Nitrogen cycle and NITROGEN FIXATION

role of nitrogen, sources of nitrogen, nitrogen fixation, nitrogen assimilation, ammonification, nitrification, denitrification, nitrate reduction.

1. NITROGEN CYCLE

2. NITROGEN Nitrogen is element of group 14 in periodic table, having 7 as its atomic number and 14 as it atomic mass. It has stable valance states ranging from -3, as in ammonia (NH 3 ), to +5, as in Nitrate (NO 3 - ), occurs in numerous oxidation states. Nitrogen makes up about 78% of our atmosphere. In atmosphere it is present in it’s elementary form, i.e. N 2 which cannot be utilised by plants and animals directly.

3. Reservoir for nitrogen (3.8 * 10 15 metric tons) is N 2 gas of the atmosphere (78%). Unavailable reservoirs of nitrogen are present in: Igneous rock (1.4 * 10 16 metric tons) Sedimentary rock (4.0*10 16 metric tons) Nonexchangeable able ammonia (Blackburn 1983). Physicochemical and biological weathering releases ammonia from unavailable reservoirs so slowly that it has little influence on yearly cycling models. Geological deposits of more readily available combined nitrogen are rear. Availability combined nitrogen is an important limiting factor for primary production in many ecosystems.

4. Roles of Nitrogen Plants and bacteria use nitrogen in the form of NH 4 + or NO 3 - It serves as an electron acceptor in anaerobic environment. Nitrogen is often the most limiting nutrient in soil and water. Following perform actively cycled reservoirs of nitrogen: The inorganic nitrogen ions, ammonium, nitrite and nitrate occur as salts that are highly water soluble and consequently are distributed in diluted aqueous solution throughout the ecosphere. Living and dead organic matter In temperate climates, stabilized soil organic matter, or humus, forms a substantial and relatively stable nitrogen reservoir.

5. Plants could not continue their photosynthetic metabolism without the availability of fixed forms of nitrogen provided by microorganisms or by synthetic fertilizer. The biogeochemical cycling of the element nitrogen is highly dependent on the activities of microorganisms . The various transformations of nitrogen bring about the circulation of nitrogen from the atmosphere through terrestrial and aquatic habitats. These movements of nitrogen through the biosphere in large part determine ecological productivity in aquatic and terrestrial habitats.

6. Nitrogen is a key element for amino acids nucleic acids (purine, pyrimidine) cell wall components of bacteria (NAM).

7. Prior to human intervention N 2 fixation and denitrification processes appeared to be in balance. With increasing anthropogenic inputs, this may no longer be the case.

8. R-NH 2 NH 4 NO 2 NO 3 NO 2 NO N 2 O N 2

9. Sources Lightning Inorganic fertilizers Nitrogen Fixation Animal Residues Crop residues Organic fertilizers

10. NITROGEN CYCLE Process by which nitrogen is converted between various chemical form. This transformation can be carried out through both biological and physical processes.

11. Nitrogen cycle consist of following steps:- Nitrogen Fixation Nitrogen Assimilation Ammonification Nitrification Denitrification

12. Ways to fix Nitrogen Bacteria convert the nitrogen gas (N2) to ammonia (NH3). Lightning strikes convert N2 to N2O or NO3. Chemical manipulation turns N3 into NH3 (Fertilizer).

13. NITROGEN FIXATION Nitrogenase is the enzyme complex responsible for nitrogen fixation. The nitrogenase enzyme system has two co proteins (1: molybdenum + iron, 2: iron). Nitrogenase is extremely sensitive to oxygen, requiring low oxygen. The conversion of free nitrogen of atmosphere into the biologically acceptable form or nitrogenous compound. The fixation of nitrogen is done by rhizobium. The rate of fixation of magnitude higher then rate exhibited by free living fixing Bactria in soil.

14. The assay is based on the fact that nitrogenase enzyme reduced acetylene to ethylene. The similarity of acetylene molecule (CH--- CH) to (N---N). The enzyme that are produced by nitrogenase is a complex of dinitrogenase reductase (Fe protein) and dinitrogenase ( MoFe Protein). The electron is transfer to the dinitrogenase reductase (Fe4S4). Then they are transfers to a P cluster of the ( Dinitrogenase reductase protein). The P clusters passes the electron to the iron molybdenum cofactors (FeMoco;Fe7S9MO-homocitrate) of the dinitrogenase and then the N2-H2 is evolved from the reaction. The nitrogenase producing bacteria converts atm. Nitrogen to fixed form or nitrogen (NH3 ) that can be used by other microorganism , plants and animals. Azatobacter and Beijerinkckia both well-established genera of free living N-F-B and other genera have been found over time to fix atmospheric nitrogen. Species such as (chormatium, rhodopseudomonas, rhodospirillum, rhodomycrobium, cholorbium, azospirillum, bacillus, clostridium, vibrio and thiobacillus etc )

