University of Baghdad Lecture: 1

College of Science Level: 4th

Soil Microbiology Lecturer: Dr. Shayma

Soil Microbiology
It is branch of science/ microbiology which deal with the study of soil
microorganisms and their activities in the soil.
 Soil: Is crucial for sustaining life, like water and air, because it is the source of
most of our food.
 Soil can be defined as; the outer region of earth crust, consist of loose
material formed by series of various processes called soil – forming factors
(SFF), include:
Topography of land,
the organisms present in the environment,
the climate under which the soil was formed,
the parent material or the original minerals that give rise to the soil,
and the time that all of these processes have been occurring.

Soil Profile
Soil profile refer to the layers of soil develops over a long period of time, the soil
profile consist of horizons, each with a distinctive features.
The following horizons are listed by their position from the top to the bottom:

1. Horizon A ( Top Soil )

 Surface layer, is the top about 25cm of soil profile, with a darker in color than
deeper layers and contain highest percentage of organic matter accumulation,
this horizon also known as the biomantle because most biological activity
occurs.
 The layer was likely formed from decomposing plant and mineral materials.
 It has a large amount of sand, and less clay.

2. Horizon B ( Subsoil)

 This horizon is found from 25 – 40cm, has a lighter color than the horizon A,
with increase in clay and mineral salts such as deposits of silicates or
aluminum that wash down through the top soil to create this layer, a process
referred to as illuviated zone.
 It also contains less microbial population, but some biological activity
extends into this layer.
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 3. Horizon C (Parent Soil)

 This third horizon has gray mottles, or patches of gray colors throughout the
soil matrix.
 With strong increase in clay percentage, it's thickness of 45cm and more may
it reach the rocky layer.
 The layer indicated with the absence of organic matter and microbial
activities.

4. Horizon D ( Rocky bed )

 This layer is found from 75cm and beyond, represent the parent material sitting
on bedrock, may be weathered to form part of soil profile.

Soil Texture and Soil Structure

 Soil texture and soil structure are both unique properties of the soil that have
a profound effect on the behavior of soil, such as water holding capacity,
nutrient retention and supply, drainage, and nutrient leaching.
 The combination influence of soil texture and structure may be best described
by the term "Soil bulk density", is a measure of percent pore space and solid
in soil.

Soil texture: referred to the proportion and distribution of mineral particles, sand,
silt, and clay present. The texture of soil can be determined when the percentage of
these three soil constituents are known. Table 1.1 illustrated soil particles size.

Table 1.1 Soil Texture Particles size

Type of Soil Particles Size range/mm

Sand 2.0 – 0.05

Silt 0.05 – 0.002
Clay Less than o.oo2

 Physicochemical feature of soil depends mainly on its texture, because it

influences plants growth by its direct effects on soil, aeration, water
infiltration, and cations exchange capacity (CEC).
 Sand, has large particle size with small surface area in comparison with
same mass of silt and clay, in accordance to this little surface area the sand
proportion of any soil has an importance as organizing skeleton for other
constituents, in case of reasonable sand proportion the soil has enough pores
which facilitate aeration and water drainage. Sandy soils are less productive
than other.
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  Silt, produced from fragmentation of rocks, also has small surface area,
but in this character it's larger than those of sand particles, with smooth
appearance look like cosmetic powders, and with low adherence capacity.
 Clay, fraction made up tiny particles, despite their small size the particles
have very large surface area relative to their volume, may be more than
thousand times the total surface area of sand particles with same mass,
resulting from the plate _ like shape of individual particles. The increase in
surface area is highly reactive and has the ability to attract and hold
positively charged nutrient ions, clay particles are somewhat flexible and
plastic because of their lattice – like design, this feature allow clay particles to
absorb water and provides many places for soil particle to retain and supply
nutrients, soil containing clay is the most productive and use fertilizers most
effectively.

Textural class a grouping of soils based upon this relative proportion.

 Soil with the finest texture is called clay soil, while soil with coarsest
texture is called sands. However, a soil that has a relatively even mixture of
sand, silt, and clay and exhibits the properties from each separate is called
loam.
There are different types of loams, based upon which soil separate is most
abundantly present. Once the sand, silt, and clay percentage of a soil are, known, the
textural class can be read from the textural triangle used to determine the soil type
(Figure 1.1)

Figure 1.1 Soil texture triangle for determination of soil

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  Soil structure; is the arrangement of soil particles into grouping. These
grouping are called pads or aggregates, which often form distinctive shapes
typically found within certain soil horizons. For example, granular soil
particles are characteristic of surface horizon.
 Soil aggregation is an important indicator of the workability of the soil. The
various types of soil structures are provided in Figure 1.2.

Figure 1.2 Types of soil structures

Soil Composition
 Soils are made up of four basic components: mineral particles, air, water,
and organic matter.
 Solid materials ( organic and inorganic ) , which represent around half of
soil content and spaces filled with air and water, each of them are important to
life, four basic components: minerals, air , water, and organic matter (Fig. 1.3).

(Figure 1.3) Soil composition

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Inorganic materials
 In most soil types inorganic materials (mineral particles) represent about
45% of total soil volume.
 Mineral portion consist of three distinct soil particles sand, silt, and clay
Particles.
 Most soil influential particles are clay, it play a significant role in
determining the availability of nutrient and water to different life forms.
 Clay particles are negatively charged, due to exchange of SiO2 and Al3+
Al3+ + SiO2 AlO 2- + Si 4+
 Net particles charge depends on soil microorganisms metabolic activities and
pH of soil solution.

Water and Air

 Soil particles pack loosely, forming a soil structure filled with pore spaces,
these pores contain soil solution (water) and gas (air).
 Water and air in soil vary significantly with soil texture, weather, and plants
uptake of water, but their percentage together in most of soil types is about 50
% of total soil volume.
 Soil pore space doesn't change, but after rain the soil pore space will have a
high percentage of of water in relation to air.

Soil water
 Comes from rain, snow, dew or irrigation.
 Soil water serve as solvent and a carrier of nutrient for 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 about 25 % total volume of soil.

Soil water amount affected by many factors:

1. Porosity:
 Soil porosity refers to the space between soil particles, which consists of
various amounts of water and air, porosity depends on both soil texture and
structure, for example, a fine soil has small but numerous pores than coarse
soil.
 A coarse soil has bigger particles than a fine soil, but it has less porosity.
Water can be held tighter in small pores than in large one, so fine soils can
hold more water than coarse soil.
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2. Infiltration:
 Water infiltration refers to the movement of water from soil surface to the
soil profile.
 Soil texture – structure, slope, and gravitation has the largest impact on
filtration rate, water move by gravity into the open pore space in the soil, the
size of soil particles and their spacing determines how much water can flow in.

3. Permeability:
 Soil permeability refers to the movement of air and water through the soil,
which is important because it affects the supply of root – zoon.
 Water holding capacity is controlled by permeability, by the combination
effects of soil texture and organic matter, soil with smaller particle (silt and
clay) have large surface area than those with large sand particles, so the first
one has a high water holding capacity and allow a soil to hold more water than
the second type.
Soil air
 Apart of soil pores which not occupied with water are filled with air.
 Compared with atmospheric air, soil is lower in oxygen and higher in
carbon dioxide, because CO2 is continuous recycled by microorganisms
during the process of decomposition of organic matter.
 Soil air comes from external atmosphere and contains nitrogen, oxygen, CO2,
and water vapor (CO2> O2).
 CO2 in soil air is (0.3 – 1.0) more than atmosphere air (0.03%).
 Soil aeration plays important role in plants growth, microbial population,
and microbial activity in soil.
 A good aerated soil types lead to complete oxidation of organic matter, and
characterized with high redox potential capacity, which offer e- and H+ donor
and acceptors, results in thrive of aerobic and facultative microorganisms.
 Poor aerated soil types (saturated soils), which featured by low redox
potential capacity cause continues release of NO3- and SO42- and
accumulation of some harmfully intermediates like CH4, that affected soil
fertility and increase of anaerobic microorganisms population.

Organic matter
 Soil organic matter (SOM) is one of the most important components of soil
ecosystem, in its broadest sense, and complex combination of living organisms
and non – living organic matter ( fresh organic residues, actively
decomposing material, and humus).
 Generally the proportion of SOM in the soil ranging from 3 – 5 % of total
soil volume.
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Non – living organic matter can be considered to exist in two distinct pools:

Nonhumic Substances
 Its particulate matter represents microbial metabolites products, all with
identifiable structure, like polysaccharides , amino acids, organic phosphorus.
This organic matter can constitute from a few percent up to 25% of total
organic matter in soil.

Humic Substances
 Carbon decomposition, successive decomposition of dead material, and
modified organic matter results in the formation of undefined organic matter
called humic substances or humus, by a process is called humification.
 Humus comprise both organic molecules of identifiable structure like
proteins and cellulose, and molecules with no identifiable structure, like
plants residues such as lignin, remains of animal carcasses (waxes, hair, nail,
wool, and feather), also humin , humic acid, and fulvic acids are major
components of humus.
 Humus is very stale, long – lived pool of organic matter in soil ( with turnover
rate of 100 – 500 years), which makes it an effective way to sequester excess
carbon.
 Humus affects soil properties, as it:
Slowly decomposes
colors the soil darker,
offer spongy appearance ,
encourages aggregate formation,
increase water and nutrient retention and contributes to N, P, S, and other
nutrients.

Soil Living organic matter (Soil Biota)

 The living part of soil organic matter includes a wide variety of organisms
such as plants, insects, earthworms, animals, and microorganisms.
 Soil is excellent culture media for the growth and the development of various
microorganisms that contains several distinct groups, and amongst them;
bacteria, fungi, actinomycetes, algae, protozoa and viruses.
 Microorganisms form a very small fraction of 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 organism or groups of organism are responsible for a specific change/
transformation in the soil.
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  The final effect of various activities of microorganisms in the soil is to make
the soil fit for growth and development of higher plants.

Living organisms present in the soil are grouped into two categories as follow:

1. Soil flora (micro flora) e.g. Bacteria, Fungi, Actinomycets and Algae.
2. Soil fauna (micro fauna) animal like e.g. Protozoa, Nematodes,
earthworms, moles, ants, rodent.

Soil Microflora

1. Bacteria
 It is the most abundant groups, and usually more numerous than others.
 Soil bacteria numbers vary between 108 – 109 cell/ gm of soil, however, in an
agriculture field their number goes about 3 × 109 cell/ gm of soil , which
accounts for about 3 tones of wet weight/ acre.

Based on its regular presence soil bacteria are divided into two groups:
1. Soil indigenous (true resident), or autochthonous.
2. Soil invaders or allochthonous.

 Bacterial number and variety influenced by soil type, microenvironment,

organic matter, cultivation processes.
 They are found in a high number in cultivated soil than virgin land, in a
maximum in rhizospheric space than in non – rhizospheric region, possibly
due to aeration and availability of nutrients.
 The inner region aggregates contained high level of G – ve bacteria ,
while the outer region contains high level of G + ve bacteria, thus may be
due to ; polymer formations, motility, surface charge, and life cycle of
bacteria.
 Bacteria do not occur freely in soil solution, but are closely attached to soil
particles by motility apparatuses (flagella and pilli), or by extracellular
polysaccharides , or may be embedded in organic matter.

Bacteria have some major roles in soil such as:

1. Mineralization of elements.
2. N2 – fixation from atmosphere.
3. Stabilization of mineral ions.
4. Biotransformation of chemicals.
5. Biogas formation.
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Example of some soil bacteria:
Agarobacteriun, Arthobacter, Bacillus, Alcaligenes, Erwinia, Corynebacter,
clostridium, Nitrosomonas, Nitrobacter, Rhizobium, Thiobacillus.

Rhizosphere
 It is a zone of increased microbial growth and activity in soil around of
plants, it may extended several inches into soil around root system of growing
plants.
 The microorganisms growing in this zone is under the influence of roots often
quantitatively and qualitatively.
 Therefore the rhizosephere is a unique subterrean habitat for m.o, the
microflora of one plant differ from the other plant.

The rhizospheric region can be divided in to two zones:

- Inner rhizosphere, which is in close vicinity of root surface.

- Outer rhizosphere, embracing the immediate adjacent soil.

 The outer epidermal walls of living roots and root hairs are covered with
mucilage and cuticle, also organic and inorganic compounds, which
accumulated in root cells cytoplasm and diffused out which is known as root
exudation, these exudates contain carbohydrates, organic acids, enzymes,
flavonones, and root hairs are continuously sloughed – off during secondary
thickening, all these compounds constitute a food base for microorganisms.
 The rhizospheric microoganisms have either beneficial or harmful effects
on developing of plants, the m.o. are intimately associated on rhizoplan,
therefore any toxic or beneficial substances produced by them has direct effect
on plants.

