extraction unit. 25

Fig: 13 Scheme of the extraction process

3rd Extraction unit:

In third extraction unit the residual BAC is completely removed and rich aqueous phase pH is adjusted to 6.2-6.5 with KOH. Then the RAS is transferred to crystallization unit. The exhaust BAC is sent to SRP for recovery.

Crystallization unit:
Crystals are highly organized inert matters. If grown without external interference, they grow in polyhedral shapes and exhibit many degrees of symmetry. Penicillin G is an odorless, colorless or white crystal, or crystalline powder. Crystallization is essentially a polishing step that yields a highly pure product. It is done through phase separation from a liquid to a solid. In crystallization unit we use a Butanol as solvent. Batch crystallization is the most the most used method for polishing antibiotics, including penicillin G. They are slowly cooled to produce supersaturation. Seeding causes nucleation and growth is encouraged by further cooling until the desired crystals are obtained. While the crystallization procedures product of very high purity, improves appearance and has a low energy input, the process can be time consuming due to the high concentration of the solutions during crystallization. It can also be profoundly affected by trace impurities and batch crystallization can often give poor quality, nonuniform product. After crystallization gets over the slurry is sent to further purification and the exhaust Butanol is sent to SRP for recovery. 26

Crystal washing:
While the penicillin G crystals we have formed are essentially pure in nature but adsorption and capillary attraction cause impurities from its mother liquor on their surfaces and within the voids of the particulate mass. Because of this the crystals must be washed and pre-dried in a liquid in which they are relatively insoluble. This solvent should be miscible with the mother solvent. For this purpose we use anhydrous l-Propanol, n-Butanol or another volatile solvent.

Dryer:
Drying is done by fluid bed dryer. Fluid bed processing involves drying, cooling, agglomeration, granulation, and coating of particulate materials. It is ideal for a wide range of both heat sensitive and non-heat sensitive products. Uniform processing conditions are achieved by passing a gas (usually air) through a product layer under controlled velocity conditions to create a fluidized state. In fluid bed drying, heat is supplied by the fluidization gas, but the gas flow need not be the only source. Heat may be effectively introduced by heating surfaces (panels or tubes) immersed in the fluidized layer. In fluid bed cooling, cold gas (usually ambient or conditioned air) is used. Conditioning of the gas may be required to achieve sufficient product cooling in an economically sized plant and to prevent pick up of volatiles (usually moisture). Heat may also be removed by cooling surfaces immersed in the fluidized layer. Agglomeration and granulation may be performed in a number of ways depending upon the feed to be processed and the product properties to be achieved. Fluid bed coating of powders, granules, or tablets involves the spraying of a liquid on the fluidized powder under strictly controlled conditions. The dry PenG powder is sent to pulverizing unit.

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3.

Butyl acetate

3-5% BAC with spent broth

Exhaust BAC

Exhaust BAC with H2O

Solvent recovery plant

Solvents used in recovery plant are costly hence these should be recycled inorder to reduce production cost, so a solvent recovery plant is been installed. The two main solvents which are used in recovery of PenG are BAC and Butanol. These solvents after extraction are sent to solvent recovery plant at different stages, these solvents are treated by different techniques and they are recycled.

3.1 Recycling of BAC:

n-Butyl acetate solvent used in L-L extraction of Penicillin is costly, so recycling of BAC is necessary to minimize the cost of production. BAC is sent to SRP plant in three stages: 1. BAC after 1st extraction process, it contains about 3-5% of BAC with spent broth 2. Exhaust BAC after 2nd extraction process, it contains only BAC 3. Exhaust BAC with H2O, this comes after L-L extraction in 3rd stage

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Fig:14 Scheme of the BAC sent to SRP BAC received at different stages are collected separately and treated in different methodologies.

3.1.1 BAC with spent broth:

BAC with spent broth is treated by (KNIK) distillation column.

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Fig: 15 Counter-current Distillation column The liquid inlet is given at the top of the column. The pressure at the top of the column will be around 100mm.Hg and at the bottom it will be around 280mm.Hg. Pressure drop is used as key in this distillation process. After vapourisation the gas phase is passed to condenser and then to a phase separator. BAC is collected in a tank and recycled back to PenG recovery plant.

