ENVIRONMENTAL MICROBIOLOGY - M. I. Cherniavska - 2016

TOPIC 4. MICROBIOCENOSES OF AIR, WATER BODIES, AND SOILS

Under natural conditions, microorganisms do not exist as pure cultures. Instead, they grow in mixed cultures, forming part of a more complex community that includes organisms from other taxonomic groups. Microorganism habitats are complex and constantly changing, driven by nutrient gradients, limiting factors (Temperature, pH, light, Water activity, etc.), and the presence of toxic compounds. The combination of these factors determines the ecological niche of a specific microorganism.

Air is an unfavorable environment for microorganisms. It lacks nutrients, a constant optimal temperature, and frequently moisture, while solar radiation exerts a lethal effect. Microorganisms enter the air primarily through dust or liquid droplets from the surfaces of soil, water, plants, animals, and transport systems. Their distribution in the atmosphere is uneven. Atmospheric air and indoor air differ significantly in their Qualitative and quantitative microbial composition. The concentration of airborne microorganisms depends on weather conditions (levels are lower during rain than in dry weather), season (higher in summer than in winter), and Location (higher over major urban areas than in rural settings). Microbial counts are low in the air above forests, gardens, meadows, lakes, oceans, and high mountain regions. The airborne microbiota is characterized by a high Abundance of coccoid forms (micrococci, diplococci, sarcinae, staphylococci), bacilli (B. subtilis, B. cereus, B. mesentericus), and Fungi (Penicillium, Aspergillus, Mucor) that are resistant to desiccation and ultraviolet radiation.

The microbial content of indoor air in residential buildings exceeds that of ambient outdoor air. When assessing the sanitary condition of indoor environments, depending on the research objective, parameters such as the total microbial count and the presence of sanitary-indicator microorganisms (staphylococci, α- and β-hemolytic streptococci) are determined. In the investigation of air in medical facilities (surgical clinics, maternity hospitals), the primary focus is on detecting pathogenic staphylococci, Pseudomonas aeruginosa, and other Gram-negative opportunistic pathogens responsible for nosocomial infections. At microbiological industry plants, detection targets include the presence and concentration of producer microorganisms (Candida in Hydrolysis-Yeast plants and protein-vitamin concentrate production facilities; Aspergillus and spore-forming Bacteria in enzyme plants; Bacillus thuringiensis and Salmonella in The production of bacterial plant protection agents).

Special differential-diagnostic media are employed to study the airborne microbiota, including the detection of pathogenic and opportunistic microorganisms. Investigations of air microbiota are conducted using several Methods:

✺ Sedimentation method (Koch's method) is based on the gravitational settling of bacteria-laden particles and droplets onto The surface of an Agar medium in an open Petri dish. The Omelyansky calculation is then applied: over a 5-minute period, the number of bacteria settling onto 100 cm² of solid medium corresponds to the bacterial load present in 10 L of air. This method lacks precision and is unsuitable for analyzing ambient atmospheric air, where high wind velocities occur;

✺ Aspiration methods are based on the forced impingement of airborne microorganisms onto the surface of a solid nutrient medium or into a trapping liquid. The Dyakonov apparatus captures bacteria in a liquid by bubbling air through it, afterization of which the liquid is plated onto various differential-diagnostic media. The Rechmensky device precipitates microbial aerosols using steam or atomized liquid. The Krotov apparatus operates on THE PRINCIPLE OF a jet impact action. A stream of air passes through a narrow wedge-shaped slit and impacts the moist surface of the nutrient medium at high velocity. As a result, aerosols suspended in the air, including dust particles and bacteria-containing droplets, are forcibly deposited onto the surface of the nutrient agar or selective media. These methods are the most accurate and reliable.

Natural water bodies contain a high abundance of microorganisms belonging to the autochthonous microbiota, as well as allochthonous microorganisms introduced via precipitation, wastewater, and other sources of contamination. The quantitative and taxonomic composition of aquatic microbiota depends heavily on environmental conditions (nutrient availability, temperature, aeration, redox conditions, pH, etc.).

