DISTRIBUTION OF MICROORGANISMS IN AQUATIC ENVIRONMENT

DISTRIBUTION OF MICROORGANISMS IN AQUATIC ENVIRONMENT

30.1 Introduction
Water occurring in nature contains dissolved salts and gases, especially sea and mineral waters. Water covers 70% of the earth’s surface, and thus, it is the most essential habitat of life. The overall volume of inland waters is estimated at 7.5 x105 km3, of seas and oceans at 1.4 x 109km3, and of glaciers and continental glaciers at 1.8 x 10km. Water makes up the most crucial component of living organisms (70-90% of cell mass) and fulfils a purpose in taking part in various biological reactions and processes.


30.2 Types of Waters Inhabited by Microorganisms
The biotopes of water microorganisms may be underground and/or surface waters as well as bottom sediments.

  • The underground waters (mineral and thermal springs, ground waters) – due to their oligotrophic character (nutrient – deficient) are usually inhabited by a sparse microflora that is represented by a low number of species with almost a complete lack of higher plants or animals.
  • The surface waters such as streams, rivers, lakes and sea waters are inhabited by a diverse flora and fauna. Microorganisms in those waters are a largely varied group. Next to the typical water species, other microorganisms from soil habitats and sewage derived from living and industrial pollution occur.
  • Bottom sediments are a transient type of habitat i.e. the soil-water habitat that is almost always typically oxygen-free in which the processes of anaerobic decomposition by microorganisms cause the release of hydrogen sulphide and methane into water. In the bottom sediment, anaerobic putrefying microflora, cellulolytic bacteria and the anaerobic chemoautotrophs develop.

30.3 Groups of Water Organisms
Microorganisms occupy surface waters in all of the zones; they may be suspended in water (plankton), cover stationary underwater objects, plants etc. (periphyton), or live in bottom sediments (benthos).

30.3.1 Plankton
The group of organisms that passively float in water not being able to resist the movement and the flow of water mass is called plankton or bioseston. These are of following types: 

30.3.1.1 Phytoplankton 
Phytoplankton are mainly microscopic algae and blue-green algae. It is a varied community in terms of the systematics and mainly composed of forms smaller than 50 μm. Sea phytoplankton are dominated by diatoms and dinophyta, whereas fresh water phytoplankton are dominated one by cryptophytes, diatoms, green algae, and blue-green algae.


30.3.1.2 Zooplankton 
Zooplankton are small water animals that occur in plankton. There are three systematic groups that occur in fresh waters: rotifers, branchiopods and copepods. The sea water plankton is composed of copepods, ctenophores, urochordata, arrow-worms as well as some species of snails. Most of them are filtrators (condense suspended particles) or predators.

30.3.1.3 Protozoa plankton
Protozoa plankton consists of protozoa which occupy the open water zones like flagellates and ciliates. They are the main consumers of bacteria. Moreover, most ciliates feed upon flagellates, algae and smaller ciliates. The protozoa itself feeds the zooplankton.

30.3.1.4 The heterotrophic bacteria plankton 
The heterotrophic bacteria plankton occupy waters which are abundant in organic compounds. The amount of bacteria in open waters varies between 105-107 cells/ml.


30.3.1.5 Virus plankton 
Virus plankton is composed of viruses which are the smallest element of plankton. Their numbers may be very high (from 108 in 1ml) in various fresh and sea water habitats. Viruses are, next to the protozoa, a crucial factor in bacteria mortality.

30.3.2 Periphyton

Periphyton occupy the shore line zones. They are a group of organisms that create outgrowths upon various objects and underwater plants. Most of the time, they usually consist of small algae – diatoms, green algae and bacteria. Moreover, various settled or semi-settled protozoa, eelwarms, oligochaetes, insect larva, and even crustaceans make up the periphyton biocenosis. Periphyton has a characteristic complex biocenosis and many ecological relationships can be observed between its components.


30.3.3 Benthos
The bottom habitat is occupied by a group of organisms called the benthos. The muddy bottom contains an abundance of organic compounds that are created as a result of dead matter decomposition (fallen parts of plants and animals). At great depths the bottom is free from any plants which, due to a lack of light cannot grow. However, the absence of oxygen supports the development of, among others, an oxygen-free putrid microflora. Among the benthos microflora the most numerous are bacteria and fungi (decomposers) as well as some animals (detritophages). Both of the above groups are responsible for decomposition of the organic matter. Benthos of shallow reservoirs may also contain some algae.


30.4 Factors Affecting Growth of Microorganisms in Water
The development of microorganisms in water is influenced by a large number of chemical and physical factors which, in various ways, interact or oppose each other. They have an influence on the size, species and composition of the microbial biocenosis as well as on their appearance and life processes. Within water ecosystems two groups of factors that have a crucial influence on the quantitative and qualitative relationships between microorganisms may be distinguished:

  • Abiotic factors – light and thermal energy, water reaction, water flow, climate and the compounds dissolved and suspended in water (dead organic matter, non-organic compounds and gasses such as oxygen, carbon dioxide, methane and others).
  • Biotic factors – all water living organisms such as plants, animals, microorganisms and the relationship between them.

30.4.1 Abiotic factors

30.4.1.1 Light energy

Light plays a major role in the process of photosynthesis. The amount of light penetrating different layers of water strictly depends on the position of the sun, transparency, colour and depth of water. The lesser the incidence angle the smaller the loss of sun rays due to reflection. Depending on the level of insolation and water turbidity, the biologically active sun rays usually penetrate water somewhere between 10-150 m. Undoubtedly, sea waters are clearer and less polluted than inland waters, thus light can penetrate much further down through these waters. Sun rays penetrate sea waters down to about 150 m creating the so called photic zone where photosynthesis takes place. Due to different light conditions the development of photoautotrophs isn’t identical throughout the entire water mass. The indicator of the illumination quantity is often the lower boundary (limit) of algae occurrence – their greatest development takes place at a depth of 0.5-2 m. Most algae possess an ability to change and adapt their colouring to the light conditions. Light is harmful to those microorganisms which are deprived of any pigments. Both the ultraviolet and the longer wavelength may have a negative effect. For instance, blue light (wave length 366-436 nm) inhibits the process of nitrite oxidation by Nitrobacter vinogradskyi. Light also has an influence upon water fungi development. Blue and green rays have a greater impact than red rays.


30.4.1.2 Temperature
The amount of thermal energy depends, just as in the case of light energy, on the incidence angle (the position of the sun in relation to the water surface). Therefore, it varies with time of day, seasons and latitude. Lotic waters such as rivers have a steady temperature throughout their mass due to constant mixing by the water flow. However, such a water habitat is characterised by daily temperature fluctuations especially in shallow rivers. In lentic (stagnant) waters such as lakes, where the water current is very weak or nonexistent, the temperature fluctuates during the annual cycle. Lakes, especially deep ones, are characterized by vertical stratification (the formation of layers that vary according to their composition and temperature). Illuminated warm and near-surface waters have a lower density than the dark and cold waters from below. The difference in density prevents mixing of the layers. The warm water layer is called the epilimnion. The cooler layers from below form a thermocline or metalimnion and become cooler with depth. The temperature falls by 1°C with each meter. In the lowest layer – hypolimnion – the water is at 4°C and has the highest density.

The thermocline works as a barrier between the epi- and hypolimnion. The upper waters do not mix throughout the year due to their different density. Water is only moved within the epilimnion layer by the wind. The biogenes present near the bottom are not available for the organisms living in the upper layers thus, in late summer; the top layer has a deficit of trophic substances. In the autumn the surface waters begin to cool down, slowly falling while pushing the warmer waters upwards, which also cool down. As the waters continue to exchange (autumn circulation) and are mixed by the wind they oxygenate and at the same time lose CO2 by releasing it to the atmosphere especially from the bottom waters. A slight inversion of temperature occurs in the winter since the water at less than 4°C has a lower density than the 4°C water and it rises towards the surface. Different circulation occurs in the spring as the surface waters warm up. Then, the entire body of water is rich in oxygen and biogenes. The mixing of water also causes organisms to move.

30.4.1.3 Water movement
Mixing of water is of great importance to both the temperature distribution and for the balance of the chemical composition (gasses, nutrients, substances that equalize the osmotic pressure, water pH etc.). The movement of water is caused by the following:

  • variations in density caused by different temperatures and contents of soluble
  • suspended compounds
  • winds
  • difference in the levels at the bottom (lotic waters)
  • specific hydraulic engineering processes.

