The project of the heat exchanger for cooling compressed air with water used in penicillin production

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MINISTRY OF EDUCATION AND SCIENCE, YOUTH AND SPORTS OF UKRAINE National Aviation University Department of Biotechnology

Course work (Explanatory Note) Discipline "Processes and apparatus for biotechnological production "

Theme: The project of the heat exchanger for cooling compressed air with water used in penicillin production

Kyiv 2013



TASKS for the course work of the student

1. Theme: The project of the heat exchanger for cooling compressed air with water used in penicillin production

2. Deadline student completed the work: "24" April 2013 3. Output to: Calculate a shell-and-tube heat exchanger for cooling 1050 m3/h (in standard conditions) of air from 140 oC to 31oC with water. The pressure of the air is p=0,35 MPa. The cooling water, which gives a deposit of scale, has a temperature of 16 oC.

4. Content of explanatory note (list of issues to be developed): theoretical part contains a description of the design of heat exchangers and producing penicillin by technical methods.

Estimated portion contains calculations and tube heat exchanger for cooling water.

5. Calendar plan:

№ п/п	Stage	Deadline	Evaluation of the performance
1	Preparation of the course work plan	14.02.2013	performed
2	Retrieval of Information	15.02.13-03.03.13	performed
3	Preparation of the theoretical part	04.03.13-04.04.13	performed
4	Preparation of the computation and analytical part	05.04.13-07.04.13	performed
5	Additions and Changes	08.04.13-15.04.13	performed
6	Defence of the course work	24.04.13	performed

6. Date of assignment:

Supervisor __________ Kuznetsova O.O.

Executor ____________ Kohut K.G.



Summary

Explanatory note to the course work "The project of the heat exchanger for cooling compressed air with water used in penicillin production":

Object of the study - heat exchanger.

Subject of research - heat exchanger.

Purpose - to examine and analyze the design of heat exchanger.

Research methods - analysis, a systematic approach of observation.

Exchangers, Selecting, antibiotics, penicillin, CALCULATION.



Introduction

An antibacterial is an agent that inhibits bacterial growth or kills bacteria. The term is often used synonymously with the term antibiotics. Today, however, with increased knowledge of the causative agents of various infectious diseases, antibiotic(s) has come to denote a broader range of antimicrobial compounds, including anti-fungal and other compounds. [2]

The term antibiotic was first used in 1942 by Selman Waksman and his collaborators in journal articles to describe any substance produced by a microorganism that is antagonistic to the growth of other microorganisms in high dilution. This definition excluded substances that kill bacteria, but are not produced by microorganisms (such as gastric juices and hydrogen peroxide). It also excluded synthetic antibacterial compounds such as the sulfonamides. Many antibacterial compounds are relatively small molecules with a molecular weight of less than 2000 atomic mass units.[2]

With advances in medicinal chemistry, most of today's antibacterials chemically are semisynthetic modifications of various natural compounds. These include, for example, the beta-lactam antibacterials, which include the penicillins (produced by fungi in the genus Penicillium), the cephalosporins, and the carbapenems. Compounds that are still isolated from living organisms are the aminoglycosides, whereas other antibacterials-for example, the sulfonamides, the quinolones, and the oxazolidinones-are produced solely by chemical synthesis. In accordance with this, many antibacterial compounds are classified on the basis of chemical/biosynthetic origin into natural, semisynthetic, and synthetic. Another classification system is based on biological activity; in this classification, antibacterials are divided into two broad groups according to their biological effect on microorganisms: bactericidal agents kill bacteria, and bacteriostatic agents slow down or stall bacterial growth. [4]

Testing the susceptibility of Staphylococcus aureus to antibiotics by the Kirby-Bauer disk diffusion method - antibiotics diffuse from antibiotic-containing disks and inhibit growth of S. aureus, resulting in a zone of inhibition.[2]

The successful outcome of antimicrobial therapy with antibacterial compounds depends on several factors. These include host defense mechanisms, the location of infection, and the pharmacokinetic and pharmacodynamic properties of the antibacterial. A bactericidal activity of antibacterials may depend on the bacterial growth phase, and it often requires ongoing metabolic activity and division of bacterial cells. These findings are based on laboratory studies, and in clinical settings have also been shown to eliminate bacterial infection. Since the activity of antibacterials depends frequently on its concentration, in vitro characterization of antibacterial activity commonly includes the determination of the minimum inhibitory concentration and minimum bactericidal concentration of an antibacterial. To predict clinical outcome, the antimicrobial activity of an antibacterial is usually combined with its pharmacokinetic profile, and several pharmacological parameters are used as markers of drug efficacy.[4]



General characteristics of antibiotics

The term "antibiotic" was proposed in 1942 by SA Waksman to describe substances produced by microorganisms and have antimicrobial activity. Subsequently, many researchers have offered their own language, putting in them sometimes too limited content or excessively expanding this concept.[1]

Currently, under the understanding chemotherapeutic antibiotic substances derived from microorganisms or other natural sources, and their semi-synthetic analogs and derivatives that have the ability to selectively suppress the patient's body of pathogens and (or) delay the development of cancer. [2]

Antibiotics are of natural origin produced by different groups of microorganisms (mostly actinomycetes, rarely bacteria), lower plants (yeast, algae, fungi, higher fungi), plants and animals. [1]

For example, the genera Micrococcus, Streptococcus, Diplococcus, Chromobacterium, Escherichia. Bacteria of the genus Bacillus form gramicidin, subtilin polymyxins. [1]

Antibiotics, which are formed by microorganisms belonging to several Actinomycetales, - streptomycin, tetracycline, novobiocin, actinomycin, etc.

