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1. The following slides are provided by Dr. Vincent O’Flaherty. Use the left mouse button to move forward through the show Use the right mouse button to view the slides in normal view, edit or print the slides

2. The Nitrogen Cycle • Growth of all organisms depends on the availability of mineral nutrients • Nitrogen required in large amounts as an essential component of proteins, nucleic acids and other cellular constituents • Abundant supply of nitrogen in the atmosphere - nearly 79% in the form of N2 gas - main reservoir of N

3. However, N2 is unavailable for use by most organisms because there is a triple bond between the two nitrogen atoms, making the molecule almost inert • Needs v. high energy or specialised enzyme complexes to break this bond • Haber-Bosch industrial process fixes N2 to NH3 - 1000˚C and 200 atm pressure • Some special microbes can carry out the process under “normal” conditions

4. In order for nitrogen to be used for growth it must be "fixed" (combined) in the form of ammonium (NH4) or nitrate (NO3) ions • This problem occurs because most plants can only take up nitrogen in two solid forms: ammonium ion (NH4+) and nitrate ion (NO3- )

5. Major reservoir of N is atmospheric N2, other major stores of nitrogen include: rocks in the earths crust and organic matter in soil and the oceans • Weathering of rocks releases these ions so slowly that it has negligible effect on the availability of fixed nitrogen • So, nitrogen often the limiting factor for growth and biomass production in all environments where suitable climate and availability of water supports life

6. Sources of N for plants and animals • Most plants obtain the nitrogen they need as inorganic nitrate from the soil solution • Ammonium is used less by plants for uptake because in large concentrations it is extremely toxic • Animals receive the required nitrogen they need for metabolism, growth, and reproduction by the consumption of living or dead organic matter containing molecules composed partially of nitrogen.

7. The Microbiology of the N-cycle • Microorganisms have a central role in almost all aspects of nitrogen availability and thus for life support on earth: • N2 gas is cycled from the atmospheric form through a number of inorganic and organic forms back to N2 - bacteria are the major organisms involved in the N-cycle, often specific species are NB

8. Some bacteria can convert N2 into ammonia by the process termed nitrogen fixation; these bacteria are either free-living or form symbiotic associations with plants or other organisms (e.g. termites) • Other bacteria carry out transformations of ammonia to nitrate, and of nitrate to N2 or other nitrogen gases

9. Many bacteria and fungi degrade organic matter, releasing fixed nitrogen for reuse by other organisms. • All these processes contribute to the nitrogen cycle. • We shall deal first with the process of nitrogen fixation and the nitrogen-fixing organisms, then consider the microbial processes involved in the cycling of nitrogen in the biosphere

10. Stages in the N-cycle • The stages in the N-cycle can be summarised as follows: • N2 fixation • Ammonification/mineralisatio • Nitrification • Denitrification

12. Nitrogen Fixation • N2 is an inert gas - must first be reduced to ammonia (nitrogen fixation),which can then be incorporated into organic molecules by microbes • A relatively small amount of ammonia is produced by lightning. Some ammonia also is produced industrially by the Haber-Bosch process, using an iron-based catalyst, very high pressures and fairly high temperature

13. But the major conversion of N2 into ammonia, and thence into proteins etc, is achieved by microorganisms- biological N-fixation • Total biological nitrogen fixation is estimated to be twice as much as the total nitrogen fixation by non-biological processes

14. Type of fixation N2 fixed (1012 g/year106 metric tons/year) Non-biological Industrial 50 Combustion 20 Lightning 10 • Total 80 Biological Agricultural land 90 Forest and non-agricultural land 50 Sea 35 • Total 175

15. Mechanism of biological nitrogen fixation • Biological N- fixation - 2 moles of ammonia produced from 1 mole of nitrogen gas, at the expense of 16 moles of ATP and a supply of electrons and protons (hydrogen ions): • N2 + 8H+ + 8e- + 16 ATP = 2NH3 + H2 + 16ADP + 16 Pi • Reaction is exclusive to prokaryotes using an enzyme complex - nitrogenase

16. Nitrogenase • Consists of two proteins - an iron protein and a molybdenum-iron protein • Reactions occur while N2 is bound to the enzyme complex. Fe protein is first reduced by electrons donated by ferredoxin. Reduced Fe protein binds ATP and reduces the Mo-Fe protein, which donates electrons to N2, producing HN=NH

17. Mechanism of biological N2 fixation • 2 further cycles of this process (each requiring electrons donated by ferredoxin) HN=NH is reduced to H2N-NH2, and this in turn is reduced to 2NH3

18. The reduced ferredoxin which supplies e’s for this process is generated by photosynthesis, respiration or fermentation (lots of energy required ATP’s) • Very strong functional conservation between the nitrogenase proteins of all nitrogen-fixing bacteria

19. Can mix the Fe protein of one species is mixed with the Mo-Fe protein of another bacterium, even if the species are very distantly related in the lab - still work • N-fixing organisms are all bacteria • Some free-living, others live in intimate symbiotic associations with plants or other organisms (e.g. protozoa)

