THE NITROGEN CYCLE
Nitrates are essential for plant growth
Plant
protein
Root
uptake
Nitrate
NO3-
© 2008 Paul Billiet ODWS
Nitrates are recycled via microbes
Animal
protein
Soil organic nitrogen
Ammonification
Plant
protein
Root
uptake
Ammonium NH4+
Nitrification
Nitrite NO2Nitrification
© 2008 Paul Billiet ODWS
Nitrate
NO3-
Ammonification

Nitrogen enters the soil through the
decomposition of protein in dead organic
matter
Amino acids + 11/2O2  CO2 + H2O + NH3

+ 736kJ
This process liberates a lot of energy which
can be used by the saprotrophic microbes
© 2008 Paul Billiet ODWS
Nitrification
This involves two oxidation processes
 The ammonia produced by ammonification is an
energy rich substrate for Nitrosomas bacteria
They oxidise it to nitrite:

NH3 + 11/2O2  NO2- + H2O
+ 276kJ
This in turn provides a substrate for Nitrobacter
bacteria oxidise the nitrite to nitrate:
NO3- + 1/2O2  NO3-
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+ 73 kJ
This energy is the only source of energy for
these prokaryotes
They are chemoautotrophs
© 2008 Paul Billiet ODWS
Nitrogen from the atmosphere
Atmospheric
fixation
Out
gassin
g
Atmospheric Nitrogen
4 000 000 000 Gt
Plant
protein
Biological
fixation
Soil organic
nitrogen
Root uptake
Nitrate NO3© 2008 Paul Billiet ODWS
Atmospheric nitrogen fixation
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Electrical storms
Lightning provides sufficient energy to split
the nitrogen atoms of nitrogen gas,
Forming oxides of nitrogen NOx and NO2
© 2008 Paul Billiet ODWS
Atmospheric Pollution
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This also happens inside the internal combustion
engines of cars
The exhaust emissions of cars contribute a lot to
atmospheric pollution in the form of NOx
These compounds form photochemical smogs
They are green house gases
They dissolve in rain to contribute to acid rain in the
form of nitric acid
The rain falling on soil and running into rivers
They contribute to the eutrophication of water
bodies
© 2008 Paul Billiet ODWS
Biological nitrogen fixation
Treatments
Yield / g
Oats
No nitrate & sterile soil
Peas
0.6
0.8
Nitrate added & sterile soil
12.0
12.9
No nitrate & non-sterile soil
0.7
16.4
11.6
15.3
Nitrate added & non-sterile soil
© 2008 Paul Billiet ODWS
Conclusion
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Adding nitrate fertiliser clearly helps the growth of
both plants
The presence of microbes permits the peas to grow
much better than the oats
The peas grow better in the presence of the
microbes than they do with nitrate fertiliser added
The difference is due to the present of mutualistic
nitrogen fixing bacteria which live in the pea roots.
© 2008 Paul Billiet ODWS
Root nodules
Alafalfa (Medicago sativa)
USDA - ARS
University of Sydney
Only prokaryotes show nitrogen
fixation
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These organisms possess the nif gene complex which make the
proteins, such as nitrogenase enzyme, used in nitrogen fixation
Nitrogenase is a metalloprotein, protein subunits being
combined with an iron, sulphur and molybdenum complex
The reaction involves splitting nitrogen gas molecules and adding
hydrogen to make ammonia
N2  2N
2N + 8H+  NH3 + H2


