Biological N2 Fixation PDF

# Nitrogen Fixation in Free-living and Associative Symbiotic Bacteria ## Nitrogen-fixing Bacteria The free-living bacteria having the ability to fix molecular nitrogen can be distinguished into obligate aerobic, facultative aerobic and anaerobic organisms. Obligate aerobic bacteria belong to the g...

# Nitrogen Fixation in Free-living and Associative Symbiotic Bacteria ## Nitrogen-fixing Bacteria The free-living bacteria having the ability to fix molecular nitrogen can be distinguished into obligate aerobic, facultative aerobic and anaerobic organisms. Obligate aerobic bacteria belong to the genera *Azotobacter*, *Beijerinckia*, *Derxia*, *Achromobacter*, *Mycobacterium*, *Arthrobacter* and *Bacillus*. Among the facultative anaerobic nitrogen-fixing bacteria are represented by the genera *Clostridium*, *Chlorobium*, *Chromatium*, *Rhodomicrobium*, *Rhodopseudomonas*, *Rhodospirillum*, *Desulfovibrio* and *Methanobacterium*. In some of these genera, nitrogen fixation takes place in a photoautotrophic manner by virtue of the presence in them of photosynthetic pigments as exemplified by the well-known genus *Rhodopseudomonas*. On the other hand, the genus *Desulfovibrio* fixes nitrogen in the process of reducing sulphates. Anaerobic nitrogen-fixing bacteria are represented by the genera *Aerobacter*, *Klebsiella* and *Pseudomonas*. Bacteria of the family *Azotobacteraceae* constitute the majority of heterotrophic free-living nitrogen-fixing bacteria. They are grouped into three genera- *Azotobacter*, *Beijerinckia* and *Derxia*. Several species of *Azotobacter* are recognized as *A. chroococcum*, mainly occurring in neutral or alkaline soils; *A. agilis*, an aquatic species; *A. vinelandii* and *A. beijerinckii*, originally isolated from North American soils: *A. insignis*, isolated from Indonesian water samples; *A. macrocytogenes*, isolated from Danish soils; and *A. paspali* from the rhizhosphere of *Paspalum spp.* originally isolated from Brazilian soils. While the genus *Beijerinckia* has three species- *B. indica*, *B. mobile* and *B. fluminensis*, the genus *Derxia* has only one, *D. gummosa*. Cell-size, flagellation, pigmentation and production of extra-cellular slime are considered as diagnostic features of these bacteria in distinguishing species, which could be summarized as follows: - *Azotobacter chroococcum* (peritrichous flagella, moderate slime and black-brown insoluble pigment): - *A. vinelandii*, *A. paspali* and *A. agilis* (peritrichous flagella, little to moderate slime and green, fluorescent and soluble pigment): - *A. beijerinckii* (no flagella, moderate slime and yellow-light brown insoluble pigment): -*A. macrocytogenes* (polar flagella, abundant slime and pink soluble pigment): -*Beijerinckia indica* (peritrichous flagella, abundant slime and light rust-brown insoluble pigment) and *Derxia gummosa* (polar flagellum, abundant slime and yellow-brown pigment). Among the different nitrogen fixing bacteria, *Clostridium pasteurianum* and *Azotobacter* (*A. chroococcum* and *A. vinelandii*) are the most intensively investigated genera. Earlier evidences mainly pointed out the ability of these bacteria to fix atmospheric nitrogen when cultured on a nitrogen-free medium. The amount of nitrogen fixed is usually estimated by the well-known Kjeldahl method. The use of 15N tracer and acetylene reduction method have however enriched our knowledge regarding the biochemical pathway between N2 and NH3 but the exact nature of intermediate products have eluded even critical investigators. Nevertheless, the overall reaction in the enzymic reduction of atmospheric nitrogen to ammonia could be postulated as follows: $2H^+\\N_2 \longrightarrow HN=NH\\2e\\(Dinitrogen) \hspace{1cm} (Di-imide)$ $2H^+\\2e\\H_2N-NH_2\\2H^+\\\longrightarrow 2NH3\\2e\\(Hydrazine) \hspace{1cm} (Ammonia) \\ [6e + 6H+ + N2 \longrightarrow 2NH3]$ ## Nitrogenase One of the significant advances made in our knowledge of biological nitrogen fixation was the discovery that cell-free extracts of *Azotobacter* and *Clostridium* could fix nitrogen in the same way as the free-living intact bacterial cells. The finding led to the initial isolation of the enzyme nitrogenase from *C. pasteurianum* and *A. vinelandii* and the subsequent finding that the enzyme is responsible for the adsorption and reduction of N2 gas. Cell-free extracts which retain the capacity to fix nitrogen have also been obtained by various workers from *Mycobacterium flavum*, *Bacillus polymyxa*, *Klebsiella pneumoniae*, *Rhodospirillum rubrum*, *Chromatium* and *Chloropseudomonas ethylicum*. Nitrogenase