Consortium for
Educational
Communication
Module on
Biogeochemical Nitrogen
Cycle
Forms of N Pools and Fluxes; process that bring about
cycling of Nitrogen through/ across various reservoirs
By
Sumira Tyub
Senior Technical Assistant
Centre of Research for
Development
University of Kashmir
9419563957
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Introduction
The terrestrial nitrogen cycle (Fig 1) comprises soil, plant and
animal pools that contain relatively small quantities of biologically
active N, in comparison to the large pools of relatively inert nitrogen
in the lithosphere and atmosphere.
Fig 1. Global Nitrogen Cycle
After carbon and oxygen, N is the next most abundant
element in plant dry matter, typically 10-30g/ kg. It is usually
acquired by plants in greater quantity from soil than any other
element. Many constituents of living cells contain nitrogen; they
include proteins, amino acids, nucleic acids, purines, pyrimidines,
porphyrin, alkaloids and vitamins. The nitrogen atoms of these
cycles eventually travel the nitrogen cycle, in which nitrogen of
the atmosphere serves as reservoir. Nitrogen is removed from
reservoir by the process of fixation and it is then returned by the
process of denitrification.
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Forms of Nitrogen
Several inorganic nitrogen compounds, as well as a myriad of
organic nitrogen compounds can be considered as components of
nitrogen cycle. The inorganic compounds include N2 gas, Ammonia
(NH3), nitrate ion (NO3-), nitrite ion (NO2-) and hydroxylamine
(NH2OH). Nitrogen atom can possess a variety of oxidation numbers.
Some of these are NO3- (+5), NO2- (+3), N2 gas (0), NH2OH (-1),
NH3 (-3). Thus in nature, nitrogen may exist either in a highly
oxidized form (NO3-) or highly reduced state (NH3).
Microbially mediated processes transform nitrogen from one
to another form. Certain bacteria can transform dinitrogen to
ammonia by a process known as nitrogen fixation. The processes
of ammonification, immobilization, nitrification, and denitrification
are responsible for moving the fixed nitrogen from one form to
another in the soil.
Reservoirs/Pools of nitrogen:
The sizes of the nitrogen pools vary over several orders of
magnitude. The relatively inert dinitrogen pool (an invisible gas)
is the largest pool of biologically active nitrogen in terrestrial
ecosystem. Soil organic nitrogen makes up the next largest pool
of nitrogen and varies widely among soil types. The variation in
soil organic nitrogen is determined largely by the factors of soil
formation, particularly temperature and precipitation. The amount
of nitrogen tied up in plant biomass is of intermediate size and
varies as a function of vegetation type, climate and soil nitrogen
availability. Naturally occurring soil organic nitrogen compounds
isolated from soil include proteins and amino acids, microbial cell
wall polymers and amino sugars; nucleic acids and a variety of
vitamins, antibiotics and metabolic intermediates.
Soil inorganic nitrogen pools are usually small generally just
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a few mg N per Kg in natural ecosystems and rarely exceeding 100
mg N per kg in the plow layer of recently fertilized agricultural soils.
Inorganic nitrogen pools in the soil which include nitrite, nitrate,
nitric oxide, ammonia etc are usually small compared to organic
nitrogen but are more important because they serve as substrates,
metabolic intermediates, alternate electron acceptors, or products
of primary biological nitrogen transformations.
Larger pools tend to be less reactive i.e. they turn over
slowly
and the smaller pools usually are more dynamic. Mean
residence time of the atmospheric nitrogen is thousands to millions
of years, decades are required to turn over the organic nitrogen
pool, and nitrogen in plant biomass often turns over annually while
as inorganic nitrogen pools may turn over once a day. The global
nitrogen reservoirs and fluxes are given in Table 1.
Table 1: Global nitrogen reservoirs and annual fluxes [Values
of reservoirs are in petagrams (1 Pg= 1x 1015 g) and those
of fluxes are in tetragrams ( 1 Tg= 1x 1012 g) nitrogen per
year]. (Based on Rosswall, 1983).
