General Microbiology - Schlegel, H. 1987

Molecular nitrogen fixation
Nitrogen fixation by symbiotic bacteria

Only prokaryotes are capable of utilizing atmospheric nitrogen reserves—that is, of fixing molecular nitrogen. Partly on their own and partly in Symbiosis with higher plants, they convert inert N₂ into Organic compounds and incorporate it (either directly or via plants) into Proteins, which ultimately end up in the soil. As a result of N₂ fixation by ROOT-nodule Bacteria in symbiosis with legumes, the soil is enriched with 100–300 kg of nitrogen per hectare annually. Free-living nitrogen-fixing microorganisms contribute 1–3 kg of nitrogen per hectare per year to the soil. In addition, significant amounts of fixed nitrogen can enter the soil from the atmosphere via precipitation. Depending on air pollution levels, this adds from 3 to 30 kg of nitrogen per hectare annually (see Section 1.3).

According to rough estimates, globally 175∙10⁶ t of nitrogen were fixed in 1974, of which 90∙10⁶ t were fixed in agricultural soils and 40∙10⁶ t industrially via the Haber-Bosch process. The majority of nitrogen is fixed by bacteria of the genus Rhizobium in root nodules.

Due to the noticeable enrichment of soil with fixed nitrogen, its symbiotic fixation has long attracted attention in various agricultural practices (fallow, crop rotation) and has come to be consciously utilized in agriculture.

Boussingault was the first to point out that clover and other legumes enrich the soil with nitrogen. The ESTABLISHMENT OF THE link between Nitrogen Fixation and legume root nodules is credited to Hellriegel and Wilfarth (1886–1888). Legumes can grow in the absence of combined nitrogen only if their roots are studded with nodules, which form as a result of root Hair infection by soil bacteria (Fig. 13.1).

Root nodules of leguminous plants. The bacteria responsible for nodule formation in legumes (nodule bacteria) belong to the genus Rhizobium. Leading a free-living existence in the soil, these aerobic Gram-negative rods grow as Saprophytes utilizing organic compounds. Based on host plant Specificity and several other characteristics, several species of root-nodule bacteria are distinguished (Rhizobium leguminosarum, R. meliloti, R. trifolii, R. phaseoli, R. lupini, R. japonicum, etc.).

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Fig. 13.1. Symbiotic nitrogen fixation in legume root nodules. A. Pea root with nodules. B. Cytology/practical/72.html">Cross section of nodules. C. Cross section of a plant Cell filled with bacteria (Rhizobium). D. Bacteria inside plant Cells acquiring an unusual shape (bacteroids, involution forms). E. Invasion of bacteria through root hair tips and growth of infection threads. All drawings are highly schematic.

Plant infection occurs exclusively through young root hairs (Fig. 13.1). Bacteria penetrate at or near the tip of the hair and grow in the form of an infection thread down to its base. These Cellulose-sheathed threads then penetrate the thin walls of young epidermal cells into the root cortex. Encountering one of the tetraploid cortical cells, the thread stimulates the division of both this cell itself and neighboring diploid cells. The infection threads branch and spread across the tetraploid cells. As a result of this tissue proliferation, induced by Rhizobium with the participation of a growth substance, nodules are formed. Bacteria inside the nodules multiply very rapidly and form large, irregularly shaped cells (bacteroids) whose volume can exceed that of free-living Rhizobium by a factor of 10–12; bacteroids are located individually or in groups, surrounded by a membrane in the Cytoplasm of plant cells. The tissue filled with bacteria has a reddish color—it contains leghaemoglobin, a pigment related to haemoglobin. Molecular nitrogen is fixed exclusively by those nodules that contain leghaemoglobin; "empty," pigment-free nodules are incapable of nitrogen fixation. The appearance of the pigment in the tissue coincides in time with the onset of N₂ fixation. When leghaemoglobin breaks down to form green Bile pigments (biliverdins), nitrogen binding also ceases. Leghaemoglobin is most likely located in The plant cell cytoplasm rather than in the spaces between bacteroids and their surrounding membranes. Pigment formation is a specific result of symbiosis: the prosthetic group (protohaem) is synthesized by bacteroids, while the protein component is produced with the participation of the plant. Leghaemoglobin is similar to Myoglobin, and in nodules it is predominantly represented by the Fe(II)-containing form. This pigment exhibits a high affinity for oxygen. It can be assumed that leghaemoglobin facilitates oxygen diffusion across the plant cell to the bacteroid. Thanks to the special properties of leghaemoglobin, bacteroids are supplied with sufficient oxygen for their growth and energy production, while avoiding an excessively high partial pressure of O₂, which is detrimental to Nitrogen fixation by bacteroids.