The autochthonous microbiota of rivers, lakes, seas, and oceans is represented by aerobic species of aquatic bacteria from diverse taxonomic and physiological groups. The total abundance of autochthonous microbiota in open water bodies depends on the water body type, the composition and concentration of organic and Inorganic Compounds, meteorological conditions, and the season. Aquatic microorganisms drive closed cycles of essential biogenic elements within water bodies because microbial communities comprise primary producers of organic matter (eukaryotic and prokaryotic phototrophs, chemoautotrophic prokaryotes), consumers (Protozoa), and reducers (the majority of heterotrophic bacteria and fungi). The surface zone of a water body is dominated by microorganisms capable of maintaining a suspended state: Cells possessing flagella, prosthecae, holdfast disks, gas vacuoles, or organisms of small size. This zone is dominated by oligotrophs and prosthecate bacteria of the genera Hyphomicrobium, Planctomyces, Blastobacter, Pasteuria, Caulobacter, Asticcacaulis, Prosthecomicrobium, Seliberia, Prosthecochloris, etc. Spirilla are classified among typical planktonic bacteria. Phototrophy, methylotrophy, and nitrification are the predominant metabolic types. Depending on environmental gradients, the water Column is subdivided into subzones of Photosynthesis, biomass production by heterotrophic and chemolithotrophic microorganisms, organic matter destruction, and the thermocline. In the aerobic water column (at depths down to 10 m), the bulk of organic matter—both synthesized within the water body and introduced externally—is degraded. Myxobacteria, alongside bacteria of the genera Flexibacter and Cellvibrio, are capable of lysing living cells of cyanobacteria and green Algae. Biopolymers derived from dead microbial biomass, phytoplankton, higher plants, and animals are decomposed by myxobacteria and Representatives of the genera Sporocytophaga, Lysobacter, Beneckea, Alginomonas, Vibrio, and Cytophaga. The surface layer of silt is inhabited by attached or gliding microorganisms: microaerophilic bacteria of the genera Flexibacter, Beggiatoa, Thiothrix, facultatively anaerobic cytophagas and bacilli, filamentous bacteria Pelonema, Peloploca, Leucothrix, Metallogenium, Hyphomicrobium, Seliberia, and green filamentous bacteria. Anaerobic decomposition of organic matter in the Superficial layer of bottom sediments is carried out by clostridia and enterobacteria. The deeper layers of silt contain sulfate-reducers and methanogens, which complete the anaerobic breakdown of PLANT AND ANIMAL debris deposited on the bottom.

In oligotrophic waters deficient in organic matter, gliding and prosthecate bacteria are present, capable of attaching to available substrates and colonizing them to form flocs.

Marine waters harbor substantial populations of Viruses, archaea, and ultramicrobacteria (nanobacteria). Archaea, traditionally found in extreme habitats, account for approximately one-third of the picoplankton (cells smaller than 2 µm). Marine ultramicrobacteria, predominantly representatives of the genus Sphingomonas, exhibit starvation resistance and extremely small dimensions, enabling them to evade predation even by nanoflagellates.

In coastal zones of water bodies, the abundance and species composition of aquatic microorganisms increase sharply. This is attributed to contamination by allochthonous microbiota washed into the water from soil via stormwater, snowmelt, and runoff. Wastewater microbiota contains inhabitants of the Human and Animal gastrointestinal tracts, including members of both normal and opportunistic microbiota. Pathogenic bacteria are poorly adapted to survival in water, where they face adverse effects from sunlight and other environmental factors, including competition from native aquatic microbiota. Nevertheless, many can persist in the aquatic environment for relatively long periods.

Microbiological methods for water analysis focus on determining the total microbial count per 1 mL of water and detecting pathogenic microorganisms (Salmonella, Vibrio cholerae, Leptospira, Shigella, and enteroviruses). Because the direct isolation of pathogenic bacteria from water requires specialized Procedures, indirect methods are used to quantify the degree of water contamination (detection of coliform bacteria, enterococci, staphylococci). Mandatory sanitary-microbiological analysis is required for centralized drinking water supplies, wells, open water bodies, and swimming pools. Water samples for sanitary-microbiological analysis from open water bodies (pools, reservoirs, etc.) are collected at a depth of 10–15 cm; for shallow depths, sampling is performed at least 10–15 cm above the bottom. Samples from ice holes are taken 10–15 cm beneath the lower surface of the ice. Water samples from specific strata of open water bodies (wells, lakes, rivers) are retrieved using a bathometer. MICROBIOLOGICAL ANALYSIS OF collected water samples must be performed no later than 2 hours after collection. If maintaining this timeframe is impossible, water analysis is permitted within 6 hours of storage provided the sample is kept at 1–6 °C. The investigation of water microbiota is carried out using the following methods:

• Determination of the total microbial count in water — the number of microorganisms per 1 mL of water. Drinking water is considered good if the total bacterial count per 1 mL does not exceed 100, doubtful at 100–150, and polluted at 500 or more;

• Determination of the coli-titer and coli-index of water. The coli-titer is the minimum volume of water (in mL) in which coliform bacteria are detected. The coli-index represents the number of coliform bacteria contained in 1 L of the tested water (or 100 mL according to international and European standards). These parameters are established using a two-stage Fermentation (titration) method or the Membrane filtration technique. The fermentation method is based on inoculating defined volumes of the analyzed water, incubating them at 37 °C in enrichment media, subsequently plating onto Endo agar, differentiating the resulting bacterial growth, and determining the most probable number (MPN) of coliform bacteria per 1 L of water using standard tables. The membrane filtration method involves concentrating bacteria from specific volumes of the analyzed water onto a membrane filter, incubating them at 37 °C on Endo medium, differentiating the colonies that develop, and calculating the concentration of coliform bacteria per 1 L of water.

