Nitrogen Fixation | Research & Encyclopedia Articles

Biological nitrogen (N₂) fixation is the reduction of atmospheric nitrogen gas to ammonia, according to the equation:

N₂+ 10H⁺ 8 e^- 16ATP ⁺2NH₄⁺ + H₂ + 16ADP + 16P_i

The reaction is mediated by an oxygen-sensitive enzyme nitrogenase and requires energy, as indicated by the consumption of adenosine triphosphate (ATP). This conversion of inert N₂ gas into a form utilized by most organisms is the second most important biological process on Earth after photosynthesis. It contributes 175 million tons of nitrogen per year to the global nitrogen economy and accounts for 65 percent of the nitrogen used in agriculture. In Brazil alone, N₂ fixation contributes the equivalent of 2.5 million tons of fertilizer nitrogen annually to agricultural production and is essential to a country with limited natural gas reserves for fertilizer nitrogen production.

This article emphasizes symbiotic N₂ fixation in grain and pasture legumes in the family Fabaceae. N₂ fixation also occurs in leguminous and actinorhizal trees, sugarcane, and rice.

The ability to fix N₂ is restricted to prokaryotic organisms. Within this group the ability occurs in many different species. These include cyano-bacteria and actinomycetes, as well as eubacteria, including heterotrophic (e.g., Azotobacter), autotrophic (Thiobacillus), aerobic (Bacillus), anaerobic (Clostridium), and photosynthetic (Rhodospirillum) species.

N₂-fixing organisms can live free in nature (e.g., Azotobacter), enter loose (associative) symbiosis with plants or animals (Acetobacter and sugarcane), or establish longer-term relationships within specialized structures provided by their host (Rhizobium and the legume nodule).

Some free-living organisms fix enough N₂ in vitro to grow without added nitrogen, but limited energy supply can limit N₂ fixation in nature. For instance, non-symbiotic organisms in primary successional areas of the Hawaii Volcanoes National Park were found to fix only 0.3 to 2.8 kilograms of N₂ per hectare per year, and non-symbiotic N₂ fixation in soil rarely exceeds 15 kilograms per hectare per year. Higher levels in tidal flats and rice paddies are largely due to photosynthetic bacteria and cyanobacteria.

The importance of energy supply for fixation can be seen by comparing these rates to those found in legumes, where the symbiotic bacteria are supplied with high-energy products from photosynthesis. Rates of symbiotic N₂ fixation in legumes vary with plant species and cultivar, growing season, and soil fertility. Some forage legumes can fix 600 kilograms per hectare per year but more common values are 100 to 300 kilograms per hectare per year. Rates for grain legumes are often lower. Inclusion of legumes in crop rotations is generally thought to improve soil nitrogen levels, but benefits depend on the level of N₂ fixed and the amount of nitrogen removed in grain or forage. A good soybean crop might fix 180 kilograms per hectare but remove 210 kilograms per hectare in the grain.

The most-studied symbiotic system is between N₂-fixing bacteria known as rhizobia and legumes such as clover and soybean. Rhizobia produce stem or root nodules on their host(s), and within these nodules receive protection from external stresses and energy for growth and N₂ fixation. The host receives most of the nitrogen it needs for growth. Six genera of rhizobia (Rhizobium, Azorhizobium, Mesorhizobium, Bradyrhizobium, Sinorhizobium, and Allorhizobium) are recognized.

Rhizobia use several different mechanisms to infect their host, but only infection via root hairs is described here. Infection is initiated with the attachment of suitable rhizobia to newly emerged root hairs and leads to localized hydrolysis of the root hair cell wall. Root hair curling and deformation results, with many of the root hairs taking the shape of a shepherd's crook. Hydrolysis of the cell wall allows rhizobia to enter their host, but they never really gain intracellular access. Plant-derived material is deposited about them, and as they move down the root hair toward the root cortex they remain enclosed within a plant-derived infection thread. Even within the nodule they are separated from their host by a host-derived peribacteroid membrane. This separation is usually seen as a mechanism to suppress plant defense responses likely to harm the bacteria.

