GROWTH AND CULTIVATION OF BIOOBJECTS - V. M. Samygin - 2016

CHAPTER 4. BASIC MECHANISMS OF METABOLISM AND ENERGY CONVERSION IN PROKARYOTES

Microbial Cells constantly undergo a multitude of biochemical reactions that collectively comprise METABOLISM—a set of processes aimed at preserving and reproducing life, supported by Nutrition and Respiration. All metabolic processes are governed by the "principle of biochemical unity" (Kluyver, 1926), according to which All living organisms on Earth are biochemically similar: they share common building blocks, a single energy accumulator—ATP, a universal Genetic Code, and fundamentally identical Major Metabolic Pathways.

The distinctive features of microbial metabolism include:

- a holophytic mode of nutrition, in which nutrients must be dissolved in a liquid;

- the uptake of nutrients across the entire Cell surface, which is extremely large relative to the size of the microorganism itself;

- an extraordinary intensity of metabolic processes (under favorable conditions, a single cell consumes 30–40 times its own body weight in food per day);

- A wide variety of Energy Sources and plastic Materials;

- a high degree of adaptation to changing environmental conditions.

Nutrients entering The Cell are utilized in two main directions. Part of them is used in Cleavage and oxidation reactions to generate energy and store it in the form of ATP or other high-energy compounds—Energy Metabolism (Catabolism). Another part is channeled into constructive, biosynthetic metabolism (anabolism). These processes are coupled and merely reflect different aspects of metabolism. The End products of these reactions are called metabolites. At certain stages of metabolism, intermediate reactions rearrange some substances into others. These are known as amphibolic reactions, and their products—amphibolites—can be used in both anabolic and catabolic processes.

Nutrients entering the cell are first broken down into small fragments and then, through amphibolic reactions, converted into a series of organic acids and phosphoric esters. These two pathways transition seamlessly into one another. Diverse low-molecular-weight compounds serve as the substrate from which "building blocks" are synthesized—namely, Amino Acids, purine and pyrimidine bases, phosphorylated sugars, organic acids, and other metabolites representing the end products of biosynthetic chains. These are used to build polymeric macromolecules, such as Nucleic Acids, Proteins, reserve substances, and Cell wall components, which make up the cell. These two stages of cellular substance Biosynthesis—the synthesis of building blocks and the synthesis of polymers—constitute the synthetic branch of metabolism, or anabolism.

The oxidation of glucose, as the primary amphibolite, generally proceeds via three pathways:

- Glycolysis, or the Embden-Meyerhof-Parnas pathway, characterized by The formation of two molecules of pyruvic acid (Pyruvate) from a single glucose molecule, the synthesis of three pyridine nucleotide molecules, and two ATP molecules;

- the Pentose Phosphate Pathway (hexose monophosphate shunt, or the Warburg-Dickens-Horecker pathway), which plays an auxiliary role in microbes. Its reactions yield pentoses for subsequent syntheses, pyruvate, three pyridine nucleotide molecules, and two ATP molecules;

- the ketodeoxyphosphogluconate (KDPG, or the Entner-Doudoroff) pathway, which is exclusive to microorganisms and leads to the formation of two pyruvate molecules, one ATP, and one NADPH from a single glucose molecule.

After a series of preliminary stages, glucose is split "in half," and the cleavage products are converted into pyruvate, which occupies a key position in Intermediary Metabolism as a starting compound in numerous Synthesis and degradation processes. As a result of pyruvate decarboxylation, C₂ compounds are formed, which first bind to an acceptor molecule (oxaloacetate) and are then gradually oxidized to CO₂ and H₂O in The Tricarboxylic Acid Cycle, also known as The Citric Acid Cycle. Oxaloacetate (oxaloacetic acid) is regenerated during this cyclic process. Hydrogen atoms (or reducing equivalents), released at various stages of organic substance oxidation, enter the ATP-regenerating System of the Respiratory Chain (Oxidative Phosphorylation). With each turn of the tricarboxylic acid cycle, two molecules of CO₂ and eight hydrogen protons are produced from a single C₂ unit (acetyl-coenzyme A—activated acetic acid). These reactions balance the tricarboxylic acid cycle.

Intermediates of the tricarboxylic acid cycle include organic acids supplied for biosynthetic processes (2-oxoglutarate, succinate, oxaloacetate). The tricarboxylic acid cycle not only participates in the terminal oxidation of nutrients but also acts as a "distributor," supplying the initial compounds for the Synthesis of the cell's basic structural units ("building blocks").

