Technology of modern antibiotics production

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MINISTRY OF EDUCATION AND SCIENCE OF UKRAINE

NATIONAL AVIATION UNIVERSITY

INSTITUTE OF ECOLOGICAL SAFETY

DEPARTMENT OF BIOTECHNOLOGY

SEMESTER PAPER

(Explanatory Note)

On the discipline: “General Biotechnology”

Theme: “Technology of modern antibiotics production”

Student: Litvin Irina

Group IES 304

Leader: Karpenko V.I.

Kyiv 2013



CONTENTS

ABSTRACT

INTRODUCTION

1. NEW APPROACHES TO RESEARCH AND CONSTRUCTION MICROBIAL PRODUCERS OF ANTIBIOTICS

2. STRATEGIES FOR MODERN ANTIBIOTICS

3. TECHNOLOGY FOR ANTIBIOTIC PRODUCTION

3.1 Receiving producers of antibiotic substances

3.2 Antibiotics biosynthesis

3.2.1 Nutrient mediums for biosynthesis

3.2.2 Preparation of inoculum

3.2.3 Development of antibiotics producer in fermenters

3.2.4 Prediction of yields in continuous antibiotic

fermentation systems

3.3 Isolation and purification

3.4 Refining and quality control

4. WASTES OF PRODUCTION AND ENVIRONMENTAL PROTECTION

5. MATERIAL BALANCE

6. PROBLEMS AND PROSPECTS OF NEW ANTIBIOTICS BIOTECHNOLOGICAL PRODUCTION

CONCLUSIONS

LIST OF REFERENCES



ABSTRACT

In this cource work, current state and development at trends in antibiotic biotechnology have been analyzed. Reasons prompting the search of novel and improvement of existing antibiotics have been elucidated. Current strategies of investigations are discussed, particularly those that are aimed to increase the efficiency of screening of new antibiotics, optimization of conditions of antibiotic biosynthesis as well as genetic and gene engineering methods of construction of antibiotic producers. Data are presented on biotechnological production of antibiotics and their practical use in the world and Ukraine.

Microbial resistance is emerging faster than we are replacing our armamentarium of antimicrobial agents. For example resistance to penicillin developed soon after it was introduced into clinical practice in 1940s. Now resistance accompanies the application of every major class of antibiotics. In healthcare facilities around the word, bacterial pathogens that express multiple resistance mechanisms are becoming common. The origins of antibiotics resistance genes can be traced in the environmental microbiota. A coordinated effort from government, public and industry is needed to deal with antibiotic resistance health care crisis. Therefore, mechanisms and origins of antibiotic resistance have been discussed.

And finally in this work was regard mechanism for new antibiotics cultivation which include obtaining a strain - producers of antibiotics suitable for industrial production, biosynthesis of antibiotics, antibiotics isolation, purification, refining and quality control. The methods for environment protection are discussed and shown material balance for modern antibiotic production.

microbial antibiotic biosynthesis



INTRODUCTION

The history of the production and use of antibiotics has more than half a century. Since the opening of Alexander Fleming in 1929 producer of penicillin

Penicillium notatum in the development of biotechnology as a science and industry began an era of antibiotics.

Antibiotics were among the first biologically active products of biotechnology, production of which has been established.

The term "antibiotic" is called substances of microbial, plant or animal origin, which can inhibit the growth of organisms and cause their death. In contrast, synthetic and semi-synthetic substances that exhibit antimicrobial activity, called "antibacterial chemotherapy."

Representatives of the latter is sulfonamides, fluoroquinolones, quaternary ammonium salts, and others [1].

Opened more than 25 thousand of antibiotic compounds which are synthesized by plants, animals, fungi and bacteria [2]. However, practical applications in medicine, veterinary medicine and agriculture found only about 150 compounds.

While in medical practice exacerbated the problems that arise when use of antibiotics and chemotherapy.

The main ones are the rapid development of resistance to antiseptics, hospital infection and formation phenomenon biofilms by microbial pathogens on surfaces of instruments, implants etc. [3, 4].

Research has established that microbial biofilms are responsible for the etiology and pathogenesis of many acute and especially chronic bacterial infections in humans.

In this case, infectious disease, which is the etiological agents are biofilms can be caused as a representative of the same species, and succession species of bacteria.

These include, for example, periodontal disease, caries, otitis media, respiratory tract infections occurring in cystic fibrosis, bacterial prostatitis, infectious endocarditis, etc. [4].

Group of diseases that constitute a particular danger are nosocomial (hospital) infections, the development of which is one of the greatest problems in long-term treatment of patients in stationary department of hospitals. Thus, approximately of 5 million cases of infectious diseases are diagnosed each year in hospitals, about 40% acquired in hospitals (hospital infectious disease) [5, 6].

The solution to these problems lies in the use of combined therapy, usage antibiotics only after the establishment of the type of pathogen and the rational use of chemotherapeutic agents [7, 8].



1. NEW APPROACHES TO SEARCH AND CONSTRUCTION MICROBIAL PRODUCERS OF ANTIBIOTICS

Today we know more than 1 million natural compounds. Most of them (50 -60%) isolated from plants, and 5% are of microbial origin.

