ECOLOGICAL MICROBIOLOGY - M. I. Chernyavskaya - 2016

TOPIC 2. AUTOECOLOGY. THE INFLUENCE OF PHYSICAL ENVIRONMENTAL FACTORS ON MICROBIAL ACTIVITY

Under natural conditions, microorganisms are exposed to abiotic factors that vary significantly in their nature and MECHANISM OF ACTION. Nevertheless, the pattern of dependence between microbial activity and the intensity of various external factors is quite similar. For any abiotic factor, There is a range of variations within which microbial activity remains virtually unchanged at a level referred to as the optimum. This interval is called the optimum zone. Lower factor levels fail to support proper microbial functioning, thereby limiting metabolic processes; the corresponding interval is designated as the limiting zone. Excessive factor levels suppress microbial activity, an interval referred to as the inhibition zone. There are extremely high and extremely low factor levels at which microbial life becomes impossible. The outer limits of factor variation that an Organism can endure are generally termed the limits of tolerance. Different microorganisms exhibit significantly varying tolerance limits to the exact same ecological factor.

In most cases, the response of a microorganism to a particular abiotic factor is represented as a growth-versus-factor-intensity graph. This involves determining the so-called cardinal points: the optimum value (or range of values) ensuring maximal growth, along with the minimum and maximum values at which growth ceases. The range between the minimum and maximum values constitutes the tolerance range; within this range (outside the optimum zone), the microorganism remains active but possesses low competitive ability and may be outcompeted by other organisms. A microorganism's tolerance range for a specific factor can be either narrow or broad. Organisms with broad tolerance limits are called eurybionts, whereas those capable of surviving within relatively narrow ranges of an ecological factor are termed stenobionts. For stenobionts, THE POSITION OF their optimum zone along the scale of possible factor variations must be taken into account. For instance, stenothermal microorganisms are divided into psychrophiles, whose Temperature optimum lies in the low-temperature region (0 — 4 °C), and thermophiles, which feature a high optimum growth temperature (70 °C and above).

The physicochemical conditions of microbial habitats in nature cover a rather wide spectrum. Commonly prevailing conditions are referred to as usual or normal, while extreme factor values are termed extremes.

Temperature is one of the most critical environmental factors influencing microorganisms. Its effect on Microbial growth stems from its impact on The rate of intracellular Chemical Reactions and the state of cellular macromolecules (membrane fluidity, protein conformation, etc.). Elevating the temperature above a critical threshold leads to the irreversible inactivation of cellular components—primarily the Denaturation of Proteins and Nucleic Acids—resulting in microbial death. To assess the lethal Effect of temperature on microbial Cells, quantitative parameters are employed: the thermal death point (TDP), which is the temperature at which a given organism is killed in 10 minutes, and the thermal death time (TDT), representing the time required to kill a given organism at a specific temperature. The lower growth limits of microorganisms are constrained by membrane "solidification," a state where the membrane loses its fluidity and ceases to function. When the temperature drops below the minimum level, microorganisms do not die and can remain viable for extended periods. Low temperatures reduce not only the growth rate but also the mortality rate of microorganisms, thereby enhancing their survival. Consequently, freezing microorganisms at low and ultra-low temperatures (-20, -70, -196 °C) is widely utilized for long-term preservation.

Microorganisms inhabit environments with diverse temperature regimes. Low-temperature habitats include the Arctic, Antarctic, tundra, and deep-ocean basins, where temperatures remain constantly around 4 °C. High-temperature habitats encompass geysers, volcanic springs, hot springs, and "black smokers" (hydrothermal vents along mid-ocean ridges)—sites where superheated volcanic gases emerge along Earth's crustal fractures, and temperatures under high pressure can reach up to 360 °C.

