Atmospheric nitrogen (N2 or N≡N) is abundant (78%) and vital element essential for growth of all living organisms on the earth. Despite its abundance, it is the most limiting nutrient available for growth of producers and consumers from all ecosystems from different habitats. This is because the gaseous N2 form of nitrogen is chemically inert and cannot be assimilated and therefore needs to be converted into soluble and assimiable forms such as ammonia (NH3), nitrites (NO2-) or nitrates (NO3-); NH3 is the most assimiable form of nitrogen by plants. The conversion of N2 to NH3 is the first and very important step of nitrogen cycle occurring on the earth. Naturally, this reaction is carried out only by prokaryotes and termed as biological nitrogen fixation. The prokaryotes which have the capacity to fix atmospheric nitrogen are symbiotic and free living bacteria and different species of cyanobacteria. Bacteria exhibiting this unique ability are termed as diazotrophic bacteria and the phenomenon of biological fixation of nitrogen is called as diazotrophy. Plants, animals, fungi and yeasts do not fix nitrogen. In absence of adequate supply of available soil nitrogen, plants depend on biologically fixed nitrogen.
The process of diazotrophy: Nitrogen fixation reaction is catalyzed by enzyme complex nitrogenase consisting of Mo-Fe cofactor. Nitrogenase is oxygen sensitive and inhibited by presence of oxygen; it means that nitrogen fixation takes place under anoxic environment. Nitrogen fixation genes (nif genes) such as nifA, nifB, nifF, nifM, nifV etc codes for various functional proteins of nitrogenase system during process of nitrogen fixation. Diazotrophic process is chemically reduction reaction requiring large amount of energy in the form of about 16 equivalents of adenosine tri phosphate (ATP). Dinitrogen is reduced to ammonia by addition of hydrogen to each N atom resulting in the cleavage of N≡N triple bond. ATP requirement is furnished by oxidation of carbohydrate source like sugar or glucose released by root exudates. Evolution of hydrogen in the form of gas is a wasteful side chain reaction in terms of energy that occurs in every diazotrophic strain during fixation of dinitrogen by nitrogenase. However, presence of other enzyme system termed as 'uptake hydrogenase' prevents loss of energy by catalyzing oxidation of H2 evolved during N2 fixation to ATP, a useful form of energy. This reaction removes hydrogen which is also a competitive inhibitor of nitrogen fixation.
Types of diazotrophic bacteria and their adaptive mechanisms: Diazotrophic bacteria can fix nitrogen either independently or in symbiotic association with plants. Independent bacteria are recognized as free living bacteria. Bacteria from genera Azospirillum, Azotobacter, Beijerinckia, Pseudomonas, Klebsiella, Bacillus and Clostridium are known to be potential free living nitrogen fixers. In addition to these, species of photosynthetic bacteria Rhodobacter, Rhodospirillum, Thiospirillum and Chromatium; different species of methanogenic archaebacteria like Methanococcus, Methanosarcina, Methanobacter and Methanospirillum; cyanobacterial species of Anabaena, Nostoc and Calothrix are also free living diazotrophs. In above examples, it is interesting to note that three physiological groups, that is aerobic, facultatively anaerobic and strictly anaerobic bacteria are diazotrophic. Since nitrogenase is inactivated by oxygen, then how do aerobic bacteria carry out nitrogen fixation? To overcome this problem, aerobic bacteria have developed special adaptive mechanisms so that temporarily anaerobic conditions are created until nitrogenase is actively involved in nitrogen fixation. Some of the mechanisms include high respiratory rate, partial uncoupling of respiratory chain, synthesis of slime or exopolysaccharides and formation of heterocysts as specialized structures found in cyanobacteria wherein nitrogen fixation takes place. Clostridium and Azospirillum can perform fixation of dinitrogen in absence of such adaptive mechanisms as being strictly anaerobic and microaerophilic respectively.
Other groups of diazotrophic bacteria are symbiotic which can fix nitrogen only when associated with root nodules of host plants. Nodules are sites for nitrogen fixation formed on plant roots. Rhizobium and Bradyrhizobium spp. are well known nodule bacteria of plants from family Leguminaceae; legumes like alfalfa, beans, clover, peas and soybean have been studied in great depth regarding their nodule bacteria and nitrogen fixation. Rhizobium and Bradyrhizobium are actually free living soil bacteria but fix nitrogen by establishing symbiotic association with host plants. Species of Bradyrhizobium are confined to legume soybean. Rhizobia are host-specific; specific rhizobial strain is able to nodulate specific host plant species. It means that Rhizobium sp. infecting alfalfa will always inhabit alfalfa only and not clover or other legume. Identification of host plant by rhizobial sp. and recognition of correct species of Rhizobium by a host plant is actually highly regulated biochemical series of specific events. After recognition, rhizobia can enter inside the plant roots and establish a microenvironment as an endosymbiont 'bacteriod' also followed by nodule formation. Nodulation is promoted by rhizobial production of cytokinin type of hormone. Nodule morphology and its nitrogen fixation products are also specific to that Rhizobium-host plant association; one can easily identify legume species or Rhizobium sp. from the observations of morphology of nodules. Nodule environment is protected from oxygen by leghemoglobin; 'leg' word in leghemoglobin stands for 'legume'. It is oxygen consuming protein similarly like human hemoglobin and it is secreted by host plant (globin part) and Rhizobium (heme portion) together during nitrogen fixation by active bacteriods. Rhizobial bacteriods actively fix nitrogen to ammonia at the expense of sugar and organic acids provided by host plant roots; ammonia is instantly converted to amino acids by bacteriods which are then assimilated by host plant for biosynthesis. Legume-Rhizobium association is obligate symbiosis and extremely important for agriculture as it is one of the major nitrogen fixing system present on the earth.
