Lab soil

 Subject objective: Each student should be able to
 To isolate antibiotic-producing microorganisms
(Bacillus, Penicillium and Actinomycetes colonies) from
soil that may be antibiotic producers.
 To determine the spectrum of the antimicrobial activity
of the isolated antibiotic against some clinical important
bacteria (Staphylococcus epidermidis as a Gram positive
and Escherichia coli as a Gram-negative bacteria).
 Biological assay for screening of antimicrobial activity.
 Reporter screening

Principle:
 The antibacterial effect of penicillin was discovered by Alexander Fleming in 1929.
He noted that a fungal colony had grown as a contaminant on an agar plate streaked
with the bacterium Staphylococcus aureus, and that the bacterial colonies around the
fungus were transparent, because their cells were lysing. Fleming had devoted much of
his career to finding methods for treating wound infections, and immediately
recognised the importance of a fungal metabolite that might be used to control bacteria.
The substance was named penicillin, because the fungal contaminant was identified as
Penicillium notatum. (See Figure A) Fleming found that it was effective against many
Gram positive bacteria in laboratory conditions, and he even used locally applied, crude
preparations of this substance, from culture filtrates, to control eye infections.
 The phenomenal success of penicillin led to the search for other antibiotic-
producing microorganisms, especially from soil environments. One of the early
successes (1943) was the discovery of streptomycin from a soil actinomycete,
Streptomyces griseus. actinomycetes are bacteria that produce branching filaments
rather like fungal hyphae, but only about 1 micrometer diameter. They also produce large
numbers of dry, powdery spores from their aerial hyphae. Actinomycetes, especially
Streptomyces species, have yielded most of the antibiotics used in clinical medicine
today. Some examples are shown in the table (1).
 Other bacteria, including Bacillus species (see Figure E: Agar plate showing inhibition of
fungal growth by a contaminating colony of Bacillus species.), have yielded few useful
antibiotics. Fungi also have yielded few useful antibiotics. Apart from penicillin, the
most important antibiotics from fungi are the cephalosporins (beta-lactams with
similar mode of action to penicillin, but with less allergenicity).

Figure E. The constant search of soils throughout the world has yielded an abundance of
antibiotics of great value for the treatment of many infectious diseases. Pharmaceutical
companies are in constant search for new strains of bacteria, molds, and Actinomyces that can be
used for antibiotic production. Although many organisms in soil produce antibiotics, only a small
portion of new antibiotics are suitable for medical use. In this experiment an attempt will be made
to isolate an antibiotic-producing Bacillus, Actinomyces and Penicillium from soil. Students will
work in group. Figure 1 illustrates the procedure.

 Antimicrobial agents: are substances that are naturally produced by a variety of
microorganisms (primarily Actinomycetes, fungi and bacteria), or have been synthesized
in the laboratory, or a combination of both.
 Antibiotic: refers only to those antimicrobial substances produced by microorganisms,
but the term is often used interchangeably with antimicrobial agent. Antimicrobial
agents have inhibitory or lethal effects on many pathogenic organisms (especially
bacteria) that cause infectious diseases.
 Antibiotic producer such as:
Antibiotics are the best known products of actinomycete. Over 5000 antibiotics have been
identified from the culture of gram positive, gram negative organisms and filamentous
fungi, but only100 antibiotics have been commercially used to treat human, animal and
plant disease. The genus Streptomycete is responsible for the formation of more than
60% of known antibiotics. While further 15% are made by number of related
Actinomycete, Micromonospora, Actinomadura, Streptoverticillium and
Thermoactinomycetes
1. Streptomyces spp.: produce (Chloramphenicol, Erythromycin, Kanamycin, Neomycin,
Nystatin, Rifampin, Streptomycin, Tetracyclines, Vancomycin)
2. Micromonospora: produce (Gentamicin)
3. Bacillus:Produce (Bacitracin, polymxins)
4. Fungi:
 Penicillium griseofulvum: produce (Griseofulvin)
 Cephalosporium: produce Cephalosporins

Some clinically important antibiotics
Site or mode of
action
ActivityProducer organismAntibiotic
Wall synthesisGram-positive bacteriaPenicillium chrysogenumPenicillin
Wall synthesisBroad spectrumCephalosporium acremoniumCephalosporin
MicrotubulesDermatophytic fungiPenicillium griseofulvumGriseofulvin
Wall synthesisGram-positive bacteriaBacillus subtilisBacitracin
Cell membraneGram-negative bacteriaBacillus polymyxaPolymyxin B
Cell membraneFungiStreptomyces nodosusAmphotericin B
Protein synthesisGram-positive bacteriaStreptomyces erythreusErythromycin
Protein synthesisBroad spectrumStreptomyces fradiaeNeomycin
Protein synthesisGram-negative bacteriaStreptomyces griseusStreptomycin
Protein synthesisBroad spectrumStreptomyces rimosusTetracycline
Protein synthesisGram-positive bacteriaStreptomyces orientalisVancomycin
Protein synthesisBroad spectrumMicromonospora purpureaGentamicin
Protein synthesisTuberculosisStreptomyces mediterraneiRifamycin

 Why the few antibiotics are clinically useful?
 Several hundreds of compounds with antibiotic activity have been isolated
from microorganisms over the years, but only a few of them are clinically useful.
The reason for this is that only compounds with selective toxicity can be used
clinically - they must be highly effective against a microorganism but have
minimal toxicity to humans. In practice, this is expressed in terms of the
therapeutic index - the ratio of the toxic dose to the therapeutic dose. The
larger the index, the better is its therapeutic value. So the antibacterial product
should be assessed by pharma and then decide to put in the market when it
passes ADME/T test
 It will be seen from the table above, that most of the antibacterial agents act
on bacterial wall synthesis or protein synthesis. Peptidoglycan is one of the
major wall targets because it is found only in bacteria. Some of the other
compounds target bacterial protein synthesis, because bacterial ribosomes
(termed 70S ribosomes) are different from the ribosomes (80S) of humans and
other eukaryotic organisms. Similarly, the one antifungal agent shown in the
table (griseofulvin) binds specifically to the tubulin proteins that make up the
microtubules of fungal cells; these tubulins are somewhat different from the
tubulins of humans.

