Several types of symbiotic biological nitrogen fixing associations are known. The most prominent among them is the legume-bacteria relationship. Species of rod-shaped Rhizobium has such relationship with the roots of several legumes such as alfalfa, sweet clover, sweet pea, lentils, garden pea, broad bean, clover beans, etc. The most common association on roots is as nodules. These nodules are small outgrowths on the roots. The microbe, Frankia, also produces nitrogen-fixing nodules on the roots of nonleguminous plants (e.g., Alnus). Both Rhizobium and Frankia are freeliving in soil, but as symbionts, can fix atmospheric nitrogen.
Uproot any one plant of a common pulse, just before flowering. You will see near-spherical outgrowths on the roots. These are nodules. If
you cut through them you will notice that the central portion is red or pink. What makes the nodules pink? This is due to the presence of
leguminous haemoglobin or leg-haemoglobin.

Nodule formation involves a sequence of multiple interactions between Rhizobium and roots of the host plant. Principal stages in the nodule formation are summarised as follows : Rhizobia multiply and colonise the surroundings of roots and get attached
to epidermal and root hair cells. The root-hairs curl and the bacteria invade the root-hair. An infection thread is produced carrying the bacteria into the cortex of the root, where they initiate the nodule formation in the cortex of the root. Then the bacteria are released from the thread into the cells which leads to the differentiation of specialised nitrogen fixing cells. The nodule thus formed, establishes a direct vascular connection with the host for exchange of nutrients.
The nodule contains all the necessary biochemical components, such as the enzyme nitrogenase and leghaemoglobin. The enzyme nitrogenase is a Mo-Fe protein and catalyses the conversion of atmospheric nitrogen to ammonia, the first stable product of nitrogen fixation.

The enzyme nitrogenase is highly sensitive to the molecular oxygen; it requires anaerobic conditions. The nodules have adaptations that ensure that the enzyme is protected from oxygen. To protect these enzymes, the nodule contains an oxygen scavenger called leg-haemoglobin. It is interesting to note that these microbes live as aerobes under free-living conditions (where nitrogenase is not operational), but during nitrogen-fixing events, they become anaerobic (thus protecting the nitrogenase enzyme). You must have noticed in the above reaction that the ammonia synthesis by nitrogenease requires a very high input of energy (8 ATP for each NH3 produced). The energy required, thus, is obtained from the respiration of the host cells.

At physiological pH, the ammonia is protonated to form NH4+ (ammonium) ion. While most of the plants can assimilate nitrate as well
as ammonium ions, the latter is quite toxic to plants and hence cannotaccumulate in them. Let us now see how the NH4+ is used to synthesise amino acids in plants. There are two main ways in which this can take place :

(i) Reductive amination : In these processes, ammonia reacts with α-ketoglutaric acid and forms glutamic acid as indicated in the
equation given below :