15. The rate of nitrogen fixation for free living soil bacteria are relatively low, these bacteria are wild spread in soil. The rate fixation by free living Bactria, such as Azotobacter and Azosprillium are within the rhizosphere then in soil lacking plant roots allowing for increased efficiency of nitrogen transfer to photosynthetic organism. Rates of nitrogen fixation by cyanobacteria are generally one to two orders of magnitude higher than by FREE LIVING NON-PHOTOSYNTHETIC soil bacteria. Nitrogen fixing cyanobacteria may of which form heterocyst. Some N-F-C form association with other organism as in: lichens, some form symbiotic association plants such as the Azolla –Anabaena association. Azotobacter and Beijerinckia can fix nitrogen at normal oxygen tension and appear to protect their nitrogenase from oxidative inactivation by a combination of complex biochemical mechanism. Other free living nitrogen fixer, such Azospirillum , fix nitrogen only at reduced oxygen tension.

16. The latter are active in anaerobic sediments and in the rhizosphere of plants growing in such sediments. As in aerobic nitrogen fixation, carbon rich, nitrogen poor substrate such as cellulose favor the process. This has been known to occur indirectly such as nitrogen fixer utilized low molecular weight products of cellulose fermentation.

17. AMMONIFICATION & ASSIMILATION Process of releasing ammonia by certain microogranisms utilizing organic compounds derived from dead organic remain of plants and animals and excreata of animals. Many plants, animals and microorganisms are capable of ammonification, a process in which organic nitrogen is converted to ammonia. Nitrogen in living and dead organic matter occurs predominantly in the reduced amino form. The release of ammonia from a simple nitrogenous organic compound, urea.

18. Some of the ammonia produced by ammonification is released from alkaline environment to the atmosphere, where it is relatively inaccessible to biological systems. Ammonium ions can be assimilated by numerous plants and many microorganisms. When they incorporated into amino acid and other nitrogen containing biochemical. The initial incorporation of ammonia into living organic matter is often accomplished either by glutamine synthase/ glutamate synthase reaction or by direct amination of an α -keto carboxylic acid from amino acid. Two important assimilation pathways vary among habitats and depend on environmental factor and species composition. The transformations of organic nitrogen-containing compounds are not restricted to microorganisms

19. NITRIFICATION Nitrification is a stage of the nitrogen cycle where ammonium is converted into nitrate by certain microorganism in soil. Nitrification is the process which is accomplished in two step. In nitrification, ammonia is first converted into nitrite and then nitrate. NH 4 + NITROSOMONAS NO 2 NITROBACTER NO 3 - (AMMONIUM) (NITRITE) (NITRATE) 2NH 3 + 3O 2 2NO 2 + 2H + + 2H 2 O

20. The nitrification process is primarily accomplishing by two groups of autotrophic nitrifying bacteria. In the first step of nitrification ammonia oxidizing bacteria oxidize ammonia to nitrite according to equation: NH 3 + O 2 NO 2 - + 3H + + 2e - Nitrosomonas is a chemoautotrophic organism found in soil and water are responsible for the oxidation of ammonium to nitrite and nitrite to nitrate. E.g. nitrococcus ,nitrospira In the second step of the process, nitrite oxidizing bacteria oxidize nitrite to nitrate. NO 2 - + H 2 O NO 3 - + 2H + 2e -

21. Nitrobacte r is the most frequently identified genus associated with this second step although other genera including Nitrospira , Nitrococcus, Nitrospina. Nitrifying bacteria are chemolithotrophs and utilize the energy derived from nitrification to assimilate CO 2 . The temperature for optimum growth nitrifying bacteria is 25° to 30°C. Nitrifying bacteria will die at 0°C. The oxidation of ammonia to nitrite and the oxidation of nitrite to nitrate are both energy yielding process. In the first reaction molecular oxygen is incorporated into the nitrite molecule. The oxidation is a multistep process and involves the generation of hydroxylamine (NH 2 OH) by ammonia mono oxygenase . The single oxygen atom incorporated into hydroxylamine and water, nitric acid and hydrogen, so the second oxygen atom in nitrite comes from water.