According to current estimates, 25 to 30 thousand natural compounds exhibit antibiotic activity. Allocated over 13 thousand antimicrobial , anticancer and antiviral compounds of plant origin, and about 7,000 antibiotic compounds that form different animal organisms, especially marine (sponges , coelenterates , tunicates, clams , etc.).

Approximately 1,700 natural compounds of microbial origin exhibit antibiotic activity.

Actinomycetes constitute 45% of bioactive microbial compounds, fungi - 38%, and bacteria - 17%.

The most well-known microbial antibiotics also produced by actinomycetes, especially members of the genus Streptomyces. Antibiotics of actinomycete origin constitute the majority among those used in medicine, veterinary medicine and agriculture (see table 1) [9, 10].

Sequencing of microbial genomes shows that they are able to synthesize 5-10 times more secondary metabolites than that found during the traditional screening. For example, in each of the sequenced genomes of actinomycetes identified more than 20 gene clusters synthesis of secondary metabolites, most of which are silent [11, 12]. Thus, the structural diversity of natural compounds is far from exhausted, and the understanding of the mechanisms of evolution of the diversity of experimental simulations have great theoretical and practical value.



Table 1

Number of antibiotics produced by different microorganisms

Source	Summary amount	Number of antibiotics used in practice (including thoseused for human therapy)
Bacteria	2900	10-12 (8-10)
Eubacteria	2170
Bacillus sp.	795
Pseudomonas sp.	610
Mixobacteria	400
Cyanobacterium	300
Actinomycetes	8700	100-120 (70-75)
Streptomyces sp.	6550
Rare actinomycetes	2250
Fungi	4900	30-35 (13-15)
Microscopic fungi	3770
Penicillium/Aspergillus	1000
Basidiomycetes	1050
Yeasts	105

The above-mentioned microorganisms and today remains the main objects for developers of new and modified antibiotics. Analyzing the literature of the last decade, there are several priority areas of research and development, which are most active work:

· Search the producers of new antibiotics;

· Antibiotic selection to solve specific problems (problems of formation of biofilms by microbial pathogens, nosocomial infections, etc.);

· Optimization of conditions biosynthesis of antibiotics known microbial producers;

· Genetic engineering designing producers with new properties.

Optimization of biosynthesis conditions makes it possible to increase yield and make its production efficient. Thus, the study of features of the biosynthesis of ribavirin culture Bacillus subtilis obtained data on the decisive influence of the temperature regime on product yield [13].

Maintain the temperature in the first 24 hours at the level of 36° C and a gradual increase every 6 hours at 2° C made ??it possible to get the most product in the 60th hour of cultivation.

Adding 0.5% Tween-80 is an additional factor in optimizing the biosynthesis of ribavirin.

About decisive, but the reverse effect of temperature on the biosynthesis of bacteriocins in Bacillus cereus according to the findings of other researchers [14]. It was found that the optimum temperature for this producer is 25-30°C and a critical decline in product synthesis observed at higher temperatures than 37°C.

Number of microbial cultures inherent significant increase in the synthesis of antibiotics or changing direction of synthesis under conditions of stress induced by components of nutrient medium.

Thus, a critical increase in the concentration of mineral salts in the culture medium Streptomyces coelicolor causes a change in the balance of the synthesis of two antibiotics - actinorodin and undecylprodygiocine that caused the expression of different genes in these conditions [15].

If "fasting" other streptomycetes Streptomyces parvullus increased synthesis of actinomycin D [16, 17]. In addition, during the optimization process of biosynthesis is suggested to use the culture of this strain immobilized in calcium alginate, and hold it in semi-batch cultivation, eliminating the constant training of seed and improves overall efficiency.

Mixing and aeration are critical factors in the biosynthesis of antibiotics by many cultures. For example, the producer of antimicrobial and antifungal antibiotic Xenorhabdus nematophila proved that these parameters optimization makes it possible to significantly improve the yield [18]. Identified three phases during which the necessary changes in the level of aeration and agitation within 30%.

Summarizing data on directions optimize the conditions of the biosynthesis of antibiotics can be noted in common - the need to change most of the gradient factors (temperature, aeration, etc.) during the different stages of cultures to obtain maximum product.

Today it is generally accepted idea that dramatically increase the effectiveness of antibiotic can only introducing new antibiotics in the clinic of the classes that had not been used, or being used very rarely. Therefore, the search for new antibiotics and modification known for their improvement is one of the main areas of modern biotechnology.

From the beginning of the era of antibiotic and still screened bacteria isolated from different ecotopes, has been a major source of new bioactive compounds, including antibiotics.

However, the discovery of our time, the antibiotic that belongs to a new chemical class or are already an advanced version of the well-known compound - an extremely rare event. Evidently, there is a need to change the approach to the search for new natural compounds.



2. STRATEGIES FOR MODERN ANTIBIOTICS

Processed number of strategies aimed at optimizing the search for new antibiotics.

Firstly, is selection of antibiotic producers of exotic and unexplored ecotypes (eg. endosymbiosis of plants and animals, those found in the rhizosphere of rare plants in the sediment cores of the seabed, etc.), and screening of new microbial taxons in synthesis of bioactive compounds. An example of the successful use of this approach is the discovery of a new chemical class of antibiotics - abisomicyne a new genus of marine actinomycetes Verruscosispora [19].