 Factors affecting antibiotic production:
1. Medium Composition:
 Carbon source
 Nitrogen source
 Inorganic phosphates
 Inorganic salts
 Trace metals
 Precursors
 Inhibitors
 Inducers
2. Fermentation Conditions:
 pH
 Temperature
 Oxygen
 How can determine the target of inhibitor molecule which may inhibit one of
the biological pathways?
 There are many different pathwaies can be applied such as reporter essay by using the
reporter strains

This lab. Consist
of three steps
Primary isolation
Colony selection
&
Inoculation
Evidence of antibiosis
&
Confirmation

FIRST STEP:
 (Primary Isolation)
Unless the organisms in a soil sample are thinned out sufficiently, the isolation of potential
antibiotic producers is nearly impossible.
Materials per group of students:
1. six large test tubes, one bottle of physiological saline solution
2. Three Petri plates of glycerol yeast extract agar, Tryptic soy agar, Sabouraud
dextrose agar.
3. L-shaped glass rod, beaker of alcohol
4. six 1 ml pipettes, one 10 ml pipette
 Procedure:
1. Label six test tubes, and with a 10 ml pipette, dispense 9 ml of saline into each tube.
2. Weigh out 1 g of soil and deposit it into tube 1.
3. Vortex mix tube 1 until all soil is well dispersed throughout the tube.
4. Make a tenfold dilution from tube 1 through tube 6 by transferring 1 ml from tube to
tube. Use a fresh pipette for each transfer and be sure to pipette-mix thoroughly before
each transfer.
5. Label three Petri plates with your initials and the dilutions to be deposited into them.
6. From each of the last three tubes transfer 1 ml to a plate of glycerol yeast extract agar.
7. Spread the organisms over the agar surfaces on each plate with an L-shaped glass rod that
has been sterilized each time in alcohol and open flame. Be sure to cool rod before using.
8. Incubate the plates at 30° C for 7 days.

 SECOND STEP
(Colony Selection and Inoculation)
 The objective in this laboratory period will be to select Bacillus, Penicillium and Actinomyces-like
colonies that may be antibiotic producers. The organisms Penicillium sp and Actinomyces will be
streaked on nutrient agar plates that have been seeded with Staphylococcus epidermidis, and
Bacillus will be streaked on nutrient agar plates that have been streaked firstly by fungi, after
incubation we will look for evidence of antibiosis. Students will continue to work in groups. Figure 2
illustrates the procedure.
 Materials per group of students:
1. four trypticase soy agar pours (liquefied)
2. four sterile Petri plates
3. TSB culture of Staphylococcus epidermidis, Bacillus firmus, and Penicillium sp.
4. 1 ml pipette
5. three primary isolate plates from previous period water bath at student station (50° C)
Procedure:
1. Place four liquefied agar pours in water bath (50°C) to prevent solidification, and then inoculate
each one with 1 ml of S. epidermidis.
2. Label the Petri plates with your initials and date.
3. Pour the contents of each inoculated tube into Petri plates. Allow agar to cool and solidify.
4. Examine the three primary isolation plates for the presence of Bacillus sp. Penicillium sp. and
Actinomyces-like colonies. Actinomyces have a dusty appearance due to the presence of spores.
They may be white or colored. Your instructor will assist in the selection of colonies.
5. Using a sterile inoculating needle, scrape spores from Penicillium sp. and Actinomyces-like colonies
on the primary isolation plates to inoculate the seeded TSA plates. Use inoculums from a different
colony for each of the four plates.
6. Incubate the plates at 30° C until the next laboratory period.

 THIRD AND FOURTH STEPS
(Evidence of Antibiosis and Confirmation)
 Examine the four plates you streaked during the last laboratory period. If you
see evidence of antibiosis (inhibition of S. epidermidis growth and Fungal
growth), proceed as follows to confirm results.
 Materials:
1. 3 Petri plates of trypticase soy agar
2. TSB culture of S. epidermidis, PDA culture of Penicillium and Aspergillus sp.
 Procedure:
1. If antibiosis is present for each of Actinomyces, Penicillium, Bacillus, use
three TSA plates and make two streaks on each of the TSA plates as shown in
figure 2. Make a straight line streak from (antibiotic producer microorganisms)
2. cross-streak with organisms from a culture of S. epidermidis and Aspergillus
sp.
3. Incubate at 30° C until the next period.

Subject objective: Each student should be able to
• Being able to determine which kind of organic compounds (carbohydrates, proteins,
and lipids) are more decomposed by soil microorganisms under different :
1. Temperatures (20,25 and 37°C)
2. Humidity (40%, 60% and 80% of field capacity)
• Effect of incubation time (7, 14, 21 and 28 days) on organic compound
decomposition
Materials per Group of Students:
• 1 kg of garden soil.
• 0.5gm of each (cellulose, starch, glucose, peptone, lipids, HCl (1N), NaOH (1N),
phenophthalene, (BacL2 )
• 5 beakers with 5 test tubes,(wax pencil, Bunsen burner, Oven, pipette with pipetter)

CARBON CYCLE```
 The concentration of carbon in living matter (18%)is almost 100
times greater than its concentration in the earth (0.19%).
 So living things extract carbon from their non-living
environment.
 For life to continue, this carbon must be recycled.