The underlying basis of host-plant specificity in Rhizobium symbiosis is not yet fully understood. According to a new hypothesis currently undergoing testing, the compatibility between partners is determined at the very first contact between bacteria and the root hair. Legumes contain Lectins—Glycoproteins capable of specifically binding Polysaccharides. Lectins are widespread in nature and apparently perform a recognition function. It is hypothesized that lectins are located on the outer surface of root hairs, while the outer layer of the Rhizobium Cell wall contains species-specific polysaccharide chains. It is possible that the interaction between root hair lectins and Rhizobium surface polysaccharides determines whether the hair becomes infected.

If the partners are compatible, a true symbiosis is established between the plant and Rhizobium. The plant provides the bacteria with nutrients (primarily sugars) and creates optimal conditions for them. When the plant dies, more viable bacteria enter the soil than could otherwise grow without the symbiotic association. N₂ fixation occurs exclusively within bacteroids, with about 95% of the fixed nitrogen being transferred in the form of ammonium ions into the host plant cytoplasm.

In addition to root nodules, stem nodules containing Rhizobium bacteroids also occur; such stem nodules are formed, for example, in the hygrophilous legume Sesbania rostrata from Central Africa.

It is unlikely that free-living soil rhizobia are capable of fixing N₂. Nitrogenase has been detected in colonies on Agar culture media for only a few strains. The formation of nitrogenase in these colonies depended on the partial pressure of O₂.

Root nodules in non-leguminous plants. Several non-leguminous plants also possess root nodules capable of fixing N₂. Here, too, Nitrogen fixation is based on symbiosis with prokaryotes. In the case of endosymbionts, these are mostly actinomycetes belonging to the genus Frankia.

Both woody and herbaceous plants can serve as hosts for symbiotic actinomycetes. They are distributed worldwide and are among the pioneer species in nitrogen-deficient soils. Nitrogen accumulation in the soil facilitated by such plants can reach 150–300 kg per hectare per year and is therefore of great agricultural importance. Plants most effective in nitrogen binding include Casuarina equisetifolia, alder (Alnus), sea buckthorn (Hippophae), and Ceanothus; less effective in this regard are bog myrtle (Myrica), mountain avens (Dryas), oleaster (Elaeagnus), and Shepherdia.

Root nodules of woody plants can reach the size of a tennis ball. They represent dense tangles of roots, branched like corals and having ceased growth. In Casuarina, nodules consist of a loose bundle of thickened roots with negatively geotropic growth. Symbiotic bacteria infect only the parenchymal cells in the root cortex. Just as in legumes, the infection penetrates the roots from the soil via root hairs; leghaemoglobin is also formed in the nodules. Only recently has a Rhizobium strain been discovered as an endosymbiont in a non-leguminous plant—Parasponia parviflora (elms). It was even possible to transfer this strain to leguminous plants. The root nodules of woody plants actively fix N₂.

Symbioses with nitrogen-fixing cyanobacteria. Cyanobacteria can also act as nitrogen-fixing partners in symbiosis with higher plants. In the aquatic fern Azolla, which grows On the surface of stagnant tropical Water bodies, cyanobacteria are housed in leaf cavities. The symbiotic partner of this plant is Anabaena azollae. While free-living Anabaena species contain very few (5%) heterocysts, 15–20% of the cells in the trichomes of symbiotic species are heterocysts. This ratio in itself indicates active nitrogen binding. Measurements of nitrogenase activity confirm this assumption. Azolla grows on The surface of flooded rice paddies and, with proper agricultural management, can fully meet the rice crop's nitrogen demand. Soil nitrogen accumulation resulting from the symbiosis between Anabaena and Azolla is about 300 kg/ha per year. A similar symbiosis is observed between liverworts (Blasia pusilla, Anthoceros punctatus, Peltigera) and Nostoc.

In the tropical shrub Gunnera macrophylla, symbiotic cyanobacteria Nostoc punctiforme reside in the lower part of the stem, specifically in specialized glands at the base of leaf petioles. This Nostoc species also forms heterocysts and nitrogenase.