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How do aerobic Rhizobium bacteria survive in root nodules while fixing atmospheric nitrogen?


I read that rhizobium carry the enzyme nitrogenase, which is irreversibly damaged upon the exposure of oxygen. Inside the root nodule of legumes leg-haemoglobin keeps a microaerophilic environment where nitrogenase enzyme can function and molecular nitrogen can be fixed (source: Brock's Biology of Microorganisms).

Shouldn't therefore Rhizobium bacteria die in such microaerophilic condition inside the root nodules?


TLDR One hypothesis for survival is nitrate respiration.

Long Answer Ampount of oxygen available in root nodules is controlled by the host plant by two ways:

  1. By presence of leghemoglobin
  2. By the diffusion resistance Under anaerobic condition the bacteria survives by making use of denitrification process which could be used to produce ATP under anaerobic conditions.

It is known for a while that, beside nitrogenase activity, in many symbiotic associations between legumes and Rhizobium the activity of nitrate reductase also exists 12. Most of the nitrate reductase ativity has been found to be concentrated in infected bacterial body i.e., bacteriod3. The bacteriod uses the process of conversion of nitrate into nitrite for creating a proton gradient which is inturn used for generation of ATP. Reference.


Transfer of Symbiotic Genes in Rhizobium

A.W.B. JOHNSTON , J.E. BERINGER , in Molecular Biology of Plant Tumors , 1982

Publisher Summary

This chapter discusses the mechanism of transfer of symbiotic genes in Rhizobium . Plasmids play an important role in determining symbiotic phenotypes, including host range, nodulation ability, and the capacity to fix nitrogen in the nodules. Indeed, some of the genes that code for nitrogenase appear to be plasmid-linked. The involvement of plasmids should not be too surprising as it is well known that in another member of the Rhizobiaceae, Agrobacterium tumefaciens, plasmids are responsible for the tumorigenicity of this species. Rhizobium and Agrobacterium have in common the ability to induce cellular proliferation on plants. In the case of R. phaseoli, the transconjugants differed from the wild type R. phaseoli in another character, namely, the ability to produce a black pigment. It was found that many field isolates of R. phaseoli produce a black pigment after prolonged incubation, 2–3 weeks, on complete medium. A number of strains of Rhizobium have been shown to be capable of nitrogen fixation in vitro.


Mechanism of Biological Nitrogen Fixation

The biological nitrogen fixation is carried out by some bacte­ria, cyanobacteria and symbiotic bacteria. In symbiotic association, the bacterium provides fixed nitrogen (NH3) to the host and derives carbohydrates and other nutrients from the latter.

Biological nitrogen fixation occurs in the presence of the enzyme nitrogenase which is found inside the nitrogen fixing prokaryote. In addition to this enzyme, a source of reducing equivalents (ferredoxin (Fd) or flavodoxin in vivo), ATP and protons are required.

The overall stoichiometry of biological nitrogen fixation is represented by the following equation:

N2 + 8H + + 8e – + 16 ATP → 2NH3 + H2 + 16 ADP + 16 Pi

The enzyme nitrogenase is in-fact an enzyme complex which consists of two metallo-proteins.

(i) Fe-protein or iron-protein component (previously called as azo ferredoxin) and

(ii) Fe Mo-protein or iron-molybdenum protein component (previously called as molybdoferredoxin). None of these two components alone can catalyse the reduction of N2 to NH3.

The Fe-protein component of nitrogenase is smaller than its other component and is an Fe-S protein which is extremely sensitive to O2 and is irreversibly inactivated by it. This Fe-S protein is a dimer of two similar peptide chains each with a molecular mass of 30-72 kDa (depending upon the micro-organism). This dimer contains four Fe atoms and four S atoms (which are labile and 12 titrable thiol groups).

The MoFe-protein component of nitrogenase is larger of the two components and consists of two different peptide chains which are associated as a mixed (α2β2 ) tetramer with a total molecular mass of 180 – 235 k Dalton (depending upon the micro-organism). This tetramer contains two Mo atoms, about 24 Fe atoms, about 24 labile S atoms and 30 titrable thiol groups probably in the form of three 24 Fe4 – S4 clusters. This component is also sensitive to O2.

i. Because nitrogenase enzyme complex is sensitive to O2, biological nitrogen fixation requires anaerobic conditions. If the nitrogen fixing organism is anaerobic than there is no such problem. But, even when the organism is aerobic, nitrogen fixation occurs only when conditions are made to maintain very low level of O2 or almost anaerobic conditions prevail inside them around the enzyme nitrogenease.

ii. Apart from N2, the enzyme nitrogenase can reduce a number of other substrates such as N2O (nitrous oxide), N3 – (azide), C2H2 (acetylene), protons (2H + ) and catalyse hydrolysis of ATP.

iii. Direct measurement of nitrogen fixation is done by mass spectroscopy. However, for comparative studies reduction of acetylene can be measured rather easily by gas chromatography method.

The electrons are transferred from reduced ferredoxin or flavodoxin or other effective reducing agents to Fe-protein component which gets reduced. From reduced Fe-protein, the elec­trons are given to MoFe-protein component which in turn gets reduced and is accompanied by hydrolysis of ATP into ADP and inorganic phosphate (Pi). Two Mg ++ and 2 ATP molecules are required per electron transferred during this process.

Binding of 2 ATPs to reduced Fe-protein and subsequent hydrolysis of 2 ATPs to 2 ADP + 2 Pi is believed to cause a conformatorial change of Fe-protein which facilitates redox (reduction-oxidation) reactions. From reduced MoFe-protein, the electrons are finally transferred to molecular nitrogen (N2) and 8 protons, so that two ammonia and one hydrogen molecule are produced (see the equa­tion and Fig. 9.4)

iv. At first glance, it might be expected that six electrons and six protons would be required for reduc­tion of one N2 molecule to two molecules of ammonia. But, the reduction of N2 is obligatorily linked to the reduction of two protons to form one H2 molecule also. It is believed that this is necessary for the binding of nitrogen at the active site.

v. The electrons for regeneration of reduced electron donors (ferredoxin, flavodoxin etc.) are provided by the cell metabolism e.g., pyruvate oxidation.

Substantial amount of energy is lost by the micro-organisms in the formation of H2 mol­ecule during nitrogen fixation. However, in some rhizobia, hydrogenase enzyme is found which splits H2 to electrons and protons (H2 → 2H + + 2e – ). These electrons may then be used again in reduction of nitrogen, thereby increasing the efficiency of nitrogen fixation.

Although scientists have tried to explain the mechanism of biological nitrogen fixation, but the precise pathway of electron transfer, substrate entry and product release and source of protons during biological nitrogen fixation have not yet been fully elucidated.

Formation of Root Nodules in Leguminous Plants:

The rhizobia occur as the free-living organisms in the soil before infecting their respec­tive host plants to form root nodules. The symbiosis between rhizobia and leguminous host plant is not always obligatory. However, under conditions of limited nitrogen supply in the soil, there is elaborate exchange of signals between the two symbionts for development of symbiotic relationship.

vi. There are separate host specific genes and rhizobial specific genes which are involved in nodule formation. The host plant genes are called as nodulin or Nod genes while rhizobial genes are called as nodulation or nod genes. Some Nod factors produced by rhizobia act as signals for symbiosis.

The rhizobia migrate and accumulate in the soil near the roots of the legume plant in response to the secretion of cer­tain chemicals such as flavonoids and be-taines by the roots. Root hairs of legume produce specific sugar binding proteins called as lectins. These lectins are activated by Nod factors to facilitate the attachment of rhizobia to the root hairs whose tips in turn become curved (Fig. 9.5 A).

Rhizobia now secrete enzymes which degrade the cell walls of root hairs at the point of their attachment for entry into the root hair. From root hairs, the rhizobia en­ter into the cells of inner layers of cortex through infection threads (tubular exten­sions of the in-folded plasma membrane pro­duced by fusion of Golgi-derived membrane vesicles).

The rhizobia continue to multiply inside infection thread and are released into cortical cells in large numbers, where they cause cortical cells to multiply and ulti­mately result in the formation of nodules on the upper surface of the roots (Fig. 9.5 A & B). After their release into cortical cells, the rhizobia stop dividing and enlarge.

Electron microscopic studies have shown groups of rhizobia to the surrounded by single membranes which originate from host cell plasma membrane. The enlarged and non motile groups of bacteria inside the membranes are called as bacteroids and the membrane surrounding them as peribacterioid membrane.

The space between bacteroids and peribacteroid membrane is called as peribacteroid space. These bacteroids are aerobic and the nitrogenase enzyme is found inside them. The bacteroides lack a firm wall and are osmotically labile. In root nodule cells of Glycine max, often groups of 4 – 6 bacteroids are enclosed in­side the peribacteroid membranes (Fig. 9.5 C)

The number of chromosomes in cortical cells infected by rhizobia which later develop into nodule is double the number of chromosome in other somatic cells of the legume (i.e., they are tetraploid) and seems to be pre-requisite for nodule formation. Apart from infected cells which are tetraploid, some unifected diploid cells are also found in nodule. The nodule has its own vascular system which is connected with vascular system of the root to facilitate transfer of fixed nitrogen i.e., NH3 to the host and carbohydrates and other nutrients from the host to the bacteroids.

In root nodules of leguminous plants, a red pigment- an oxygen binding heme protein which is very much similar to hemoglobin of red blood corpuscles is found. This pigment is called as leg-hemoglobin and occurs in cytosol of infected nodule cells. Leg-hemoglobin gives pinkish-red colour to the nodules. The globin part of this pigment is synthesized in host plant genome in response to the bacterial infection, while its heme portion is synthesized by bacte­rial genome.

Although a correlation has been found between the concentration of hemoglobin and the rate of nitrogen fixation, but this pigment does not play a direct role in nitrogen fixation. It (i) protects the nitrogenase inside the bacteroids from deterimental effect of oxygen and (ii) main­tains adequate supply of oxygen to the bacteroids, so that through respiration ATPs continue to be generated which are required for nitrogen fixation.

After its formation inside bacteroids, ammonia (or NH4 + ) is released into cytosol of infected nod­ule cells where it is converted into amides (chiefly asparagine and glutamine) or ureids (chiefly allantoic acid, allantoin and citrulline). These amides or ureids are then translocated to shoots of host plant through xylem, where they are rapidly catabolized to NH4 + for entry into mainstream of ammonium assimilation.


Nitrogen Metabolism

The nitrogen cycle depicts the different ways in which nitrogen, an essential element for life, is used and converted by organisms for various purposes. Much of the chemical conversions are performed by microbes as part of their metabolism, performing a valuable service in the process for other organisms in providing them with an alternate chemical form of the element.