Another strategy - is the use of screening programs with high sensitivity. This high sensitivity inherent in screening based on antisense technologies. When the intracellular level of the target, to which acts the required antibiotic reduced as a result of the relevant antisense-RNA test strains are more sensitive to the antibiotic. Thus we can identify compounds that by standard conditions did not inhibit the growth of test strains wild type. With the help of this method revealed a new class of antibiotics, which include platensimycine, producing strain Streptomyces platensis [20].

Another example is the sensory system for the rapid detection of three-, penta- and hexaglycosided landomycin that exhibit high antitumor activity. It used a strain of Streptomyces albus, carrying plasmid reporter gene with resistance to neomycin and kanamycin neo. It is under the transcriptional control of the promoter PlanK-J gene and lanK, originating from the biosynthetic gene cluster landomycin A S. cyanogenus S136. The lank gene encodes a repressed protein in the absence of cell polyglycosilided landomycin binds to promoter PlanK-J and suppresses the expression of genes that are transcribed from it. When three-, penta-and hexaglycolysilided landomycin accumulate in the cell above a certain threshold, they bind to the repressor and deny it the ability to interact with PlanK-J.

As a result of the rapid development of genetic engineering not only accumulated a lot of information about the structure and function of genes and genomes, but also established methods for the manipulation of large DNA molecules, as well as whole chromosomes, based on the process of homologous and site-specific recombination [21]. These methods are widely used for manipulating genomes bacteria-antibiotic producers. In particular, the constructed strains "super hosts" to clone entire biosynthetic gene cluster of antibiotics with different types of actinomycetes. In the future, we plan to use these strains as universal biotech producers of various antibiotics. Perform "optimize" the genomes of these strains by removing one genomic islands, which usually contain genes that are not required for the existence of bacteria in a relatively stable environment for industrial fermenter, and mobile genetic elements.

Obviously, creating strains of antibiotic-overproducers impossible without the significant changes in the management of the biosynthesis of antibiotics. Production of antibiotics usually begins in early stationary phase of growth in liquid culture and coincides with the beginning of morphological differentiation in culture growing on agar medium [22].

We now know that genes regulating the biosynthesis of secondary metabolites form a hierarchical system which includes both pleitropic genes global regulation affecting the biosynthesis of many antibiotics and genes that specifically regulate the biosynthetic pathway specific antibiotic. The latter plays a crucial role in the initiation of synthesis of the antibiotic, and their mutations can completely stop it.

All this suggests that the development of genetics and genomics of industrial microorganisms, as well as improved methods of genetic engineering led to a qualitatively new level of design biotech producers of antibiotics.



3. TECHNOLOGY FOR ANTIBIOTIC PRODUCTION

Figure 3.1 The scheme of antibiotic production in the microbial biosynthesis



Production of antibiotics involves four basic steps (Figure 3.1):

1) obtaining a strain - producers of antibiotics suitable for industrial production;

2) biosynthesis of antibiotics;

3) antibiotics isolation and purification ;

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

Antibiotics are produced industrially by a process of fermentation, where the source microorganism is grown in large containers (100,000-150,000 liters or more) containing a liquid growth medium . Oxygen concentration, temperature, pH and nutrient levels must be optimal, and are closely monitored and adjusted if necessary. As antibiotics are secondary metabolites, the population size must be controlled very carefully to ensure that maximum yield is obtained before the cells die [23].

Once the process is complete, the antibiotic must be extracted and purified to a crystalline product. This is simpler to achieve if the antibiotic is soluble in organic solvent. Otherwise it must first be removed by ion exchange, adsorption or chemical precipitation.

3.1 Receiving producers of antibiotic substances

The first task when searching for producers of antibiotics - extraction them from natural sources. Producers of antibiotics may be isolated from a wide variety of substrates: soil, decaying plant and animal residues, sludge, water, lakes and rivers, air and other sources. Most rich in microorganisms producing antibiotics is soil.

To isolate microorganisms - producers of antibiotics from natural habitat used a variety of methods. The basis of most of the techniques based on the principle of isolating pure cultures of the microbe and direct test it against used test organisms. However, as noted above, essential value in the formation of antibiotic substances have a mixed culture. About this fact is also necessary remember when searching for producers of antibiotic substances [24].

However, for obtaining the most active strain is widely used method of changing the genome of selected antibiotic producers by mutagenesis and genetic engineering. Mutation is often used, and is encouraged by introducing mutagens such as ultraviolet radiation, x-rays or certain chemicals. Selection and further reproduction of the higher yielding strains over many generations can raise yields by 20-fold or more. Another technique used to increase yields is gene amplification, where copies of genes coding for enzymes involved in the antibiotic production can be inserted back into a cell, via vectors such as plasmids (Figure 3.1.1). This process must be closely linked with retesting of antibiotic production and effectiveness.

Figure 3.1.1 Plasmids used in genetic engineering are called vectors.

3.2 Antibiotics biosynthesis

The basis of most methods for extraction of the producers lies on the principle of obtaining a pure culture of the microbe and its direct testing in relation to the used test organisms. Most saprophytic bacteria is well developed in the rich composition natural environments (meat peptone agar, potato agar, wort-agar, etc.) at pH of about 7.0 and a temperature of 30-37° C. Under these conditions develops actinomycetes and some fungi, but they are less favorable than those for bacteria [25].

