Nitrogen Fixation

Nitrogen fixation
Nitrogen fixation

Nitrogen fixation is the process whereby elemental nitrogen from the atmosphere is converted to ammonium, an ionic form of nitrogen available to higher plants.

Nitrogen, the fourth most abundant element in most organisms, can account for as much as 4 percent of a plant’s dry weight. The majority of this nitrogen is present as a constituent of protein structure, but it is also a component of numerous other biological compounds, such as the chlorophylls and nucleic acids. Thus, for normal plant growth and development, nitrogen must be maintained at fairly high levels in the soil.

The earth’s atmosphere is about 79 percent nitrogen, and the vast majority of atmospheric nitrogen exists in the elemental state. Unfortunately, the elemental form of nitrogen is of no direct value to higher plants; they must acquire their nitrogen in the form of either ammoniumor nitrate. These two forms of nitrogen can be supplied to the soil as fertilizer by humans or by nature, as the product of microbial action.


There are three microbial processes that render nitrogen into forms usable by higher plants. These are ammonification, nitrification, and nitrogen fixation.

Ammonification is the process whereby various forms of organic nitrogen, such as is present in the proteins in plant and animal residues and animal wastes (manures), are converted to ammonium. Nitrification is the process by which ammonium is converted to nitrate. Both these processes are carried out by populations of free-living soil microorganisms.

In the nitrogen fixation process, atmospheric nitrogen is converted to ammonium. While some of the nitrogen fixers are free-living microbes, bacteria that live symbiotically within the roots of a number of plant species are also responsible for much of this conversion in terrestrial ecosystems.

Nitrogen-Fixing Bacteria

Nitrogen-fixing bacteria have been shown to coexist with a variety of lower plants, including lichens, liverworts, mosses, and ferns.

Among the more advanced seed-bearing plants, nitrogen fixers have been found to be associated with some tropical grasses and a number of shrubs and trees, such as the alders. Agriculturally, the legumes are the most important group of plants coexisting with nitrogen-fixing bacteria.

Some fifteen hundred species of legumes, including peas, beans, clover, and alfalfa, have been shown to live symbiotically with nitrogen-fixing bacteria called Rhizobium. A different species of Rhizobium infects each different species of legume.

The bacteria penetrate the root by entering the filamentous projections of epidermal cells called root hairs. The epidermal cells respond to the invasion by enclosing the bacterium in a threadlike structure referred to as an infection thread.

The infection thread begins to grow and branch, and, as it does so, the Rhizobia reproduce numerous times inside the thread. After penetrating several layers of cells, the infection thread eventually reaches the root cortex, where it ruptures and releases the encased bacteria.

The release of the bacteria induces the secretion of plant hormones that stimulate specialized root cortical cells to divide several times. As these cells divide, the Rhizobia are encapsulated, and a nodule is formed.

Within the cytosol of the nodule cells, the bacteria become nonmotile, increase in size, and accumulate in groups of four to eight bacteroids. These bacteroids are responsible for the biochemical conversion of elemental nitrogen to ammonium.

Chemistry of Nitrogen Fixation

Chemically, the fixation of nitrogen requires that six electrons and eight hydrogen ions be transferred to the atmospheric nitrogen molecule. This reaction is an energy-requiring process; therefore, adenosine triphosphate (ATP), the cell’s form of stored energy, must be available for the reaction to take place. The electrons, hydrogen ions, and ATP are supplied by the cellular respiration process that takes place in the root cells.

The electrons and hydrogen ions are transferred to the atmospheric nitrogen atom by an enzyme called nitrogenase. This enzyme consists of two subunits. One subunit takes the electrons and hydrogen ions from the respiratory products and transfers them to the other subunit. The ATP binds with part II of the nitrogenase, and the hydrolysis of the ATP releases the energy stored in the molecule.

This energy drives the reaction and makes it even easier to pass the electrons and hydrogen ions on to the second subunit. In the last step, the nitrogenase transfers the electrons and hydrogen ions to the nitrogen atom.

This final transfer results in the production of ammonium. The ammonium moves out of the bacteroids into the cytosol, where it is converted to an organic form of nitrogen that can be transported throughout the plant.

Rates of Fixation

Not all species fix nitrogen at the same rate. A number of factors can account for these differences. Some plants form nodules much more abundantly than others. Because of their more extensive nodule formation, these plants will fix more nitrogen than those that produce fewer nodules.