Nitrogen Cycle.

Nitrogen Fixation

Nitrogen fixation describes the conversion of the relatively inert dinitrogen gas (N2) into ammonia (NH3), a much more useable form of nitrogen for most life forms. The process is performed by diazotrophs, a limited number of bacteria and archaea that can grow without an external source of fixed nitrogen, because of their abilities. Nitrogen fixation is an essential process for Earth’s organisms, since nitrogen is a required component of various organic molecules, such as amino acids and nucleotides. Plants, animals, and other organisms rely on bacteria and archaea to provide nitrogen in a fixed form, since no eukaryote is known that can fix nitrogen.

Nitrogen fixation is an extremely energy and electron intensive process, in order to break the triple bond in N2 and reduce it to NH3. It requires a particular enzyme known as nitrogenase, which is inactivated by O2. Thus, nitrogen fixation must take place in an anaerobic environment. Aerobic nitrogen-fixing organisms must devise special conditions or arrangements in order to protect their enzyme. Nitrogen-fixing organisms can either exist independently or pair up with a plant host:

  1. Symbiotic nitrogen-fixing organisms: these bacteria partner up with a plant, to provide them with an environment appropriate for the functioning of their nitrogenase enzyme. The bacteria live in the plant’s tissue, often in root nodules, fixing nitrogen and sharing the results. The plant provides both the location to fix nitrogen, as well as additional nutrients to support the energy-taxing process of nitrogen fixation. It has been shown that the bacteria and the host exchange chemical recognition signals that facilitate the relationship. One of the best known bacteria in this category is Rhizobium, which partners up with plants of the legume family (clover, soybeans, alfalfa, etc).
  2. Free-living nitrogen-fixing organisms: these organisms, both bacteria and archaea, fix nitrogen for their own use that ends up being shared when the organisms dies or is ingested. Free-living nitrogen-fixing organisms that grow anaerobically do not have to worry about special adaptations for their nitrogenase enzyme. Aerobic organisms must make adaptations. Cyanobacteria, a multicellular bacterium, make specialized cells known as heterocysts in which nitrogen fixation occurs. Since Cyanobacteria produce oxygen as part of their photosynthesis, an anoxygenic version occurs within the heterocyst, allowing the nitrogenase to remain active. The heterocysts share the fixed nitrogen with surrounding cells, while the surrounding cells provide additional nutrients to the heterocysts.

Assimilation

Assimilation is a reductive process by which an inorganic form of nitrogen is reduced to organic nitrogen compounds such as amino acids and nucleotides, allowing for cellular growth and reproduction. Only the amount needed by the cell is reduced. Ammonia assimilation occurs when the ammonia (NH3)/ammonium ion (NH4+) formed during nitrogen fixation is incorporated into cellular nitrogen. Assimilative nitrate reduction is a reduction of nitrate to cellular nitrogen, in a multi-step process where nitrate is reduced to nitrite then ammonia and finally into organic nitrogen.

Nitrification

As mentioned above, nitrification is a 2-step process performed by chemolithotrophs using a reduced or partially reduced form of nitrogen as an electron donor to obtain energy. One group of chemolithotrophs can perform the first part of the nitrification process, ammonia oxidation, while a different group of chemolithotrophs can perform the nitrite oxidation that occurs in the second part of nitrification. A non-nitrogen compound would serve as the electron acceptor. ATP is gained by the process of oxidative phosphorylation, using an ETC, PMF, and ATP synthase.

Denitrification

Denitrification refers to the reduction of NO3- to gaseous nitrogen compounds, such as N2. Denitrifying microbes perform anaerobic respiration, using NO3- as an alternate final electron acceptor to O2. This is a type of dissimilatory nitrate reduction where the nitrate is being reduced during energy conservation, not for the purposes of making organic compounds. This produces large amounts of excess byproducts, resulting in the loss of nitrogen from the local environment to the atmosphere.

Anammox

Anammox or anaerobic ammonia oxidation is performed by marine bacteria, relatively recently discovered, that utilize nitrogen compounds as both electron acceptor and electron donor. Ammonia is oxidized anaerobically as the electron donor while nitrite is utilized as the electron acceptor, with dinitrogen gas produced as a byproduct. The reactions occur within the anammoxosome, a specialized cytoplasmic structure which constitutes 50-70% of the total cell volume. Just like denitrification, the anammox reaction removes fixed nitrogen from a local environment, releasing it to the atmosphere.

Key Words

chemolithotrophy, hydrogen oxidizers, hydrogenase, sulfur oxidizers, sulfite oxidase, nitrogen oxidizers, nitrification, iron oxidizers, chemolithoautotroph, reverse electron flow, chemolithoheterotroph, mixotroph, nitrogen fixation, diazotroph, nitrogenase, symbiotic nitrogen-fixing organisms, Rhizobium, legume, free-living nitrogen-fixing organisms, Cyanobacteria, heterocyst, assimilation, ammonia assimilation, assimilative nitrate reduction, denitrification, dissimilatory nitrate reduction, anammox, anaerobic ammonia oxidation, anammoxosome.


Symbiotic and Non-Symbiotic Nitrogen Fixing Bacteria

The heterotrophic bacteria that fix di-nitrogen gas (N2) from the atmosphere in plant root nodules (symbiotic bacteria) have a mutually beneficial relationship with their host plants. Legumes (pod-bearing plants such as peas, beans, alfalfa and clovers etc.) had a beneficial effect upon both companion and whatever crop was planted next in the same soil. It is evident that the fixation of atmospheric nitrogen in the legume is due to the formation of root nodules.

Symbiotic bacteria initially start by infecting root hairs, causing an invagination (enclosing-like sheaths) inward through several cells. Surrounding plant cells proliferate quickly, perhaps because of auxin, a phytohormone produced by the infecting bacteria.

As the bacteria enter the nodule cells, they form enclosing membranes and produce meta-hemoglobin, an oxygen-carrying pigment (the nodule may be pink in cross-section). The hemoglobin like material may be an oxygen sink or trap to keep the bacteria in an anaerobic environment, which is necessary for N2 fixation.

The di-nitrogen (N2) fixation is performed by the enzymes nitrogenase. This enzyme lowers the activation energy (the energy requires to perform the reaction). The fixation proceeds in reduction stages from di-nitrogen (N = N) through uncertain intermediates HN=NH and H2N-NH2 to produce 2 NH3.

Finally, the ammonium is transformed into some organic compounds such as amino acids. All of this will take place when the nitrogen is bonded to the enzyme(s).

The lifetime of a bacterium may be only a few hours and the bodies of a portion of the bacterial population are continuously dying, decomposing, and releasing NH4 + and NO3 – ions for the utilization by the host plant. Most of the nitrogen fixed is excreted by the bacteria and made available to the host plant and to the other plants growing nearby. The well-known symbiotic bacteria belong to the genus Rhizobium.

Symbiotic heterotrophic bacteria specific to the crop to be grown are frequently applied or inoculated, in a dried powdered from to the crop seed to ensure that nitrogen fixing organisms are present. The same bacterial species will not inoculate all legumes. Sesbania rostrata (dhaincha) was found to form nodules both in roots and stems and it is most important host plant for the symbiotic N2-fixation.

Recently some plants have been found to have symbiotic relationship with different N2-fixing bacteria, including blue green bacteria (cyanobacteria), are Digitaria (grass species), water fern e.g. azolla (with blue green bacteria), Gunnera macrophylla (with blue green bacteria).

It has been also reported that bacteria of the genus Klebsiella have been found to be associated in N2-fixation with various grasses (non-legumes) but none has yet proven to be symbiotic. In addition, may other non-leguminous plants have symbiotic N2-fixing nodulation (e.g. Alnus spp., Casuaraina equisetifolia etc.)

Since the number of host plants is limited, cross inoculation groups have been established. A cross-inoculation group refers to a collection of leguminous species that are capable of developing nodules when exposed to bacteria obtained from the nodules of any member of that particular plant group. Some cross-inoculation groups and Rhizobium-Legume associations are shown in table 18.1.

Although the cross-inoculation classes are not solely considered for the description of the nodulating performance of many root nodule organisms.

Non-Symbiotic N2-Fixing Bacteria:

The non-symbiotic nitrogen fixing bacteria do not require a host plant. In 1891, Winogradsky observed that when soil was exposed to the atmosphere, the nitrogen content of the soil was recorded to be increased.

The anaerobic bacterium Clostridium pasteurianum was found responsible for such an increase of the nitrogen content in soil. In 1901, Beijerinck proved that there were also free-living aerobic bacteria, Azotobacter chroococcum that could fix atmospheric nitrogen.

Another bacterial group, Granulobacter (purple colour) obtains nitrogen directly from the atmosphere. The amounts of atmospheric nitrogen fixed by these bacteria are largely variable because of divergent nature of soils.

In aerobic soils of tropical climatic regions, the acid tolerant N2-fixer Azotobacter beijerinckia is most abundant Azospirillum spp. also fix N2-non-symbiotically and help to many crops for their growth and yield.


Hidden conflict in the mutually beneficial relationship between legumes and rhizobia

A cluster of nodules on the roots of the plant Lotus japonicus. Bacterial rhizobia are housed within root nodules and supplied with carbohydrates from the host plant. The carbohydrates are used by rhizobia in exchange for the fixation of nitrogen then used by the plant. Credit: K. Quides

The mutually beneficial relationship between legumes and rhizobia, the nitrogen-fixing soil bacteria that make their home in legume root nodules and create nutrient-rich fertilizer for them, is one of the most well-known and agronomically important examples of symbiosis. New research from Dr. Kenjiro Quides, a Postdoctoral Teaching and Research Fellow in the Grand Challenges Initiative at Chapman University, tested the boundaries of this relationship—and found that it's not always as perfectly harmonious as previously thought.

The results are reported in a new paper in the journal Evolution.

Legumes provide carbohydrates for rhizobial bacteria that live in root nodules, while the rhizobia fix atmospheric nitrogen into a form that's usable for the legume (nitrogen is often a limiting nutrient for plants). In theory, if legumes have greater root nodule growth, they should be able to host more rhizobia, which should produce more nitrogen and enable larger plant growth in general.

The research, carried out by Quides and colleagues at UC Riverside during his doctorate, tested the relationship between root nodule growth and rhizobia using a smaller relative of Soybeans, named Lotus japonicus. By using multiple genetic variants that formed a low, medium, and high number of nodules, the study showed that legumes grew to maximum size when a low and medium number of nodules formed, but legumes that formed a high number of nodules had drastically reduced growth.