30.4.1.4 Pressure
Pressure is an important ecological factor that strongly influences the life of microorganisms among other things by affecting the activity of the cells enzymatic systems. In water the hydrostatic pressure gradually increases with depth at 1 atm per 10 m. Thus, in large oceans and some deep lakes the pressure is quite high – in most seas it’s at about 100 atm and in some Pacific trenches it may reach even 1100 atm. The group of abyssal microorganisms, which occur at depths of 10,000 m, are called barophilic. They grow and develop not only under great pressures, but also at very low temperature (3 -5°C) but their growth is very slow. Most fresh water and soil bacteria do not develop when the pressure exceeds 200 atm (barophobic microorganisms).

30.4.1.5 pH of water
An optimal pH for water-bacteria is between 6.5 and 8.5. The pH of most lakes is 7.0, rivers – 7.5, and the surface layer of the seas 8.2. Because of the high content of carbonates and their buffer properties, the pH of water does not usually fluctuate significantly. But when there is a rapid growth of photosynthesising organisms the pH may increase rather considerably. Some mineral springs and inland waters with a high content of humus compounds may be acidic. In such conditions the number of acidophilic fungi increases. Relatively large changes in pH can be observed in eutrophic lakes where the pH varies between 7-10, which has an obvious influence on the populations of bacteria and fungi.

30.4.1.6 Salinity
Most microorganisms that live in clean rivers and lakes are halophobic and in natural conditions do not live in waters in which the salinity exceeds 10%. There aren’t many halophilic organisms which may grow in waters of higher salinity. Due to the salinity, sea is thought of as a separate (distinct) biotope; the predominant number of bacteria and fungi living in seas are halophilic. Their life processes depend on a specific concentration of NaCl thus, most of the organisms living in such habitats cannot survive anywhere else. The major mass of salt (99%) is composed of the following elements: Cl, Na, S, Mg, Ca and K. The concentration of salt in sea water is on average 35%. The optimal salinity range for most halophilic bacteria and fungi varies between 25-40%. In the oceans is on average 32-38%, however in closed seas (salty lakes) the range is much greater. For instance, the Caspian Sea contains a low level of salt (1.1-1.3%), whereas the Dead Sea’s salinity ranges up to 28%. An increase in salinity has an influence on the generation cycle of bacteria and fungi, and on their morphological and physiological properties. Lakes with a high concentration of salts are extreme biotopes and their biotic groups are low in species variation (the main microorganisms are the bacteria, blue-green algae, flagellates).


30.4.1.7 Other non-organic substances
The life cycle of water microorganisms is also dependent on non-organic substances other than NaCl, among which phosphorus and nitrogen compounds play a major role.
Besides free nitrogen, many mineral compounds of this element, such as nitrates, nitrites and ammonium salts, occur in surface waters. Algae and heterotrophic bacteria most often use nitrates and ammonium salts. The maximum amounts of nitrogen which are tolerated by various algae species are different. For instance, diatoms (such as Asterionella) may reproduce at high concentrations – even at 100 μg N/l, whereas the maximum level for Pediastrum algae is only 2 μg N/l. It is similar for the bacteria – the maximum amount is different for various species. The most important element which limits the development of algae is phosphorus. Its content in water is rather low (0.01-0.1 mg P2O5/l). Mineral phosphorus occurs in waters in diluted forms (orthophosphate) and in the form of insoluble salts – calcium phosphate, magnesium phosphate etc. Algae may store phosphorus in their cells in amounts exceeding their requirement. The influence of an increasing phosphate concentration by the introduction of pollutants is a reason for water blooming. In oligotrophic lakes as well as in seas that are nutrient-deficient, it is difficult to detect any presence of ammonium ions, nitrites, nitrates and phosphates since these elements are utilized by phytoplankton immediately after their production. Within the photic zones of many tropical seas the deficiencies in nitrogen and phosphorus compounds last throughout the year, whereas in temperate zones it undergoes seasonal changes.

On the other hand, in the deep waters of some large lakes and seas the accumulation of nitrates and phosphates occurs as a result of heterotrophic microorganism activities.


Ammonium ions and nitrites are the energy substrates for the nitrification bacteria whereas, oxygen combined in nitrates may be utilized by a number of denitrifying bacteria to oxidize the organic substances in anaerobic conditions. Other life essential salts are the compounds of S, Mg, Ca, K, Fe and Si. They are utilized by microorganisms to build cell structures and for the activation of enzymes.

30.4.1.8 Gases-In water reservoirs
Gases-In water reservoirs, besides salts and organic substances, small quantities of diluted gas can be found. Water possesses an ability to dilute gases but the solubility decreases as the temperature and salinity increase; it is lower in sea waters than in the fresh water basins. It mainly concerns oxygen, carbon dioxide and nitrogen. The main source of the above gases is the atmosphere from which gases diffuse into the upper layers of water until a state of saturation is obtained. In addition, gases diluted in water and sediments may be created during biochemical processes. In this way oxygen is released by green plants as a result of photosynthesis, COduring respiration, free nitrogen during denitrification, hydrogen sulphide as a result of desulfurication, and hydrocarbons as a result of fermentation processes.

30.4.1.9 Organic substances

Organic substances are either secreted by living cells or the products of their autolysis. However, the greatest amounts of organic compounds are introduced into water by sewage. Organic compounds occur in water in the form of solutions or as suspended matter. First of all they serve as food for heterotrophic bacteria and fungi. Microorganisms that often occur on the surface of the suspensions, especially upon the particles of the detritus which absorb the organic substances from water, enjoy favourable feeding conditions. The development and metabolic changes of microorganisms are influenced, more by the content of readily available organic compounds (such as carbohydrates, organic acids, proteins and lipids) rather than the amount of the organic substances in general. Their depletion from water occurs rather quickly. When there is a lack of organic substances bacteria do not reach their proper size and their cell division is slowed down.


30.4.1.10 Trophicity of surface waterTrophicity is the water’s abundance of biogenic elements and soluble simple organic compounds. Trophicity determines the primary production rate and the size of the biomass. Major indicators of water trophicity are: phosphorus and nitrogen concentration, chlorophyll concentration, water transparency and oxygen conditions near the bottom. With reference to the water trophicity, the following kinds of water reservoirs may be distinguished: oligotrophic (low nutrient concentration), mesotrophic (medium nutrient concentration), eutrophic (rich in nutrients/fertile), and hypertrophic (very rich in nutrients). The abundance of nutrients in water reservoirs changes with time.

This process of water fertility increases from oligothrophic through mesotrophic to eutrophic waters in a process called eutrophication. When the above process is moderate and its effects are beneficial, then it is considered to be a fertilization process.

However when the process is excessive and its effects aren’t beneficial, then it is considered to be biogenic substance pollution. Nutrient deficient low-fertile waters are those which have a low content of phosphorus and nitrogen – it is these two elements that are the most crucial. Therefore the waters contain low numbers of phyto- and zooplankton organisms and they are clean and clear. In waters which contain a low amount of plankton very little dead matter falls to the bottom. Therefore, its decomposition does not deplete the oxygen reserves near the bottom. Up to a certain level, the increase in water fertility in turn causes an increase in the number of most organisms in water and consequently an intensification of life manifestations. When the level of nutrients is too high the organic matter produced disturbs the ecosystem’s homeostasis. Some enzymes released by bacteria cause decomposition of the other bacteria and algae. Owing to a release of organic substances the plankton microorganisms grow abundantly. The increased use of oxygen causes a deficit of oxygen in deeper waters and consequently, the development of anaerobic microorganisms and the appearance of methane and hydrogen sulphide. Thus, high vitality means increased production in water basins, biomass development of phytoplankton and at the same time lower oxygen concentration in deeper layers. Water blooming is a consequence of eutrophication and it is caused by the reproduction of algae in the upper layers of water. The above process causes changes in water colour, its turbidity, water quality deteriorates, and toxic compounds are produced. The process of natural eutrophication proceeds very slowly – it takes up to a few thousand years. Accelerated eutrophication, however, is caused by human activities. As a result, excess amounts of nitrogen and phosphorus get into waters from various sources such as industrial and municipal wastes, as well as fields that have been fertilized with phosphorus and nitrogen fertilizers. Such activities greatly increase the concentration of biogenes creating favourable conditions for algae reproduction. In such polluted waters, faecal and pathogenic bacteria may survive for a longer period of time. Precipitation in highly industrial and polluted areas has also had an influence upon eutrophication.

30.4.2 Biotic factors
Mutual interactions exist between individual members of the biocenosis that inhabit surface waters. As a result, the organisms may support each other (synergism) or inhibit each other (antagonism).