has been isolated from the following genera of free-living nitrogen-fixing microorganisms: *Clostridium*, *Bacillus*, *Klebsiella*, *Chloropseudomonas*, *Chromatium*, *Rhodospirillum*, *Anabaena*, *Gloeocapsa*, *Plectonema*, *Azotobacter* and *Mycobacterium*. The enzyme consists of two protein fractions- the Mo-Fe containing protein (mol. weight 220,000–270,000) and the Fe containing protein (mol. weight 55,000–66,800). Active nitrogenase can be reconstituted by the addition of purified Mo-Fe and Fe proteins of different microorganisms. For examples, proteins of *Klebsiella pneumoniae* and *Bacillus polymyxa* and those of blue-green algae and photosynthetic bacteria having been combined to reconstitute active nitrogenases, capable of reducing acetylene to ethylene. The Mo-Fe protein has been designated as dinitrogenase because nitrogen binds to the protein moiety whereas the Fe protein has been referred to as the dinitrogen reductase since the second moiety serves the specific function of reducing the Mo-Fe protein. During catalysis by nitrogenase, protons and nitrogen compete for electrons. Therefore, in an atmosphere containing nitrogen, hydrogen evolution occurs simultaneously with ammonia formation. This evolution of hydrogen diverts 25-35 per cent of the total reductants available for the nitrogenase reaction, which is regarded as an intracellular wastage of energy in the overall process of nitrogen fixation, and the reactions can be summarized as follows: $e$ $ENERGY\\FROM\\METABOLISM$ $FERREDOXINS\\AND OTHER\\CARRIERS$ $Mg \hspace{1mm} ATP \hspace{1mm} + \hspace{1mm} P^{¹}$ $Mg \hspace{1mm} ATP$ $e$ $Fe \hspace{1mm} PROTEIN$ $2H$ $H_2$ $Mo-Fe \hspace{1mm} PROTEIN$ $6H + N2 \longrightarrow 2NH3$ In *Azotobacter vinelandii*, two additional nitrogenases have been recognized, in addition to the nitrogenase described earlier. These nitrogenases have been studied in mutants of *A. vinelandii*. One of these contains vanadium instead of molybdenum and the other has neither molybdenum nor vanadium. The characterization of these nitrogenases, has posed fresh problems in pinpointing evidences to demonstrate the essentiality of molybdenum for nitrogen fixation and characterization of the site at which nitrogen binds to nitrogenase. ## Mechanism of Nitrogen Fixation The nitrogenase reaction has two essential steps: 1) electron activation by a suitable donor or adenosine di-phosphate (ADP) and 2) substrate reduction. These two steps of the reaction take place at different sites on the nitrogenase molecule but are interdependent. Purified preparations of nitrogenases are highly sensitive to oxygen, specially the Fe protein part of the enzyme. However, it is believed that an undefined respiratory system exists in *Azotobacter* near the site of nitrogen fixation which actively 'scavenges' oxygen so as to prevent the inactivation of nitrogenase. Energy requirements for nitrogenase reaction come from the cellular metabolic cycles in the form of adenosine triphosphate or ATP (roughly 12 to 20 moles of ATP per mole of molecular nitrogen reduced). Pyruvate functions both as an electron donor and an energy source. In the phosphoroclastic reaction, pyruvate forms acetyl phosphate which in the presence of adenosine diphosphate or ADP gives rise to ATP. The reductants are the strongly reducing naturally occurring electron carrier proteins, ferredoxin and flavodoxin. Dithionite (Na₂S₂O₂) and certain dyes such as methyl viologen and benzyl viologen can also serve as artificial extracellular sources of electron donors. Since all nitrogen-fixing microorganisms contain hydrogenase, this enzyme system in cells catalyzes the transfer of electrons from pyruvate or hydrogen to ferredoxin or flavodoxin. Ferredoxins are naturally occurring iron-sulphur (Fe-S) electron carrier proteins capable of undergoing reversible oxidation and reduction. They have been isolated from plants, blue-green algae and bacteria such as *Clostridium pasteurianum*, *Azotobacter vinelandii*, *Rhizobium japonicum*, *Anabaena cylindrica*, *Bacillus polymyxa*, *Chromatium sp.* and *Desulfovibrio gigas*. Ferredoxins differ in molecular weight, iron and sulphide contents and biological activity. Such electron carrier proteins isolated from several nitrogen-fixing organisms can react not only with the nitrogenase of specific microorganisms but also be effectively interchanged with other microorganisms. Flavodoxin is a flavoprotein first isolated from *Clostridium pasteurianum* in media containing low concentrations of iron and was found to replace ferredoxin as a electron carrier in a large number of reactions. Most of the nitrogen-fixing microorganisms are now known to possess flavodoxins. Subsequently, such electron carriers have been isolated from other anaerobic bacteria like *Peptostreptococcus elsdenii* and *Desulfovibrio spp.