Feed in

K N I K

Condenser Phase separator

Vent condenser Vacuum pump

BAC
To waste treatment plant

Fig: 16 Recycling of BAC

3.1.2 Rectification of exhaust BAC:

In this stage of BAC the water will be present in addition; this can be rectified by using same strategy. Here a process heater is attached in order to vaporize the heat. Then it is passed to distillation column then to condenser and then to a phase separator. BAC and water can be collected separately from the phase separator.

Distillation column

Condenser Phase separator 30

Process heater

Feed inlet

BAC Water

Fig: 17 Recycling of BAC and water

3.1.3 Stripping of exhaust BAC:

When the feed contains low amount of BAC and high amount of water this kind of procedure is used. This kind of mixture is received after the third extraction unit. In this BAC concentration will be lower when compared to feed received after second extraction. Usually BAC concentration will be less than1%. First the exhaust BAC is sent to the process heater and then it is sent the distillation column then to the condenser and then to the phase separator. The water from the phase separator is recycled back to the distillation column. Until the clear separation is achieved recycle is continued.

Distillation column

Condenser Phase separator

BAC Process heater Water

Feed inlet

Fig: 18 Stripping of BAC 31

3.2 Recovery of Butanol:

Butanol used in crystallization and crystal wash procedures should be recovered and recycled to PenG recovery plant. Like BAC Butanol is also received in different conditions. This can be categorized by the quantity of the water present with the Butanol and also with the presence of BAC.

Butanol present in the effluent of PenG recovery process can be categorized into 4 classes of feed and they are: 1. Low exhaust Butanol: In this butanol-water concentration ratio will be around 20:80. This can be recycled by stripping unit. This feed is got during drying process. 2. High exhaust Butanol: In this feed the composition of Butanol and water will be around 80:20. This can be recycled by using rectification unit. Feed filtration after crystallization has this property. 3. Mother liquor: Water will be present in butanol in trace amounts (93:7); the complete recycling can be achieved by using a rectification unit. This feed is got after crystal wash. 4. Ternary phase: In this feed BAC is present along Butanol and water (10:75:15). This forms a tertiary phase and these can be separated by hydrolysis.

3.2.1 Stripping of low exhaust Butanol:

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Stripping of Butanol is done by distillation and phase separation techniques as used in the recovery BAC. The difference in boiling points is exploited for this stripping process. The pressure in the column is reduced so that the boiling point difference can be used as a tool for removing Butanol from the water. As the water concentration is more we have to recycle the water to distillation column until complete stripping of Butanol takes place.

This can be schematically represented as:

Distillation column

Condenser Phase separator High exhaust Butanol

Process heater

Water
Low exhaust butanol

Fig: 19 Stripping of Low exhaust Butanol

3.2.2 Rectification High exhaust Butanol:

In exhaust butanol water amount will be low, so the after phase separation we have to recycle the feed so that the water is removed completely. Initially after phase separation we will get another high exhaust butanol on continuous recycling we will get butanol alone. We also get some low exhaust butanol which has to be sent to stripping column for further treatment. Distillation column Condenser 33

Phase separator Process heater Butanol

High exhaust Butanol Low exhaust Butanol

High exhaust butanol

Fig: 20 Rectification unit for high exhaust Butanol

3.2.3 Rectification of mother liquor:

In mother liquor the water concentration is low hence the recovery can be achieved by simple distillation column setup. Recycle of condensate is not necessary but when water concentration is high we can recycle and butanol can be recovered.

Distillation column

Condenser

Process heater Mother liquor

Butanol

Fig: 21 Schematic representation of rectification column

3.2.4 Ternary phase:

In this feed BAC is present along water and Butanol. Dissocitive extraction is used. Dissociation extraction is the process of using chemical reaction to force a solute to transfer from one liquid phase to another. One example is the use of a sodium hydroxide solution to extract phenolics, acids, or mercaptans from a hydrocarbon stream. 34

The opposite transfer can be forced by adding an acid to a sodium phenate stream to spring the phenolic back to a free phenol that can be extracted into an organic solvent. Similarly, primary, secondary, and tertiary amines can be protonated with a strong acid to transfer the amine into a water solution, for example, as an amine hydrochloride salt. Conversely, a strong base can be added to convert the amine salt back to free base, which can be extracted into a solvent. This procedure is quite common in pharmaceutical production.