The investigation then turned to the rhizobia. The size of the rhizobial population, a standard measure of bacterial growth, was found to continue to increase as the number of nodules formed increased. This suggests a hidden conflict in the symbiotic relationship. It seems that the legume and rhizobia interests are only aligned until the host optimum is reached, a point at which their interests diverge. This provides support for the conclusion that in the symbiotic relationship, rhizobia have an evolutionary advantage.

The results demonstrate that to avoid conflict in symbiotic relationships, hosts must tightly regulate their investment into symbiotic organs (like legumes' root nodules) to maximize their own benefit-to-cost ratio of associating with their symbiotic partner.

"Legumes seem to play a balancing act to maximize their growth, but rhizobia continue to grow and that is a really exciting result," Dr. Quides said.

He noted that this study opens the door to more research. "Although we found diminishing returns for the host from nodulation, the fact that rhizobia population size continued to increase is promising. We found the costs outweigh the benefits at high nodule numbers. However, if we can increase the number of nodules and therefore the rhizobia population size while minimizing the cost to the plant, we have the potential to increase the productivity of legume crops in the future."

The title of the paper is "Dysregulation of host-control causes interspecific conflict over host investment into symbiotic organs."


EICOSANOID ACTIONS IN INSECT IMMUNOLOGY

DAVID W. STANLEY , JON S. MILLER , in Insect Immunology , 2008

3.5 EICOSANOIDS IN HEMOCYTE SPREADING AND MIGRATION

Microaggregation and nodulation reactions are large-scale, visible cellular defense reactions to microbial challenge. We take these large-scale reactions to be the culmination of an unknown number of small-scale, relatively invisible reactions. Plasmatocyte spreading on surfaces may be regarded as one of the component actions that comprise the overall nodulation process. Because of the important role in nodulation, Miller (2005) investigated the hypothesis that eicosanoids mediate cell spreading in primary hemocyte cultures prepared from tobacco hornworms.

Primary hemocyte cultures were prepared by pericardial puncture from hornworms that had been injected with EtOH (the drug vehicle for control larvae) or with a selected EBI diluted in EtOH. Hemocyte preparations were applied to glass cover slips and allowed to settle for 7 min. After washing and allowing hemocytes to settle for selected times, the hemocytes were fixed in formaldehyde. Digital images ( Fig. 3.5 ) were analyzed using ImageJ software. Because plasmatocyte length, but not width, changed with incubation times the main focus was directed to length of the cells.

FIGURE 3.5 . A photomicrograph of a plasmatocyte from an untreated tobacco hornworm, Manduca sexta, after spreading on a glass cover slip for 1 h. The red lines represent digital measurements of the cell dimensions. This photograph was taken through confocal optics at 400×.

(Prepared and photographed by Jon S. Miller.)

Plasmatocytes from control hornworms elongated to about 41 μm after 60 min incubations. The most rapid cell elongation took place during the first 30 min and length did not significantly increase in longer incubation periods. The elongation reactions were severely truncated in primary hemocyte cultures prepared from hornworms that had been treated with Dex and the Dex effect was expressed in a dose-related manner. The inhibitory influence of Dex was reversed by injecting AA into Dex-treated hornworms. Miller (2005) also found that inhibitors of COX and LOX pathways resulted in truncated elongation. Hence, the outcomes of these experiments allow the conclusion that eicosanoids mediate one of the smaller-scale steps in the overall nodulation process, namely plasmatocyte elongation.

Continuing in this line, Kwon et al. (2007) considered the influence of bacterial infection on plasmatocyte elongation. We found that hemocytes prepared from tobacco hornworms 15 and 60 min after infection were altered in size. Specifically, all hemocytes were smaller than 15 μm and none of the hemocytes exhibited cell spreading. On the idea that this change could result from an adventitious influence of S. marcescens, our standard bacterial challenge species, we performed these experiments with three additional bacterial species, Escherichia coli, Bacillus subtilis and Micrococcus luteus. The results were similar for all four species. In another set of experiments, we found that the influence of bacterial infection on cell spreading declined with time after infection. As seen before, the retarding influence of Dex was reversed by AA, PGH2 and CM. For these experiments, we considered the possibility that CM prepared in the presence of bacteria could be contaminated with bacterial molecules that passed through the filter step. The appropriate control experiment was to prepare CM using only bacterial cells in the absence of hemocytes. We showed that CM prepared in the absence of hemocytes did not influence cell behavior. Overall, Kwon et al. (2007) demonstrated that bacterial infection exerts a strong effect on eicosanoid mediate cell spreading reactions to challenge.

As a final word in this section, we mention on emerging work on hemocyte chemotaxis. Chemotaxis is the ability to register and move toward or away from a chemical source, following a chemical gradient. This is a fundamental property of many cells, including bacteria and other single-celled organisms. In metazoans, chemotaxis is important in development and immunity, among other important areas. We used a modern form of a Boyden apparatus ( Boyden, 1962 ) in which a lower chamber is charged with a chemotactic molecule and an upper chamber, separated from the lower by a porous membrane, is charged with cells. In our work, primary hemocyte cultures prepared from tobacco hornworms were placed in the upper chamber. We found that hemocytes are able to migrate toward the source of a bacterial peptide and to demonstrate that the chemotactic response depended on eicosanoids. A full report on this work is forthcoming.


Energy supply to nitrogenase: the O2 paradox

Rhizobia are obligate aerobes, thus needing oxygen for their energy metabolism, also in SNF. On the other hand, both nitrogenase proteins and the protein-free FeMo-Co are sensitive to oxygen with the sensitivity in the order FeMo-co»>Fe protein» MoFe protein (Mortenson and Thorneley, 1979 ). The protection of nitrogenase is mediated by several mechanisms. Vance and Heichel ( 1991 ) summarized the importance of oxygen and carbon for nodule physiology and uncover several factors/processes with importance for efficiency. The nodule inner cortex, which surrounds the central zone of infected cells, serves as a diffusion barrier which limits the flux of O2 to the bacteroids. Together with leghaemoglobin, this barrier regulates the flux of O2 to nitrogenase. Further, they list plant redirection of glycolysis to malate with subsequent reductive formation of succinate under microaerobic conditions and bacteroid ATP formation coupled to a high-O2-affinity terminal oxidase as factors regulating the O2 flux.

Leghaemoglobin is invariably associated with nitrogen-fixing nodules in legumes and has been used as an index of fixation potential (Virtanen et al., 1955 ). This is due to the pigment being present in fairly constant cellular concentration within the nitrogen-fixing cells of nodules. Its nodular concentration is therefore an index of the amount of nitrogen-fixing tissue which is present (Bergersen, 1961 ). Appleby ( 1984 ), being a pioneer in nodule physiological research, summarized knowledge on leghaemoglobin and rhizobial respiration accumulated thus far, confirming the role of leghaemoglobin as a facilitator of O2 flux to the vigorously respiring, phosphorylating, N2-fixing rhizobium bacteroids, albeit at a stabilized O2 tension (10 n m in soybean nodules). However, not until 2005 was it demonstrated that leghaemoglobins are required for efficient nitrogenase activity. Plant reverse genetics employing RNAi was then used by Ott et al. ( 2005 ) in the model legume Lotus japonicus to create plants carrying nodules abolished in leghaemoglobin synthesis, causing an increase in nodule free oxygen, loss of nitrogenase and absence of SNF, whereas growth on combined nitrogen was not affected. The non-legume Parasponia seemed to be the only exception to N2-fixing nodules having leghaemoglobin, but van Velzen et al. ( 2018 ) in a comprehensive study of this plant showed that this non-legume has indeed recruited the class I leghaemoglobin HB1 for balancing oxygen levels in the nodules, whereas legumes and the actinorhizal Casuarina use class 2 haemoglobins for this purpose.

Following the demonstration of the bacteroid as the nitrogen-fixing organelle (Bergersen and Turner, 1967 ), the energy provision for nitrogenase has been a long-standing and complicated issue that has received the attention of scientists over the years (e.g. Bergersen, 1971 Appleby, 1984 Kaminski et al., 1996 Marchal and Vanderleyden, 2000 ). The respiratory machineries in different rhizobia are diverse and complex. Fischer ( 1994 ) gives a thorough account of nif and fix genes in S. meliloti, B. japonicum and A. caulinodans identified by then, showing gene arrangements and regulatory networks. Genes, mutations in which caused loss of N2 fixation, were named fix genes. Important fix genes are the microaerobically induced fixNOQP, which encode a membrane-bound cytochrome oxidase. fixGHIS genes, which map immediately downstream of the fixNOQP operon, encode the symbiotically essential cbb3-type haem-copper oxidase complex (Preisig et al., 1996 ). The fixABCX operon, also required for nitrogenase activity, was later shown to encode an electron-transferring-flavoprotein (ETF) dehydrogenase (fixABC), whereas FixX shows similarity to ferredoxins. Delgado et al. ( 1998 ) reviewed genes involved in the formation and assembly of rhizobial cytochromes and their role in symbiotic nitrogen fixation. However, still many features of rhizobial respiration are unknown. The genome of B. japonicum encodes as many as eight different terminal oxidases for respiration in oxic conditions (Youard et al., 2015 ). Thus, even though respiration is crucial for efficiency, no definite answers to whether respiratory activity or nitrogenase function limits efficiency of N2 fixation have so far been identified in this complex system.

Modern molecular approaches now enable genome comparisons and phylogenetic approaches to be employed for renewed studies on symbiotic processes with a wider scope. Degli Esposti and Martinez Romero ( 2016 ) present an interesting advance regarding the respiratory chain of symbiotic rhizobia. They depict a current view on rhizobial energy provision to nitrogenase (Fig. 2). The respiratory complex I is a NADH: ubiquinone oxidoreductase (Nuo), and one of the largest membrane protein assemblies known. Complex I has a central role in energy production in mitochondria. Whereas the mitochondrial complex I consists of 45 subunits, the prokaryotic enzyme consists of 14 ‘core’ subunits, conserved from bacteria to humans (Efremov et al., 2010 Degli Esposti, 2015 ). Degli Esposti and Martinez Romero ( 2016 ) compiled data from literature into a figure with enzyme expression or protein data of 16 enzymes (nitrogenase and respiratory) from Frankia and seven rhizobial species representing five genera. They proposed that in rhizobia the NUO14 complex 1 was downregulated in symbiosis in comparison with non-symbiotic conditions, and the so-called green complex I, an ancient form of the enzyme, was upregulated in Rhizobium and Sinorhizobium, but absent from the other genera. They also studied the evolution of respiratory complex I which showed that about 70 Alphaproteobacteria, predominantly Rhizobiaceae, possessed two different operons for respiratory complex I, namely the standard NUO14 and another called the green complex I (Degli Esposti and Martinez Romero, 2016 ). The peculiar rearrangement of gene order of green complex I was discovered in about half of the Rhizobium and Sinorhizobium strains studied by Degli Esposti and Martinez Romero ( 2016 ). The complex was always upregulated in the symbiosis of strains that possessed it. The authors thus proposed that the function of the hypothetical green complex I is to increase the efficiency of N2 fixation in symbiotic nodules.


