Metabolism of microorganisms

Metabolism is a set of biochemical processes that occur in the cell and ensure its vital activity. Cellular metabolism consists of two oppositely directed processes: energy metabolism (catabolism) and constructive metabolism (anabolism).

Energy metabolism (catabolism) is a set of oxidation reactions of various reduced organic and non-organic organic compounds accompanied by the release of energy accumulated by the cell in the form of phosphate bonds.

Constructive metabolism (anabolism)- this is a set of biosynthesis reactions, as a result of which, due to substances coming from outside, and intermediate products formed during catabolism, the substance of cells is synthesized. This process is associated with the consumption of free energy stored in ATP molecules or other energy-rich compounds.

Constructive and energy metabolism consists of a series of successive enzymatic reactions, the course of which can be conditionally represented as follows. At the initial stage, molecules are exposed chemical substances, which serve as initial substrates for both types of metabolism. Subsequent transformations include a number of enzymatic reactions and lead to the synthesis of intermediate products. The end products of the constructive pathways formed at the last stages are used to build the substance of the cells, and the energy pathways are released into the environment.

Constructive and energy processes occur in the cell simultaneously. In most prokaryotes, they are closely related. In the process of anabolism, numerous enzymes are synthesized that are involved in energy metabolism. On the other hand, catabolism reactions produce not only energy for biosynthetic purposes, but also many intermediate products that are necessary for the synthesis of substances that make up cellular structures.

The metabolism of prokaryotes, both energetic and constructive, is extremely diverse. This is the result of the fact that bacteria can use the widest range of organic and inorganic compounds as sources of energy and carbon. This ability is due to differences in the set of cellular peripheral enzymes, or exoenzymes, belonging to the class of hydrolases, which are released to the outside and destroy the macromolecules of the original substrates to substances with a low molecular weight. The substances formed as a result of the action of such enzymes enter the bacterial cell and are exposed to the action of intermediate metabolism enzymes.

General characteristics of energy metabolism. In relation to energy sources, all microorganisms are divided into two groups: chemotrophic and phototrophic. The synthesis of ATP molecules from ADP and phosphates can occur in two ways:

Phosphorylation in the respiratory or photosynthetic electron transport chain. This process in prokaryotes is associated with membranes or their derivatives, therefore it is called membrane phosphorylation. ATP synthesis in this case occurs with the participation of ATP synthase:

Phosphorylation at the substrate level. In this case, the phosphate group is transferred to ADP from a substance (substrate) richer in energy than ATP: This method of ATP synthesis is called substrate phosphorylation In the cell, substrate phosphorylation reactions are not associated with membrane structures and are catalyzed by soluble enzymes of intermediate metabolism.

All redox reactions of energy metabolism in chemotrophic microorganisms can be divided into three types:

Aerobic respiration, or aerobic oxidation;

Anaerobic respiration;

Fermentation.

The main process of energy metabolism in many prokaryotes aerobic respiration, in which the donor of hydrogen or electrons are organic (rarely inorganic) substances, and the final acceptor is molecular oxygen. The main amount of energy during aerobic respiration is formed in the electron transport chain, i.e. as a result of membrane phosphorylation. Oxidative phosphorylation is preceded by glycolysis and the tricarboxylic acid cycle (Krebs cycle)

Anaerobic respiration- a chain of anaerobic redox reactions, which are reduced to the oxidation of an organic or inorganic substrate using non-molecular oxygen as the final electron acceptor, but others not organic matter(nitrate, nitrite, sulfate, sulfite, CO: etc.), as well as organic substances (fumarate, etc.). ATP molecules in the process of anaerobic respiration are formed mainly in the electron transport chain, i.e. as a result of membrane phosphorylation reactions, but in a smaller amount than during aerobic respiration.

In anaerobic respiration, the final electron acceptor in the electron transport chain is inorganic or organic compounds. For example, if SO 4 2- is the final electron acceptor, then the process is called sulfate breath, and bacteria are sulfate-reducing or sulfite-reducing . If the final electron acceptor is NO 3 - or NO 2 -, then the process is called nitrate breath or denitrification, and the bacteria that carry out this process - denitrifying . CO 2 can act as the final electron acceptor, the process is respectively called carbonate respiration, and the bacteria methanogenic (methane-forming) . One of the few examples where organic matter serves as the final acceptor is fumarate respiration.

Bacteria capable of anaerobic respiration have shortened electron transport or respiratory chains; they do not contain all the transporters characteristic of respiratory chains functioning under aerobic conditions. In addition, in the respiratory chains of anaerobes, cytochrome oxidase is replaced by the corresponding reductases. In strict anaerobes, the Krebs cycle does not function, or it is broken and performs only biosynthetic, but not energy functions. The main amount of ATP molecules during anaerobic respiration is synthesized in the process of membrane phosphorylation.

Fermentation- a set of anaerobic redox reactions in which organic compounds serve as both donors and acceptors of electrons. As a rule, electron donors and acceptors are formed from the same fermented substrate (for example, from carbohydrate). Various substrates can be fermented, but carbohydrates are used better than others. ATP during fermentation is synthesized as a result of reactions of substrate phosphorylation.

In its biological essence, fermentation is a way of obtaining energy, in which ATP is formed as a result of anaerobic oxidation of organic substrates in reactions of substrate phosphorylation. During fermentation, the products of cleavage of one organic substrate can simultaneously serve as both donors and acceptors of electrons.

During the fermentation of carbohydrates and a number of other substances, products such as ethanol, milk, formic, succinic acid, acetone, CO 2 , H 2 , etc. Depending on which products prevail or are especially characteristic, alcohol, lactic acid, formic acid, butyric, propionic and other types of fermentation are distinguished.

Aerobic respiration is the most beneficial type of redox reactions in bacteria, which generates the largest amount of energy in the form of ATP molecules. The least beneficial type of energy-producing reactions is fermentation, which is accompanied by a minimum yield of ATP.

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Introduction

1. General concepts of metabolism and energy

2. Constructive metabolism

3. Nutrient requirements of prokaryotes

3.1 Carbon sources

3.3 Requirements for sulfur and phosphorus sources

3.4 The need for metal ions

3.5 Need for growth factors

4. Types of metabolism of microorganisms

5. Energy metabolism of phototrophs

6. Energy metabolism of chemotrophs using fermentation processes

7. Energy metabolism of chemoorganotrophs using the process of respiration

8. Energy metabolism of chemolithoautotrophs

Conclusion

This course work contains basic information about the constructive and energy metabolism of bacteria. The work is done on 37 sheets. Contains 5 figures and 1 table.