22. The two hydrogens from water are converted back to water by a terminal oxidase using atmospheric oxygen. The second step of nitrification obtains the oxygen for formation of nitrate from a water molecule. Both steps of nitrification are aerobic. Nitrite oxidation is a single step process and yields low amount of energy. Other bacteria capable of oxidizing ammonia to nitrite are found in the genera Nitrospira and Nitrosococcus, Nitrosolobus and Nitrosovibrio . Nitrobacter member of the genera nitrospira, nitrospina and Nitrosococcus are able to oxidize nitrite to nitrate. Nitrosococcus and Nitrosolobus are found in soil habitats. Some other microorganism, including heterotrophic bacteria and fungi, are capable of a limited oxidation of nitrogen compounds, but heterotrophic nitrification does not appear to make major contribution to the conversion of ammonia to nitrite and nitrate ions (shown in table).

23. The process of nitrification is especially important in soils, because the transformation of ammonium ions to nitrite and nitrate ions results in a change in charge from positive to negative.   Positively charged ions tend to bound by negative charged clay particles in soils, but negatively charged ions freely migrate in the soil water.  

24. NITRATE REDUCTION Nitrate can be incorporate by variety of microorganisms into organic matter through assimilatory nitrate reduction. Heterogeneous group of microorganisms are capable of assimilatory nitrate reduction. Assimilatory nitrate reduction: Reduction of nitrate into ammonia and its organic material. This process involve several enzyme: Nitrate and nitrite reductase to form ammonia, which can subsequently incorporate into amino acid. Enzyme involves several metalloprotein and reduced cofactor including NADPH. Assimilatory reductase enzyme does not result in accumulation of high concentration of extracellular ammonium ion, because ammonia is incorporated relatively rapidly into organic nitrogen

25. Dissimilatory nitrate reduction: In absence of oxygen, nitrate ion act as formal electron acceptor. Under anaerobic condition nitrate is converted in nitrite by facultative chemo-organotrophic anaerobic microorganism. E.g . Alcaligenes, Nocardia, Spirillum, Vibrio. Dissimilatory nitrate reduction do not inhibited by ammonia, this ammonia can be excreted in relatively high concentration. Some of these microorganisms will reduced nitrite via hydroxylamine to ammonia (nitrate ammonification).These microorganisms do not produce gaseous nitrogen product that why they do not denitrify. Nitrate ammonification play role in stagnant water, sewage water Denitrifying nitrate reducer such as Paracoccus denitrificans, Thiobacillus denitrificans and many more complete reduction pathway- NO 3 NO 2 NO N 2 O N 2

26. In soil primary denitrifying genera are Pseudomonas and Alcaligens. Many other are Rhizobium, Rhodopseudomonas, and Propionibacterium. The proportion of denitrifying product dependent upon both denitrifying microorganism and on environmental condition. Lower the pH of soil, greater the proportion of nitrous oxide formed. Simultaneously with denitrification, organic matter is oxidized. Utilization of glucose through nitrate reduction by Pseudomonas denitrificans is- C6H12O16 + 14NO3- 6CO2 +6H20 + 2N2 Enzyme involved is dissimilatory nitrate and nitrite reductase. Dissimilatory nitrate reductase is membrane bound, competitively inhibited by oxygen and not inhibited by ammonia. Dissimilatory nitrite reductase is soluble type, inhibited by ammonia, not substantially inhibited by oxygen .

27. DENITRIFICATION Occur strictly in anaerobic condition or reduced oxygen tension. Some time may occur in aerobic condition if these contain anoxic micro inhabitants. Denitrification most common in standing water condition. Denitrification rate is typically higher in hypolyminion of eutrophic lake during summer and winter stratification then during fall and spring turnover. DENITRIFICATION ACTIVITY OF Paracoccus denitrificans Activity restricted to some anaerobic condition. On change from aerobic to anaerobic respiration, culture of P.denitrificans enter unstable transition pathway, during which denitrification pathway induced. On this, phase is formed by a 15-45 fold increase of mRNA level for individual denitrification enzyme. All mRNA accumulate for short period of time, after which overall concentration decline to reach the stable value slightly higher then observed under aerobic steady-state condition. M-RNA formed for nitrate and nitrous oxide reduction.

28. When anaerobic culture somewhat switch back to aerobic culture, denitrification of cell stop at once. Although sufficient nitrite reductase is still present. Strains of Aeromonas, Maraxella, Pseudomonas (gram-ve) express nitrate reductase, show significant rate of nitrite respiration in the presence of oxygen when assayed with physiological electron donor. Arthobacter (gram+ve) able to suppress nitrate in presence of oxygen but different type of nitrate reductase. Thus, it show co-respiration of nitrate and oxygen make significant contribution to flux the nitrate to nitrite in environment.

29. THANK YOU