In the previous sections, you have already seen some of the techniques for working with microorganisms and had the opportunity to try these techniques on experience. Moving from laboratory scale to industrial scale, biotechnologists must solve many problems in various branches of science, including bioengineering, chemistry and biology. When making decisions about the industrial production of bacteria, it is important to consider both economic, social and ethical aspects. In this section, we will touch on some of the practicalities of large-scale production, and in the following sections we will look at specific examples of microbiological production and related problems.

Use of microorganisms in industrial production is possible for the following reasons:
1) microorganisms have simple nutritional needs;
2) in fermenters (large vessels in which microorganisms grow), the growth conditions can be very precisely controlled;
3) microorganisms are characterized by high growth rates;
4) reactions can be carried out at lower temperatures than in conventional chemical plants; energy charges are reduced accordingly;
5) microorganisms provide a higher product yield and higher specificity than conventional chemical production;
6) a wide range of chemical compounds can be used and produced;
7) some complex chemical compounds can be produced, such as hormones and antibiotics, which are difficult to obtain by other methods, as well as specific isomers (such as L-amino acids);
8) the genetics of microorganisms is relatively simple, and methods of genetic manipulation with them are constantly evolving.

However, the need to use special methods such as sterilization methods and sophisticated separation techniques can result in significant increases in technical requirements to the process.

Screening

We know that for microorganisms characterized by a huge variety of chemical reactions that they can carry out, and the products that they form. However, only a small part of their potential is used in industrial production. Commercial companies, especially drug manufacturers, are constantly looking for microorganisms that may be useful. Hoping to discover new commercially important products or more effective ways obtaining available products, microorganisms are collected and cultivated from all over the world, from a wide variety of habitats. Very often this is purely empirical work in the sense that chance plays an essential role in any discovery. Checking for microorganisms in this way is called screening. Good example is an ongoing screening that is done to find new antibiotics. The first antibiotic was discovered in 1928 by Alexander Fleming and was named penicillin after the name of the fungus Rep. That produces it. Natural antibiotics are chemicals synthesized by microorganisms that kill other microorganisms or inhibit their growth. Since 1928, more than 5,000 different antibiotics have been isolated from microorganisms, including a number of different penicillins that differ slightly in structure and activity. Most of the antibiotics found are unsuitable for medical purposes, mainly due to their high toxicity. However, members of the genus Streptomyces have proven to be an extremely rich source of various antibiotics, including streptomycin.

Antibiotics are used to treat bacterial or fungal diseases in humans and pets. Some of them also inhibit the growth of cancerous tumors. Apparently, antibiotics are products of secondary metabolism. With systematic screening, there is always hope to find a new "miracle drug" or microorganism that produces a known antibiotic, but with improved properties.

Of more than 100 thousand known microorganisms, only a few hundred species are used in industry, since an industrial strain must meet a number of strict requirements:

1) grow on cheap substrates;

2) have a high growth rate or give a high product yield in a short time;

3) show synthetic activity towards the desired product; the formation of by-products should be low;

4) be stable in terms of productivity and to the requirements of cultivation conditions;

5) be resistant to phage and other types of infections;

6) be harmless to people and the environment;

7) thermophilic, acidophilic (or alcophilic) strains are desirable, since it is easier to maintain sterility in production with them;

8) anaerobic strains are of interest, since aerobic strains create difficulties in cultivation - they require aeration;

9) the product formed must have economic value and be easily distinguished.

In practice, strains of four groups of microorganisms are used:

- yeast;

- filamentous fungi (mold);

- bacteria;

- ascomycetes.

The term "yeast" in the strict sense has no taxonomic meaning. These are unicellular eukaryotes belonging to three classes: Ascomycetes, Basidiomycetes, Deuteromycetes.

Ascomycetes include, first of all, Saccharomyces cerevisiae, certain strains of which are used in brewing, winemaking, bread production, and ethyl alcohol.

Ascomycetes Saccharomyces lipolytica degrade oil hydrocarbons and are used to obtain protein mass.

Deuteromycete Candida utilis is used as a source of protein and vitamins and is grown on non-food raw materials: sulfite lye, wood hydrolysates and liquid hydrocarbons.

Deuteromycete Trichosporon cutaneum oxidizes many organic compounds, including toxic ones (for example, phenol), and is used in wastewater treatment.

Filamentous fungi are used by:

- in obtaining organic acids: citric (Aspergillus niger), gluconic (Aspergillus niger), itaconic (Aspergillus terreus), furmaric (Rhizopus chrysogenum);

- in obtaining antibiotics (penicillin and cephalosporin);

- in the production of special types of cheeses: Camembert (Penicillium camamberti), Roquefort (Penicillium roqueforti);

- cause hydrolysis in solid media: in rice starch when making sake, in soybeans when making tempeh, miso.