Based on their temperature requirements, microorganisms are categorized into several groups (Fig. 2):

Class="center">Fig. 2. Growth curves of various microbial groups in relation to temperature

• mesophiles — grow at moderate temperatures. For many of them, the temperature optimum is close to the body temperature of warm-blooded animals (30 — 37 °C) or slightly lower (20 — 25 °C). The maximum growth temperature for free-living mesophiles is 45 — 50 °C, which aligns with peak soil heating temperatures. The majority of known microorganisms are mesophiles, including the favored object of microbiological research, Escherichia coli;

• psychrophiles — grow at temperatures below 20 °C, down to sub-zero temperatures, with an optimum growth temperature below 15 °C. Psychrophilic microorganisms include Representatives of the species Bacillus psychrophilus and iron Bacteria of the genus Galionella. Psychrophiles inhabit stably cold environments and are extremely sensitive even to slight temperature increases. One of the reasons for psychrophily is the thermal denaturation of cellular proteins at moderate temperatures (above 20 °C). Psychrophiles feature a specialized membrane composition with a lowered freezing point; they contain more unsaturated, short-chain, and branched Fatty acids, and fewer cyclic fatty acids. The temperature optimum for enzyme activity in psychrophiles is lower than that of mesophilic microorganisms, and their protein-synthesizing machinery Functions efficiently at low temperatures;

• psychrotrophs — are capable of growing at 0 °C, yet, compared to psychrophiles, they exhibit higher optimal (20 — 30 °C) and maximal (35 °C) growth temperatures. This group includes representatives of certain Pseudomonas and Arthrobacter species. Psychrotrophs are adapted to seasonal climate shifts and hold selective advantages over stenothermal species because they remain metabolically active during both warm and cold seasons. Many of them are typical refrigerator inhabitants responsible for the spoilage of frozen foods. The adaptation of psychrotrophs to lower temperatures is manifested in altered membrane compositions (increased unsaturated fatty acid content) and the synthesis of intracellular cryoprotectants (such as glycerol). Another adaptation mechanism involves accumulating large amounts of vital Enzymes within the cells, enabling The Cell to maintain sufficient activity even at suboptimal temperatures. Psychrophilic and psychrotrophic microorganisms play a vital role in natural processes within cold and temperate climate zones. During a Study of the tundra methanogenic community, a team of Russian researchers discovered, for the first time, a temperature-driven shift in the community's trophic pathway caused by A change in the dominant microbial group. At temperatures above 15 °C, methanogenesis served as the primary terminal process in the community, whereas below 15 °C, acetate formation became dominant;

• thermophiles — microorganisms whose optimal growth temperature exceeds 50 °C. Depending on the cardinal points of their temperature range, they are subdivided into the following groups: thermotolerant (temperature maximum of 45 — 50 °C), facultative (maximum of 50 — 65 °C), obligate (maximum growth temperature up to 70 °C), and extreme (optimal growth temperature of 70 — 75 °C, maximum of 90 °C) thermophiles. Examples of thermotolerant microorganisms include bacteria of the species Methylococcus capsulatus; facultative thermophiles include Homofermentative lactic acid bacteria of the genus Lactobacillus; obligate thermophiles are represented by Bacillus stearothermophilus; and extreme thermophiles include bacteria of the genera Thermus, Thermomicrobium, and Thermoplasma. The survival record at elevated temperatures belongs to archaea, certain cultures of which grow on nutrient media at temperatures above 110 °C. For instance, Methanopyrus kandleri grows at 122 °C, representing the record-high temperature for all known organisms. Thermophiles contain high-temperature-stable proteins and thermostable Ribosomes; their Membrane Lipids are enriched in Glycolipids and high-melting saturated fatty acids, and their DNA and RNA have a high GC-content. Thermophilic enzymes display greater heat resistance compared to their mesophilic counterparts, which is primarily due to modifications in the Introduction/19.html">Primary Structure of the protein molecule.

Thermophilic microorganisms are of immense practical importance. They serve as active producers of enzymes, Vitamins, organic acids, and Single-Cell Protein, and are utilized in the biological Treatment of domestic waste with biogas generation.

Hydrostatic pressure. Most microorganisms living on the land or Water surface do not experience significant pressure fluctuations and grow at approximately 1 atm. However, certain habitats feature pressures that deviate considerably from atmospheric pressure. Elevated pressures in nature occur in deep oil reservoirs (typically characterized by high sulfur content) and deep-ocean zones, which generally combine low temperatures with nutrient scarcity. Microorganisms have even been discovered at the deepest point of the World Ocean—the Mariana Trench—under a pressure of ~1016 atm. The highest artificially induced pressure that microorganisms can endure is 1400 atm.