Strictly speaking the “total carbon” of the soil comes from two principal
sources:
 Inorganic carbon Carbon dioxide in the atmosphere and dissolved in water
(forming bicarbonate - HCO3, Carbonate rocks(lime stone and coral - Ca CO3,
 Organic carbon (only slightly processed organic residues of plant and animal
origin, humus, charcoal, petroleum, fossil organic matter, Dead organic matter,
e.g., humus in the soil, microorganisms). In the majority of methods, the gas
phases present in the atmosphere of the soil (CO2 linked with biological
activity, CH4).Soil organic matter (SOM) can be of plant, animal, or microbial
origin and the terms “soil organic matter” and “humus” are considered
synonyms.
Organic matter is anything that contains carbon compounds that were
formed by living organisms. Four main components are:
 1-dead forms of organic material - mostly dead plant parts (85%)
 2-living parts of plants - mostly roots (10%)
 3-living microbes and soil animals
 4-Partly decayed organic matter is called humus

Organic matter is the vast array of carbon compounds in soil. Originally created by plants,
microbes, and other organisms, these compounds play a variety of roles in nutrient,
water, and biological cycles. For simplicity, organic matter can be divided into two major
categories: stabilized organic matter which is highly decomposed and stable, and the
active fraction which is being actively used and transformed by living plants, animals,
and microbes. Two other categories of organic compounds are living organisms and
fresh organic residue. These may or may not be included in some definitions of soil
organic matter.
Organic matter plays a determining role in pedogenesis and can drastically modify the
physical, chemical, and biological properties of soil (structure, plasticity, color, water
retention). The fundamental processes of evolution include phenomena of
mineralization and immobilization and, in particular, of carbon and nitrogen.
 Mineralization: allows the transformation of organic residues into inorganic
compounds in the soil, the atmosphere, and the hydrosphere, these are usable by flora
and by micro-organisms.
Carbon returns to the atmosphere by
1. respiration (as CO2)
2. burning
3. Decay (producing CO2 if oxygen is present, methane (CH4) if O2 is absent.
Immobilization: is the transformation of organic matter into more stable organic and
organomineral compounds with high molecular weights that are fixed in the interlayer
spaces of clays. These processes are summarized by the following diagram

 Major steps in the degradation of organic matter and their
types:
1. The dead organic matter is colonized by microbes and degraded
with help of microbial enzymes
2. Macromolecules are broken down into simpler units and
further degraded into constituent elements.
 Breakdown of compounds that are easy to decompose (e.g. sugars,
starches and proteins)
 Breakdown of compounds that may take several years to decompose
(cellulose and lignin)
 Breakdown of compounds that may take 10 years (e.g. waxes and phenols)
 Compounds that may take 100-1000’s of years (e.g. humus like
substances, which are very complex)

Atmospheric
CO2
CO2 from plant and
animal respiration
Assimilation of
CO2 by plants
CO2 from
degradation
of lignin
Fungal mycelia
Glucose from degradation of
cellulose transferred to fungivores
like insect larvae, ants, and squirrels

Decomposition
 When organisms die and decay, the carbon molecules in them
enter the soil.
 Microorganisms break down the molecules, releasing CO2
• Oxygenic photosynthesis:
CO2 + H2O (CH2O) + O2
• Respiration:
(CH2O) + O2 CO2 + H2O

Procedure:
1. After knowing the volume of water that need for obtaining 60% of soil
humidity, we add 0.5gm of different organic compound (Cellulose, glucose,
starch, peptone) to each beaker respectively, with remaining 5th beaker
without addition of organic compound it act as a control.
2. Vertically fix or put test tube containing (15ml) of NaOH (1N) in each soil
sample, then put cover on each beaker to avoid reaction of NaOH with air
CO2.
3. Incubate the samples at 25°C for 3 weeks (interval= 1 week)
4. At the end of each week we estimate volume of released CO2 form organic
compound decomposition by titrating NaCH (1N) test tube with HCl (1N)
after addition of BaCl2 and some drops of phenolphthalein as an indicator
for determination end point of reaction between HCl and NaOH by changing
their color from pink to colorless.

After titration calculation is done by the following steps:
we designate the letter (X) for the (ml) of NaOH that reacted with CO2 in controlled
test tube.
X=15 ml of NaOH- (?)ml of NaOH reacted with HCl= (?)
we designate the letter (Y) for the (ml) of NaOH that reacted with CO2 in a different
test tube
Y=15 ml of NaOH- (?)ml of NaOH reacted with HCl= (?)
We designate the letter (Z) for the volume of NaOH that reacted with released CO2
form decomposed of organic compounds.
Z = Y – X = (? ) ml of NaOH purely reacted with released CO2 from decomposition of
studied organic compound
Amount of CO2 released from = Volume of NaOH that reacted with CO2 = CO2 (mg)
organic compound decomposition

Amount of CO2 (mg) = Equivalent weight × Z(1?) = (2?)
Equivalent weight (CO2)= Molecular weight / equivalent= 12+ 2×16 / 2=
22=2/44=
Amount of CO2 (mg) = Equivalent weight × Z = ?
22 × (2?) = (3?)
C2CO
M.wt. 44 12
Mg (3?) X
X= (3?)×12 / 44= (4?) mg of C that released from the 1st week and so on for the next
week.
Then at the end of three weeks carbon (C) measurements draw a diagram showing C
mg and time as follow:

Time by week
Cellulose
Peptone
Glucose
Starch
1 2 3

0
20
40
60
80
100
120
140
160
180
200
incubaction time (days)
AccunulativemineralizedC/2gm
ofdifferentcarbohydrates
Glucose 87 111.3 131.7 142.5 151.5 159.3 164.1 167.1 170.8 172.1
Maltose 60 91.5 117.9 138.9 153.9 165.9 176.7 179.7 185.7 187.9
Lactose 36 63.2 84.2 102.2 118.4 132.9 143.7 152.7 160.2 163.7
Cellulose 0 0.9 8.7 17.1 29.2 38.4 46 52.9 57.6 60.6
Starch 0 0.9 29.7 48.3 59.4 67.6 74.6 77.7 80.2 82.3
3 6 9 12 15 18 21 24 27 30
Cumulative carbon dioxide released from soils treated with different
carbohydrates (polymers and monomers) at 25°C, (60% humidity) in (30 days).