Among the most significant factors influencing the expression of antibiotic properties of microorganisms include the composition of the medium, its active acidity, redox conditions, cultivation temperature, joint cultivation of two or more microorganisms and other factors.

Actinomycetes grow more slowly than bacteria, they can use such a power source that is not well assimilated by bacteria. The pH of the medium after sterilization is set within 6,8-7,1. Filamentous fungi develop better in environments with slightly low pH (4.5-5.0), which do not grow a lot of bacteria and actinomycetes.

3.2.1 Nutrient mediums for biosynthesis

For antibiotic producers cultivation commonly used such nutrient mediums:

1. Meat peptone broth (MPB) in which in addition to meat and peptones add sodium chlorine, potassium phosphate, sometimes glucose or sucrose.

2. Potato medium with glucose and peptone is used in laboratory for cultivation of many types of bacteria and actinomycetes.

3. Nutrient medium with corn extract, soybean meal and other substances, which include ammonium sulphate, calcium carbonate, phosphates, glucose, sucrose, lactose.

Such nutrient mediums are natural and therefore successfully used in industry because they are cheaper and ensure the development of microorganisms high yield of antibiotics.

Because the natural nutrient mediums does not allow for rigorous quantitative data for study the physiological and biochemical characteristics of the organism, use synthetic mediums that is selected for individual producers individually.

Carbon source can be organic acids, alcohols, carbohydrates and combination of different compounds. In industrial production of some antibiotics as a source of carbon is used potato starch, corn flour and other plant materials.

Sources of nitrogen greatly influence the formation of antibiotics by microorganisms. Usually in a media for culturing microorganisms as a source of nitrogen are nitrates, ammonium salts of organic and inorganic acids, amino acids, proteins and products of their hydrolysis.

The source of mineral nutrients are phosphorus, sulfur and other micro-and macroelements. Most microorganisms easy to use as a source of phosphorus orthophosphate. Antibiotic producers in relation to the concentration of phosphorus in the environment can be divided into 3 groups:

1) High sensitive producers for which the optimal concentration of phosphorus in the medium is less than 0,01 % (producers of nystatin, florimycin, tetracycline, vancomycin).

2) Producers of average sensitivity, for which the optimal concentration of phosphorus in the medium is 0,010-0,015 % (producers of streptomycin, erythromycin, cycloserin, neomycin).

3) Insensitive producers for which the optimal concentration of phosphorus in the medium is 0,018-0,20 % (producers of novobiocyn, gramicidin, oleandomycin).

Sulfur is a part of some antibiotics formed by fungi, bacteria and actinomycetes. Usually sulfates are the source of sulfur in the medium.

3.2.2 Preparation of inoculum

Preparation of inoculum is one of the most crucial operations in a cycle of biotechnological method of obtaining antibiotics. The quantity and quality of the material depends on both the seed culture growth in the fermenter, and the biosynthesis of the antibiotic. Producer of antibiotics commonly grown on the rich composition of natural mediums, capable of providing the highest physiological activity of microorganisms. Preparation of inoculum is a multistage process (Figure 3.2.1).

Figure 3.2.1. Scheme of multistage preparation of inoculum. A) growing in bottles; B) Cultivation in flasks.

1 - preserved raw material; 2 - generation at slanting agar in a test tube; 3 - II spore generation on solid medium in the vessel; 3a, 3b - I and III generating in liquid medium in a flask; 4 - fermenter for prior inoculation; 5 - fermenter for inoculation; 6 - major fermenter.

Microorganisms are cultured on agar medium in vitro (Fig.3.2.1, 2), and then make from the tube sowing into flasks with liquid nutrient medium and spend two generations in submerged culture for 2-3 days at each generation 3a, 3b. The second generation of the culture in the flask make sowing small (10 liters) inoculators 4, then a well-developed culture was transferred to a larger inoculator 5, (100-500 liters), from which seeded in the main fermenter 6.



3.2.3 Development of antibiotics producer in fermenters

The process of development of the microorganism in fermenters passes under the strict control of all its stages, very accurately performed devepoled regulation conditions for the development of an organism - the producer of the antibiotic.

Much attention is paid to maintaining the desired temperature of cultivation, the active medium acidity (pH), degree of aeration, and the speed of mixer. In the process of development of an organism is carried out biological control, accounted consumption by organisms of nutrient substrate (carbon, nitrogen, phosphorus), closely monitoring the formation of the antibiotic.

Considerable attention in the development of the producer in fermenters pay for defoaming process. While blowing air through the culture of the microorganism occurs copious foam, which gives substantially the entire process flow.

The main reason for the occurrence of large amounts of foam - the presence of proteins in the medium and high viscosity due to the abundant biomass accumulation.

To counter foam in fermentors use various surfactants such as animal oils, vegetable oils, alcohols and high fatty acids. Sometimes used mechanical methods of defoaming (foam suction through a special pipe, collapse of foam bubbles by strong streams of liquid, vapor or gas) [33].

The general scheme of the production of antibiotics to the stage of isolation and chemical treatment is represented in Figure 3.2.2.



