Process of Nitrogen Fixation in Microorganisms | Microbiology

In this article we will discuss about the process of nitrogen fixation in microorganisms.

The symbiotic association of cyanobacteria with fungi (lichen), cyanobacteria with bryophytes (Anthoceros), with pteridophytes, (Azolla) with gymnosperms (coralloid root of Cycas) and bacteria (Rhizobium Brady-rhizobium, Azorhizobium, Sino-rhizobium, Ensifer and Mesorhizobium etc.) with leguminous plants are under mutual beneficial relationship (symbiosis) in which both the host and bacteria are benefitted.

There are number of other angiosperms (excluding legumes) which have the symbiotic association with nitrogen-fixing microorganisms. About 15 angiospermic plants are of non-legumes category which fix atmospheric nitrogen, for example, Alnus, Myrica, Purshia, etc. The symbiotic microorganisms are not only bacteria but also comprise actinomycetes such as Frankia which fixes nitrogen.

There are some indications of the existence of haemoglobin like pigments in the root nodules of Alnus, Elaeganus, Shepherdia and Hippophae. In such cases, the hyphal threads of the endophyte fill the cortical cells which increase in volume resulting into a primary nodule recognizable on the root as a ‘swelling’.

The lateral roots arise in the vicinity of the primary nodule. Their meristem undergoes branching and gets infected with the endophyte results in the formation of a typical adult nodular structure referred to as a ‘rhizothamion’.

The occurrence of “leaf nodules” is confined to the families of Rubiaceae and Myristicaceae. The bacteria are isolated and identified as Mycobacterium rubiacearum, Mycoplana rubra, Flavobacterium species, Phyllobacterium rubiacearum and Kleb­siella rubiacearum. It is interesting to note that these bacterial isolates do not fix nitrogen.

Several species of Podocarpus possess numerous small nodules on the root system. The most common endophyte is a non- septate fungus resembling the fungal component of endotrophic mycorrhizae. This nodulated root system demonstrates that it performs the process of nitrogen fixation very slowly.

1. Root Modulating Symbiotic Bacteria:

Rhizobium forms nodules and participates in the symbiotic acquisition of nitrogen. The rod shaped bacteria, 0.5 – 0.9 × 1.2 – 3.0 µm long, motile. Gram – negative, non-spore forming, utilize organic acid salts as carbon sources without gas formation; while the cellulose and starch are not utilised. The growth is optimum at 27°C (pH 6.8) and colonies appeared as circular, convex, semi-translucent, raised and mucilaginous, usually 2 – 4 mm in diameter.

Production of an acid reaction occurs in mineral salt medium. Some strains of rhizobia and agrobacteria show a close relationship in DNA base composition. All species (except Agrobacterium radiobacter, syn. Rhizobium radiobacter), incite hypertrophies on plant roots.

Nodules are incited by strains of rhizobia on root of leguminous plants and leaves of certain plants in the families Myristicaceae and Rubiaceae by strains of Phyllo-bacteria. The strains of Rhizobium are fast-growing, where generation time lasts about 6 h besides showing some other differences with rest of the members of family Rhizobiaceae.

Some plants bear stem nodules (Sesbania species) by Azorhizobium caulinodans. The strains bear flagella; hence cells are motile (peritrichous flagella on solid medium but one lateral flagellum in liquid medium). They also fix nitrogen. These are oxidase and catalase positive and cannot oxidize mannitol.

The Brady rhizobium strains are slow growers where generation time is about 12 h or more. The motility occurs by one polar or sub-polar flagellum. The growth on carbohydrate medium is accompanied by exopolysacchaiide (EPS) slime. Some strains can grow chemolithotrophically (utilize inorganic salts) in the presence of H₂, CO₂ and low level of O₂.

The bacteroids in root nodules are slightly swollen rods with rare branching or coccus forms. Their main symbiotic partner is soybean, while other Brady rhizobia produce nodules in the plants such as Lotus, Vigna, Lupinus, Ornithopus, Cicer, Leucaena, Mimosa, Lahlab, Acacia and Dalbergia. Now the strains of Brady rhizobia are designated as name of the host plant in parentheses e.g. Brady rhizobium (Lotus) species.

Recently, it has been observed that some rhizobial strains which are fast growers nodulate soybean, (generally, Brady rhizobia nodulate soybean). These fast growers are identified as a separate genus Sino rhizobium which are rod shaped; usually contain poly-β-hydroxybutyric acid (PBHA) granules, non-spore forming. Gram-negative, motile, aerobic. Most strains grows at 35°C (pH 6- 8). Recently several new species have been added (Table 14.2).

Most of the rhizobia are discovered only in last decade. Hence, it is not surprising if more and more host which bear bacterial nodules, may not contain the traditional strains of Rhizobium or Brady rhizobium.

Mesorhizobium, a new genus of the family Rhizobiaceae has been named on the basis of whole sequence studies of 16S rRNA. Some of the species of Rhizobium namely, R. loti, R. huakii R. ciceri, R. Mediterranean and R. tianshanense now known as Mesorhizobium.

All the rhizobia live freely in soil in the root region of both leguminous and non-leguminous plants. Generally, they can enter into symbiosis only with legumes. If legumes are bigger partner in this process and rhizobia are smaller, such a relationship develops which is named as micro-symbiosis and strains are called micro-symbionts. The nodules become senescent after a long period.

Breznak (1973) and French (1976) demonstrated nitrogen fixing activity in the termite gut. It could be accomplished with the aid of nitrogen fixing bacteria e.g. Enterobacter sp. and Desulfovibrio species.