0
10
20
30
40
50
60
70
80
90
100
Incubation time (days)
mgofmineralizedC/2gmof
differentcarbohydrates
Glucose 87 24.3 20.4 10.8 9 7.8 4.8 3 3.7 1.3
Maltose 60 31.5 26.4 21 15 12 10.8 3 6 2.2
Lactose 36 27.2 21 18 16.2 14.5 10.8 9 7.5 3.5
Cellulose 0 0.9 7.8 8.4 12.1 9.2 7.6 6.9 4.7 3
Starch 0 0.9 28.8 18.6 11.1 8.2 7 3.1 2.5 2.1
3 6 9 12 15 18 21 24 27 30
CO2 efflux by soil microorganisms, mean (mean=3) respiration among different
polymers and monomers carbohydrates in different time intervals (3-days).

0
50
100
150
200
250
AccumulativeofmineralizedC/2gm
ofdifferentaminoacidsandproteins
Alanine 48 78.6 107.4 134.4 156.4 175.2 191.4 203.4 211.6 216.4
Lysine 46.8 78.6 105.8 127.8 146.4 160.2 168.2 172.2 175.3 176.2
Albomine 19.8 92.7 123.9 145.5 163.5 179.7 192.7 198.4 203.2 205.4
Casein 44 69 90 105 111.3 116.2 120 122.9 125 126.2
Peptone 69 104.4 131.4 156 174 183 188 191.8 193.2 194.4
3 6 9 12 15 18 21 24 27 30

0
10
20
30
40
50
60
70
80
mgofmineralizedC/2gmof
differentaminoacidsandproteins
Alanine 48 30.6 28.8 27 22 18.8 16.2 12 8.2 4.8
Lysine 46.8 31.8 27.2 22 18.6 13.8 8 4 3.1 0.9
Albomin 19.8 72.9 31.2 21.6 18 16.2 13 5.7 4.8 2.2
Peptone 69 35.4 27 24.6 18 9 5 3.8 1.4 1.2
Casein 44 25 21 15 6.3 4.9 3.8 2.9 2.1 1.2
3 6 9 12 15 18 21 24 27 30
CO2 efflux by soil microorganisms, mean (mean=3) respiration among different
polypeptides and amino acids different in different time intervals (30 days).

0
20
40
60
80
100
120
140
160
180
200
1 2 3 4 5 6 7
Incubaction time ( weeks )
mgofC/2.5gmofplantresidue
100% of FC
80% of FC
60% of FC
40% of FC
Cumulative C mineralized (mean; n = 3) in different humidity conditions of soils, at
10°C and in different durations.

0
50
100
150
200
250
1 2 3 4 5 6 7
Incubation time ( weeks )
mgofC/2.5gmofplantresidue
100% of FC
80% of FC
60% of FC
40% of FC
Cumulative C mineralized (mean; n = 3) in different humidity conditions of soils,
at 15°C and in different durations.

General purposes:
1- To make the students aware with the role of microbes in
maintaining environment, existing microbial interactions
and recycling of nutrients in nature:
2- A technique for the isolation of a free living soil bacterium
Azotobacter.
3- A technique for the isolation of root nodule Bacterium
Rhizobium sp.

Cycling can be studied at different scales

What is nitrogen?
Or nitrogen cycle?

By traveling through one of the four
processes in the Nitrogen Cycle!
(1) Nitrogen Fixation
(3) Nitrification (2) Ammonification
(mineralization)
(4) Denitrification
Nitrogen
Cycle

•modified from Goldman and Horne. 1994. Limnology. McGraw Hill.
Nutrients- The Nitrogen Cycle

Why does atmospheric
nitrogen need to be
converted?
N
N
N
N
N
N

It is one of nature’s great
ironies…
Nitrogen is an essential component of DNA, RNA,
and proteins—the building blocks of life.
why is N fixation important?
• atmospheric N2 is inert – biotically unavailable.
• availability of fixed N is often the factor most
limiting to plant growth

How could atmospheric nitrogen
be changed into a form that can
be used by most living organisms?
N
N

There are three ways that nitrogen
could be “fixed”!
(a) Atmospheric Fixation
(b) Industrial Fixation
(c) Biological Fixation
Bacteria

Atmospheric Fixation
(Only 5 to 8% of the Fixation
Process)
The enormous energy of lightning
breaks nitrogen molecules apart
and enables the nitrogen atoms to
combine with oxygen forming
nitrogen oxides (N2O). Nitrogen
oxides dissolve in rain, forming
nitrates. Nitrates (NO3) are carried
to the ground with the rain.
Lightning “fixes” Nitrogen!
Nitrogen
combines
with Oxygen
Nitrogen oxides forms
Nitrogen
oxides dissolve
in rain and
change to
nitrates
Plants use
nitrates to grow!
(NO3)
N
N O
(N2O)

Industrial Fixation
Under great pressure, at a
temperature of 600 degrees
Celsius, and with the use of a
catalyst, atmospheric nitrogen
(N2) and hydrogen are combined
to form ammonia (NH3).
Ammonia can be used as a
fertilizer.
Industrial Plant combines
nitrogen and hydrogen
Ammonia is formed
Ammonia is used a fertilizer in soil
(NH3)
NN
H
N
H3

