MICROBIOLOGY Study Guide - 2012

CHAPTER 7. THE IMPACT OF ENVIRONMENTAL FACTORS ON MICROORGANISMS

7.2. CHEMICAL FACTORS

Medium reaction (pH) is determined by the concentration of hydrogen [Н⁺] and hydroxyl [OH^-] ions in an aqueous solution. To quantitatively characterize the reaction of the medium, the pH value—the hydrogen ion exponent—is introduced. The reaction of the medium is one of the key Factors Determining the bacterial growth rate. Changes in medium pH can alter enzyme activity and, consequently, The rate of biochemical reactions occurring within The Cell. Furthermore, it can shift the biochemical pathways of transformations carried out by microorganisms. For instance, the same Yeast species will produce ethanol and carbon dioxide from sugar in an acidic environment, whereas in an alkaline environment, the ethanol yield decreases while glycerol content increases.

Alterations in the medium reaction affect the electrical charge of the cell surface, leading to changes in Cell wall permeability for various molecules and ions of the nutrient substrate, thereby disrupting normal metabolic processes.

The vital activity of each microbial species is possible only within a specific pH range. The limiting pH values for microorganisms range from 4.0 to 9.0. The most favorable growth pH values lie within a relatively narrow range. Based on their optimal pH requirements, microorganisms are classified into acidophiles (growing in acidic environments), neutrophiles (growing at neutral pH), and alkaliphiles (growing in alkaline environments). Molds and Yeasts prefer an acidic environment with a pH of 4.0–6.0. The majority of pathogenic and putrefactive Bacteria thrive best at a neutral pH of 6.5–7.5. Some bacterial species can multiply in highly alkaline environments; for example, urobacteria grow at a pH of around 9.0. Bacteria that produce acid during their metabolic activity are more resilient to drops in medium pH. These include acetic acid and lactic acid bacteria, which are classified as acid-tolerant.

The RESISTANCE OF MICROORGANISMS to acidic and alkaline environments is explained by the specific chemistry of their cellular barriers. The cytoplasmic membrane of microorganisms has low permeability to hydrogen and hydroxyl ions. In addition, during METABOLISM, microbes are capable of pumping [Н⁺] or [OH^-] ions out of the cell, maintaining an intracellular pH within the neutral range despite wide fluctuations in the pH of the surrounding medium.

The NEGATIVE IMPACT OF elevated medium acidity on most microorganisms is utilized in the food industry as a preservation method through marinating or pickling. In the former case, a small amount of acetic or citric acid is added to the product; in the latter, lactic acid bacteria are allowed to proliferate, accumulating lactic acid and thereby inhibiting the growth of putrefactive bacteria.

Redox conditions of the medium. Numerous oxidation-reduction processes continuously occur within living Cells, the rate of which is determined by the redox potential (E_h). This parameter characterizes the tendency of a solution to donate or accept electrons and is expressed as the negative logarithm of the partial pressure of molecular hydrogen in the solution. It reflects the degree of oxygen or hydrogen saturation of the medium. If an aqueous solution is saturated with hydrogen, E_h = 0, whereas if it is saturated with oxygen, E_h = 41. For anaerobic microorganisms, energy generation proceeds without the participation of free oxygen, and their vital activity manifests at E_h values ranging from 0 to 12, reaching maximum multiplication rates at E_h 3–5. Aerobic microorganisms multiply within an E_h range of 14–36.

By regulating the redox conditions of the medium, one can influence the rate of microbial multiplication. Through their metabolic activity, microorganisms can alter the redox conditions of the medium by secreting various metabolic byproducts, thereby adapting the environment to their own needs. E_h is measured by immersing an inert electrode (typically platinum) into the solution and recording the potential difference between the platinum electrode and a reference electrode.

Oxygen. Significant differences exist among prokaryotes regarding their relationship with molecular oxygen. Based on this characteristic, they are divided into several physiological groups.

1. Obligate aerobes are capable of growing only in the presence of atmospheric oxygen (pseudomonads, bacilli, acetic acid bacteria, Mycobacterium tuberculosis). Many obligate aerobes can withstand oxygen concentrations of about 40–60%. However, this group also includes bacteria that require molecular oxygen in negligible amounts—no more than 2%. Such microorganisms are termed microaerophiles (brucellae, leptospires).

2. Facultative anaerobes can grow both in the presence and absence of oxygen. Many prokaryotes belonging to this group have adapted to exist depending on the availability or absence of atmospheric oxygen and can switch from one metabolic pathway to another—i.e., from Respiration to Fermentation and vice versa. Representatives of this group include enterobacteria (family Enterobacteriaceae). Under aerobic conditions, they obtain energy via respiration because they possess Respiratory Chain Enzymes. Under anaerobic conditions, enterobacteria carry out mixed-acid fermentation.