*. An electron carrier named azotoflavin has been isolated from *Azotobacter vinelandii* possessing biological activity similar to ferredoxins. The role of pyruvate and ferredoxin in nitrogenase reactions can be illustrated as follows: $Acetate\\ \uparrow \\Acetyl \hspace{1mm} Phosphate \\ N_2 \\ 1 \\ ATP\\ Nitrogenase \\ ADP \\ Pyruvate\\\downarrow\\2H^+ + 2e \\\swarrow\\H_2\\\downarrow\\2NH3$ Nitrogenase, in addition to reducing atmospheric N2 to NH3, can also reduce certain other compounds, as follows: $C2H2 \longrightarrow C2H4; \hspace{1cm} HCN \longrightarrow CH4 + HN3; \hspace{1cm} H^+ \longrightarrow H2;\\ HN3 \longrightarrow N2 + HN3;\hspace{1cm} N2O \longrightarrow N2 + H2O.$ According to Hardy and his associates of the Du Pont Laboratory, U.S.A., the active site of the enzyme for substrate reduction is believed to be composed of an Mo-Fe dinuclear site bridged by sulphur, having the proper size and electron characteristics to provide Mo-Fe distance of about 3.8 A (Fig. 40). This distance is specific so as to accommodate various nitrogenase substrates including N₂ and to exclude others. The first reaction in nitrogen reduction is the formation of a linear complex of N2 with the Fe of nitrogenase. This is followed by transfer of electron from Mo which is the end point of the electrons activating system, resulting in the formation of diimide which is stabilized by hydrogen bonding from the protein as well as the metal-nitrogen bonds. Successive addition of electrons produce hydrazine followed by cleavage of NN bond to yield 2 moles of NH3. The increase in the NN bond length during reduction is accompanied by compensating changes in MNN angles so that Mo-Fe distance remains constant. # Nitrogen Fixation by Rhizobium in a Free-living State It was generally agreed upon that Rhizobium can fix atmospheric nitrogen only in the root nodules of legumes and that too when it is in the bacteroid stage of its life cycle. All attempts to obtain nitrogen fixation by pure cultures of Rhizobium with or without extracts of host plants failed in the past and it was postulated that some of the genes determining Rhizobium's ability to fix atmospheric nitrogen (nif genes) reside in the host plant and hence the need for symbiosis between the host and the bacterium in the bacteroid tissue of the root nodule. Interesting experimental findings have emerged some years ago which show that Rhizobium possesses the entire complement of genes for nitrogen-fixation which is normally latent and become active only under special conditions. One of the first evidences came from the successful transfer of nif genes from Rhizobium trifolii to a non-nitrogen-fixing strain of Klebsiella aerogenes. Secondly, a strain of Rhizobium sp. cowpea group was induced to fix atmospheric nitrogen in the presence of diffusible substances from callus tissue of leguminous as well as non-leguminous plants, thereby indicating that plant tissues contain some substances which stimulate bacteria to fix nitrogen. The third set of evidences have come forth simultaneously from two groups of Australian and one group of Canadian workers. A simple synthetic medium was evolved which contains besides other ingredients, certain key metabolites such as a pentose sugar (arabinose or xylose), a dicarboxylic acid (such as succinate) and a relatively small amount of fixed nitrogen (such as glutamine, glutamate or nitrate) to induce nitrogen fixation by a strain of cow-pea type of Rhizobium in a free-state on a solid agar medium. The ability of fix nitrogen has been verified by C₂H₂ reduction test as well as by 15N enrichment procedures. These results lead us to believe that we may have to reconsider the recognition of legume-Rhizobium association within the nodules as a true instance of symbiosis. In a more restricted way, the term symbiosis is valid since the specialised structure of nodule may be meant only to restrict the access of oxygen to rhizobia for proper functioning of the enzyme nitrogenase. # Nitrogen Fixation by Free-living Blue-Green Algae ## General Aspects Blue-green algae constitute an important group of microorganisms capable of fixing atmospheric nitrogen. They comprise unicellular, colonial and filamentous types (Fig. 43, Table 20). Some fossil forms have also been discovered which date back to pre-cambrian periods. Most of the nitrogen-fixing blue-green algae belong to the orders *Nostocales* and *Stigonematales* under the genera *Anabaena*, *Anabaenopsis*, *Aulosira*, *Chlorogloea*, *Cylindrospermum*, *Nostoc*, *Calothrix*, *Scytonema*, *Tolypothrix*, *Fischerella*, *Hapalosiphon*, *Mastigocladus*, *Stigonema* and *Westiellopsis*. In pure cultures, blue-green algae fix varying amounts of nitrogen ranging from 5.2 to 14.48 mg/100 ml of the medium, depending upon the incubation time. In general, nitrogen fixation is associated with forms possessing heterocysts, although there are reports of fixation by unicellular and filamentous non-heterocystous strains. The plankton of lakes contains species of nitrogen-fixing algae which are invariably heterocystous forms such as *Anabaena*. The number of heterocysts could be taken as a rough parameter to indicate the nitrogen-fixing capacity of blue-green algae in spite of the fact that the amount of nitrogen fixed is dependent on physiological and environmental factors such as intensity of light, concentration of inorganic nitrogen sources, concentration of dissolved organic nitrogen compounds, temperature and aeration of the substrate. There is also a diurnal fluctuation in the quantity of nitrogen fixed by a given species of blue-green alga. ## Heterocysts Heterocysts are large, thick-walled, apparently empty cells appearing amidst normal pigmented cells (Fig. 43). However, when viewed through an electron microscope they seem to have a complex lamellar network. A large body of evidence has accumulated showing that heterocysts are the sites of nitrogen fixation and recent reports provide further direct evidence of nitrogenase activity in preparations made from isolated heterocysts. A mature heterocyst is surrounded by a multilayered envelope and shows an elaborate cytoplasmic membrane system devoid of granular inclusions. Heterocysts provide a congenial environment for the effective functioning of nitrogenase, to generate energy and reductant required for nitrogen fixation, to bring the nitrogen fixed into organic combination and to maintain a dual transport system for getting carbon and sending out nitrogen into the vegetative cells. The transition or metamorphosis of a vegetative cell of a blue-green alga into a heterocyst is a gradual process from a CO2 fixing and O2 evolving cell into an anaerobic cell conducive for active nitrogen fixation. The heterocyst has a multilayered cell wall connected by cytoplasmic bridges to neighbouring photosynthetic vegetative cells. These bridges regulate the flow of molecules between the two types of cells and therefore a series of regulated physiological and biochemical changes lead to nitrogen fixation and assimilation. Experiments carried out with *Anabaena variabilis* have shown that under light, nitrogenase activity in isolated heterocysts is dependent on a supply of H2 whereas in dark the activity is dependent on a supply of O2 and H2. The principal product of fixation of nitrogen which is translocated from heterocysts to vegetative cells is glutamine. The formation of glutamine in heterocysts is dependent on the transfer of glutamate from vegetative cells. These conclusions have been made by conducting experiments using N13 labelled nitrogen. A disaccharide probably maltose, a product of photosynthesis, moves from vegetative cells to the heterocysts where it is metabolized to glucose 6 phosphate and oxidized by the oxidative pentose pathway. It is believed that pyridine nucleotide (NADPH) reduced by this pathway can combine with O2 and thus provide conducive environment for the reduction of electron carrier ferredoxin (Fd). Heterocysts lack photosystem II activity but photosystem I which is present can also reduce ferredoxin. The reduced ferredoxin can donate electrons to the nitrogenase which reduces N2 to NH4+ as well as release H2. Uptake hydrogenase, capable of recycling H2 is present only in heterocysts. Heterocysts have high levels of glutamine synthetase (GS) and low levels of glutamine oxoglutarate amido transferase (GOGAT). Glutamate formed in vegetative cells and which gets transferred to the heterocysts reacts with NH4+ to form glutamine. The latter moves into the vegetative cells where it reacts with alphaketoglutarate (alpha KG) to provide 2 molecules of glutamine. It is believed that glutamine or a metabolite of glutamine other than NH4+ is a component of the system that represses heterocysts differentiation. These results have come from the work of Wolk and colleagues of the Michigan State University, East Lansing, Michigan and Haselkorn of the Chicago University, USA and schematically reproduced in Fig. 44. ## Algal Nitrogenase Considerable progress has