30.4.2.1 Competition for food

The organisms which most efficiently find and take in food may have an advantage over others. For a given habitat with a typical supply of nutrients, the number of microorganisms quickly increases. However, in many cases, the abundant production of products of metabolism (inhibitors) decreases the number of competitors, sometimes eliminating them entirely. Such situations occur, for instance, when the pH is significantly altered by acidification or alkalization, and when antibiotic substances are released.

30.4.2.2 Co-operation
In feeding and growth processes, co-operation between the microorganisms is often observed. It allows quicker development of mixed microorganism cultures. Biodegradation is a multistage process when consecutive reactions are conducted by different specialized microorganisms. The process prevents the accumulation of the metabolism by-products. Owing to this co-operation, the biodegradation of persistent organic compounds (ligninocellulose) becomes possible.


30.4.2.3 Predation
Bacteria and fungi are food for lower animals. This is why in some water reservoirs their numbers may vary a lot. Most protozoa feed on bacteria. It has been confirmed that their biomass increases along with the increase in bacterial numbers. Numerous multi-cellular organisms also utilize bacteria as their food. These mainly include filtrating animals such as sponges. In bottom sediments many animals feed upon fungi. Blue-green algae which are a part of the benthos are often eaten by turbellarians, nematodes, crustaceans and insect larva. Blue-green algae are eaten by zooplankton, without the latter water blooming and release of toxic substances would occur.

30.4.2.4 Parasitism
Water microorganisms are attacked and destroyed by viruses, bacteria and fungi. The presence of bacteriophages has been affirmed in inland and sea waters. They are especially numerous in sewage and are probably the reason for a quick depletion of bacteria in rivers, lakes and in inshore waters that were polluted with sewage. Another reason for the limitation of bacterial numbers is the presence of the Vibrio bacteria which belong to Bdellovibrio genera and lead a parasitic existence. They attach themselves to host cells and reproduce utilizing their energy and consequently digest the cells content. After lysing the host’s cell wall, they free themselves and infect further bacteria.

MICROBIOLOGY OF AIR

28.1 Introduction

The earth’s atmosphere is teeming with airborne microorganisms. These organisms are thought to exhibit correlations with air pollution and weather. Most airborne bacteria originate from natural sources such as the soil, lakes, oceans, animals, and humans. Many ‘unnatural’ origins are also known, such as sewage treatment, animal rendering, fermentation processes, and agricultural activities which disturb the soil. Viable airborne microorganisms are not air pollutants, but should be considered as a factor affecting air quality. Air is an unfavourable environment for microorganisms, in which they cannot grow or divide. It is merely a place which they temporarily occupy and use for movement.

There are 3 elementary limiting factors in the air

  • A lack of adequate nutrients
  • Frequent deficit of water (desiccation)
  • Solar radiation

The atmosphere can be occupied for the longest time by those forms which, due to their chemical composition or structure, are resistant to desiccation and solar radiation. They can be subdivided into the following groups:

  • Bacterial resting forms,
  • Bacterial vegetative forms which produce carotenoidal dyes or special protective layers (capsules, special structure of cell wall),
  • Spores of fungi,
  • Viruses with envelopes

28.2 Resting forms of Bacteria

Endospores are the best known resting forms. These structures evolve within cells and are covered by a thick multi-layer casing. Consequently, endospores are unusually resistant to most unfavourable environment conditions and are able to survive virtually endlessly in the conditions provided by the atmospheric air. They are only produced by some bacteria, mainly by Bacillus and Clostridium genera. Because each cell produces only one endospore, these spore forms cannot be used for reproduction.

Another type of resting form is produced by very common soil bacteria, the actinomycetes. Their special vertical, filiform cells, of the so-called air mycelium, undergo fragmentation producing numerous ball-shaped formations. Due to the fact that their production is similar to the formation of fungal, they are also called conidia. Contrary to endospores, the conidia are used for reproduction. There are also other bacterial resting forms, among others, the cysts produced by azotobacters – soil bacteria capable of molecular nitrogen assimilation.

28.3 Resistant Vegetative Cells of Bacteria

The production of carotenoidal dyes ensures cells with solar radiation protection. Carotenoids, due to the presence of numerous double bonds within a molecule (-C=C-), serve a purpose as antioxidants, because, as strong reducing agents, they are oxidizedby free radicals. Consequently, important biological macromolecules are being protected against oxidation (DNA, proteins etc.). Bacteria devoid of these dyes quickly perish due to the photodynamic effect of photooxidation. That explains why the colonies of bacteria, which settle upon open agar plates, are often colored. The ability to produce carotenoids is possessed especially by cocci and rod-shaped actinomycetes. Rod-shaped actinomycetes, e.g. Mycobacterium tuberculosis, besides being resistant to light, also demonstrate significant resistance to drying due to a high content of lipids within their cell wall. High survival rates in air are also a characteristic for the bacteria which possess a capsule, e.g.Klebsiella genus, that cause respiratory system illnesses.

28.4 Fungal Spores

Spores are special reproductive cells used for asexual reproduction. Fungi produce spores in astronomical quantities, for example the giant puffball (Calvatia gigantea) produces 20 billion spores, which get into the air and are dispersed over vast areas. A very common type of spores found in air is that of conidia.

Conidia are a type of spore formed by asexual reproduction. They form in the end-sections of vertical hyphae called conidiophores and are dispersed by wind. The spores of common mould fungi such as Penicillium and Aspergillus are examples of the above. Spore plants such as ferns, horsetails and lycopods also produce spores. Plant pollen is also a kind of spores.

28.5 Resistant Viruses

Besides cells, the air is also occupied by viruses. Among those that demonstrate the highest resistance are those with enveloped nucleocapsids, such as influenza viruses.
Among viruses without enveloped nucleocapsids, enteroviruses demonstrate a relatively high resistance.

Of course, besides the previously mentioned resistant forms, the air is also occupied by more sensitive cells and viruses, but their survival is much shorter. It is believed, that among vegetative forms, gram-positive bacteria demonstrate greater resistance than Gram negative bacteria (especially for desiccation), mainly due to the thickness of their cell wall. Viruses are usually more resistant than bacteria.


28.6 Factors Affecting Growth of Microorganism in Air 
There are several factors which influence the ability of a bioaerosol to survive in air:

  • Particular resistance for a given microorganism (morphological characteristics)
  • Meteorological conditions (inter alia, air humidity, solar radiation),
  • Air pollution,
  • The length of time in air.

28.6.1 Resistance of microorganisms
It is a species dependent feature, which relies on the microorganism’s morphology and physiology. 

28.6.2 Relative humidity

The content of water in air is one of the major factors determining the ability to survive. At a very low humidity and high temperature cells face dehydration, whereas high humidity may give cells protection against the solar radiation. Microorganisms react differently to humidity variations in air, but nevertheless most of them prefer high humidity. The morphology and biochemistry of cell-surrounding structures, which may change its conformation depending on the amount of water in air, are crucial. Actually, an exact mechanism of this is not known. Forms of resting spores with thick envelopes (e.g. bacterial endospores) are not particularly susceptible to humidity variations. Gram-negative bacteria and enveloped viruses (e.g. influenza virus, myxo) deal better with low air humidity which is contrary to gram-positive bacteria and non-enveloped viruses (e.g. enteroviruses) that have higher survival rates in high air humidity.

28.6.3 Temperature

Temperature can indirectly affect cells by changing the relative-air humidity (the higher the temperature, the lower the relative humidity) or a direct affect, causing, in some extreme situations, cell dehydration and protein denaturation (high temperatures) or crystallization of water contained within cells (temperatures below 0°C). Therefore, it can be concluded that low temperatures (but above 0°C) are optimal for the bioaerosol. According to some researchers the optimal temperatures are above 15°C.


28.6.4 Solar radiation
Solar radiation has a negative affect on the survival rate of the bioaerosol, both visible as well as ultraviolet (UV) and infrared radiation due to the following factors:

  • Causes mutation,
  • Leads to the formation of free radicals, which damage important macromolecules.
  • Creates a danger of dehydration.

Visible light rays of about 400-700 nm wavelength, create the so-called photodynamic effect, which produces free radicals within cells, especially compounds such as peroxy and hydroxyl radicals. These radicals demonstrate strong oxidizing activities and may cause damage to crucial macromolecules, e.g. DNA or proteins.


UV radiation has a much larger affect on cells than visible light does, especially the rays of 230-275 nm wavelengths. The mechanism of this effect is based on changes to DNA, both directly (e.g. by creating thymine dimer and consequently causing mutation), as well as indirectly, by creating free radicals as in the case of the visible light.