Non-leguminous but nitrogen fixing symbiotic associations have also been investigated. Soil bacteria Frankia from family Actinomycetes are diazotrophic, nodulating and found associated with different angiosperms and gymnosperms such as Alnus and Cycas respectively. Other nitrogen fixing but symbiotic bacteria include the genera of cyanobacteria. Association between Azolla and Anabaena from paddy fields is representative of such association. Anabaena spp. are found with water fern Azolla spp. Azolla is grown in rice paddies early in the season. As the rice grows above water surface, it shades out the fern, which dies, releasing the stored nitrogen. In this way, paddy gets richly fertilized with organic nitrogen and application of chemical fertilizers is no longer necessary.
In vitro detection of diazotrophy: Practically, it is almost impossible to measure nitrogenase activity by directly measuring nitrogen converted to ammonia. However, enzyme nitrogenase is found to reduce number of substrates (cyanides, azides, nitrous oxides) other than nitrogen and acetylene gas was detected as one of the substrates of nitrogenase; reduction of acetylene to ethylene gas could provide a direct measurement for nitrogenase activity. No enzyme other than nitrogenase reduces acetylene to ethylene and requirements for acetylene reduction are similar to those for nitrogen fixation. Derepressed condition because of nitrogen starvation, lag or log phases of active enzyme or temperature-time dependent nitrogen activity can be determined by Acetylene reduction test (ART). Experimental analyses using ART have proved that nitrogenase complex is active at low temperatures and this has been supported by reports on the presence of diazotrophs from Arctic regions. It can be concluded that nitrogenase shows adaptability at cold temperatures retaining its functional state. Other two methods of measurement of nitrogenase activity, N15 isotope dilution and Kjeldhal method are also utilized but they are laborious enough to carry out during routine laboratory estimations. Now-a-days, genes from set of nif gene system can be directly detected to indicate nitrogenase activity and diazotrophy of particular bacterial strain. Estimation of diazotrophy also presents valuable data regarding functioning of complex nitrogenase enzyme system. It has been detected that active hydrogenase activity is co-linked with nitrogenase activity. The detection of hydrogenase activity can also be done which indirectly shows the presence of active nitrogenase in diazotrophic bacteria.
Importance of diazotrophy: Fixed nitrogen is required by plants to synthesize nucleotides, proteins; the plant nitrogen is then circulated gradually in ecosystem via different levels of consumers for their utilization. Besides biological nitrogen fixation, nitrogen fixation is also carried out by non-biological processes; naturally by lightning and industrially by Haber-Bosch process. However, lightning process alone cannot furnish the demand of this limiting nutrient on the earth. On the other hand, Haber-Bosch process which is used for industrial production of ammonia and other nitrogenous fertilizers like urea and ammonium nitrate from nitrogen and hydrogen employing catalysts like ruthenium and nickel. The process is very expensive as high fossil fuel energy and costly catalysts are required to break triple bonds of inert N2. Detrimental of chemical nitrogen fertilizers are many; eutrophication is the one of them which has initiated problem like algal blooms and their toxic effects. Chemical fertilizers are being used to supplement nitrogen requirements of crops but it has been proved that applied dose is never able to fulfill crop's need but instead it is washed out in soil and aquifers. It has also led to the increased acidity of soils and imbalance of ecosystems.
Biofertilizer potential of diazotrophs: The biological nitrogen fixation is the first known property of plant growth promoting rhizobacteria (PGPR). It is the most important process on which the growth of all types of plants present on the earth is relied. Nitrogenous fertilizers are the oldest one known for their effect on plants and increased crop yield. Nitrogen fixing species of PGPR Azotobacter, Azospirillum and Rhizobia have been utilized since the century as biofertilizers by the farmers worldwide to increase the crop yield. In 1895, the first rhizobial inoculant 'Nitragin' was commercialized by Nobble and Hiltner to increase crop yield. This was followed by commercialization of Azotobacter containing bioinocula 'Azotogen' and 'Azotobaktrin' in India, Europe, USA, New Zealand, Soviet Union and Australia. Nitrogen is the nutrient most limiting the plant growth especially in agricultural systems; application of nitrogen biofertilizers thus becomes mandatory to retain soil fertility as well as to protect environment from overuse of chemical nitrogen fertilizers. Today's agricultural practices are still dependent on nitrogen fixation by legumes and their associated rhizobia; legumes crops are also used as green manure and in rotational cropping to supplement natural nitrogen fertilization of soil.
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