been made in studies on the nitrogenase enzyme obtained from cell-free extracts of nitrogen-fixing algae. The first report of nitrogenase activity came from preparations of *Anabaena cylindrica*. Subsequently, nitrogenase activity was shown in isolated heterocysts and also in extracts from non-heterocystous *Plectonema*. The soluble nitrogenase from both heterocystous and non-heterocystous algae appear very similar. One distinctive feature of algal nitrogenase is its high oxygen sensitivity, which can be overcome by exclusion of oxygen from the medium containing cell-free extracts. Blue-green algae evolve oxygen during photosynthesis and obviously, a built-in mechanism must exist at the cellular level to protect the highly sensitive nitrogenase from oxygen. Nitrogenase enzyme from filamentous forms such as *Plectonema* is more sensitive to oxygen than that from heterocystous algae indicating the protective mechanism afforded by the heterocyst which prevents the oxygen inactivation of the nitrogen-fixing system under aerobic conditions. However, it is not yet clear whether the nitrogenase enzyme is exclusively located in the heterocyst, since many workers have also detected nitrogenase activity in normal vegetative cells of heterocyst bearing algae. Nitrogenase of BGA is very similar to that of other N2 fixing bacteria. It catalyses the reduction of protons, cyanide and C₂H₂ besides N2. The enzyme requires ATP, reductant and Mg2+ and is oxygen sensitive. As in other bacteria, the Fe-protein component binds to MgATP and transfers electrons to the Moke protein which binds to the substrate and reduces it. The MoFe and Fe proteins of *Anabaena cylindrica* and *Plectonema boryanum* are known to cross react with the Fe proteins of *Azotobacter* and *Clostridium*. The O2 sensitivity of nitrogenase is overcome in serval ways, the chief among which is the structural modification of some of the vegetative cells into thick walled heterocysts. Further the physiological environment in heterocysts is conducive for N₂ fixation, due to the absence of photosystem II, the lack of photosynthetic O2 evolution and the presence of uptake hydrogenase coupled with high activity of oxidative pentose pathway. In non-heterocystous forms, compartmentalization or separation of photosynthesis and N₂ fixation has been suggested from studies in *Plectonema* and *Trichodesmium* as a means to overcome O2 sensitivity of nitrogenase. Other explanations include the presence of antioxidants, Or linked H2 uptake or the presence of certain internal membrane systems to serve as intracellular O2 protective mechanisms. Such hypotheses are merely speculative and need confirmation. In heterocystous BGA, the source of reductant to nitrogenase can come from the photosystem I-dependent reaction in light, from oxidative pentose pathway in light or dark and likely from glycolysis or Kreb's cycle or both. Since the heterocysts lack ribulose biphosphate (RuBP) carboxylase and Photosystem II, they receive carbon from photosynthetic vegetative cells. Indeed the transfer of photosynthates from vegetative cells to heterocysts has been shown but the nature of substances translocated is not clear. Substrates such as maltose, sucrose and erythrose have been suggested as likely sugars that are received by the heterocysts. Fixation of N2 in dark for limited periods can take place in heterocystous BGA and the source of reductant to generate reduced ferredoxin in dark has to come from non-photosynthetic means. No inactivation of the Ferredoxin-NADP+ reductase in heterocysts of *Anabaena cylindrica* by light has been demonstrated, suggesting that a light-independent flow of electrons from glucose-6-phophate to NADP+ and then to ferredoxin takes place in light as well as in dark. The possibility of hydrogen as another source of reductant for the supply of electrons to nitrogenase via an uptake hydrogenase system has also been suggested. The major source of ATP in light for N2 fixation is cyclic photophosphorylation as shown in *Anabaena cylindrica* and *Anabaenopsis circularis* in the presence of adequate supply of fixed carbon. Non-cyclic photophosphorylation cannot be the source of ATP since heterocysts lack photosystem-II. In dark, oxidative phosphorylation can supply ATP under aerobic surroundings. In summary, the supply of ATP for N2 fixation by BGA may come from photophosphorylation, cyclic photophosphorylation, oxidative phosphorylation or from substrate level phosphorylation, as mentioned above. Nitrogenase in BGA functions as an ATP dependent hydrogenase to generate hydrogen which can be recycled back in the main