Beneficial bacteria are eubacteria.

Lactic acid bacteria of the genera Lactobacillus, Leuconostoc, Lactococcus have been used industrially for a long time.

Acetic acid bacteria of the genera Acetobater, Gluconobacter convert ethanol into acetic acid.

The bacteria of the genus Bacillus are used for the production of toxins harmful to insects, as well as for the synthesis of antibiotics and amino acids.

The bacteria of the genus Corynebacterium are used for the production of amino acids.

The most representative actinomycetes are Streptomyces and Micromonospora, which are used as antibiotic producers. When growing on solid media, actinomycetes form a thin mycelium with aerial hyphae, which differentiate into chains of conidiospores.

Currently, the following compounds are synthesized with the help of microorganisms:

- alkaloids,

- amino acids,

- antibiotics,

- antimetabolites,

- antioxidants,

- proteins,

- vitamins,

- herbicides,

- enzyme inhibitors,

- insecticides,

- ionophores,

- coenzymes,

- lipids,

- nucleic acids,

- nucleotides and nucleosides,

- oxidants,

- organic acids,

- pigments,

- surfactants,

- polysaccharides,

- antihelminthic agents,

- antineoplastic agents,

- solvents,

- plant growth hormones,

- sugar,

- sterols and converted substances,

- factors of iron transport,

- pharmacological substances,

- enzymes,

- emulsifiers.

2 PRODUCTION OF SINGLE CELL PROTEINS

ORGANISMS

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2.1 Feasibility of using microorganisms for

protein production

In accordance with the norms of nutrition, a person should receive daily from 60 to 120 g of complete protein with food.

To maintain the vital functions of the body, build cells and tissues, a constant synthesis of various protein compounds is required. If plants and most microorganisms are able to synthesize all amino acids from carbon dioxide, water, ammonia and mineral salts, then humans and animals cannot synthesize some amino acids (valine, leucine, isoleucine, lysine, methionine, threonine, tryptophan and phenylalanine). These amino acids are called essential. They must come from food. Their deficiency causes serious human diseases and lowers the productivity of farm animals.

Currently, the world protein deficit is about 15 million tons. Microbiological synthesis is the most promising. If for cattle it takes 2 months to double the protein mass, for pigs - 1.5 months, for chickens - 1 month, then for bacteria and yeast - from 1 to 6 hours. The world production of food protein products due to microbial synthesis is more than 15 thousand tons per year.

Consider an example: the doubling time of E. coli is 20 minutes, then after 20 minutes two daughter cells are formed from one cell, after 40 minutes - four "granddaughters", after 60 minutes - eight "great-grandchildren", after 80 minutes - 16 "great-granddaughters". After 10 hours 40 minutes, more than 6 billion bacteria will be formed from one bacterium, which corresponds to the population of the Earth, and after 44 hours from one bacterium weighing 1 10 -12 g, biomass is formed in the amount of 6 10 24 g, which corresponds to the mass of the Earth.

The use of various microorganisms as sources of protein and vitamins is due to the following factors:

A) the possibility of using various chemical compounds for the cultivation of microorganisms, including industrial waste;

B) a relatively simple technology for the production of microorganisms, which can be carried out year-round; the possibility of its automation;

C) high protein content (up to 60 ... 70%) and vitamins, as well as carbohydrates, lipids in microbial preparations;

D) increased content of essential amino acids in comparison with vegetable proteins;

D) the possibility of directed genetic influence on chemical composition microorganisms in order to improve the protein and vitamin value of the product.

For industrial production food products on the basis of microorganisms, careful biomedical research is required. Such products must undergo a comprehensive check to identify carcinogenic, mutagenic, embryotropic effects on humans and animals. Toxicological studies, assimilation of microbial synthesis products are the main criteria for the expediency of their production technology.

For the production of proteins, yeast, bacteria, algae and filamentous fungi are used.

The advantage of yeast over other microorganisms is their manufacturability: resistance to infections, ease of separation from the medium due to the large size of the cells. They are able to accumulate up to 60% of the protein rich in lysine, threonine, valine and leucine (these amino acids are scarce in plant feed). The mass fraction of nucleic acids is up to 10%, which has a harmful effect on the body. As a result of their hydrolysis, many purine bases are formed, which are then converted into uric acid and its salts, which are the cause of urolithiasis, osteochondrosis and other diseases. The optimal rate of addition of yeast mass to feed for farm animals is from 5 to 10% of dry matter. Yeast is used for food and feed purposes.