3. Obligate anaerobes are capable of living and multiplying only in the absence of atmospheric oxygen. In the cells of obligate anaerobes, substrate oxidation occurs without oxygen participation, yielding a small amount of energy, with oxidized mineral compounds serving as hydrogen acceptors. Strict anaerobes include methanogenic bacteria, sulfate-reducing bacteria, the causative agents of tetanus and botulism, butyric acid bacteria, etc.

Lactic acid bacteria are classified as facultatively anaerobic microorganisms; however, they possess exclusively anaerobic-type metabolism. Although they can grow in the presence of atmospheric oxygen, they are unable to utilize it and are therefore assigned to a separate group of aerotolerant anaerobes.

Oxygen derivatives exert a toxic effect on prokaryotes. When Flavoproteins are oxidized by oxygen, hydrogen peroxide is formed as one of the toxic byproducts:

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Hydrogen peroxide is produced by all aerobes and anaerobes growing under aerobic conditions, making its generation in Prokaryotic Cells a natural process. Aerobic and aerotolerant bacteria contain the enzyme catalase, which breaks down hydrogen peroxide, thereby protecting the cell from its toxic effects:

Only a single group of bacteria capable of growing in the presence of atmospheric oxygen lacks catalase—the lactic acid bacteria. However, they do not accumulate significant amounts of hydrogen peroxide because it is decomposed within them by peroxidase enzymes.

Anaerobic bacteria lack both catalase and peroxidase; consequently, H₂O₂ accumulates in the medium and exerts a toxic effect on them.

If molecular oxygen is reduced stepwise, The transfer of a single electron to O₂ yields the superoxide anion:

Exogenously generated superoxide anions can penetrate the cell and participate in reactions leading to various intracellular damages. Many prokaryotes possess specific defense mechanisms against superoxide anions in the form of the enzyme superoxide dismutase (SOD), which catalyzes The conversion of this radical into oxygen and hydrogen peroxide:

Thus, superoxide dismutase, catalase, and peroxidase protect the cell against the toxic products of oxygen metabolism.

Chemical agents. The effects of chemical agents on microorganisms can vary: growth-stimulating, microbostatic (growth-inhibiting), and microbicidal (leading to cell death). The resulting effect depends on The Nature of the chemical compounds themselves, their concentration, and accompanying environmental factors such as Temperature, pH, etc.

Chemical agents that exert a lethal effect on microorganisms are called antiseptics. The action of chemical agents that results in the destruction of microorganisms is termed disinfection.

Inorganic substances possessing bactericidal properties include heavy metals and their salts (silver, mercury, lead, copper, etc.). Heavy Metal Ions cause the death of microorganisms even at low concentrations, exhibiting what is known as the "oligodynamic effect" (from the Greek oligos—small, dynamic—power). The oligodynamic properties of silver have long been utilized for the disinfection of drinking Water. Upon penetrating the cell, heavy metal ions act as potent enzyme poisons by binding to SH-groups, thereby profoundly altering the Tertiary and Quaternary structures of enzyme Proteins.

Effective antiseptics include strong oxidizing agents: chlorine-, bromine-, and iodine-containing compounds, as well as potassium permanganate and hydrogen peroxide. Chlorine-based preparations include bleaching powder, chloramines, pantocid, neopantocid, sodium hypochlorite, chlordeysin, sulfochlorantine, etc. Promising preparations based on iodine and bromine include iodopyrine and dibromantine. The bactericidal action of these substances is based on the impairment of enzyme systems and microbial cell proteins. Liquid chlorine is widely used for disinfecting drinking water, whereas 0.5–5.0% aqueous solutions of bleaching powder and chloramine are employed for the disinfection of industrial equipment, premises, etc.

Hydrocyanic acid and its salts (KCN) act as respiratory poisons. By binding iron, they block the function of the terminal respiratory enzyme, cytochrome oxidase. Carbon monoxide (CO) inhibits respiration by competing with free oxygen for cytochrome oxidase.

Acids and alkalis also exhibit antimicrobial activity. They primarily cause the Hydrolysis of cellular proteins. In food Processing plants, the cleaning and Treatment of equipment are carried out using acid and alkali solutions of specific concentrations.

Organic compounds such as ethanol, cresol, phenol and its derivatives, and formaldehyde possess antimicrobial activity.

Ethanol at a concentration of 70–80 vol. % induces the coagulation of microbial cell proteins and exerts a bactericidal effect. Phenol, cresol, neutral soaps, and surface-active agents (detergents) damage the Surface structures of microbial cells. Phenol and its derivatives find particularly widespread application; their action dissolves the Lipids of the cytoplasmic membrane, thereby disrupting its semipermeability and ultimately leading to cell death. Most microbes are destroyed by the action of a 1–5% solution of phenol (carbolic acid).

Diethyl ether, acetone, and alcohols, which function as organic Solvents, disrupt the lipids of the cytoplasmic membrane.