Soil is a favorable environment for microbial proliferation, densely populated by microorganisms and serving as the primary reservoir for their dissemination. Soil provides all the essentials for microbial activity: organic and mineral compounds, moisture, and protection from lethal solar ultraviolet radiation. The abundance of soil microorganisms and their taxonomic composition depend on various edaphic and climatic factors, including soil texture, water-holding capacity, acidity, tillage practices, and season. Soils are rich in rod-shaped bacteria, actinomycetes, and Molds. When analyzing soil microbiota, one must account for the fact that only 1–10% of soil microorganisms are culturable under standard laboratory conditions. Unculturable forms are identified using molecular genetic techniques: the Polymerase Chain Reaction (PCR) and its various modifications, the ligase chain reaction (LCR), DNA-DNA Hybridization, and total cellular RNA hybridization.

Soils contain numerous surfaces that influence nutrient availability, while variable pore sizes dictate differential accessibility for microbial colonization. Soils are composed of sand, clay, silt, and other particulate matter. Organic matter, continually supplied via plant and animal residues, is gradually transformed into nutrient-rich humus. These components form heterogeneous aggregates, or soil peds, penetrated by a complex pore network. Bacteria and fungi employ distinct strategies to gain a competitive advantage in this physically complex substrate. On the surfaces of soil particles, bacteria typically occur as microcolonies, whereas in the soil solution within pores, they exist in suspension. Bacteria require close proximity to water and nutrients. Mycelial fungi grow on or between soil aggregates and can form bridges spanning separate aggregates, granting access to sites with higher moisture availability. Protozoa inhabit the thin water films.

Soils develop under diverse environmental conditions. The colonization of newly exposed geological material by microorganisms begins following events such as earthquakes or volcanic eruptions. The pioneers in this process are cyanobacteria, which are capable of photosynthesis and Nitrogen Fixation. Gram-positive bacteria—specifically coryneform and nocardioform bacteria, as well as actinomycetes—play a major role in soil formation and functioning.

The vertical DISTRIBUTION OF MICROORGANISMS along the soil profile is critical: 1) at the soil surface within the litter and detritus layer; 2) in the aerated zone featuring a developed plant ROOT system; 3) below the water table.

Decomposing plant litter represents a zone of proliferation for hydrolytic aerobic microorganisms and serves as an optimal environment for saprotrophic fungi, given that the litter is primarily composed of lignocellulose. Characteristic saprotrophic fungi in the litter layer include Alternaria and Cladosporium, whereas Penicillium, Trichoderma, and Fusarium predominate in the underlying humus layer. The products of litter decomposition by fungi, alongside senescent mycelium, provide a substrate for the growth of mycophilic bacteria and actinomycetes.

Associations between soil microorganisms and plant root systems are of major interest. Roots reside in the soil horizon most enriched in organic matter. Interactions between microorganisms and plant roots encompass:

1) the rhizosphere, the zone of soil adjacent to plant roots influenced directly by root exudates;

2) the rhizoplane, the root surface itself;

3) the root tissue.

In the rhizosphere, the action of root exudates—which contain various organic substances (CARBOHYDRATES, Amino Acids, organic acids)—and root litter can be observed. The Diversity of microorganisms near the root is determined by the influx of various compounds, trophic interactions among microorganisms, and METABOLISM/18.html">The Influence of specific plant-derived substances. A wide spectrum of organotrophic aerobic bacteria, whose nutritional requirements are geared toward these exudates, is found here. Typically, this microbial habitat is characterized by an excess of organic carbon alongside limiting levels of nitrogen and phosphorus. On leaf surfaces within the phyllosphere, organisms that interact specifically with the plant develop, along with parasitic bacteria and fungi.

S. N. Vinogradsky proposed terming saprotrophic microorganisms responsible for mineralizing organic litter as zymogenic microbiota, and those decomposing soil humus as autochthonous microbiota. Microorganisms that thrive on minimal concentrations of organic substances and complete the Mineralization of organic litter in the soil are designated as oligotrophic microbiota. Within this group, a distinction is made between oligonitrophiles, which require minimal concentrations of organic nitrogen-containing compounds, and oligocarbophiles, which consume residual organic carbon-containing compounds. Microorganisms that utilize CO₂ or carbonates as a carbon source and derive energy from The oxidation of mineral compounds are grouped together as autotrophic microbiota.