Some of possible effects are briefly:

1. The microorganisms catalyze the reactions in rhizosphere and produce CO2, and
form organic acids that in turn solubolize the inorganic nutrients to plants.

2. Aerobic bacteria utilize O2 and produce CO2, therefore lower O2 and increase CO2
tension that reduce roots elongation and nutrient and water intake.

3. Some of rhizospheric m.o. produces growth – stimulating substances and release

elements in organic forms through the process of mineralization.

4. Some of rhizospheric m. o. secretes plant regulators such as; indole acetic acid,
gibberellins, cytokinnens.
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5. They influence phosphorus availability through immobilization, however, when
plants suffer from nutrient scarcity during summer in tropical area, the rhizospheric
microflora release the immobilized nutrients, therefore they act as a sink between soil
and plant roots in nutrient poor systems.

6. Rhizospheric m. o. changes the availability or toxicity of sulfur in soil.

7. The products of rhizosrheric zone m. o. metabolism sometimes have toxic effects

on plants developing, these termed as the phytotoxins.

2. Fungi
 In most aerated and cultivated soil, Fungi share a major part of total microbial
biomass, because of their large diameter and extensive network of mycelia,
however, population of soil fungi ranging from 2×10 4 – 1×106 cell/ gm of dry
soil.
 Fungi drive their growth nutrient from organic matter, lining animals
(including; protozoa, arthropods, nematodes, etc.), and from living plants,
establishing different types of relationships.
 The most important relationship between fungi and plants in soil is
Mycorrhiza, which is a symbiotic relationship that occurs in plant root
systems.

There are two types of association between fungi and plant:

1. Endotrophic: The fungi mycelium grows into the root tissue of associated plant.
2. Ectotrophic: The fungi mycelium unsheathes the root system, or can form like
tubercles around rootlets, with limited penetration of hyphae into root tissue.

Some of soil fungi are:

Alternaria, Aspergillus, cladosporium, Helimenthosporium, Humicola, Fusarium,
Phytophthra, Plasmodiophora, Pythium, Rhizoctonia.

Fungi in soil play variety roles, some of these roles are:

1- Mycorrizal fungi supplies minerals to associated plants, likewise fungi receives
benefit from plants exudation, like some carbohydrates.
2- Production of fungi static products including antibiotics.
3- Support soil microenvironments bio balance by their feeding practices on protozoa
and nematodes.
4- Contributes in soil aggregation that protects soil particles from weathering effects.
5- Degrade some of tough plant residues, like lignine's.
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3. Actinomycetes
 Actinomycetes share the characteristics of both bacteria and fungi, they are
commonly known as "ray fungi", because of their close affinity with fungi.
 Actinomycetes are G+ve, and release antibiotic substances, however the
earthy odor of newly wetted soil has been found to be a volatile growth
products of actinomycetes.
 Their population remains greater in grass land than in the cultivated land.
 The number of actinomycetes ranging from 105 – 108 cell/ gm of soil.
 The most limiting factor is the soil pH which governs their abundance in soil,
its luxuriant growth favored by neutral and alkaline pH (6.0 to 8.0).

The important members of actinomycetes are:

Actinomyces, Streptomyces, Nocordia, Micromonospora, Actinoplans,
Thermoactinomycetes.

Roles of soil actinomycetes:

1- Contributes mainly in humus substances formation.
2- Decompose plant and animal residues that are resisting to bacterial and fungal
decomposition.
3- Maintain soil bio balance for their ability to produce antibiotics, particularly
Streptomyces spp. and the production of lysis enzymes, such as lysozyme.

4. Algae
 Algae grow where adequate amount of moisture and light present due to their
need of photosynthesis process, most of them prefer growth in neutral to
alkaline soil (pH 7 – 10).
 The prominent genera in soil are ; Anabaena, Nostoc, Calothrix,
Oscillotoria, Scytonema.

Algae roles in soil can be listed as the follow:

1- Revolutionized the failed of agriculture due to their photosynthetic capacity,

that act as a source of carbonic and nitrogenous organic matter in soil.
2- Many of algae species used commercially as bio fertilizers.
3- Soil algae used in reclamation of sodas soil and alkaline soil types.

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University of Baghdad Lecture: 2
College of Science Level: 4th
Soil Microbiology Lecturer: Dr. Shayma

Cycles of Elements

 All elements that are essential component of protoplasm undergo cyclical

alteration, between an inorganic state, free in nature, and combined state in
living organisms. This repeated transformation of elements from living
protoplasm to free state in nature constitutes the cycle of elements in nature.
 Among the essential elements undergoing biotransformation (recycling) is:
Carbon, Hydrogen, Nitrogen, Sulfur, and phosphorus.
 When the above elements (except phosphorus) are incorporated into
protoplasm, there is usually a change in the oxidation state. In protoplasm
many of these elements are in reduced state, but as they are returned, they
frequently are in oxidized state.
 Therefore these elements not only served as a source of energy (for
chemolethotrophes) by oxidation, others may serve as e- (H+) acceptors in
redox reactions.

The Carbon Cycle

 Carbon recycling is one of several recycling processes, but it may be the most
important cycle affects all life on earth, since is known to the basic building
block of life.
 Cycling is the process in which the carbon atoms are recycled over and over,
take place within Earth's biosphere and between living things and nonliving
environment.

Carbon occur in nature into two main states:

1. Complex carbonated organic compounds (C reduced form).

2. Inorganic carbon, most of inorganic carbon is in form of CO2 (C more
oxidized state). So CO2 is the source of all carbonated compounds, both in
living organism and fossil deposits.

Carbon dioxide constitutes about 0.0 45% of atmosphere gases, in this rate is more
less than the requirements of photosynthetic organisms, so CO2 release continuously
from respiration and decomposition to maintain carbon balance in nature.

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Biology plays an important roles in the carbon cycle through three types of
participation:
1. Producers
They represent the beginning of all food chains in nature, the producers fix
atmospheric CO2 and its subsequent assimilation into organic molecules through
photosynthesis process.
2. Consumers
Carbon is transferred from organism to other, when plants are eaten by herbivores,
which are in turn eaten by carnivores along the food chain.
Primary Secondary Tertiary.
Consumers get their required carbon either directly by eating plants or indirectly by
eating animal that have eaten plants, to become their cellular materials or to release
to atmosphere through respiration, in which a considerable portion of
carbohydrates is oxidized to yield CO2, H2O, and energy as follows:
Respiration
C6H12O6 + 6O2 6CO2 + 6H2O + energy
3. Decomposers
Decomposers are m.o. that lives mostly in soil, but also in water, they contribute
substantially to carbon pool by their feeding and processing of rotting remains of
other organisms. Their job is to consume both waste products and dead organic
matter of land or ocean. Decomposers not only play key role in carbon cycle, but
also break down, remove, and recycle what might be called nature's garbage.

Microorganisms play a key role in carbon recycling; they contribute to the

carbon cycle by:
1. Fixation of CO2 (Photosynthesis); an important step in carbon cycle is the
fixation of atmospheric CO2 and its subsequent assimilation into organic molecule.
Autotrophic organisms are able to covert CO2 into organic molecules via
photosynthesis process; include plants, algae, some bacteria, and some archaea.
The cyanobacteria and specific soil bacteria have the ability to conduct
photosynthesis, H2S and other reduced compounds serve as e- donor to reduce CO2.

Bacterial photosynthesis:
Light
CO2 + 2 H2A (CH2O)x + H2O + 2A
bactochlorophyll
2. Decomposition
It is a biological process that includes physical breakdown and biochemical
transformation of complex organic molecules of dead materials into simpler
organic and inorganic molecules.
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  The major function of soil m.o. in the Carbon Cycle is as decomposers –
degrader of complex organic matter that would otherwise permanently
sequester carbon, keeping it from being useful to organisms.
 Each organic compound is utilize in a slightly different manner, and each
yield different products when degrade, but many of these decomposers also
release CO2, contributing to the rising concentration in the atmosphere.
 Bacteria and fungi are particularly effective in breaking down of organic
matter, while actinomycetes are responsible in degradation of tough remains,
like lignin and chitin.

The rate of decomposition is affected by:

a. Soil temperature b. moisture c. aeration d. food availability

Mainly decomposition is either aerobic or anaerobic:

A. Aerobic decomposition, Most of heterotrophic microbes easily utilize
aerobically soil organic compounds for both energy metabolism and as a carbon
source:

Mineralization: This is the process by which organic matter is decomposed to

release simpler, inorganic compound (e.g. CO2, NH4+, CH4, H2).
Immobilization: The nutrients that are converted into biomass become temporarily
"tied up" from nutrient recycling, until the organism dies, at which time the C
released back into the environment via decomposition.
B. Anaerobic decomposition, The anaerobic degradation of carbonaceous matters
is a collaborative effort involving numerous bacteria, these bacteria are
responsible for the bulk of Co2 and methane that released to the atmosphere.

Anaerobic decomposition H2 + CH4 + alcohol + organic acid

 Decomposition of organic matter under anoxic conditions such as deep
compacted mud, differs considerably from oxygen availability, in that reduced
end products accumulated including organic acids, and CH4. Accomplished
by certain soil bacteria known as Methanogenic bacteria, are biologically
very primitive, strict anaerobic, and sensitive to pH.
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Main soil methanogens are:

Methanococcus, Methanobacteria, Methanosarcina.

 To complete the recycling pattern another group of methane bacteria called

Methanotrophes (literally methane eaters) are able to reoxidize released CH4
again to CO2, like Pseudamonas and Methylomonas. This conversion also
yields water and energy.
 Other soil autotrophic bacteria are able to participate in the cycling of carbon
by oxidizing carbon monoxide CO. This gas is relatively rare under ordinary
condition, released from some activities, commonly from partial combustion.
 Exceedingly poisonous for most aerobic organisms, including man, its relished
as a source of energy and carbon by at least one bacterial species
Carboxydomonas that oxidize CO to CO2

CO + 1\2 O2 CO2

 There is an increase of Co2 in atmosphere about one – third, and it’s continue
to rise.
 Methane concentration is likewise increasing about 1% per year, from 0.7 to
1.7 ppm.
 These two gases (CO2 & CH4) in combination with H2O (water vapor), O3
(ozone), and N2O (nitrous oxide), represents the greenhouse gases, the term
describes the ability of these gases to trap heat within Earth's atmosphere, in
that correlated with global temperature change, a phenomenon known as
global warming.
 Soil microorganisms play a role in the generation of each of these gases.

Cellulose decomposition

 Cellulose is the most abundant chemical constituent of plant cells, its

polysaccharides contain glucose units linked by ᵝ -1-4 linkage. Total amount
of cellulose in plants tissues varies from 15 to 60 %.
 Cellulose are not tough for decomposition, variety microbes presents in
millions per 1 millimeter of soil are capable to breakdown cellulose under
different circumstances, oxic and anoxic.
 In availability of oxygen cellulose decomposes into CO2, while in anoxic
condition cellulose incompletely decomposes with release of many
intermediates like organic acids and alcohols.

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Three different enzymes involve in cellulose breakage, they collectively termed
cellulases , each enzyme participate in certain stage of cellulose decomposition and
produced by different microorganisms, m.o. that are able to biosynthesize all three
enzymes called true cellulytic microorganism.
 Initial stages of cellulose decomposition take place by cleavage of cellulose by
extracellular enzymes then,
 The cleaved pieces are transported into the decomposers cell for energy
generation (catabolism) or production of biomass (anabolism) and
manipulated by the two rest intacellular lytic enzymes.

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University of Baghdad Lecture: 3
College of Science Level: 4th
Soil Microbiology Lecturer: Dr. Shayma

Nitrogen Recycling

 Nitrogen is a part of vital and essential organic compounds in organisms, such

as amino acids, proteins and nucleic acids the building block of life.
 Although the majority of air we breathe is nitrogen as it make up 78% of the
atmosphere air, but most of atmospheric nitrogen is unavailable to use by
organisms, this is because of the strong triple bond between the nitrogen
atoms in nitrogen molecules make it relatively inert, in order for plant and
animals to be able to utilize nitrogen, first N2 must be converted to more a
chemically available forms, such as ammonium NH4+, nitrate NO3, or
organic nitrogen e.g. urea.
 Nitrogen exists in nature in both organic and inorganic forms, as well as in
many different oxidation states. The movement of N2 between the atmosphere,
biosphere, and geosphere, in different forms is described by the Nitrogen
Cycle, microorganisms are the key element in the cycle, provide different
forms of nitrogen compounds by their metabolic activities.
The Main features of N2 cycle are:
• Nitrogen Fixation
• Nitrogen Mineralization
• Ammonification
• Nitrification and Denitrification

Nitrogen Fixation
 Nitrogen fixation is natural process, it occurs either biologically or
abiotically, by which the gaseous nitrogen (N2) in the atmosphere is converted
into combined form e.g. ammonia NH3 or other nitrogen organic forms,
which are more reduced than when it is free, and become more available to
farm corps either directly or through further microbial action.