Reservoirs
Atmosphere
N2
39 x 105
N2O
1.4
NH3 + NH4
174 x 10-5
NO + NOx
6 x 10-4
NO3
1 x 10-4
Plants
Animals
Microbes
Dissolved detritus
Particulate detritus
0.30
0.17
0.02
530
3-240
Organic N
N2 (dissolved)
22000
N2O
NO3
Nitrite
NH4
0.2
570
0.5
7
1x 10-3
Ocean
Fluxes
Biological N fixation
Atmospheric fixation (lightning)
Industrial fixation
Industrial combustion and fuel burning
Land
Plants
11- 14
Animals
0.2
Microbes 0.5
Detritus
1.9-3.3
Soil
300
organic
matter
Soil
160
inorganic
44-200 (land), 1-130 (ocean)
0.5 – 30
60
10-20
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Fire
Biogenic NOx production
Denitrification
Denitrification N2O
Nitrification N2O production
Ammonia volatilization
Dry and wet NH3/NH4 deposition
Dry and wet NOx deposition
Dry and wet deposition of organic N
River runoff
10-200
0-90
43-390 (land), 0-330 (ocean)
16-69 (land), 5-80 (ocean)
4-10 (ocean)
36-250
110-240
40-116
10-100
13-40
The nitrogen cycle can be divided into three subcycles which
function in concert:
Elemental: emphasizing the biological oxidation reduction
reactions that interconvert nitrogen and dinitrogen into various
chemical forms.
Phototrophic: driven by plant nitrogen uptake, which is fueled
by photosynthesis and converts inorganic nitrogen into organic
nitrogen (plant constituents).
Heterotrophic: linked to decomposition processes and driven
by the need of heterotrophic organisms for preformed carbon.
Biological Nitrogen Fixation
The fixation of gaseous nitrogen in forms usable for plants
involves breaking of the triple bond of molecular nitrogen (N≡N),
and is particularly energy consuming. Nitrogen fixation which takes
place in nature and in which the atmospheric nitrogen is converted
into ammonia through biological processes is called biological
nitrogen fixation.
In biological nitrogen fixation, N2 is fixed by some specialized
microscopic prokaryotic algae and bacteria. In this process N2 and
H2O combine to form NH3. This strongly endergonic reaction is
catalyzed by nitrogenase enzyme system, and may require from
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50 to 200g glucose per g of nitrogen fixed. The nitrogen fixing
organisms may be either free living (non symbiotic), symbiotic or
associative. Free living fixers are less efficient than symbiotic fixers
and may fix 0.1 to 2 Kg N/ha/yr in subarctic and arctic regions and
50-70 Kg N/ha/yr in warm and damp environments. The symbiotic
bacteria (e.g. Rhizobium, the nodule bacteria of legume) are more
efficient and may fix about 200 or more Kg N/ha/yr. Free living
bacteria colonizing root network and mycorrhizae, and maintaining
associative symbiosis with host plants may fix 2-25 Kg N/ha/yr.
Groups of nitrogen fixing organisms
1. Free living bacteria: This group includes both aerobic (e.g.
Azotobacter) and anaerobic bacteria (e.g. Clostridium), purple
bacterium (Rhodospirillum) and some other photosynthetic
bacteria and soil bacteria like Pseudomonas.
2. Symbiotic nodule bacteria on legume plants: Example Rhizobium
3. Symbiotic nodule actinomycetes on non legume plants: Example Frankia.
4. Blue green algae (Cyanobacteria): Examples Anabaena, Nostoc
5. Bacteria living on leaves of certain plants: e.g Gunnera
6. Unicellular filamentous Cyanobacteria in aquatic environments
Some economically important microbes involved in biological
nitrogen fixation are given in Table 2.
Table 2: Some economically important microbes involved in
biological nitrogen fixation
Organisms
Azotobacter
Azospirillum
General properties
Aerobic, nitrogen fixers
Aerobic, N fixers in association with roots of grass
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Rhizobium
Frankia
Anabaena
Fixes N in legume, Rhizobium symbiosis
Fixes N in symbiosis with woody trees- Alder, Myrica
Fixes N in aquatic and terrestrial ecosystems
Nitrogen in lesser quantity is also fixed in the atmosphere with
the aid of lightning and sunlight energy through electrochemical
and photochemical fixation respectively. In these processes
nitrogen combines with oxygen to form nitrogen dioxide (NO2). N
compounds in the atmosphere are returned to the soil in rainfall
as NH3, NO3, NO2, nitrous oxide and organic nitrogen. Soil has a
capacity for adsorbing NH3 gas from the atmosphere. The NO3
is taken up by plant roots when it is washed to the ground with
rain and snow. Nitrogen fixation through these processes is of
the order of 35 mg/m2/yr.
Nitrogen fixation is also achieved by industrial chemical
processes for manufacture of fertilizers. This type of nitrogen fixation
is called industrial or chemical nitrogen fixation. The industrial
fixation of nitrogen is the most important source of nitrogen as a
plant nutrient in which H2 and N2 reacts to form NH3.