20. Nitrogenase and oxygen • Nitrogenase is highly sensitive to oxygen - inactivated if exposed to oxygen, reacts with the iron component of the proteins • Major problem for aerobes - orgs have various methods to overcome the problem • E.g. Azotobacter sp. have the highest known rate of respiratory metabolism of any organism, so might protect enzyme by maintaining a v. low level of oxygen in their cells

21. Azotobacter species also produce copious amounts of extracellular polysaccharide • By maintaining water within the polysaccharide slime layer, these bacteria can limit the diffusion rate of oxygen to the cells • In the symbiotic nitrogen-fixing organisms such as Rhizobium, the root nodules can contain oxygen-scavenging molecules such as leghaemoglobin

22. Examples of nitrogen-fixing bacteria (* denotes a photosynthetic bacterium) Free living: Aerobic • Azotobacter • Beijerinckia • Klebsiella (some) • Cyanobacteria (some)* Anaerobic • Desulfovibrio • Purple sulphur bacteria* • Purple non-sulphur bacteria* • Green sulphur bacteria*

23. Symbiotic with plants: Legumes • Rhizobium Other plants • Frankia • Azospirillum • Clostridium (some) • Frankia

24. Symbiotic nitrogen fixation 1. Legume symbioses • Most NB examples of nitrogen-fixing symbioses are the root nodules of legumes (peas, beans, clover, etc.). • Bacteria are Rhizobium species, but the root nodules of soybeans, chickpea and some other legumes are formed by small-celled rhizobia termed Bradyrhizobium

25. Bacteria "invade" the plant and cause the formation of a nodule by inducing localised proliferation of the plant host cell • Chemicals called lectins act as signal molecules between Rhizobium and its plant host - v. specific • Bacteria form an “infection thread” and eventually burst into the plant cells - cause cells to proliferate - form nodules

28. Bacteria always separated from the host cytoplasm by being enclosed in a membrane • In nodules - plant tissues contain the oxygen-scavenging molecule - leghaemoglobin • Function of this molecule is to reduce the amount of free O2, protects the N-fixing enzyme nitrogenase, which is irreversibly inactivated by oxygen

29. Bacteria are supplied with ATP (80%), substrates and an excellent growth environment by the plant -carry out N-fixation • Bacteria provide plant with fixed N - major advantage in nutrient poor soils

30. Other symbiotic associations • 2. Frankia form nitrogen-fixing root nodules (sometimes called actinorhizae) with several woody plants of different families, such as alder • 3. Cyanobacteria often live as free-living organisms in pioneer habitats such as desert soils (see cyanobacteria) or as symbionts with lichens in other pioneer habitats

31. The nitrogen cycle • Diagram shows an overview of the nitrogen cycle in soil or aquatic environments • At any time a large proportion of the total fixed nitrogen will be locked up in the biomass or in the dead remains of organisms

32. So, the only nitrogen available to support new growth will be that which is supplied by NITROGEN FIXATION from the atmosphere (pathway 6) • or by the release of ammonium or simple organic nitrogen compounds through the decomposition of organic matter (pathway 2 (AMMONIFICATION/MINERALISATION)

33. Other stages in this cycle are mediated by specialised groups of microorganisms - NITRIFICATION AND DENITRIFICATION

34. Nitrification • Nitrification - conversion of ammonium to nitrate (pathway 3-4) • Brought about by the nitrifying bacteria, specialised to gain energy by oxidising ammonium, while using CO2 as their source of carbon to synthesise organic compounds (chemoautotrophs) • The nitrifying bacteria are found in most soils and waters of moderate pH, but are not active in highly acidic soils

35. Found as mixed-species communities (consortia) because some - Nitrosomonas sp. - are specialised to convert ammonium to nitrite (NO2-) while others - Nitrobacter sp. - convert nitrite to nitrate (NO3-) • Accumulation of nitrite inhibits Nitrosomonas, so depends on Nitrobacter to convert this to nitrate, and Nitrobacter depends on Nitrosomonas to generate nitrite • Nitrate leaching from soil is a serious problem in Ireland

36. Denitrification • Denitrification - process in which nitrate is converted to gaseous compounds (nitric oxide, nitrous oxide and N2). • Several types of bacteria perform this conversion when growing on organic matter in anaerobic conditions • Use nitrate in place of oxygen as the terminal electron acceptor. This is termed anaerobic respiration and can be illustrated as follows:

37. In aerobic respiration (as in humans), organic molecules are oxidised to obtain energy, while oxygen is reduced to water: • C6H12O6 + 6 O2 = 6 CO2 + 6 H2O + energy • In the absence of oxygen, any reducible substance such as nitrate (NO3-) could serve the same role and be reduced to nitrite, nitric oxide, nitrous oxide or N2

38. Conditions in which we find denitrifying organisms: (1) a supply of oxidisable organic matter, and (2) absence of oxygen but availability of reducible nitrogen sources • Common denitrifying bacteria include several sp. of Pseudomonas, Alkaligenes and Bacillus. Their activities result in substantial losses of N into the atmosphere, roughly balancing the amount of nitrogen fixation that occurs/year

39. Microbial N-Fixation