Distillation column

Condenser

Phase separation

Process heater

Butanol + BAC + water + NaOH

Water

High exhaust Butanol

Fig: 22Butanol, BAC recovery by water hydrolysis The NaOH is first mixed with the feed, here phase dissociation takes place. Then it is passed to process heater and then to distillation column then to condenser then to phase separation. Till separation is finished the butanol phase is recycled. Butanol is recovered as high exhaust butanol and this is sent as feed to the high exhaust butanol rectification unit.

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4. Effluent treatment plant

Effluent Treatment Plant or ETP are used in the pharmaceutical and chemical industry to purify water and remove any toxic and non toxic materials or chemicals from it. These plants are used by all companies for environment protection. Importance of ETP Plants The treatment of effluents in pharmaceutical industry is essential to prevent pollution of the receiving water. The effluent water treatment plants are installed to reduce the possibility of pollution; biodegradable organics if left unsolved, the levels of contamination in the process of purification could damage bacterial treatment beds and lead to pollution of controlled waters. The COD reduction is mainly carried over in ETP; due to addition of formaldehyde organisms are dead and hence the BOD value will be appropriate so there is no need for the methods to reduce BOD. COD - Chemical Oxygen Demand The chemical oxygen demand (COD) is commonly used to indirectly measure the amount of organic compounds in water. Most applications of COD determine the amount of organic pollutants found in surface water (e.g. lakes and rivers), making COD a useful measure of water quality. It is expressed in milligrams per liter (mg/L), which indicates the mass of oxygen consumed per liter of solution. Class A water: COD level is above 7000 36

Class B water: COD level is below 4000 Class C water: COD level is above 400

Normally the effluent coming from plant has following properties its pH is around 2-2.5 and its temperature is around 50-60C. Total solids in present in the effluent will be in the range of 20-25%. The effluent is first sent to decanter or evaporator and then effluent is transferred to double drum dryer. In this step about 80% of moisture is reduced to 20%. Then temperature of the effluent is then reduced by using suitable heat exchangers. There are about two UASB systems and an anaerobic sludge digestion system is present. The intention is to reduce the COD of the effluent. In addition to this a Multi effect evaporator is present.

4.1 UASB:
Upflow anaerobic sludge blanket (UASB) technology, normally referred to as UASB reactor, is a form of anaerobic digester that is used in the treatment of waste water. UASB uses an anaerobic process whilst forming a blanket of granular sludge which suspends in the tank. Wastewater flows upwards through the blanket and is processed (degraded) by the anaerobic microorganisms. The upward flow combined with the settling action of gravity suspends the blanket with the aid of flocculants. The blanket begins to reach maturity at around 3 months. Small sludge granules begin to form whose surface area is covered in aggregations of bacteria. In the absence of any support matrix, the flow conditions create a selective environment in which only those microorganisms, capable of attaching to each other, survive and proliferate. Eventually the 37

aggregates form into dense compact biofilms referred to as "granules". A picture of anaerobic sludge granules can be found here. The blanketing of the sludge enables a dual solid and hydraulic (liquid) retention time in the digesters. Solids requiring a high degree of digestion can remain in the reactors for periods up to 90 days. Sugars dissolved in the liquid waste stream can be converted into gas quickly in the liquid phase which can exit the system in less than a day. A properly designed UASB can reduce upto 75-85% COD.

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Fig: 23 UASB plant

4.2 Anaerobic digestion:

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Anaerobic digestion is a series of processes in which microorganisms break down biodegradable material in the absence of oxygen, used for industrial or domestic purposes to manage waste and/or to release energy. It is widely used as part of the process to treat wastewater. As part of an integrated waste management system, anaerobic digestion reduces the emission of landfill gas into the atmosphere. Anaerobic digestion is widely used as a renewable energy source because the process produces methane and carbon dioxide rich biogas suitable for energy production, helping to replace fossil fuels. The nutrient-rich digestate which is also produced can be used as fertilizer. The digestion process begins with bacterial hydrolysis of the input materials in order to break down insoluble organic polymers such as carbohydrates and make them available for other bacteria. Acidogenic bacteria then convert the sugars and amino acids into carbon dioxide, hydrogen, ammonia, and organic acids. Acetogenic bacteria then convert these resulting organic acids into acetic acid, along with additional ammonia, hydrogen, and carbon dioxide. Finally, methanogens convert these products to methane and carbon dioxide.