Antibiotics, which are formed by imperfect fungi: penicillin - Penic. Chrysogenum; griseofulvin - Penic. Griseofulnum; trihotsetin - Tricholecium roseum.

Antibiotics, which are formed by fungi, belonging to the classes of basidiomycetes and ascomycetes: termofillin, lenzitin, hetomin.

Lichens, algae and lower plants are capable of forming acid and usnic hlorellin, higher plants - almitsin, rafanin.

Antibiotics are of animal origin: lysozyme ekmolin, krutsin, interferon.[2]

Classification of antibiotics. By the nature of the impact of antibiotics on bacterial cell can be divided into three groups:

- Bacteriostatic (bacteria are alive but unable to reproduce)- Bactericide (bacteria were sacrificed, but still physically present in the medium)

- Bacteriolytic (death to the bacteria, and bacterial cell walls are destroyed).On the mechanism of biological action of antibiotics are divided into:

1. Antibiotics that inhibit the synthesis of the bacterial wall (penicillins, cephalosporins, bacitracin, vancomycin).

2. Antibiotics disrupt the cytoplasmic membrane (polypeptides, polyenes, gramicidin).

3. Antibiotics destroy the ribosomal subunit and inhibiting protein synthesis (tetracyclines, hlormitsetiny, aminoglycosides, macrolides).

4. Antibiotics that selectively inhibit the synthesis of nucleic acids:

- Inhibitors of RNA (actinomycin, griseofulvin, kanamycin, neomycin, novobiocin, etc.);

- Inhibitors of DNA synthesis (bruneomitsin, sarkomitsin).Antibiotics have a selective effect, only active against microorganisms while maintaining the viability of the host cells and do not act at all, but in some genera and species of microorganisms. Since selectivity is closely related to the concept of the breadth of the spectrum of activity of antibiotics. Traditionally, the spectrum antimicrobial effects of antibiotics are divided into narrow-spectrum drugs and broad:

1. Narrow spectrum antibiotics are only a certain type of bacteria. These include penicillin, oxacillin, erythromycin.

2. Antibiotic with a broad spectrum of activity are effective in killing not only Gram-positive and Gram-negative bacteria, but also spirochetes leptospira, rickettsia, large viruses (trachoma, psittacosis, and others). These include tetracyclines (tetracycline, oxytetracycline, chlortetracycline, glitsiklin, methacycline, morfotsiklin, doxycycline), and chloramphenicol.

Expression values of the biological activity of antibiotics is usually produced in units contained in 1 ml (U / ml) or 1 mg (U / mg). One unit of antibiotic activity take the minimum number of antibiotic that can suppress or delay the development of the growth of the standard strain of test organisms in a given volume of medium. [1]

Use of antibiotics. Antibiotics are the most numerous group of drugs. They are used to prevent and treat inflammation caused by bacterial microflora. Now there are hundreds of drugs that selectively act on various disease. Scope of antibiotics - is rapidly progressing infection or bacterial infection of vital organs, which the immune system can not handle myself. Antibiotics are essential for the development of acute disease - angina and pneumonia, as well as in infectious inflammation, which is localized in closed cavities (otitis media, sinusitis, osteomyelitis, abscess, cellulitis). Currently, the company is actively working to find a new generation of antibiotics that are effective in the treatment of viral diseases and cancer.

Antibiotics are used in agriculture, mainly as medical drugs in livestock, poultry farming, beekeeping and plant, and some antibiotic substances - as growth promoters of animals.

Some antibiotics have been successfully used in the food and canning industry in the preservation of perishable goods (fresh fish, meat, cheese, various vegetables).[2]

Peculiarities of antibiotics

The process of getting an antibiotic includes the following main stages:

1. receive appropriate strain - producer antibiotic suitable for industrial production;

2. biosynthesis of antibiotics;

3. isolation and purification of the antibiotic;

4. concentration, stabilization of antibiotic and the finished product.

The first task of finding producers of antibiotics - their separation from natural sources. Biosynthesis of antibiotics - the hereditary characteristics of an organism, which is manifested in the fact that each species (strain) is capable of forming one or more well-defined and strictly specific to his antibiotic substances.

Identifying potential form during the life of antibiotics is associated with conditions of cultivation of organisms. In some conditions, the body forms an antibiotic, in other circumstances the same body, with good growth will not have the ability to synthesize an antibiotic substance. Education antibiotics will only occur during the development of an organism in a specific environment and under specific environmental conditions. By changing the culture conditions can be more or less out of antibiotic, or to create the conditions in which no antibiotic will be formed. It is also possible by changing the culture conditions of the producer to achieve a preferential biosynthesis of antibiotics, provided education studied organism several antibiotic substances or to obtain new forms of antibiotics, but only within the limits of those compounds that can be synthesized by this organism.[3]

Among the most important factors that influence the expression of antibiotic properties of microorganisms, are part of the environment, its active acidity, redox conditions, cultivation temperature, joint cultivation of two or more micro-organisms and other factors.

Media for culturing microorganisms. Natural (complex) environment consisting of natural compounds and have an indefinite chemical composition (of green plants, animal tissue, malt, yeast, fruit, vegetables, manure, soil, etc.), contain all the components needed for growth and development most types of microorganisms. The following environments:

- Meat medium, composed simultaneously with beef extract and peptone are sodium chloride, potassium phosphate, sometimes glucose or sucrose, is typically used in laboratories.