Biological Nitrogen Fixation

The synthesis of organic nitrogenous compounds from atmospheric nitrogen by certain microorganisms is called biological nitrogen fixation. Higher plants cannot directly utilize molecular nitrogen of the atmosphere. But certain micro-organisms can utilize atmospheric nitrogen. There are two types of nitrogen fixing micro-organisms: Asymbiotic and symbiotic.

(a) ASYMBIOTIC NITROGEN FIXATION

The free living nitrogen fixing organisms are called are asymbiotic – organisms. There are following organisms which fix the atmospheric nitrogen.

They are saprophytic organisms. Therefore, they fix nitrogen only in the soil. This soil must have good supply of organic manure. The example of anaerobic bacteria is Clostridium and the example of aerobic bacteria is Azotobacter.

Blue green algae are autotrophic photosynthetic organisms. They grow in waterlogged and wet soil like paddy (rice) fields. Members of families Nostocales and Stigonematales are important nitrogen fixture. Some of its examples are:

(a) Anabaena is an important member. It consists of branched
filaments. These filaments arc made up of two types of cells

• Green photosynthetic cells: They fix the atmospheric carbon dioxide and release oxygen during pltotosynthesis.

• Heterocyst: They are slightly enlarged colourless cells. These

are non-photosynthetic. They can fix atmospheric nitrogen aerobically. Some of the blue green algae do not have heterocysts. But they can fix nitrogen. Their mechanism is not understood.

(b) Blue green algae living in higher plants: The blue-green algae are free living in the soil. Some blue green algae enter into the tissues of higher plants. They permanently live inside. For example Nostoc lives in the mucilage cavities of Anthoceras. Some anabaena lives in the leaf cavities of the xvater fern. The nitrogen fixing ability of the blue green algae increases in higher plants. They produce more number of heterocysts. Nitrogenous substances regularly pass from the alga to the higher plant and carbohydrates pass from plant to cyanobacteria. Therefore, it may be called a symbiotic association.

(c) Lichens: Nostoc and Scytonema develops symbiotic association with fungi. This association is called lichens. The algal components fix the molecular nitrogen in the form of organic compounds.

(b) SYMBIOTIC NITROGEN FIXATION

It is the most important metlioa of nitrogen fixation. These are of two types:

Several species of Actinomycetes develop mycorrhizal association with the root of Casurina. Pinus and other plants. These mycorrhizae may be ectotropic and endotropic mycorrhizae. These fungi have ability to fix atmospheric nitrogen.

It is most important nitrogen fixation. Its mechanism is fully known. A large number of leguminous plants like bean, pea, gram and soybean develop root nodules. All the plants of family Papilionacae develop nodules. 30 per cent of the species belongs to Caesalpinacae. Leguminous plants increase the soil fertility. German Bacteriologist, Beijerinck identified that Rhizobium infect the root system and they develop nodules.

Bacteria mainly fix atmospheric nitrogen in the form of amino acids. These amino acids are transported by transpiration stream to different parts of the plant. In turn, bacteria get their food (carbohydrates) from the legume plant. Therefore, this relationship is symbiosis. The nodules are not formed on the root system without bacteria. Similarly bacteria themselves can fix atmospheric nitrogen.

Nodules are formed by following process:

I. Infection thread formation: Nodule causing bacteria are present in

the soil. The root system of leguminous plants secretes some sugars and amino acids. It attracts the bacteria. The bacteria multiply near the root zone. They develop a sufficient bacterial population. Then they infect the root hairs. A number of changes take place in the root hair cell. Cytoplasmic streaming movement increases. The nucleus of the root hair become doubles in size. The cell wall loosened. The plasmalemma invaginates and engulfs the bacterium. The root hair becomes curled. The bacterium becomes threadlike inside the root hair This is called infection thread.


Process of Nitrogen Cycle (With Diagram)

The nitrogen cycle consists of four processes. The processes are: (1) Biological Nitrogen Fixation (2) Ammonification (3) Nitrification and (4) Denitrification.

The main processes comprising this cycle of changes are as follows:

I. The transformation of atmospheric gaseous nitrogen by micro-organisms and certain photo- synthetic bacteria and blue-green algae into the combined form of NH4 + nourishing the bacteria and the green plant.

This process is generally known as biological nitrogen fixation and is accomplished largely by three sets of organisms, the first one consisting of micro-organism living in endo- or ectosymbiotic association with a number of plants, the second consisting of organisms living in the soil independent of host plants (Azotobacter and Clostridium, the frequently chemo-synthetic Desulphovibrio, etc.) and the last consisting of a group photo- synthetic bacteria and the blue-green algae.

II. The transformation in soil of organic nitrogenous compounds arising from decomposition and autolysis of all forms of biological materials, or from excreta of animals or from products of metabolism of living soil organisms into NH4 + ions.

The process is accomplished by a great variety of micro-organisms which may be anaerobes or aerobes, and which break down the organic nitrogenous compounds into ions as a part of their metabolism or the formation of NH4+ ions may be brought about by the action of hydrolytic and oxidation enzymes present in the micro­organisms.

III. The conversion of ammonium cations to nitrite and nitrate anions.

The process is generally referred to as soil nitrification and is accomplished largely by two groups of organisms—Nitrosomonas (also Nitrosocystic and Nitrosospira) which form nitrites from NH4 ions and Nitrobacter which converts nitrites to nitrates.

IV. The reduction of nitrates into nitrites and finally into NH4 ions or into free molecular nitrogen which goes back to the atmosphere.

Reduction of NO3 – into NO2 – and NHS and to gaseous nitrogen is accomplished by various micro-organism. Several bacteria can reduce nitrate to NH4 + for assimila­tion purposes. However, there are a group of bacteria which reduce nitrate to the level of gaseous nitrogen, which usually escapes into the atmosphere. This process is known as denitrification.

Examples of denitrifying bacteria include Bacillus denitrificans, Pseudomonas stutzeri, Ps. denitrificans and Thiobacillus denitrificans.

Process # 1. Biological Nitrogen Fixation:

(A) Symbiotic—Root Nodule-Legume-Bacteria System:

For several centuries it has been a matter of common observation and agricultural practice that soils impoverished by the growth of the cereal crops can be revitalized by growing leguminous plants. Yet, not until the end of nineteenth century were adequate explanations forthcoming as to why these procedures caused such beneficial effects.

It was in 1837, that the first observation was made that the fixation of atmospheric nitrogen takes place during the growth of legumes, such as clover, peas, lucerne, etc., whereas no such fixation occurs, during the growth of cereals, like wheat or, oats.

In 1857, it was demonstrated that plots of lands cropped to non-legumes without addition of artificial manures, gave rise to only low yields while plots cropped to legumes main­tained relatively high yields even without any manurial treatment.

Moreover if a non- legume followed a legume in a rotation [crop rotation such as wheat (1st year) → potato, beet, etc., (2nd year) → barley, oat, etc., (3rd year) → legume (4th year) → wheat (again)] the yield was as high as if the field has been previously left uncropped (fallow).

And this occurred in spite of the fact that large quantities of nitrogenous com­pounds were removed by the preceding leguminous crop. By 1866, it was definitely estab­lished that certain bacteria in soil infect legumes forming nodules which enabled the nodule-bacteria system to fix atmospheric nitrogen these bacteria have no infecting power on cereals.

The supply of available nitrogen is so commonly a limiting factor in plant growth that the almost unique ability of leguminous plants to fix and to draw on atmospheric nitrogen (hence to be largely, if not entirely independent of soil nitrogen supply) is of the greatest importance in the development of economic cropping systems in the rotation of crops.

The organisms responsible are known as root nodule bacteria. They are species of the genus Rhizobium, once collectively known as Bacillius radicicola. They were first iso­lated in the pure state by Beijerinck.

The exact mechanism of nitrogen fixation by the root nodule bacteria is not well understood on the other hand great deal is now known of the complex interrelations between Rhizobium and its host.

It has been established that fixation of N2 occurs in the nodules and that its amount is proportional to the volume and duration of life of the bacterial cells, so that it appears that the bacteria are, in fact, the actual agents of fixation.

When bacteria and the host plant are in close symbiotic association nitrogen fixation can take place actually. Rhizobium cells growing some distance away from cultures of even non-legumes have been reported to be capable of fixing nitrogen.

In 1975 it was demonstrated independently in five different laboratories that Rhizo­bium, particularly slow growing rhizobia belonging to the soybean or cow pea group, can fix nitrogen in laboratory culture media supplemented with pentozes like xylose, and arabinose, galactose, succinate and a small amount of glutamine.

The site of nitrogen fixation within the root nodule is the bacteroid. These bacteroids are usually X- or Y-shaped and are not found in labo­ratory cultures unless treated with some alkaloids or manc acid.

There are some biochemical differ­ences between Rhizobium and its bacteroid form. A bacteroid transferred to laboratory cultures pro­duces cells having the usual morphology of Rhizo­bium. Bacteroids are stable only within the root nodule.

The genetic basis or the bacteroid form is not understood. Nitrogen fixation is controlled by the nif (nigrogen fixation) gene responsible for the synthesis and function of the enzyme—nitrogenase which has two components—one of which contains iron alone and the other iron and molybdenum. Both iron and molybdenum are essential for nitrogen fixation.

For vigorous fixation, it is essential that the host plant should be intact and healthy nodules excised from the roots, for example, rapidly lose their powers of fixation, although in recent years it has been conclusively demonstrated by subject­ing only soluble nitrogenous compounds of the nodules to isotopic analysis with 15 N, that excised nodules taken from legumes grown in the fields (not in the greenhouse) and performing the experiments immediately after detachment from the roots, are capable of continuing nitrogen fixation for quite some time.

It has been found that under certain conditions, nitrogenous products formed in the nodules may be excreted from the roots and become available to other plants and it is true, that it is sometimes possible to grow successfully mixed cultures of legumes and non-nigrogen-fixing plants such as oats without supplying any combined nitrogen.