In addition, infrared (IR) radiation may have a negative effect upon cells contained in air – heating up and consequently dehydration.

28.6.5 Biological aerosols

Microorganisms in air occur in a form of colloidal system or the so-called bioaerosol. Every colloid is a system where, inside its dispersion medium, particles of dispersed phase occur whose size is halfway between molecules and particles visible with the naked eye. In the case of biological aerosols, it’s the air (or other gases) that has the function of the dispersion medium, whereas microorganisms are its dispersed phase. However, it is quite rare to have microbes independently occurring in air. Usually, they are bound with dust particles or liquid droplets (water, saliva etc.), thus the particles of the bioaerosol often exceed microorganisms in size and may occur in two phases:

  • Dust phase (e.g. bacterial dust) or
  • Droplet phase (e.g. formed as the result of water-vapour condensation or uring sneezing).

The dust particles are usually larger than the droplets and they settle faster. The difference in their ability to penetrate the respiratory tract is dependent on the size of the particles; particles of the droplet phase can reach the alveoli, but dust particles are usually retained in the upper respiratory tract. The number of microorganisms associated with one dust particle is greater than in the droplet phase.

The average size of bioaerosols ranges from about 0.02 μm to 100 μm. The sizes of certain particles may change under the influence of outside factors (mainly humidity and temperature) or as a result of larger aggregates forming. By using size criterion, the biological aerosol can be subdivided into the following:

  • Fine particles (less than 1μm) and
  • Coarse particles (more than 1μm)

Fine particles are mainly viruses, endospores and cell fragments. They possess hygroscopic properties and make-up the so-called nucleus of condensation of water vapour. At high humidity water collects around these particles creating a droplet phase.

Then, the diameter of the particles increases. Coarse particles consist mainly of bacteria and fungi, usually associated with dust particles or with water droplets.

Biological aerosols as a human hazard source .

  • What types of dangers are connected to the presence of microorganisms in air?
  • Infectious diseases (viral, bacterial, fungal and protozoan),
  • Allergic diseases,
  • Poisoning (exotoxins, endotoxins, mycotoxins).

Bioaerosols may carry microorganisms other than those which evoke respiratory system diseases. The intestinal microorganisms contained in aerosols may, after settling down, get into the digestive system (e.g. by hands) causing various intestinal illnesses.


28.7 Infectious Airborne Diseases

The mucous membrane of the respiratory system is a specific type of a ‘gateway’ for most airborne pathogenic microorganisms. Susceptibility to infections is increased by dust and gaseous air-pollution, e.g. SO2 reacts with water that is present in the respiratory system, creating H2SO4, which irritates the layer of mucous. Consequently, in areas of heavy air pollution, especially during smog, there is an increased rate of respiratory diseases.

Bioaerosols may, among other things, carry microbes that penetrate organs via the respiratory system. After settling, microbes from the air may find their way onto the skin or, carried by hands, get into the digestive system (from there, carried by blood, to other systems, e.g. the nervous system). Fungi that cause skin infections, intestinal bacteria that cause digestive system diseases or nervous system attacking enteroviruses are all examples of the above.


28.7.1 Viral diseases

After penetrating the respiratory system with inhaled air, particles of viruses reproduce inside the cuticle cells of both the upper and lower respiratory system. After reproduction some of the viruses stay inside the respiratory system causing various ailments (runny nose, colds, bronchitis, pneumonia), whereas others leave the respiratory system to attack other organs (e.g. chickenpox viruses attack the skin). The most noteworthy viruses are:


Influenza (orthomyxoviruses) Influenza, measles, bronchitis, mumps and pneumonia among newborns (paramyxoviruses)

  • German measles (similar to paramyxoviruses)
  • Colds (rhinoviruses and koronaviruses)
  • Cowpox and true pox (pox type viruses)
  • Chickenpox (cold sore group of viruses)
  • Foot-and-mouth disease (picorna type viruses)
  • Meningitis, pleurodynia (enteroviruses)
  • Sore throat, pneumonia (adenoviruses)

28.7.2 Bacterial diseases
Similarly to viruses, some bacteria that find their way to the respiratory system may also cause ailments of other systems. Especially staphylococcus infections assume various clinical forms (bone marrow inflammation, skin necrosis, intestinal inflammation, pneumonia). Often, a susceptible base for development of various bacterial diseases is first prepared by viral diseases, e.g. staphylococcus pneumonia is usually preceded by a flu or mumps. Bacterial airborne diseases include:

  • Tuberculosis (Mycobacterium tuberculosis),
  • Pneumonia (Staphylococcus, PneumococciStreptococcus pneumoniae, less frequently chromatobars of Klebsiella pneumoniae),
  • Angina, scarlet fever, laryngitis (Streptococcus),
  • Inflammation of upper and lower respiratory system and meningitis (Haemophilus influenzae),
  • Whooping cough (chromatobars of Bordetella pertussis),
  • Diphtheria (Corynebacterium diphtheriae),
  • Legionnaires disease (chromatobars of Legionella genus, among others L. pneumophila),
  • Nocardiosis (oxygen actinomycetes of Nocardia genus).

28.7.3 Fungal diseases

Many potentially pathogenic airborne fungi or the so-called saprophytes live in soil. They usually have an ability to break down keratin (keratinolysis) – difficult to decompose proteins found in horny skin formations, e.g. human or animal hair, feathers, claws. Some of the keratinolytic fungi, the so-called dermatophytes, cause mycosis of the outer skin (dermatosis), as the break down of keratin enables them to penetrate the epidermis. Other fungi, after penetrating the respiratory system, cause deep mycosis (organ), e.g. attacking lungs. The following are examples of airborne fungi diseases:

  • Mycosis (Microsporum racemosum),
  • Deep mycosis: aspergillosis (Aspergillus fumigatus), cryptococcus (Cryptococcus neoformans).

28.7.4 Protozoan diseases

Some protozoa, which are able to produce cysts that are resistant to dehydration and solar radiation, may also infect humans by inhalation. The most common example of the above is Pneumocystis carinii which causes pneumonia. Dangers connected with pathogenic bioaerosols do not concern only human diseases. Other significant diseases are those that attack cultivated plants or farm animals. The following are examples of the above:

  • Blight – grain disease caused by Puccinia graminis, and
  • Aphthous fever – very infectious disease that attacks artiodactylous animals.

28.8 Basic Sources of Bioaerosol Emission
There are two basic sources of bioaerosol:

Natural sources: These are mainly soil and water, from which microorganisms are being lifted up by the movement of air, and from organisms such as fungi, that produce gigantic amounts of spores that are dispersed by the wind. Therefore, there are always a given number of microorganisms in the air, as a natural background. It is estimated, that the air is considered to be clean, if the concentration of bacteria and fungi cells does not exceed 1000/m3 and 3000/m3 respectively. This latter statement is only true when the concentration of microorganisms consists of saprophytic organisms, not pathogenic organisms. If the concentration of microorganisms in the air exceeds the above values, or contains microorganisms dangerous to humans, then such air is considered to be microbiologically polluted.

Human activities: From the hygienic point of view, living sources of bioaerosols related to human activity, are more important than the natural sources. The emissions from these sources are dangerous due to the following two reasons

  • They may distribute pathogenic microorganisms,
  • They often cause a high increase of microorganisms in the air, significantly exceeding the natural background.

The emission sources of biological aerosols can have a localized character (e.g. aeration tank) or a surface character (e.g. sewage-irrigated field).


The most important sources of bioaerosol emission are:

  • Agriculture and farming-food industry,
  • Sewage treatment plants,
  • Waste management.

28.9 Microbiology of Inside Air

Bacteria are microscopic organisms found in indoor environments typically come from human sources (skin and respiration) or from the outdoors. Like mold, most of the bacteria found in the air in buildings are saprobes meaning they grow on dead organic matter. As far as building envelopes are concerned the primary concern is about bacteria colonies that may grow in damp areas. Most of the bacteria are shed from human skin surfaces. It is not surprising to find hundreds of thousands of bacteria per gram of dust in carpets. As long as the bacterial types are a mixture of those listed below, there is generally no cause for concern. Bacteria may also enter with outdoor air or floodwater and grow in indoor environmental reservoirs. Common indoor reservoirs are water systems, air handling unit and wet organic material. Inadequately maintained air handling system is an important source for bacterial exposure that may lead to allergic type disease. Air handling system must be check for the contaminated water where chest tightness, cough, and fever are associated with a particular indoor environment.