Ammonification/ Mineralization
The production of inorganic nitrogen both ammonium and
nitrate is termed as nitrogen mineralization. The increase or
decrease in inorganic nitrogen is most often called as net nitrogen
mineralization and it represents sum of concurrent ammonium
production and consumption processes. Biological transformation
of organic nitrogen to ammonium is also referred as ammonification
or gross nitrogen mineralization.
The conversion of organic nitrogen compounds to ammonium
is mediated by enzymes produced by microbes and soil animals.
Production of ammonium often involves several steps. Extracellular
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enzymes first breakdown organic nitrogen polymers and the
resulting monomers pass across the cell membrane and are further
metabolized with the resulting production of ammonium which
is released in the soil solution. The major extracellular enzymes
produced by microorganisms depolymerize proteins, amino
polysaccharides and nucleic acids and hydrolyze urea (Table 3).
Table 3: Extracellular enzymes involved in microbial nitrogen
mineralization
Substrates
Proteins
Peptides
Chitin
Chitobiose
Peptidoglycan
Enzymes
Proteinases, Proteases
Peptidases
Chitinases
Chitobiase
Lysozyme
DNA and RNA
Urea
Endonucleases and Exonucleases
Urease
Products
Peptides, Aminoacids
Aminoacids
Chitobiose
N-acetylglucosamine
N-acetylglucosamine and
N-acetyl muramic acid
Nucleotides
NH3 and CO2
In most cases, the final production of ammonium occurs within
microbial cells through the action of intracellular enzymes. Some
of these intracellular enzymes may become “extracellular’ when
microbial cells are lysed.
Immobilization/ Assimilation
Immobilization on the other hand is the conversion of ammonium to
organic nitrogen, primarily as a result of assimilation of ammonium
by microbial biomass and in turn renders nitrogen unavailable
for plants and microbes. The assimilation of nitrate by microbial
biomass is referred as nitrate immobilization. However, nitrate
assimilation requires nitrate reduction to ammonium before it can
be incorporated into cell constituents.
Microbes and other organisms assimilate ammonium by two
primary pathways
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i.
ii.
Glutamate dehydrogenase, and
Glutamine synthetase-glutamate synthase (GOGAT).
When ammonium is present in relatively high concentrations (>
0.1 mM or about 0.5 mg N kg-1 soil), glutamate dehydrogenase,
acting with NADPH2 as a coenzyme, can add ammonium to
α-ketoglutarate to form glutamate.
In most soils, ammonium is present at rather low concentrations,
which results in low intracellular ammonium concentrations. Under
these conditions, the second ammonium assimilation system is
operable. The GOGAT pathway is complex. The first step requires
ATP to add ammonium to glutamate to form glutamine. The second
step transfers the ammonium from glutamine to α -ketoglutarate
to form two glutamates. Once ammonium has been incorporated
into glutamate, it can then be transferred to other carbon skeletons
by various transaminase reactions to form additional amino acids.
Several factors influence whether there is net production or
consumption of ammonium by microorganisms in soil. The general
principle is that net immobilization of ammonium occurs if the
availability of nitrogen is limiting; otherwise, net production occurs.
When organic amendments with C/N ratios below 20/1 are added
to soils, net ammonium production results; at wider C/N ratios,
inorganic nitrogen from the soil is immobilized.
In addition to the mineralization/immobilization cycle,
ammonium has several other fates in soil. It can be chemically
held on cation exchange sites or become fixed in the lattice of
clay minerals (ammonium fixation), such as illite and vermiculite.
Ammonium may react chemically with organic compounds, such as
quinones, or it may be volatilized at high pH. Major biological fates
are plant uptake, microbial assimilation, or oxidation to nitrate by
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nitrifying microorganisms.
Nitrification:
Nitrification is the process that converts ammonia to nitrite
and then to nitrate and is another important step in the global
nitrogen cycle. Most nitrification occurs aerobically and is carried
out exclusively by prokaryotes.
Autotrophic nitrification is the two step, two organism
process of oxidizing ammonium to nitrate, in which the inorganic
nitrogen serves as the energy source for nitrifying bacteria.
The first step of autotrophic nitrification is ammonia oxidation
i.e. conversion of ammonium (actually ammonia at enzyme level) to
nitrite by ammonium oxidizing bacteria. Aerobic ammonia oxidizers
convert ammonia to nitrite via the intermediate hydroxylamine,
a process that requires two different enzymes, ammonia
monooxygenase and hydroxylamine oxidoreductase. The process
generates a very small amount of energy relative to many other
types of metabolism; as a result, nitrosofiers are very slow growers.