Biological nitrogen fixation (BNF):

 Nitrogen fixation is utilized by numerous prokaryotes, Microorganisms that fix
nitrogen are called Diazotrophs, which are diverse group of prokaryotes
including free – living and symbiotic bacteria, cyanobacteria and
actinomycetes.
 Common N2 – fixing microorganism is given in table 3.1, and the
phenomenon of this activity is known as Diazotrophy.
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  The biological conversion of atmospheric nitrogen taken place with the help
of an enzyme called Nitrogenase that combines gaseous nitrogen with
hydrogen to produce ammonia, which is then further converted by bacteria
to make their own organic compounds.
 Nitrogenase in fact is a complex enzyme system, contains multiple metal –
containing prosthetic groups, act in anaerobic conditions that require a
reducing powers such as; NADH and NADPH, each one of these work with a
certain type of bacteria, and the fixation process requires such a large amount
of energy, about 5 – 6 ATP molecules, this energy produced by substrate
level phosphorylation in anaerobic microorganisms and via oxidative
phosphorylation in aerobic one.

Table 3.1 N2 – fixing microorganism

A. Free living Microorganisms Name of Microorganisms

Aerobic Azotobacter spp. , Beijerinckia spp. ,

Trichodesmium spp.

Microaerophilic Xanthobacter spp., Azospirillum spp. ,

Thiobacillus ferooxidance

Facultative anaerobic Klebsiella pneumonia, Erwinia spp.

Bacillus polymyxa

Anaerobic Bacteria Closteridium pasteuriunum,

Propionibacter, Desulfovibrio spp.

B. Symbiotic Microorganisms Rhizobium spp., Burkhoderia spp. ,

Mesorhizobium, Frankia, Anabena

Symbiotic N2 – Fixation Processes

 There are many symbiotic N2 – fixation bacteria, a few actinomycetes, and
cyanobacteria, but the most common symbiotic bacterium is Rhizobium spp.
which live free in soil and are able to infects legume root nodules when come
in contact with suitable legume.
 Rhizobium are G – ve rods, motile, non – spore forming, utilize organic salts
as carbon source, and fast growing bacteria with generation time lasts about 6h.
2
Process of root nodules formation
 Infection of Rhizobium typically occurs in root hair of legumes.
 Many rhizobia and host plant are highly specific and legumes can either
attracted rhizobia to root hairs directly by excretion compounds or by
induction of nod gene activity in bacteria.
 The communication between legume and rhizobium stared by releasing of
flavonoids from host root.
 Flavonoids are at the highest concentration at the root and bind bacterial
protein product of nod gene, in that rhizobia colonize the soil in the vicinity
of the root hair in response to the flavonoids. This binding induces encoding
several enzymes that are required for nodulation of appropriate host plants.

Generally nodulation start from the following stages:

1. Curling and Deformation of Root hairs

 Invasion of Rhizobium occurs through root hairs, but they are unable to
hydrolyze the cellulose of plant cell walls, they appear to find non-cellulosic
points at the tips of root hairs of legumes which called hyaline spots, through
these points inter to root tissues.
 The penetrated bacteria grow in form of infection tube inside the root hair
cells, this tube surrounded by a cellulose coat produced by infected plant cells,
after 4h. of infection, the bacterial cells arrange themselves side by side like
thread, so termed infection thread, the formation of this thread lead to the
curling of root hair then deformation of infected hair.

2. Formation of nodules
 The infection thread inside host cortical cells bulges and then rupture, the
bacteria released into cytoplasm and after the bacteria undergoes alteration
morphologically into larger forms called bacteriods, infected cells in turn
rapidly divided to form a tumor like nodules of bacteriods – packed cells.

3. Structure of root nodules

 The root nodule is formed due to tissue proliferation induced by the action of
growth promoters of rhizobial in origin, probably cytokininis.
 The core of mature nodule constitutes the "bacteriod zone" which is
surrounded by several layers of cortical cells.
 The bacteroids singly or in groups, surrounded by peribacteroid membranes,
inhabit the cytoplasm of plant cell.

3
  The effective nodules are generally large and pink due to presence of
legheamoglobine with well-developed and organized tissue. After the
senescence, when the nodules dies the stationary – phase Rhizobium are
released into the soil.

4. Function of nodules
 Bacteroids are the site of N2 – fixation. The isotopic (15N) studies indicated
that bacteroids are unable to utilize sugars, and secrete ammonia, which are
apparently incorporated into organic matter as amines by glutamine synthetase
enzyme present in the surrounding plant cells.
 This amine N – atoms are eventually introduced into protein, nucleic acids and
other biomolecules.
 N2 – fixation process is an energy consuming process, for every mole of N2
fixed about 22 moles of glucose is utilized as seen in the following equation:
N2 + 8 H+ + 8 e- 2NH3 + H2
This process indicates the involvement of true mutual symbiosis in which role
played by leghaemoglobin in the fixation of nitrogen is much more significant. The
formation of leghaemoglobin is specific effect of the symbiosis. The relative
capacity of plant – bacterial association , once established , to assimilate molecular
nitrogen is called effectiveness.

5. Leghaemoglobin
 A red pigment similar to blood hemoglobin is found in the legume plant
infected nodules between bacteroids and the membrane envelopes
surrounding them.
 This pigment or protein enhance the transport of oxygen at low partial
pressure to the nodules and maintains a steady supply of oxygen at low
concentration of the nodule.
 In fact the presence of leghaemoglobin seems provide full protection to
nitrogenase against oxygen damage, since nitrogenase is very sensitive to
O2 and must be protected from oxidizing conditions.

Nonsymbiotic N2 – fixation
 Many free living microorganisms are capable of converting molecular
nitrogen to cellular nitrogen independently of other living organisms , they
are primitive, live freely in soil and water, operate under poor aeration
condition process by reduction process.
 Azotobacter is the most studied and best example of free living aerobic N2 –
fixers.

4
  Azotobacter evades the harmful effects of O2 on its nitrogenase by having an
exceedingly high rate of respiratory metabolism, thus preventing the retention
of O2 inside the cell, thus protecting enzyme complex.
 It produces FeS protein which complexes with two nitrogenase proteins to
form a three membrane oxygen stable and inactive complex, when they do this
start N2 fixation.
Ammonification
 When plant and animal dies, or animal expels wastes, the initial form of
nitrogen is organic.
 Bacteria, or in some cases fungi convert the organic nitrogen within the
remains back into ammonium ion (NH4+), and water soluble ammonium
salts, a process called ammonification or mineralization.
 In fact the amino group (NH2) is split off to form ammonia (NH3) through
series of enzymes reactions. Usually ammonification carries out under oxic
conditions as follow:

 A diverse soil microflora activated the ammonification process, this

includes bacteria, e.g. Pseudomonas, Proteus, Micrococcus, Serratia,
Closteridium, etc., and Fungi, e.g. Alternaria, Mucor, Aspergillus, Rhizopus,
etc.
 The most abundant nitrogenous organic compounds in soil are proteins,
which decompose by proteolytic m.o. They synthesize extracellular proteases
degrade proteins to their structural subunits, amino acids through series of
subsequent reactions:
Proteinase Peptidase
Protein Peptides amino acids
Released amino acids are subjected to Deamination either oxidatively or reductively:

5
  If the protein decomposition proceed under anaerobic conditions the
process termed " Putrefaction" some of released amino acids converted to
offensive Oder producing amines and related compounds, and in the
presence of air the produced amines and related compounds are again
oxidized with liberation of ammonia.
 Ammonium is soluble, or capable of being dissolved, in water and often is
used as fertilizer. It is attracted to negatively charged surfaces of clay and
organic matter in soil and therefore tends to become stuck in one place
rather than moving around, as nitrate does.
 In acidic soils, typically plants receive their needed nitrogen from
ammonium, but most nonacidic soils can use only nitrate.
 Ammonium may be combined with nitrate to form ammonium nitrate, a
powerful fertilizer.
 NH4 predominant in well – aerated soil and rich in organic matter, in acidic
soil types there is less ammonia production, since decomposition is carried
out by fungi, and if the soil is rich by carbohydrates wastes ammonia
formation also is low , since the m.o. prefer to utilize carbohydrates than
nitrogenous wastes.
Nitrification
In soil the liberated ammonia during ammonification pathway is rapidly oxidized to
nitrate by some soil highly specialized bacterial groups of strictly aerobic
chemolithtrophes, this oxidation process termed Nitrification, which occurs into
two stages:
The primary stage, involves the oxidation of ammonia (NH3) to nitrite (NO2-), so
the process called Nitrosofication.
2 NH4+ + 3 O2 2 NO2- + 2H+ + H2O
This stage performed by soil bacteria such as Nitrosomonas, Nitrosobacter ,
Nitrosovibrio.
The secondary stage, involves the oxidation of liberated nitrite into nitrate (NO3-)
2NO2- + O2 2 NO3- + energy
Other bacterial species are responsible for this oxidation such as Nitrobacter,
Nitrococcus, Nitrospira.
 It is important for the nitrites to be converted to nitrates because accumulated
nitrites are toxic to plant. Some plants get nitrogen from the soil, and by
absorption via their root in form of either nitrate ions or ammonium ions , it
is reduced to nitrite ions and then ammonium ions for incorporation into
amino acids, nucleic acids, and chlorophyll, this pathway mention nitrogen
assimilation process.
 Due to their high solubility, nitrates can enter groundwater. Elevated
nitrate in groundwater is a concern for drinking water use because nitrate
6
 can interfere with blood oxygen levels in infants and cause
methemoglobinemia or blue – baby syndrome.
 Where groundwater recharges stream flow, nitrate – enriched ground
water can contribute to eutrophication, a process leading to high algal,
especially blue – green algal population and the death of aquatic life due to
excessive demand for oxygen.
Factors affecting nitrification:
1. Acidity: The nitrites are extremely susceptible to acidity even if they produce
acids. Thus nitrification proceeds slowly in acid soil.
2. Oxygen: It is an obligate requirement, Nitrification occurs even in submerged
soils (paddy fields) in the upper few centimeters since the diffused oxygen present in
waters helps nitrification in such soil.
3. Moisture: is needed for nitrification since the nitrifies cannot tolerate arid
conditions.
4. Temperature: Nitrate production is high during 30° C – 35°C, since nitrifiers are
mesophiles.
Denitrification
 The nitrate reduction is biologically facilitated process, performed by a large
group of heterotrophic facultative anaerobic bacteria.
 Denitrifying microbes require a very low oxygen concentration less than
10%, as well as organic C for energy.
 The denitrification generally proceed through a series of intermediates;
nitrogen dioxide (NO2), nitric oxide (NO), nitrous oxide (N2O), finally
resulting N2 completing the nitrogen cycle.
 The products of pathway are volatile products, therefore are lost to the
atmosphere and fail to enter the cell structure.

Denitrification can lower leaching of No2 to ground water; it can be strategically

used to treat swage and municipal wastewater or animal residues of high nitrogen
content. Denitrification allows for the production of N2O, which is greenhouse gas
that can have a considerable influence on global warming.
Denitrification is essentially a respiratory mechanism in which nitrate replace the
molecular oxygen, therefore denitrification may be termed as nitrate respiration
(dissimilatory nitrate reduction), the reduction process mediated by nitrate
reductase enzyme , nitrate serve as the e- acceptor, which is carried out according to
the following reaction : 2No3- + 10 e- + 12H+ N2 + 6 H2O
7
University of Baghdad Lecture: 4
College of Science Level: 4th
Soil Microbiology Lecturer: Dr. Shayma

Sulfur Recycling

 Sulfur is one of essential nutrient for all organisms that make up proteins and
vitamins, is important element for the functioning of proteins and enzymes.
 Sulfur exists most abundantly in earth crust in low concentration, and in its
native forms is unavailable to plants.
 It occurs in nature and in soil as organic forms such as proteins of animal
excretory products, and inorganic forms, such as dissolved sulfate (SO42-),
thiosulfate (S2O3), thiocyanate, and hydrogen sulfide gas (H2S), these elements
undergoes familiar alteration between organic and inorganic forms, and
between oxidative and reductive states.
 Like nitrogen, sulfur in its more oxidized form is most available to plants.
 The environmental Sulfur cycle involves many physical, chemical and
biological agents. A simplified schematic diagram of the cycle is shown in
Figure 4.1, which has been prepared to show the major phases of Sulfur
cycling with relation to mineral deposits.
 As such, the figure indicates the relationships between Sulfur (S), hydrogen
Sulfide (H2S), Sulphur dioxide (SO2) and Sulfate ion (SO42-). In mineral form
Sulfur may be present as sulphides e.g. pyrite (FeS2), chalcopyrite,
(FeS.CuS), pyrrhotite (FeS) and/or sulphates e.g. gypsum (CaSO4.2H2O)
and barite (BaSO4).