28.9.1 The most abundant bacteria present include

28.9.1.1 Micrococcus sp

Micrococcus species are human shed bacteria and are caused by the normal shed of skin. It is found in areas of higher occupant density and/or inadequate ventilation. Micrococcus species are generally regarded as being harmless bacteria. Normally, these bacteria are removed through ventilation systems or cleaning procedures such as mopping or vacuuming.

28.9.1.2 Bacillus sp
Bacillus sp mainly associated with soil and dust. Appropriate temperature and moisture with deposited dust on hard surfaces allow for ideal growing conditions. Most are not serious pathogens.

28.9.1.3 Staphylococcus sp
Staphylococcus sp is an inhabitant and shed from of the skin surfaces. Among the Staphylococcus species that are commonly found indoors isStaphylococcus aureus, which is an important pathogen in hospital environments. It shouldn’t be a matter of concern unless it is the predominating colony found on air or surface samples in indoor environment.

28.9.1.4 Gram positive rod
Gram positive rod bacteria mainly associated with soil and dust. Appropriate temperature and moisture allow for ideal growing conditions on carpet, wall, furniture’s etc. Most are not serious pathogens. These bacteria can be removed by good house keeping practice and adequate ventilation systems.

28.9.1.5 Gram negative rod

These organisms are rare in indoor environments, if they found in higher concentration may be related to the bio aerosol of contaminated water or other contamination of wet/moist surfaces or materials, or possibly air handling units systems in which they are proliferating. Some Gram negative bacteria (or endotoxin extracted from their walls) have been shown to provoke symptoms of fever. Occasionally, growth in air handling units has been great enough for aerosols to be generated which contained sufficient allergenic cells to have caused the acute pneumonia like symptoms. If there has been a sewage spill or flood, then Gram negative bacteria are to be expected and such environments should be thoroughly cleaned with disinfectant.

Identification of bacteria by cultural analysis is based on morphology (e.g., spherical, rod-shaped, etc.), by staining reactions (e.g. Gram positive or negative) and by the pattern of results from a series of biochemical tests.

MICROBIOTA OF SOIL

26.1 Introduction 

Soil microbiology is branch of microbiology which deals with study of soil microorganisms and their activities in the soil. Soil is the outer, loose material of earth’s surface which is distinctly different from the underlying bedrock and the region which support plant life. Agriculturally, soil is the region which supports the plant life by providing mechanical support and nutrients required for growth. From the microbiologist view point, soil is one of the most dynamic sites of biological interactions in the nature. It is the region where most of the physical, biological and biochemical reactions related to decomposition of organic weathering of parent rock take place. 

26.2 Components of Soil
Soil is an admixture of five major components viz. organic matter, mineral matter, soil-air, soil water and soil microorganisms/living organisms. The amount/proposition of these components vary with locality and climate.


26.2.1 Mineral/Inorganic matter

It is derived from parent rocks/bed rocks through decomposition, disintegration and weathering process. Different types of inorganic compounds containing various minerals are present in soil. Amongst them the dominant minerals are Silicon, Aluminum and iron and others like Carbon, Calcium Potassium, Manganese, Sodium, Sulphur, Phosphorus etc. are in trace amount. The proportion of mineral matter in soil is slightly less than half of the total volume of the soil. 


26.2.2 Organic matter (components)

These are derived from organic residues of plants and animals added in the soil. Organic matter serves not only as a source of food for microorganisms but also supplies energy for the vital processes of metabolism which are characteristics of all living organisms. Organic matter in the soil is the potential source of N, P and S for plant growth. Microbial decomposition of organic matter releases the unavailable nutrients in available from. The proportion of organic matter in the soil ranges from 3-6% of the total volume of soil.

26.2.3 Soil water

The amount of water present in soil varies considerably. Soil water comes from rain, snow, dew or irrigation. Soil water serves as a solvent and carrier of nutrients for the plant growth. The microorganisms inhabiting in the soil also require water for their metabolic activities. Soil water thus, indirectly affects plant growth through its effects on soil and microorganisms. Percentage of soil-water is 25% total volume of soil. 


26.2.4 Soil air (Soil gases)

A part of the soil volume which is not occupied by soil particles i.e. pore spaces are filled partly with soil water and partly with soil air (Fig.26.2). Most microbes are in micro colonies on soil particle. They may escape predator activities by refuge in these small pores. These two components (water and air) together, accounts for approximately half the soil’s volume. Compared with atmospheric air, soil is lower in oxygen and higher in carbon dioxide, because CO2 is continuous recycled by the microorganisms during the process of decomposition of organic matter. Soil air comes from external atmosphere and contains nitrogen, oxygen, CO2 and water vapor (CO2 > oxygen). CO2 in soil air (0.3-1.0%) is more than atmospheric air (0.03%). Soil aeration plays important role in plant growth, microbial population, and microbial activities in the soil.


26.2.5 Soil microorganisms

Soil is an excellent culture media for the growth and development of various microorganisms. Soil is not an inert static material but a medium pulsating with life. Soil is now believed to be dynamic or living system. Soil contains several distinct groups of microorganisms and amongst them bacteria, fungi, actinomycetes, algae, protozoa and viruses are the most important. But bacteria are more numerous than any other kinds of microorganisms. Microorganisms form a very small fraction of the soil mass and occupy a volume of less than one percent. In the upper layer of soil (top soil up to 10-30 cm depth i.e. Horizon A), the microbial population is very high which decreases with depth of soil. Each organisms or a group of organisms are responsible for a specific change transformation in the soil. The final effect of various activities of microorganisms in the soil is to make the soil fit for the growth and development of higher plants. 


26.3 Types of Microorganisms in Soil

Living organisms both plants and animals, constitute an important component of soil. The pioneering investigations of a number of early microbiologists showed for the first time that the soil was not an inert static material but a medium pulsating with life. The soil is now believed to be a dynamic or rather a living system, containing a dynamic population of microorganisms. Cultivated soil has relatively more population of microorganisms than the fallow land, and the soils rich in organic matter contain much more population than sandy and eroded soils. Microbes in the soil are important to us in maintaining soil fertility/productivity, cycling of nutrient elements in the biosphere and sources of industrial products such as enzymes, antibiotics, vitamins, hormones, organic acids etc. At the same time certain soil microbes are the causal agents of human and plant diseases.

The soil organisms are broadly classified in to two groups viz. soil flora and soil fauna, the detailed classification of which is as follows.

26.3.1 Soil flora

a) Microflora

  • Bacteria
  • Fungi, Molds, Yeast, Mushroom
  • Actinomycetes, Streptomyces
  • Algae e.g. BGA, Yellow Green algae, Golden brown algae.

Bacteria is again classified in

I) Heterotrophic e.g. symbiotic and non – symbiotic N2 fixers, Ammonifier, Cellulose Decomposers, Denitrifiers
II) Autrotrophic e.g. Nitrosomonas, Nitrobacter, Sulphur oxidizers, etc.

b) Macroflora

  1. Microfauna: Protozoa, Nematodes
  2. Macrofauna: Earthworms, moles, ants and others. As soil inhabit several diverse groups of microorganisms, but the most important amongst them are: bacteria, actinomycetes, fungi, algae and protozoa.

Relative proportion/percentage of various soil microorganisms are: bacteria – aerobic (70%), anaerobic (13 %), actinomycetes (13%), fungi/molds (3%) and others (algae, protozoa, viruses) 0.2-0.8 %.
Major groups of microorganisms are discussed in following sections:

26.4 Bacteria


Amongst the different microorganisms inhabiting in the soil, bacteria are the most abundant and predominant organisms. These are primitive, prokaryotic, microscopic and unicellular microorganisms without chlorophyll. Morphologically, soil bacteria are divided into three groups viz. cocci (round/spherical), bacilli (rod-shaped) and spirilla (cells with long wavy chains). Bacilli are most numerous followed by cocci and spirilla in soil. The most common method used for isolation of soil bacteria is the ‘dilution plate count’ method which allows the enumeration of only living cells in the soil. The size of soil bacteria varies from 0.5 to 1.0 µ in diameter and 1.0 to 10.0 µ in length. They are motile with locomotory organs flagella. Bacterial population is one-half of the total microbial biomass in the soil ranging from 1,00000 to several hundred millions per gram of soil, depending upon the physical, chemical and biological conditions of the soil.