The advantages of bacteria are a high growth rate and the ability to synthesize up to 80% of protein. The resulting protein contains many deficient amino acids: methionine and cysteine. The disadvantages are the small size of the cells and their low concentration in the culture medium, which complicates the isolation process. Some bacterial lipids can contain toxins. Mass fraction of nucleic acids up to 16%. Used for feed purposes only.

The advantages of algae are a high content of protein, complete in amino acid composition, accumulating in an amount of 65%, easy isolation of algae from the culture medium, a low content of nucleic acids - 4% (for comparison - in higher plants 1 ... 2%). Algae are used for food and feed purposes.

Filamentous fungi are traditionally used as a food product in African countries, India, Indonesia, China, etc. They accumulate up to 50% of protein, which is close to animal protein in amino acid composition, rich in B vitamins. Cell walls are thin and easily digested in the gastrointestinal tract. intestinal tract of animals. The mass fraction of nucleic acids is 2.5%.

Since 1985, microbial protein has been used in the food industry for the manufacture of various products and semi-finished products.

In food production, three main uses of microbial protein are considered:

1) whole mass (without destruction of cell walls);

2) partially purified biomass (destruction of cell walls and removal of unwanted components is provided);

3) proteins isolated from biomass (isolates).

The WHO (World Health Organization) concluded that the protein of microorganisms can be used in food, but the permissible amount of nucleic acids introduced together with protein into the diet of an adult should not exceed 2 g per day. The introduction of microbial protein does not cause negative consequences, but there are manifestations of allergic reactions, stomach diseases, etc.

Microorganism production

Microorganisms in the production of protein nutrients

Microorganisms help humans produce effective protein nutrients and biogas. They are used in the application of biotechnical methods of air and wastewater purification, in the use of biological methods for the destruction of agricultural pests, in the receipt of medicinal products, in the destruction of waste materials. Bacteria, fungi, algae, lichens, viruses, protozoa play a significant role in human life. Since ancient times, people have used them in the processes of baking, making wine and beer, in various industries. Currently, in connection with the problems of obtaining valuable protein substances, increasing soil fertility, cleaning the environment from pollutants, obtaining biological products and other goals and objectives, the range of study and use of microorganisms has expanded significantly.

Microorganisms in food production

Many microorganisms, including yeast-like and some types of microscopic fungi, have long been used in the transformation of various substrates to obtain different types food products. For example, the use of yeast to obtain porous bread from flour, the use of fungi of the genera Rhisopus, Aspergillus for the fermentation of rice and soybeans, the production of lactic acid products using lactic acid bacteria, yeast, etc.

Auxotrophic mutants of Candida guillermondii are used to study flavinogenesis. Hyphalous fungi assimilate well the carbons of oil, paraffin, n-hexadecane, and diesel fuel.

For different degrees of purification of these substances, species of the genera Mucorales, Penicillium, Fusarium, Trichoderma are used.

For the utilization of fatty acids, strains of Penicillium are used, and fatty secondary alcohols are better processed in the presence of strains of Penicillium and Trichoderma. Species of fungi Aspergillus, Absidia, Cunningham, Ella, Fusarium, Mortierella, Micor, Penicillium, Trichoderma, Periconia, Spicaria, are used in the utilization of paraffin wax oils, diesel fuel, aromatic hydrocarbons, polyhydric alcohols, fatty acids. Penicillium vitale is used to obtain a purified glucose oxidase preparation that inhibits the development of pathogenic dermatomycetes Microsporum lanosum, Achorion gypseum, Trichophyton gypseum, Epidermophyton kaufman.

The industrial use of microorganisms for the production of new food products contributed to the creation of such industries as bakery and dairy, the production of antibiotics, vitamins, amino acids, alcohols, organic acids, etc.

A variety of microorganisms. Biotechnology of dairy products. Environmental biotechnology.

Microbiological synthesis of various substances plays a key role in biotechnological production. The beginning of modern industrial microbiology was laid in the 40s, when the production of penicillins by fermentation methods was established. Currently, microorganisms produce dozens of types of compounds - amino acids, antibiotics, proteins, vitamins, lipids, nucleic acids, polysaccharides, pigments, sugars, enzymes, etc.

The diverse world of microorganisms includes prokaryotes (unicellular organisms that do not contain formed nuclei) - bacteria, actinomycetes, rickettsiae lower eukaryotes (unicellular and multicellular organisms that have formed nuclei, in which chromosomes are surrounded by a special porous membrane (lipoprotein nature), - yeast, filamentous fungi , protozoa and algae.Of more than 100 thousand species of microorganisms known in nature, only a few hundred are used in biotechnological processes.The microbiological industry imposes strict requirements on producers that are important for production technology: high growth rate, use of cheap substrates for life and resistance to infection extraneous microflora.