Figure 3.2.2 Scheme of antibiotic production:

I - preparation of inoculum; II - inoculators for inoculums increasing; III - medium sterilizer for large fermenter; IV - setting for the biosynthesis of antibiotics: a - medium sterilization in flasks, b - cooling and sowing of producer into the flask, c - culture growth alone, d - culture growth in shaker, e - inoculators with sterile medium, f - inoculators with medium that sowed by producer culture, g - filters and compressors, h - reservoir with compressed air, i - heating of air, j - fermenter, A - shell for cooling fermenter.



3.2.4 Prediction of Yields in Continuous Antibiotic Fermentation Systems

· Cell growth

Since cell growth is autocatalytic, cell proliferation should be limited chiefly to the first stage of a seed tank system to insure most efficient operation of this phase of the process. To achieve this, a point relatively high along the cell growth curve should be chosen for operation as indicated by A in Figure 3.2.3. The holdup time necessary for maintenance of the desired cell density can be determined graphically.

Figure 3.2.3 Data of a novobiocin fermentation

· Antibiotic formation

The antibiotic level in the first stage will be relatively low and the spread of

residence times of the cells leaving this stage relatively narrow and can therefore be neglected. The approximate antibiotic titer to be expected in the cell generator

is indicated graphically at the point of intersection B (see Fig. 3.2.3) where a straight line drawn perpendicular to the abscissa through point A intersects the antibiotic formation curve.

This value will be slightly too high due to the negligence of the spread of residence times of the cells in the first stage. Since by operational means growth is limited essentially to stage 1, antibiotic formation in any subsequent stages will be primarily a function of the spread of residence times of the mycelium in the remaining stages. The yield to be expected in these stages can therefore be determined by applying the graphical method.

Mass-balance equation:

Rate of input + rate of increase = rate of formation - rate of output.

3.3 Isolation and purification

After three to five days, the maximum amount of antibiotic will have been produced and the isolation process can begin. Depending on the specific antibiotic produced, the fermentation broth is processed by various purification methods. For example, for antibiotic compounds that are water soluble, an ion-exchange method may be used for purification.

In this method, the compound is first separated from the waste organic materials in the broth and then sent through equipment, which separates the other water-soluble compounds from the desired one. To isolate an oil-soluble antibiotic such as penicillin, a solvent extraction method is used. In this method, the broth is treated with organic solvents such as butyl acetate or methyl isobutyl ketone, which can specifically dissolve the antibiotic. The dissolved antibiotic is then recovered using various organic chemical means. At the end of this step, the manufacturer is typically left with a purified powdered form of the antibiotic, which can be further refined into different product types.



3.4 Refining and quality control

Antibiotic products can take on many different forms. They can be sold in solutions for intravenous bags or syringes, in pill or gel capsule form, or they may be sold as powders, which are incorporated into topical ointments. Depending on the final form of the antibiotic, various refining steps may be taken after the initial isolation. For intravenous bags, the crystalline antibiotic can be dissolved in a solution, put in the bag, which is then hermetically sealed. For gel capsules, the powdered antibiotic is physically filled into the bottom half of a capsule then the top half is mechanically put in place. When used in topical ointments, the antibiotic is mixed into the ointment. From this point, the antibiotic product is transported to the final packaging stations. Here, the products are stacked and put in boxes. They are loaded up on trucks and transported to various distributors, hospitals, and pharmacies. The entire process of fermentation, recovery, and processing can take anywhere from five to eight days.

Quality control is of utmost importance in the production of antibiotics. Since it involves a fermentation process, steps must be taken to ensure that absolutely no contamination is introduced at any point during production. To this end, the medium and all of the processing equipment are thoroughly steam sterilized. During manufacturing, the quality of all the compounds is checked on a regular basis. Of particular importance are frequent checks of the condition of the microorganism culture during fermentation. These are accomplished using various chromatography techniques. Also, various physical and chemical properties of the finished product are checked such as pH, melting point, and moisture content [26].



4. WASTES OF PRODUCTION AND ENVIRONMENTAL PROTECTION

The main waste generated as a result of the isolation and purification of chemical antibiotics are the following: waste native solutions, aqueous and washings solutions, aqueous solutions of acids and alkalis after regeneration of ion exchange resins, spent activated carbon, bottoms after solvent recovery. In these waste harmful portion is made by antibiotics and their degradation products, as well as organic solvents. The principal task of improving the technology of antibiotics from native solutions in terms of reducing waste production are to increase the yield of the desired products and thereby reducing the loss of the antibiotic, reducing costs of raw materials at the stages of regeneration and improving the effectiveness of organic solvents.

Significant reduction of losses of antibiotics during their separation can be achieved by solving complex problems: improving the fermentation process in order to improve the quality of culture fluids, conducting efficient purification of native solutions from impurities that hinder the process of allocating antibiotics, reducing the number of process steps, a reduction in duration of processes, the use of effective high-efficiency equipment. Main gas emissions from companies producing antibiotics containing harmful substances include air emissions and local general dilution ventilation and technological air emissions in the biosynthesis of antibiotics, boiler emissions and some other auxiliary productions. Various purification methods can capture about 60% of the harmful substances from all sources of pollution.

