GENERAL MICROBIOLOGY - T.P. Pyrog - 2004

6. MICROBIAL GROWTH

6.1. EFFECT OF ENVIRONMENTAL FACTORS ON MICROORGANISMS

6.1.2. Chemical factors

Hydrogen ion concentration (medium pH). H^- and OH ions are the most mobile of all ions, which is why even the slightest changes in their concentration strongly affect microorganisms. Therefore, establishing and maintaining the optimal pH value of the medium is of great importance for the growth of a particular microorganism.

Most organisms grow best at a neutral pH or in media with pH values close to neutral (pH 6-7) [Table 6.1]. Such microorganisms are called neutrophils.

Class="center">Table 6.1.

Relation of microorganisms to the pH of the medium

Micromycetes, Yeasts, and certain bacteria develop well in acidic environments (pH 5-6) and are called acidophiles. Among bacteria, these include lactobacilli, bacteria of the genus Acetobacter, and some species of Thiobacillus. Many bacteria (such as nitrifiers and actinomycetes) grow better in alkaline environments and are termed alkaliphiles. For instance, the cholera vibrio has an optimal pH of 9,0.

Maintaining pH during growth is especially crucial for microorganisms that synthesize acids yet are not tolerant to them (e.g., lactobacilli: the lactic acid they produce inhibits their own growth). Therefore, buffered media are used in such cases.

The Effect of pH on a microbial Cell can be either direct or indirect. In the latter case, pH affects not the microorganism itself, but certain Components of the medium whose dissociation depends on pH, which in turn influences the penetration of compounds into The Cell. The production and activity of microbial Enzymes also depend on the pH value.

Oxygen and aeration, Redox Potential of the medium. Molecular oxygen is vital and essential for all microorganisms (though to varying degrees). Based on their oxygen requirements, all microorganisms are divided into several groups:

obligate aerobes — able to obtain energy solely through Respiration and therefore require oxygen, which serves as the terminal electron acceptor in aerobic respiration. They are incapable of obtaining energy via Fermentation;

obligate anaerobes — can grow only in an oxygen-free environment. Moreover, oxygen is toxic to them. Many enzymes of these microorganisms are denatured upon contact with molecular oxygen. The lethal effect of oxygen on obligate anaerobes is due to The formation of hydrogen peroxide in the cell in the presence of oxygen, which in high concentrations can cause cell death. Aerobes possess catalase or peroxidase enzymes that decompose hydrogen peroxide, whereas anaerobes lack these enzymes;

facultative anaerobes — grow both in the presence and absence of oxygen. Two types are distinguished among them: aerotolerant lactic acid bacteria can grow in the presence of oxygen but cannot utilize it, obtaining energy exclusively through fermentation. Facultative anaerobes of the second type (yeasts, Escherichia coli) can switch from respiration (in the presence of oxygen) to fermentation (in the absence of oxygen);

microaerophiles — thrive at low oxygen concentrations. They require oxygen for energy production but cannot tolerate the partial pressure of O₂ present in the air (0,02 MPa): they require between 0,001 and 0,003 MPa.

Aeration. For obligate aerobic microorganisms growing on Agar media, oxygen is sufficient. In liquid media with a large volume of liquid, aerobic bacteria can grow only On the surface, as conditions approach anaerobic with increasing distance from the surface. To ensure normal growth of aerobes in deep layers of liquid culture, aeration is necessary. Microorganisms can only utilize dissolved oxygen, but its solubility is very low. For example, 1 L of Water at 20 C in equilibrium with atmospheric air contains only 6,2 mL of oxygen (0,28 mmol). This amount of oxygen is sufficient to oxidize only 8,3 mg of glucose (i.e., one-thousandth of the total amount of glucose contained in a standard nutrient medium).

The rate of oxygen dissolution increases with a larger gas-liquid interfacial area and a higher partial pressure of oxygen in the gas phase. The following techniques are used to increase the interfacial area:

cultivation in a thin layer;

liquid agitation by shaking (linear or rotary); rotation of a horizontal vessel around its longitudinal axis; sparging air through the liquid using a gas distributor (Glass filters, Kluyver flasks); mechanical agitation.

