Nitrogen fixation by Microorganisms

Air contains approximately 79% nitrogen, but plants cannot use it. Nitrogen is the most limiting nutrient for plant growth. The conversion of N2 into the compound which can be absorbed by the plants by biological means is termed as nitrogen fixation. Nitrogen is the major part of proteins and nucleic acids which are the basis of all life forms. It is also present in chlorophylls, alkaloids, and cytochromes. Nitrogen from the soil is continually depleted by soil erosion, chemical voltalisation, and denitrification etc. The bacteria present in the root nodules of the leguminous plant helps in restoring N2 in the soil. Significance of biological nitrogen fixation as the major mechanism of recycling of nitrogen from the unavailable atmospheric form to available forms in the biosphere is well documented.

Nitrogen fixation process is also influenced by soil temperature. The optimum temperature for nitrogen fixation is 55 to 80°C. N2 fixation takes place mainly in the specialized cell called heterocyst of the cyanobacteria but non-heterocystous cyanobacteria have also been reported to fix N2. The factors which affect this process is the level of soil nitrogen, the rhizobia strain to infect the legume, amount of legume plant growth and the length of growing season. The rate of nitrogen fixation depends upon the rate of plant growth. We may define N2 fixation as the conversion of atmospheric N2 into nitrogenous salt which is readily absorbed by the plant. Nitrogen present in the atmosphere in the dinitrogen (N=N) form, therefore, the microorganism which fixes nitrogen is called diazotrophs. Nitrogen fixation by the activity of microorganisms is called biological N2 fixation and the microorganisms are called biofertilizers. But N2 -fixation also occurs spontaneously due to lightening. The dinitrogen reduction process is mediated by nitrogenase. In the case of photosynthetic bacteria, nitrogen fixation with nitrogenase frequently involves hydrogen generation as a byproduct. Such hydrogen production by photosynthetic bacteria may be a viable source for fuel cells, a possible next-generation energy source that is, independent of fossil fuels (Wakayama et al., 2000).

Aerobic diazotrophs—require oxygen for growth and fix nitrogen in the presence of oxygen (low concentration) (Azotobacter, methane-producing bacteria).

Free-living diazotrophs—fix nitrogen both in aerobic and anaerobic conditions (Bacillus and Klebsiella).

Symbiotic diazotrophs—fix nitrogen only by the formation of nodules (Rhizobium, Bradyrhizobium and Sinorhizobium).

Biological nitrogen fixation can be explained in two ways i.e., symbiotic and asymbiotic N2 fixation.

Non-biological

Lightning heats the air around it breaking the bonds of N₂ starting the formation of nitrous acid. Nitrogen can be fixed by lightning that converts nitrogen and oxygen into NOx (nitrogen oxides). NOx may react with water to make nitrous acid or nitric acid, which seeps into the soil, where it makes nitrate, which is of use to plants. Nitrogen in the atmosphere is highly stable and nonreactive due to the triple bond between atoms in the N₂ molecule. Lightning produces enough energy and heat to break this bond allowing nitrogen atoms to react with oxygen, forming NOx. These compounds cannot be used by plants, but as this molecule cools, it reacts with oxygen to form NO₂ This molecule, in turn, reacts with water to produce HNO₃ (nitric acid), or its ion NO−3 (nitrate), which is usable by plants.

Biological

Biological nitrogen fixation was discovered by German agronomist Hermann Hellriegel and Dutch microbiologist Martinus Beijerinck. Biological nitrogen fixation (BNF) occurs when atmospheric nitrogen is converted to ammonia by a nitrogenase enzyme. The overall reaction for BNF is:

N₂+6e^-+12ATP+12H₂O                                 2NH4++12ADP+12Pi+4H⁺

                                        Nitrogenase complex 

The process is coupled to the hydrolysis of 16 equivalents of ATP and is accompanied by the co-formation of one equivalent of H₂. The conversion of N₂ into ammonia occurs at a metal cluster called FeMoco, an abbreviation for the iron-molybdenum cofactor. The mechanism proceeds via a series of protonation and reduction steps wherein the FeMoco active site hydrogenates the N₂ substrate. In free-living diazotrophs, nitrogenase-generated ammonia is assimilated into glutamate through the glutamine synthetase/glutamate synthase pathway. The microbial nif genes required for nitrogen fixation are widely distributed in diverse environments. Nitrogenases are rapidly degraded by oxygen. For this reason, many bacteria cease the production of the enzyme in the presence of oxygen. Many nitrogen-fixing organisms exist only in anaerobic conditions, respiring to draw down oxygen levels, or binding the oxygen with a protein such as leghemoglobin.

Microorganisms

Diazotrophs are widespread within domain Bacteria including cyanobacteria (e.g. the highly significant Trichodesmium and Cyanothece), as well as green sulfur bacteria, Azotobacteraceae, rhizobia and Frankia. Several obligately anaerobic bacteria fix nitrogen including many (but not all) Clostridium spp. Some archaea also fix nitrogen, including several methanogenic taxa, which are significant contributors to nitrogen fixation in oxygen-deficient soils.

Cyanobacteria inhabit nearly all illuminated environments on Earth and play key roles in the carbon and nitrogen cycle of the biosphere. In general, cyanobacteria can use various inorganic and organic sources of combined nitrogen, such as nitrate, nitrite, ammonium, urea, or some amino acids. Several cyanobacteria strains are also capable of diazotrophic growth, an ability that may have been present in their last common ancestor in the Archean eon. Nitrogen fixation by cyanobacteria in coral reefs can fix twice as much nitrogen as on land—around 660 kg/ha/year. The colonial marine cyanobacterium Trichodesmium is thought to fix nitrogen on such a scale that it accounts for almost half of the nitrogen fixation in marine systems globally.

Marine surface licing[check spelling] and non-photosynthetic bacteria belonging in Proteobacteria and Planctomycetes fixate significant atmospheric nitrogen.

Root nodule symbioses

Legume family

Plants that contribute to nitrogen fixation include those of the legume family—Fabaceae— with taxa such as kudzu, clover, soybean, alfalfa, lupin, peanut and rooibos. They contain symbiotic rhizobia bacteria within nodules in their root systems, producing nitrogen compounds that help the plant to grow and compete with other plants. When the plant dies, the fixed nitrogen is released, making it available to other plants; this helps to fertilize the soil. The great majority of legumes have this association, but a few genera (e.g., Styphnolobium) do not. In many traditional and organic farming practices, fields are rotated through various types of crops, which usually include one consisting mainly or entirely of clover or buckwheat (non-legume family Polygonaceae), often referred to as "green manure".

Non-leguminous

Other nitrogen fixing families include:

• Parasponia, a tropical genus in the family Cannabaceae, which are able to interact with rhizobia and form nitrogen-fixing nodules.
• Actinorhizal plants such as alder and bayberry can form nitrogen-fixing nodules, thanks to a symbiotic association with Frankia bacteria. These plants belong to 25 genera distributed across eight families.

The ability to fix nitrogen is present in other families that belong to the orders Cucurbitales, Fagales, and Rosales, which together with the Fabales form a clade of eurosids. The ability to fix nitrogen is not universally present in these families. For example, of 122 Rosaceae genera, only four fix nitrogen. Fabales were the first lineage to branch off this nitrogen-fixing clade; thus, the ability to fix nitrogen may be plesiomorphic and subsequently lost in most descendants of the original nitrogen-fixing plant; however, it may be that the basic genetic and physiological requirements were present in an incipient state in the most recent common ancestors of all these plants, but only evolved to full function in some of them.