The totality of the processes of transformation of matter in a living organism, accompanied by its constant renewal, is called metabolism or metabolism.

The most important properties of living organisms are the ability to self-reproduce and their closest relationship with the environment. Any organism can exist only under the condition of a constant influx of nutrients from the external environment and the release of waste products into it.

Nutrients absorbed by the cell are converted into specific cellular components as a result of complex biochemical reactions. The totality of biochemical processes of absorption, assimilation of nutrients and the creation of structural elements of the cell due to them is called constructive metabolism or anabolism. Constructive processes go with the absorption of energy. The energy needed for the biosynthetic processes of other cellular functions, such as movement, osmoregulation, etc., the cell receives due to the flow of oxidative reactions, the totality of which is energy metabolism, or catabolism (Fig. 1).

All living organisms can only use chemically bound energy. Every substance has a certain amount of potential energy. Its main material carriers are chemical bonds, the breaking or transformation of which leads to the release of energy.

Energy level chemical bonds unequal. For some, it has a value of the order of 8-10 kJ. Such connections are called normal. Other bonds contain much more energy - 25-40 kJ. These are the so-called macroergic bonds. Almost all known compounds with such bonds include phosphorus and sulfur atoms involved in the formation of these bonds.

Adenosine triphosphate (ATP) plays an important role in cell life. Its molecule consists of adenine, ribose and three phosphoric acid residues: (Appendices Fig 2)

ATP occupies a central place in the energy metabolism of the cell. Macroergic bonds in the ATP molecule are very fragile. Hydrolysis of these bonds leads to the release of a significant amount of free energy:

ATP + H20 → ADP + H3P04 - 30.56 kJ

Hydrolysis proceeds with the participation of specific enzymes, providing energy for biochemical processes that take place with the absorption of energy. In this case, ATP plays the role of an energy supplier. Having a small size, the ATP molecule diffuses into various parts of the cell. The supply of ATP in cells is continuously renewed due to reactions of addition of a phosphoric acid residue to an adenosine diphosphoric acid (ADP) molecule:

ADP + H3P04 → ATP + H20

The synthesis of ATP, like hydrolysis, occurs with the participation of enzymes but is accompanied by the absorption of energy, the methods of obtaining which in microorganisms, although diverse, can be reduced to two types:

1) use of light energy;

2) use of the energy of chemical reactions.

In this case, both types of energy are transformed into the energy of chemical bonds of ATP. Thus, ATP acts as a transformer in the cell.

Anabolism and catabolism are inextricably linked, making up a single whole, since the products of energy metabolism (ATP and some low molecular weight compounds) are directly used in the constructive metabolism of the cell (Fig. 6.1).

In the cells of microorganisms, the ratio between energy and constructive processes depends on a number of specific conditions, in particular, on the nature of nutrients. Nevertheless, catabolic reactions usually outnumber biosynthetic processes in volume. The interrelation and conjugation of these two types of metabolism is manifested primarily in the fact that the total volume of constructive processes depends entirely on the amount of available energy obtained in the course of energy metabolism.

Constructive metabolism is aimed at the synthesis of four main types of biopolymers: proteins, nucleic acids, polysaccharides and lipids.

The generalized conditional scheme of the biosynthesis of complex organic compounds is shown below, where the following main stages are distinguished: the formation of organic precursors from the simplest inorganic substances (I), from which “building blocks” (II) are synthesized at the next stage. In the future, building blocks, binding to each other by covalent bonds, form biopolymers (III): Applications (Fig. No. 3)

The presented scheme of biosynthetic processes does not reflect the complexity of the transformation of low molecular weight precursors into high molecular weight building blocks. In fact, the synthesis proceeds as a series of successive reactions with the formation of various metabolic intermediates. In addition, the levels of development of the biosynthetic abilities of microorganisms are very different. In some microbes, constructive metabolism includes all the stages shown in the diagram, in others it is limited to the second and third or only the third stage. That is why microorganisms differ sharply from each other in their nutritional needs. However, the elemental composition of food is the same for all living organisms and must include all the components that make up the cellular substance: carbon, nitrogen, hydrogen, oxygen, etc.

Depending on the carbon sources used in constructive metabolism, microorganisms are divided into two groups: autotrophs and heterotrophs.

Autotrophs (from the Greek "autos" - itself, "trophe" - food) use carbon dioxide as the only source of carbon and synthesize all the necessary biopolymers from this simple inorganic precursor compound. Autotrophs have the highest biosynthetic capacity.

Heterotrophs (from the Greek "heteros" - another) need organic sources of carbon. Their nutritional requirements are extremely varied. Some of them feed on the waste products of other organisms or use dead plant and animal tissues. Such microorganisms are called saprophytes (from the Greek "sapros" - rotten and "phyton" - plant). The number of organic compounds used by them as carbon sources is extremely large - these are carbohydrates, alcohols, organic acids, amino acids, etc. Almost any natural compound can be used by one or another type of microorganism as a source of nutrition or energy.

Nitrogen is required for the synthesis of cellular proteins. In relation to the sources of nitrogen nutrition among microorganisms, autoaminotrophs and heteroaminotrophs can be distinguished. The former are able to use inorganic nitrogen (ammonium, nitrate, molecular) or the simplest forms of organic (urea) and build various proteins of their body from these compounds. In this case, all forms of nitrogen are first converted to the ammonium form. This most reduced form of nitrogen easily transforms into an amino group. Heteroaminotrophs need organic forms of nitrogen - proteins and amino acids. Some of them require a complete set of amino acids, others create the necessary protein compounds from one or two amino acids by converting them.

Many microorganisms that are heterotrophic with respect to carbon are autoaminotrophs. These include bacteria involved in wastewater treatment.

Microorganisms satisfy the need for oxygen and hydrogen for constructive exchange at the expense of water and organic nutrients. The sources of ash elements (P, S, K, Mg, Fe) are the corresponding mineral salts. The need for these elements is small, but the presence in the environment is mandatory. In addition, for the normal functioning of microbes, microelements are necessary - Zn, Co, Cu, Ni, etc. Some of them are part of natural nutrition microbes, part is absorbed by them from mineral salts.