Gaseous pollutants are composed essentially of carbon monoxide (77.4%), sulfur dioxide (15,2%) and nitrogen oxides (7.4%).Specific for antibiotics producing liquid and gaseous products include vapors of organic solvents is 24.3% of the total amount of emitted substances (Table 2).



Table 2. Qualitative and quantitative composition of organic solvents in air emissions of the production of antibiotics

Names and classes of substances	Solvent content of the emission in %
1. Alcohols	55,27
Ethanol	26,26
Butanol	16,69
Methanol	8,20
Isopropyl alcohol	4,00
Propyl alcohol	0,07
Isooctyl alcohol	0,05
2. Esters	32,22
Butyl acetate	30,66
Ethyl acetate	1,56
3. Acetone	9,26
4. Chlorine derivatives of carbohydrates	2,88
Methylene chloride	2,37
Carbon tetrachlorine	0,39
Trichloroethylen	0,09
Chloroform	0,03
5. Carbohydrates	0,32
Petrol	0,27
Benzene	0,05
6. Ethers	0,05
Diethyl ether	0,04
Dimethyl ether	0,01

In addition, air emissions in a variety of impurities present vapors of different substances constituting 0.4 % of the total amount released into the atmosphere of liquid and gaseous products. Among the predominantly hydrogen chloride, hydrochloric acid vapors, formaldehyde and tricresol. Nonspecific to produce antibiotics solids trapped gas emissions dust treatment plants by 90%, gaseous emissions boilers scattered through tall chimneys. Specific antibiotics for the production of solids from air emissions by 92.5%, organic solvent - 10%, neutralized 5.4% by volume of air emissions during biosynthesis of antibiotics.



5. MATERIAL BALANCE

During receiving of 3 tons of antibiotic 70-80% of the byproduct is formed - minerals, complex mixture of organic substances, including proteins, polypeptides, low molecular weight nitrogenous compounds, carbohydrates, and various organic acids, depending on the producer strain, or that the amount of pigment and irrecoverable loss is 5%.

Accepted byproducts = 70%

The material balance equation is:

C1 - Fungus of producer strain;

C2 - an antibiotic;

C3 - byproducts;

C4 - irrecoverable losses.

С1 = (С2+ С3)+ С4

For example for peniciline material balance is С1 = 3000+8400+600= 12000

Taken	kg	Received	kg
Penicillium chrysogenum	12000	Penicillin	3000
		By-products	8400
		Irrecoverable loss	600



6. PROBLEMS AND PROSPECTS OF ANTIBIOTICS BIOTECHNOLOGICAL PRODUCTION

Scope of biotech sector of the world market increasing annually by 10-15% over past years. Considerable part of the structure of the global biotech sector accounted for the production of pharmaceuticals [27].

At present, biotech leader in the pharmaceutical industry is the U.S., where produced almost 50 % of world production of this industry. That is the U.S. market is the main base for the introduction of new biopharmaceutical products. This is facilitated by less strict U.S. legislation on registration of biopharmaceutical products, understanding significant benefits that are based on biotechnology over traditional drugs. The fundamental difference is also significant (almost 5-fold) increase in financing the biotech industry over the past 10 years [28].

The largest segment of biotech drugs in the world - is an antibiotic for the treatment of human and animal diseases, as well as feed additives and premixes. In 2010, cost of sales of antibiotics in the world was 42 billion U.S. dollars, accounting for 46% of the market of anti-infective agents, which includes antiviral medicines and vaccines, as well as 5% of the global pharmaceutical market.

Over the past five years, the market of antibiotics increased by an average of 4% compared with growth on 16.7 and 16.4% market share of antiviral drugs and vaccines, respectively. The largest market share of antibiotics (28%, 11.9 billion in 2011) accounts for cefalosporins, mostly due to the widespread use of the latest generation of drugs in this class - cefcapen (flomocs, Shionogi), ceftriaxone (rotsefin, Roche) and cefuroxime (zinnat, GlaxoSmithKline). In second place in terms of sales (19%, 7.9 billion dollars) is a broad-spectrum penicillins market action, and the third - the fluoroquinolones (17%, 7.1 billion dollars). Interestingly, in some countries there is a positive tendency to reducing the use of antibiotics. In particular, France and Japan for the period 2000-2009 years of antibiotic use decreased by 21 and 15%, respectively. The main reason is to try to fight with the spread of multi-resistant pathogens through the rational use of antibiotics - only after these pathogens isolated and tested on antibiotic sensitivity or resistance [29].

In Ukraine, the development of modern biotechnology and their application in pharmacy only begins, although traditional biotechnology used in the manufacture of medical products for over 20 years. Currently, out of 418 agents required immunobiotechnological drugs domestic companies produce only 40 (9%), and 62 drugs of microbial origin, belonging to priority medicines produce only 10-12 items (19%) [30, 31].

The structure of the pharmaceutical biotech market in Ukraine differs on the structure of world market. Thus, the largest market segments that we have are drugs - probiotics, vaccines and serum. At present, the Ukrainian pharmaceutical market prevail imported probiotics. Foreign firms account for over 70% of our pharmaceutical market in this area. Only about 20-30% of it accounts for Ukrainian producer.