Dairy Biotechnology.

The spectrum of food products obtained with the help of microorganisms is extensive. These are products obtained as a result of fermentation - bread, cheese, wine, beer, cottage cheese, and so on. Until recently, biotechnology was used in the food industry with the aim of improving the mastered processes and more skillful use of microorganisms, but the future here belongs to genetic research to create more productive strains for specific needs, to introduce new methods in fermentation technology.

Obtaining dairy products in the food industry is built by fermentation processes. Milk is the backbone of the biotechnology of dairy products. Milk (secretion of the mammary glands) is a unique natural nutrient medium. It contains 82 - 88% water and 12 - 18% dry residue. The milk solids composition includes proteins (3.0 - 3.2%), fats (3.3 - 6.0%), carbohydrates ( milk sugar lactose - 4.7%), salts (0.9 - 1%), minor components (0.01%): enzymes, immunoglobulins, lysozyme, etc. Milk fats are very diverse in their composition. The main proteins of milk are albumin, casein. Thanks to this composition, milk is an excellent substrate for the development of microorganisms. Usually they take part in fermenting milk streptococci and lactic acid bacteria... By using the reactions that accompany the main process of lactose fermentation, other milk processing products are also obtained: sour cream, yogurt, cheese, etc. The properties of the final product depend on the nature and intensity of fermentation reactions. Those reactions that accompany the formation of lactic acid usually determine the special properties of the products. For example, secondary fermentation reactions occurring during the ripening of cheeses determine the taste of some of their varieties. These reactions involve peptides, amino acids and fatty acids found in milk.

Microzyme. Environmental biotechnology.

In nature, which is not subject to human intervention, the ecosystem is tuned to self-purification, that is, nature itself copes with the processing of no longer needed (dead) organic material. The utilization of organic matter involves soil containing natural biota (microorganisms, edapon) - a living component represented by various representatives of the flora and fauna. One gram of garden soil contains tens of millions of microorganisms - saprophytes, and ctenomycetes, fungi, oligonitrophils, azotobacters and nodule bacteria, bacteria decomposing fiber, ammonifiers, nitrifiers, denitrifiers, anaerobic nitrogen fixers. Together, microorganisms make up the soil microflora responsible for metabolism, as a result of which dead organic matter is processed into fertile humus. Human activities have a powerful anthropogenic impact on the environment, in particular, by contamination of soil and water with industrial and life waste, where organic pollutants account for a significant share. As a result of soil and water pollution with organic substances, the natural biota is suppressed, the ratio between individual groups of microorganisms changes and, in general, the direction of metabolism changes, and natural self-purification processes are disrupted. In areas of constant pollution, the soil microflora in pollutant substrates amounts to no more than several thousand CFU per 100 grams of substrate, some groups of microorganisms retain their presence, while the number of others is critically reduced, soil formation processes are disrupted, non-decomposable waste accumulates in the soil and water. In a polluted ecosystem with a suppressed beneficial microflora, harmful and pathogenic microorganisms develop - in water bodies contaminated with nutrients of nitrogen and phosphorus, blue-green algae, dangerous to the ecology of the reservoir, are rapidly developing, causing water poisoning and death. Technogenic and anthropogenic violations of the ecological balance change the sanitary state in the place of their formation, worsen the living conditions of people.

The development of the most rational methods of using microbes in human economic activity and the conscious selection of microbes became possible only after the development of microscopic methods for studying and clarifying the methods of dispersal and reproduction of microorganisms. Pathways of microbial origin with increased resistance or with reduced requirements for nutrients as in natural conditions under the influence natural selection, and in artificial conditions as a result of the activities of breeders, are of very important practical importance. The person is interested in getting as quickly as possible useful forms microbes. The intensity of natural selection strongly affects the speed of emergence of resistant forms, and the more severe this selection, the faster resistant forms are identified. With the help of stepwise selection, new strains of microorganisms are obtained that can grow and give high productivity in conditions of environmental pollution. New highly effective strains can be isolated from the environment, for example, from natural and man-made biotopes, contaminated areas and treatment facilities, as well as obtained through targeted selection.

Many environmentally hazardous pollutants are complex organic substances. For their processing, microorganisms synthesize enzymes into the external environment - special protein bioactive substances that play a key role in the destruction of complex organic substrates: cellulose, lignin, starches, lipids, hydrocarbons, to simple molecular structures that are freely absorbed and mineralized by bacteria or other microorganisms, for example, mushrooms. Biotechnology uses this ability of microorganisms and bacteria in particular when applied to specific environmental problems.