nitrogenase reaction in strains possessing an uptake hydrogenase system. As in Rhizobium, the presence uptake hydrogenase in BGA is beneficial as it helps to enhance N2 fixation. ## Ammonia Assimilation As mentioned earlier, N2 fixation results in NH4 formation in heterocysts which reacts with glutamate translocated from vegetative cells to form glutamine with the aid of glutamine synthetase. Most of this glutamine gets back into vegetative cells where it is metabolized into glutamate and other amino acids by glutamate synthase or glutamine oxoglutarate amidotransferase (GOGAT) located in vegetative cells. The glutamate formed may eventually form a substrate for more glutamine synthesis or else be transported back into heterocysts. In this way, the first stable nitrogen fixation is NH4+ and the first organic product of assimilation of NH4+ is glutamine followed by glutamate. Alanine may be formed through alanine dehydrogenase or transamination reaction in some species of BGA and aspartate in other species by transamination reaction probably from glutamate. # Lichens There are some blue-green algae which exist in association with fungi, liverworts, ferns and flowering plants. Some of them fix atmospheric nitrogen. The alga-fungus association to form lichens (Fig. 46) occurring on soils, rocks and tree-tops is yet another instance of symbiosis wherein the genus Nostoc, Calothrix or other unidentified blue-green algae fix nitrogen and, in turn, obtain protection and space from the fungal partner. The ability of lichens to fix nitrogen has been proved by the use of 15N in genera of lichens such as Collema, Stereocaulon, Leptogium, Lichina and Peltigera. (Recently, the list of lichens capable of fixing nitrogen has been expanded to include Lobaria, Massalongia, Nephroma, Pannaria, Parmeliella, Placopsis, Placynthium, Polychidium and Sticta which reduce acetylene to ethylene and thus demonstrate nitrogenase activity. Apart from releasing the nitrogen fixed, the alga Nostoc is known to provide biotin, riboflavine, thiamine, nicotinic acid and pantothenic acid to support the growth of the fungal partner. Fixation of nitrogen by lichens is dependent on the intensity of light and the moisture content of the thallus although it is well known that lichens can withstand very severe environmental stress. Lichens appear as crusts, foliose or shrubby fruiticose growths on rocks, walls, trees or on ground. There are about 13,500 species of lichens. Lichen fungi are mostly ascomycetes and are known as mycobionts. Lichen growth is a slow process even with plentiful nutrients, the reasons for which are not clearly understood. The algal partner of a lichen is either a true alga also known as 'phycobiont (85 per cent of lichens) or a cyanobacterium also known as 'cyanobiont (15 per cent of lichens). Together, the cyanobiont and the phycobiont are called 'photobionts' and there are many unidentified photobionts. One particular genus of a lichen usually has one genus or species of a photobiont but in some instances two photobionts (one a true alga and the other a cyanobacterium) coexist in the same lichen species. Of the two photobionts, one situated in the thallus of the lichen happens to be a true green alga while the other is a blue-green alga situated in special nitrogen fixing structures known as cephalodia that are either within or on the surface of the thallus. The commonest photobionts found ## Blue-green Algal Association with Bryophytes Certain mosses and liverworts are also known to be inhabited by blue-green algae. The lower surface of the thallus of Anthoceros contains a species of Nostoc which stimulates the formation of papillae within the cavities of the host. The papillae provide space for the formation of compact colonies of the alga, which in turn, help in the nitrogen nutrition of the host plant. Nostoc sphaericum inhabits the cavities occurring on outgrowths near the edges of the lower surface of Blasia and Cavicularia (genera of liverworts). The dependence of the host on the lower symbiont for its nitrogen nutrition has been established in the case of Blasia, using 15N) Another instance of an association between a species of Hapalosiphon and Sphagnum (a moss) has also been cited in literature together with 15N data on translocation of fixed nitrogen. ## Blue-green Algal Association with Higher Plants The only angiosperm to develop a symbiotic association and fix nitrogen (Table 22) with a nitrogen-fixing blue-green alga is Gunnera. This genus has about 40 species which are herbaceous and widely distributed in the southern hemisphere. The plants have mucilage-filled