Figure 4.1 Sulfur Recycling in Nature

1
The Role of Microorganisms in the Sulfur Cycle
Microorganisms (most frequently bacteria) are often integrally involved in the
chemical alteration of sulfur or intermediate products of their decomposition, may be
directly or indirectly necessary to their metabolism.

Sulfur is microbiologically metabolized in soil through different transformation

processes:

1. Mineralization (decomposition) of organic Sulfur to the inorganic form,

hydrogen sulfide: (H2S).
2. Oxidation of sulfide and elemental sulfur (S) and related compounds to sulfate
(SO42–).
3. Reduction of sulfate to sulfide.
4. Microbial immobilization of the sulfur compounds and subsequent
incorporation into the organic form of sulfur.

Decomposition of organic Sulfur compounds

Many heterotrophic bacteria decompose organic compounds into smaller units
and finally into inorganic compounds, (mineralization), as given below:

Oxidation of inorganic Sulfur

 Many bacteria are able to oxidize various forms of sulfur for energy gain,
especially hydrogen sulfide (H2S) to sulfate, as the major oxidation producer,
sulfur oxidizing prokaryotes are phylogenetically divers,
 In the domain archaea sulfur oxidation restricted to Sulfolobus, and in the
domain bacteria sulfur is oxidized by aerobic lithotrophes, like genera
Thiobacillus, Thiomicrospira, (they produce sulfuric acid i.e. hydrogen ions,
H+, and sulphate ions, SO42-), or anaerobic photosynthetic bacteria
belonging to the family Chromatiaceae (purple sulfur bacteria, used H2S as
electron donors for anoxygenic photosynthesis).
 Lithotrophic sulfur oxidizing bacteria are known to accelerate the
generation of Acid Rock Drainage (ARD) from pyritic (FeS2) and
pyrrhotitic (FeS) rocks under suitable conditions.
 The bacteria catalyzed sulfide oxidation reaction may have a reaction rates
six orders of magnitude (i.e. 1,000,000 times) greater than the same reactions
in the absence of bacteria.
2
Sulfate Reduction
 As in nitrogen cycle, sulfate produced by sulfur – oxidizing bacteria may be
reduced to hydrogen sulfide by a few anaerobic bacteria species.
 Sulfur – reducing bacteria are distributed in anoxic conditions, for example,
these bacteria have been found in sewage, polluted water and sediment of
lakes, sea and marine mud's, and the rumen of bovine animals.
4 H2 + CaSO4 H2S + Ca ( OH)2 + 2 H2O

 The direct reduction of sulfate ions to hydrogen sulfide is affected in nature

by specialized strictly anaerobic bacteria of the genera:

1. Desulfovibirio desulfuricans

The best known species of reducers, G – ve, pleomorphic, curved rods (vibrio –
like), motile with polar flagella, anaerobic, these bacteria use molecular hydrogen
in sulphate reduction.

2. Desulfotomaculum.

Obligate anaerobic, spore forming rods and heterotrophic (cell carbon from
organic compounds). The bacteria utilize sulfate, thiosulfate S2O32-, sulfite SO32-, or
other reducible sulfur-containing ions as terminal electron acceptors in their
respiratory metabolism. In the process these sulfur-containing ions are reduced to
hydrogen sulfide.
 Due to flooding, increase in temperature and addition of organic matter
when the O2 levels decrease, so the level of sulfides considerably increases,
and sometimes increase above 150 ppm. Consequently the No. of sulfate
reducer also increases.
 Released H2S, S- and S2O3 from sulfate reduction or amino acids
decomposition are readily oxidized microbiologically aerobically by colored
photosynthetic bacteria belonging to the genera Chlorobium (green sulfur
bacteria) and Chromatium (purple sulfur bacteria) .

The oxidation process follows the equation:

CO2 + H2 S ( CH2 O)x + H2O + 2 S
 These bacteria consume CO2 but do not release oxygen because H2S serves
as reducing agent (electron donor), instead of water, which they give off sulfur
particles inside their cells rather than oxygen in anoxygenic photosynthesis
process.
 Oxidation of H2S in soil lead to pH reduction due to released H2+, so sulfur
fertilizers added to alkaline soil types as treatment practices.
3
 Phosphorus Cycle

 Phosphorus; is an essential nutrient for all organisms in form of ions PO4 3-

and HPO4 2-
 It is a part of DNA – molecules, energy storage molecules ATP and GTP, and
cellular membranes. Phosphorus also a building block of certain parts of
human and animal body, such as the bones and teeth.
 Phosphorus can be found on earth in water, soil and sediments, especially
rock deposits which is the major store house of phosphorus in nature.
 One unique characteristic of P is its low availability due to low diffusion and
high fixation in soil.

Several transformation processes recycled phosphorus in nature,

Microorganisms integrated in these processes through different jobs:

1. Inorganic phosphorus solubolization

 Phosphorus is the most important key element in the nutrition of plants, next
to nitrogen. It plays an important role in virtually all major metabolic
processes in plant including photosynthesis, energy transfer, signal
transduction, macromolecular biosynthesis and respiration.
 Although P is abundant in soils in both inorganic and organic forms, it is a
major limiting factor for plant growth as it is in an unavailable form for root
uptake.
 Inorganic P occurs in soil, mostly in insoluble mineral complexes, some of
them appearing after frequent application of chemical fertilizers. These
insoluble precipitated forms cannot be absorbed by plants.
 Plants can use only a small amount of this P since 75–90% of added P is
precipitated by metal–cation complexes, and rapidly becomes fixed in soils
because they combined to form insoluble salts.
 Organic matter is also an important reservoir of immobilized P that
accounts for 20–80% of P in soil.
 Microorganisms are an integral component of the soil P cycle and are
important for the transfer of P between different pools of soil P.
 Phosphate Solubilzing Microorganisms (PSM) through various mechanisms
of solubilization and mineralisation are able to convert inorganic and
organic soil P respectively into the bioavailable form facilitating uptake by
plant roots.
 The main P solubilization mechanisms employed by PSM include, release of
organic acids as a side products of microorganisms activities, these acids
4
 acts on conversion insoluble salts to more available to plants, such as
tricalcium phosphate [Ca3 (PO4)2] is converted to dicalcium phosphate.

[Ca3 (PO4)2] Ca (H PO4)2

[Ca3 (PO4)2] + 2 HNO3 2 Ca (HPO4)2 + Ca (NO3)2
[Ca3 (PO4)2] + H2SO4 2 Ca (HPO4)2 + CaSO4
 Liberation of hydrogen sulfide from sulfur reduction by bacteria facilitate
reaction with ferric phosphate, (other insoluble P compounds are also
abundant in soil) to yield ferrous sulfide (FeS), which made phosphorus
more available to plants absorption.
Fe PO4 + 3 H2S FeS + 2 H3PO4
 P dissolution rates, depending on size of mineral particles and soil pH, with
increasing soil pH, solubility of Fe and P increases but solubility of Ca
phosphate decreases.
 PSM are abundant in root surface of plants, and a counts about 105 – 107 cell/
gm of soil.
 Many microbial species are associated with phosphorus conversion;
Pseudomonas, Bacillus, Micrococcus, Mycobacterium, Flavobacterium,
Penicillium, Fusarium, Aspergillus, etc..

2. Mineralization of organic compounds

 Mineralization mean decomposition of organic compounds with the release
of inorganic phosphate.
 Liberation of phosphate depends upon: the rate of microbial assimilation,
PH, temperature, and availability of organic phosphate.
 Phosphatase enzymes catalyze split of phosphorus from organic
compounds.

 In soil 15 to 80 % of total phosphorus are in organic form, so soil rich with

organic matter contains abundant of organic phosphate, therefore a good
correlation exist the concentration of organic phosphorus, organic carbon, and
total nitrogen.
The C: N: P mineralization ratio in soil in steady state is about 90: 8: 1.
3. Immobilization of Phosphorus
Conversion of inorganic, available phosphate ions into cell compounds.
5
Soil Microbiology: Lecture 5
Equation of biodegradation:
Organic molecules Inorganic molecules

CO2, H2O and other

metabolic by-
Biodegradable products such as
methane, methanol,
substance ethanol.
Biodegradation by
Microorganisms
Under suitable conditions:
• Optimum pH
• Temperature
• Water
• O2
Examples of compounds that have produced environmental
problems:
1. Alkyl benzyl sulfonates: (Laundry detergents).
2. Chlorinated hydrocarbons: (Pesticides).
3. Hydrocarbon pollutants: (Oil or Petroleum).
4. Cellulose and lignin.
5. Solid waste.
 radation Dr. 6hadah Al-Quraishi
:\\,.\" 11,R
L.c1{ 4
T
In trotlrr ction:

human erploitation of tbssil lire:l reser\.'es and the production of manl' nolel
s.r,ndretic compornds har:e introduced into the environrnent mall--v compounds that
ruricroorganisms normally do no1 c-.ncounter and thus are not prepared to biodegrade .

Many of these compounds are toxic to living s),stems, and there presence in aquatic and
terrestrial habitats often has serious ecological consoquences, including major kills of
indigenous biota. The disposal or accidental spillage of these compounds has oreated
serious modern environmental pollution problems, particularly q,hen microbial
biodegradation activities fail to resolve the pollutants qlickly enough to prevent
em,ironmental damage. se.lgage treatment and water puriflcation systems are usually
incapable of removing these substances if they enter municipal water supplies u'hen
they pose a potential human hazard. Elimination of a wide range of pollutants and
wastes from the environment without adversely affecting the environment is an absolute
requirement to promote a sustainable development of our society' . Microbes are
excellent candidates that can be used to remove contaminants as they are catabolically
' versatile and are capable of degrading or converting hazardous compounds into
], ,' harmless ones.

BIODEGRADATION:
Mic.r'oorganisms have an inherent ability. to degrade , transform, or accumulate a
rvide range of compounds, including hydrocarbons (eg., oil), polychlorinated biphenls
I
,l (PCBs), polyaromatic hydrocarbons (PAHs), pharmaceutical substances, radionuclides,.
l
and metals. Nutrients and energy thus produced are used for growth and multiplication
of the microbe. This breakdoun of organic or inorganic compounds by living organisms
is called biodegradation. Most biodegradation occurs at temperatures berween l0 and
3 5 "C in the presence of rvater.

The term biodegradation is often used u,ith reference to solid waste *a its ,r*u!3rrl".rt,
removal of pollutants from the environment (bioremediation).It is related to
biomineralization, in which organic matter is converted into minerals, CO2, water, and
nutrients. Incomplete biodegradation results in the formation of molecules u,hich are
smaller than the original ingredient. These new molecules are called metabolites and
have commercial appli cations.
,{
'Biodegradation , Dr. Ghadah Al-Quraishi

u
Substances rhat can be de-c.raded b-v microbial activifi" are termed hiodegradable. In
I nature, this occur spontaneously. fliodegradation can occur under aerobic and under
anaerobic conditions. I)uring microbial breakdoun, the first substance to be degraded is

I carbohl'drates, folloq'ed b,v proteins. Microorganisms respond to the presence of orgaric

materials b-, grou,ing rapidl-v and using the easy availabie parts of the organic material.
The more resistant parl-s such a-s c.ellulose, lieniq and other compounds ate degraded
I larer. Although biodegradation processes vary greatly. In most cases the fural product of
the degradation is (CO2) or methan.
I Biodegradation
I Organic
.t1olecules lnorganic
nutrients

I \
-Ga=-
.,' tt- \-.t, a .i rarl!'-1r r

I w
{llE
"reQr*
Biodegradation by Microbes

I
I
I
I 1. Alkyl Bebzyl Sulfonates : (laundry detergents).