The use of microscopic soil inhabitants for the biological disposal of organic waste and the neutralization of pollutants is called bioremediation(bio - life, remedio - cure). High concentrations of specially selected different types of microorganisms that make up the community, which were previously isolated from the soil, selected and multiplied in the form of a ready-to-use preparation, are introduced into the environment to be cleaned or into the waste being disposed of.

As a result, in the right place at the right time, useful microbiological activity is purposefully created, which consists in the assimilation and processing of dead organic matter by microbes into metabolic products: carbon dioxide (carbon dioxide, CO2), water (H2O), methane (CH4), humus, various forms of nitrogen ( from mineral to gaseous). Such measures make it possible to neutralize with high efficiency the inhibitory effect of pollutants on the natural processes of self-purification of soil and water, to stimulate microbiological metabolism, to activate the corresponding aboriginal microflora and natural processes of self-purification, soil formation, and respiration.

The advantages of bioremediation include the possibility of targeted and dosed application of technology in the right place at the right time, a fairly high rate and ecologically significant efficiency of assimilation and processing by microorganisms of organic waste and pollution, technologically specified characteristics of purification or processing processes, environmental and hygienic safety. For example, biological wastewater treatment uses biotechnology in cases where certain substances contained in the wastewater are not biodegradable by activated sludge flocs.

Then specially selected microorganisms that can effectively destroy a complex pollutant, for example fats, polymers, to molecular structures that do not harm the activated sludge of treatment facilities, come to the rescue.

Bioremediation- biological cleaning of soil and water from oil and oil products pollution is based on the ability of microorganisms to gradually metabolize complex petroleum hydrocarbons to obtain simpler molecular hydrocarbon structures until they are completely neutralized as an environmentally hazardous pollutant.

Disposal and neutralization of faeces, cleaning of household wastewater is based on the ability of microorganisms to metabolize organic matter in the faeces and suppress the growth of pathogenic microflora due to competition for a food source. The destruction of odors, the deodorization effect is based on several abilities of bacteria to metabolize smelling volatile organic compounds or prevent their formation, metabolize fatty acids.

Methane gas production(biogas) from organic waste directly depends on the vital activity of methanogenic microorganisms. At the same time, biotechnology closely interacts with environmental engineering. For example, biological rehabilitation of water bodies in situ (consideration of the phenomenon exactly in the place where it occurs, that is, without moving to a special environment) is based on the theory of practice of the role of communities of bacteria and microorganisms in the whole biological ecosystem of a reservoir, trophic relationships of the aquatic ecosystem.

The main link in the biotechnological process, which determines its entire essence, is a biological object capable of carrying out a certain modification of the feedstock and forming one or another necessary product. Such objects of biotechnology can be cells of microorganisms, animals and plants, transgenic animals and plants, as well as multicomponent enzyme systems of cells and individual enzymes.

The basis of most modern biotechnological industries is still microbial synthesis, that is, the synthesis of various biologically active substances with the help of microorganisms. Unfortunately, for a number of reasons, objects of plant and animal origin have not yet found such widespread use.

Regardless of the nature of the object, the primary stage in the development of any biotechnological process is to obtain pure cultures of organisms (if these are microbes), cells or tissues (if these are more complex organisms - plants or animals). Many stages of further manipulations with the latter (i.e., with plant or animal cells), in fact, are the principles and methods used in microbiological production. Both cultures of microbial cells and tissue cultures of plants and animals practically do not differ from cultures of microorganisms from a methodological point of view.

The world of microorganisms is extremely diverse. Currently

Relatively well characterized (or known) more than 100 thousand of their different species. These are primarily prokaryotes (bacteria, actinomycetes, rickettsiae, cyanobacteria) and part of eukaryotes (yeast, filamentous fungi, some protozoa and algae). With such a wide variety of microorganisms, a very important, and often complex, problem is the correct choice of exactly the organism that is capable of providing the required product, that is, serving industrial purposes. Microorganisms are divided into industrial and non-industrial, these are those microorganisms that are used in industrial production - industrial, and those that are not used - non-industrial.

The basis of industrial production is a few, but deeply studied groups of microorganisms that serve as model objects in the study of fundamental life processes. All other microorganisms have not been studied at all or have been studied to a very limited extent by geneticists, molecular biologists, and genetic engineers. The former include Escherichia coli (E. coli), hay bacillus (Bac. Subtilis) and baker's yeast (S. cerevisiae).