Additionally, aerobic ammonia oxidizers are also autotrophs, fixing
carbon dioxide to produce organic carbon, much like photosynthetic
organisms, but using ammonia as the energy source instead of
light. Unlike nitrogen fixation that is carried out by many different
kinds of microbes, ammonia oxidation is less broadly distributed
among prokaryotes. Until recently, it was thought that all ammonia
oxidation was carried out by only a few types of bacteria in the
genera Nitrosomonas, Nitrosospira, and Nitrosococcus. However, in
2005 an archaeon was discovered that could also oxidize ammonia.
Since their discovery, ammonia-oxidizing Archaea have often been
found to outnumber the ammonia-oxidizing Bacteria in many
habitats.
The second step in nitrification is the oxidation of nitrite
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(NO2-) to nitrate (NO3-). This step is carried out by a completely
separate group of prokaryotes, known as nitrite-oxidizing
Bacteria. Some of the genera involved in nitrite oxidation
include Nitrospira, Nitrobacter, Nitrococcus, and Nitrospina. Similar
to ammonia oxidizers, the energy generated from the oxidation of
nitrite to nitrate is very small, and thus growth yields are very
low. In fact, ammonia- and nitrite-oxidizers must oxidize many
molecules of ammonia or nitrite in order to fix a single molecule of
CO2. For complete nitrification, both ammonia oxidation and nitrite
oxidation must occur.
Ammonia-oxidizers and nitrite-oxidizers are ubiquitous in
aerobic environments. They have been extensively studied in
natural environments such as soils, estuaries, lakes, and openocean environments. However, ammonia- and nitrite-oxidizers also
play a very important role in wastewater treatment facilities by
removing potentially harmful levels of ammonium that could lead
to the pollution of the receiving waters.
Anammox
Traditionally, all nitrification was thought to be carried out under
aerobic conditions, but recently a new type of ammonia oxidation
occurring under anoxic conditions was discovered (Strous et al.
1999). Anammox (anaerobic ammonia oxidation) is carried out by
prokaryotes belonging to the Planctomycetes phylum of Bacteria. The
first described anammox bacterium was Brocadia anammoxidans.
Anammox bacteria oxidize ammonia by using nitrite as the electron
acceptor to produce gaseous nitrogen.
Heterotrophic Nitrification
Several
heterotrophic
microorganisms
oxidize
either
ammonium or organic nitrogen to nitrite or nitrate. Heterotrophic
nitrifiers include both fungi (e.g., Aspergillus) and bacteria (e.g.,
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Alcaligenes, Arthrobacter spp., and some actinomycetes. Unlike
the autotrophic nitrifiers, heterotrophic nitrifiers gain no energy
through this activity.
Assimilatory Nitrate Reduction
Nitrate can be assimilated by plants and microorganisms. The
process of assimilatory nitrate reduction requires energy for the
conversion of nitrate to ammonium and subsequent incorporation of
ammonium into amino acids. Consequently, this process is regulated
by nitrogen availability, and nitrate utilization is expected when
energy is in excess relative to the concentrations of ammonium or
organic-nitrogen compounds. For this reason assimilation of nitrate
is also called nitrate immobilization, as nitrogen is made unavailable
to other organisms by soil microorganisms.
Dissimilatory Nitrate Reduction/ Denitrification:
The major form of dissimilatory nitrate reduction in soil is
respiratory denitrification, more commonly known as denitrification.
This refers to the reduction of nitrate to gaseous nitrogen products,
principally dinitrogen and nitrous oxide, coupled to energy
production via oxidative phosphorylation. It is an example of
anaerobic respiration, where an alternate electron acceptor other
than oxygen is used.
Unlike nitrification, denitrification is an anaerobic process,
occurring mostly in soils and sediments and anoxic zones in lakes and
oceans. Similar to nitrogen fixation, denitrification is carried out by a
diverse group of prokaryotes, and there is recent evidence that some
eukaryotes are also capable of denitrification. Some denitrifying
bacteria include species in the genera Bacillus, Paracoccus
and Pseudomonas. Denitrifiers are chemoorganotrophs and thus
must also be supplied with some form of organic carbon.
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Denitrification is important in that it removes fixed nitrogen
(i.e., nitrate) from the ecosystem and returns it to the atmosphere
in a biologically inert form (N2). This is particularly important in
agriculture where the loss of nitrates in fertilizer is detrimental and
costly. However, denitrification in wastewater treatment plays a very
beneficial role by removing unwanted nitrates from the wastewater
effluent, thereby reducing the chances that the water discharged
from the treatment plants will cause undesirable consequences
(e.g., algal blooms).