With culture independent methods using oligonucleotide probes specific for nitrogenase, the presence of nitrogen fixing bacteria from different systematic positions has also been demonstrated. Koustiane (2001) have isolated bacterial strains related to Rhizobium which were found to be related to Rhizobium fredii and R. meliloti.

2. Process of Root Nodule Formation:

The ‘rhizobia’ live freely in soil and as soon as they come in contact with suitable host, starts the process of infection. There is an initial contact between the bacteria and host which depends upon recognition.

Recent evidences suggest that polysaccharides on the surface of invasive bacteria are involved in binding of these cells to constituents (lectins) on the surface of the roots. The factors or proteins located in the nodules are called nodulins while on bacterial surfaces, named as bacteriocins which help in nodulation.

Generally, nodulation starts from the following processes:

(i) Curling and Deformation of Root Hairs:

Invasion of rhizobia occurs through root hairs. Fine studies of infected root hairs showed the continuation of the wall of the infection thread with the cell wall of the root hair which lends support to the invagination hypothesis.

The physiological events leading to infection can be summarised below:

(ii) Formation of Infection – thread and Formation of Nodule:

It is interesting to note that such bindings occurs between compatible (bacteria – host) partners. The tip of curled root hair bends and the bacteria (rhizobial polysaccharide and DNA) penetrate and grow in the form of an infection tube.

Meanwhile, the polysaccharides react with a component of root hair cell to form an ‘organizer’. The ‘organizer’ induces the production of polygalacturonase (PG) followed by de-polymerisation of cell wall pectin.

In such process, incorporation of rhizobia into cell wall occurs which participate in ‘intussusception’ i.e. taking in of rhizobia by root hair and its conversion into organic tissues. The infection tube or thread branches into the central portions of the nodule, and the bacteria released into their symbiont’s cytoplasm multiply. The nucleus of the root hair cell guides the rhizobia.

(iii) Development of Nodule:

Immediately, at the time of release of rhizobia into cytoplasm of the host cortical cells, rapid cell division (called hyperplasia) takes place in the cortical cells. Inside these cells, the bacteria alter their morphology into larger forms called bacteroids.

The root cells are stimulated due to this infection to form a tumor like nodule of bacteroid-packed cells (Fig. 14.1). The host cells chromosome number of the area become double. The doubling of the chromosome number occurs in the nodules of polyploids as well as diploid legumes.

(a) Structure of root nodule:

The root nodule is formed due to tissue proliferation induced by the action of growth promoters of rhizobial in origin, probably cytokinesis. The core of a mature nodule constitutes the ‘bacteroid zone’ which is surrounded by several layers of cortical cells.

The bacteroids, singly or in groups, surrounded by peribacteroid membranes, inhabit the cytoplasm of the plant cell. The effective nodules are generally large and pink due to presence of leghaemoglobin with well developed and organised tissue. After the senescence, when the nodule dies, stationary- phase rhizobia are released into the soil.

(b) Function of the nodule ‘bacteroids’:

The present evidences cited the fact that bacteroids are the sites of nitrogen fixation. The isotopic (¹⁵N) studies indicated that bacteroids are the primary sites of nitrogen fixation. Further, in contrast to the free-living rhizobia, the bacteroids are unable to utilise sugar, and secrete ammonium ions which are apparently incorporated into organic compounds by glutamine synthetase present in the surrounding plant cell.

This process indicates the involvement of true mutual symbiosis in which role played by leghaemoglobin in the fixation of nitrogen is much more significant. The formation of leghaemoglobin is a specific effect of the symbiosis. The relative capacity of the plant – bacterial association, once established, to assimilate molecular nitrogen is called ‘effectiveness’.

Leghaemoglobin:

A red pigment similar to blood haemoglobin is found in the nodules between bacteroids and the membrane envelopes surrounding them. Leghaemoglobin, the prefix ‘leg’ indicating its presence in legume root nodules, is a haemoprotein having a haeme moiety synthesised by the bacteria attached to a peptide chain which represents the globin part of the molecule, is encoded by a plant gene.

The molecular weight of leghaemoglobin is about 16,000-17,000 Daltons while the blood haemoglobin has around 66,000 Daltons mol wt. The prosthetic group protohaem is synthesized by the bacteroids, while the synthesis of the protein part involves the plant cell. The induction of leghaemoglobin enhances the transport of oxygen at a low partial pressure to the nodules and regulates a steady supply of oxygen at low concentration of the nodule.

It is not analysed in cyanobacterial symbiotic system or in other higher plants such as Frankia and Parasponia which fix nitrogen without leghaemoglobin. The presence of the leghaemoglobin seems to provide full protection against oxygen damage to the N₂ fixing enzyme (Table 14.3).

(c) Free-living nitrogen fixing bacteria:

There are 54 genera comprising in 22 families as well as 3 thermophilic archaeobacteria which fix nitrogen in free-living form. Winogradsky in 1893 demonstrated biological nitrogen fixation in Clostridium pasteurianum, and in 1901 Beijerinck detected in Azotobacter.

About 30 genera of cyanobacteria representing 10 families have been shown to fix atmospheric nitrogen. Cyanobacteria are heterocystous, filamentous, nonheterocystous filamentous, unicellular, reproducing by binary fission or budding, and by multiple fission.

Other N₂ fixing symbionts:

(i) Lichens (Cyanobacteria – fungus association)

(ii) Liverworts (Anabaena – Anthoceros)

(iii) Pteridophytes (Anabaena azollae – Azolla)

(iv) Gymnosperms (Nostoc – Cycads)

(v) Angiosperms (Nostoc – Gunnera)

(vi) Termites (with Citrobacters)

(vii) Human intestine (Klebsiellae)