The Nitrogen Cycle

1. The Nitrogen Cycle • All life requires nitrogen-compounds, e.g., proteins and nucleic acids. • Air, which is 79% nitrogen gas (N2), is the major reservoir of nitrogen. • But most organisms cannot use nitrogen in this form. • Plants must secure their nitrogen in "fixed" form, i.e., incorporated in compounds such as: • nitrate ions (NO3−) • ammonia (NH3) • urea (NH2)2CO • Animals secure their nitrogen (and all other) compounds from plants (or animals that have fed on plants).

2. The Nitrogen Cycle

3. Four processes participate in the cycling of nitrogen through the biosphere: • nitrogen fixation • decay • nitrification • denitrification • Microorganisms play major roles in all four of these.

4. Nitrogen Fixation • The nitrogen molecule (N2) is quite inert. To break it apart so that its atoms can combine with other atoms requires the input of substantial amounts of energy. • Three processes are responsible for most of the nitrogen fixation in the biosphere: • atmospheric fixation by lightning • biological fixation by certain microbes — alone or in a symbiotic relationship with plants • industrial fixation

5. Atmospheric Fixation • The enormous energy of lightning breaks nitrogen molecules and enables their atoms to combine with oxygen in the air forming nitrogen oxides. These dissolve in rain, forming nitrates, that are carried to the earth. • N2 + O2 2NO • 2NO + O2 2NO2 • 2NO2 + H2O HNO3 • Ca or K + HNO3 CaNO3 or KNO3 (absorbed by roots plant) • Atmospheric nitrogen fixation probably contributes some 5– 8% of the total nitrogen fixed.

6. Industrial Fixation • Under great pressure, at a temperature of 600°C, and with the use of a catalyst, atmospheric nitrogen and hydrogen (usually derived from natural gas or petroleum) can be combined to form ammonia (NH3). Ammonia can be used directly as fertilizer, but most of its is further processed to urea and ammonium nitrate (NH4NO3).

7. Biological Fixation • The ability to fix nitrogen is found only in certain bacteria. • Some live in a symbiotic relationship with plants of the legume family (e.g., soybeans, alfalfa). • Some establish symbiotic relationships with plants other than legumes (e.g., alders). • Some nitrogen-fixing bacteria live free in the soil. • Nitrogen-fixing cyanobacteria are essential to maintaining the fertility of semi-aquatic environments like rice paddies. • Biological nitrogen fixation requires a complex set of enzymes and a huge expenditure of ATP. • Although the first stable product of the process is ammonia, this is quickly incorporated into protein and other organic nitrogen compounds.

9. Mechanism of biological nitrogen fixation • Biological nitrogen fixation can be represented by the following equation, in which two moles of ammonia are produced from one 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 • This reaction is performed exclusively by prokaryotes (the bacteria and related organisms), using an enzyme complex termed nitrogenase. This enzyme consists of two proteins - an iron protein and a molybdenum-iron protein, as shown below.

10. The reactions occur while N2 is bound to the nitrogenase enzyme complex. The Fe protein is first reduced by electrons donated by ferredoxin. Then the reduced Fe protein binds ATP and reduces the molybdenum-iron protein, which donates electrons to N2, producing HN=NH. In two 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. • Depending on the type of microorganism, the reduced ferredoxin which supplies electrons for this process is generated by photosynthesis, respiration or fermentation.

12. The nitrogen-fixing organisms

13. Symbiotic nitrogen fixation • 1. Legume symbioses • The most familiar examples of nitrogen-fixing symbioses are the root nodules of legumes (Paraserianthes falcataria, Accacia spp, peas, beans, clover, etc.).

14. Part of a crushed root nodule of a pea plant, showing four root cells containing colonies of Rhizobium. The nuclei (n) of two root cells are shown; cw indicates the cell wall that separates two plant cells. Although it cannot be seen clearly in this image, the bacteria occur in clusters which are enclosed in membranes, separating them from the cytoplasm of the plant cells. • In nodules where nitrogen-fixation is occurring, the plant tissues contain the oxygen-scavenging molecule, leghaemoglobin (serving the same function as the oxygen-carrying haemoglobin in blood). The function of this molecule in nodules is to reduce the amount of free oxygen, and thereby to protect the nitrogen-fixing enzyme nitrogenase, which is irreversibly inactivated by oxygen

15. 2. Associations with Frankia • 2. Associations with Frankia • Frankia is a genus of the bacterial group termed actinomycetes - filamentous bacteria that are noted for their production of air-borne spores. Included in this group are the common soil-dwelling Streptomyces species which produce many of the antibiotics used in medicine (see Streptomyces). Frankia species are slow-growing in culture, and require specialised media, suggesting that they are specialised symbionts. They form nitrogen-fixing root nodules (sometimes called actinorhizae) with several woody plants of different families, such as alder (Alnus species), sea buckthorn (Hippophae rhamnoides, which is common in sand-dune environments) and Casuarina (a Mediterranean tree genus). Figure A (below) shows a young alder tree (Alnus glutinosa) growing in a plant pot, and Figure B shows part of the root system of this tree, bearing the orange-yellow coloured nodules (arrowheads) containing Frankia.

17. 3. Cyanobacterial associations • The photosynthetic 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. They also form symbiotic associations with other organisms such as the water fern Azolla, and cycads.The association with Azolla, where cyanobacteria (Anabaena azollae) are harboured in the leaves, has sometimes been shown to be important for nitrogen inputs in rice paddies, especially if the fern is allowed to grow and then ploughed into the soil to release nitrogen before the rice crop is sown. A symbiotic association of cyanobacteria with cycads is shown below. The first image shows a pot-grown plant. The second image shows a close-up of the soil surface in this pot. Short, club-shaped, branching roots have grown into the aerial environment. These aerial roots contain a nitrogen-fixing cyanobacterial symbiont.