The significant contribution of soil microorganisms to the transformation of major biogenic elements in nature is due to the complex Structure OF THE soil microbiocenosis. The decomposition of organic litter entering the soil begins with the zymogenic microbiota, represented by various saprotrophic microorganisms. During the initial stages, easily accessible Organic compounds are mineralized by non-spore-forming bacteria of the genera Pseudomonas, Proteus, and others. The subsequent process of profound organic matter mineralization is accompanied by microbial succession. Non-spore-forming bacteria are replaced by various species of bacilli (B. subtilis, B. mesentericus, etc.). Partially, plant and animal litter products as well as microbial metabolites are converted into humus, which is gradually mineralized by the autochthonous microbiota. The latter represents a specific subgroup of saprotrophic microorganisms possessing a more powerful enzymatic apparatus capable of oxidizing complex cyclic compounds. Such microorganisms include actinomycetes, coryneform, and nocardioform bacteria. The final stages of mineralizing residual products of organic matter breakdown and humus at minimal concentrations are carried out by oligotrophic microorganisms. This group of microorganisms includes A number of specific

species of saprotrophic microorganisms adapted to growing on nutrient-poor substrates. Inorganic compounds (NH₃, H₂S, H₂, etc.) generated during the mineralization of organic matter are transformed through the METABOLIC ACTIVITY OF autotrophic microorganisms. The latter utilize mineral compounds as Energy Sources, oxidizing them during cellular Energy Metabolism. The ratio of the aforementioned microbial groups determines the characteristic microbiocenosis structure of each zonal soil type. When compiling a microbiological profile of soil, one must account for the uneven distribution of microorganisms across soil microloci, the high dynamics of abundance and qualitative COMPOSITION OF THE soil microbiota, and the insufficient development of Taxonomy and identification for the majority of soil microorganism species.

In addition to autochthonous microbiota, the soil contains representatives of the normal human and animal microbiota that have ended up there, as well as pathogenic microorganisms. Allochthonous microorganisms in soil typically do not survive for long; however, certain members of the normal human microbiota can integrate into the soil biocenosis, and some species become permanent residents. The survival of pathogenic bacteria in soil is influenced by soil composition and type, temperature, humidity, precipitation, and the degree and nature of contamination (organic, chemical, or microbial). Pathogenic microorganisms found in soil are divided into three groups:

✵ those permanently residing in the soil (e.g., Clostridium botulinum, representatives of the genus Actinomyces—agents of subcutaneous mycoses, and certain mycotoxicosis agents);

✵ spore-forming species for which the soil serves as a secondary reservoir (e.g., Bacillus anthracis, Clostridium tetani, and certain Clostridium species causing anaerobic infections). These bacteria enter the soil via human and animal excreta, as well as animal carcasses. Under favorable conditions they can multiply, while under unfavorable conditions they persist in the soil as spores for extended periods;

✵ non-spore-forming species that enter the soil with human and animal excreta and persist relatively briefly—from several weeks to months (e.g., bacteria of the genera Salmonella, Shigella, Vibrio, Brucella, Francisella, Mycobacterium, Leptospira, Pseudomonas).

The sanitary condition of soils is assessed using a set of indicators: the total count of saprotrophic microorganisms is calculated, and the presence of sanitary-indicator microorganisms (coliforms, Clostridium perfringens, etc.) is determined. A high abundance of saprotrophic microbiota indicates organic pollution, whereas microbial contamination is dominated by sanitary-indicator microorganisms. When necessary, the composition of nitrifying and ammonifying bacteria, actinomycetes, fungi, and cellulolytic microorganisms is also examined.

Review Questions

1. What are the Specific characteristics of air as a habitat for microorganisms?

2. Which groups of microorganisms constitute the air microbiota?

3. What methods are used to study the air microbiota?

4. Which groups of microorganisms represent the autochthonous microbiota of water bodies?

5. What factors influence the qualitative and quantitative composition of aquatic microorganisms?

6. What microbiological methods are applied to water analysis?

7. What are the characteristics of soil as a microbial habitat?

8. Name the main physiological and taxonomic groups of microorganisms inhabiting the soil.

9. Into which groups are pathogenic microorganisms found in soil divided?

10. What indicators are used to evaluate the sanitary condition of soils?

Class="center">Laboratory Work 6. Investigation of Air, Soil, and Water Microbiota

Objective: to master Methods for determining the qualitative and quantitative composition of air, soil, and water microbiota.