I Alkyl ben4'1 sulfonates (ABS) are the major componetrts of anionic laun'dry
detergents. Cleaning occurs when ABS molecules from a monolayer around

I lipophilic droplets or particles that make up most stains or dirt on clothing , forming
an emlrlsion that can be rinsed out of the fabric with water.
, ,r.,.,, ..r1,.

I 2. Chlorinated Hydroearbons : (Pesticides).

Pesticides : are dre chemical substances that kill pests . In the contexl of soil, pests
I are fungi, bacteria, insects, worms. and nematodes etc. that cause damage to field
crops. Although u,ide-scale application of pesticides is an essential parl of
t
I
I
I
fi
Dr. Ghadah Al-Quraishi

!
augmenting crrop ),'ields: excessir,e use of these chemicals leads to the mic.robia.l
I imbalance, c:nvironmental pollution and health hazards. An ideal pestic-.ide:

I 1. should have the abiliq' to destro-v target pest quickl-v

2.
.

should be able to be degraded into non-toxic substances as quickll' as possible.

I ElTects of pesticides: Pesticides reaching the soil in significant quantities have direct
effect on soil microbiological aspects, which in tum influence plant grou.th. Some of

t the most important effects caused by pesticides are :

(1) alterations ofecological balance of the soil microflor4

T (2) continued application of large quantities of pesticides may cause ever lasting
changes in the soil microflora,
I (3) adverse effect on soil fertility and crop productivity,

T (4) inhibition of N2 fxing soil microorgarrisms such x Rhilobium, Azatobacter,

Azospirillum etc. and cellulolytic and phosphate solubilizing microorganisms,

I (5) suppression of nitrifing bacteri4 Nitrosomonas and Nitrobacter.

I Persistence of pesticides in soil: How long a pesticide persists in soil is ofgreat

importance in relation to pest management and environmental pollution. Persistence of
pesticides in soil for longer period is undesirable because ofthese reasons:
t a),accumrilation of t}le chemicals in soil to highly toxic levels, , ,
\ . ,,'

I b) may be assimilated by the plants and get accrrmulated in edible plant products, ti

c) accumulation,ln the edible portions of the root crops,

I 't
d) to be get eroded with soil particles and may enter into the water streams, and finally
leading to the soil, water and air pollutions. The effective persistence of pesticides in
t soil varies from a week to several yeaf,s depending upon structure and pfepeflLigg of the
constituents in the pesticide and availabilitv of moisture in soil. For instance, the highly
toxic phosphates do not persist for more than three months while chlorinated
T hydrocarbon insecticides (eg. DOT, aldrin, chlordane etc) are known to persist at least
for 4-5 years and some times more than 15 years.
t Biodegradation of Pesticides in Soil: Many soil microorganisms have the ability
to act upon pesl-icides and convert them into simpler non-toxic c-ompounds. Not all
T

I
n
I
E
H
Dr. 6hadah Al-Quraishi

I
i)tsticjdcs renchjil!- to the sr-rii are hiocle graL.lable lurd such r:i'lcrnieiil: th:it slrlit ri{)1r,ir,lr1L:

I resi:rt.ancc to biiide !.riidaiioll ilre callird "retaicil ral1".

Il iode!.radatic)n o1'pesticides is greatlf ilfluencc:d br the si.iil lactors like lttoirrrtre.

T tall.]lrc'!.a1ure, [,IJ and organic matler coutect. rn addition to mict'obia] l-..)i)uli]tiirn 3nd
pesticide -rolubiliry. Optimunr telnperarure. moisrure. and or g.anic matfer in -soll provide
congenial enl irorlrrent for the lrlcrrk dortn ut retenTi()rl of anr pcsljciilr uLllrd itr ilre
I soil. N{ost ol the organic pesticides degr-ade within a sirort periotl (3-6 monihs) under
tropic:al conditions. Metabolic activities of bacte.riE fungi and actinom)'cretes har.e the
I significant role in the degnadation ofpesticides.

!--or successful biodegradation of pesticide in soil, lollowing aspects must be taken into
I consideration.

1) Organisms must have necessary catabolic activify required for degradation of

I contaminant atfast rate to
bring down the concentration
contaminant must
of
contaminant.
be
2) the tsrget bioavailability.
3) soil conditions must be congenial for microbial /plant grorn'th and enz).rnatic activig,.
I 4) cost of biodegradation must be less than other technologies of removal of
contaminants.
I 3. Hydrocarbon pollutants : ( Oil or Petroleum )
, . '."., )-
I Components.of petroleum as well as its refine.d products are cornmon environmental
contaminants, as they are important components of gasoline and are also widely used as
solvents and irtteqnediates in
chemical synthesis. Certatn microbes use these
I compomds as thES,lg sources of carbon and energy for example: Pseudomonous
putida , P. aeruginosa, P. mildenbergii, P. oleovorans, Ag-O6obactg-1 spL, Salmo ella
I g.phimurilmt, Moraxellc, Arthrobacter, and Nocard.ia .[qpj_,optfdUacteria such as
Thiohaeillus and Desutfovibrto can metabolize the sulfur component of crude oil.
Thiobacillas metabolizes sulfur to sulfirric acid and under anaerobic condition
I Desulfovibiia metabolizes it to sul-fide . Several yeasts as well as mycelia frmgi , such
as Trichoderma , Aspergillus , and Cladosparitmt , arc active in soils as well as water
systems , and bring about hydrocarbon degradation .
I
Hydrocmbons are degraded onl,v in water since bacteria and flmgi cannot grorv in
I hydrocarbons. They grow in water present within or surrounding the hydrocarbons. Oil
and water form an emulsion which is an important part of hydrocarbon degradation.
Thus , despite its toxicity , a considerable fraction of petroleum oil entering marine
I systems is eliminated by the hydrocarbon degrading activities of microbial
-
communities , in particular by a remarkable discovered group of specialists , the so-
called hydrocarbonoe lastic tracteria (HCB). Genomic analysis of Alcanivara-r
I barkamensis , the most important global oil degrade.r (IICB) , has revealed several

I
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i
i t-t
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Dr. Ghadah Al-Quraishi

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insights into il,s capacit_v for h-vdrocarhorr degradation and has hrlped in desiqnil,g
I strategies fcrr the niirigarion of ecological damage caused bv ttil pollution of n.iarine
habitats. I{BC also have potential bioreclmological applications in rhe arca.s of
bioplastics.
I 4. lliodegradation of (lellulose antl l-ignin:
I A rvide range of bacteria and fungi are capable of degrading celiulose under a r.ariet-y
of environmental conditions . Some important cellulolltic microbes are:
I Bacteria: Slreplom.l'ces, Cllophaga, Cellulomonas, Nocardia, l;/ibrilt.and Clostridium.
Fungi : Trichoderma, Chaetornium, Aspergilttts, Fusarium, and Ph.oma.
I Microbial degradation of cellulose is brought about by tlie action of extracellular
cellulose etlztrne compiex, which is made up of three types of enzymes :
I . CI
;.i..1 !

enzyme that cleaves native cellulose, not partially degraded cellulose.

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t Cx enzlme that cleaves partially degraded cellulose molecules. The endo 1-4
glucanase t5pe break the cellulose internally at random and the exo 1-4 glucanase
attack ttre ends of the chain resulting in cellubiose molecules.
I o p - glucosidase degrade cellubiose and other small fragments into glucose.

These enzymes act in a sequential manner , and the end product of one become the
I substrate of the other.

Rec*lcitrant substances are resistant to biodegradation in the sense that they are not
t rapidly degraded in nature. One of the most important substances of this group is
Iignin. Biodegradation of lignin molecules is brought about by :

t White-rot fungi: members of the Agaricaceae, hydnaeeae, Polyporaceae , and the

Thelephoraceae that include : Chatomium sp., Paecilomyces, and AII esch eria sp.
I Bacteria: Pseudamonas, Xanthomonas, Acinitabscter, Bacillus, Arthrobacler,
trficrococcus, Aera toflas, Ckramobacteriatn and Flavobocterium .
T
5. Biodegradation of solid \Yaste:
T Solid waste is a material that is not of any value to the person responsible for it . it is
generated by domestic, commercial, industrial, healthcare, agricultural and mineral
I extraction activities and accumulates in streets and public places. It is referred to as
"garbage" , "trash" , "refuse" , and "rutrbish". Accumulation of u'aste creates
se'r,eral problems , such as ugly dumps and landfills, which not only appear as a scare on
I the face of the city but also become breeding grounds for flies , mosquitoes , rodents ,

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and other suoh disease car.Lsing a.sent.s as u,ell as street dogs , stra) cattle: ect.
l)ecornposing v,astes give out a foui odor and also cause clogging and flooding of
drains , resulting in unsanitary conditions. Water flon ing fi'onr u aste dumps and
disposal sites causes rvater pollution . Buming of q'aste result in air pollution . The
products of combustion include dioxins , which are particularly hazardous. NIuch of the
\\iaste acoumulate in the streets and open spaces oreates urh,vgienic conditions is
dornestic $'aste or waste generated in our homes. It is mostl-v composed of both
biodegradable (substances u'hich decompose naturally by the action of microorganisms)
and non - biodegradable (substances which can not be decomposed b.v the action of
microorganisms).

I Biodegradable substances :

I . Kitchen scraps: unprocessed or uncooked food , meat, diary products,

spoiled food vegetable and fruit peelings, tea bags and tea leaves, ect.
grease,

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Garden residue: weeds, leaves, grass, and other clipping fi'om the garden.
Floor sweepings: Tobacco, cigarettes, hair, paper, ect.
o Miseellrieous: Clothing, rugs, wood scraps, newspap er and magazines, glass,
t rubber tires, ect.

t Non-Biodegradable substances :

T r Plastics: Bottles, bags, jars, ect.

o l\l[etaL Cans , aluminum foil , medicine material, ect.
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I Strategies for Biodegradation:
contaminant following strategies are needed:
For the successful biodegradation of a given

I 1. Composting.
2. Bioremediation.
3. Biotransformation.
I 4. Bioleaching.
5. Biodeterioration.
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Biodegradation Dr. Ghadah Al-Quraishi

Conrposting

Iliodeg,radable \1 a-ste can be conr erled inll irseill na,r',rre ( .rrf l, tlr r r\ ,1 ltiilllt-8]
process in u,hicb decornposers hrcakdo\\ll kjlchcn, lasl. anrl garden rrali:rials into .t
dark. carthr'. soil-ljlie uraterial callcd "crnrpr.,-{i'. {lrj,s.iid pri.)rlrirt is rrr r: ccllcat soil
conditioner tbr plant-s. gardens and larurs. Cornposting is a u at to help reduce the
amount of u,aste materials going to our landh1l. fhe tinished compost c,att be used as a
soil amendment or mulch to improve most soils . I)ecomposition of organic material irr
the compost pile is dependent on maintaining the microbial activity.

Beneflts of eompost :

l.Compost acts like a sponge , helping soil to retain moisture and nutrient

2. helps breakdown heavy clay soils or help to bind sandy soils ,

3. reduce the need for expensive chemical fertilizers by returning nutrients back into the
soil ,

4. it is an ecofriendly method of waste management and results in flre generation of

good quality manure.

Materiak sdt*ble for compastine:

Composting requires carbon , nitrogen , oxygetr and water. Carbon provides enerry,
nitrogen is required for growth and reproduction, oxygen is needed for oxidation of
carbon whereas water maintain activif without creating anaerobic conditions.

A. Greens : Items contain nitrogen are generally green such as green grass,
greerr leaves, vegetable/fruit peels, flowers, ecI.

B. Browns: Items contain carbon are generally brown such as autumn leaves, dry grass
clippings, hair, ect.

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Dr. 6hadah Al-Quraishi

I BROlA'N
+*-ffik.: GRE EN
- COM POS-i
Leavas. sterv. Grasg food scraps,
v.,ood). matenals rnanureS
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Earttn .orrn3 iltsecrts- etc
MICRO.ORGANISMS
Eiacle^a. fungi. microt!€s

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Materials that should not he used to make compost :
I Certain substances are avoided since they cause bad odor or attract pests, such as :
.4r- mea! bones, fish scraps, and diary products attract rats and other animals to the site.
I B. plants or grass clrppings that have been treated with chemicals , or pesticides.
C. human or pet feces cannot be used as they may transmit disease, and diseased plants.