Winogradsky (1925), on the basis of ecological characteristics classified soil microorganisms in general and bacteria in particular into two broad categories i.e. autochnotus (Indigenous species) and the zymogenous (fermentative). Autochnotus bacterial population is uniform and constant in soil, since their nutrition is derived from native soil organic matter (e.g. Arthrobacter and Nocardia, whereas zymogenous bacterial population in soil is low, as they require an external source of energy, e.g. Pseudomonas and bacillus. The population of zymogenous bacteria increases gradually when a specific substrate is added to the soil. To this category belong the cellulose decomposers, nitrogen utilizing bacteria and ammonifiers. As per the system proposed in the ‘Bergey’s Manual of Systematic Bacteriology’, most of the bacteria which are predominantly encountered in soil are taxonomically included in the three orders, Pseudomonadales, Eubacteriales and Actinomycetales of the class Schizomycetes. The most common soil bacteria belong to the genera Pseudomonas, Arthrobacter, Clostridium Achromobacter, Sarcina, Enterobacter etc. The another group of bacteria common in soils is the Myxobacteria belonging to the genera Micrococcus, Chondrococcus, Archangium, Polyangium, Cyptophaga.

Bacteria are also classified on the basis of physiological activity or mode of nutrition, especially the manner in which they obtain their carbon, nitrogen, energy and other nutrient requirements. 

They are broadly divided into two groups i.e. a) Autotrophs and b) Heterotrophs 

  • Autotrophic bacteria are capable synthesizing their food from simple inorganic nutrients, while heterotrophic bacteria depend on pre-formed food for nutrition. All autotrophic bacteria utilize CO2 (from atmosphere) as carbon source and derive energy either from sunlight (photoautotrophs, e.g. Chromatrum. Chlorobium. Rhadopseudomonas or from the oxidation of simple inorganic substances present in soil (chemoautotrophs e.g. Nitrobacter, Nitrosomonas, Thiaobacillus).
  • Majority of soil bacteria are heterotrophic in nature and derive their carbon and energy from complex organic substances/organic matter, decaying roots and plant residues. They obtain their nitrogen from nitrates and ammonia compounds (proteins) present in soil and other nutrients from soil or from the decomposing organic matter. Certain bacteria also require amino acids, B-vitamins, and other growth promoting substances also.

26.4.1 Functions/role of bacteria

Bacteria bring about a number of changes and biochemical transformations in the soil and thereby directly or indirectly help in the nutrition of higher plants growing in the soil. The important transformations and processes in which soil bacteria play vital role are: decomposition of cellulose and other carbohydrates, ammonification (proteins ammonia), nitrification (ammonia-nitrites-nitrates), denitrification (release of free elemental nitrogen), biological fixation of atmospheric nitrogen (symbiotic and non-symbiotic) oxidation and reduction of sulphur and iron compounds. All these processes play a significant role in plant nutrition.

26.5 Actinomycetes

These are the organisms with characteristics common to both bacteria and fungi but yet possessing distinctive features to delimit them into a distinct category. In the strict taxonomic sense, actinomycetes are clubbed with bacteria the same class of Schizomycetes and confined to the order Actinomycetales. They are unicellular like bacteria, but produce a mycelium which is non-septate (coenocytic) and more slender, tike true bacteria they do not have distinct cell-wall and their cell wall is without chitin and cellulose (commonly found in the cell wall of fungi). On culture media unlike slimy distinct colonies of true bacteria which grow quickly, actinomycetes colonies grow slowly, show powdery consistency and stick firmly to agar surface. They produce hyphae and conidia/sporangia like fungi. Certain actinomycetes whose hyphae undergo segmentation resemble bacteria, both morphologically and physiologically.

Actinomycetes are numerous and widely distributed in soil and are next to bacteria in abundance. They are widely distributed in the soil, compost etc. Plate count estimates give values ranging from 104 to 108 per gram of soil. They are sensitive to acidity/low pH (optimum pH range 6.5 to 8.0) and waterlogged soil conditions. The population of actinomycetes increases with depth of soil even up to horizon ‘C’ of a soil profiler They are heterotrophic, aerobic and mesophilic (25-30°C) organisms and some species are commonly present in compost and manures are thermophilic growing at 55-65°C temperature (e.g. Thermoatinomycetes, Streptomyces). Actinomycetes belonging to the order of Actinomycetales are grouped under four families viz. Mycobacteriaceae, Actinomycetaceae, Streptomycetaceae and Actinoplanaceae. Actinomycetous genera, which are agriculturally and industrially important, are present in only two families of Actinomycetaceae and Strepotmycetaceae. In the order of abundance in soils, the common genera of actinomycetes are Streptomyces (nearly 70%), Nocardia and Micromonospora although Actinomycetes, Actinoplanes, Micromonospora and Streptosporangium are also generally encountered.

26.5.1 Functions of actinomycetes

  • Degrade all sorts of organic substances like cellulose, polysaccharides, protein fats, organic-acids etc.
  • Soil, added with organic residues/substances, is first attacked by bacteria and fungi and later by actinomycetes, because they are slow in activity and growth than bacteria and fungi.
  • They decompose/degrade the more resistant and indecomposable organic substance/matter and produce a number of dark black to brown pigments which contribute to the dark color of soil humus.
  • They are also responsible for subsequent further decomposition of humus (resistant material) in soil.
  • They are responsible for earthy/musty odor/smell of freshly ploughed soils.
  • Many genera species and strains (e.g. Streptomyces, if actinomycetes synthesize number of antibiotics like Streptomycin, Terramycin, Aureomycin etc.
  • One of the species of actinomycetes, Streptomyces scabies causes disease ‘potato scab’ in potato.

26.6 Fungi 
Fungi in soil are present as mycelial bits, rhizomorph or as different spores. Their number varies from a few thousand to a few million per gram of soil. Soil fungi possess filamentous mycelium composed of individual hyphae. The fungal hyphae may be aseptate/coenocytic (Mastigomycotina and Zygomycotina) or septate (Ascomycotina, Basidiomycotina and Deuteromycotina). As observed by C. K. Jackson (1975), most commonly encountered genera of fungi in soil are; Alternaria, Aspergillus, Cladosporium, Cephalosporium Botrytis, Chaetomium, Fusarium, Mucor, Penicillium, Verticillium, Trichoderma, Rhizopus, Gliocladium, Monilia, Pythium, etc. Most of these fungal genera belong to the subdivision deuteromycotina/fungi imperfeacta which lacks sexual mode of reproduction. As these soil fungi are aerobic and heterotrophic, they require abundant supply of oxygen and organic matter in soil. Fungi are dominant in acid soils, because acidic environment is not suitable for the existence of either bacteria or actinomycetes. The optimum pH range for fungi lies-between 4.5 to 6.5. They are also present in neutral and alkaline soils and some can even tolerate pH beyond 9.0

26.6.1 Functions/role of fungi

  • Fungi plays significant role in soils and plant nutrition.
  • They plays important role in the degradation/decomposition of cellulose, hemi cellulose, starch, pectin, lignin in the organic
  • Matter added to the soil.
  • Lignin which is resistant to decomposition by bacteria is mainly decomposed by fungi.
  • They also serve as food for bacteria.
  • Certain fungi belonging to sub-division Zygomycotina and Deuteromycotina are predaceous in nature and attack on protozoa and nematodes in soil and thus, maintain biological equilibrium in soil.
  • They also plays important role in soil aggregation and in the formation of humus.
  • Some soil fungi are parasitic and causes number of plant diseases such as wilts, root rots, damping-off and seedling blights e.g. Pythium, Phyiophlhora, Fusarium, Verticillium etc.
  • Number of soil fungi forms mycorrhizal association with the roots of higher plants (symbiotic association of a fungus with the roots of a higher plant) and helps in mobilization of soil phosphorus and nitrogen e.g. Glomus, Gigaspora, Aculospora, (Endomycorrhiza) and Amanita, Boletus, Entoloma, Lactarius (Ectomycorrhiza).

26.7 Algae
Algae are present in most of the soils where moisture and sunlight are available. Their number in soil usually ranges from 100 to 10,000 per gram of soil. They are photoautotrophic, aerobic organisms and obtain CO2 from atmosphere and energy from sunlight and synthesize their own food. They are unicellular, filamentous or colonial. Soil algae are divided in to four main classes or phyla as follows:

  • Cyanophyta (Blue-green algae)
  • Chlorophyta (Grass-green algae)
  • Xanthophyta (Yellow-green algae)
  • Bacillariophyta (diatoms or golden-brown algae)

Out of these four classes/phyla, blue-green algae and grass-green algae are more abundant in soil. The green-grass algae and diatoms are dominant in the soils of temperate region while blue-green algae predominate in tropical soils. Green-algae prefer acid soils while blue green algae are commonly found in neutral and alkaline soils. The most common genera of green algae found in soil are: Chlorella, Chlamydomonas, Chlorococcum, Protosiphon etc. and that of diatoms are Navicula, Pinnularia. Synedra, Frangilaria. Blue green algae are unicellular, photoautotrophic prokaryotes containing Phycocyanin pigment in addition to chlorophyll. They do not posses flagella and do not reproduce sexually. They are common in neutral to alkaline soils. The dominant genera of BGA in soil are: Chrococcus, Phormidium, Anabaena, Aphanocapra, Oscillatoria etc. Some BGA posses specialized cells known as ‘Heterocyst’ which is the sites of nitrogen fixation. BGA fixes nitrogen (non-symbiotically) in puddle paddy/water logged paddy fields (20-30 kg/ha/season). There are certain BGA which possess the character of symbiotic nitrogen fixation in association with other organisms like fungi, mosses, liverworts and aquatic ferns Azolla, e.g. Anabaena-Azolla association fix nitrogen symbiotically in rice fields.