An increase in hydrostatic pressure leads to the disruption of Cellular Structures and Protein Denaturation. Under elevated pressure, microbial cells cease dividing, fail to separate after division, and adopt a filamentous Morphology.

Based on their response to high pressure, microorganisms are classified into the following groups:

• piezosensitive (barosensitive) — microorganisms (typically possessing gas vacuoles) that halt growth under elevated hydrostatic pressure;

• piezotolerant (barotolerant) — microorganisms capable of withstanding pressures up to 400 atm while retaining The ability to grow at normal pressure;

• piezophilic (barophilic) — microorganisms that require elevated pressure for growth. Moderate barophiles tolerate pressures up to 850 atm, whereas extreme barophiles withstand pressures exceeding 1000 atm.

Microorganisms are susceptible to various types of electromagnetic radiation. Depending on wavelength, electromagnetic radiation is categorized into ionizing (up to 10 nm), ultraviolet (10 — 400 nm), infrared (700 — 1100 nm), and the visible spectrum (300 — 700 nm). Radiation can exert the following effects on microorganisms:

1) physiological;

2) lethal and mutagenic;

3) thermal and mechanical.

Physiological effects are exerted by near-ultraviolet light, visible light, and infrared rays (350 — 400 — 800 — 1100 nm). Infrared rays exhibit a thermal effect on microorganisms and are utilized by green and purple bacteria during Photosynthesis. The visible part of the spectrum is used for photosynthesis by cyanobacteria and other phototrophic bacteria. Different phototrophic microorganisms absorb light of varying wavelengths. The Limits of the usable light spectrum are determined, on the one hand, by The Need for energy to drive photochemical reactions and, on the other hand, by the need to prevent pigment destruction. Electromagnetic waves are crucial for phototaxis. Photosependent syntheses also occur in non-photosynthetic microorganisms (e.g., The formation of carotenoids in mycobacteria). However, visible light and infrared radiation do not always have a positive impact on microorganisms. Infrared rays can cause cell overheating, while visible light under aerobic conditions leads to the formation of singlet oxygen, which induces photooxidation of cellular enzymes. As a defense mechanism, microorganisms synthesize carotenoids, which act as singlet oxygen quenchers.

Depending on wavelength and dose, ultraviolet radiation can exert a lethal or mutagenic effect on microorganisms. The highest lethal effect of UV rays is observed at a wavelength of 260 nm, which corresponds to the maximum absorption of this radiation by DNA molecules. The lethal action of ultraviolet light is primarily caused by alterations in DNA Structure (breaking of Hydrogen Bonds, Cleavage of bonds between deoxyribose and phosphate, formation of cyclobutane thymine dimers located on the same strand), leading to the inhibition of Replication and METABOLISM/31.html">Transcription processes. Thymine dimers can be eliminated via two pathways: photoreactivation and dark repair. In the first case, DNA damage is repaired by a single enzyme activated by visible light, which breaks the bonds between thymine bases in the dimers. In the second case, light is not required, and several enzymes are involved: a nuclease (which excises the damaged region), a DNA polymerase (which synthesizes the correct structure using the complementary strand as a template), and a ligase (which restores the phosphodiester bond).

Ultraviolet light with a wavelength of 325 — 400 nm is also harmful to microorganisms because, alongside the formation of thymine dimers, it causes the destruction of Tryptophan and the generation of its toxic photoproducts that act as Chemical Mutagens. Microorganisms whose cells contain carotenoids are the most resistant to UV exposure. In heterotrophic microorganisms, carotenoids serve as a protective system that minimizes nucleic acid damage, whereas in phototrophic bacteria, they protect bacteriochlorophyll from photooxidation.