I
I Factors th*.t affect composting:

1. Air:Oxygen is required for microbes to decompose fie organic wastes

I efEcienfly. Decomposition in the absence of oxygen is slow and leads to the
development of foul odors.
2. Moisture: is necessary to maintain a steady decomposition rate.
I 3. I{eat decomposition of the organic material raises the tempe.rature , the compost
should be warm . Temperature of 55oC is desAable because it kills weed seeds

I 4.
and speeds up the composting process.
Particle size: breaking the waste into smaller particles speeds up the composting
process. Small particles have more surface area that can be easily attacked by the
I micrcbes , it also reduce ihe volume of the compost pile.

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Dr. Ghadah Al-Quraishi

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modification ends in rnineral cotnporurds sLrch as Ct )1. Nl-Ii. or ll2O, h iotransfolr r rui.l'on
is called mineralizalion .
I
Bacterial biotransformation can be used fr-rr the biore mediation of severlal xenobiotics
exploiting tl,e abilitv of microbes to degr:ade, 1r'ansfrrmr, or accumulate compounds
I b1,
such as h)drocarbons (e.g., oil), phannaceulical subst.ances and nretals, ect. lhis process
also has industrial biotechnological applications. For example. methanolrophic bact,:ria
I can be used to remediate 'ICE ( tetrachloroetltane, an industrial solvert) .

The te.rm xenobiotic is derived from Greek u,ords "xenos" meaning foreigner. stranger
I and "bios" meaning life .

Man made chemicals present in the nature at high concentrations polluting the
I environrnent is known as Xenobiotic compounds. These compounds are not commonly
produced by nature. Some microbes have been seen to be capable of breaking dolr.n of
I xenobiotics to some extent. But most of the xenobiotic compounds are non degradable
in nahrre. Such compounds are known to be recalcitrant in
nature.
For example, in humar-rs, most drugs axe part of this category, since people don't
I produce them naturally, or consume tiem under normal circumstances. Xenobiotics can
also be deflrned as substances that are present in higher-than-normal concentralions, or

I ones that are entirely artificial and did not exist before they rvere produced synthetically
by humans.

I The properfies ofxenobiotic compounds attribudng to its recalcitrant properties are:

(i) Non recognizable as subskate by microbes to act upon and degrade it.
I (ii) It does not contain permease which is needed for transport into microbial cell.
(iii) Large molecular nature makes it diffrcult to enter microbial cell.
(iv) They are highly stable and insolubility to $,ater adds to this property.
T (v) Mostly toxic in nature.

I Ilazards posed by xenobiotic compounds \. ]

The hazards posed by xenobiotics are huge. These compounds are highly ioxic in nature
and can affect survival of lower as well as higher ffiaryotes. It also poses health
I hazards to humans like various skin'problems, reproduclively and even known as a
trigger for causing bfirter. These compounds are persistent and remain in the
environment for many years leading to bioaccutnulation or biomagniJication. They
I also find a way into the food chains and the concentrations of such compounds was t'
found to be high even in organisms that do not come in contact with xenobiotics

I d irectly.

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 Dr.,Ghadah Al-Quraishi

Ived in triodegradation of xenotriotics:

owing to its recalcitrant nature, is hard to break dorlrr and
itv of its chemic.al composition adds to this. For breaking donl
erzymes act on certain groups present in the compound. Enz-vrnes
i maior role, The bonds like ester-, amide-, or ether bonds present
I,rs1 attacked lcadtng to,breakurg do*'tl of cornf''oillttls l-hc silt''
rend-s on tht: ecliott ol eitzr me. i1s clrtcenlralioll illlrl illi
Often it is seen that the renobiotics do not act as a source of
as a result t1-iev are not deggaded. 1he presence o1 a 'cuitable
:akdown. llris substrate is knou'n as co - rfielubollfe arld the
wn as c{, melabolism.

t Biomagnification:
is a process in-whiqh.pbroigal,9ghg1g9eg*baeOaoc-gereSo+centrated higher
T {1^94ch
trophic levql. Such chemical substances tetrd to be difficutt to be removed &om aniriial
and plant Jisrsues. The result is that when plants have the substance on them, they are
I eaten bv
accumulates in the
As each herbivore eats more of the planq--the s-@stance
.: when rhe herbivores are eaten uv-fCiffiiiiffiJ tle
'*.*ar!{a*gJ-_l
substance accumuht& in the cainivo.". As more herbivores are eate4 tfu arffiiinttG ihe
I chemical substance increases in the camivore. This scenario repeats itself at hi€llier
trophic levels, so that the top consumer has very high levels of the substance in its
I tissues. Often, such high levels of chemical substances causes mutations, cancers and
eventually death.

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I Dr. Ghadah Al-Quraishi

I ocY

I !na.ts lFi tn,lr'.r,!

o! OOI co.rc.cfirnlrdr r

.B="'"
;:0 (,-r

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t Bioleaching

Bioleaching is the extraction of specific metals fiom their ores tlrough the use of
'la t4b

I materials native to the environment , \ryater, afu. and microorganisms. It is frequently

used to extract miiibials such as gold and copper, from their ores. Mic.roorganisms gain
energy by breaking dou'n minerals inro their constituent elements. The;, L"4 un
I nutrients in minerals , thereb-v separating the metal , s'hich is then collected in a
solution.

I Organisms used for Bioleachinq: , ,.

The ferrous and sulfur oxidizing bacteria used for this process belong to the genus
T Thiobaciltus and were fust isolated from acid mihe rvaters. Various species used are
Thiobacillus ferroo-ridans and Thiobacillus lhiooxidans, Leplospirill uttt ferrooxidans,
and thermophilic species , Sulfolobus brierleyi and salfolobus metallicas . to date,
approximately, 30 naturally occurring strains of microorgzufsms have been screened as
being useful in bioleaching. Two fungal strains ( Aspergiltus niger art,d penicillium
I simplicissimun r) have also been reportedly used for bioleaching .

I Biodeterioration
I Biodegradative abilities of fi:ngi are not always beneficial. 'Ihese saprotr:ophic fungi
are also capable of contaminating our food sources and destoving coltsutner goods such
I as paper and paper products , leather, wooden items, and many similar things

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 Dr. Ghadah AI-Quraishi

manufacnred fi'om natural ra$, material-c. lor erample, "dn rot" fungLrs, .i,:11:ti/ii
l*r:4'ma;is, afiacks \\'ood ard tirnbers Ltsed in [Lre construction of buildings.

Any undesirable change in the properries of materials caused b1'rhe \.ital activiries of
organisms is called biodete riora tio n. Frequc'.ntly, in moist conditions fimgus g;lou,s on
shol,er curlains, shoes, clothes, q,ooden cupboards, ect. Sucrh biodet.erioration is cailed
fouling or soiling. and doe-s not causc se\ere damage to the nraterial. In some c-ases, the
material is used as somce of nutrients and energ),. this is knou,n as Chg-4qcgl
. ?;siUurlatory rype of biodeterioration and danages the rnater.iel. Example, food
spoilage. I_n gheryigal dissimilatory t1'pe of biodeterioration , substance is not used as
carbon and energy source, but is damaged in the process, for example: acid u,aste
plod]:cts, 9o!LlgguY, ect. Agents responsibie for biodeterioiation inpludq : exffiieliular
en'qimes,S-dcGfra fungi, insects., Igrms. The effects may include co6'bion, fouling,
'-'' rotting, 'decay, infection, disfiguremen! toxification. One of the most important
cellulytic molds h biodeterioration is Chaetomium. lt is commonly found in soil, on
paper, cloth, cotton and other cellulose containing substrates. It causes severe problems
in libraries, arc[ive-q, and food industry. Because of th eir 6iodeteriorarion ability ,
several strains of this firngus me used in testing materials for mold growth resistance.

Biofertilizres refer to living, microbial inoculants that ale added to the soil. They are
100% natural and organic that help to provide and keep in the soil all the nutrients and
microorganisms required for the benefits of the plants. Biological rvastes. biologically
active products, or microbial inoculants of bacteria, algae, and fungi (separately or in
combination), which may help in biological nitrogen fixation for the benefit of the
plants, are used as biofertilizers.

Commercial biofertilizers, also knolr,n as Microbial inoculants, are preparations

containiugligs or !3|9qt.9911-s of efficient strains of nitrogen - fiii"g , ph-o5phale-
-so]ubilizing or cellull'tic microorganisms used for application to seed or soil improving
soil fertility. Microbial inoculants can be classified as follorv:

J- Nitrogen fixers: *-hich are either: Symbiotic (Rhizobian) or Non-S),mbiotic

(aerobic bacteria like Azotohacter, Azomonas, lzospirillum ; anaerobic bacteia
like Clostridium and Chlorobium ).
) Symbiotic green algae: ,,4 n{tbfiena, A nabaenopsis, Nosfar, ect.
.3. Phosphate-solubilizing microorganisms: B n <:i ! ! u s rrt eg t li e r i u t1t.
4. Cellulytic and lignolytic microorganisms '.R nntinarocti:s sp.
5 Sulfur-dissolving bacteria : fhibacillus sp.

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Aquatic Microbiology Lectures: 1, 2, 3
Level: 4th

An aquatic ecosystem is an ecosystem in a body of water.

The two main types of aquatic ecosystems:
Marine ecosystems and Freshwater ecosystems.
Communities of organisms that are dependent on each other and on their environment
live in aquatic ecosystems.
Aquatic microbiology is the science that deals with microscopic living organisms in
fresh or salt water systems.
While aquatic microbiology can encompass all microorganisms, including bacteria,
viruses, and fungi and their relation to other organisms in the aquatic environment.

Bacteria, viruses and fungi are widely distributed throughout aquatic environments.
They can be found in fresh water rivers, lakes, and streams, in the surface waters and
sediments of the world's oceans, and even in hot springs.

Microorganisms live in these diverse environments must deal with a wide range of
physical conditions, and each has specific adaptations to live in the particular place it
calls home. For example, some have adapted to live in fresh waters with very
low salinity , while others live in the saltiest parts of the ocean. Some must deal with
the harsh cold of arctic waters, while those in hot springs are subjected to intense
heat. In addition, aquatic microorganisms can be found living in environments where
there are extremes in other physical parameters such as pressure, sunlight, organic
substances, dissolved gases, and water clarity.

Aquatic microorganisms obtain nutrition in a variety of ways. For example, some

bacteria living near the surface of either fresh or marine waters, where there is often
abundant sunlight, are able to produce their own food through the process
of photosynthesis. Bacteria living at hydrothermal vents on the ocean floor where
there is no sunlight can produce their own food through a process known
as chemosynthesis, which depends on preformed organic carbon as an energy source.
Many other microorganisms are not able to produce their own food. Rather, they obtain
necessary nutrition from the breakdown of organic matter such as dead organisms.

Importance of aquatic microorganisms

Aquatic microorganisms play a vital role in the cycling of nutrients within
their environment, and thus are a crucial part of the food chain. Many
microorganisms obtain their nutrition by breaking down organic matter in dead
plants and animals. As a result of this process of decay, nutrients are released in a form
usable by plants.
1
 These aquatic microorganisms are especially important in the cycling of the
nutrients nitrogen , phosphorus , and carbon. Without this recycling , plants would
have few organic nutrients to use for growth.

In addition to breaking down organic matter and recycling it into a form of

nutrients that plants can use, many of the microorganisms become food themselves.
There are many types of animals that graze on bacteria and fungi. For example,
some deposit-feeding marine worms ingest sediments and digest numerous bacteria
and fungi found there. Therefore, these microorganisms are intimate members of the
food web in at least two ways.

Humans have taken advantage of the role these microorganisms play in nutrient cycles.
At sewage treatment plants, microscopic bacteria are cultured and then used to
break down human wastes.
Specific zonations , microbial flora and microbial activity in water Column

Figure 1 :Specific zonations on the basis of light and temperature variations at

different depths of the water column
Water surface: 22-25 ° C

Epilimnion: 20-22° C

Metalimnion( Thermocline): 7 ° C

Hypolimnion: -5° C

Sediments
2
 However, in addition to the beneficial uses of some aquatic microorganisms,
others may cause problems for people because they are pathogens, which can cause
serious diseases. For example, bacteria such as Salmonella typhi, S. paratyphi, and the
Norwalk virus are found in water contaminated by sewage can cause illness. Fecal
coliform (E. coli ) bacteria and Enterococcus bacteria are two types of microorganisms
that are used to indicate the presence of disease causing microorganisms in aquatic
environments.

A-Water surface and Epilimnion: Microbial flora consists of:

1- Photosynthetic bacteria. 2- Cyanobacteria.
3- Mesophilic contaminating bacteria. 4- Psychrotrophic.