In Ukraine, a sufficiently large number of companies manufactures pharmaceutical biotech products are "Enzyme" (Ladyzhin), "Dniprofarm" (Dnepropetrovsk), "Biolik" (Kharkiv), NPK "Farmbiotek" (Kyiv), "Biostimulyator" (Odessa), "Indar" (Kyiv), "Biopharma" (Kyiv). The leading among them is "Indar" (Kyiv), which specializes in production of insulin, "Biolik" (Kharkiv) and "Biopharma" (Kyiv) that produce drugs, normobiotics, proteins of human blood, vaccines and serums for prevention and treatment of many diseases, and various diagnosticums [31].

Biotechnology production of non-medical purpose antibiotics is realized on such Ukrainian enterprises as plant feed additives in Novograd Volyn, and Zaporozhye plant biochemical research group "Ukrmedprom."

There are many reasons that hinder the development of new antibiotics. One of them - is the complexity and high cost of scientific development to create new drugs with a new mechanism of action. The second reason - commercial. Investment in the development of antibacterial drugs bring low income, as they are designed for short-term treatment of certain acute diseases. Absence of investments led to stopping the development of antimicrobial drugs by many companies. According the data published in January 2011 in “Clinical Infectious Diseases” journal only 5 the largest pharmaceutical companies - “GlaxoSmithKline”, “Novartis”, “AstraZeneca”, “Merck” and “Pfizer” still yet realize programs on development antimicrobial drugs. In recent years observed significant growth of staphylococcal and streptococcal infections caused by strains resistant to all в-lactam antibiotics (penicillins, cephalosporins, carbapenems and monobactams) and to macrolides, aminoglycosides, tetracyclines and other antibiotics. In clinical practice, this means that a number of known diseases caused by these pathogens, defies traditional scheme of treatment. The emergence of "super bacteria" indicates a return "to the antibiotic era."

In 2010, the volume of the Ukrainian pharmaceutical market in producer prices, calculated by the formula "production + imports - exports", was 22.4 billion UAH of which the lion's share by end of 2010 accumulates segment imports - 17 billion UAH [32]. Thus, the domestic pharmaceutical market is still dependent on imports.

Development of Biotechnology in Ukraine declared as one of the main directions of the country's innovation. In this framework, to be implemented major necessary measures for the development of biotech sector: establishment preferential treatment in biotechnology (scientific and industrial) sector, providing long term credits for new technologies, identify sources of public and private financing of priority areas of biotechnology.



CONCLUSION

Antibiotic resistance it is a growing unsolved medical problem. To prevent crisis of healthcare new family of antibiotics should appear regularly in the pharmaceuticals market. Use in practice analogues existing antibiotics active against resistant bacteria, prolong the life of this family of drugs for several decades, but new classes of antimicrobial agents are essential needed.

Over the next 10 years, screening of libraries of natural and synthetic compounds may lead to creation of new antibiotic agents. Genomics, non-culturable bacteria and bacteriophages can also be a source of new active compounds. Antiseptics and non antibiotic antimicrobial agents should be used wherever possible for local application, so that only resorted to antibiotics in the treatment of serious systemic infections. New government and private initiatives are needed to stimulate the research against antibiotic-resistant pathogens. Finally, it should impose restrictions on the use of antibiotics in agriculture.

Since the development of a new drug is a costly proposition, pharmaceutical companies have done very little research in the last decade. However, an alarming development has spurred a revived interest in the development of new antibiotics. It turns out that some of the disease-causing bacteria have mutated and developed a resistance to many of the standard antibiotics. This could have grave consequences on the world's public health unless new antibiotics are discovered or improvements are made on the ones that are available. This challenging problem will be the focus of research for many years to come.

Currently, cultivation of strains-producers opens up new opportunities for a wide range of biologically active compounds, including antibiotics. In this regard, prospective biotechnological processes, which are used to increase the efficiency of resistant strains producers. The growing role of antibiotics in medicine raises the important task of finding suitable and economic producers.

The continued development of antibiotic-resistant organisms has started to reveal weaknesses in the armoury of drugs available to treat serious hospital and community acquired infections. Thus the focus in drug discovery has shifted away from the identification of broad spectrum antibiotics to new entities that target the resistant organisms. A focus on this market niche, however, reduces the potential market for these new narrow-spectrum agents and has an impact on business decisions whether or not to proceed with discovery efforts. In addition, the emphasis on cost savings by the health-care industry continues to drive the expansion of use of generic broad spectrum antibiotics where they are effective.



LIST OF REFERENCES

1. Ланчини Д., Паренти Ф. Антибиотики. Москва: Мир, 1991. 270 с.

2. Berdy J. Bioactive microbial metabolites. J. Antibiot, 2005; 58(1): 1-26.

3. Aiello A.E., Larson E. Antibacterial cleaning and hygiene products as an emerging risk factor for antibiotic resistance in the community. Lancet Infect. Dis, 2003, 3(8): 501-506.

4. Stewart P.S. Mechanisms of antibiotic resistance in bacterial biofilms. Int. J. Med. Microbiol, 2002; 292: 107-113.

5. Demain A.L., Elander R.P.. The b-lactam antibiotics: past, present and future. Antonie van Leeuwenbook, 1999; 75: 5-19.