If these acids were continuously removed from the cycle, the regeneration of the acceptor molecule would fail, disrupting the cycle. However, anaplerotic reaction sequences ensure a continuous supply of new intermediate compounds to the tricarboxylic acid cycle to replace those consumed in biosynthesis. These sequences are particularly crucial for organisms growing on simple one- or two-carbon compounds.

All vital processes of the microbial cell are associated with energy consumption and require its replenishment, which is accomplished through exothermic biochemical Reactions Involving the oxidation of various organic and inorganic substances. These processes of energy metabolism (catabolism), which restore cellular energy, are driven by respiration. They utilize the same compounds that are used to build the Structural components of the cell—sugars, alcohols, organic acids, fats, etc.

The oxidation of substances can occur via different pathways:

- direct, when oxygen is added to a substance. In this case, oxidases mediate the direct oxidation of an inorganic substrate—molecular hydrogen, carbon monoxide, or sulfur—by atmospheric oxygen. Direct oxidation is observed in the majority of Saprophytes;

- indirect, through the removal of two hydrogen atoms or two electrons (e^-) from the substrate. This process is called dehydrogenation. The hydrogen (e^-) abstracted from the oxidized substrate-donor is transferred to another substance, which is thereby reduced. The substance accepting the hydrogen (e^-) is called the acceptor. The process of hydrogen and electron transfer is equivalent and is accompanied by the release of energy, which is utilized by the cell and accumulated in high-energy compounds (primarily ATP). The terms "hydrogen donor" and "electron donor" are frequently used as synonyms; similarly equivalent are "hydrogen acceptor" and "electron acceptor," as well as "oxidation" and "dehydrogenation," and "reduction" and "hydrogenation."

Enzyme proteins that cleave hydrogen atoms from substrates are called dehydrogenases. In Bacteria, this process involves

several dehydrogenases, which are named after their hydrogen donor (e.g., Lactate dehydrogenase, malate dehydrogenase). They transfer hydrogen to one of two Coenzymes—nicotinamide adenine dinucleotide (NAD⁺) or nicotinamide adenine dinucleotide phosphate (NADP⁺). These coenzymes readily dissociate from the dehydrogenase protein and, after binding to another dehydrogenase, transfer the hydrogen to a different acceptor. Therefore, they are also referred to as hydrogen carriers.

Energy-requiring processes are powered by ATP. ATP (adenosine triphosphate) is the primary carrier of biologically usable energy (the cell's "energy currency"), acting as a universal carrier of chemical energy between energy-yielding and energy-consuming reactions. All energy processes in living cells are directly or indirectly coupled with The conversion of ATP to ADP and inorganic phosphate (P). The cleavage of phosphate bonds in ATP releases a large amount of energy, which is then stored in the reaction products.

There are Two main mechanisms of ATP generation in cells:

- Membrane-bound oxidative phosphorylation, which proceeds by removing electrons from an organic or inorganic substrate (its oxidation) and transferring them to terminal acceptors, which are thereby reduced. The terminal acceptors are Inorganic Compounds—oxygen (in aerobes), sulfates, nitrates, carbonates, etc. (in anaerobes). This electron transfer occurs in mesosomes—membrane structures where respiratory Enzymes are localized—via Electron transport along the Electron Transport Chain (ETC). A classic example of this ATP synthesis mechanism is the Krebs cycle. As noted, a single turn of this cycle yields two molecules of carbon dioxide, eight reducing equivalents, and one molecule of ATP;

- Substrate-level phosphorylation (Fermentation). In this pathway, electron Donors and acceptors are Organic compounds (most commonly hexoses), the oxidation of which takes place in cyclic processes (the tricarboxylic acid cycle and its variants, The pentose phosphate cycle, etc.).