The methods of obtaining food, i.e., the methods of nutrition of microorganisms, are very diverse. There are three main types of nutrition: holophyte, saprozoic, holozoic.

Holophyte nutrition (from the Greek "holo" - whole, "fit" - a plant) is carried out according to the type of plant photosynthesis. Such nutrition is inherent only to autotrophs. Among microorganisms, this method is characteristic of algae, colored forms of flagellates and some bacteria.

To understand the complex metabolic processes of microorganisms, it is necessary to consider the chemical composition of the microbial cell and the arsenal of enzymes that it has.

Chemical composition microbial cells.

The chemical composition of microbial cells is the same as that of higher plants. They contain 75-85% water and 15-25% dry matter of the total cell mass.

Water is a necessary component of the cell - chemical processes take place in it, minerals dissolve and complex organic substances - proteins, carbohydrates, fats - are broken down. Proteins and nucleic acids have the most importance during cell reproduction and growth. Carbohydrates are found in significant quantities in the cells of yeast and fungi. These are polysaccharides - glycogen, dextrin, glucose. There are few carbohydrates in bacterial cells.

Fats and fat-like substances (lipids) are found mainly in the surface layer of the cytoplasm. Lipids account for an average of 3-7% of the dry matter of the cell (in tubercle bacillus - 20-40%, in Endomyces fungi - 50-60%).

Minerals are contained in the cells of microorganisms in small quantities (only 3-10%), but their role is great - they affect the speed and direction of chemical reactions. The most important of them are potassium, magnesium, calcium, iron, etc. The content of proteins, fats, carbohydrates and minerals depends on the type of microorganism and the conditions of its existence.

microbial cell enzymes.

Enzymes are complex organic substances that catalyze chemical reactions. Cells produce (produce) them for the implementation of physiological processes. A cell can contain many enzymes (the fungus Aspergillus, for example, has about 50), so that various chemical reactions can occur simultaneously. The most common enzymes produced by microorganisms are carbohydrases and proteases.

Carbohydrases - break down starch, fiber and other polysaccharides with the participation of water. These include amylases (break down starch into simple carbohydrates), maltase (breaks down carbohydrate maltose), lipase (hydrolyzes fats and oils to form fatty acids). These enzymes contain most microorganisms.

Proteases catalyze the breakdown of proteins and polypeptides. These enzymes are produced by putrefactive bacteria, mold fungi, actinomycetes.

Each enzyme has a specific action, that is, it can only cleave certain compounds. In addition, for the action of each enzyme, there are cardinal points in relation to temperature, pH and other conditions.

Metabolism.

Every living cell needs a constant influx of energy - it receives this energy in the process of metabolism. Metabolism (metabolism) is the totality of all chemical reactions occurring in the cell during its life.

Metabolism is carried out in two main directions.

One of them is a building exchange. It is necessary for a living cell for biosynthetic activity, i.e. for building a cell, replacing worn parts, growth and reproduction. The cell receives the necessary building material in the form of food coming from outside. Nutrients enter the microbial cell in two ways. The first is osmosis (diffusion) of nutrients from the external environment, where their concentration is higher than in the cell. The driving force in this case is the difference in osmotic pressure between the cell and the external environment. The second way is the active transfer of nutrients into the cell with the help of special enzymes. In both cases nutrients penetrate the cell membrane into the cytoplasm of the cell. The process of nutrition is the most important physiological function of the microbial cell. The essence of the nutrition process is that under the action of cell enzymes, high-molecular organic compounds are broken down to low-molecular ones: sugars, amino acids, organic acids, and from them the substances of the microorganism cell itself are synthesized: cytoplasm, cell wall, nucleic acids, etc.

In addition to nutrients for building biosynthetic activity, the cell needs energy. Therefore, the second side of the metabolism of microorganisms is energy metabolism, i.e., providing the cell with energy. Microorganisms obtain energy by oxidizing organic substances (carbohydrates, fats and other energy materials) during respiration, a very important physiological function. At different organisms the process of respiration proceeds differently depending on their relationship to oxygen. Thus, aerobes use gaseous oxygen and obtain energy by oxidizing organic matter (respiration). This is possible due to the presence in the cells of aerobes of certain enzymes - cytochromes. In anaerobes, these enzymes are absent and the process of obtaining energy proceeds without the participation of oxygen. In relation to oxygen, anaerobes are divided into three groups. Strict anaerobes (for example, butyric acid bacteria) cannot live at all in the presence of oxygen. They receive energy by conjugated oxidation - reduction of the substrate (for example, fermentation processes). Facultative anaerobes (not strict) in the presence of oxygen use it for oxidative processes (for respiration), and in its absence they receive energy without the participation of oxygen (yeast).

The oxidative processes of anaerobes consist in the removal of hydrogen from the oxidized compound (dehydrogenation). Hydrogen joins other substances (hydrogen acceptors). This process of oxygen-free respiration is called fermentation. Energetic material for fermentation are substances with a large supply of energy.

Thus, nutrients are consumed by the cell in two directions: for the synthesis of body substances and for providing the body with energy. The processes of nutrition and respiration are closely related and are carried out by the cell simultaneously. They provide all the vital functions of the cell. The resulting metabolic products are released from the cell into the external environment. The metabolism is shown in Scheme 1 below.

Scheme 1. Metabolism in microorganisms.

According to the type of nutrition, microbes are divided into two groups: autotrophs and heterotrophs.

Autotrophs- microorganisms that synthesize the substances of their body from inorganic elements. The paths for this synthesis can be different. Some microorganisms, for example, purple sulfur bacteria, like green plants, use photosynthesis, but other substances play the role of chlorophyll in them. Other energy for these synthetic processes is obtained in the course of redox reactions. In this case, inorganic substances serve as electron donors, and carbon dioxide serves as a source of carbon.

Heterotrophs- these are microorganisms that need ready-made organic compounds, using carbohydrates, alcohols and organic acids as carbon sources, and proteins and their decay products as nitrogen sources. The vast majority of bacteria, yeasts and molds are heterotrophs.

oxidative metabolism. Bacteria with an oxidative metabolism obtain energy by breathing.