Many biotechnological processes use a limited number of microorganisms that are classified as GRAS (generally recognized as safe). These microorganisms include the bacteria Bacillus subtilis, Bacillus amyloliquefaciens, other types of bacilli and lactobacilli, Streptomyces species. This also includes the species of fungi Aspergillus, Penicillium, Mucor, Rhizopus and the yeast Saccharomyces, etc. GRAS microorganisms are non-pathogenic, non-toxic and generally do not form antibiotics, therefore, when developing a new biotechnological process, one should focus on these microorganisms as basic objects of biotechnology.

The microbiological industry today uses thousands of strains from hundreds of types of microorganisms, which were initially isolated from natural sources based on their beneficial properties, and then (mostly) improved using various methods. In connection with the expansion of production and the range of products, more and more representatives of the world of microbes are involved in the microbiological industry. We should be aware that in the foreseeable future none of them will be studied to the same extent as E. coli and Bac.subtilis. And the reason for this is very simple - the colossal laboriousness and high cost of this kind of research.

The most common biotechnological objects are:

Bacteria and cyanobacteria;

Seaweed;

The simplest;

Plant and animal cell cultures;

Plants - lower (anabena-azolla) and higher - duckweed.

Subcellular structures (viruses, plasmids, DNA).

Bacteria and cyanobacteria

The biotechnological functions of bacteria are diverse.

Acetic acid bacteria, genera Gluconobacter and Acetobacter.

Gram-negative bacteria that convert ethanol to acetic acid and acetic acid to carbon dioxide and water.

Representatives of the genus Bacillus - B. subtilis B. thuringiensis are used to obtain probiotics, substances that have an antibiotic effect on other microorganisms, as well as on insects (B. thuringiensis). They belong to gram-positive bacteria that form endospores.

B.subtilis is a strict aerobic, while B. thuringiensis can live in anaerobic conditions.

The anaerobic spore-forming bacteria are represented by the genus Clostridium. C. acetobutylicum ferments sugars into acetone, ethanol, isopropanol and n-butanol (acetobutanol fermentation), other species can also ferment starch, pectin and various nitrogen-containing compounds.

Lactic acid bacteria include representatives of the genera Lactobacillus, Leuconostoc and Streptococcus, which do not form spores, are gram-positive and insensitive to oxygen.

Heterofermentative bacteria of the genus Leuconostoc convert carbohydrates into lactic acid, ethanol and carbon dioxide.

Homofermentative bacteria of the genus Streptococcus produce only lactic acid.

Representatives of the genus Lactobacillus provide a variety of foods along with lactic acid.

Representative of the genus Corynebacterium, immobile gram-positive cells of C. glutamicum serve as a source of lysine and sodium glutamate.

Other types of corynebacteria are used for microbial leaching of ores and disposal of mining waste.

This property of some bacteria is widely used, such as diazotrophy, that is, the ability to fix atmospheric nitrogen.

There are 2 groups of diazotrophs:

Symbionts: without root nodules (mainly lichens), with root nodules (legumes);

Free-living: heterotrophs (azotobacter, clostridium, methylobacter), autotrophs (chlorobium, rhodospirillum and amoebobacter).

Bacteria are also used for genetic engineering purposes.

Cyanobacteria have the ability to fix nitrogen, which makes them very promising protein producers. A product close to glycogen is deposited in the cytoplasm of cells.

Such representatives of cyanobacteria as nostok, spirulina, trichodesmia are edible and directly eaten. The sock forms crusts on the barren lands, which swell when moistened. In Japan, the local population uses the layers of nostok formed on the slopes of the volcano for food and calls them Tengu barley bread (Tengu is a good mountain spirit).

Spirulina (Spirulina platensis) comes from Africa - the region of Lake Chad.

Spirulina maxima grows in the waters of Lake Texcoco in Mexico. The Aztecs also collected it from the surface of the lakes and eaten it.

Spirulina was used to make biscuits, which were a dried mass of spirulina.

Analysis showed that spirulina contains 65% protein (more than soybeans), 19% carbohydrates, 6% pigments, 4% lipids, 3% fiber and 3% ash. Proteins are characterized by a balanced amino acid content. The cell wall of this alga is well digested.

Spirulina can be cultivated in open ponds or in a closed system of polyethylene pipes. The yield is very high: up to 20 g of dry mass of algae is obtained from 1 m 2 per day, which is about 10 times higher than the yield of wheat.

The domestic pharmaceutical industry produces the drug "Splat" based on the cyanobacterium Spirulina platensis. It contains a complex of vitamins and microelements and is used as a tonic and immunostimulating agent.

Escherichia coli

Escherichia coli Is one of the most studied organisms. Over the past fifty years, it has been possible to obtain comprehensive information about genetics, molecular biology, biochemistry, physiology and general biology. Escherichia coli... It is a gram-negative, movable shelf less than 10 microns in length. Its habitat is the intestines of humans and animals, but it can also live in soil and water. Usually, E. coli is not pathogenic, but under certain conditions it can cause disease in humans and animals.