Microbial activities of this part of water column are:

1- Photosynthesis 2- Aerobic nitrogen fixation
3- Aerobic decomposition of organic matter

B- Thermocline: Microbial flora consist of Psychrophilic facultative anaerobic

bacteria .
Microbial activity of this part of water column are Aerobic and Anaerobic
decomposition .

C- Hypolimnion : Microbial flora consist of extrem psychrophilic anaerobic

bacteria. Microbial activity are:
1- Anaerobic nitrogen fixation bacteria (Clostridium pasteurianum),
2- Anaerobic decomposition of organic matter , 3- production of CH4 ,H2S,NH3.

D-Sediments: Microbial flora consist of:

1- Barophilic bacteria 2- Anaerobic bacteria ,H2S ,CH4 producing bacteria.
Microbial activity is Anaerobic decomposition of sediments.

Microbial Water Pollution

Water pollution is the contamination of water bodies (e.g. lakes, rivers, oceans
and ground waters). Water pollution occurs when pollutants are discharged directly or
indirectly into water bodies without adequate treatment to remove harmful compounds.
Water pollution is a major global problem which requires ongoing evaluation
and revision of water resource policy at all levels. It has been suggested that it is the
leading worldwide cause of deaths and diseases, and that it accounts for the deaths of
more than14,000 people daily. An estimated 1,000 Indian children die of diarrheal
sickness every day. Some 90 % of China’s cities suffer from some degree of water
pollution, and nearly 500 million people lack access to safe drinking water.
3
Main Sources of Water Microbial Pollution:
1-Sewage:
Most treated wastewater goes into rivers, lakes, and oceans. Occasionally, heavy
rains overwhelm sewer systems, causing them to overflow increasing the risk of water
borne-diseases. This can put communities at risk from high concentrations of microbial
pollutants in raw, untreated sewage. The mian source of water pollution with all
microorganisms through dumping untreated sewage into water bodies. This is very
dangerous because they contaminate the environment and water bodies and bring
many deadly diseases.
2-Agricultural and Animal Wastes
Over the past few decades, the increase in population and advances made in
farming technology has increased the demand for crops and livestock from the
agricultural industry. This growth in agricultural production has resulted in an increase
in contaminants polluting waterways.
Poorly managed animal feeding operations and dropping of dead animals
into water bodies also poorly managed and ineffective application of irrigation
water, and fertilizer(manure) are important sources of water pollution with
microorganisms. Nearly 1.4 billion tons of animal manure are produced annually in
the United States. These animal wastes carry concentrations of microbes as high as a
billion organisms per gram of feces. Most are part of normal bodily flora, but some are
potentially harmful to humans.
3- Industrial pollution: Most of industrial wastes polluted the water with chemicals;
however some of these industries such as medical and food industries polluted the
water with microorganisms. Unesco classified the water pollution according to the
bacterial pollution and organic pollution to seven classes as in the following Table:

Pollution level Bacterial pollution Organic pollution

(bacteria per ml water
Hypersaprobic 108 Heavy polluted
Polysaprobic β <106 Clearly polluted
α-mesosaprobic 105-106 polluted
β- mesosaprobic 102-105 Semi polluted
Oligosaprobic >102 Little polluted
Antisaprobic None Polluted with toxic
chemicals
Katharotic Very low Low organic matter

Low metals

(spring water)

4
Water-associated diseases
Water-associated diseases can be classified under 4 different categories:
1- Water-borne diseases
Definition: water-borne diseases are diseases caused by the ingestion of water
contaminated by human or animal faeces or urine containing pathogens.
Many bacteria, viruses, protozoa and parasites can cause disease when ingested. The
majority of these pathogens derive from human or animal faeces, and are transmitted
through the faecal-oral route.

2- Water-washed diseases
Definition: water-washed diseases are diseases caused by inadequate use of water
(water scarcity) for domestic and personal hygiene.
Control of water-washed diseases depends on: the quantity of water than the
quality. Most of the diarrhoeal diseases should be considered to be water-washed as
well as water-borne.
Four types of water-washed diseases are considered here:
A- soil-transmitted helminths B- acute respiratory infections (ARI)
C- skin and eye diseases D- diseases caused by fleas, lice, mites or ticks.
For all of these, washing and improved personal hygiene play an important role in
preventing disease transmission.
These types of diseases are more common in tropical, 3rd world countries where water
supplies may be scarce.

3- Water-based diseases
Definition: water-based diseases are infections caused by parasitic pathogens found
inaquatic host organisms ,or diseases caused by pathogens that have a complex life-
cycle which involves an intermediate aquatic host.
All of these diseases are caused by worms, e.g. Schistosomiasis caused by the
Schistosoma worm which uses aquatic snails as an intermediate host, also the Guinea
worm (Dracunculus medimensis) which uses a small crustacean as an intermediate host.

4- Water-related diseases
Definition: water-related diseases are caused by insect vectors which either breed in
water or bite near water.Very difficult to control and diseases are very severe .
Examples:
1-Yellow fever (viral disease) is transmitted by the mosquito.
2-Dengue (viral) carried by the mosquito (breeds in water).
3-Malaria is caused by a protozo and is also spread by a mosquito.
4-Trypanosomiasis (Gambian sleeping sickness) is also caused by a protozoan
transmitted by the riverine Tsetse fly.
5
Indicators of microbial water quality
To determine if a given water supply is safe, the source needs to be protected and
monitored regularly. There are two broad approaches to water quality monitoring
for pathogen detection.
The first approach is direct detection of the pathogen itself, for example, the
protozoan Cryptosporidium parvum. While it will be more accurate and precise if
specific disease-causing pathogens are detected directly for the determination of water
quality, there are several problems with this approach. First, it would be practically
impossible to test for each of the wide variety of pathogens that may be present in
polluted water. Second, even though most of these pathogens can now be directly
detected, the methods are often difficult, relatively expensive, and time-consuming.
Instead, water monitoring for microbiological quality is primarily based on a second
approach, which is to test for indicator organisms.

This concept of indicator organisms was introduced in 1892 and is the basis for most
microbiological quality standards in water today.

Indicator Microorganism: A non pathogenic microorganism whose presence

suggests the presence of enteric pathogens.
Indicator organisms are used because pathogens themselves are frequently difficult to
detect in drinking water and wastewater low numbers, difficult, time consuming, or
expensive to culture.

The US Environmental Protection Agency (EPA) lists the following criteria

for an organism to be an ideal indicator of fecal contamination:
1- An indicator should always be present when pathogens are present.
2- Indicators and pathogens should have similar persistence and growth characteristics.
3- Indicators and pathogens should occur in a constant ratio so that counts of the
indicators give a good estimate of the numbers of pathogens present.
4- Indicator concentrations should far exceed pathogen concentration at the source of
pollution.
5 - The indicator should not be pathogenic and should be easy to quantify.
6- Tests for the indicator should be applicable to all types of water.
6
7- The test should detect only the indicator organisms thus not giving false-positive
reactions.
8-Should survive longer in the environment than the toughest enteric pathogen.
9-Should be a member of the normal intestinal microflora of warm-blooded animals.

Types of indicators
1- Coliform Organisms (Total Coliform)
Coliform bacteria are metabolically defined as gram-negative, rod-shaped
bacteria capable of growth in the presence of bile salts and able to ferment lactose at an
optimum 35ºC.
The main reason is because they are easy to detect and enumerate in water and
are representative enough for determining microbial contamination of drinking water.
Besides the criteria discussed previously in regard to the choice of indicator
organisms, there are numerous reasons for their use. Waterborne pathogens such as
Vibrio cholerae and Salmonellaspp. usually die very quickly and are present in very low
numbers. These characteristics make their isolation and detection difficult and
impractical.

2-Thermotolerant Coliform Bacteria

This group of bacteria comprises the bacteria genus Escherichia, and to a lesser
extent, Klebsiella, Enterobacter, and Citrobacter. They are defined as a group of
coliform organisms that are able to ferment lactose at 44 to 45ºC. Sometimes, this group
is also called Fecal Coliform (FC) to specify coliforms of fecal origin.
However, concentrations of thermotolerant coliforms are usually directly related
to that of E.coli and thus can be used as a surrogate test for E.coli.
Escherichia coli (E.coli)
Escherichia coli is a specific subset of the thermotolerant coliform bacteria . They
are found abundantly in human feces and warm-blooded animals. Usually, E.coli
cannot multiply in any natural water environment and they are, therefore, used as
specific indicators for fecal contamination. Both WHO Guidelines and EPA
standards require zero E.coli to be found per 100 ml of drinking water sample.
Relationships among Total and
Fecal Coliforms and E. coli
Total Coliforms

Fecal Coliforms

Escherichia coli
• All total and fecal coliforms and E. coli
possess -galactosidase; they can
hydrolyze and and ferment lactose
• E. coli also possesses -
glucuronidase and hydrolyzes
glucuronide substrates

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3- Fecal Streptococci
Most of the species under the genus Streptococcus are of fecal origin and can be
generally regarded as specific indicators of human fecal pollution. However, certain
species may be isolated from the feces of animals. Fecal streptococci seldom multiply in
polluted water and they are more persistent than coliform and E.coli bacteria. Therefore,
they are generally useful as additional indicators of treatment efficiency. This indicator
organism is commonly tested with E.coli for evidence of recent fecal contamination.
Four key points in favour of the faecal streptococci are:
(1) Relatively high numbers in the excreta of humans and other warmblooded animals.
(2) Presence in wastewaters and known polluted waters.
(3) Absence from pure waters, virgin soils and environments having no contact with
human and animal life.
(4) Persistence without multiplication in the environment.

4- Sulfite-Reducing Clostridia
Sulfite-reducing clostridia are gram-positive, anaerobic, spore-forming bacteria.
Clostridial spores can resist treatment and disinfection processes better than most
pathogens. One of the members, Clostridium perfringens, like E.coli, is normally
present in feces, but in much smaller numbers. However, they are not exclusively of
fecal origin and can be found in other environmental sources , Clostridial spores can
survive in water much longer and resist disinfection better than other coliform groups
However, they are not recommended for routine monitoring of distribution systems
(on other word they tend to accumulate and are detected long after pollution has
occurred, thus giving rise to false alarms).

5- Bacteriophages
Bacteriophages (phages) are viruses that infect and replicate in specific bacteria. The
ability to identify phages (coliphages) of E.coli, also detects fecal contamination. This is
because the presence of coliphages also indicates the presence of E.coli. The
significance of coliphages as indicators of sewage contamination, and their greater
persistence compared to bacterial indicators make them useful as additional indicators
of treatment efficiency.

6- Heterotrophic Bacteria
Heterotrophic bacteria are members of a large group of bacteria that use organic
carbon for energy and growth. Many laboratories measure heterotrophic bacteria
(Pseudomonas, Aeromonas, Klebsiella, Flavobacterium, Enterobacter,) by the
heterotrophic plate count (HPC) Varies from 1 to 104 CFU/mL, and depend on
temperature, residual chlorine concentration, and availability of organic nutrients.

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 The presence of heterotrophic bacteria does not indicate the likelihood of
pathogen presence. However, a sudden increase in HPC may suggest a problem with
treatment or water disinfection. HPC > 500 CFU/mL indicates poor water quality.

Current methods of detection:

1- Most Probable Number (MPN)

2- Heterotrophic Plate Counts (HPC)

3- Membrane filtration and culture on selective media

Indicator bacteria can be cultured on media which are specifically formulated to

allow the growth of the species of interest and inhibit growth of other organisms.
Typically, environmental water samples are filtered through membranes with small
pore sizes and then the membrane is placed onto a selective agar. It is often necessary to
vary the volume of water sample filtered in order to prevent too few or too many
colonies from forming on a plate.

Figure 2 Membrane filtration method

Emerging methods of detection:

A- Fast detections using chromogenic substances

The time required to perform tests for indicator organisms has stimulated research
into more reliable and faster methods. One result is the use of chromogenic compounds,
which may be added to the conventional or newly devised media used for the isolation
of the indicator bacteria.
These chromogenic substances are modified either by enzymes (which are typical
for the respective bacteria) or by specific bacterial metabolites. After modification the
chromogenic substance changes its colour or its fluorescence, thus enabling easy
detection of those colonies displaying the metabolic capacity.
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In this way these substances can be used to avoid the need for isolation of pure
cultures and confirmatory tests. The time required for the determination of
different indicator bacteria can be cut down to between 14 to 18 hours.