- Potato medium with glucose and peptone, often used in the laboratory for the cultivation of many species of actinomycetes and bacteria;

- Protection from corn extract, soy flour, bards and other substances, which include ammonium sulfate, calcium carbonate, calcium phosphate, glucose, sucrose, lactose or other carbohydrates and other compounds, from successfully used in industry, because are cheap and offer good growth of microorganisms with a high yield of antibiotics.[1]

Since the natural environment do not provide strict quantitative data to study the physiological and biochemical characteristics of the organism, used synthetic media that is selected for individual producers individually.

Carbon sources are organic acids, alcohols, carbohydrates, a combination of carbon-containing compounds. With the industrial production of antibiotics as carbon sources is often used potato starch, corn meal, or other plant materials.

Nitrogen sources have a great influence on the formation of antibiotic substances by microorganisms. Usually in the media for the cultivation of microorganisms are a source of nitrate nitrogen (less nitrous acid), ammonium salts of organic and inorganic acids, amino acids, proteins and their hydrolysates.

Typically, the most favorable for microorganisms is the ratio C / N = 20. But for the antibiotic, this ratio is not always optimal. Therefore, for each producer must select the appropriate ratio of carbon and nitrogen.

Sources of mineral nutrients are phosphorus, sulfur and other macro-and micronutrients. [1]

The producers of antibiotics in relation to the concentration of phosphorus in the environment can be divided into three groups:- Highly sensitive producers, for which the optimal concentration of phosphorus in the medium is less than 0.01% (producers nystatin, tetracycline florimitsina, vancomycin);- Producers of high sensitivity, for which the optimal concentration of phosphorus is 0,010-0,015% (producers of streptomycin, erythromycin, cycloserine, neomycin);- Insensitive producers, for which the optimal concentration of phosphorus is 0,018-0,020% (producers of novobiocin, gramicidin, oleandomitsina).Sulfur is part of some antibiotics, produced mushrooms (penicillin, cephalosporin, gliotoksin, etc.), bacteria (bacitracin, subtilin, lowlands) and actinomycetes (ehinomitsiny, a group of thiostrepton). Usually, the source of sulfur in the environment are sulfates. However, the biosynthesis of penicillin, the best source of sulfur for the producer is sodium thiosulfate.

In addition, for the biosynthesis of antibiotics are needed and some trace elements. Thus, the producer albomitsina S. subtropicus forms antibiotic with significant concentrations of iron in the medium. Iron is necessary for the formation of chloramphenicol and other antibiotics.[3]

The influence of pH. Many bacterial organisms that synthesize antibiotics, better developed at a pH of about 7.0, although some, such as lactic streptococci producing lowlands develop better in the medium at pH = 5,5 ч 6,0.Most actinomycetes grow well under the initial pH values ??ranging from 6.7 to 7.8, in most cases, the viability of actinomycetes at a pH below 4.0-4.5 suppressed.

Temperature. For most bacterial organisms temperature optimum in the range of 30-37 ° C. For a producer of gramicidin with optimum temperature for growth and biosynthesis is 40 ° C.

Actinomycetes are usually cultivated at 26-30 ° C, although some species can develop as Streptomyces at low (0 to 18 ° C) and elevated (55-60 ° C) temperatures.

For most filamentous fungi optimum temperature is 25-28 ° C.

Aeration. Most producers of antibiotics studied are aerobes. For the biosynthesis of many antibiotics (penicillin, streptomycin, etc.), the maximum accumulation occurs when the degree of aeration, equal to one in which a certain amount of protection for 1 min purged the same volume of air.[2]

In the development of antibiotic producer in industrial conditions the body's need for oxygen varies with the stage of development, the culture fluid viscosity and other factors. At certain stages of the situation may arise related to anoxia producer. In these circumstances, additional measures should be taken, such as increasing the concentration of the oxidant addition of hydrogen peroxide.

The most promising method of micro-organisms - producers of antibiotics found submerged culture method using batch processes. In the deep cultural development of the organism and antibiotic synthesis takes place in two phases.

In the first phase of development of culture, or as it is sometimes called, tropofaze (phase balanced growth of the microorganism), there is an intense accumulation of biomass producer associated with the rapid consumption of the main components of the medium-and high-oxygen absorption.

In the second phase of development, called idiofazoy (phase unbalanced growth of microorganism), biomass accumulation slowed or even reduced. During this period the products of metabolism of the microorganism is only partially used for the construction of cellular material, they are mainly directed to the biosynthesis of the antibiotic. Usually, the maximum production of antibiotic in the medium occurs after the maximum biomass accumulation. A detailed description of the process by the example of the production of penicillin is given in the following chapter. [1]

Production of penicillin

Penicillin was discovered in 1929 by Alexander Fleming, and was isolated in crystalline form in 1940. Found that penicillin has antimicrobial activity against certain gram-positive bacteria (stafillococci, streptococci, diplococci and some others) and virtually inactive against Gram-negative species and yeast.

Ability to form penicillin is common among many of fungi belonging to the genera Penicillium and Aspergillus. However, this property is more characteristic of Penicillium notatum-chrysogenum. First isolated from natural substrates strains as the most active producers of penicillin produced not more than 20 units (12 micrograms) antibiotic for 1 ml of the culture fluid. As a result of extensive research on the selection of new active producing strains of penicillin obtained various strains of Penicillium chrysogenum, which, in contrast to the original strains are highly productive and are used in industry. At present, the industrial environment are cultural liquids containing more than 15,000 units of penicillin / ml, and some strains are able to synthesize an antibiotic in an amount up to 25 thousand units / ml.