- 669 kJ
+ 54 kJ
This is extremely energy expensive requiring 16 ATP molecules
for each nitrogen molecule fixed
The microbes that can fix nitrogen need a good supply of energy
© 2008 Paul Billiet ODWS
The nitrogen fixers
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Cyanobacteria are nitrogen fixers that also
fix carbon (these are photosynthetic)
Rhizobium bacteria are mutualistic with
certain plant species e.g. Legumes
They grow in root nodules
Azotobacter are bacteria associated with the
rooting zone (the rhizosphere) of plants in
grasslands
© 2008 Paul Billiet ODWS
The human impact
Atmospheric Nitrogen
Atmospheric
fixation
Out
gassin
g
Industrial
fixation
Plant
protein
Biological
fixation
Soil organic
nitrogen
Ammonium
NH4+
Nitrate NO3© 2008 Paul Billiet ODWS
Industrial N-Fixation
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The Haber-Bosch Process
N2 + 3H2  2NH3
- 92kJ
The Haber process uses an iron catalyst
High temperatures (500°C)
High pressures (250 atmospheres)
The energy require comes from burning fossil
fuels (coal, gas or oil)
Hydrogen is produced from natural gas
(methane) or other hydrocarbon
© 2008 Paul Billiet ODWS
The different sources of fixed nitrogen
Sources of fixed nitrogen
Production / M tonnes a-1
Biological
175
Industrial
50
Internal Combustion
20
Atmospheric
10
© 2008 Paul Billiet ODWS
Eutrophication
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Nutrient enrichment of water bodies
Nitrates and ammonia are very soluble in
water
They are easily washed (leached) from free
draining soils
These soils tend to be deficient in nitrogen
When fertiliser is added to these soils it too
will be washed out into water bodies
There algae benefit from the extra nitrogen
This leads to a serious form of water pollution
© 2008 Paul Billiet ODWS
Eutrophication
Fertilisers washed into river or lake
Sewage or
other organic
waste
© 2008 Paul Billiet ODWS
New limiting factor imposes itself
Making things worse!
Hot water
from industry
(Thermal
pollution)
Increased Biochemical
Oxygen Demand (BOD)
Reduction in dissolved O2
© 2008 Paul Billiet ODWS
Pollution
from oil or
detergents
The death of a lake
Reduction in dissolved O2
Increased nitrite
levels
NO3-  NO2-
Death/emigration
of freshwater
fauna
© 2008 Paul Billiet ODWS
Methaemoglobinaemia in infants
Stomach cancer link
(WHO limit for nitrates 10mg dm-3)
The future of industrial nitrogen
fixation
 Food production relies heavily upon synthetic
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fertilisers made by consuming a lot of fossil
energy
Food will become more expensive to produce
Nitrogen fixing microbes, using an enzyme
system, do the same process at standard
temperatures and pressures essentially using
solar energy
Answer: Genetically engineered biological
nitrogen fixation?
© 2008 Paul Billiet ODWS
Making things better
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The need for synthetic fertilisers can be reduced by
cultural practices
Avoiding the use of soluble fertilisers in sandy (free
draining soil) prevents leaching
Rotating crops permits the soil to recover from
nitrogen hungry crops (e.g. wheat)
Adding a nitrogen fixing crop into the rotation cycle
Ploughing aerates the soil and reduces
denitrification
Draining water logged soil also helps reduce
denitrification
© 2008 Paul Billiet ODWS
Return to the atmosphere:
Denitrification
Nitrates and nitrites can be used a source of
oxygen for Pseudomonas bacteria
 Favourable conditions: Cold waterlogged
(anaerobic) soils
2NO3-  3O2 + N2providing up to 2385kJ
2NO2-  2O2 + N2 
 The liberated oxygen is used as an electron
acceptor in the processes that oxidise
organic molecules, such as glucose
 These microbes are, therefore, heterotrophs

© 2008 Paul Billiet ODWS
Atmospheric Nitrogen
4 000 000 000 Gt
Atmospheric
fixation
Out
gassin
g
Industrial
fixation
Animal
protein
Plant protein
3500 Gt
Biological
fixation
Denitrification
Soil organic nitrogen
9500 Gt
Root
uptake
Ammonification
Dissolved in water
6000 Gt
Ammonium NH4+
Nitrification
Nitrite NO2Nitrification
Nitrate
NO3-
Leaching
Sediments 10 Gt
© 2008 Paul Billiet ODWS