26.7.1 Functions/role of algae or BGA 

  • Plays important role in the maintenance of soil fertility especially in tropical soils.
  • Add organic matter to soil when die and thus increase the amount of organic carbon in soil.
  • Most of soil algae (especially BGA) act as cementing agent in binding soil particles and thereby reduce/prevent soil erosion.
  • Mucilage secreted by the BGA is hygroscopic in nature and thus helps in increasing water retention capacity of soil for longer time/period.
  • Soil algae through the process of photosynthesis liberate large quantity of oxygen in the soil environment and thus facilitate the aeration in submerged soils or oxygenate the soil environment.
  • They help in checking the loss of nitrates through leaching and drainage especially in un-cropped soils.
  • They help in weathering of rocks and building up of soil structure.

26.8 Protozoa
These are unicellular, eukaryotic, colorless, and animal like organisms (Animal kingdom). They are larger than bacteria and size varying from few microns to a few centimeters. Their population in arable soil ranges from l0,000 to 1,00,000 per gram of soil and are abundant in surface soil. They can withstand adverse soil conditions as they are characterized by ‘cyst stage’ in their life cycle. Except few genera which reproduce sexually by fusion of cells, rest of them reproduces asexually by fission/binary fission. Most of the soil protozoa are motile by flagella or cilia or pseudopodia as locomotors organs. Depending upon the type of appendages provided for locomotion, protozoa are 

  • Rhizopoda (Sarcondia)
  • Mastigophora
  • Ciliophora (Ciliata)
  • Sporophora (not common inhabitants of soil)

Rhizopoda consists protozoa without appendages usually have naked protoplasm without cell-wall, pseudopodia as temporary locomotory organs are present some times. Important genera are Amoeba, Biomyxa, Euglypha, etc. 

Mastigophora Belongs flagellated protozoa, which are predominant in soil. Important genera are: Allention, Bodo, Cercobodo, Cercomonas, Entosiphon Spiromonas, Spongomions and Testramitus. Many members are saprophytic and some posses chlorophyll and are autotrophic in nature. In this respect, they resemble unicellular algae and hence are known as ‘Phytoflagellates’.

Ciliophora are characterized by the presence of cilia (short hair-like appendages) around their body, which helps in locomotion. The important soil inhabitants of this class are Colpidium, Colpoda, Balantiophorus, Gastrostyla, Halteria, Uroleptus, Vortiicella, Pleurotricha etc.

Protozoa are abundant in the upper layer (15 cm) of soil. Organic manures protozoa. Soil moisture, aeration, temperature and pH are the important factors affecting soil protozoa.


26.8.1 Function/role of protozoa

  • Most of protozoans derive their nutrition by feeding or ingesting soil bacteria belonging to the genera Enterobacter, Agrobacterium, Bacillus, Escherichia, Micrococcus, and Pseudomonas and thus, they play important role in maintaining microbial/bacterial equilibrium in the soil.
  • Some protozoa have been recently used as biological control agents against phytopathogens.
  • Species of the bacterial genera viz. Enterobacter and Aerobacter are commonly used as the food base for isolation and enumeration of soil protozoans.
  • Several soil protozoa cause diseases in human beings which are carried through water and other vectors, e.g. Amoebic dysentery caused by Entomobea histolytica.

ENUMERATION OF MICROORGANISMS IN AIR, CONTROL OF AIRBORNE MICROORGANISM


29.1 Enumeration of Microorganisms in Air

Various methods commonly applied for enumeration and detection of microorganisms can be subdivided into:

  • Microscopic methods
  • Culture methods
  • Combination of both

29.2 Microscopic Methods

These consist of

  • Letting air through a membrane filter or placing a glass coated with a sticky substance (e.g. vaseline), in the path of air
  • Staining of the trapped microorganisms and
  • Microscopic testing consisting of cell counting

Staining with acridine orange and examination under a fluorescence microscope is often applied. The final result is given as a total number of microbes in 1 mof air. The advantage of this method is that it allows the detection of live and dead microbes in air, as well as those, which do not abundantly flourish in culture media. Due to this, the number of microbes determined is usually higher by one order of magnitude than in culture methods. In addition, it is possible to detect and identify other biological agents e.g. plant pollen, allergenic mites, abiotic organic dust (fragments of skin, feathers, plants, etc.).

However the methods have a serious drawback: inability to determine the species of microbes (bacteria, fungi, viruses).


29.3 Culture Methods

These methods consist of transferring microbes from air onto the surface of the appropriate culture medium. After a period of incubation at optimal temperature, the formed colonies are counted and the result is given as cfu/m3 of air (colony forming units). Because a colony can form not only from a single cell, but also from a cluster of cells, the air may contain more microbes than suggested by the CFU result. Besides, the method allows the detection of only the cells that are viable and those which are able to grow upon the medium used. Microbes transferred to the culture medium require resuscitation as they were subjected to the influence of unfavourable conditions. Therefore it is recommended to supplement the culture mediums are required to be supplemented with components such as betaine and catalase. Betaine, the methylic derivative of the glycine amino acid, is utilized by bacteria to maintain osmotic balance, and as a donor of methylic groups it is essential during the processes of biosynthesis. Catalase however breaks down harmful peroxides created in air as a result of UV radiation.


However, testing of viruses differs significantly from the methods utilized for other organisms because:

  • They may develop only in living cells, therefore they require tissue cultures (e.g. the epithelium of human trachea or monkey’s kidney) or, in the case of bacteriophages, bacterial cultures,
  • Species identification of detected viruses is meticulous and, among other things, consists of performing electrophoresis or utilizing antiserum that contains antibodies of common viruses,
  • Drawing large quantities of air is essential (over 1000 dm3, at least one order of magnitude higher than in the case of bacteria), as the amount of viruses in air is rather small (this especially concerns the enteroviruses).

After transferring the viruses onto the surface of a single-layer culture, the viruses penetrate the cells, reproduce in them, and after their destruction attack the neighboring cells. Consequently, the areas around the initial places of the cell infections get cleared of cells – this clearing is called plaques. Therefore, the number of viruses detected is given as the number of units that form the plaques, in short pfu/m3(plaque forming units). It has to be pointed out though, that such a method only allows the detection of viruses capable of infecting the utilized cells.


29.4 Sampling of Air 
There are four basic ways of sampling the air for use in culture methods:

  • Koch’s sedimentation method
  • Filtration method (also used in microscopic methods)
  • Centrifugation
  • Impact methods

29.5 Sedimentation Method

This ‘Settling Plate Technique’ based on this approach is the simplest and is often used by air microbiologists. The principle behind this method is that the bacteria carrying particles are allowed to settle onto the medium for a given period of time and incubated at the required temperature. A count of colonies formed shows the number of settled bacteria containing particles. In this method petridishes containing an agar medium of known surface area are selected so that the agar surface is dry without any moisture. Choice of the medium depends upon the kind of microorganisms to be enumerated. For an overall count of pathogenic, commensal and saprophytic bacteria in air blood agar can be used. For detecting a particular pathogen which may be present in only small numbers, an appropriate selective medium may be used. Malt extract agar can be used for molds. The plates are labeled appropriately about the place and time of sampling, duration of exposure etc. Then the plates are uncovered in the selected position for the required period of time. A Petri dish containing agar medium is kept covered and, at the time of sampling, the cover is removed from the Petri dish so that the agar surfaces is exposed to air for a few minutes. The Petri dish is now incubated. One can see a certain number of colonies developing on agar medium (Fig. 29.1). Each colony represents a particle carrying microorganisms which has fallen on the agar surface. The optimal duration of exposure should give a significant and readily countable number of well isolated colonies, for example about 30-100 colonies. Usually it depends on the dustiness of air being sampled. In occupied rooms and hospital wards the time would generally be between 10 to 60 m. During sampling it is better to keep the plates about I metre above the ground. Immediately after exposure for the given period of time, the plates are closed with the lids. Then the plates are incubated for 24 hrs at 37°C for aerobic bacteria and for 3 days at 22°C for saprophytic bacteria. For molds incubation temperature varies from 10-50°C for 1-2 weeks. After incubation the colonies on each plate are counted and recorded as the number of bacteria carrying particles settling on a given area in a given period of time.