Under the name "penicillin" combined a large group of substances that are N-acyl derivatives of the heterocyclic amino acids. Of natural penicillins are used penicillin and penicillin. [1]

Statement of the process

Preparation of inoculums

Preparation of seed comprises the following steps:

1) the cultivation of seed mycelium 1st generation devices in a small container (inoculator);

2) the cultivation of seed mycelium 2nd generation in large-capacity vehicles.

Spore culture used to inoculate inoculator is grown on millet in glass vials, dried and stored in this form at room temperature. Seeding produces dry spores of 2-3 bottles.

The basis for the cultivation of the producer of penicillin in sowing machines in preparation of inoculum is the rapid acquisition of large mass of mycelium that can provide for reseeding the fermenter intensive growth and high yield of the antibiotic. To fulfill this task producer must grow in media rich digestible nutrients in good aeration at optimum temperature for growth of the microorganism. [1]

As an easily assimilated carbon acts glucose, sucrose, etc. As a second source of carbon used in small amounts of lactose, whose presence in the environment for growing seed mycelium desirable for the following reasons: its consumption does not start immediately, but after a period of adaptation (addiction), during which the formation of an enzyme that breaks down lactose. Sowing mycelium grown on a medium containing lactose, has a high enzymatic activity against lactose and faster to use it, which has a positive effect on the course of fermentation.

The need of the fungus is easily satisfied in nitrogen mineral nitrogen - ammonium or nitrate. In addition to inorganic nitrogen in the crop of media used in the industry, is an organic nitrogen-rich plant material such as corn steep liquor. Vegetable materials is characterized not only by the presence of organic nitrogen, it contains an additional carbon, which is part of amino acids, peptides, proteins, and trace minerals, vitamins and growth substances.

In addition to the carbon and nitrogen for the growth of a microorganism requires phosphorus, sulfur, magnesium, potassium and trace elements - manganese, zinc, iron, copper. Most of the known cultivated media contains almost all of the above elements, but in different proportions. Table 1 shows an example of the medium used for growing seeds.

Table 1 - The composition of one of the media for the cultivation of seed

Substance	%
Corn extract	2 (dry weight)
Glucose	2
Lactose	0,5
Ammonium nitrate	0,125
Monobasic potassium phosphate	0,2
Magnesium sulphate	0,025
Sodium sulphate	0,05
Chalk	0,5

Growing seed mycelium continues 36-50 hours to produce biomass of medium thickness. Mycelium grown in inoculator, transferred to 10% by volume in the seed sets, which were cultured for 12-18 hours, and then transferred to large fermenters in an amount of 15-20%. The process of growing plants for the mycelium of the 1st and 2nd generation is carried out at a temperature of 24-26 °.

Penicillium - producers of penicillin are common aerobes and require for their growth and development, the availability of oxygen. To obtain high-yielding seed mycelium along with optimal nutrient and is necessary to ensure an adequate supply of the fungus. In the process of growing plants for the production of penicillin mycelium performed with continuous stirring and uninterrupted supply of air into the apparatus in an amount of 1.2-1.5 air to 1 volume of medium per minute. [4]

Preparation of inoculum - one of the most important operations in the cycle biotechnological method for producing antibiotics. The number and quality of seeds depends on the development of culture in the fermenter, and antibiotic biosynthesis. PRODUCER usually grown on rich composition of natural environments, capable to provide the highest physiological activity of microorganisms.

Preparation of inoculum - multistep process (Figure 1).

Fig. 1 - Scheme of multi-cooking seed A - growing in vials B - growing in flasks on rocking: 1 - conserved starting material, 2 - spore generation on beveled agar in vitro, 3 - II spore generation on solid medium; 3a and 3b - I and III generation on liquid medium in the flask, 4 - fermenter prior 5 - fermenter inokulation 6 - primary fermenter

Microorganism previously grown on agar medium in vitro (1, 2), then make a tube hanging in flasks with liquid nutrient medium, spend two generations by deep cultivation on rocking for two to three days for each generation (3a and 3b). With the second generation culture flask makes planting in a small (10 liter) inokulyator 4, then a well-developed culture is transferred to a larger inokulyator 5 (100 - 500 l), which also makes planting in the main fermenter 6. For planting in the main fermentor using 5 to 10% inoculum (inoculum) [2].

Fermentation

Fermentation is a major step in the production of penicillin, which forms the desired product. In industry, the method of submerged fermentation, in which the culture of the microorganism is grown in a nutrient medium, filling its entire volume. Different strains of the need for power supplies varies. Therefore, the composition of matter is not constant and universal for all producers that make penicillin, and changes with the emergence of new strains.

The fermentation medium should be designed in such a way as to develop a culture, consuming nutrients and extracting metabolites, she created the necessary conditions and the transition from the growth phase to the phase of the mycelium. It is desirable that the second phase was longer and the process of fermentation stopped before autolysis.

For this, as for growing seed to the simultaneous presence in the environment is easy carbohydrate. Carbohydrate provides rapid growth and the formation of abundant biomass carbohydrate creates conditions favorable for the biosynthesis of the antibiotic.