3. Biological Fixation:
a. Non-symbiotic bacteria) Free Living Bacteria: (“fixes” 30% of N2)
Highly specialized bacteria live in the soil and have the ability to combine
atmospheric nitrogen with hydrogen to make ammonia (NH3).
Such as Azotobacteraceae
b.Symbiotic Relationship Bacteria: (“fixes” 70% of N2)
Bacteria live in the roots of legume family plants and provide the plants with ammonia
(NH3).
Among the most beneficial microorganisms of the soil are those that are able to convert
gaseous nitrogen of the air to “fixed forms” of nitrogen that can be utilized by other
bacteria and plants. Without these nitrogen-fixers, life on this planet is probably
disappear within a relatively short period of time. The utilization of free nitrogen gas
by fixation can be accomplished by organisms that are able to produce the essential
enzyme nitrogenase. This enzyme, in the presence of traces of molybdenum,
enables the organisms to combine atmospheric nitrogen with other elements to form
organic compounds in living cells.
Such as Rhizobiaceae.
Other organisms of less importance that have this ability are a few strains of Klebsiella,
some species of Clostridium, the cyanobacteria, and photosynthetic bacteria. In
this exercise we will concern ourselves with two activities: the isolation of
Azotobacter from garden soil and the demonstration of Rhizobium in root nodules of
legumes.

Biological Fixation
There are two types of “Nitrogen Fixing Bacteria”
Free Living Bacteria
(“fixes” 30% of N2)
Symbiotic Relationship Bacteria
(“fixes” 70% of N2)

Free Living Bacteria
Highly specialized bacteria live in the soil and have the ability
to combine atmospheric nitrogen with hydrogen to make
ammonia (NH3).
Free-living bacteria live
in soil and combine
atmospheric nitrogen
with hydrogen
Nitrogen changes
into ammonia
N
N
H
NH3
(NH3)
Bacteria

Symbiotic Relationship Bacteria
Bacteria live in the roots of
legume family plants and
provide the plants with
ammonia (NH3) in exchange for
the plant’s carbon and a
protected-home.
Legume plants
Roots with nodules
where bacteria live
Nitrogen changes into
ammonia.
NH3
N
N

Nitrogen Fixation
The nodules on the roots
of this bean contain
bacteria called
Rhizobium that helps by
converting nitrogen in
the soil into a form the
plant can utilize it.
14

Mechanism of N-fixation:
The general chemical reaction for the fixation of nitrogen (N + 3H2 + Energy -> 2NH3) is
identical for both the chemical and the biological processes. The triple bond of N must
be broken and three atoms of hydrogen must be added to each of the nitrogen atoms.
Living organisms use energy derived from the oxidation ("burning") of carbohydrates to
reduce molecular nitrogen (N2) to ammonia (NH3).
 AZOTOBACTERACEAE
Azotobacteraceae that fix nitrogen as free-living organisms under aerobic conditions:
Azotobacter and Azomonas. Both are large gram-negative motile rods that may be
ovoid or coccoidal in shape, (pleomorphic). The free-living Azotobacteraceae are
beneficial nitrogen-fixers, their contribution to nitrogen enrichment of the soil is
limited due to the fact that they would rather utilize NH3 in soil than fix nitrogen. In
other words, if ammonia is present in the soil, nitrogen fixation by these organisms is
suppressed.
 RHIZOBIACEAE
The symbiotic nitrogen-fixers of genus Rhizobium, family Rhizobiaceae, are the principal
nitrogen enrichers of soil. Three genera in family Rhizobiaceae: Rhizobium,
Bradyrhizobium, and Agrobacterium. Although the three genera are related, only genus
Rhizobium fixes nitrogen. This genus of symbiotic nitrogen-fixers contains only three
species:
 R. leguminosarum: peas, beans.
 R. meliloti: sweet clover.
 R. loti: trefoil.
 All three of these species are gram-negative pleomorphic rods (bacteroids), often X-, Y-,
star-, and clubshaped; some exhibit branching. All are aerobic and motile.

Examples of nitrogen-fixing bacteria (* denotes a photosynthetic bacterium)
Symbiotic with plantsFree living
Other plantsLegumes
Anaerobic (Winogradsky
column)
Aerobic
Frankia
Azospirillum
Rhizobium
Clostridium (some)
Desulfovibrio
Purple sulphur bacteria*
Purple non-sulphur bacteria*
Green sulphur bacteria*
Azotobacter
Beijerinckia
Klebsiella (some)
Cyanobacteria (some)*

Procedure for isolation of AZOTOBACTERACEAE
FIRST PERIOD (ENRICHMENT)
Proceed as follows to inoculate a bottle of the nitrogen free glucose medium with a sample of garden soil.
Materials:
 1 bottle (50 ml) N2-free glucose medium (Thompson-Skerman) or Azotobacter agar
 rich garden soil (neutral or alkaline)
 spatula
1. with a small spatula put about 1 gm of soil into the bottle of medium. Cap the bottle and shake it sufficiently to mix
the soil and medium.
2. Loosen the cap slightly and incubate the bottle at 30° C for 4 to 7 days. Since the organisms are strict aerobes, it is
best to incubate the bottle horizontally to provide maximum surface exposure to air.
SECOND PERIOD (PLATING OUT)
During this period a slide will be made to make certain that organisms have grown on the medium. If the culture has
been successful, a streak plate will be made on nitrogen-free, iron-free agar. Proceed as follows:
Materials:
Microscope slides and cover glasses microscope, 1 agar plate of nitrogen-free, iron-free glucose medium
1. After 4 to 7 days incubation, carefully move the bottle of medium to your desktop without agitating the culture.
2. Make a wet mount slide with a few loopfuls from the surface of the medium and examine under oil immersion,
Look for large ovoid to rod-shaped organisms, singly and in pairs.
3. If the presence of azotobacter-like organisms is confirmed, streak an agar plate of nitrogen-free, iron-free medium,
using a good isolation streak pattern. Ferrous sulfate has been left out of this medium to facilitate the detection of
water-soluble pigments.
4. Incubate the plate at 30° C for 4 or 5 days.