Nodulated legumes require illumination for the host for nitrogen fixation and nodule slices showed enhanced fixation when supplied with sucrose, fructose and glucose in that order, which are well known to be prominent photosynthetic products.

Interest with regard to the symbiotic system is centred around the question why association with leguminous plants should endow one or the other of symbionts with the ability of nitrogen fixation.

When the seed of a legume develops in a soil containing Rhizobium leguminosarium, the latter are attracted to the region of developing root hairs. The result of the presence of the bacteria in the near neighbourhood of the root hairs is to produce
a ‘curling’ or deformation of the hairs.

Specific chemical substance, possibly the growth- promoting auxin, indoleacetic acid is responsible for this curling for bacterial extracts in absence of rhizobia produce the same effect. (For the curling of the root hairs, a conc. of 0.01 p.p.m. of indoleacetic acid is sufficient.)

The response, however, is bio­logically non-specific, i.e., may be induced by extracts of bacteria other than rhizobia. At the site of deformation of the root hair, rhizobia invade the root tissue and proli­ferate within the hair in the form of a thread directed towards the cells of the roots (Fig. 688) and a sheath is laid down by the cells of the host separating the infected tissue from the rest of the plant.

Cell division is stimulated, the newly formed tissues are invaded by more bacteria and thus a nodule is formed. Bacterial cells are included in a thread-like structure known as the infection thread. Many cells in the vicinity of the infection thread are triploid and some are tetraploid.

Direct vascular connection of nodules formed with the host plant is maintained as long as fixation takes place. The local stimulation of host cells to divide is also due to the specific hormonal subs­tances secreted by the bacteria.

Both the bacteria and the host cells fail to proliferate as the nodule becomes older, and finally the whole nodular tissue becomes necrotic. The nodule softens, its interior is digested and finally falls off. The Rhizobia return to the soil.

The exact site of fixation inside leguminous nodules remains obscure. Some investi­gators believe that fixation occurred on a membrane surrounding single bacterium or groups of them in the nodule.

Rhizobium induces nodule formation in a restricted number of leguminous plants. There is a number of different species of Rhizobium, and each can function satisfactorily on certain legumes only.

The species of Rhizobium concerned in N2 fixation are: R. meliloti, R. trifolii, R. leguminosarium, R. phaseoli, R. japonicum and R. lupini. For con­venience, leguminous crops are divided into cross inoculation groups, each group capable of being infected by the specific strain of bacteria belonging to that group only.

Eight cross inoculation groups have been identified:

(1) Alfa alfa group (alfalfa, lucern), (2) clover group (clover, berseem), (3) soybean group (soybean), (4) pea group (pea, lentil, cicer etc.), (5) cowpea group (cowpea, sunhemp), (6) lupin group, (7) bean group and (8) only specific strain group.

Thus if only cowpea group bacteria are present in the soil, effective nodules are formed in all the plants belonging to that group leading to a successful crop of plants of that group.

The cropping of legumes of other groups, as for example clover or soybean, will not, however, be profi­table because the particular strain either does not form any nodule on clover or soybean, plants of different groups, or even if the nodules are formed they are ineffective, for the quantity of nitrogen fixed by ineffective nodules per unit volume of the nodular tissue compared to those produced by effective strains of bacteria is insignificant.

A noticeable feature of the association of the bacteria with the legume is the pre­sence of a red haemoglobin-like pigment (leghaemoglobin), similar to the vertebrate blood pigments, in the effective, healthy and mature nodules only.

This leghaemoglobin (LHb) shows the characteristic absorption spectrum (in red), identical with myoglobin, a variety of haemoglobin specifically found in muscle fibres of animals. This haemoprotein was first isolated and crystallised from soybean root nodules as two proteins each having one haeme (about 0.34% Fe) group but differing in molecular weight—15,400 and, 16,800—and amino acid composition.

LHb is localised outside of the bacteroids. LHb thus can conclusively be eliminated as an integral part of nitrogenase enzyme, as it (enzyme) is only found inside the bacteroids.

There also seems to be a complete lack of requirement for added LHb for the actual nitrogen fixation process. This haemoglobin is never formed in either partner when alone. It can easily be seen that mature nodules on a healthy pea plant are pink, whereas those on plants which are unhealthy or which have been kept in the dark for some days, are white or green.

The infecting rhizobium apparently not only induces growth and multiplication of the root cells of the legume but also supplies the proliferating cells, directly or indirectly with a factor or factors necessary for the synthesis of this haemoglobin. The fact at first pointed naturally to the possibility that in the root nodule, haemoglobin itself or the mechanism connected with its synthesis may be directly linked with nitrogen fixation.

A more probable explanation was there all the time that haemoglobin may act only indirectly by securing in the root nodule necessary optimal conditions for oxidative processes with which nitrogen fixation may be associated by regulation of oxygen diffusion.

The demonstration of inhibition of nitrogen fixation by nodules and its bacteroid suspen­sions by 2-4 dinitriphenol (DNP) at a conc. normally used for uncoupling phos­phorylation, gives strong indications that oxidative phosphorylation is the major source of ATP for nitrogen fixation in the nodulated system.

The intensity of nitrogen fixation is proportional to the amount of haemoglobin present. The pigment is only formed after the nodules have been properly established and concurrent with the change of the red colour of the pigment to a green colour, fixation of N2 by the nodular bacteria declines.

The rhizobia certainly need a continuous supply of oxygen to enable them to fix nitrogen efficiently. Rhizobium is a strict aerobe. It has been demonstrated that rhizobia in the root nodules possess a high rate of respiration, much more intense than that of the other parts of host plant.

It might, at first sight, seem that the O2 evolved in photosynthesis of green host plant would possibly more than suffice for the purpose. It is, however, not possible for this to occur because photosynthesis occurs in the host plant only in cells containing chlorophyll, that is, in the aerial parts and O2 evolved must be transported from these parts to the nodular bacterial tissue and also there is no photo­synthesis at night.

The transport of O2 from the exterior to the nodular bacteria, as also between the nodule and host plant is greatly hampered in many nodules due to the presence of common endodermis characterised by the presence of complete suberin lamella in the outer tissue of the nodule, in addition to individual endodermal sheath around the vascular strands of the nodule.

This considerable reduction in the direct communication between the nodule tissue and the exterior results especially in a greatly reduced oxygen supply to the tightly packed rhizobia inhabiting nodular tissue.

Thus, it is evident that coupled with high rates of respiration of the bacteria in the nodules and the difficulty of supplying oxygen from exterior or from the host, due to the common endodermal sheath, the aeration of the nodules is greatly restricted and the nodular bacteria are in a state of partial anaerobiosis. This is, however, useful for Rhizobium, since the enzyme nitrogenase is highly sensitive to O2.

In order that N2 fixation in the root nodules may continue at a normally high rate and the supply of O2 does not become a limiting factor in the fixation mechanism, the nodules certainly need an efficient means of absorbing and transporting O2 to the grow­ing bacterial cells.

In the course of biological evolution, the problem of efficient oxygen absorption arose early and because of its urgency it was solved first by certain worms, such as earthworm by synthesising haemoglobin, the most efficient oxygen carrier of all.

Similarly in order that nitrogen fixation can proceed uninhibited, some device must be adopted by legume host plant which can enhance the supply of oxygen to the deep- seated bacteria.

The net result apparently was believed to be the formation of haemo­globin in the nodules from the porphyrin precursors, apparently always available in plants as in animals. It has been proved that within the nodules the leghaemoglobin displays the usual property of reversible oxygenation, characteristic of vertebrate blood.

Some investigators believed that the rhizobia in the root nodules of legumes may meet at least a part of their oxygen requirement by the same biochemical mechanism as used by all vertebrates, including man himself, although it has been definitely proved that haemoglobin-bound oxygen in the nodules would suffice for only less than 4 minutes of respiration of an average-sized nodule. Thus there is really very little evidence that the red pigment in the nodule can function as an oxygen store for the nodular tissue.

The role of LHb which now appears to be most probable (1971) is that proposed by Yocum (1964). Scholander (1960) more than a decade ago, demonstrated that the diffusion of O2 through a membrane which was saturated with a solution of a haemo- protein—both haemoglobin and myoglobin—was much faster than the diffusion of N2 in the same system.

It has been proposed that this process of facilitated diffusion was an important way in which O2 could be transported through tissues which had a high oxygen consumption. The LHb seems to be suited for the role in facilitated O2– diffusion, under low conc. of O2 which, as we know, obtains in the nodules.

No doubt, in legume root nodules then O2 is required for bacteroid respiration and consequent provision of ATP for nitrogen fixation. Without LHb, it would appear that the diffusion of O2 through the dense nodular tissue would be completely inadequate to meet the necessary ATP-requirement. (Bergersen, 1971).

It is interesting to note that formation of haemoglobin in the plant kingdom, may be only under special circumstances and identified as yet only in the root nodules, solves one more discrepancy and supplies us with one more evidence that the world of living organisms, whether of animal or plant, is one and there is a splendid natural arrange­ment governing the both.

The presence of combined nitrogen, nitrates or ammonium salts, in the soil in which the host legume is growing, apparently makes the legume resistant to attack by rhizobia the combined nitrogen impede the development of nodules, fewer root hairs are deform­ed and as a result fewer nodules are formed.

The net effect is a great reduction in the quantity of nitrogen fixed and under certain circumstances this quantity is almost nil. Organic nitrogenous compounds such as proteins which have to be broken down before they can be assimilated, inhibit nitrogen fixation to a much lesser extent.

Molybdenum appears to be of special significance in all organisms which fix atmospheric nitrogen. However, whilst molybdenum undoubtedly influences the growth of all nitrogen-fixing organisms there is now direct evidence that it is specifically concerned with the fixation mechanism itself.

Recent work with the nitrogen-fixing blue-green algae Anabaena, indicates that Mo may also be concerned in phosphate metabolism. There is a unique requirement for cobalt for the nitrogen-fixing system copper may also be essential. It is well known that carbon monoxide gas reduces the fixation of nitrogen in nodulated legume.

This effect of CO on the fixation process in legumes and on vertebrate animal respiration may thus be analogous, due to the presence of Cytochromes or haemoglohin or both. It is interesting to point out here that recently there has been strong speculative suggestion that the antipernicious anaemia factor, vitamin B12 may play a similar role in the nitrogen-fixing mechanism in the nodulated legume, as in human blood.

But this view fails to explain how CO affects fixation in Azotobacter and in the blue-green algae, which certainly contain no such haemoproteins and are less likely than the rhizobia to live in conditions of any anaerobiosis.