Materials and equipment: saline solution, PDA (potato dextrose agar), Endo agar, Ashby's medium, Czapek's medium, casein-glycerol agar (CGA), sterile distilled water, sterile test tubes, sterile Petri dishes, sterile flasks (500 ml capacity), 1–2 ml and 5–10 ml pipettes, spreaders, spirit burner, incubator, Microscope slides, Gram staining Reagents.

Procedure

1. Investigation of air microbiota using Koch's sedimentation method.

1) PDA and Endo agar Petri dishes are uncovered in the test room (classroom, laboratory, corridor, canteen, staircase) and left open for 5 min, after which they are covered with a lid and placed in a thermostat at 28 — 30 °С for 24 h.

2) The Morphological Characteristics of the formed colonies are described.

3) Smears are prepared from morphologically distinct colonies, Gram-stained, and examined microscopically.

4) Based on the morphological diversity and the number of grown colonies, Conclusions are drawn regarding the qualitative and quantitative composition of the airborne microbiota.

5) The number of microorganisms per 1 m³ of air for each studied room is calculated using the formula

where М is the number of CFU in 1 m³ of air; а is the number of colonies formed on the dish; V₁ is the volume of air (l) from which microorganisms settle onto the medium surface with an area of S₁ (cm²) (V₁ = 10 l, S₁= 10 cm²); V₂ is the volume of air for which the calculation is performed (l); d is the diameter of the Petri dish.

6) The results are entered into a table (Table 6).

Table 6. Qualitative and quantitative composition of indoor air microbiota

2. Analysis of water microbiota.

1) Water from a water body (400 — 500 ml) is collected into sterile 500 ml bottles immediately before inoculation. Sampling is carried out at a depth of at least 10 — 15 cm from the bottom.

2) Tap water (400 — 500 ml) is sampled from a tap previously wiped with a cotton swab soaked in alcohol, after letting the water run for 10 — 15 min. When analyzing chlorinated water, a dechlorinator (10 mg of sodium hyposulfite) is added to the sampling bottle prior to sterilization.

3) Serial tenfold dilutions (up to 10^-4) are prepared from each water sample using sterile water.

4) Aliquots of 1 ml of the initial water sample and respective dilutions (10^-1 — 10^-4) are added to sterile Petri dishes (2 series of dishes per dilution), poured with 20 ml of PDA melted and cooled to 45 — 50 °С, and thoroughly mixed with circular motions.

5) After the agar solidifies, one series of dishes is placed in a thermostat at 37 °С for 24 h, and the second series at 20 °С for 48 h.

6) The number of colonies grown On the surface and in the depth of the agar is counted, and the microbial count of the water is calculated. Conclusions are drawn regarding the quality of the tap water and the water body sample.

7) Fixed smears are prepared from colonies of various morphotypes, Gram-stained, and examined microscopically. Based on the Morphological diversity of the grown colonies, conclusions are drawn regarding the qualitative composition of the water microbiota. The results are entered into a table (Table 7).

Table 7. Qualitative and quantitative composition of water microbiota

3. Analysis of soil microbiota.

1) Soil samples (200 — 300 g) are collected with a sterile knife at a depth of 10 — 15 cm and placed in a sterile jar or bag.

2) A 10 g subsample is weighed out from the soil samples, transferred to a sterile mortar, mixed with 2 — 3 ml of sterile water, and ground to a paste-like consistency.

3) The resulting soil suspension is transferred to a flask with sterile water (90 ml), stirred thoroughly for 5 min, and allowed to settle for 30 min. This represents the first dilution (10^-1) of the tested soil sample.

4) A series of tenfold dilutions (up to 10^-6) is prepared.

5) Spread 0.1 ml of the 10^-4 — 10-⁶ dilutions onto the surface of nutrient media using a hockey stick spreader (inoculating 4 plates per dilution):

✵ PDA — to determine the total bacterial count;

✵ Czapek's medium and PCA — to enumerate and isolate actinomycetes;

✵ Ashby's medium — to enumerate and isolate Azotobacter.

6) Incubate the inoculated Petri dishes in a thermostat at 28 °C. Count the total bacteria on PDA after 1 — 5 days, and actinomycetes and Azotobacter after 5 — 7 days.

7) Prepare fixed smears from colonies with different morphologies, Gram-state them, and examine under a microscope.

Table 8. Qualitative and quantitative composition of soil microbiota

8) Based on the morphological diversity and the number of grown colonies, draw a Conclusion regarding the qualitative and quantitative composition of the soil microbiota. Record the results in the table (Table 8).