Nitrogen cycle/nitrogen fixation Nitrogen is an important constituent of biological molecules. The availability of N c

1. Nitrogen cycle/nitrogen fixation Nitrogen is an important constituent of biological molecules. The availability of N can affect plant growth and thus primary Production. Microbes are intimately involved in this process.

4. Nitrogen is a very stable and common molecule. A lot of energy is required to break the NN bond. Most organic nitrogen is recycled from the more easily available forms nitrate and NH4+. But N fixation is a critically important process in the environment and in agriculture

5. Denitrification Dissimilatory (anaerobic process), nitrate is used as an electron acceptor, producing N2. It is beneficial in waste water treatment, removing nitrate, thus reducing algal growth (blooms), eutrophication. Note they will grow aerobically using O2 as an electron acceptor if it is available (THIS IS NOT FERMENTATION). Pseudomonas denitrificans 2NO3- + 10e- +2H+ N2 + 6H2O The electrons come from metabolism of carbohydrates etc. NO3- NO2- NO  N2O N2 Thought to be an enzyme for each stage. Nitric and nitrous oxide can be released into the atmosphere causing potential problems.

6. Nitrification It is oxidation of ammonia to nitrate (via nitrite). Occurs in well drained, aerated soils by two nitrifying bacteria, Nitrosomas and Nitrobacter together (example of syntrophism). Manure and sewage promote nitrification. Nitrate is rapidly absorbed by plants but as it is very soluble it is easily leached out by rain, so it is not always of benefit. Ammonia at neutral and acid pH, is cationic and is absorbed by clay minerals. Anhydrous ammonia is used as a fertiliser, chemicals are added to inhibit nitrification. This (nitrapyrin) increases efficiency of the fertiliser and reduces run off water pollution.

7. Nitrification is a two stage process The process in energetically fairly inefficient, generating few ATP molecules the bacteria grow slowly. Nitrosomas NH4 + 3/2O2- NO2- +2H+ + energy Nitrobacter NO2- + 3/2O2 – NO3- + energy The energy generated is used to fix CO2

8. Assimililatory is is the conversion of nitrate (or NH3) to NH3 and then to nitrogenous compounds like amino acids.

9. Nitrogen Fixation This reaction is very important. N2 is very stable and there is a large a reservoir of N in the atmosphere. It requires a lot of energy to break the triple bond, and only a small number of organisms can do it, all prokaryotes. Both free living and in symbiotic associations.

10. Examples of nitrogen fixers Free living aerobes Azotobacter, Azomonas, Cyanobacteria (some) Free living anaerobes Closridium, Rhodobacter etc Symbiotic Rhizobium and Bradyrhizobium with legumes (Soya, peas, clover etc) Frankia with woody shrubs Anabaena with azolla (fern) in paddy fields

11. Biochemistry of Nitrogen fixation Nitrogen has a triple bond and this requires a lot of energy to break it. 940 kj compared to 493kj for O2. 6 electrons are required to reduce N2 to 2NH3. This reduction process is catalysed by Nitrogenase. Made up of two proteins – dinitrogenase and dinitrogen reductase. Both contain Fe and DR also has Mo. In DR the Fe and Mo are contained in a cofactor FeMo-co and the reduction of N2 occurs here. The formula is MoFe7S9. Two molecules of FeMo-co per molecule. FeS forms a cage.

12. FeMo co complex

13. Properties of Nitrogen fixation Nitrogen fixation is inhibited by O2. As it is a highly reducing process. Both enzymes are rapidly and irreversibly inactivated by O2. In aerobic bacteria, nitrogenase is protected from O2 either by rapid respiration, production O2 retarding slime or production of special cells (heterocysts), or O2 is removed by special chemicals. For every molecule of N2 fixed. 16-24 molecules ATP are used.

14. Electron Flow Ferredoxin, flavodoxin or low potential iron-sulphur protein are the electron donors. They transfer electrons to dinitrogen reductase. For each cycle of e- transfer, dintrogen reductase binds two ATP, which is then able to interact with dinitrogenase and transfer electrons to it. ATP is hydrolysed and the two proteins disassociate to begin another cycle of reduction. Only 6 electrons used in the useful reduction, another two are wasted to make H2, which can back react withN2H2.

16. Steps in Nitrogen Fixation

17. Assay Nitrogenase activity by acetylene to ethylene Artificial substrate HCCH  H2C=CH2

18. Soybean Root Nodules

20. Leguminous plants are at an advantage in poor soils. These bacteria are unable to fix, N2 alone, they need the plant. In the nodule O2 levels are controlled by leghaemolobin. The bacteria and plant form this iron containing compound which binds O2. Bound: free O2 is 10,000:1

21. Stages in Root Nodule Formation • 1 Recognition and attachment of bacterium to root hair • 2 Invasion of root hair, by formation of an infection thread. • 3 Travel to main root • 4 Formation deformed cells, bacteroids to get to N fixing state • 5 Formation of Nodule

22. Rhizobia grow well in the rhizopsphere, responding well to plant secretions. Rhicadhesin on the surface of bacterium may bind calcium complexes on the root hair surface. Invasion of the root hair is via the tip as a result of the action of bacterial encoded nod factors, inducing formation of a cellulosic tube, the infection thread. Root cells adjacent to this thread also become infected. Nod factors stimulate plant cell division. Bacteria multiply rapidly in the root. Bacterial cells become swollen into bacteroids, become surrounded singly, or in groups by plant cell membrane to become symbiosome. Only then does nit fix take place. When the plant dies, nodule deteriorate, bacteroid cannot divide, but some dormant rods always there which proliferate on the products released from the dying nodules. The fixed N is released to the soil

27. Stem nodulating legumes in tropics – Sesbania (water plant). Soils get leached because of high microbial activity.

28. Azolla pinnata (left) 1cm. Anabaena from crushed leaves Of Azolla.