A most important feature of nitrogen fixation is that the process only occurs in growing organisms. Unlike photosynthesis which can continue after cessation of all growth, nitrogen fixation by the nodulated legumes ceases with the active growth of rhizobia. The nitrogenous compounds formed are possibly excreted through the roots of the host plant.

Taken overall, biological nitrogen fixation must be an endergonic reaction and, therefore, to put it in terms which the biologist understands, energy released, as indicated before, in the form of ATP, during respiration must be supplied to make it work.

All nitrogen-fixing organisms, including the rhizobia in the root nodules, generally grow best in neutral or slightly alkaline medium and there appears to be an inhibition of nitrogen fixation under acid conditions.

Ammonia is the most obvious candidate for the position of key intermediate in the mechanism of biological nitrogen fixation for it is often excreted by nitrogen-fixing organisms and its formation from elementary nitrogen by addition of hydrogen is a reaction of apparent simplicity. In experiments with isotopic 15 N with various nitrogen- fixing organisms, it is always found that the heavy 15 N appears in the greatest extent in glutamic acid just as it does if 14 N is supplied.

Glutamic acid is well known to be the main substance through which NH3 enters into the general metabolism of the cells and thus this would strongly suggest that NH3 is the key intermediate in nitrogen fixation by various micro-organisms.

Ammonia perhaps is produced from elementary nitrogen in several stages, one of which stage may conceivably be represented by hydroxyl- amine, NH2OH, even now believed by some investigators, as the key intermediate in the nitrogen-fixing mechanism.

The leguminous genera Cassia, Cercis, Gleditschia, Gymnocladus, etc., contain species, not known to form any nodules in the roots. As a matter of fact it seems that effective root nodules are only formed in species belonging to Papilionaceae species of the two other subfamilies of Leguminosae—Caesalpinieae and Mimosae—are rarely known to form symbiotic association with bacteria.

Gleditschia belongs to a small group of legumes known from Cretaceous and early tertiary strata and this probably suggests that the association between the legume and rhizobia began later than that time. Rhizobium may form nodules on the roots of some non-leguminous plants also, e.g., Trema can- nabina of Ulmaceae, which grows in New Guinea. The nodules are pink in colour, but the pigment is not leghaemoglobin.

(B) A Few Micro-Organisms Other than Rhizobium also Form Nodules on Roots and Leaves of Non-Leguminous Plants:

Some of these associations are mentioned below:

(i) SYMBIOTIC—ROOT NODULE—MYRICA—ACTINOMYCES SYSTEM. (Frankia)

(ii) SYMBIOTIC – ROOT NODULE – ALNUS – ACTINOMYCES SYSTEM.

(iii) SYMBIOTIC – ROOT NODULE – CASUARINA – BACTERIA SYSTEM.

(iv) LEAF NODULE – PAVETTA, DIOSCOREA, etc., – KLEBSIELLA SYSTEM. (Fixation doubtful)

The agricultural importance of legumes has resulted in a concentration of atten­tion upon their relations with Rhizobium and a neglect of other nitrogen-fixing symbiotic associations. Such symbioses are perhaps not so highly developed as the legume- bacteria system but their contribution in maintaining the fertility of the earth is by no means negligible.

Azospirillum (=Spirillum lipoferum) also forms endosymbiotic association with maize roots. Dobereiner in Brazil has claimed that the maize plants benefit substantially from this association in so far as its nitrogen nutrition is concerned.

Some strains of Bacillus sp. also fix nitrogen in association with certain genotypes of wheat. Azospirillum, Azotobacter and Beijerinckia have been detected in the rhizosphere of several plants, particularly grasses including sugarcane. They utilize root exudates.

Several nitrogen fixing bacteria grow in ectosymbiotic association with the leaves of plants, particularly in the tropics. The organisms include Klebsiella, Beijerinckia and Azotobacter. Some active strains have been found to improve the growth of crop plants, when sprayed on their foliage.

These organisms derive the energy for N2 fixation and for their life activities from the organic matter leached out from the leaves when they come in contact with water, as for example, when it rains or when the leaves collect dew drops.

The bog plants, Myrica gale and Myrica cerifera (Fam. Myricaceae) have root nodules produced by a species of Actinomycetes (filamentous organisms classified with bacteria occupies a position somewhere between bacteria and fungi.

Many antibiotics, such as streptomycin, chloromycin, terramycin are produced by species belonging to this group of soil micro-organisms) and thus infected, the plant is able to fix atmospheric nitrogen.

Free-living Actinomycetes alone are incapable of fixing nitrogen, so are the Myrica, if uninfected by Actinomycetes. Root nodule Actinomycetes of Myrica gale can fix nitrogen when the reaction of the soil or water in which they live is distinctly acid.

Except for a few species of Azotobacter which can fix nitrogen in acid medium, all the nitrogen-fixing mechanisms seem to be inhibited by acid conditions. It has been shown that Myrica has got a molybdenum requirement for N2 fixation. Nodulation occurred with or with­out added molybdenum, but N2 fixation was increased tenfold by a trace of Mo.

An interesting feature of the root nodules of Myrica is the development of negatively geo- tropic pneumatophores from the nodules which come above the soil water. The produc­tion of pneumatophores is certainly a biological adaptation for obtaining oxygen for the Actinomycetes inhabiting the nodular tissue and suffering like rhizobia, from an acute shortage of oxygen.

The same difficulty of suboptimal oxygen conditions of the acti­nomycetes is effectively surmounted by the production of pneumatophores in the root nodules from Myrica from the upper surface of the nodules. The root nodules of Myrica are somewhat brownish in colour the pigment is most probably a flavone glycoside.

The alder, Alnus, also develops root nodules as a result of infection by an endophytic actinomycete the alder is then able to grow in the absence of a supply of combined nitrogen. Fixation rate is high and it is probably extracellular to the endophyte.

Trees of the subtropical genus Casuarina (and also Podocarpus, Eleagnus, etc.) are also evidently able to fix nitrogen by virtue of symbioses with a root-nodule-forming as yet unidentified bacterium.

In some cases, micro-organisms which alone can fix nitrogen may enter into symbi­oses with others which cannot. These organisms appear to be able to fix nitrogen when isolated and grown in culture. It is not clear how much benefit is derived by the micro­organism partner from this type of association.

If it is symbiosis, it is evident that it is neither so highly developed nor so successful as the legume bacteria system. Evidences of such associations are afforded by the nodules in the leaves of some tropical shrubs, such as Pavetta belonging to the family Rubiaceae (also Chomelia and Psychotria) and also by the monocotyledon, Dioscorea.

Altogether, evidences have accumulated during recent years that more than 400 spp. of non-leguminous plants harbour bacteria in the leaf nodules. Psychotria exhibits leaf nodules which are the seats of nitrogen fixation. The endophyte has been isolated and it has been established that it belongs to the genus Klebsiella.

These bacteria isolated from the leaf-knots do not fix nitrogen. The leaf nodules of Pavetta are economically important they are widely utilised as ‘green manures’ in the tropics, particularly in Ceylon.

Species of sensitive aquatic Mimosaean genera, Neptmia, are known to form beautiful nodules harbouring rhizobia, in their floating stems which root at the nodes.

(C) Non-Symbiotic Nitrogen Fixation:

(i) By free-living nitrogen-fixing bacteria of azotobacter and clostridium groups. Winogradsky discovered an anaerobic soil micro-organism, Clostridium pasteurianum which will fix free molecular nitrogen, when supplied with carbohydrate, the amount of nitrogen fixed, being roughly proportional to the amount of carbohydrate broken down.

About 10 years after, Beijerinck isolated from soil two aerobic organisms capable of fixing atmospheric nitrogen. They were Azotobacter chroococcum (the common non- motile type) and Azotobacter agilis (the motile variety).

These nitrogen-fixing bacteria were found to differ strikingly in that, Clostridium (rod-shaped) is obligately anaerobic, i.e., able to grow only in the absence of oxygen whereas Azotobacter species are aerobes, growing only under condition of good aeration (obligate aerobes).

Azotobacter species have a world-wide distribution although it seems they are absent from some arctic soils. Species of Clostridium are more widespread for they can thrive well where anaerobic conditions prevail and in an examination of 15 different species of the genus, only three have been found unable to fix nitrogen.

As far as it is known, in the sea, biological nitrogen fixation does not occur to any appreciable extent. Azotobacter and Clostridium species have been isolated from marine habitats but there is no evidence that these or any other nitrogen-fixing organisms occur in anything but low numbers in the open waters of the ocean.

Compared with legume bacteria, they are relatively unimportant sources of nitrogen for higher plants, fixing perhaps not more than about 15% of the total nitrogen fixed in the soil or aquatic surface per year.

Optimal condition of the soil necessary for maximum intensity of nitrogen fixation by Azotobacter and Clostridium as well as by most other known nitrogen-fixing organisms is, as we know, neutral or only slightly alkaline. Clostridium under certain circumstances can tolerate a pH of approximately 5, but the fixation of nitorgen is greatly reduced.

The growth of Azotobacter completely ceases at a pH less than 6. Thus it is evident if the soil is distinctly acidic in nature, all types of fixation of nitrogen come to a halt sooner or later except perhaps, as we have discussed before, in root nodule-Myrica-actinomycete system which can tolerate distinctly acid conditions of bog soils.

It appears that the actual fixation process is specifically inhibited under acid conditions for Azotobacter supplied with N03 may still grow under slightly acid conditions that totally inhibit growth of the bacteria in presence of molecular nitrogen only.

Recently, however, several species of nitrogen-fixing bacteria closely resembling Azotobacter, except in details of cell form, have been grouped under a newly created genus Beijerinckia.

Like the nodulated Myrica, they are very tolerant of distinctly acid conditions of the medium of the soil or water in which they live. Is there, then, a physiological difference between the nitrogen-fixing mechanisms of acid tolerant organisms and the vast majority of the nitrogen-fixers, which certainly prefer a neutral or alkaline medium?

It does not necessarily imply that, for it is quite possible that alkaline conditions may be maintained in certain parts of the cell interior, even though the environment and the bulk of the protoplasm are acid in reaction.

The most important single factor influencing nitrogen fixation in soils is the presence of NO – 3 or NH + 4 salts. With both Azotobacter and Clostridium, the presence of utilisable nitrogenous compounds diminishes the rate of nitrogen fixation, NH + 4 or NO – 3 salts being most effective in this way.

Inhibition of fixation by Azotobacter is complete in the pre­sence of NH4-nitrogen at a conc. of 0.5 mg nitrogen per 100 ml. The same is true for root nodule bacteria also, as we have seen before.