When assessing the dependence of microbial survival on the dose of UV irradiation, the density of the bacterial suspension is of great importance. UV rays are intensely absorbed by the bacterial cell; therefore, at high concentrations, cells can shield one another. This factor does not play a significant role in bacterial Suspensions where the density does not exceed 10⁸ cells/mL. When using a dense bacterial suspension during irradiation, it must be continuously mixed. The bacterial suspension should be spread in a thin layer because UV rays are characterized by low penetrating power, meaning that cells located deeper are not exposed to their action. Cell survival under UV radiation depends on the COMPOSITION OF THE suspending medium. Irradiation is best carried out in Buffer solutions. A liquid nutrient broth absorbs UV rays more intensively than buffer solutions, thereby reducing the dose received by the cells. At the same time, irradiation in a nutrient medium can produce toxic products that enhance the lethal effect of UV rays, complicating the Interpretation of Results. The lethal effect of UV rays depends on the physiological state, primarily the age, of the bacterial culture. Cells are more sensitive to UV radiation during the exponential growth phase.

Ionizing radiation consists of very short, high-energy waves. Low levels of ionizing radiation can induce Mutations in microorganisms, while high levels almost invariably lead to cell death. The primary cellular damages caused by ionizing radiation include the disruption of Hydrogen bonds and ring structures of biological molecules, as well as their polymerization. Unlike UV rays, ionizing radiation affects Biopolymers indirectly rather than directly, by inducing the formation of free radicals and organic peroxides that react with Nucleic Acids and Proteins, leading to single- and double-strand DNA breaks, alterations in nitrogenous bases, and The oxidation of protein sulfhydryl groups into Disulfide Bonds. The presence of oxygen significantly enhances The Effect of ionizing radiation, most likely due to the generation of hydroxyl radicals (OH•). Microorganisms from various taxonomic groups differ substantially in their sensitivity to ionizing radiation. For instance, Clostridium botulinum bacteria remain viable at a dose of 1.5 Mrad, whereas Escherichia coli tolerate 0.18 Mrad. Certain microorganisms isolated from irradiated foods, nuclear Reactor water, and uranium ore deposits—such as Deinococcus radiodurans and Schizosaccharomyces pombe—are resistant to ionizing radiation doses of 2 — 3 Mrad, which is attributed to the presence of robust DNA Repair systems in their cells. Since ultraviolet and ionizing radiation are lethal to microorganisms at certain doses, they are employed for sterilization purposes.

Ultrasound represents high-frequency (~25 kHz) mechanical vibrations of an elastic medium that are imperceptible to the human ear. When acting on microorganisms, ultrasound creates a large pressure differential across different PARTS OF THE cell, damaging it: the Cytoplasm liquefies and foams, and the cell contents mix with the external environment. The sensitivity of microorganisms to ultrasound is proportional to the oscillation frequency and the duration of exposure, and depends on the structural features and physiological state of the cell. The larger the cell, the more sensitive it is to ultrasound; rods and spirilla are more sensitive than cocci. Prolonged ultrasound exposure results in the complete destruction of microorganisms, which is utilized for sterilization. Ultrasound is also applied to disrupt bacterial cells in order to extract BIOLOGICALLY ACTIVE SUBSTANCES from them.

Microbial development is affected by changes in magnetic field strength. This factor is currently regarded as an ecological parameter that governs many biological processes. Microorganisms containing magnetosomes in their cells are particularly sensitive to changes in magnetic field strength.

It should be emphasized that in nature, microorganisms experience the influence not of a single factor, but of a multitude of abiotic factors (temperature, light, pressure, etc.); therefore, the interaction among these factors must be taken into account. For every abiotic factor that causes irreversible alterations in the Biomolecules of most microorganisms, there exists a group of highly specialized prokaryotes that thrive optimally at extreme values of that specific factor.

Review Questions

1. Into what physiological groups based on temperature are microorganisms divided?

2. Provide examples of low-temperature and high-temperature habitats for microorganisms.

3. What is The Mechanism of action of high and low temperatures on microorganisms?

4. List the morphological and biochemical features of thermophiles and psychrophiles.

5. Into what groups based on hydrostatic pressure are microorganisms classified?

6. List the types of cellular damage in microorganisms caused by elevated hydrostatic pressure.

7. What effects do radiations of different wavelengths exert on microorganisms?

8. List the primary damages to the Prokaryotic Cell caused by UV radiation and their repair mechanisms.

9. Name the MAIN MECHANISMS OF the damaging action of ionizing radiation on microorganisms.

10. What factors influence the sensitivity of microorganisms to ultrasound?

11. Which microorganisms are most sensitive to changes in magnetic field strength?

12. Provide an example of a natural microbial habitat that combines extreme values of several abiotic factors.

Laboratory Work 2. Effect of Temperature on MICROBIAL GROWTH AND Metabolism

Objective: To study the effect of cultivation temperature on the growth and metabolism of microorganisms from various systematic groups.