cavities called glands near the bases of petioles. Nostoc cells penetrate the cells of the gland when infection takes place naturally. Two glands known as apical papillate glands, just below the point of cotyledonary attachment are formed which produce mucilage enabling the growth of Nostoc. From the mucilage, Nostoc cells penetrate the interior of the glands and then the host cells. Other blue-green algal species are also capable of forming associations with Gunnera under artificial inoculated conditions. An excellent example of algal association with higher plants is the occurrence of endophytes Anabaena or Nostoc in the coralloid masses on the roots of Cycadaceae (Figs. 48 and 49). Cycads produce stubby apogeotropic profusely dichotomously branching tannin-rich coral-like (coralloid) root masses which become infected with cyanobacteria. These roots appear in addition to normal ordinary roots which are often very tuberous. The functions of coralloid roots are not clearly understood. It has been suggested that lenticels present on these roots facilitate gas exchange between the plant and the atmosphere. Cyanobacteria may enter through lenticels or through breaks in the root's dermal layers. The cyanobacteria are restricted to intercellular spaces of the cyanobacterial zone although occasional penetration of the cortex has been reported in Cycas revoluta. Invariably the cyanobacteria are heterocystous belonging to the genus Nostoc. In some cases *Anabaena* and *Calothrix* have been encountered. Endophytes have been found in distinct zones in the cortex of the coralloid nodules on roots of genera such as *Cycas*, *Encephalartos*, *Zamia*, *Ceratozamia*, *Macrozamia* and *Stangeria*. The blue-green alga, *Nostoc cycadeae*, isolated from *Cycas*, *Encephalartos* and *Macrozamia* have been shown to fix atmospheric nitrogen in the free state as well as in association with their host plants, as revealed by experiments using 15N. When washed coralloid and normal roots were exposed to 15N labelled gas, coralloid roots showed enrichment with 15N while normal roots showed no such enrichment. ## Azolla-Anabaena Associaton A species of *Anabaena* (*A. azollae*) is associated with the aquatic fern *Azolla* occurring in a ventral pore in the dorsal lobe of each vegetative leaf. The endophyte fixes atmospheric nitrogen and resides inside the tissues of the water fern. Azolla is being used as green compost for rice cultivation in North Vietnam. An added advantage is that the plant multiplies fast and provides higher yields of green compost (200-300 t ha/yr) than conventional green manure plants such as *Sesbania*, *Crotalaria*, and *Tephrosia* which are known to yield 30-50 t ha/yr. The disadvantages are that the plant is susceptible to parasites and sensitive to fluctuations in temperature, the most favourable range of temperature being 20-28°C. It is also necessary to prevent algal growth in rice fields for utilization of *Azolla* since algae tend to overgrow and inhibit the proliferation of the water weed) It is reported from Vietnam that a 10-ton layer of *Azolla* enables the rice yield to increase by 10-25% over corresponding Azolla-free rice fields/The benefit from Azolla growth to the associated rice crop has been variously estimated) estimated) It ranges from 95 kg N/ha/yr to 670 kg N/ha/yr, depending on the method used in determining the amount of nitrogen fixed. The common species of *Azolla* in India is *A. pinnata* (Fig. 50A). It is recommended that *Azolla* nurseries are raised in small plots (50-100 sq m) or in concrete tanks with 5-10 cm deep water (pH 7-8) containing super-phosphate at 4-8 kg P2O5/ha after seeding the plots with *Azolla* inoculum at the rate of 0.1 to 0.4 kg per sq m. These nurseries have to be planned several weeks ahead of the date set for transplanting rice seedlings (Fig. 50B). At the end of 2-3 weeks, when full growth of *Azolla* takes place, the water is drained and the *Azolla* growth is incorporated into the rice fields by ploughing the mass (10-20 t/ha) into the puddled rice field. This is followed by transplanting of rice seedlings within 7 days. Alternatively, *Azolla* is grown as a dual crop with the main crop of rice. As and when the *Azolla* mat formation takes place, it is ploughed into the field, a process which can be repeated depending on the growth of *Azolla*. Field experiments in India have demonstrated that 10 t/ha of *Azolla* is equivalent to 25 to 30 kg N/ha and similarly, an application of 20 kg N/ha as ammonium sulphate with *Azolla* is equivalent to 40 kg N/ha as ammonium sulphate (Table 23). # Rhizobium and Legume Root