Breath - the process of obtaining energy in oxidation-reduction reactions associated with oxidative phosphorylation reactions, in which organic (in organotrophs) and inorganic (in lithotrophs) compounds can be electron donors, and only inorganic compounds can be an acceptor.
In bacteria with an oxidative metabolism, the electron (or hydrogen (H+)) acceptor is molecular oxygen. In this case, pyruvate is completely oxidized in the tricarboxylic acid cycle to C 2 . The tricarboxylic acid cycle functions both as a supplier of precursors for biosynthetic processes and hydrogen atoms, which, in the form of reduced NAD, is transferred to molecular oxygen through a series of carriers that have a complex structural multienzyme system - respiratory chain. respiratory chain in bacteria, it is localized in the CPM and in intracellular membrane structures.
Carriers that carry out the transport of hydrogen (electrons) to molecular oxygen belong to 4 classes of dehydrogenases, the coenzymes of which are NAD, flavoproteins, quinones and cytochromes. Protons (electrons) move from one carrier to another in the direction of increasing redox potential. A typical circuit looks like this:

TTK -> NAD (H 2) -> flavoprotein -> quinone ---> cytochromes: c -> c --> a - O 2

Among bacterial cytochromes, cytochromes b, c, a and a 3 are distinguished. The final step in the transfer of electrons (protons) along the respiratory chain is the restoration of cytochromes a - a3 (cytochrome oxidase). Cytochrome oxidase is the terminal oxidase that donates electrons to oxygen. In the process of electron transfer along cytochromes, the valency of iron, which is part of the porphyrated iron group, changes. The electron transfer is completed by the reaction O 2 + 4F 2+ 2O 2 + 4F 3+. The protons formed during the oxidation of FAD or quinones are bound by O 2 " ions to form water.

The formation of ATP in the respiratory chain is associated with the chemoosmotic process. The special orientation of carriers in the CPM leads to the transfer of hydrogen from the inner to the outer surface of the membrane, resulting in a gradient of hydrogen atoms, which manifests itself in the presence of the membrane potential. The energy of the membrane potential is used for the synthesis of ATP localized in the membrane by ATPase.

At this time, in eukaryotes, the respiratory chain enzymes have a relatively constant composition, while in bacteria there are variations in the composition of the respiratory chain. Thus, many bacteria have naphthoquinones instead of ubiquinones, and the composition of cytochromes may depend on the growth conditions of the bacteria. Some bacteria do not have cytochromes, and upon contact with oxygen, hydrogen is directly transferred to oxygen with the help of flavoproteins, and the end product is hydrogen peroxide - H 2 O 2.

In addition to carbohydrates, prokaryotes are able to use other organic compounds, in particular proteins, as an energy source, oxidizing them completely to CO 2 and H 2 O.

Amino acids and proteins can also act as energy resources. Their use is associated, first of all, with certain enzymatic transformations of a preparatory nature. Proteins are first cleaved outside the cell by proteolytic enzymes into peptides, which are absorbed by the cell and cleaved by intracellular peptidases to amino acids. Amino acids can be used in constructive metabolism, and in ammonifying bacteria they can serve as the main material in energy processes during oxidative deamination, as a result of which ammonia is released and the amino acid is converted into a keto acid, which enters constructive metabolism through the tricarboxylic acid cycle:

2R-CHNH 2 -COOH + O 2 -> 2R-CO-COOH + 2NH 3

The process of ammonification is known as "rotting", and there is an accumulation of products that have an unpleasant specific odor of the primary amines formed during this.

Putrefactive bacteria carry out protein mineralization, decomposing it to CO 2, NH 3, H 2 S. Putrefactive bacteria include Proteus, Pseudomonas, Bacillus cereus.

Fermentation (enzymatic) metabolism.

Fermentation, or fermentation,- the process of obtaining energy, in which hydrogen split off from the substrate is transferred to organic compounds.

Oxygen does not take part in the fermentation process. Reduced organic compounds are released into the nutrient medium and accumulate in it. Carbohydrates, amino acids (with the exception of aromatic ones), purines, pyrimidines, polyhydric alcohols can be fermented. Aromatic hydrocarbons, steroids, carotenoids, fatty acids are not able to ferment. These substances decompose and oxidize only in the presence of oxygen; they are stable under anaerobic conditions. Fermentation products are acids, gases, alcohols.

During hexose (glucose) fermentation, pyruvate is only partially oxidized in the tricarboxylic acid cycle. The latter performs only the functions of a supplier of precursors for biosynthetic processes. Energy in the form of 2 ATP molecules is formed as a result of substrate phosphorylation, which occurs during the oxidation of triose phosphate to pyruvate. The hydrogen cleaved from the substrate, which is in the form of reduced NAD, is transferred to pyruvate, turning it into ethanol, acids, and gases in a chain of reactions. Based on the nature of the end products, several types of carbohydrate fermentation are distinguished.

Alcoholic fermentation. It is found mainly in yeast. The end products are ethanol and CO 2 . Fermentation of glucose occurs via the FDF pathway under anaerobic conditions. With the access of oxygen, the fermentation process weakens, it is replaced by respiration. The suppression of alcoholic fermentation by oxygen is called the Pasteur effect.

Alcoholic fermentation is used in the food industry: baking, winemaking.

Lactic acid fermentation. There are two types of lactic acid fermentation: homofermentative and heterofermentative.

At homofermentative type, the breakdown of glucose proceeds along the FDF pathway. The hydrogen from the reduced NAD is transferred to pyruvate by lactate dehydrogenase, producing lactic acid. Homofermentative lactic acid fermentation occurs in S. pyogenes, E. faecalis, S. salivarius in some species of the genus Lactobacillus: L. dulgaricus, L. lactis.

heterofermentative lactic acid fermentation is present in bacteria that lack the FDF pathway enzymes: aldolase and triose phosphate isomerase. Cleavage of glucose occurs via the PP pathway with the formation of phosphoglyceraldehyde, which is further converted to pyruvate via the FDP pathway and subsequently reduced to lactate. Additional products of this type of fermentation are also ethanol, acetic acid. Heterofermentative lactic acid fermentation occurs in various representatives of bacteria of the genera Lactobacillus And Bifidobacterium.