Materials and equipment: Peptone-Yeast Agar (PYA), peptone-yeast broth (PYB), sterile Petri dishes, sterile test tubes, sterile 1 — 2 mL and 5 — 10 mL pipettes, automatic pipette (2 — 20 µL), pipette tips (2 — 200 µL), microbiological loop, sterile pins, spirit lamp, incubators set at 4, 10, 18, 28, 37, 42, 55 °C, vortex mixer, spectrophotometer or photocolorimeter.

Procedure

1. Production of the red pigment (prodigiosin) by Serratia marcescens bacteria as a function of cultivation temperature.

1) Inoculate the S. marcescens culture into two tubes with PDA slants.

2) Place one tube in a thermostat at 28 °C and the second at 37 °C, then incubate for 24 — 48 h.

3) Compare The production of the red pigment (prodigiosin) at different cultivation temperatures.

4) Record the results in a laboratory journal and draw a Conclusion on the effect of cultivation temperature on prodigiosin production.

2. Determination of the temperature range and optimal growth temperature of bacteria.

1) The day before the class, inoculate the test bacterial cultures of the genera Bacillus, Enterobacter, Pseudomonas, Rhodococcus, Serratia, and Sarcina into 2 mL of PDB. Incubate them at 28 — 37 °С for 18 h.

2) Dispense 2 mL of PDB into six sterile tubes.

3) Add 20 µL of each thoroughly vortexed bacterial culture to the tubes containing PDB. The initial cell concentration in each of the 6 tubes must be identical to accurately assess the bacterial growth rate at various cultivation temperatures.

4) Place the inoculated tubes in thermostats set to the respective temperatures (4, 10, 18, 28, 37, 42, 55 °C) and incubate for 24 — 48 h.

5) Determine the optical density of the culture at a wavelength of X = 600 nm (OD₆₀₀). An optical density value of OD₆₀₀ ≥ 0.2 is considered positive growth.

6) Based on the spectrophotometric measurements, plot the dependence of bacterial culture growth on the cultivation temperature (Fig. 3).

Fig. 3. Growth of the tested bacterial cultures as a function of cultivation temperature:

— culture 1; ...culture 2; ---culture 3

7) Conclude on the temperature range and optimal growth temperature of the tested bacterial cultures.

3. Determination of the temperature range for bacterial growth.

1) Label PDA Petri dishes According to the incubation temperature (4, 10, 18, 28, 37, 42, 55 °C) and divide them into sectors, indicating the name of the test bacterial culture on each sector. Prior to inoculation, place the dishes in a thermostat at the corresponding temperature for 15 — 20 min.

2) Inoculate the bacterial cultures onto the corresponding sector of the Petri dish using a bacteriological loop or inoculating needles.

3) Place the dishes in thermostats at the appropriate temperatures and incubate for 7 days.

4) Record the results daily by comparing bacterial growth at different temperatures. Enter the results into a table (Table 1).

Table 1. Growth of the tested microorganism cultures at various temperatures

Note: "++++" — abundant growth, "+++" — good growth, "++" — moderate growth, "+" — slight growth, "-" — no growth.

Laboratory Work 3. Effect of Heat Stress on Microorganisms

Objective: To study the effect of extremely high temperature (heat stress) on microorganisms from various taxonomic groups.

Materials and equipment: PDB, PDA, 1 — 2 ml sterile pipettes, microbiological loop, spreader, water bath, thermometer, spirit lamp, 28 and 37 °C incubators.

Procedure

1. The day before the practical class, bacterial cultures of Bacillus licheniformis, Staphylococcus aureus, and Escherichia coli are inoculated into 10 ml of PDB and cultivated at 28 — 37 °C for 24 h.