The nitrogenase of all rhizobial species has a tendency to transfer electrons to hydrogen ions rather than to nitrogen. As a result, hydrogen gas, which escapes into the atmosphere, is produced. This represents a loss of electrons that could have been used to produce ammonium.


Some Rhizobia species, however, have a second enzyme, called hydrogenase, which uses the hydrogen gas to produce water. ATP is produced as a byproduct of this process. Consequently, these rhizobial species are more efficient because less energy is wasted.

In addition, the fixation rate and the amount of nitrogen fixed will vary with age or the stage of plant development. Inmost cases, fixation rates are highest when the fruits and seeds are being produced.

The seeds of many plants, and especially legumes, are high in protein. Hence, nitrogen fixation and transport out of the nodules must be higher at the time the seeds are developing. In fact, more than 85 percent of the total nitrogen fixation in legumes occurs at such times.

Plant-Bacteria Mutualism

The nitrogen-fixing bacteria exist in a symbiotic relationship with their plant hosts. The bacteria supply the plant with much-needed nitrogen, while the plant supplies the bacteria with carbohydrates and other nutrients.

Some of the energy, derived from the plant-supplied carbohydrates, is used in nitrogen fixation, but there is ample left over to supply the bacteria with all the energy necessary for their survival.

Providing Nitrogen

Plant production throughout the world is limited more by the shortage of nitrogen than by any other nutrient. The root zone that encompasses the upper 15-centimeter layer of soil contains from 100 to 6,000 kilograms of total nitrogen per hectare. This includes all forms of nitrogen, many of which are not available to plants.

The total nitrogen content is determined by a number of factors, such as the minerals making up the soil, the kinds of vegetation, and the extent to which these factors are affected over time by climate, topography, and the presence of people.

Ammonium and nitrate are the only forms readily available to plants. These two molecules, in addition to those such as organic nitrogen compounds that can easily be converted to the available forms, are the only ones of ecological or agricultural importance. Unfortunately, these forms of nitrogen are continually being removed from the soil.

Crop removal, leaching (the removal of minerals as water percolates through the soil), denitrification (the process by which anaerobic microbes convert nitrates to gaseous nitrogen-containing compounds that escape the soil), and erosion account for a total loss of approximately 125 kilograms per hectare annually.

While some of the lost nitrogen can be replaced by available forms falling to the earth in rain, that amount is much too low to be of value in plant growth. Microbial fixation and the application of fertilizers are the only sources that supply sufficient nitrogen for plant growth.

Research Applications

The non symbiotic nitrogen fixers are of extreme ecological importance, especially in non agricultural soils. Forest, desert, and prairie ecosystems are dependent on nitrogen fixation by free-living species to replace the annual nitrogen loss.

Without it, growth of a number of plant species would suffer, and food chains in these ecosystems would soon be disrupted. A number of studies have investigated the advantages of incorporating free-living nitrogen fixers into nonlegume crop production, but clear benefits are uncertain.

On the other hand, knowledge of symbiotic nitrogen fixation have resulted in definite improvements in the production of legume crops. When Rhizobium is included with seeds as they are planted, increased yields have been observed in nearly every case.

There is considerable interest in enhancing the efficiency of the nitrogen-fixing process by increasing nodulation in the roots of some species or by incorporating the hydrogenase system into species that do not have it. An increase in biological nitrogen fixation could enhance the nitrogen content of the soil and decrease the dependency on commercial nitrogen fertilizers.

The application of nitrogen fertilizers to nonleguminous crops was once one of the best investments a farmer could make. Commercial fertilizers, however, have become very expensive because of increased energy costs. In addition, a number of environmental problems have developed from the accumulation of nitrates from fertilizers in rivers, ponds, and lakes.

Consequently, there is a renewed interest in symbiotic nitrogen fixation. For years, before the extensive use of nitrogen fertilizers, farmers planted legume crops alternately with other crops. The legume crops were plowed under to supply nitrogen to the soil.

There has been a resurgence in this technique. In fact, there is considerable interest in growing plants containing symbiotic nitrogen fixers in the same fields with plants lacking symbiotic nitrogen fixers, to improve the natural nitrogen balance in certain environments.

There are a number of studies directed at incorporating nitrogen fixation into nonlegume crop plants; this research is difficult, however, because the genetics of the process is so complex.