Cultivation of anaerobic cultures. To cultivate strict anaerobic cultures, oxygen access must be eliminated. The technique for handling anaerobic cultures involves:

using boiled nutrient media and vessels sealed without air bubbles;

creating an oxygen-free atmosphere in vacuum desiccators; using oxygen absorbents (pyrogallol, dithionite, monovalent copper chloride);

adding reducing agents to the medium (ascorbic acid, thioglycolate, Cysteine, or even sulfide if tolerated by the Organism);

continuous purging of nitrogen or an inert gas (such as argon) through culture vessels (contact between the medium and air can be prevented even during inoculation). This is known as the Hungate technique;

use of anaerobic chambers filled with nitrogen, hydrogen, or argon;

application of color indicators (remazurin turns pink in the presence of oxygen and is colorless under anaerobic conditions; methylene blue is also decolorized under anaerobic conditions).

Redox potential. The degree of aerobiosis or anaerobiosis of a medium can be quantified using the redox potential (rН₂). In an aqueous solution fully saturated with oxygen, rН₂ = 41, whereas under conditions of complete saturation with hydrogen, it is 0. Thus, the scale from 0 to 41 characterizes any degree of aerobiosis. The lower limit of rH₂ for obligate aerobes is 10, although values close to 30 are unfavorable for them. Obligate anaerobes remain viable at rH₂ levels not exceeding 18–20, but they can multiply only at rH₂ values no higher than 3–5. Facultative anaerobes maintain metabolic activity across a wide range of rH₂ — from 0 to 30.

Chemical compounds. The effect of chemicals on microorganisms can be stimulating (promoting GROWTH AND REPRODUCTION), bacteriostatic, fungistatic (inhibiting the growth and reproduction of bacteria and Fungi, respectively), and bactericidal or fungicidal (causing the death of bacteria and fungi, respectively). An example of a stimulating effect on microorganisms is the action of Vitamins and other growth factors.

Many chemical substances exert a detrimental effect on microorganisms, and are thus called antimicrobial agents. They can be of organic origin (ethyl alcohol, formaldehyde, phenol) or inorganic origin (salts of heavy metals such as lead, mercury, silver, and copper). Antimicrobial agents used in practice to suppress pathogenic microbes are called disinfectants (0.5–5.0% chlorinated lime, 2% iodine solution, 1–5% phenol solution, i.e., carbolic acid), and their application is referred to as disinfection.

Antimicrobial agents cause the following types of cellular damage:

damage to surface structures or cell envelopes (ethanol, phenol, cresols, detergents, Polypeptide Antibiotics such as polymyxin, bacitracin, subtilin);

damage to enzymes and metabolic disruption (heavy metals bind to SH-groups of Proteins, thereby profoundly altering their Tertiary and Quaternary structures; cyanide, a respiratory poison, binds to iron and blocks the function of cytochrome c oxidase; arsenate inhibits substrate-level phosphorylation; fluoracetate blocks The Tricarboxylic Acid Cycle);

disruption of cellular component synthesis. There are compounds known as structural analogs, or antimetabolites. They are structurally similar to normal cellular metabolites. A normal metabolite competes with its structural analog for the catalytic site of an enzyme. For instance, sulfonamide is a structural analog of p-aminobenzoic acid, which in turn is a component of the coenzyme tetrahydrofolic acid. In most bacteria, tetrahydrofolic acid is synthesized from simpler components. Upon entering the cell, sulfonamide is incorporated into Folic acid. This results in the formation of a non-functional coenzyme, thereby arresting cell growth;

inhibition of Protein Synthesis by antibiotics. The action of antibiotics in prokaryotes targets the function of 70S Ribosomes. Streptomycin and neomycin inhibit amino acid binding, erythromycin disrupts the function of the 50S subunit, and chloramphenicol suppresses amino acid incorporation into proteins;

inhibition of nucleic acid synthesis by antibiotics. Mitomycin C prevents DNA Synthesis, actinomycin D disrupts RNA Synthesis, and rifampicin targets DNA-dependent RNA polymerase, thereby inhibiting Messenger RNA synthesis in bacteria;

inhibition of Cell wall synthesis. Peptidoglycan synthesis is inhibited by penicillin, Cephalosporins, and bacitracin.