5. Utility and Instrumentation

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5.1 Utility:
Utility plant provides the steam for sterilization, De-mineralized water, cold water for heat exchanging purposes and a nitrogen plant for production of nitrogen. The accessories present in this plant are boilers, chillers, compressors and nitrogen plant.

DM water plant: Dm water is used to avoid corrosion and to have control over process feed inputs. Entire plant uses demineralized water hence it should be produced economically. The water is got from SIPCOT and this is sent to pretreatment plant and then to aeration tank. Inorder to convert ferrous iron to ferric iron we are aerating it, such that it is converted to ferric iron and in the presence of coagulating agent like aluminium sulphate ferric is coagulated and this is removed by filters and sent to reservoir. Water from the reservoir is transferred to DM-Plant. In DM plant it is first passed through filter then through Strong acid cation filter. SAC filter contains strong acid cation resin (Duolite C-20). This resin filters ions like Ca 2+, Mg2+, Na2+ etc. Then this water is transferred to weak base anion filter. WBA filter has weak base anion resin (Duolite A-368). This can filter Cl -, SO4-, CO2 then this water is passed to degasser where CO2 is reduced. After water passed to degasser it is passed to Strong base anion filter to ensure that SiO2 and CO2 are completely removed. This demineralized water is then passed to process plants.

Boilers:

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Steam for sterilization and for various processes should be produced in large volume but economically, since this is a most power consuming process power consumption should be taken care. There 3 boilers in the plant: 8Tonne/hr coal boiler 15tonne/hr coal boiler 15tonne/hr fuel boiler Chiller: Cold water for heat exchange purposes and for various processes is produced by chilling unit. The water should be cooled down to 5C. This water after fulfilling its purpose and gaining heat it will be around 11-15C. This water is then processed in chilling unit and recycled back. Brine chillers are used to chill down the brine (Methyl ethylene glycol). This is done because the brine doesnt lose chillness readily. There are two brine chilling units. There are two kinds of chillers: Water chiller Brine chiller There are two cooling towers one is utility cooling tower and other one is for process cooling tower for cooling process water.

Nitrogen plant: Nitrogen is a cool, dry, inert, gas that can displace Oxygen in a situation where Oxygen may provide the catalyst for combustion or where it may affect the quality of a product. Two nitrogen plants are there in the plant: Capacity 40Nm3/hr: 6.5Kg/Cm2 Capacity 60 Nm3/hr: 6Kg/Cm2 42

5.2 Instrumentation department:

Instrumentation is the branch of engineering that deals with measurement and control. The department should check its accuracy, reliability and validation of the instruments. An instrument is a device that measures or manipulates variables such as flow, temperature, level, or pressure. Instruments include many varied contrivances which can be as simple as valves and transmitters, and as complex as analyzers. Instruments often comprise control systems of varied processes. The control of processes is one of the main branches of applied instrumentation. Control instrumentation includes devices such as solenoids, valves, circuit breakers, and relays. These devices are able to change a field parameter, and provide remote or automated control capabilities. Instrumentation plays a significant role in both gathering information from the field and changing the field parameters, and as such are a key part of control loops. Pressure measurement: Pressure is an effect which occurs when a force is applied on a surface. Pressure is the amount of force acting on a unit area. Type: Pressure guage Draft guage Diaphragm seal transmitter Differential pressure instrument

Flow measurement: The volumetric flow rate in fluid dynamics and hydrometry, (also known as volume flow rate or rate of fluid flow) is the volume of fluid which passes through a given surface per unit time (for example cubic meters per second [m3 s-1] in SI units, or cubic feet per second [cu ft/s]). It is usually represented by the symbol Q. Type: Orifice plate Venturimeter 43

 Rotameter Temperature measurement: Temperature is a physical property that underlies the common notions of hot and cold. Something that feels hotter generally has a higher temperature, though temperature is not a direct measurement of heat. Temperature is one of the principal parameters of thermodynamics. If no net heat flow occurs between two objects, the objects have the same temperature; otherwise, heat flows from the object with the higher temperature to the object with the lower one. This is a consequence of the laws of thermodynamics. Type: Resistance temperature devices Thermocouples

Level measurement: Level Measurement refers to instrumentation techniques designed to measure the height of a fluid or solid within a containing vessel. Type: Magnetic float Float switch Differential type

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