In the industrial biosynthesis of penicillin as the most common carbohydrate was glucose or hydrological. Carbohydrate is lactose. Lactose is the only carbohydrate that provides a full course of phase. High yield antibiotic receive only when the medium of lactose as the main source of carbohydrate. Lactose is in the culture medium during the process of fermentation, so the mycelium provided sugar, biomass for formation of penicіllins slowly growing, and the accumulation of the antibiotic is maximized. [4]

The composition of the fermentation medium is organic and mineral nitrogen. An excellent source of organic nitrogen is corn steep liquor, but it can be successfully replaced by wheat extract, various cake, soy flour, gluten and other vegetable raw materials rich in nitrogen.

Source of mineral nitrogen are usually the ammonium nitrate, ammonium sulphate and other salts. The assimilation of ammonium fungus released anions of these salts, acids, which contribute to some acidification.

An extremely important role in the metabolism of the cells is phosphorus, which is essential not only for normal growth and development of the fungus, but also for the process of biosynthesis of penicillin. For the formation of penicillin required much higher concentration of phosphorus in the environment than for the growth of the fungus.

Mandatory component of the fermentation medium is sulfur, which is part of the essential amino acids and enzymes. Sulfur is necessary also because it is part of the penicillin molecule. In the culture medium is introduced in the form of sulfur salts of sulfuric acid and hyposulphate.

Of the other elements necessary for the normal functioning of the fungus and the formation of the antibiotic, it should be noted potassium, magnesium, zinc, iron, manganese and copper.[2]

The optimum concentration of the precursor in the medium is established, depending on the efficiency of its use for the biosynthesis of penicillin to the strain.

The main indicator, the end of fermentation, are the complete disappearance of carbohydrates in the culture medium and the cessation of antibiotic biosynthesis. The fermentation process is carried out in a production environment at a temperature of 26 ± 10C and usually lasts 120-125 hours.

The intensity of the biosynthesis of penicillin depends on the amount of mycelium formed during fermentation. Much more biomass forms of penicillin, so the content of carbohydrates, nitrogen, phosphorus, and sulfur in the environment must be sufficiently high to ensure maximum education mycelium. However, most of the biomass does not guarantee a high yield of the antibiotic. The fungus is not only necessary to ensure enough nutrients, but necessary amount of oxygen. Mushroom nutrition and aeration are two sides of the same process - the more nutrients in the environment, the more oxygen is required for oxidation. On the other hand, increasing the concentration of nutrients in the environment leads to increased biomass for respiration which requires a proportionately larger amount of oxygen. The composition of the culture medium and aeration interdependent. The maximum number of penicillin can be obtained only in products with a high concentration of the components in an adequate supply of dissolved oxygen culture [1].

For the biosynthesis of penicillin is most favorable pH neutral. To maintain a certain level of culture fluid pH should adjust it with the automatic addition of acid or alkali or by establishing the proper ratio of the components of the medium. In synthetic media as pH regulators are most often used organic acids in complex environments - chalk. To obtain the maximum yield of penicillin main components of the environment must be a member of a well-defined in the ratios and concentrations. Part of some media, used in the production of penicillin is given in Table 2.

Table 2 - Composition of media used for penicillin medium components

Components	Medium
	Corn	Oilcake	Fat
Corn extract	2,0 - 3,0	-	2,0 - 3,0
Oilcake	-	2,0 - 4,0	-
Lactose	5,0	5,0	1,0
Glucose	1,5	1,5	1,5
Vegetable oil	0,5 - 0,1	0,5 - 0,1	2,5 - 3,5
Ammonium nitrate	0,4	0,4	0,4
Glauber`s salt	0,05	0,05	0,05
Potassium phosphate monobasic	0,4	0,4	0,4
Magnesium sulphate	0,025	0,025	0,025
Hyposulphate	0,2	0,2	0,2
Chalk	0,5 - 1,0	0,5 - 1,0	0,5 - 1,0
Precursor	0,3 - 0,4	0,3 - 0,4	0,3 - 0,4

An important condition for the success of the biosynthesis of penicillin is a strict aseptic conditions, as the penetration of foreign microorganisms can dramatically reduce the output of the antibiotic. Many common microorganisms capable of producing penicillase enzyme that breaks down penicillin. Contact with even a small number of bacteria capable of producing penicillase, leads to complete inactivation of penicillin, because what should emphasize sterile culture media, air and auxiliary materials.

The need to ensure the conditions for sterile process technology ties units each collector system load growth media, transfer of inoculum inoculators in fermenters imposes greater demands on the level of automation of these processes.[2]

The general scheme of antibiotics to the point of extraction and chemical treatment is shown in Fig. 2.

Fig. 2 - Scheme of antibiotics: I - making seed; II - inokulyatory for increasing seed; III - sterilizer environment for large fermenters; IV - setting for the biosynthesis of antibiotics and - sterilization medium in flasks, b - cooling and seeding crops producer in the flask; in - growth of culture in resting g - growth of culture in rocking and e - inokulyator with a sterile environment, e - inokulyator with the environment, sown crop producer, is - fermenter



The first stage of the process - growing standard colony strains mold Penicillium chrysogenum - made in inokulyator on nutrient medium where the process is ~ 30 hours. The prepared inoculum transferred to sowing machine volume is ~ 10 times more volume inokulyatora. In sowing machine is also sterilized nutrient medium. The process of growth here goes ~ 15 - 20 hours, and then seed fed to fermentation in larger reactors - fermenter volume of 100 m3. Fermentation process lasts ~ 70 hours at 23 - 24 ° C, pH 6 - 6.5 and intensive aeration (1 units. Air volume for 1 min at 1 m. Environment volume) [2].To stabilize the reaction medium using chalk. When the number reaches a maximum penicillin fermentation ceased. Dynamics of mycelium penicillin biosynthesis and consumption of lactose from the environment shown in Fig. 6.Rice. 6. Dynamics of the biosynthesis of penicillin mycelium formation and assimilation of lactose: X - dry mycelium (mg/100 g), P - concentration of penicillin (1000 units. / Ml), S - lactose concentration (mg / ml) .