The products of lactic acid fermentation play an important role in the formation of colonization resistance by bacteria of the genus Lactobacillus And Bifidobacterium, constituting the obligate intestinal flora.

Lactic acid bacteria are widely used in the dairy industry for the production of lactic acid products, as well as in the creation of probiotics.

Formic acid (mixed) fermentation. Found in families Enterobacteriaceae Vibrionaceae. Glucose is cleaved by the FDF pathway, gluconate is cleaved by the KDPG pathway.

Depending on the fermentation products released under anaerobic conditions, two types of processes are distinguished:
1. In one case, pyruvate is cleaved to form acetyl coenzyme A and formic acid, which, in turn, can be cleaved into carbon dioxide and molecular hydrogen. Other fermentation products formed through the chain of reactions are ethanol, succinic and lactic acids. Strong acid formation can be detected by reaction with an indicator methyl mouth, which changes color in a strongly acidic environment.
2. In another type of fermentation, a number of acids are formed, but the main products of fermentation are acetoin And 2,3-butanediol. Acetoin is formed from two molecules of pyruvate followed by twofold decarboxylation. Upon subsequent reduction of acetoin, 2,3-butanediol is formed. These substances, when interacting with al-naphthol in an alkaline medium, cause the formation of a brown color, which is revealed Voges-Proskauer reaction used in the identification of bacteria.

Butyric fermentation. Butyric acid, butanol, acetone, isopropanol and a number of other organic acids, in particular acetic, caproic, valeric, palmitic, are products of the fermentation of carbohydrates by saccharolytic strict anaerobes. The spectrum of these acids, determined by gas-liquid chromatography, is used as an express method for the identification of anaerobes.

Protein fermentation. If proteins serve as an energy source for bacteria with a fermentative metabolism, then such bacteria are called peptolytic. Some clostridia are peptolytic, in particular C. histolyticum, C. botulinum. Peptolytic bacteria hydrolyze proteins and ferment amino acids. Many amino acids co-ferment with others, with one serving as a hydrogen donor and the other as a hydrogen acceptor. The donor amino acid is deaminated to a keto acid, which is converted to a fatty acid by oxidative decarboxylation.
5 classification of bacteria in relation to oxygen. Features of cultivation of anaerobes.

Oxygen, widely distributed in nature, is in a free and bound state. In cells, it is in a bound state in the composition of water and organic compounds. In the atmosphere, it is present in the free state in the form of a molecular form, the volume fraction of which is 21%.

In relation to oxygen, as well as its use in the processes of obtaining energy, microorganisms are divided into 3 groups: obligate aerobes, obligate anaerobes, facultative anaerobes.

obligate aerobes.
They grow and reproduce only in the presence of oxygen. They use oxygen to produce energy through oxygen respiration.

Energy is obtained by oxidative metabolism, using oxygen as the terminal electron acceptor in the reaction catalyzed by cytochrome oxidase.

Obligate aerobes are subdivided into strict aerobes, which grow at the partial pressure of the air atmosphere, and microaerophiles, which, using oxygen in energy production processes, grow at its reduced partial pressure.

This is due to the fact that microaerophiles have enzymes that are inactivated upon contact with strong oxidizing agents and are active only at low values of oxygen partial pressure, for example, the hydrogenase enzyme.

obligate anaerobes.
They do not use oxygen for energy.
The type of metabolism they have is fermentative, with the exception of the metabolism of two types of bacteria: Desulfovibrio And Desulfomaculum, which belong to chemolithotrophs and have sulfate respiration. Obligate anaerobes are divided into two groups: strict anaerobes and aerotolerant.

Strict anaerobes characterized by the fact that molecular oxygen is toxic to them: it kills microorganisms or limits their growth.

Strict anaerobes obtain energy by butyric fermentation. Strict anaerobes include, for example, some clostridia (C. botulinum, C, tetani), bacteroids.

Aerotolerant microorganisms do not use oxygen for energy, but can exist in its atmosphere.

This group includes lactic acid bacteria that obtain energy by heterofermentative lactic acid fermentation.

Methods of cultivation of anaerobes.
For the cultivation of anaerobes, it is necessary to lower the redox potential of the environment, to create conditions for anaerobiosis, i.e., a low oxygen content in the environment and its surroundings. This is achieved by using physical, chemical and biological methods.

Physical methods. Based on the cultivation of microorganisms in an airless environment, which is achieved:
1) sowing in media containing reducing and easily oxidized substances;
2) inoculation of microorganisms in the depth of dense nutrient media;
3) mechanical removal air from vessels in which anaerobic microorganisms are grown;
4) replacement of air in vessels by some indifferent gas.

As a reducing agent usually use pieces (about 0.5 g) of animal or plant tissues (liver, brain, kidneys, spleen, blood, potatoes, cotton wool). These tissues bind oxygen dissolved in the environment and adsorb bacteria. To reduce the oxygen content in culture medium, it is boiled for 10-15 minutes before sowing, and then quickly cooled and poured on top with a small amount of sterile vaseline oil. The height of the oil layer in the test tube is about 1 cm.

As easily oxidized substances glucose, lactose and sodium formate are used.

The best liquid culture medium with reducing substances is the Kitt-Tarozzi medium, which is used successfully for the accumulation of anaerobes during the initial inoculation from the test material and to maintain the growth of the isolated pure culture of anaerobes.

Sowing microorganisms in the depth of dense media produced according to the Vinyal-Veillon method, which consists in the mechanical protection of anaerobe crops from atmospheric oxygen. Take a glass tube 30 cm long and 3-6 mm in diameter. One end of the tube is pulled into a capillary in the form of a Pasteur pipette, and a constriction is made at the other end. A cotton plug is inserted into the remaining wide end of the tube. The test material is inoculated into test tubes with melted and cooled to 50°C nutrient agar. The inoculated agar is then sucked into sterile Vinyal-Veyona tubes. The capillary end of the tube is sealed in a burner flame and the tubes are placed in a thermostat. This creates favorable conditions for the growth of the most severe anaerobes. To isolate a separate colony, the tube is cut with a file, observing the rules of asepsis, at the level of the colony, broken, and the colony is captured with a sterile loop and transferred to a test tube with a nutrient medium for further cultivation and study in its pure form.