B-Gene sequence-based methods

Advances in molecular biology in the past 20 years have resulted in a number of new
detection methods, which depend on the recognition of specific gene sequences. Such
methods are usually rapid and can be tailored to detect specific strains of
organisms on the one hand or groups of organisms on the other.
The new methods will influence epidemiology and outbreak investigations
more than the routine testing of finished drinking water.
1- PCR (polymerase chain reaction)
With the polymerase chain reaction and two suitable primer sequences (fragments
of nucleic acid that specifically bind to the target organism) trace amounts of DNA can
be selectively multiplied. In principle, a single copy of the respective sequence in the
assay can produce over a million-fold identical copies, which then can be detected and
further analysed by different methods.
One problem with PCR is that the assay volume is in the order of some micro-
litres, whereas the water sample volume is in the range of 100–1000 ml. Bej et al.
(1991) have published a filtration method to concentrate the sample, but another
problem is that natural water samples often contain inhibitory substances (such as
humic acids and iron) that concentrate with the nucleic acids.
Hence, it is critical to have positive (and negative) controls with each
environmental sample PCR to check for inhibition and specificity. It may also be
critical to find out whether the signal obtained from the PCR is due to naked nucleic
acids or living or dead micro-organisms .
One solution has been established by using a three-hour pre-incubation period in
a selective medium so that only growing organisms are detected .
2- FISH (fluorescence in situ hybridization)
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Microbiological standards for water
The microbiological examination of water is used worldwide to monitor and control the
quality and safety of various types of water. These include potable waters (water
intended for drinking or use in food preparation), treated recreational waters (swimming
pools, spa pools, and hydrotherapy pools), and untreated waters used for recreational
purposes such as sea, river, and lake water.
Various standard and guideline values have been introduced over the years. Many of
these have become legally enforceable, while others have been recommended by
appropriate bodies and trade associations.

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Water quality standards: are regulations or rules that protect lakes, rivers, streams
and other surface water bodies from pollution.

Drinking Water Standards: Defined as water quality parameters established for public
water supplies by regulatory authorities to define the limiting concentrations of various
constituents.
- Limiting concentrations are those that can be tolerated for the intended use ,are
revised periodically.

Standards Classifications
- Primary Standards: are health related and enforceable which includes such
parameters as: – Coliforms, turbidity, toxic inorganic and organic chemicals.

- Secondary Standards: are non-health related used for aesthetic purposes which
includes: – Color, odor, chloride ion, sulfate ion, dissolved solids, manganese, pH,
copper, and zinc.

Some Important World Standards

- The World Health Organization (WHO)
- Set up some guidelines for drinking-water quality which are the international reference
point for standards setting and drinking-water safety.
- Unesco :united nation education ,science and culture organization
- The European Union (EU)
- US Environmental Protection Agency (EPA)
-Icsqc :Iraqi center of standardization and quality control

For example :DrinkingWater Standards

Fecal coliform counts should be zero per 100 mL of sample (0/100 mL) in all
water supplies, piped or unpiped, treated or untreated.
Total coliform counts should be 0 per 100 mL in piped, treated water
supplies with an occasional occurrence of two organisms per 100 mL being allowed.
In unpiped water supplies, total coliform counts of 10 organisms per 100 mL
can occur infrequently.
Frequent occurrences of high coliform counts signify the need for an
alternative water source, or sanitary protection of the current source. A “boil water”
order is needed when emergency water supplies fail to meet a criterion of zero (0) fecal
and total coliforms.

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 Microbiological guidelines and standards for drinking water

Water Treatment
The principal objective of water treatment is to provide potable water that is
chemically and biologically safe for human consumption. It should also be free from
unpleasant tastes and odors.

Steps of water treatment:

1-Flocculation
Flocculation refers to water treatment processes that combine small particles
(clay, organic material, metals, microorganisms) into larger particles, which often quite
small and so will not settle out from the water column without assistance.
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 To help the settling process along, "coagulating" compounds are added to the
water, and suspended particles "stick" to these compounds and create large and
heavy clumps of material. Alum and iron salts or synthetic organic polymers (used
alone or in combination with metal salts) are generally used to promote coagulation.

2-Sedimentation
The water is left undisturbed to allow the heavy clumps of particles and
coagulants to settle out.

3-Filtration
The water is run through a series of filters which trap and remove particles still
remaining in the water column. Typically, beds of sand or charcoal are used to
accomplish this task.
Many water treatment facilities use filtration to remove all particles from the
water. Those particles include clays and silts, natural organic matter, precipitates from
other treatment processes , filtration clarifies water and enhances the effectiveness of
disinfection.

5-Disinfection (chlorination/ozonation)
Water is often disinfected before it enters the distribution system to ensure that
potentially dangerous microbes are killed. Chlorine, chloramines, or chlorine dioxide
are most often used because they are very effective disinfectants, not only at the
treatment plant but also in the pipes that distribute water to our homes and businesses.
Ozone is a powerful disinfectant, and ultraviolet radiation is an effective disinfectant
and treatment for relatively clean source waters, but neither of these are effective in
controlling biological contaminants in the distribution pipes.

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***To produce water that is safe to drink, pleasant in taste and could be used for various
purposes, treatment processes should be selected to remove specific constituents from
raw water. The basis for selecting treatment process alternatives is established by the
characteristics of raw water and the finished water quality goals.
***In addition to these five steps of water treatment, there are other steps:
1-Fluoridation
2- remove of iron and manganese.
3-water softening (remove of hardness)
4-remove of dissolved salts ,phosphprus ,nitrogen
5-remove of color ,odor, taste,etc.
Post treatment water contamination
The treated drinking water may be exposed to the contamination in distribution system
(pipes) of water due to :
1- Main breaks, repairs and installation in WTP and distribution system, operation and
maintenance deficiencies and cross-connections cause entering the pollutants from the
surrounding soil .
2- From the sewers through leaks or flooding of sewers.
3-Connection the new pipes from new buildings.
4-Biofilm in the old pipes are reduced the chlorine content in the distribution system
which lead to growth of resistant bacteria.

Water chlorination
In water treatment, disinfection is the most important treatment step. The most
important oxidant used for disinfection is chlorine Cl2.
 Chlorine is rather soluble in water: solubility decrease at rising temperature.

 It is very applicable and very effective for the deactivation of pathogenic

microorganisms.

 Chlorine can be easily applied, measures and controlled. It is relatively cheap.

Chlorination
Chlorination can be achieved by using liquefied chlorine gas, sodium
hypochlorite solution or calcium hypochlorite granules and on-site chlorine
generators. Chlorine, whether in the form of chlorine gas from a cylinder, sodium
hypochlorite or calcium hypochlorite, dissolves in water to form hypochlorous acid
(HOCl) and hypochlorite ion (OCl-).
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Free “active” chlorine= Hypochloric acid (HOCl) & hypochlorite ion (OCl-).
Hypochloric acid is the most reactive and a stronger disinfectant because it’s neutral.

Chlorination is employed primarily for microbial disinfection. However, chlorine also

acts as an oxidant and can remove or assist in the removal of some chemicals for
example, decomposition of easily oxidized pesticides.

A disadvantage of chlorine: is its ability to react with natural organic matter and
plant phenolic compounds to produce carcinogenic such as trihalomethane and
chlorophenolic compounds respectively.
When chlorine is added to water, some of the chlorine reacts first with organic
materials and metals in the water and is not available for disinfection (this is called the
chlorine demand of the water). The remaining chlorine concentration after the
chlorine demand is accounted for is called total chlorine.

Total chlorine is further divided into:

1) the amount of chlorine that has reacted with nitrates and is unavailable for
disinfection which is called combined chlorine.
2) the free chlorine , which is the chlorine available to inactivate disease-causing
organisms, and thus a measure to determine the potability of water.

Breakpoint chlorination
It is a point in which the chlorine dose is sufficient to rapidly oxidize all the ammonia
nitrogen in the water and to leave suitable free residual chlorine available to protect the
water against reinfection from the point of chlorination to the point of use.

Cl2 + H2O HOCl + H+ + Cl–

Monochloramine:

Dichloramine:

Trichloramine or nitrogentrichloride:

Whole ammonia removed from water when reaching the breakpoint reaction, this
point is achieved when the ratio of chlorine to ammonia –nitrogen will be ten to one
(10/1).

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The steps of chlorine action in water:
How chlorine kills/deactivates
Chlorine disinfects water through two mechanisms:
 oxidizing power of free oxygen

 chlorine substitution reactions

Free oxygen is produced by the breakdown of chlorine compounds in water
HOCl HCl + O
Oxygen attacks the lipids in cell walls and destroys the enzymes and structures inside
the cell which either destroys or inactivates it.

Chlorine substitution

 Chlorine breaks chemical bonds in enzyme molecules and replace some hydrogen
atoms with chlorine.
 This changes the shape or destroys the molecules and makes it difficult for the
enzymes to function properly.

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Post-chlorination
The development of an excessive growth of bacteria can be prevented by the
application of a water treatment system proper for taking out the nutrients necessary for
bacterial growth and by the maintenance of free chlorine residual in the water to be
distributed.
In post chlorination so much chlorine is added to the already disinfected water
that drinking water entering the distribution system contains a free chlorine residual of
0.3to 0.5g Cl2/m3.
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Biological Wastewater Treatment
Wastewater: is simply that part of the water supply to the community or to the industry
which has been used for different purposes and has been mixed with solids either
suspended or dissolved.Wastewater is 99.9% water and 0.1% solids. The main task
in treating the wastewater is simply to remove most or all of this 0.1% of solids
People excrete 100-150 grams wet weight of feces and 1-1.3 liters of urine per person
per day.

The amount of organic matter in domestic wastewater determines the degree of

biological treatment required.
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Three tests are used to assess the amount of organic matter:
 Biochemical Oxygen Demand (BOD)

 Chemical oxygen demand (COD)

 Total Organic Carbon (TOC)

Measurements of organic matter

Many parameters have been used to measure the concentration of organic matter in
wastewater.

The following are the most common used methods:

1. Biochemical oxygen demand (BOD)
BOD: is the oxygen equivalent of organic matter. It is determined by measuring the
dissolved oxygen used by microorganisms during the biochemical oxidation of organic
matter in 5 days at 20oC.
2. Chemical oxygen demand (COD): It is the amount of oxygen necessary to oxidize
all the organic carbon completely to CO2 and H2O. Is measured by oxidation with
potassium dichromate (K2Cr2O7) in the presence of sulfuric acid and silver and
expressed in milligram per liter.
3. Total organic carbon (TOC)This method measures the organic carbon existing in
the wastewater by injecting a sample of the wastewater in special device in which the
carbon is oxidized to carbon dioxide then carbon dioxide is measured and used to
quantify the amount of organic matter in the wastewater . This method is only used for
small concentration of organic matter.

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Biofilms in Drinking Water Distribution Systems
Biofilm is a complex mixture of microbes, organic and inorganic material accumulated
amidst a microbially produced organic polymer matrix attached to the inner surface of
the distribution system pipe. The inner surface of a water pipe may have a continuous
biofilm, but usually biofilms are quite patchy.

Factors causing biofilm growth

1- Presence of microbial nutrients in water.
2- Characteristics of pipe wall such as roughness.
3- Microbial and chemical quality of water entering the system.
4- Water temperature and pH.
5- Low chlorine level in water.
6- Velocity of water.

Biofilms in drinking water pipe networks can be responsible for a wide range of
water quality and operational problems there are:
1- Biofilms can be responsible for loss of distribution system disinfectant residuals.
2- Increased bacterial levels, bacteria may be sloughed from the biofilm into the
water column due to changes in the flow rate,as a result, biofilms can act as a
slow-release mechanism for persistent contamination of the water.
3- Reduction of dissolved oxygen.
4- Reduce the utility of total coliforms as indicator organisms
5- Taste and odor changes, red or black water problems due to iron or sulfate-
reducing bacteria.
6- Microbial-influenced corrosion, hydraulic roughness, and reduced materials life .

Many different microbes have demonstrated the ability to survive in the distribution
system, with some possessing the ability to grow and/or produce biofilms. Some of
these organisms may be primary pathogens (i.e., those that cause disease in healthy
individuals), while others may be opportunistic pathogens (i.e., those that cause
disease in individuals with underlying conditions that may facilitate infection).

The biofilm can protect microbes from disinfection and allow microbes injured by
environmental stress and disinfectants to recover and grow.

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 Processes involved in the biofilm growth

Opportunistic Bacterial Pathogens Detected in the Distribution System and/or

Biofilms

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