Sulfur dioxide, sulfurous acid, and its salts (the sulfitation method) are used for the processing of fresh fruits and berries, fruit semi-finished products, and fruit and berry juices.

Benzoic and sorbic acids are used as preservatives to prevent food spoilage by mold.

The preservation of food products through smoking is based on THE PRINCIPLE OF antisepsis. Smoke contains antiseptic substances: Phenolic Compounds, formaldehyde, and organic acids. Nowadays, smoking liquid containing similar antiseptics is used instead of smoke. Smoking is employed for preserving meat, fish products, and certain types of cheese.

Spherical forms of bacteria are more resistant to the action of chemical agents; rod-shaped and spiral forms perish more rapidly. Bacterial spores are characterized by the highest resistance to chemical agents due to their dense coat and the absence of free water.

Antimetabolites. The inhibition of Microbial growth is caused by structural analogs (antimetabolites) of cellular components. Structural analogs prevent the incorporation of normal metabolites into cellular metabolism, thereby disrupting the synthesis of individual cellular components, which results in growth inhibition and cell death. Specifically, the antimetabolite malonate competes with the normal metabolite succinate for the catalytic center of the enzyme succinate dehydrogenase. As a result of this competitive inhibition, the enzyme becomes inactive. A competitive analog of Para-aminobenzoic Acid (PABA) is sulfonamide (a derivative of sulfanilic acid).

The MECHANISM OF ACTION of sulfonamides on microorganisms was discovered in 1940 by D. Woods. He established that PABA participates in The Biosynthesis of tetrahydrofolic acid, a substance essential for bacterial vital activity. When sulfonamide is added to the medium, it penetrates the cell unhindered and is incorporated into Folic acid in place of p-aminobenzoic acid, leading to the synthesis of a non-functional enzyme and ultimately to the inhibition of microbial growth.

Antibiotics (from the Greek anti—against, bios—life). These are highly active metabolites of microorganisms, animal cells, and plants that possess the selective ability to inhibit the growth of microorganisms and suppress The Development of malignant neoplasms.

The first antibiotic, penicillin, was discovered by the English microbiologist A. Fleming in 1929. Fleming failed to isolate a pure preparation from the culture fluid of Penicillium notatum. In 1940, American scientists H. Florey and E. Chain obtained purified penicillin, and in 1945, A. Fleming, H. Florey, and E. Chain were awarded the Nobel Prize for this discovery. According to their source of origin, antibiotics are divided into the following groups:

✵ antibiotics obtained from Fungi. Penicillin is obtained from molds of the genus Penicillium, fumigacin from the genus Aspergillus, cephalosporin from the genus Cephalosporium, clavicin from the genus Mucor, etc.;

✵ antibiotics obtained from branching bacteria—actinomycetes. Actinomycetes of the genus Streptomyces are the primary producers of such antibiotics as streptomycin, erythromycin, levomycetin, nystatin, and many others;

✵ antibiotics obtained from bacteria. The antibiotic gramicidin S is produced by B. brevis, subtilin by B. subtilis, and polymyxin by B. polimyxa;

✵ antibiotics of animal origin—Lysozyme (found in egg white, saliva, lacrimal fluid, nasopharyngeal mucus, human milk, and colostrum);

✵ antibiotics of plant origin, known as phytoncides. The strongest phytoncides are secreted by onions, garlic, horseradish, mustard, etc. They have not been obtained in pure form because they are extremely unstable compounds. Many plants also possess antimicrobial activity: chamomile, St. John's wort, sage, calendula, etc.

The Mechanism of action of antibiotics on microorganisms varies. Depending on the mechanism of action, antibiotics are divided into four groups.

1. Antibiotics that inhibit the synthesis of Introduction/37.html">Bacterial cell wall peptidoglycan. This group includes Penicillins, Cephalosporins, and others.

2. Antibiotics that disrupt the Molecular Structure and Synthesis of cell membranes. These include nystatin, polymyxins, and others.

3. Antibiotics that inhibit Protein Synthesis. This is the largest group of antibiotics, represented by Tetracyclines, erythromycin, chloramphenicol, neomycin, streptomycin, and kanamycin. The action of these antibiotics aims to disrupt protein synthesis at various stages (for instance, erythromycin impairs the function of 50S subunits, tetracyclines prevent the binding of aminoacyl-tRNA to Ribosomes, etc.).

4. Antibiotics that inhibit nucleic acid synthesis. This category includes actinomycins, which suppress RNA Synthesis, and rubomycin, which suppresses DNA Synthesis. Most of them exhibit anticancer properties.

The Use of antibiotics and antiseptics for food preservation is extremely limited. Only a few antibiotics are approved in small doses for use in the food industry for specific products. Primarily, these include nisin, a bacteriocin synthesized by certain strains of lactic acid bacteria of the species Lactococcus lactis ssp. lactis.