Air removal produced by its mechanical pumping out of special devices - anaerostats, in which cups with anaerobic sowing are placed. A portable anaerostat is a thick-walled metal cylinder with a well-ground lid (with a rubber gasket), equipped with a drain cock and a vacuum gauge. After placing the seeded cups or test tubes, the air is removed from the anaerostat using a vacuum pump.

Air replacement indifferent gas (nitrogen, hydrogen, argon, carbon dioxide) can be produced in the same anaerostats by displacing it with gas from the cylinder.

Chemical methods. They are based on the absorption of atmospheric oxygen in a hermetically sealed vessel (anaerostat, desiccator) by substances such as pyrogallol or sodium hydrosulfite Na 2 S2O 4 .
biological methods. Based on the joint cultivation of anaerobes with strict aerobes. To do this, a strip of agar about 1 cm wide is cut out from a frozen agar plate along the diameter of the dish with a sterile scalpel. Two agar half-discs are obtained in one dish. An aerobe is inoculated on one side of the agar plate, for example S.aureus or Serratiamarcescens is often used. Anaerobe is seeded on the other side. The edges of the cup are sealed with plasticine or poured with molten paraffin and placed in a thermostat. In the presence of suitable conditions, aerobes will begin to multiply in the cup. After all the oxygen in the space of the cup is used by them, the growth of anaerobes will begin (after 3-4 days). In order to reduce the air space in the cup, the nutrient medium is poured as thick as possible.
Combined methods. Based on a combination of physical, chemical and biological methods creating anaerobiosis.

6 bacterial enzymes. Their classification. Enzymatic activity of microbes and its use for identification of bacteria.
At the heart of all metabolic reactions in a bacterial cell lies the activity of enzymes that belong to 6 classes: oxidoreductases, transferases, hydrolases, ligases, lyases, isomerases. Enzymes formed by a bacterial cell can be localized both inside the cell - endoenzymes, and be released into the environment exoenzymes. Exoenzymes play an important role in providing the bacterial cell with sources of carbon and energy available for penetration. Most hydrolases are exoenzymes that, when released into the environment, break down large molecules of peptides, polysaccharides, and lipids into monomers and dimers that can penetrate into the cell. A number of exoenzymes, such as hyaluronidase, collagenase, and others, are aggression enzymes. Some enzymes are localized in the periplasmic space of the bacterial cell. They are involved in the transfer of substances into the bacterial cell. An enzymatic spectrum is a taxonomic feature that is characteristic of a family, genus and, in some cases, species. Therefore, the determination of the spectrum of enzymatic activity is used to establish the taxonomic position of bacteria. The presence of exoenzymes can be determined using differential diagnostic media; therefore, special test systems have been developed for the identification of bacteria, consisting of a set of differential diagnostic media.

Identification of bacteria by enzymatic activity.

Most often, enzymes of the class of hydrolases and oxidoreductases are determined using special methods and media.

To determine proteolytic activity microorganisms are inoculated into a column of gelatin by injection. After 3-5 days, the crops are viewed and the nature of the liquefaction of the gelatin is noted. When protein is decomposed by some bacteria, specific products can be released - indole, hydrogen sulfide, ammonia. To determine them, special indicator papers are used, which are placed between the neck and a cotton plug in a test tube with BCH and/or peptone water inoculated with the studied microorganisms. Indole (a decomposition product of tryptophan) turns a strip of paper soaked in a saturated solution of oxalic acid pink. Paper impregnated with lead acetate solution turns black in the presence of hydrogen sulfide. For the determination of ammonia use red litmus paper.

For many microorganisms, the taxonomic feature is the ability to decompose certain carbohydrates with the formation of acids and gaseous products.. To detect this, Hiss media containing various carbohydrates (glucose, sucrose, maltose, lactose, etc.) are used. For acid detection Andrede's reagent is added to the medium, which changes its color from pale yellow to red in the pH range of 7.2-6.5, so the set of Hiss media with the growth of microorganisms is called the "variegated row".

For gas detection floats are lowered into liquid media or semi-liquid media with 0.5% agar are used.

In order to determine the intense acid formation, characteristic of mixed fermentation, methyl red indicator, which is yellow at pH 4.5 and higher, and red at lower pH values, is added to a medium with 1% glucose and 0.5% peptone (Clark's medium).

Hydrolysis of urea determined by the release of ammonia (litmus paper) and alkalization of the medium.

When identifying many microorganisms, the Voges-Proskauer reaction for acetoin is used.- an intermediate in the formation of butanediol from pyruvic acid. A positive reaction indicates the presence of butanediol fermentation.

Detect catalase it is possible by oxygen bubbles, which begin to be released immediately after mixing microbial cells with a 1% hydrogen peroxide solution.

To determine cytochrome oxidase reagents are used: 1) 1% alcohol solution of ss-naphthol-1; 2) 1% aqueous solution of N-dimethyl-p-phenylenediamine dihydrochloride. The presence of cytochrome oxidase is judged by the blue color that appears after 2-5 minutes.

For the determination of nitrites Griess reagent is used: The appearance of a red color indicates the presence of nitrites.

7 growth and reproduction of bacteria. Temperature limits of growth. Phases of bacterial reproduction on liquid nutrient media.
Bacterial activity is characterized by growth- the formation of structural and functional components of the cell and the increase in the bacterial cell itself, and also breeding- self-reproduction, leading to an increase in the number of bacterial cells in the population.
bacteria multiply by binary fission in half, less often by budding. Actinomycetes, like fungi, can reproduce by spores. Actinomycetes, being branching bacteria, reproduce by fragmentation of filamentous cells. Gram-positive bacteria divide by ingrowth of the synthesized division partitions into the cell, and gram-negative bacteria divide by constriction, as a result of the formation of dumbbell-shaped figures, from which two identical cells are formed.
Cell division preceded replication of the bacterial chromosome according to a semi-conservative type (the double-stranded DNA chain opens, and each strand is completed by a complementary strand), leading to the doubling of the DNA molecules of the bacterial nucleus - the nucleoid.
DNA replication occurs in three stages: initiation, elongation, or chain growth, and termination.
Reproduction of bacteria in a liquid nutrient medium. Bacteria seeded in a certain, unchanging volume of the nutrient medium, multiplying, consume nutrients, which subsequently leads to the depletion of the nutrient medium and the cessation of bacterial growth. The cultivation of bacteria in such a system is called periodic cultivation, and the culture is called periodic. If the cultivation conditions are maintained by continuous supply of fresh nutrient medium and the outflow of the same volume of culture fluid, then such cultivation is called continuous, and the culture is called continuous.