Thus, when excess combined nitrogen is available in the soil, little or no fixation of atmospheric nitrogen takes place. This inhibition, however, may be partly counteracted by an increase in the carbohydrate concentration of the medium.

There is no doubt now that NH3 is the key intermediate product in the nitrogen fixing mechanism of all micro-organisms including Azotobacter and Clostridium. The enzyme system responsible for nitrogen fixation has been identified and the whole enzyme system has once been termed azotose, the individual enzyme, first reacting with elementary nitrogen being called nitrogenase.

Azotobacter also possesses a hydrogenase. Certain chemicals have been found to have a specific inhibitory effect on the nitrogen- fixing enzymes. We have seen that carbon monoxide is one of them and the other is gaseous hydrogen.

However, hydrogen has no marked inhibitory effect on nitrogen fixation by Clostridium or by photosynthetic bacteria (e.g., Rhodospirillum). Excretion of organic nitrogenous compounds by roots, has been reported before from nodulated legumes excretion of basically similar nature has also been observed in Clostridium.

The key intermediate, NH – 4 must represent the end of fixation reaction and the start of assimilation of the fixed nitrogen into the organic molecules of the organism.

(ii) By free-living coloured photosynthetic bacteria. a most interesting dis­covery has been that coloured photosynthetic bacteria are nitrogen-fixing. All these bacteria are anaerobic organisms, carrying out photosynthesis only in complete absence of oxygen.

These bacteria are widely distributed in marine and also in fresh water habitats. They have been studied in laboratory cultures for over 60 years before it was noticed that they could assimilate elementary nitrogen.

The genera which are known to be definitely nitrogen-fixing are Rhodospirillum, Rhodopseudomonas, Rhodomicrobium, belong­ing to the family Athiorhodaceae although these species can use inorganic sulphur com­pounds, they prefer organic reductants for assimilation of CO2 in presence of light they can best be described as facultative sulphur bacteria.

Green sulphur bacteria, such as Chlorobium and Chlorobacterium, and the purple sulphur bacteria Chromatium are also N2-fixers. In Rhodospirillum, nitrogen fixation appears to be closely associated with photosynthesis, for although this bacterium can grow in the dark if provided with suitable carbohydrate supply, assimilation of nitrogen is then really very slight.

Although the contribution of photosynthetic bacteria to the total amount of nitro­gen fixed in soil and water may not be considerable, it cannot be denied that in the geological eras, their nitrogen-fixing activities must have been much more significant.

The photosynthetic bacterium Rhodopseudomonas capsulatus abundantly found in tropical paddy fields, can certainly fix considerable quantities of nitrogen, thereby increasing the fertility of such soil.

(iii) By free-living colourless sulphur bacteria. These are one of the most interesting groups of recently discovered sulphate-reducing bacteria. These are obligate anaerobes, i.e., quite unable to grow in air.

Instead of using oxygen to oxidise their food (as in normal aerobic respiration), they use sulphate and as a by-product, the sulphide is formed and energy liberated. This exergonic energy could be utilised for the forma­tion of the cell material of the bacteria in presence of carbonates which are available in the water in which the bacteria live.

If carbohydrates are not available for oxidation by sulphate, Desulphovibrio desulphuricans can utilise hydrogen equally, if not with more facility. It is only very recently established beyond all doubt that Desulphovibrio is also capable of fixing elementary nitrogen.

It may be pointed out here that extreme thermophilic Desulphovibrio must be occupying a unique position in the hierarchy of living organisms—for it can survive and evolve on a planet completely devoid of oxygen and also of sunlight!

And life perhaps will not disappear from this planet of ours when sunlight and oxygen be in short supply and insufficient for highly specialised forms, such as ourselves (in another 3,000 million years?). A comforting thought, indeed!

(iv) By free-living yeast cells. There have been many reports that certain yeasts and also other fungi are able to fix atmospheric nitrogen. But the reported gains in nitrogen have generally been so small, as to make one suspect that errors of technique, e.g., absorption of NH3 or oxides of N2 from air, have occurred rather than fixation of the element.

Recently, however, conclusive evidence has been obtained by means of Kjeldahl and other methods and also by use of isotopic techniques with 15 N, of fixa­tion of N2 by a variety of yeast, isolated from heath soils (waste flat land, usually covered with shrubs).

It seems quite possible, also, that the capacity of nitrogen fixation is present in some fungi on first isolation, but this capacity is rather easily and rapidly lost if cultured in artificial media for any length of time.

Some free-living nitrogen-fixing bacteria isolated from soil are Pseudomonas radio- bacter and Flavobacterium fulvum in mixed culture.

(D) Nitrogen Fixation by Blue-Green Algae (Myxophyceae):

Towards the end of last century, even before the isolation of Clostridium by Winogradsky, it was claimed that some of the blue-green algae are able to fix atmospheric nitrogen.

However, the purity of such cultures was doubted by most of the investigators for the gelatinous sheath with which most members of Cyanophyceae (=Cyanobacteria) are invested, provides a natural medium for the development of bacteria and renders the removal of these bacteria by ordinary culture methods extremely difficult.

Plausi­bility was lent to the prevalent idea by the actual isolation of free-living nitrogen-fixing bacteria from the algal sheath.

So the idea—that blue-green algae are unable to fix nitrogen and where they were observed apparently to grow in absence of nitrates or ammonium salts, they were in close association with nitrogen-fixing bacteria—prevailed until about 1928, when the fixation of nitrogen was definitely shown to take place in absolutely pure cultures of the algae (pure cultures are nowadays obtained easily by using ultra-violet light of suitable irradiation, which would kill the bacteria but not the algae).

Recently, however, conclusive evidence of considerable nitrogen fixation by members of blue-green algae has been given by the use of heavy isotopic 15 N. Of some 40 species of algae, many of them belonging to the family Nostocaceae tested so far, for nitrogen-fixing capacity, more than half have been found to possess it.

Fixation of nitrogen, perhaps not quite as efficient as some species of Nosto­caceae, has also been demonstrated in species of family other than Nostocaceae, e.g., Tolypothrix sp. (Scytonemataceae), Calothrix (Rivulariaceae), Mastigocladus laminosus (Stigo- nemataceae), etc.

Some of the fixed nitrogen, not all, is liberated into the medium. Blue-green algae are common in soil and fresh water and have a world-wide distri­bution, being particularly abundant in the tropics.

Blue-green algae may be the most important nitrogen fixers in certain types of fresh water and where anaerobic condi­tions prevail, Clostridium or the photosynthetic bacteria may also fix appreciable quan­tities.

These algae are common in alkaline or near neutral soil but are rarely present under acid conditions. They are certainly more abundant in the surface layers of soil exposed to light but the demonstration that some species are capable of heterotrophic nutrition in presence of carbohydrate suggests that they are not necessarily confined to such regions only.

Like all other nitrogen-fixing organisms, blue-green algae have a high nitrogen content, about 8% of the total dry wt.

Nitrogen fixation by the blue-green algae occurs only under certain well-defined conditions, such as:

(1) Fixation has never been found to take place in resting material active growth is essential,

(2) as with the ease of all other nitrogen-fixing bacteria, assimilation of free nitrogen does not take place in presence of readily available combined N2, NO – 3 NO + 4or salts,

(3) traces Of molybde­num are necessary, like all other N2-fixing organisms, for fixation of nitrogen by the algae,

(4) as in the case of nodulated legume and Azotobacter, a slightly alkaline medium is the most favourable for the growth of the algae—no fixation takes place below a pH of approximately 5.7 and (5) like those of Azotobacter and the symbiotic system, the enzyme system of the blue-green algae is specifically inhibited by GO and O2.

As the cyanophycean algae arc photosynthetic organisms capable of synthesising carbohydrate, they are able to flourish in situations such as bare rock faces, open sea, etc., where nitrogen-fixing bacteria cannot thrive due to lack of carbohydrate.

On the other hand, environments into which light cannot penetrate, such as all but superficial layers of soils and water, although suitable for bacteria, would appear unsuitable for the algae.

In combining the nitrogen-fixing and photosynthetic modes of nutrition, blue-green algae resemble root nodule-bacteria symbiotic system. However, in the latter, the seat of photosynthesis (in the green aerial parts) and nitrogen fixation (in the root nodules) are separated in space if we regard the whole system as a composite organism.

In the algae, nitrogen and carbon assimilation may proceed side by side in the same cell, indi­cating the close relation between the two mechanisms. It may very well be that there are common intermediate compounds.

(We know that in higher green plants, the same intermediate compound of photosynthesis, the phosphoglycerates may be tapped and a part may be preferentially elaborated to proteins in young growing cells.)

Recent evidences in studies on cell-free extracts on Anabaena cylindrica support the conclusion that N2 fixation is localised together with photosynthesis on the chromato- plasmic lamellae.

The blue-green algae as well as the photosynthetic bacteria must have undergone their greatest development at a stage when the shortage of combined nitrogen on the earth was really becoming acute.

Higher plants and animals appear to have evolved from non-nitrogen-fixing stocks but the frequency with which symbioses have arisen between higher photosynthetic plants and nitrogen-fixing organism, photosynthetic and heterotrophic, certainly to a large extent, testifies to the decided biological advan­tage of the combination (both nitrogen-fixing and photosynthetic at the same time) found in the blue-green algae and photosynthetic bacteria.

The nitrogen fixed by blue-green algae, like all others, eventually passes into general circulation and is assimilated by plants, unable to fix it for themselves.

Two possible ways, which are true also for other nitrogen-fixing flora, in which (i) the nitrot gen compounds formed in the cell released are excreted into the surrounding medium during the life of the algae and (ii) they may be released by death and subsequent decomposition of the algae.

While blue-green algae are of general occurrence in soils of agricultural value in temperate regions, it may be doubted whether they are all nitrogen-fixing, contribu­ting to the fertility of soil, or that they occur in appreciable number.

In certain tropical soils, blue-green algae appear to play an important part in the maintenance of the fertility of the soil. In India, rice may be grown on the same land for 1 many years without addition of fertilisers to the soil.

During the season of growth of rice (the monsoon), the rice fields are flooded by incessant rain and luxuriant growth of blue- green algae, including many species that have been shown to be nitrogen-fixing, occurs in the waterlogged fields.

The principal nitrogen-fixing blue-green algae in Indian rice fields are Aulosira fertilissima (Microchaetaceae). The chroococcalean, non-heterocystous algae, Chlorogloea fritschii which occur abundantly in paddy fields, where rice is culti­vated, also fix nitrogen.