In bacteria, the most frequent oxidation pathway is dehydrogenation. Here, hydrogen (e^-) transfer is mediated by oxidation-reduction enzymes (oxidoreductases), and the terminal acceptor of H₂ (e^-) can be oxygen or another reducible substance:

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Based on their Biological Oxidation pathways, bacteria are divided into several groups:

- Obligate aerobes — bacteria for which free molecular atmospheric oxygen serves as the terminal hydrogen (e^-) acceptor;

- Microaerophiles — aerobic bacteria whose GROWTH AND REPRODUCTION are optimal at reduced oxygen partial pressures. These include Leptospira, Actinomyces, and others. Some microaerophilic bacteria (e.g., Neisseria, Brucella) grow better at elevated CO₂ concentrations;

- Obligate anaerobes — bacteria that obtain energy by oxidizing organic or inorganic (hydrogen, sulfur, iron, hydrogen sulfide, etc.) substances in the absence of free atmospheric oxygen, or through anaerobic Photosynthesis;

- Facultative anaerobes, which are capable of extracting energy from substrates via both aerobic and anaerobic biological oxidation pathways depending on the oxygen source.

In energetic terms, the aerobic type of Respiration is the most efficient. Under aerobic conditions, the substrate is oxidized to CO₂ and Water or hydrogen peroxide. This multi-step process proceeds with the participation of A number of enzymes (cytochrome oxidase and the heme-containing cytochrome system) with the formation of various intermediates. The electrons removed in the process are transferred along the respiratory chain through a series of oxidative stages to activated oxygen. Free oxygen acts as the terminal acceptor for the removed electrons. This process releases energy and is accompanied by ATP synthesis. Aerobic microorganisms include mycobacteria, vibrios, bacilli, and others. They are cultivated using nutrient media with high aeration, and during bacterial identification, the presence of respiratory enzymes—oxidases, catalase, Cytochromes, etc.—is determined.

Anaerobic respiration refers to biological oxidation pathways in which organic (fumarate) or inorganic substances (nitrates, sulfates, sulfur, iron, carbonates), rather than free atmospheric oxygen, serve as the TERMINAL ELECTRON ACCEPTORS. All types of anaerobic respiration are classified According to the terminal electron acceptor utilized (Table 2).

Table 2. Types of anaerobic respiration

Nitrate respiration is carried out via nitrate ammonification or denitrification. Nitrate ammonification yields ammonia, which is utilized for the synthesis of Amino Acids and other nitrogen-containing compounds. This type of respiration is known in E. coli, B. subtilis, P. vulgaris, and others. Nitrate denitrification results in the formation of molecular nitrogen. This respiration pathway is found in Escherichia, staphylococci, pseudomonads, and certain other microorganisms. During nitrate respiration, organic substrates are oxidized to carbon dioxide and water. The synthesis of enzymes involved in nitrate reduction (nitrate and nitrite reductases) is induced only under anaerobic conditions. The potential energy yield with nitrate as the hydrogen acceptor is approximately 10% lower than that of aerobic respiration. Nitrate respiration is characteristic of facultative anaerobes.

Sulfate respiration has been identified in a small group of obligate anaerobes (sulfate-reducing bacteria and archaea) that use sulfate as the terminal hydrogen acceptor, resulting in The production of hydrogen sulfide:

8Н + SO₄ —> H₂S + Н₂O + 2OН^-

These microorganisms utilize organic acids or alcohols (chemoorganoheterotrophs) or molecular hydrogen (chemolithotrophs) as hydrogen donors. The organic substrates are not always oxidized completely; the end product is typically acetic acid. Therefore, such bacteria can be employed as producers of acetic acid. In natural environments, sulfate-reducing bacteria are found in hydrogen sulfide-rich sludge where anaerobic breakdown of organic matter occurs. These bacteria are responsible for the high hydrogen sulfide concentration in the deep layers of the Black Sea, as well as for the anaerobic corrosion of metal and concrete.

Sulfur respiration can be observed in volcanically active areas rich in abiogenic elemental sulfur under anaerobic conditions. Most sulfur bacteria are obligate chemolithotrophs.

Iron respiration involves the REDUCTION OF Fe³⁺ to Fe²⁺; however, ferric iron compounds are practically insoluble and precipitate outside the cell. Consequently, chelating agents—siderophores—are involved in The transport of such iron.

Carbonate respiration refers to respiration where carbon dioxide (CO₂) serves as the terminal electron acceptor. This type of respiration is known in methane-producing microorganisms (methanogens) and homoacetogenic bacteria. These are morphologically diverse methanogenic archaea—anaerobes that oxidize H₂. Methanogenic archaea inhabit swamps, where they produce marsh gas, as well as sludges and wastewater Treatment plants. Approximately 70% of humans harbor methanogens in their intestinal microbiota.