When growing bacteria on a liquid nutrient medium, near-bottom, diffuse, or surface (in the form of a film) culture growth is observed. The growth of a periodic culture of bacteria grown on a liquid nutrient medium is divided into several phases, or periods:
1. lag phase;
2. phase of logarithmic growth;
3. phase of stationary growth, or maximum concentration of bacteria;
4. phase of bacterial death.
These phases can be depicted graphically as segments of the bacterial reproduction curve, which reflects the dependence of the logarithm of the number of living cells on the time of their cultivation.

Lag phase- the period between sowing bacteria and the beginning of reproduction. The duration of the lag phase is on average 4-5 hours. At the same time, bacteria increase in size and prepare for division; the amount of nucleic acids, protein and other components increases.
Logarithmic (exponential) growth phase is a period of intensive division of bacteria. Its duration is about 5-6 hours. Under optimal growth conditions, bacteria can divide every 20-40 minutes. During this phase, bacteria are the most vulnerable, which is explained by the high sensitivity of the metabolic components of a rapidly growing cell to inhibitors of protein synthesis, nucleic acids, etc.
Then comes the stationary growth phase., at which the number of viable cells remains unchanged, constituting the maximum level (M-concentration). Its duration is expressed in hours and varies depending on the type of bacteria, their characteristics and cultivation.
The death phase completes the process of bacterial growth, characterized by the death of bacteria in conditions of depletion of the sources of the nutrient medium and the accumulation of metabolic products of bacteria in it. Its duration varies from 10 hours to several weeks. The intensity of growth and reproduction of bacteria depends on many factors, including the optimal composition of the nutrient medium, redox potential, pH, temperature, etc.
Reproduction of bacteria on a dense nutrient medium. Bacteria growing on dense nutrient media form isolated rounded colonies with even or uneven edges (S- and R-forms), of different consistency and color, depending on the bacterial pigment.

Water-soluble pigments diffuse into the nutrient medium and color it. Another group of pigments is insoluble in water but soluble in organic solvents. And, finally, there are pigments that are insoluble neither in water nor in organic compounds.

The most common pigments among microorganisms are carotenes, xanthophylls, and melanins. Melanins are insoluble black, brown or red pigments synthesized from phenolic compounds. Melanins, along with catalase, superoxide cismutase, and peroxidases, protect microorganisms from the effects of toxic oxygen peroxide radicals. Many pigments have antimicrobial, antibiotic-like effects.

8 principles of bacterial cultivation. Methods for isolating pure cultures of bacteria, purpose.
Universal Tool

Crops "lawn"

pure culture is a population of bacteria of one species or one variety, grown on a nutrient medium. Many types of bacteria are divided according to one characteristic into biological variants - biovars. Biovars that differ in their biochemical properties are called chemovars, according to antigenic properties - serovars, according to sensitivity to phage - fagovars. Cultures of microorganisms of the same species, or biovar, isolated from various sources or at different times from the same source, called strains, which are usually denoted by numbers or symbols. Pure cultures of bacteria in diagnostic bacteriological laboratories are obtained from isolated colonies by looping them into test tubes with solid or, more rarely, liquid nutrient media.

The colony is a visible isolated accumulation of individuals of one type of microorganisms, formed as a result of the reproduction of one bacterial cell on a dense nutrient medium (on the surface or in its depth). Colonies of bacteria of different species differ from each other in their morphology, color and other characteristics.

A pure culture of bacteria is obtained for diagnostic studies - identification , which is achieved by determining the morphological, cultural, biochemical and other characteristics of the microorganism.

Morphological and tinctorial features bacteria are studied by microscopic examination of smears stained with different methods, and native preparations.

cultural properties characterized by nutritional requirements, conditions and type of bacterial growth on solid and liquid nutrient media. They are established according to the morphology of the colonies and the characteristics of the growth of the culture.

Biochemical signs bacteria are determined by a set of constitutive and inducible enzymes inherent in a particular genus, species, variant. In bacteriological practice, saccharolytic and proteolytic enzymes of bacteria, which are determined on differential diagnostic media, are most often of taxonomic importance.

When identifying bacteria to the genus and species, attention is paid to the pigments that color the colonies and the culture medium in a variety of colors. For example, the red pigment is formed by Serratia marcescens, the golden pigment is formed by Staphylococcus aureus (Staphylococcus aureus), the blue-green pigment is Pseudomonas aeruginosa.

To establish a biovar(chemovar, serovar, phagotype) conduct additional studies to identify the appropriate marker - the definition of enzyme, antigen, sensitivity to Fans.

Methods for isolating pure cultures of bacteria.

Universal Tool for the production of crops is a bacterial loop. In addition to it, a special bacterial needle is used for inoculation with an injection, and metal or glass spatulas are used for inoculation on Petri dishes. For inoculation of liquid materials, Pasteur and graduated pipettes are used along with the loop. The former are pre-made from sterile fusible glass tubes, which are pulled out on a flame in the form of capillaries. The end of the capillary is immediately sealed to maintain sterility. For Pasteur and graduated pipettes, the wide end is covered with cotton wool, after which they are placed in special cases or wrapped in paper and sterilized.

When reseeding the bacterial culture take a test tube left hand, and with the right, grasping the cotton plug IV and V with fingers, they take it out, passing it over the burner flame. Holding the loop with the other fingers of the same hand, they collect the inoculum with it, and then close the test tube with a stopper. Then, a loop with inoculum is introduced into the test tube with slant agar, lowering it to the condensate in the lower part of the medium, and the material is distributed in a zigzag motion over the slant surface of the agar. After removing the loop, burn the edge of the test tube and close it with a cork. The loop is sterilized in the flame of a burner and placed in a tripod. Tubes with cultures are labeled, indicating the date of sowing and the nature of the seed (study number or culture name).