The use of settle plates is not recommended when sampling air for fungal spores, because single spores can remain suspended in air indefinitely. Settle plates have been used mainly to sample for particulates and bacteria either in research studies or during epidemiologic investigations. Results of sedimentation sampling are typically expressed as numbers of viable particles or viable bacteria per unit area per the duration of sampling time (i.e. CFU/area/time); this method can not quantify the volume of air sampled. Because the survival of microorganisms during air sampling is inversely proportional to the velocity at which the air is taken into the sampler, one advantage of using a settle plate is its reliance on gravity to bring organisms and particles into contact with its surface, thus enhancing the potential for optimal survival of collected organisms. This process, however, takes several hours to complete and may be impractical for some situations.


29.5.1 Limitation
Though the method has the advantage of simplicity, it has certain limits. 

  • In this method only the rate of deposition of large particles from the air, not the total number of bacteria carrying particles per volume, is measured.
  • Growth of bacteria in the settled particles may be affected by the medium used since not all microorganisms are growing well on all media.
  • Moreover since air currents and any temporary disturbances in the sampling area can affect the count, many plates have to be used.
  • Since only particles of certain dimensions tend to settle on to the agar surface and, also, the volume of air entering inside the Petri dish is not known, this technique gives only a rough estimate and can be used only to isolate air-borne microorganisms.
  • However, one can gather information about the kind of air-borne microbes occurring in a particular area by repeated use of settling plate technique for a fixed period of time.

29.6 Filtration Methods

The methods consist of using an aspirator to suck in a given volume of air, passing it through a sterile absorbing substance (liquid or solid) and transferring the filtered microbes onto the appropriate culture medium. After a pre-determined time of incubation the resulting colonies are counted. Most often, a membrane filter or a physiological solution (0.85% NaCl) is utilized for the filtration of air. Filtration using liquids (sometimes classified as the impact method) is one of the most often used and highly valued techniques of sampling bioaerosol (Fig. 29.2). It results in high output of microbe isolation as well as significant survival of the filtered microbes. The method may be utilized in virus testing as long as the remaining microbes are neutralized (e.g. with chloroform) and the liquid is concentrated before its introduction into the cell culture.

The filtration process through membrane filters allows the utilization of both culture methods (filters containing microbes are placed directly upon the culture media or are rinsed and then inoculated) as well as the microscopic methods (filters are stained and observed under a microscope).

These are simple methods for collecting particles from air. The filter can be made of any fibrous or granular material like sand, glass fibre and alginate wool (in phosphate buffer). However, recovery of organisms for culture is not so easy. The membrane filter devices are adaptable to direct collection of microorganisms by filtration of air. These methods are also rather inexpensive and not complicated; they possess two significant advantages over the sedimentation methods:

  • The volume of the air tested is known,
  • It is possible to detect the very small aerosol that creates the respiratory fraction (nevertheless it is still impossible to determine its size

29.6.1 Tube sampler
This is one of the oldest devices for collecting and enumerating microorganisms in the air. It consists of a tube with an inlet at the top and an outlet at the bottom which is narrower than the top end. Near the bottom there is a filter of wet sand which is supported by a cotton plug below. The entire device can be sterilized. After sterilization the air to be sampled is allowed to pass through the sand and cotton. Microorganisms as well as dust particles containing microorganisms in the air are deposited in the sand filter as the air passes through it. Later the sand is washed with broth and a plate count is made from the broth by taking aliquotes of the broth.


29.6.2 Millipore filter

This type of filters is made of pure and biologically inert cellulose ethers. They are prepared as thin porous, circular membranes of about 150 µm thickness. The filters have different porosity. The assemblage contains a funnel shaped inlet and a tube like outlet. In between these two the filter is fitted. The outlet may be connected to a vacuum pump to suck known amount of air. After collecting required volume of air through the filter, it can directly be placed onto the surface of a solid medium. After incubation colonies formed can be counted.

However, the disadvantage of this method is that it has a significantly low output as the process of passing the air through pores of the filter creates resistance. That’s why the method is not recommended for microbe testing, but is routinely put to use in detection of endotoxins in air.


29.7 Centrifugation Methods

29.7.1 Air centrifuge

The first primitive type of air centrifuge was developed by Wells in 1993. The principle of air centrifuge is that the particles from air are centrifuged onto the culture medium. In his air centrifuge sampled air was passed along a tube which was rotated rapidly on its long axis. The inner surface of the tube was lined with culture medium and any bacteria containing particle deposited on it grew into a colony on incubation. A modern version of this centrifuge is the Reuter centrifugal air sampler, which is portable and battery powered. It resembles a large cylindrical torch with an open ended drum at one end. The drum encloses impeller blades which can be rotated by battery power when switched on. A plastic strip coated with culture medium can be inserted along the inner side of the drum. Air is drawn into the drum and subjected to centrifugal acceleration. This causes the suspended particles to impact on the culture medium. After sampling the strip is removed from the instrument and incubated at 37°C for 48 h. Later the colonies can be counted. Advantage of this sampler is that it is very convenient for transportation and use. However, the disadvantage is that it is less efficient than the slit sampler in detecting particle below 5 mm in diameter. More over the size of the air being sampled cannot be accurately controlled. 


29.7.2 Impact methods

These methods consist of using an aspirator to suck in a pre-determined amount (volume) of air, which collides with the nutrient agar at high speed. It causes the microbes in the air to stick to the surface, which after a specific time of incubation, form colonies. The impact methods are the most highly valued and most often used methods of detecting microbes in air. Their biggest advantage is the possibility of detecting and determining the respiratory fraction of the bioaerosol, in other words, determining the size distribution of its particles. The methods can be utilized to test viruses (trapped microbes are swept from the surface of the culture medium and, after the elimination of other microbes with chloroform, introduced into the cell culture).

A disadvantage for the impact method is a decline in the microbes viability caused by the shock of a sudden collision with nutrient agar and also a possibility of the nutrient culture getting overgrown in cases of high air pollution. The above stated methods are usually not cheap. The most widely known device that is based on the impact technique is the Andersen’s apparatus, in which the air is drawn in passes through six vertically positioned sieves. A petri dish with nutrient agar is placed underneath each sieve. The speed of the passing air increases as it passes through the consecutive sieves, consequently causing greater impact force as it collides with the sieves. As a result, the heaviest (largest) particles settle upon the first sieve, whereas the lighter (smaller) ones are drawn in by the current of the passing air. As they pass through the consecutive sieves, the increasingly smaller and faster particles collide with the nutrient agar. Consequently the particles of the biological aerosol are sorted according to their size and the colonies are then derived from particles of particular size. This way, by counting the colonies upon the consecutive plates, it is possible to determine the ratio of particles which settle in the upper (higher positioned plates) and lower respiratory system (lower plates).


29.7.2.1 Sampling of measured volume of air

An improvised method wherein a measured volume of air is sampled has also been developed (Fig. 29.3). These are sieve and slit type devices. A sieve device has a large number of small holes in a metal cover, under which is located a petridish containing an agar medium. A measured volume of air is drawn, through these small holes. Airborne particles impinge upon the agar surface. The plates are incubated and the colonies counted. In a slit device the air is drawn through a very narrow slit onto a petridish containing agar medium. The slit is approximately the length of the petridish. The petridish is rotated at a particular speed under the slit. One complete turn is made during the sampling operation


29.7.2.2 Selection of air sampler

The following factors must be considered when choosing an air sampling instrument:

  • Viability and type of the organism to be sampled
  • Compatibility with the selected method of analysis
  • Sensitivity of particles to sampling
  • Assumed concentrations and particle size
  • Whether airborne clumps must be broken (i.e. total viable organism count vs. particle count)
  • Volume of air to be sampled and length of time sampler is to be continuously operated
  • Background contamination
  • Ambient conditions
  • Sampler collection efficiency
  • Effort and skill required to operate sampler
  • Availability and cost of sampler, plus back-up samplers in case of equipment malfunction
  • Availability of auxiliary equipment and utilities (e.g. vacuum pumps, electricity, and water)

Manoj

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