2. Prior to the experiment, 0.1 ml of each test culture is spread onto PDA Petri dishes (control).

3. A 2 ml volume of each test culture is transferred into six sterile test tubes and placed in a water bath at 60, 80, or 100 °C (two test tubes per temperature). One test tube is opened, and a thermometer is inserted to monitor the temperature (control tube), while the second tube is reserved for sampling (experimental tube).

4. Once the temperature in the control tube reaches the target value, the starting time of the experiment is recorded.

5. At 10, 20, 30, and 40 min after THE START OF the experiment, samples (0.1 ml each) are taken from the experimental tube and spread onto PDA Petri dishes.

6. The inoculated Petri dishes are incubated at 28 — 37 °C for 48 h.

7. Results are recorded (Table 2), and the thermal death time (TDT) and thermal death point (TDP) of the studied bacteria are determined.

Table 2. Survival of the studied bacterial cultures under heat stress

Note: "+++" — good growth, "++" — moderate growth, "+" — weak growth, "-" — no growth.

Laboratory Work 4. Effect of Ultraviolet Radiation on Microorganisms

Objective: To investigate the UV sensitivity of microorganisms from various taxonomic groups and to determine how microbial survival depends on the duration of UV exposure.

Materials and equipment: PDB, PDA, physiological saline, 1 — 2 ml pipettes, microbiological loop, spreader, forceps, sterile thick paper discs, DB-15 bactericidal lamp, incubator.

Procedure

1. STUDY OF BACTERIAL sensitivity to UV radiation.

1) The day before the practical class, bacterial cultures are inoculated into 5 ml of PDB and cultivated with aeration at 28 — 37 °C for 24 h.

2) A 0.1 ml aliquot of each test bacterial culture is spread onto PDA Petri dishes.

3) A thick paper disc is placed in the center of the agar surface in each Petri dish to act as a shield protecting bacterial cells from UV rays.

4) Irradiation is performed in open Petri dishes using a DB-15 bactericidal lamp for 3 min at a distance of 40 cm.

5) Upon completion of irradiation, the paper disc is removed with sterile forceps, and the Petri dishes are covered and placed in an incubator at the optimal temperature.

6) Results are evaluated after 24 h of cultivation. A bacterial culture is considered sensitive to UV light if confluent growth is observed only in the area previously covered by the disc, whereas isolated colonies or no growth are noted on the rest of the agar surface.

2. Dependence of bacterial survival on UV radiation dose.

1) The day before the experiment, the bacterial cultures under study are inoculated into 5 ml of PDB and cultivated with aeration at 28 — 37 °C for 18 h.

2) Separately, 27 ml of sterile PDB is poured into a small flask and placed in an incubator at the appropriate temperature.

3) The bacterial culture (3 ml) is transferred to the flask containing 27 ml of PDB and cultivated with aeration for 2 h.

4) The bacterial cells are washed free of the nutrient medium by centrifugation and resuspended in 30 ml of saline.

5) Portions of 5 ml of the bacterial suspension are transferred into five sterile Petri dishes, which are then placed under a UV lamp at a distance of 40 cm.

6) The Petri dishes are uncovered, and the UV lamp is turned on. The start of the experiment is recorded immediately after turning on the lamp.

7) The bacterial culture is irradiated for a specified time (30, 60, 90, 120, and 240 s), gently swirling the Contents of the dishes.

8) To determine the Number of viable cells, 0.5 ml samples are taken from each dish and from the unexposed suspension, added to 4.5 ml of saline, and serial tenfold dilutions are prepared.

9) Aliquots of 0.1 ml from the 10^-3 — 10^-5 dilutions of the bacterial culture (according to Table 3) are plated onto The surface of PDA in duplicate Petri dishes.

Table 3. Bacterial viability under UV irradiation

10) The dishes are incubated at the optimal temperature for 48 h.

11) The formed colonies are counted, and the cell titer and survival rate of the UV-irradiated cells are determined.

12) The results are recorded in the table (see Table 3). Plot a graph of bacterial survival as a function of exposure time (Fig. 4).

Fig. 4. Survival rate of the studied bacterial cultures as a function of UV exposure duration