Crops "lawn" produced with a spatula on nutrient agar in a Petri dish. To do this, having slightly opened the lid with the left hand, the inoculum is applied to the surface of the nutrient agar with a loop or pipette. Then they pass the spatula through the flame of the burner, cool it about inside covers and rub the material over the entire surface of the medium. After the incubation of the inoculation, a uniform continuous growth of bacteria appears.

Module 2. The concept of metabolism, homeostasis, physiological adaptation of a person.

Morpho-functional characteristics of a neuron (soma, dendrites, axon, axon transport, metabolism). Types of nerve cells. Functional classification of neurons.

The life of the human body is a very complex and unique phenomenon, however, it has such mechanisms that support its existence and at the same time they can be disassembled into the simplest components that are accessible to everyone. Here, first of all, it must be said about the metabolism of bacteria, which is only conditionally complex, in fact, such a process as the metabolism of bacteria is quite simple. The science of microbiology helps to get acquainted in detail with the process of metabolism of microorganisms. The processes being studied help shape new forms of treatment for a variety of ailments.

If we talk about the general picture of the metabolic bacterial process, then we are talking about a certain reaction cycle, and some reactions are responsible for providing the human body with energy, and as for others, they are ways to replenish the body with matter, that is, in fact, they are a kind of building material. If we talk about the metabolism of bacterial cells, then it is impossible to find differences from biological principles. general type. Bacteria are the basis of the supporting mechanism of the life process of living cells.

There are 2 types of such a process that depend on metabolic products:

catabolism destructive type or destructive reaction. This type of metabolism can be provided by oxidative respiration. The fact is that when the respiratory process is carried out, elements of the oxidative type flow into the human body, which begin to oxidize chemical compounds of a certain type when ATP energy is released. Such energy is available in cells in the form of phosphate-type bonds.
Anabolism constructive type or a creative reaction. We are talking about the process of biosynthesis that organic molecules undergo, they are necessary character to keep the cell alive. The whole process goes like a reaction chemical type, substances and products of the intracellular type take part in such reactions. Such reactions receive energy due to the fact that the stored energy stored in ATP is consumed.

Most of the processes of the metabolic type take place in a cell of the prokaryotic type, and such a process is of a one-time nature, all this has the form of a closed-type cycle. When the metabolic process takes place, products begin to form, which are accompanied by cell-type structures, then a biosynthetic reaction begins to start, in which certain enzymes take part, they carry out the process of synthesis of an energy nature. These types of microbial metabolism are not the only ones, there are others.

The metabolism of microorganisms refers to the substrate, here we are talking about several stages:

peripheral stage when the substrate is processed by enzymes that are produced by bacteria;
intermediate stage when products of an intermediate type begin to be synthesized in the cell;
final stage- it begins the process of release of final products into the environment that surrounds it.

All the features of this process are due to the fact that there are two types of enzymes (we are talking about protein-type molecules that can accelerate reactions in the cellular structure:

First of all, it must be said about exoenzymes, which are protein-type molecules, when the cell begins to be produced outside, and the external substrate begins the process of destruction to molecules of the original type.
Separately, we talk about endoenzymes, which are also protein-type molecules that act inside the cell, and then a joint reaction begins with substrate molecules that come from outside.

It should be noted that there are a number of enzymes that are produced by the cellular structure on an ongoing basis (of a constitutive nature), and there are also those that carry out production in the form of a reaction to when a certain substrate appears.

Energy type metabolism

Such a process in bacteria is carried out in certain ways of a biological type:

The first way is chemotrophic, when energy is obtained in the course of chemical reactions.
The second way is phototrophic (here we are talking about the energy of photosynthesis).

If we talk about how bacteria breathe in a chemotrophic way, then there can be 3 ways:

oxygen character oxidation;
oxidation without the use of oxygen;
fermentation process.

Features of bacterial metabolism

Such processes are characterized by extreme speed and intensity. Within just one day, one bacterium is able to process such an amount of nutrients that exceeds its own weight by 40 times!
To all external conditions, even the most unfavorable bacteria adapt very quickly.
As for the nutritional process, it occurs through the entire cell surface. It is noteworthy that prokaryotes are not able to swallow nutrients, inside cell structure they are not able to be digested, their splitting is carried out outside the cell, and chemosynthesis of cyanobacteria is also observed.

How do microorganisms grow and reproduce?

It should be noted that growth is the process when an individual increases in size, and as for the reproduction process itself, this is when the population begins to increase.

It is noteworthy that bacteria are able to multiply in such a way that binary fission is simply carried out, however, this method is far from the only one, there is also budding. If the bacteria have a gram-positive form, then there is a formation of a partition from a cell-type wall and a cytoplasmic-type membrane, which is capable of growing inward. If the bacteria are gram-negative, then a constriction begins to form, after which the cell splits into a couple of individuals.

The speed of the breeding process is noteworthy, it can be different. If we talk about the vast majority of bacteria, then they divide every half hour. And there are tuberculosis mycobacteria, the process of division of which is slower, suffice it to say that for one division it may take at least 18 hours. Spirochetes also do not divide quickly, about 10 hours, so you can see how the metabolism of microorganisms differs.

If you sow bacteria in a liquid nutrient medium, taking a certain volume, and then take a sample every hour, then bacterial growth has the form of a curved line.

Such substances grow in several phases:

the latent phase, in which bacteria have the ability to quickly adapt to the nutritional environment, and as for their number, it does not increase;
a logarithmic growth phase, when the bacterial count begins to increase exponentially;
the growth phase of a stationary type, when as many new substances appear as they die, and living microorganisms remain constant, all this can reach a maximum level. A term such as M-concentration is used here, this is a value that is characteristic of all bacterial types;
the dying phase is a process in which the number of dead cells becomes greater than the number of viable cells. This happens because metabolic products accumulate in the body and the environment is depleted.

In conclusion, it should be noted that the metabolism of all bacteria and microbes may have certain differences; a variety of factors may take place here. Great importance have individual characteristics of the human body. As for such a process as the regulation of metabolism, it began to be studied even in prokaryotes, and specifically in prokaryotes (these are the intestinal coli operons).

To date, there are a variety of research methods. If sulfur bacteria are studied, then the study has its own characteristics, and other methods can be used to study bacterial changes. And iron bacteria deserve special attention, which have a unique feature of oxidizing ferrous iron.