Azotobactereace on different media : a) Brown-agar medium, b)
Winogradsky solution, c) smoothed soil paste–plate surface, d)
mannitol-agar, e ,f, g, h) differential LG agar medium (different species
and components.

Procedure for isolation for isolation of RHIZOBIACEAE:
 Materials:
1. washed nodules from the root of a legume
2. methylene blue stain
3. microscope slides
 pink nodules were selected from the root of a legume and washed by water,
then kept in (MgCl2) for period of time, and washed again by water
 Place a nodule on a clean microscope slide and crush it by pressing
another slide over it. Produce a thin smear by sliding the top slide over the
lower one.
 After air-drying and fixing with heat, stain the smear with methylene blue
for 30 seconds.
 Examine under oil immersion.

A.Questions:
1. What enzyme is responsible for nitrogen fixation? By which
mechanism level of O2 regulated to obtain maximum nitoginase
activity?
2. Why is nitrogen fixation so important?
3. from the standpoint of amount of nitrogen fixation, is this group of
nitrogen-fixers Rizobacteriaceae more or less important than the
Azotobacteraceae?
4. On your opinion does it possible to increase fixation in unamended
soil by addition of high populations of bacteria (soil inoculation)?
5. Draw some of the organisms on the Laboratory Report. Look for
typical bacteroids of various configurations.

Subject objective: Each student should be
able to:• Learn about the important of ammonification process in the
nature.
• Obtain an evolution of the ammonification potentials in
different soil samples.
• Determine protease activity through detecting ammonium
using broth culture with garden soil and through using soil
extract.

By traveling through one of the four processes
in the Nitrogen Cycle!
(2) Ammonification
Nitrogen
Cycle

Ammonification: Bacteria, fungi, actinomycetes decomposers
break down amino acids from dead animals and wastes into
nitrogen ammonium.
Bacteria decomposers break down amino acids into ammonium

Principle The nitrogen in most plants and animals exists in the form of protein.
When these organisms die, the protein is broken down to amino acids,
which in turn are deaminated to liberate ammonia. This process of the
production of ammonia from organic compounds is called
ammonification. Soil bacteria (e.g. Bacillus, Proteus, and
Pseudomonas) produce the proteases that accomplish ammonification.
Once ammonia is released into the soil it dissolves in water to form the
ammonium ion (NH4
+). Some of these ions are used by plants and
microorganisms to synthesize amino acids.
 In this exercise, peptone is used as an organic nitrogen substrate. The
ability of different bacteria and the organisms in a soil sample to break
down the organic nitrogen and release ammonia will be examined.
Ammonia can be detected by adding Nessler’s reagent to samples – if
ammonia is present, the samples will turn yellow – brown.

A. Mineralization (Ammonification) – the conversion of organic nitrogen (proteins,
amino sugars, nucleic acids, chitin) to ammonium (NH4
+), a mineral form
who? heterotrophic bacteria and fungi – ‘decomposers’
generic equation:
B. Immobilization – microbial uptake of inorganic nitrogen and incorporation into
organic forms who? heterotrophic bacteria and fungi – ‘decomposers’
1) Ammonium assimilation

Why is
ammonification
necessary?

Because plants cannot use the organic forms of
nitrogen which are in the soil as a result of:
(1) wastes (manure and sewage)
(2) compost and decomposing roots and leaves

Very few plants can use
ammonia (NH3)…
…but, fortunately the
second process
Ammonification can help!
(2) Ammonification

What happened to
Ammonium
in soil?

The ammonium is either:
1. taken up by the plants (only in a few types of plants)
2. ammonium can be adsorbed and fixated (stuck) on to the negatively charged soil
particles or be taken up by plants.
3.Ammonium (NH4) Stored in the soil up to later be changed into inorganic nitrogen, the
kind of nitrogen that most plants can use.
Ammonium (NH4) is
stored in soil.
Bacteria converts organic nitrogen to
ammonium (NH4)
Ammonium (NH4) is used by
some plants

First Procedure for Estimation of Ammonia in soil:
FIRST PERIOD: (Inoculation)
 Materials:
1. 2 tubes of peptone broth
2. Rich garden soil
3. Broth cultures of Bacillus, Proteus and Pseudomonas
Procedure:
1. Inoculate one tube of peptone broth with 1gm of soil sample, save the other tube for a control.
2. Incubate the tube at room temperature for 3–4 days and 7 days.
SECOND AND THIRD PERIODS: (Ammonia Detection)
After 3 or 4 days, test the medium for ammonia with the following procedure. Repeat these tests again
after a total of 7 days of incubation.
 Materials:
1. Nessler’s reagent, Spot plate
2. pH-meter or pH paper.
Procedure:
1. Deposit a drop of Nessler’s reagent into two separate depressions of a spot plate.
2. Add a loopful of the inoculated peptone broth to one depression and a loopful from the sterile
uninoculated tube in the other, then add 1-2 dopes of nessler's reagent. Interpretation of ammonia
presence is as follows:
 Faint yellow color—small amount of ammonia
 Deep yellow—more ammonia
 Brown precipitate—large amount of ammonia
3. Check the pH of the two tubes by pH-meter or pH paper.