Due to the ability to multiply by simple division on media containing only ions Na +, K +, Mg 2+, Ca 2+, NH 4 +, Cl -, HPO 4 2- and SO 4 2-, trace elements and a carbon source (for example, glucose ), E. coli became a favorite subject of scientific research.

In cultivation E. coli on enriched liquid nutrient media containing amino acids, vitamins, salts, trace elements and a carbon source, the generation time (i.e., the time between the formation of a bacterium and its next division) in the logarithmic growth phase at 37 ° C is approximately 22 minutes.

E. coli can be cultivated in both aerobic (in the presence of oxygen) and anaerobic (without oxygen) conditions. However, for optimal production of recombinant proteins E. coli usually grown aerobically.

If the purpose of the cultivation of bacteria in the laboratory is the synthesis and isolation of a certain protein, then the cultures are grown on complex liquid nutrient media in flasks. To maintain the desired temperature and ensure sufficient aeration of the culture medium, the flasks are placed in water bath or a thermostatted room and shaken continuously. This aeration is sufficient for cell proliferation, but not always for the synthesis of a specific protein.

The growth of cell mass and protein production are limited not by the content of carbon or nitrogen sources in the nutrient medium, but by the content of dissolved oxygen: at 20 ° C it is equal to about nine ppm. This becomes especially important in the industrial production of recombinant proteins. To ensure optimal conditions for maximum protein production, special fermenters and aeration systems are designed.

For each living organism, there is a certain temperature range that is optimal for its growth and reproduction. At too high temperatures, proteins are denatured and other important cellular components are destroyed, leading to cell death. At low temperatures, biological processes slow down significantly or stop completely due to structural changes that protein molecules undergo.

Based on the temperature regime that certain microorganisms prefer, they can be subdivided into thermophiles (from 45 to 90 ° C and above), mesophylls (from 10 to 47 ° C) and psychrophiles (from -5 to 35 ° C). microorganisms that actively reproduce only in a certain temperature range can be a useful tool for solving various biotechnological problems. For example, thermophiles often serve as a source of genes encoding thermostable enzymes that are used in industrial or laboratory processes, and genetically modified psychrotrophs are used for biodegradation of toxic waste contained in soil and water at low temperatures.

In addition to E. coli, many other microorganisms are used in molecular biotechnology (Table 1). They can be divided into two groups: microorganisms as sources of specific genes and microorganisms created by genetic engineering methods to solve specific problems. Specific genes include, for example, a gene encoding a thermostable DNA polymerase, which is used in the widely used polymerase chain reaction (PCR). This gene was isolated from thermophilic bacteria and cloned into E. coli... the second group of microorganisms includes, for example, various strains Corynebacterium glutamicum that have been genetically modified to enhance the production of industrially important amino acids.

Table 1. Some genetically modified microorganisms used in biotechnology.

Acremonium chrysogenum Bacillus brevis Bacillus subtilis Bacillus thuringiensts Corynebacterium glutamicum Erwinia herbicola Escherichia coli Pseudomonas spp. Rhizoderma spp. Trichoderma reesei Xanthomonas campestris Zymomonas mobilis

At the present stage, the problem of developing a strategy and research tactics arises that would make it possible to extract from the potential of new microorganisms everything that is most valuable when creating industrially important producer strains suitable for use in biotechnological processes. The classical approach is to isolate the desired microorganism from natural conditions.

1. Samples of material are taken from the natural habitats of the alleged producer (material samples are taken) and inoculated into an elective environment that ensures the predominant development of the microorganism of interest, that is, so-called enrichment cultures are obtained.

2. The next step is the isolation of a pure culture with further differential diagnostic study of the isolated microorganism and, if necessary, an approximate determination of its productive capacity.

There is another way to select producer microorganisms - this is the selection of the desired species from the available collections of well-studied and thoroughly characterized microorganisms. This, of course, eliminates the need to perform a number of time-consuming operations.

The main criterion when choosing a biotechnological object (in our case, a producer microorganism) is the ability to synthesize the target product. However, in addition to this, additional requirements may be laid in the technology of the process itself, which are sometimes very, very important, not to say decisive. In general terms, microorganisms should:

Have a high growth rate;

1. Single-celled organisms, as a rule, are characterized by higher rates of growth and synthetic processes than higher organisms. However, this is not the case for all microorganisms. There are some of them (for example, oligotrophic) that grow extremely slowly, but they are of known interest, since they are capable of producing various very valuable substances.