Nitrogen cycle can be summarized as
1. Most nitrogen in soil is in organic forms. Organic nitrogen serves
as a reservoir of nitrogen, slowly supplying the more dynamic
and much smaller inorganic nitrogen pools.
2. The conversion of organic nitrogen to inorganic nitrogen is
called mineralization. The first step of this process is the production of ammonium by ammonification, which is carried out
by a wide variety of soil microorganisms and soil animals. Ammonification is always counterbalanced by the opposite pattern
of immobilizing ammonium into the soil biomass through assimilation.
3. A major controlling factor determining whether net mineralization or immobilization of nitrogen occurs is the C/N ratio of the
decomposing organic matter.
4. The mineralization process is continued further by the conversion of ammonium to nitrate by the nitrifiers, which are a relatively restricted group of bacteria. Ammonia oxidizing bacteria
perform the first step of this two-step process, the transformation of ammonium to nitrite. Nitrite is further oxidized to nitrate by the nitrite-oxidizing bacteria. The ammonia and nitrite
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oxidizers are autotrophic bacteria that gain their energy from
these inorganic oxidations.
5. Nitrate has many fates in the soil environment. It is readily
taken up by plants and can also be immobilized by heterotrophic microorganisms. Because nitrate is relatively mobile, it
can be readily leached, which represents not only a loss from
the system, but also a potential environmental problem.
6. A final fate of nitrate is to be lost to the atmosphere through
denitrification. Denitrification occurs under anaerobic conditions, which can exist as microsites even in well aerated soils.
The reduction of nitrate to gaseous nitrous oxide and dinitrogen is accomplished by a wide range of bacteria, most of which
normally function as aerobic heterotrophs.
7. Denitrification is a relatively benign loss of nitrogen when dinitrogen is the dominant product; however, nitrous oxide production can be an environmental concern because it acts as a
greenhouse gas and has been implicated in the destruction of
ozone in the stratosphere.
The nitrogen cycle is closed by the process of N2 fixation, which
is ultimately the source of all the nitrogen that is transformed within
the soil ecosystem.
Ecological Implications of Human Alterations to the Nitrogen
Cycle
Many human activities have a significant impact on the
nitrogen cycle. Burning fossil fuels, application of nitrogen-based
fertilizers, and other activities can dramatically increase the amount
of biologically available nitrogen in an ecosystem. And because
nitrogen availability often limits the primary productivity of many
ecosystems, large changes in the availability of nitrogen can lead
to severe alterations of the nitrogen cycle in both aquatic and
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terrestrial ecosystems. Industrial nitrogen fixation has increased
exponentially since the 1940s, and human activity has doubled the
amount of global nitrogen fixation.
In terrestrial ecosystems, the addition of nitrogen can lead to
nutrient imbalance in trees, changes in forest health, and declines
in biodiversity. With increased nitrogen availability there is often a
change in carbon storage, thus impacting more processes than just
the nitrogen cycle.
In agricultural systems, fertilizers are used extensively to
increase plant production, but unused nitrogen, usually in the form
of nitrate, can leach out of the soil, enter streams and rivers, and
ultimately make its way into our drinking water.
The process of making synthetic fertilizers for use in agriculture
by causing N2 to react with H2, known as the Haber-Bosch process,
has increased significantly over the past several decades. In fact,
today, nearly 80% of the nitrogen found in human tissues originated
from the Haber-Bosch process (Howarth, 2008).
Much of the nitrogen applied to agricultural and urban areas
ultimately enters rivers and nearshore coastal systems. In nearshore
marine systems, increases in nitrogen can often lead to anoxia (no
oxygen) or hypoxia (low oxygen), altered biodiversity, changes in
food-web structure, and general habitat degradation.
One common consequence of increased nitrogen is an increase
in harmful algal blooms (Howarth, 2008). Toxic blooms of certain
types of dinoflagellates have been associated with high fish and
shellfish mortality in some areas.
Even without such economically catastrophic effects, the
addition of nitrogen can lead to changes in biodiversity and species
composition that may lead to changes in overall ecosystem function.
Some have even suggested that alterations to the nitrogen cycle may
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lead to an increased risk of parasitic and infectious diseases among
humans and wildlife (Johnson et al. 2010). Additionally, increases
in nitrogen in aquatic systems can lead to increased acidification in
freshwater ecosystems.