The first phase - the growth of mycelium, antibiotic yield is low. Always present in corn extract producer of lactic acid consumed at maximum speed, lactose consumed slowly. Oxygen consumption - high. Growing nitrogen metabolism, resulting in an environment appears ammonia and dramatically increasing pH.

Temperatures during the first phase should be 30 ° C, pH during the growth of the fungus should be below 7.0.The second phase - the maximum penicillin formation is due to the rapid consumption of lactose and ammonium nitrogen. pH remains virtually unchanged, a slight increase in the mass of mycelium, oxygen consumption decreases. Temperatures during the second phase should be 20 ° C.

The third phase - reducing the concentration of antibiotics in the environment in connection with the beginning of autolysis of mycelium and release as a result of this process, ammonia, accompanied by an increase in pH.[5]



Filtering

Usually to separate the mycelium from the culture medium used vacuum drum filters are continuous. Filtering begin before autolysis of mycelium, because the filtering Autolyzed culture mycelium does not form a film on the surface of the filter drum and sticks in the form of individual thin lumps that do not depart in the zone of "stripping" of the filter, and they have to be removed manually. The duration of filtration is increased by 2 - 3 times, the yield drops sharply filtrate, and the filtrate is very turbid.

We must carefully observe the conditions that prevent the destruction of penicillin during filtration - native solution cooled to 4-6 ° C and systematic (every boot) filter processing, communications and collections antiseptics, such as chloramine. The filter should also systematically sterilized with saturated steam.[1]

Pre-treatment of native solution.

Native solution (culture filtrate) is a more or less turbid, colored yellow-brown or greenish-brown liquid. The pH of the medium, depending on the strain of the producer, the medium composition and the duration of the fermentation process typically ranges from 6.2 to 8.2.A very important characteristic of the native solution is the content of proteins, determined by precipitation with trichloroacetic acid, or other appropriate means.

Used several methods pretreatment native solution to free from contaminating proteins: precipitation polyvalent metal salts (for example, A13 + Fe3 + or Zn2 +), coagulation tannin, thermal coagulation at 60-75 ° C and pH 5.5 - 6.0, the deposition impurities cationic detergents such as quaternary ammonium compounds. Application of these methods results in a loss of antibiotic. Usually as a result of coagulation and subsequent filtration or separation is lost between 5 and 15%) of penicillin. In this case, coagulation with metal salts can remove up to 50% of total proteins [4].



Extraction and purification of penicillin

Native solution contains 3-6% solids. On minerals account for 30-40% of total solids, 15 to 30% for penicillin, and the rest is a complex mixture of organic substances, including proteins, polypeptides, low molecular nitrogen compounds, carbohydrates, and various organic acids, depending on the strain of the producer, a certain amount of pigment. To isolate penicillin from this complex mixture can use methods based on adsorption, extraction or precipitation.

In industry, the extraction of the active substance from the native solution is based on the extraction of water-immiscible solvent in suppressing the dissociation of the carboxyl group of penicillin. In thinner than penicillin, passes most of the organic acids. Chemical pollution, most of the nitrogen compounds and other organic substances remain in the aqueous phase, so that the purity of the product by extraction increased by 4-6 times.

To solvents used for the extraction of penicillin, must meet the following basic requirements:

1) low solubility in water;

2) the lack of interaction with penicillin;

3) low vapor pressure at the temperature of 5-30 ° C;

4) the possibility of regeneration at a temperature of 120 - 140 °;

5) low cost.

At acidic pH, penicillin is unstable, so the extraction of penicillin in the organic solvent is necessary to strictly control the pH, keeping it in the 1.9-2.0 range, extraction is carried out in the shortest possible time, the cooling fluid.

In the extraction of penicillin from the native form of the solution is very persistent due to the presence of the native solution of surface-active substances. Usually used for this purpose anionic detergents, for example sulfonated fatty or naphthenic acid. Usually the choice of detergent is determined by its availability and economic considerations. For the separation of the emulsion in the extractors, separators, usually enough to add to the native solution of 0.05-0.1% detergent.

The extraction step of penicillin native solution using either multi-extractors separators "Luvesta" and "Russia" or the two-stage scheme of extraction (contacting a native solution acidified with butyl acetate in a special mixer and separation of emulsions such as centrifugal separators Sage-3). The use of efficient centrifugal extractors, separators (with a capacity of 4000-5000 liters / hour), providing at least two stages of extraction in one car and a good phase separation, minimizes the residence time of penicillin in acidic aqueous medium and, therefore, increases the yield of the antibiotic. The use of two-stage scheme for the extraction of penicillin from the native solution, of course, is undesirable not only because of the longer residence time of penicillin in adverse conditions, in this case, but also due to the fact that the use of separators Sage-3 (whose performance is in the range 800 - 1000 l / h) does not always provide a fairly complete phase separation. This entails a deterioration of butyl acetate extract (pollution native solution) and an increase in loss of butyl acetate with used native solution. Phase relationship during Butyl extraction of penicillin from the native solution is 1,0:0,3-0,45, temperature 4-3 ° C.