Presence of the rhizobia causes multiplication and enlargement of root cortical cells and gives the nodule a characteristic shape and structure: either round as in soybean or elongated as in alfalfa or clover. Such nodules have several distinct regions. The area of active N₂ fixation is either pink or red in color due to the presence of hemoglobin needed for oxygen transport. In most legumes nodules are visible within six to ten days of inoculation; N₂ fixation as evidenced by improved plant growth and coloration of the nodules can occur within three weeks.

The signs of infection are paralleled at a molecular level by signaling between host and rhizobia. Nodulation genes in Rhizobium are borne on extra-chromosomal (plasmid) deoxyribonucleic acid (DNA). They include both common genes found in all rhizobia and host-specific genes involved in the nodulation of specific legumes. Most are only expressed in the presence of a suitable host. Substances termed flavonoids present in the root exudate trigger this response, with legumes differing in the flavonoids each produced. Rhizobia also differ in their response to these compounds.

More than fifty nodulation genes have been identified. Some are involved in the regulation of nodulation, but most function in the synthesis of a chitin -like lipo-chito-oligosaccharide or nod factor. These molecules all have the same core structure (coded for by the common nodulation genes), but they vary in the side chains each carries, affecting host range.

They are powerful plant hormones, which at low concentration can initiate most of the changes found during nodule development.

Interaction of host and rhizobia is also accompanied by the expression of nodule-specific proteins or nodulins. Several nodulins have now been found in actinorhizal and mycorrhizal symbiosis, and together with pea mutants that neither nodulate nor form mycorrhizal associations indicate some common elements in symbiosis.

Nodulin expression can vary temporally and spatially. Early nodulins are involved in infection or nodule development and may be expressed within six hours of inoculation. Later nodulins are involved in nodule function, carbon and nitrogen metabolism, or to O₂ transport. Nodule hemoglobin is an obvious example of this group.

Given the complex signaling involved, specificity in nodulation is to be expected. Each rhizobium has the ability to nodulate some, but not all, legumes. Host range can vary, with one rhizobia only nodulating a particular species of clover, for example, while another will nodulate many different legumes. A consequence of this specificity is that legumes being introduced into new areas will usually need to be inoculated with appropriate rhizobia before seeding. In the early 1900s this was often achieved by mixing seed with soil from an area where the crop had been grown before. Today, more than one hundred different inoculant preparations are needed for the different crop, tree, and pasture legumes used in agriculture and conservation. Most are grown in culture and sold commercially. The legumes for which inoculant preparations are available, and the methods used to prepare, distribute, and apply these cultures, are detailed on the Rhizobium Research Laboratory Web site (http://www.Rhizobium.umn.edu).

When properly carried out, legume inoculation should result in abundant nodulation and high levels of N₂ fixation. Reinoculation should not be necessary because large numbers of rhizobia will be released from nodules at the end of the growing season and establish themselves in the soil. Problems with the culture used and environmental and soil factors can limit response, especially in the lesser-developed countries. Common concerns include:

• poor-quality inoculant strains weak in N₂ fixation and noncompetitive or nonpersistent in soil
• inoculants with low rhizobial numbers because of problems in production or packaging or during shipment
• inappropriate use of fertilizer or pesticides injurious to the rhizobia
• soil acidity, drought, or temperature conditions that affect strain survival or nodulation and N₂ fixation.

Because of earlier problems in inoculant production and quality, many countries have now developed regulations governing the quality of inoculant cultures. In the United States, inoculant quality control still rests with the producer.

Atmosphere and Plants; Biogeochemical Cycles; Cyanobacteria; Eubacteria; Fabaceae; Fertilizer; Flavonoids; Mycorrhizae; Nutrients; Roots.

Graham, P. H. "Biological Dinitrogen Fixation: Symbiotic." In Principles and Applications of Soil Microbiology, eds. D. Sylvia et al. Upper Saddle River, NJ: Prentice-Hall, 1998.

Young, J. P. W. "Phylogenetic Classification of Nitrogen-Fixing Organisms." In Biological Nitrogen Fixation, eds. G. Stacey et al. New York: Chapman & Hall, 1992.

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