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THE NITROGEN CYCLE
Nitrates are essential for plant growth
Root
uptake
Nitrate
NO3
-
Plant
protein
© 2008 Paul Billiet ODWS
Nitrates are recycled via microbes
Nitrification
Nitrification
Ammonium NH4
+
Ammonification
Nitrite NO2
-
Soil organic nitrogen
Animal
protein
Root
uptake
Nitrate
NO3
-
Plant
protein
© 2008 Paul Billiet ODWS
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
-
+ 73 kJ
 This energy is the only source of energy for
these prokaryotes
 They are chemoautotrophs
© 2008 Paul Billiet ODWS
Root uptake
Nitrate NO3
-
Plant
protein
Soil organic
nitrogen
Nitrogen from the atmosphere
Biological
fixation
Atmospheric
fixationOut
gassin
g
Atmospheric Nitrogen
4 000 000 000 Gt
© 2008 Paul Billiet ODWS
Atmospheric nitrogen fixation
 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
 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 Peas
No nitrate & sterile soil 0.6 0.8
Nitrate added & sterile soil 12.0 12.9
No nitrate & non-sterile soil 0.7 16.4
Nitrate added & non-sterile soil 11.6 15.3
© 2008 Paul Billiet ODWS
Conclusion
 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
University of Sydney
Alafalfa (Medicago sativa)
USDA - ARS
Root nodules
Only prokaryotes show nitrogen
fixation
 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 - 669 kJ
2N + 8H+
→ NH3 + H2 + 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
 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
Nitrate NO3
-
Atmospheric
fixation
Out
gassin
g
Plant
protein
Atmospheric Nitrogen
Ammonium
NH4
+
Soil organic
nitrogen
The human impact
Biological
fixation
Industrial
fixation
© 2008 Paul Billiet ODWS
Industrial N-Fixation
 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
 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
Fertilisers washed into river or lake
New limiting factor imposes itselfSewage or
other organic
waste
Eutrophication
© 2008 Paul Billiet ODWS
Increased Biochemical
Oxygen Demand (BOD)
Hot water
from industry
(Thermal
pollution)
Pollution
from oil or
detergents
Reduction in dissolved O2
Making things worse!
© 2008 Paul Billiet ODWS
The death of a lake
Death/emigration
of freshwater
fauna
Methaemoglobinaemia in infants
Stomach cancer link
(WHO limit for nitrates 10mg dm-3
)
Increased nitrite
levels
NO3
-
→ NO2
-
Reduction in dissolved O2
© 2008 Paul Billiet ODWS
The future of industrial nitrogen fixation
 Food production relies heavily upon synthetic
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
 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
Sediments 10 Gt
Nitrification
Root
uptake
Biological
fixation
Nitrification
Ammonium NH4
+
Ammonification
Nitrite NO2
-
Dissolved in water
6000 Gt
Denitrification
Leaching
Nitrate
NO3
-
Soil organic
nitrogen 9500 Gt
Atmospheric
fixation
Out
gassin
g Industrial
fixation
Plant
protein
3500 Gt
Animal
protein
Atmospheric Nitrogen
4 000 000 000 Gt
© 2008 Paul Billiet ODWS

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N- CYCLE

  • 2. Nitrates are essential for plant growth Root uptake Nitrate NO3 - Plant protein © 2008 Paul Billiet ODWS
  • 3. Nitrates are recycled via microbes Nitrification Nitrification Ammonium NH4 + Ammonification Nitrite NO2 - Soil organic nitrogen Animal protein Root uptake Nitrate NO3 - Plant protein © 2008 Paul Billiet ODWS
  • 4. 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
  • 5. 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 - + 73 kJ  This energy is the only source of energy for these prokaryotes  They are chemoautotrophs © 2008 Paul Billiet ODWS
  • 6. Root uptake Nitrate NO3 - Plant protein Soil organic nitrogen Nitrogen from the atmosphere Biological fixation Atmospheric fixationOut gassin g Atmospheric Nitrogen 4 000 000 000 Gt © 2008 Paul Billiet ODWS
  • 7. Atmospheric nitrogen fixation  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
  • 8. Atmospheric Pollution  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
  • 9. Biological nitrogen fixation Treatments Yield / g Oats Peas No nitrate & sterile soil 0.6 0.8 Nitrate added & sterile soil 12.0 12.9 No nitrate & non-sterile soil 0.7 16.4 Nitrate added & non-sterile soil 11.6 15.3 © 2008 Paul Billiet ODWS
  • 10. Conclusion  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
  • 11. University of Sydney Alafalfa (Medicago sativa) USDA - ARS Root nodules
  • 12. Only prokaryotes show nitrogen fixation  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 - 669 kJ 2N + 8H+ → NH3 + H2 + 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
  • 13. The nitrogen fixers  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
  • 14. Nitrate NO3 - Atmospheric fixation Out gassin g Plant protein Atmospheric Nitrogen Ammonium NH4 + Soil organic nitrogen The human impact Biological fixation Industrial fixation © 2008 Paul Billiet ODWS
  • 15. Industrial N-Fixation  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
  • 16. 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
  • 17. Eutrophication  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
  • 18. Fertilisers washed into river or lake New limiting factor imposes itselfSewage or other organic waste Eutrophication © 2008 Paul Billiet ODWS
  • 19. Increased Biochemical Oxygen Demand (BOD) Hot water from industry (Thermal pollution) Pollution from oil or detergents Reduction in dissolved O2 Making things worse! © 2008 Paul Billiet ODWS
  • 20. The death of a lake Death/emigration of freshwater fauna Methaemoglobinaemia in infants Stomach cancer link (WHO limit for nitrates 10mg dm-3 ) Increased nitrite levels NO3 - → NO2 - Reduction in dissolved O2 © 2008 Paul Billiet ODWS
  • 21. The future of industrial nitrogen fixation  Food production relies heavily upon synthetic 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
  • 22. Making things better  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
  • 23. 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
  • 24. Sediments 10 Gt Nitrification Root uptake Biological fixation Nitrification Ammonium NH4 + Ammonification Nitrite NO2 - Dissolved in water 6000 Gt Denitrification Leaching Nitrate NO3 - Soil organic nitrogen 9500 Gt Atmospheric fixation Out gassin g Industrial fixation Plant protein 3500 Gt Animal protein Atmospheric Nitrogen 4 000 000 000 Gt © 2008 Paul Billiet ODWS