After the extraction of penicillin from Butyl native solution is extracted penicillin from butyl acetate extract of an aqueous solution of sodium bicarbonate or buffer at pH 6,6-7,2. At this stage it is also used multistage extraction machine or use a two-stage countercurrent extraction with separation of emulsion separators with attitude solvent-aqueous phase 1.0:0,35. The output buffer for Butyl and extractions is about 90-92%.For further purification of penicillin re-extracted from the organic solvent extract buffer (usually butyl acetate or chloroform) at pH 2.0. The process is similar to butyl acetate extraction from native solution. This stage of technology is made also with the use of multi-stage extraction machines or takes the form of a two-stage countercurrent extraction phase separation on separators. The yield is about 86% of the penicillin contained in the native solution.

The whole extraction process of extraction and chemical treatment of penicillin carried out by a continuous scheme [5].



Isolation of crystalline salts of penicillin

The most reliable methods that provide a good quality of crystalline penicillin, penicillin is the allocation of butyl acetate extract in the form of a concentrated aqueous solution of the potassium salt followed by evaporation of water with butanol under vacuum, resulting in the crystallization of the potassium salt of butyl alcohol.

This process has the following technological sequence:

1. Butyl acetate extract of dehydration by cooling to -16-18 ° C, followed by filtration of ice. Removal of pigment pollution treatment and activated carbon filtration is cold druk-filter.

2. The concentrate of the potassium salt of benzylpenicillin extraction 0,56-0,6 N sodium hydroxide.

3. Sterilizing filtration of concentrate of potassium salt and evaporation under vacuum with butyl alcohol (2.5 volume) at 16-26 ° and a residual pressure of 5-10 mm Hg. Art. Volume bottoms should be no more than 60-80% of the loaded concentrate. Adding butanol to concentrate by evaporation under vacuum, due to the fact that the butanol with water forms a mixture boiling at a lower temperature than the boiling point of water. Distillation of water held in relatively mild conditions, resulting in the possibility of inactivation of penicillin decreases. After removal of the water and most of the butanol benzylpenicillin potassium salt crystallizes.

4. Filtering the precipitate of the potassium salt of benzylpenicillin on the centrifuge and filter cake washing anhydrous butyl alcohol.

5. Granulation and drying a paste of potassium chloride in a vacuum oven at a temperature of 75-80 ° and a residual pressure of 10-20 mm Hg. Art. This yields the potassium salt of penicillin as a white fine crystalline powder with an activity of penicillin content about 95% and a yield of 70% of the antibiotic in the native solution.

The most important requirement for a dry powder obtainable penicillin, is its complete sterility. Heat treatment of the drug is not enough. Sterility can be achieved only during the final stages of the process in a strictly aseptic conditions, precluding the possibility of product contamination by micro-organisms and their spores. Therefore, starting from the sterile filtration of concentrate and butanol, all operations are conducted in an isolated clean room and sterile equipment. To ensure aseptic conditions are of all necessary sanitary and technical measures.

Before regeneration, butyl acetate and butanol, used in the process of separation and chemical treatment, penicillin, washed with alkali to remove impurities acids [3].

Calculation of a shell-and-tube heat exchanger

Calculate a shell-and-tube heat exchanger for cooling of V=1050 m3/h (in standard conditions) of air from 140 oC to 31oC with water. The pressure of the air is p=0,35 mPa. The cooling water, which gives a deposit of scale, has a temperature of 16 oC. Solution. We assume that the water in the heat exchanger becomes heated to 35 oC. The temperature scheme of the heat exchanger with counter flow is (hot fluid - air; cold fluid - water):

thi=140 oC; tho=31 oC;

tco=35 oC; tci=16 oC.

The temperature difference:

the greater temperature difference - Дgr= thi - tco=140 - 35=105 oC;

the smaller temperature difference - Дsm= tho - tci=31 - 16=15 oC.

The mean logarithmic temperature drop:



The mean temperature of the water is:

The mean temperature of the air:

The amount of heat transferred from the air to the water is:

where сair - the density of air in standard conditions (t=0 oC, p=101325 Pa); cair - the mean specific heat of air.

We find the density of air in standard condition from Table 1: сair=1,293 kg/m3.

The mean specific heat of air is about cair=1000 J/(kg•K).

Substituting the values to the latest formula we obtain:

The specific heat of water at twat=15 oC from Table 2 is cwat=4190 J/(kg•K).

The mass flow rate of the water is:



We determine approximately the required surface area of the shell-and-tube heat exchanger. Since scale may be deposited on the surface of the tubes at the side of the water being heated and this greatly hinders the transfer of heat. It is necessary to provide for the possibility of cleaning the heat exchanger. For this reason, the water should flow through the tubes, whose internal surface in a shell-and-tube heat exchanger is easily accessible for cleaning.

To increase the coefficient of heat transfer of the air (it is much lower than that for water), we adopt the design of the heat exchanger with cross flow of the air around the tubes, i.e. with baffles on the shell side.

We adopt approximately an overall heat transfer coefficient U=60 W/(m2•K). Hence the required heat exchange surface area is:

According to the data of Table 3, we adopt a single-pass shell-and-tube heat exchanger (with the nearest heat transfer area - 14,8 m2) with a shell diameter of 400 mm, and with 121 tubes 1,5 m long. The diameter of the tubes is 25x2 mm (the inner diameter di=0,021 m; the outer diameter do=0,025 m; the width of a tube - д=0,002 m) and their pitch (the distance between their axes) is 32 mm.