Second procedure for estimation of ammonia and ammonium in soil samples:
Inorganic nitrogen (NH+4) and nitrogen process rate measurements:
Accumulation of inorganic nitrogen is measured by extracting each soil sample with 80 ml of 2M KCl.
After adding KCl and shaking each container by hand to suspend the soil, sample containers were
placed on a rotary shaker at speed (100rpm/min.) for 1 h, and then shaken again by hand to re-
suspend the soil. Samples were filtered (Whatman No. 42 filter paper) All soil extracts were frozen
at (-20°C) to prevent secondary formation of nitrite ions by microbial or chemical redox reactions
from ammonium ions or nitrate ions. Gross N mineralization was measured on whole soil samples.
 Titration method used for ammonium (NH+4) measurement:
Extracted NH+4 from soil samples were determined by titration method (19) by treating (20 ml) of
extracted soil with five drops of methyl red reagent and titration was done with (0.05 N) of
H2SO4 until the end point of reaction yellow to red, then sample boiled off to room temperature and
same step of titration was repeated to the same color, and distilled water corresponding of the blank
test serves as the control sample.
 Following equations were used for determination of NH+4 in soil water extract:
 N1 × V1 = N2 × V2
N1: unknown N of (NH+4)?
V1: 20ml water extract of soil sample.
N2: 0.05N of (H2SO4)
V2: (ml) of (0.05N) H2SO4 correspond to (ml) of NH+4 at the endpoint of reaction.
a: consumption of 0.05N H2SO4 in ml for the water extract of soil sample.
b: consumption of 0.05N H2SO4 in ml for the distillated water (blank).
Then this equation is used for converting known (NH+4) N from first equation to ppm.
 ppm = Known (NH+4)N × 18 × 1000
Molecular weight of (NH+4) = 18

What happens to ammonium
(NH4) stored
in the soil?

 What are the important and how Nitrification take place in the
nature?
 Obtaining an evolution of (Nitrification) in different soil
sources.
 Practical Detection of nitrite and nitrate compounds, through
using broth medium inoculated by standard bacteria or
different garden soil samples.
 Identification of nitrifier bacteria

Nitrogen Cycle!
(4) Denitrification
Nitrogen
Cycle

(4) Denitrification
Nitrogen
Cycle
Nitrates in Soil
Ammonia is converted
to nitrites and nitrates.
Organic nitrogen is
converted to ammonium.
(a)
(b)
(c)
N2
NH3
NO3
N2O

Now we will
take a “closer
look” at the
Nitrification
Process

 NITRIFICATION:
In an aerobic environment, ammonia is liberated into the soil by the ammonification which is a part of
the cycle. It does not accumulate there. If it is not used as a nitrogen source by plants or
microorganisms, it is oxidized to nitrates by a two-step process called nitrification. Nitrification is
the conversion of NH+4 to NO-3, this process carried out through two-step process in which
ammonia (NH+3) is first oxidized to nitrite (NO-2) by chemoautotrophs: Nitrosomonas, and
the nitrite (NO-2) is subsequently oxidized to nitrate (NO-3) by chemoautotrophs: Nitrobacter.
The nitrate released into the soil is available to plants and microorganisms for protein synthesis.
This process like nitrogen fixation, this process is uniquely associated with bacteria. Nitrate is much
more readily leached from soils than is ammonia. If excessive amounts of nitrate are leached from
soils, reducing soil fertility and it can accumulate in runoff water and in wells.
Ammonium sulfate broth and nitrite broth are used in this part of the exercise to demonstrate the
oxidation of ammonia to nitrate. Ammonia and nitrite serve as energy sources in the respective
broths.
 Maximum nitrification rates occur at:
1. Neutral pH
2. High temperatures
 NH+ 4 + 1,1/2 O2 ………………. NO-2 + 2H+ + H2O + 66 Kcal. (Four genera make it)
 NO-2 + 1/2 O2 …………… NO-3 + 17.5 Kcal (Nitrobacter sp., Nitrosospira sp., Nitrosococcus sp.)
(Factors that favor the bacteria involved in this process belong to family Nitrobacetriaceae:
 Nitrosomonas sp.
 Nitrosococcus sp.
 Nitrosolobus sp.
 Nitrosospira sp.

Characters of nitrifying bacteria
ammonia-oxidizing (AOB) bacteria
 Aerobic
 Alkaline pH
 Temperature 20-30 °C
 Motile (Flagella)
 Grame negative
 Different cell shape such as spindly and bacilliform

Materials for Nitrification: Garden soil
 1 x ammonium sulfate broth (20ml)
 1 x nitrite broth (20ml)
 Nesslers reagent
 Trommdorfs reagent
 Diphenylamine
 Spot plate
 Sulfuric acid (1 part conc. Sulfuric acid to 3 parts water)

 Method:
1. Inoculate the ammonium sulfate and nitrite broth bottles with pinches of
soil (1g). Label the bottles and shake vigorously for 5 minutes.
2. Shake the bottles for 7 days at room temperature.
3. Place a drop of sulfuric acid and 3 drops of Trommsdorf’s reagent in a well
on a spot plate. Add a drop of culture from the ammonium broth and mix.
Use a Pasteur pipette and not an inoculating loop. A blue – black color
indicates the presence of nitrite.
4. Test the ammonium broth for ammonia with Nessler’s reagent (see
ammonification).
5. Test the nitrite broth for residual nitrite.
6. If no blue black color was present, test for nitrate. Add 1 drop of
diphenylamine, 2 drops of sulfuric acid and 1 drop of nitrite broth culture
in a well on the spot plate and mix. A blue black color indicates the presence
of nitrate.
7. Grams stain the organisms in the broth cultures. Record your results.

 What is the important and how Denitrification take places in
the nature?
 Obtaining an evolution of (Denitrification) in different soil
sources.
 Practical detection of nitrogen gas from reducing nitrate
compounds, through using broth medium inoculated with
standard bacteria or different garden soil samples.

How does
nitrogen reenter
the atmosphere
in the nitrogen
cycle?