Dispose of cheap substrates necessary for their life;

2. Particular attention as objects of biotechnological developments is represented by photosynthetic microorganisms that use the energy of sunlight in their life. Some of them (cyanobacteria and photosynthetic eukaryotes) utilize CO2 as a carbon source, and some representatives of cyanobacteria, in addition to all that has been said, have the ability to assimilate atmospheric nitrogen (that is, they are extremely unpretentious to nutrients).

Photosynthetic microorganisms are promising as producers of ammonia, hydrogen, protein, and a number of organic compounds. However, a problem in their use due to the limited fundamental knowledge about their genetic organization and molecular biological mechanisms of life, most likely, should not be expected in the near future.

To be resistant to extraneous microflora, that is, to be highly competitive.

3. Certain attention is paid to such objects of biotechnology as thermophilic microorganisms growing at 60–80 ° C. This property is an almost insurmountable obstacle to the development of extraneous microflora during relatively non-sterile cultivation, that is, it is a reliable protection against contamination. Producers of alcohols, amino acids, enzymes, and molecular hydrogen were found among thermophiles. In addition, their growth rate and metabolic activity are 1.5–2 times higher than that of mesophiles. Enzymes synthesized by thermophiles are characterized by increased resistance to heat, some oxidants, detergents, organic solvents and other unfavorable factors. At the same time, they are not very active at ordinary temperatures. Thus, proteases of one of the representatives of thermophilic microorganisms are 100 times less active at 200 C than at 750 C. The latter is a very important property for some industrial production.

All of the above provides a significant reduction in the cost of producing the target product.

Selection

An integral component in the process of creating the most valuable and active producers, that is, in the selection of objects in biotechnology, is their selection. And the general way of selection is the conscious construction of genomes at each stage of the selection of the desired producer. In the development of microbial technologies, methods based on the selection of spontaneously emerging modified variants characterized by the necessary useful traits have played (and still continue to play) a very important role. With such methods, stepwise selection is usually used: at each stage of selection, the most active variants (spontaneous mutants) are selected from the population of microorganisms, from which new, more effective strains are selected at the next stage.

The selection process for the most effective producers is significantly accelerated when using the method of induced mutagenesis.

As mutagenic effects, UV, X-ray and gamma radiation, certain chemicals, etc. are used. However, this technique is also not without drawbacks, the main of which is its laboriousness and lack of information about the nature of changes, since the experimenter selects according to the final result.

Thus, the current trend is the deliberate design of strains of microorganisms with desired properties on the basis of fundamental knowledge about the genetic organization and molecular biological mechanisms of the basic functions of the organism.

The selection of microorganisms for the microbiological industry and the creation of new strains are often aimed at enhancing their productive capacity, i.e. the formation of a particular product. The solution of these problems is to one degree or another associated with changes in the regulatory processes in the cell.

Changes in the rate of biochemical reactions in bacteria can be carried out in at least two ways. One of them is very fast (realized within seconds or minutes) is to change the catalytic activity of individual enzyme molecules. The second, slower (realized over many minutes), consists in changing the rates of enzyme synthesis. Both mechanisms use the same principle of systems control - the feedback principle, although there are also simpler mechanisms for regulating the activity of cell metabolism. The simplest way to regulate any metabolic pathway is based on the availability of a substrate or the presence of an enzyme. A decrease in the amount of a substrate (its concentration in the medium) leads to a decrease in the flow rate of a particular substance through a given metabolic pathway. On the other hand, increasing the concentration of the substrate leads to the stimulation of the metabolic pathway. Therefore, regardless of any other factors, the presence (availability) of the substrate should be considered as a potential mechanism of any metabolic pathway. Sometimes an effective means of increasing the yield of the target product is to increase the concentration in the cell of a particular precursor.

The most common way to regulate the activity of metabolic reactions in a cell is retroinhibition-type regulation.

The biosynthesis of many primary metabolites is characterized by the fact that with an increase in the concentration of the final product of a given biosynthetic pathway, the activity of one of the first enzymes of this pathway is inhibited. The presence of such a regulatory mechanism was first reported in 1953 by A. Novik and L. Szillard, who studied tryptophan biosynthesis by E. coli cells. The final stage of the biosynthesis of this aromatic amino acid consists of several stages catalyzed by individual enzymes.

These authors found that in one of the E. coli mutants with impaired tryptophan biosynthesis, the addition of this amino acid (which is the end product of this biosynthetic pathway) dramatically inhibits the accumulation of one of the precursors, indole glycerophosphate, in cells. Even then, it was suggested that tryptophan inhibits the activity of some enzyme that catalyzes the formation of indole glycerophosphate. This has been confirmed.