We perform more accurate calculations of the heat exchange surface area. We assume that the Reynolds number for air is Reair=20000 and calculate the overall heat transfer coefficient for a triangular pitch of the tubes (Prair -is the Prandtl number at a temperature tair; from Table 1). We have:

The thermal conductivity of air from Table 1 for tair=71,65 oC is about kair=0,03 W/(m•K). Hence, the convective heat transfer coefficient for air is:



We determine the conditions of flow of the water in the tubes.

The velocity of the water is:

The dynamic viscosity of water at twat=25,5 oC from Table 2 is мwat =902•10-6 Pa•s. The Reynolds number is:

We have a laminar flow. To find the convective heat transfer coefficient for water, we must calculate the product (taking the value of Prandtl number from Table at a temperature twat):

If the than we can use the formula:

where мwall - is the dynamic viscosity of water at the wall temperature, from Table 2 (assume, that the mean temperature of the wall is twall=(twat+tair)/2).



We have that So, we use the formula:

Nuwat=5,17*1,04=5,38

So, the corresponding value of convective heat transfer coefficient:

where kwat - the thermal conductance of water at a temperature twat (Table 2 ).

The overall heat transfer coefficient is:

The required heat transfer area:

We adopt the heat exchanger with greater surface area - 18 m2 from Table 3 (with the length of - 2 m; the inner diameter of shell - 400 mm; number of tubes - 121; the diameter of the tubes - 25x2 mm)



Table 3 - The thermal properties of air (p=760 mm Hg)

Temperature t, oC	Density, с, kg/m3	Specific heat, c, J/(kg•K)	Thermal conductivity, л, W/(m•K)	Kinematic viscosity, з, m2/s	Prandtl number Pr
0	1,293	1004,8	0,0245	13,28•10-6	0,707
10	1,247	1004,8	0,0252	14,16•10-6	0,705
20	1,205	1004,8	0,026	15,06•10-6	0,703
30	1,165	1004,8	0,0268	16,00•10-6	0,701
40	1,128	1004,8	0,0276	16,96•10-6	0,699
50	1,093	1004,8	0,0284	17,95•10-6	0,698
60	1,060	1004,8	0,0291	18,97•10-6	0,696
70	1,029	1009	0,0297	20,02•10-6	0,694
80	1,00	1009	0,0306	21,09•10-6	0,692
90	0,972	1009	0,0314	22,10•10-6	0,690
100	0,946	1009	0,0322	23,13•10-6	0,688
120	0,898	1009	0,0335	25,45•10-6	0,686
140	0,854	1013,2	0,0349	27,80•10-6	0,684
160	0,815	1017,4	0,0364	30,09•10-6	0,682

penicillin fermentation purification salt

Table 4 - The thermal properties of water (on saturation line)

Temperature t, oC	Density, с, kg/m3	Specific heat, c, J/(kg•K)	Thermal conductivity, л, W/(m•K)	Dymamic viscosity м, Pa•s	Prandtl number Pr
0	999,9	4230	0,551	1790•10-6	13,67
10	999,7	4190	0,574	1310•10-6	9,52
20	998,2	4190	0,599	1000•10-6	7,02
30	995,7	4180	0,618	804•10-6	5,42
40	992,2	4180	0,638	657•10-6	4,31
50	988,1	4180	0,648	549•10-6	3,54



Table 5 - Basic characteristics of shell-and tube heat exchangers with tubes having a diameter of 25x2 mm. Tube pitch is 32 mm



Conclusion

Consequently, industrial production process benzylpenicillin passes the following stages:

1. Selection of high-producer strain.

2. Preparation of inoculum and nutrient medium.

3. Phase biosynthesis.

4. Phase pretreatment culture fluid.

5. Phase extraction and purification of the antibiotic.

6. Phase obtain the finished product.

7. QA benzylpenicillin.

The manufacture benzylpenicillin pay special attention to the inclusion in the culture medium of precursor substances, ie compounds that producer will use for the synthesis of benzylpenicillin. These include phenylacetic acid, phenylacetamido, fenoksyotstova acid.In modern conditions of production shall take measures to minimize the cost of drugs through the intensification of all stages of the manufacturing process and, above all, increasing the efficiency of the first stage - the biosynthesis of antibiotic substances.

To do this:

a) the introduction of the most high strains-producers of antibiotics;

b) the establishment and maintenance of favorable conditions for the development of antibiotic producers at a relatively cheap media;

c) extensive use of mathematical methods of planning, the development of the body and electronic computers for optimization and simulation conditions for its cultivation, ensuring maximum yield antibiotic;

d) the use of modern equipment at all stages of the process with automated systems that control the basic parameters of the organism and stages of the biosynthesis of the antibiotic.



Reference

1. Прищеп Т.П., Чучалин В.С., др. Основы фармацевтической биотехнологии: Учебное пособие. - Ростов н/Д.: Феникс; Томск: Издательство НТЛ, 2006. - 256 с. - (Высшее образование).

2. Тимощенко Л.В., Чубик М.В. Основы микробиологии и биотехнологии: Учебное пособие. - Томск: Изд-во Томского политехнического университета, 2009. - 194 с.

3. Елинов Н.П. Химическая микробиология: Учеб для студентов химикотехнол., технол., фармац., и др. ин-тов, аспирантов и практ. работников. - М.: Высш. шк., 1989. - 448 с.

4. Бекер М.Е. Введение в биотехнологию. Пер. с латышского. - М.: издательство «Пищевая промышленность», 1978. - 228 с.

5. Елинов Н.П. Общие закономерности строения и развития микробов-продуцентов биологически активных веществ. - М., 1977. - 288 с/

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