Through the fourth process
called denitrification!
(2) Nitrification(3) Ammonification
(4) Denitrification

DENITRIFICATION:
Denitrification is defined as the reduction of nitrates to nitrites an eventually to nitrogen
gas. (NO-3) to gaseous dinitrogen (N2O, NO, and N2) these gases escape (volatilize)
into Earth's atmosphere and are not available for plant use. Because oxygen is not
necessary for denitrification to occur, this is a form of anaerobic respiration in which
the nitrates serve as electron acceptors for the denitrifying bacteria in their energy
metabolism. Denitrification takes place most rapidly in waterlogged anaerobic soil.
The four steps in the denitrification process are as follows:
Bacteria that makes this process like:
 Pseudomonas denitrificans
 Paracoccus denitrificans
 Thiobacillus denitrificans
 Micrococcus denitrificans
 Serratia sp.
 Achromobacter sp.
 (Thermophilic denitrifier) has even been isolated from a hot spring.
The most favorable environments for these organisms are:
1. Heavily fertilized agricultural soils.
2. Sewage where nitrogenous compounds abound in considerable quantity.

Denitrification converts nitrates (NO3) in the soil to
atmospheric nitrogen (N2) replenishing the
atmosphere.
Nitrates (NO3)
in Soil
Nitrogen in atmosphere (N2)

How does the
denitrification
process work?
Nitrates in soil

Denitrifying bacteria live deep in soil and in aquatic
sediments where conditions make it difficult for them to
get oxygen. The denitrifying bacteria use nitrates as an
alternative to oxygen, leaving free nitrogen gas as a
byproduct. They close the nitrogen cycle!
Denitrifying bacteria live
deep in soil and use
nitrates as an alternative
to oxygen making a
byproduct of nitrogen gas.
Nitrogen in atmosphere
closes the nitrogen cycle!
(NO3)
(N2)

Other ways that nitrogen returns
to the atmosphere…
Emissions from industrial combustion and
gasoline engines create nitrous oxides
gas (N2O).
Volcano eruptions
emit nitrous oxides
gas (N2O).

Denitrifying microorganism
 Anaerobic to reduce the nitrate to gaseous form of nitrogen.
 Room tmperature
 The predominant saturated and unsaturated fatty acids in all
denitrifying isolates are generally n-hexadecanoic acid (16:0) and cis-11-
octadecenoic acid (18:1 ω7c).
 Microscopically :Grame negative whit rod shape.
 Biochemical test and API test are probably used for identification
 16S RNA sequencing is more reliable for characterization.
 Medium for nitrification should contain nitrate and incubated in
mesophile temperature range

Materials for denitrification:
 Garden soil
 Broth culture of Pseudomonas
 2 nitrate broth tubes containing Durham tubes
 2 nitrate free broth tubes containing Durham tubes
 -napthylamine reagent
 sulfanilic acid
 powdered zinc
 Blenders, fresh soil sample, 90 ml distilled water
 Graduate 1 ml pipette, 1 Petri plate of nitrate agar,
GasPak anaerobic jar, generator envelopes

Method: To isolate denitrifiers from a soil sample, the following conditions must be met in
the growth medium:
1. In this exercise a medium containing a nitrate substrate is used for gas formation and a
Durham tube is used to detect gas (N) production.
2. Some nitrate must be available, which will provide the only terminal electron acceptor
for the generation of ATP.
3. Some peptone must be present to provide essential amino acids needed by some
denitrifiers. The next step is to demonstrate the ability of the organism to generate
visible nitrogen gas. An isolate that grows on nitrate media and generates gas can be
presumed to be a denitrifier.
Procedure:
 First Period: The nitrate agar used in the Petri plate is essentially nutrient agar to which
0.5% KNO3 is added.
 Procedure:
1. Add 10 grams of soil to 90 ml of water.
2. Blend for 2 minutes.
3. Label the bottom of a nitrate agar plate with your name and date of inoculation.
4. Pipette 1.0 ml of the blended mix onto the surface of a plate of nitrate agar.
5. Spread the inoculum over the surface of the agar with a bent glass rod.
6. Incubate the plate, inverted, at 30° C for 3 to 5 days in a GasPak anaerobic jar.

Second Period
 During this period, nitrate agar plates will be examined to select colonies that have developed
during the incubation period. Since the presence of growth doesn’t necessarily mean that the
organism is a denitrifier, it will be necessary to see if any of the isolates are nitrogen gas producers;
thus, Durham tube nitrate broths must be inoculated and incubated anaerobically. Nitrate broth
consists of nutrient broth plus (0.5% KNO3).
1. Inoculate one tube of nitrate broth containing a Durham tube with 1g of soil. DO NOT SHAKE THE
CULTURE TUBES DURING INCUBATION.
2. Inoculate the second tube with a loopful of Pseudomonas.
3. Repeat steps 1 and 2 with the nitrate free broth tubes.
4. Label all tubes and incubate at room temperature for 7 days.
5. Observe the tubes for gas formation.
6. Add 1 ml  - naphthalene reagent and 1 ml sulfanilic acid reagent to each of the culture tubes and
mix. The development of a red color within 30 seconds indicates that nitrites are present.
7. After carrying out step 6, any tube that fails to develop a red color could still have its full supply of
nitrate (i.e. lacks bacteria to reduce it) or it could have undergone denitrification without nitrite
being further converted to nitrogen. To distinguish between the two possibilities, a pinch of zinc
must be added to any tube that did not turn red. The zinc catalyses the reduction of nitrate to
nitrite and produces a red color within minutes if nitrate was present. Lack of a red color indicates
the absence of nitrate (and possibly the presence of nitrite).
8. Record your results.
Third Period
This period of inoculations is in preparation of trying to do a definitive identification of a denitrifier.
From an isolated colony a nutrient broth is inoculated and a gram-stained slide is made. After
incubation, the broth culture can be used as a stock culture for doing further tests to identify your
isolate. The slide will reveal the morphological nature of your organism.

Final Lab. About
Nitrogen cycle

Lab soil

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