All living organisms need nitrogen in one form or another to build cellular constituents necessary for life, but most of them can assimilate only mineral nitrogen. In leguminous plants, a symbiotic relationship between a bacterium (called Rhizobium or Bradyrhizobium) and the plant enables the plant to benefit directly from the air as a source of nitrogen. In this natural Biological Nitrogen Fixation (BNF) process, the bacteria form nodules on the root system and convert the nitrogen in the air (nitrogen gas) into molecules (mineral nitrogen) that can be absorbed by the plant.
Importance of nitrogen for life The element 'Nitrogen' is an essential nutrient for all organisms' growth. It enters in large amounts in the form of many basic chemical compounds, such as proteins, nucleic acids and other cellular constituents. The earth's atmosphere is very abundant in nitrogen in the form of N2 gas (nearly 79% N2, 21% O2). However this free gaseous nitrogen cannot be used directly by animals or by higher plants to build the chemicals necessary for growth and reproduction. In fact, the N2 molecule is composed of two atoms of nitrogen linked by a very strong triple bond that requires a large amount of energy to be broken. Therefore the N2 molecule is quite chemically unreactive. To be incorporated by living organisms the atmospheric N2 must first be combined with oxygen or hydrogen into biological compounds such as ammonia (NH3, NH4+) or nitrates (NO3-). This conversion process is commonly referred to as nitrogen fixation that may be accomplished chemically or biologically. In this latter case, it is known as Biological Nitrogen Fixation (BNF).
N-fixation mechanism The general chemical reaction (which is a 'reduction' reaction) for the fixation of nitrogen can be represented by the following equation: N + 3H2 + Energy -> 2NH3 The molecular nitrogen (N2) is reduced to ammonia (NH3) by the breaking of the triple N bond and the addition of hydrogen to each nitrogen atom. This reaction requires a lot of energy and, in the case of biological fixation (BNF), energy is provided by ATP, which is a high-energy molecule derived from the burning ('oxydation') of carboxyhydrates. The BNF reaction can be represented in the following way: N2 + 8H+ + 8e- + 16 ATP = 2NH3 + H2 + 16ADP + 16 Pi 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).
This BNF reaction requires an enzyme complex termed nitrogenase to catalyse the re-action. The ability of the biological system to fix N2 is thus dependent of the presence of this particular enzyme system. Nitrogenase has so far been detected only in certain micro-organisms ('procaryotes'). No eukaryotic cells can do this, but they can benefit from ammonia provided by N-fixing bacteria.
Rhizobia, N-fixing organisms
Some of nitrogen-fixing bacteria are independent of other organisms – the so-called free-living nitrogen-fixing bacteria, whereas others live in close associations with plants or with other organisms (e.g. termites, protozoa). The best-studied example and the most important for agriculture is the association between legumes and bacteria belonging to the genus Rhizobium. (Other symbiotic associations occur but are less important.) Rhizobia are Gram-negative bacilli (rod-shaped) that live freely in the soil, especially where legumes have been grown. Although some other soil bacteria (e.g., Azotobacter) can fix atmospheric nitrogen by themselves, rhizobia cannot until they have invaded the roots of the appropriate legume. Clearly rhizobia and legumes are mutually dependent. In this symbiotic (mutually beneficial) relationship, each organism receives something from the other and gives back something in return. The plant supplies some micronutrients and the necessary energy source (carboxyhydrates, photosynthesis derived sugars) that enable the bacteria to fix gaseous N from the atmosphere and the rhizobia provide NH3 (resulting from the N2 fixation) to the plant for use in producing nitrogen containing compounds (such as amino acids). Rhizobium was first isolated in roots of legumes in 1888 by the Dutch microbiologist Martinus Beijerinck. The exact biochemistry of BNF is only partially understood.
Rhizobia–legume symbiosis Soon after a legume begins to grow, common soil bacteria of the genus Rhizobium or Bradyrhizobium ‘invade’ the roots, chiefly through the tiny root hairs, and proliferate within the cortex cells of the host plant. As a reaction to this infection, the legume roots form tumour-like growths, called nodules, on the root surface.
Within the nodules the bacteria develop into forms called bacteroids, which live in a symbiotic relationship with the host plant. Bacteria take air from the soil and fix appreciable amounts of nitrogen into ammonia (NH3) absorbed by the plant. Their abundance of nitrogen is beneficial not only to the legumes themselves, but also to the plants around them. The bacteria always remain separate from the host cytoplasm by being enclosed in a membrane – a necessary feature in symbiosis. Each bacterium becomes connected by the xylem and phloem to the vascular system of the plant. The rhizobia only fix N2 when in a strictly controlled micro-aerophilic environment. Oxygen is required to generate sufficient respiratory energy to drive N2 fixation, but too much oxygen inactivates nitrogenase.
Within a week after infection, small nodules are visible with the naked eye. In the field, small nodules can be seen 2-3 weeks after planting, depending on the legume species and germination conditions. Nodule shapes vary. Nodules on annual legumes, such as beans, groundnuts and soyabeans, are round and can reach the size of a large pea. Nodules on annuals are short-lived and will be replaced constantly during the growing season.
Symbiotic nitrogen fixation or biological N-fixation is the vital biological process that allows atmospheric molecular dinitrogen (N2), the gaseous nitrogen, to be converted into mineral nitrogen (NH3) which can then be assimilated by living organisms. This process is carried out by specific N-fixing bacteria which are either free-living in soil or water or associated with the root nodules of legume plants. The symbiotic association between legumes and bacteria (Rhizobium or Bradyrhizobium) takes place inside nodules located on the plant roots: with energy provided by the plant, the bacteroid (the symbiotic form of the bacterium) is able to fix the atmospheric nitrogen into ammonium nitrogen which can then be assimilated by the plant into amino acids to produce proteins'
N-fixation efficiency Most, but not all, legumes have the capacity to fix nitrogen (N). The quantity of nitrogen fixed by legumes can range from almost none to over 22,400 kg/ha and depends on several factors, such as the level of soil nitrogen and other nutrients (P, K, Mg, Ca, Fe, Mo, Cu, B), the kind of legume (1), the rhizobium strain infecting the legume (2), the effectiveness of the N-fixing bacteria (3), the way the legume is managed, and the length of growing season.
(1) The kind of legume Some legumes are better at fixing nitrogen than others. Common beans are poor fixers (less than 560 kg/ha) and fix less than their nitrogen needs. Other grain legumes, such as groundnuts, cowpeas, soyabeans and faba beans are good nitrogen fixers and will fix all of their nitrogen needs other than that absorbed from the soil.
(2) Presence of the right strain The plant roots must encounter the appropriate bacteria strains in the soil. A specific Rhizobium is required for a legume species. For example, bacteria infecting and nodulating kura clover (Trifolium ambiguum) will not nodulate lucerne effectively.
(3) The effectiveness of the N-fixing bacteria Rhizobium species (associated with many temperate pasture legumes such as lucerne, clovers, medics, trefoils, vetches, and lespedezas) are fast-growing, whereas Bradyrhizobium species (associated with soyabean and many tropical legumes such as cowpeas, carpon desmodium, stylosanthes, aeschynomene, and Vigna ) are slower-growing.
(4) The plant growth rate The rate of N2 fixation is related directly to the legume plant growth rate. Any stress that reduces plant activity, such as drought, low temperature, limited plant nutrients, or disease, will also reduce N2 fixation.
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