Nodulation ## Leguminous Plants Leguminous plants are classified into three major botanical subfamilies of the family Leguminoseae- the Ceasalpinioideae, the Mimosoideae and the Papilionoideae. There are nearly 750 genera and 18,000-19,000 species of leguminous plants of which 500 genera and approximately 10,000 species belong to the subfamily Papilionoideae. Not all legumes bear nodules on their root system and it is known that certain tree forms do not possess them at all. Hardly 16% of Leguminoseae have so far been examined for nodulation of which 95% of Mimosoideae, 26% of Ceasalpinioideae and 90% of Papilionoideae possess root nodules. The origin of leguminous plants and the evolution of bacterial symbiosis are largely speculative. Evidence from fossil legumes does not provide much help in judging the exact time of origin of Leguminoseae. These plants are likely to have originated in sub-humid tropical, subtropical or temperate regions. The type of soil in which the first symbiotic legumes developed is also conjectural. It might have been in acid, neutral, alkaline or calcareous soil. Nodule bacteria were probably free-living nitrogen fixers before they became symbiotic under conditions of low availability of essential soil nutrients. It is likely that the slow growing, symbiotically promiscuous cowpea-type Rhizobium was the ancestral type of nodule bacterium which has persisted till today in association with many modern genera of Leguminoseae. ## Infection Studies on clover and lucerne have revealed that the first reaction of the root system to the presence of rhizobia is the curling and deformation of root hairs. The formation of a typical "Shepherd's crook" on the root hair is generally considered as a necessary prelude to the formation of a thread-like structure visible inside the root hair called "infection thread" (Figs 56, 57). The curling effect has been attributed to indole acetic acid (IAA) produced in the root region by rhizobia. IAA is also known to be produced by seveal microorganisms other than rhizobia. In view of this, a specific root hair curling factor, believed to be a water soluble polysaccharide produced by rhizobia has been implicated in the typical curling of root hairs in which infection threads are formed. There appears to be an intense interaction between the nucleus of root hair cell and the infection thread originating at the tip of the curled portion of the root hair. The nucleus guides the path of the infection thread in the hair as is evident from the fact that in the event the nucleus gets disorganised, the growth of the thread also ceases. If the nucleus moves to the distal end of the hair and then wanders towards the proximal end near the cortex, the infection thread also traverses up and down before entering the cortex. Obviously, some kind of a message or impulse is transferred by the nucleus of the host to the contents of the infection thread. Rhizobia are incapable of producing pectinase or cellulase in culture medium amended with pectin or cellulose. Recently, evidences have been provided to show that legume roots liberate pectic enzymes, in response to the presence of homologous rhizobia in the root region. The validity of such induced production of pectinase by plant/homologous rhizobia associations has been discounted by other workers since results have not always been reproducible. However, detailed investigation on the role of enzymes in the mechanism of infection of legume roots by Rhizobium may throw more light on the problem. ## Structure of the Nodule The core of a mature nodule constitutes the 'bacteroid zone' which is surrounded by several layers of cortical cells. The relative volume of bacteroid tissue (16 to 50% of the dry weight of the nodules) is much greater in effective nodules than in ineffective ones. The volume of bacteroid tissue in effective nodules has a direct positive relationship with the amount of nitrogen fixed.) Ineffective nodules produced by ineffective strains are generally small and contain poorly developed bacteroid tissue associated with structural abnormalities. In all ineffective associations, it has been shown that starch accumulates in the uninfected cell and dextran in the infected cell with glycogen in the bacteroid. (Effective nodules are generally large and pink (due to leghaemoglobin) with well developed and organised bacteriod tissue) A fully-developed bacteroid has no flagella and is surrounded by three unit membranes. There exists an intracytoplasmic membrane system in the bacteroids of nodules in subterranean clover. The nuclear region of bacteroids appears fragmented and is