In fumarate respiration, the organic compound fumarate—an intermediate in the oxidation of acetyl-CoA in the tricarboxylic acid cycle—serves as the terminal electron acceptor. Virtually all microorganisms possessing an electron transport chain with succinate dehydrogenase are capable of this type of respiration:

2Н⁺ + fumarate —> succinate.

Enterobacteria, vibrios, peptostreptococci, propionic acid bacteria, and several other groups are capable of fumarate respiration.

Energy-yielding processes in which organic compounds act simultaneously as both electron donors and acceptors are called fermentation. Oxygen is not involved in fermentation processes. ATP during fermentation is generated solely via substrate-level phosphorylation without the participation of the respiratory chain. Fermenting bacteria belong to facultative or obligate anaerobes. Facultative anaerobes (e.g., enterobacteria) grow as aerobic heterotrophs in the presence of oxygen. During aerobic dehydrogenation, two toxic compounds can be formed as byproducts: hydrogen peroxide and the peroxy radical. These are degraded by the enzymes catalase and dismutase. In such bacteria, metabolism shifts to fermentation under anaerobic conditions. The cells of obligate anaerobes lack catalase and superoxide dismutase; therefore, they cannot tolerate the presence of oxygen and perish in the air. Clostridia are Examples of obligate anaerobes.

Depending on the predominant or most characteristic end products, fermentation is classified into alcoholic (carried out by Yeasts and Mucorales Fungi), lactic acid (caused by bacteria of the family Lactobacteriaceae), butyric acid (caused by the genus Clostridium), and propionic acid (caused by the genus Propionibacterium). Alcoholic Fermentation yields two molecules of ethanol, two molecules of carbon dioxide, and 22 kcal of energy. In Lactic acid fermentation, cells derive two molecules of lactic acid and 18 kcal of energy from glucose.

Microorganisms expend a portion of the consumed energy on the sequential synthesis of macromolecules for their own cells. These macromolecules are built from corresponding subunits: proteins from amino acids, Polysaccharides from Monosaccharides, Lipids from Fatty acids and glycerol, and nucleic acids from NUCLEOTIDES, phosphoric acid, and ribose (or deoxyribose).

The synthesis of these essential macromolecules requires about 70 different precursors, as well as a number of compounds that play a catalytic role. These include approximately 20 coenzymes and a series of electron carriers. In total, the formation of a new cell requires about 150 different small molecules. The most important such intermediates are sugar phosphate esters, pyruvic, acetic, oxaloacetic, succinic, and α-ketoglutaric acids.

A bacterial cell is capable of synthesizing several thousand different protein molecules, requiring only 20 amino acids for their synthesis. Protein Biosynthesis requires not only enzymes and monomers (amino acids), but also a template (mRNA molecule) that determines The sequence of amino acid addition to the growing chain, as well as a specific carrier to activate the monomer and select it according to the specified code (tRNA). The reaction forming a new peptide chain takes place on the ribosome.

In nucleic acid biosynthesis, the building blocks are purine and pyrimidine nucleotides. The formation of nucleic acids is carried out via template-directed synthesis during Replication and Transcription. DNA replication occurs on each strand of the double-stranded parent DNA serving as a template, involving DNA polymerases and utilizing deoxyribonucleoside 5'-triphosphates as substrates. The template for transcription is one of the DNA strands, and the substrates are ribonucleotide 5'-triphosphates. RNA Synthesis is mediated by RNA polymerases enzymes.

In the synthesis of fatty acids that make up bacterial lipids, an important role is played by the acyl carrier protein (ACP), on which the carbon chain of the forming fatty acid is elongated. The synthesis of fatty acids with an odd number of carbon atoms begins with the Formation of Acetyl-ACP and malonyl-ACP followed by their Condensation. As a result, the growing ACP-bound compound is extended by two carbon atoms. The synthesis of fatty acids with an even number of atoms differs only in the initial reaction, which involves the condensation of propionyl-ACP with malonyl-ACP.

Polysaccharide synthesis is carried out from glucose, its derivatives, and other sugars by joining them together.

The regulation of activity and coordination of individual Metabolic pathways are crucial for the survival of organisms. Because The chemical composition of the surrounding environment is constantly changing, regulatory processes must continuously adapt to new conditions. Such regulation is essential for maintaining a balance between energy-yielding and biosynthetic reactions. It is extremely complex and involves a wide range of interacting regulatory mechanisms operating within the cell.