6. Demain A.L, Sanchez S. Microbial drug discovery: 80 years of progress. J. Antobiotics, 2009; 62: 1-12.

7. Дебабов В.Г. Селекция микроорганизмов на заре XXI века. Биотехнология, 2005; 4: 3-19.

8. Тец Г. В., Артеменко К. Л. Совместное действие антибиотиков и дезоксирибонуклеазы на бактерии. Антибиотики и химиотерапия, 2006; 6: 11-14.

9. А.Б. Стрептоміцети - продуценти антибіотиків. Мікробіол. журн, 2003; 65(1-2): 168-181

10. Donadio S., Maffioli S., Monciardini P. et al. Antibiotic discovery in the twenty-first century: current trends and future perspectives. J. Antibiotics, 2010; 63: 423-430.

11. Bentley S.D., Chater K.F., Cerden-o-Tarraga A.M. et al. Complete genome sequence of the model actinomycete Streptomyces coelicolor A3(2). Nature, 2002; 417: 141-147.

12. Komatsu M., Uchiyama T., Omura S. et al. Genome-minimized Streptomyces host for the heterologous expression of secondary metabolism. Proc. Natl. Acad. Sci. USA, 2010; 107(6): 2646-2651.171 Сучасний стан і перспективи біотехнологічного виробництва антибіотиків Біологічні Студії / Studia Biologica * 2011 * Том 5/№1 * С. 159-172

13. Ning Chen, Chen-Guang Xing, Xi-Xian, Qing-Yang Xu. Optimization of technical conditions of producing ribavirin by Bacillus subtilis. Annals of Microbiology, 2009; 59: 525-530.

14. D., Brandelli A. Influence of media and temperature on bacteriocin production by Bacillus cereus 8A dyring bath cultivation. Appl. Microbiol. Biotechnol, 2004; 65: 158-162.

15. Sevcikova B., Kormanec J. Differential production of two antibiotics of Streptomyces coelicolor A3 (2), actinorhodin and undecylprodigiosin, upon salt stress conditions. Arch. Microbiol, 2004; 181: 384-389.

16. Dalili M., Pao C. Production of actinomycin D with immobilized Streptomyces parvullus under nitrogen and carbon starvation conditions. Biotechnology Letters, 1988; 10(5): 331-336.

17. Singh V., Khan M., Khan S., Tripathi C.K.M. Optimization of actinomycin V production by Streptomyces triostinicus using artificial neural network and genetic algorithm. Appl. Microbiol. Biotechnol, 2009; 82: 379-385.

18. Young-Hong Wang, Xing Zhang. Influence of agitation and aeration on growth and antibiotic production by Xenorhabdus nematophila. World J. Microbiol. Biotechnol, 2007; 23: 221-227.

19. Bister B., Bischoff D., Strobele M. et al. Abyssomicin C--a polycyclic antibiotic from a marine Verrucosispora strain as an inhibitor of the p-aminobenzoic acid/tetrahydrofolate biosynthesis pathway. Angew. Chem. Int. Ed, 2004; 43: 2574-2576.

20. Singh, S.B., Phillips, J.W., Wang, J. Highly sensitive target-based whole-cell antibacterial discovery strategy by antisense RNA silencing. Curr. Opin. Drug Discov. Devel, 2007; 10: 160-166.

21. Осташ Б. Сайт-специфічні рекомбінази у генетичній інженерії: новітні технології in vivo. Цитология и генетика, 2010; 44(4): 61-69.

22. Bibb M.J. Regulation of secondary metabolism in streptomycetes. Curr Opin Microbiol, 2005; 8(2): 208-215.

23. Бирюков В.В. Основы промышленной биотехнологии. - Москва, «Химия» - «Колос», 2004 г. - 296 с.

24. Егоров Н. С. Основы учения об антибиотиках: Учебник. - 6-е изд., 2004. - 528 c.

25. Виестур У.Э. Культивирование микроорганизмов / У.Э. Виестур, Н.Ж. Кристансонс. - М.: Пищевая промышленность, 1980.

26. http://www.madehow.com/Volume-4/Antibiotic.html

27. Стрельников Л.С., Стрилец О. П., Щербак О. В. и др. Перспективы и пути развития производства биотехнологических лекарственных препаратов в Украине. Annals of Mechnikov Institute, 2006; 4: 3-8.

28. Костюк Р.В. Розвиток інноваційної діяльності біотехнологічних підприємств у сучасних умовах. Актуальні проблеми економіки, 2009; 8(98): 79-84.

29. Hamad B. The antibiotics market. Nature Reviews Drug Discovery, 2010; 9: 675-676.

30. Новіков В., Сидоров Ю., Швед О. Тенденції розвитку комерційної біотехнології. Вісн. НАН України, 2008; 2: 25-39.

31. Реєстр ЛЗ на території України. http://mozdocs.kiev.ua/

32. Сергиенко О. Ралли на фармрынке! Аптечные продажи лекарств в Украине: итоги 2010 г. Часть I. Аптека.ua Online, 2011; 4. http://www.apteka.ua/article/70328

33. Егоров Н. С. Основы учения об антибиотиках: Учеб. Для студентов биолог. спец. ун-тов. - 4 изд., перераб. и доп. - М.: Высш. шк. 1986. - с.416-418.

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