The nitrogen fixed by the algae becomes available to the rice plants when the algae decay and there is good evidence that the algae are the main agents responsible for maintaining the level of fertility of rice fields (it is true, that the level, at least in India is not very high).

It has also been shown that the growth of the blue-green algae on waterlogged soil substantially increases the content of organic matter and combined nitrogen, either organic or inorganic of the soil and when the rice plants and the blue-green algae are grown together, each seems to do better than if grown separately.

This quasi symbiosis between rice and Aulosira or Anabaena is certainly interesting from physiological point of view. The claim that uninfected rice plants can fix

N2, has not been substantiated. There also appear to be considerable potentialities for increased use of the blue-green algae in the tropics in land reclamation.

As we have seen before, blue-green algae are abundant in fresh water and copious growth of planktonic forms of microscopic plants, including blue-green algae in fresh water produces a phenomenon which is known as ‘water bloom’. The growth of the algae is sometimes so luxuriant that fishing there becomes hazardous.

Some members of the blue-green algae produce nodules on roots or stems. Root nodules are found in several gymnosperms and stem nodules are formed on the angiosperm Gunnera.

Probably the most remarkable symbiotic association between a blue- green alga and a green plant is the Azolla—Anabaena azollae association. A. azollae fixes N2 inside the leaf cavities of the water fern Azolla.

When the dead remains of Azolla decay, the soil accumulates the nitrogen which is utilized by crop plants. In Viet Nam, China and Indonesia Azolla is grown extensively on standing water in rice fields. Rice plants gain substantial quantities of nitrogen in this way.

The technology is simple and its judicial use may enhance crop yield considerably. Use of Azolla is now recom­mended for adoption in this country on a large scale.

It may be interesting to point out here that all the species of blue-green algae (e.g., Anabaena, Nostoc, Cylindrospermum, Aulosira, etc.) known definitely to be nitrogen-fixing, possess the enigmatic structure called heterocysts (except perhaps Chlorogloea fritschi and perhaps also one or two Nostoc species) whereas those as yet known to be quite unable to assimilate free nitrogen are for the most part non-heterocystous.

We do not as yet know the significance of the presence of heterocysts in blue-green algae. It is possible that heterocysts may in some way or other is functional in the mechanism of nitrogen fixa­tion in these algae.

The thick wall of heterocysts provide the anaerobic environment required for nitrogenase the heterocyst and the adjacent cells are believed to be func­tional in nitrogen fixation.

However, a few non-heterocystous unicellular forms, e.g., Gloeocapsa and Aphanothece also fix nitrogen thus, the presence of heterocysts is not essential for nitrogen fixation by the blue-green algae.

Although higher plants, such as rice and barley uninfected by bacteria, aphids and man himself have been reported from time to time to be capable of assimilating ele­mentary nitrogen, critical experiments have always failed to substantiate these asser­tions.

Thus it seems highly probable, inspite of incompleteness of our knowledge, that the property of nitrogen fixation is confined to microorganisms only. And again among microorganisms, a capacity for nitrogen fixation apparentiy occurs quite independently
of other nutritional characteristics.

Thus blue-green algae are photosynthetic and Desulphovibrio is frequently a chemautotroph, while Azotobacter requires a source of organic carbon, thus, given a suitable source of energy , these and all other nitrogen- fixing organisms are able to synthesise all their complex organic requirements from inorganic substances.

Nitrogen fixation has never been demonstrated in holozoic orga­nisms, as for example in parasites, saprophytes or insectivorous plants and in fact there appears to be definite incompatibility between morphological specialisation and nitro­gen fixation.

There is now more or less definite evidence that the mechanism of nitrogen fixa­tion is basically the same in different organisms, be it Azotobacter or nodulated legume, blue-green algae or other nitrogen-fixing organisms.

In heterotrophs the electrons required for reduction of nitrogen are provided by organic compounds. In Clostridium pasteurianum where N2-fixation has been demonstrated in cell-free systems, pyruvate is oxidised to acetyl phosphate releasing 2H+ and 2e.

The acetyl phosphate in presence of ADP is then cleaved to acetate with the concomitant formation of ATP, the energy being provided by the hydrolysis of the carboxyl phosphate bond (a high energy bond) of acetyl phosphate.

This ATP then is used by nitrogenase to split the triple bonds con­necting the two N atoms in the N2 molecule. The electrons of pyruvate are accepted by ferredoxin (in some systems flavodoxin is used), the redox potential of which is very close to that of the hydrogen electrode the enzyme hydrogenase may also provide electrons from molecular hydrogen.

Reduced ferredoxin then transfers the electrons to the enzyme which finally reduces N2 in several steps to NH3. The postulated inter­mediates include HN=NH (diimide), hydrazine (H2N-NH2) and NH2OH (hydroxyl amine). 16 N2 supplied to nitrogen-fixing cells or tissues is recovered inNH3 as the first stable intermediate and in amino acids, particularly glutamic acid. (Fig. 690a).

Although glutamic acid is the key intermediate in amino acid metabolism glutamic dehydrogenase which catalyses the reaction NH3 + α—oxoglutarate + NADPH + H + ↔ Glutamate + NADP + + H2O does not play a major role here, since it acts only in presence of relatively high concentrations of ammonia.

The enzyme which is involved here is glutamine synthetase which converts glutamic acid to glutamine glutamine then donates the —NH2 group to α-oxoglutarate forming two molecules of glutamate as follows:

The amino group of glutamate is then transferred to various keto acids, by trans aminases, forming a variety of amino acids which are then utilized for synthesis of proteins or other cellular metabolites.

The nif gene which codes for nitrogenase is plasmid (extra chromosomal DNA) carried in several nitrogen-fixing microorganisms. Attempts are now being made in many laboratories round the world to introduce the nif gene into eukaryotic cells.

If this is achieved, this will be a major break-through since it will no longer be necessary to apply nitrogenous fertilizers to such plants. However, there are many obtstacles which have to be crossed before them.

One more interesting point before we conclude: Isn’t it rather misleading to speak of ‘fixing nitrogen’? Elementary nitrogen is chemically inert and undergoes appreciable reaction with other substances only at high temperature or pressure, which is quite outside the range to be found in the living cells.

We have discussed before that nitrogen fixation is an endothermal or endergonic process, in which energy is absorbed and this energy must be supplied from outside. Thus, it is evident that the fixation of nitrogen requires an investment of energy.

Before nitrogen can be fixed, it must be ‘activated’, which means that each molecular nitrogen must be split into two atoms of free nitrogen. This step requires at least 160,000 cal for each mole of nitrogen (equivalent to 28 grs).

The actual fixation primary step, in which two atoms of nitrogen combine with 3 molecules of hydrogen, to form two molecules of ammonia is this, however, releases 13,000 calories. Thus the fixation of a molecule of nitrogen requires a net energy supply or input of at least 147,000 calories (equivalent to about 18 ATP molecules).

For photosynthetic bacteria and blue-green algae, this energy requirement can be met directly from sunlight which is a photo phosphorylation for Rhizobium, from host leguminous plant, and for free-living Azotobacter and Clostridium, from cellular respiration.

Demonstration of the Presence of Root Nodule Bacteria (Rhizobia) In Legumes:

Roots of gram, bean, pea, etc. Wash the dug-up roots carefully and note that the roots are studded with small pink nodules. Cut through a large root nodule with a blade or razor and put the cut surface on the centre of a perfectly clean slide and press.

Spread the pink juice which comes out and allow the smear to dry and then heat it gently over a Bunsen flame or pass it thrice through the flame. The slide should feel hot when placed on the back of the hand but not too hot to bear.

The heating fixes the bacteria to the slide, but it is imperative not to overheat. When the slide is cool, stain the film with methylene blue for 2-3 min wash the stain off gently with water and blot the slide dry by careful pressure with a filter paper.

When quite dry, mount in Canada balsam. Bacteria, like minute rods, are distributed singly or in groups—very variable in length, sometimes mere dots some branched Y-shaped forms are also present (see Fig. 688 c, D & E).

Process # II. Ammonification:

Nitrogen compounds in the plant and animal residues are decomposed in the soil to form NH3 so long as the ratio of carbon to nitrogen in the organic matter does not gready exceed 10: 1.

Proteins and other organic nitrogenous compounds are broken down in soil by a variety of soil micro-organisms, the end product being NH3. Whether NH3 appears or not, depends on the rate of growth of organisms in the soil themselves requiring NH3 for their own anabolic operations.

If ample utilisable carbohydrates are present, NH3 will not appear, as it is entirely used for building up fresh bacterial or fungal cells.

The ammonification of a molecule of glycine in presence of oxygen to ammonia, carbon dioxide and water releases 176,000 calories (about 22 ATP).

Many soil micro-organisms are involved in NH3 formation, the mechanism of which varies from one organism to other. Proteins are broken down to peptides and then amino acids and the amino acids may yield NH3, by the action of a host of enzyme systems, e.g., tyrosinase.

Process # III. Nitrification:

The metabolic process whereby NH3 is finally converted to N03 is called nitrification. Nitrosomonas and Nitrobacter are the two most important genera of strictly aerobic chemo- synthetic autotrophs, which respectively obtain their energy by oxidation of NH3 to nitrite (NO2) and nitrite to nitrate (NO3) (see later).

These nitrifiers prefer a slightly alkaline environment — Nitrosomonas is unable to grow in organic media growth of Nitrobacter is stimulated in certain cases by a small amount of peptone.

Nitrifying bacteria generally grow on the surface of the soil where NH3 or NH4 ions may be held (adsorbed). It has also been found that the adsorbed NH4+ ions are preferentially nitrified by micro-organisms on the surface of the soils.

KClO3 in minute traces inhibits the growth of Nitrobacter but not Nitrosomonas. Chloromycetin, an antibiotic (specific against typhoid and typhus pathogens) contain­ing a nitro group is especially active against soil organisms like Nitrobacter, oxidising NO2 to NO3.

Process # IV. Denitrification:

In contrast to the limited number of organisms capable of oxidising NH3 and NO2, several species accomplish the reverse process, namely, the reduction of nitrate and nitrite to N2O and molecular N2.

While NH3 may be retained in the organism or in the medium, gaseous products such as N2O and N2 pass readily into the atmosphere with the result that the overall nitrogen content of the soil is decreased.

This biological reduction of NO3 and NO2 is often termed denitrification. Examples of organisms known to reduce NO3, include Pseudomonas fluorescence, Pseudomonas denitrificans, Pseudomonas stutzeri, Bacillus sp. Thiobacillus denitrificans, etc.

Denitrification is generally, but not always, encouraged by poor drainage and lack of aeration due to water logging as well as by a plentiful supply of organic matter. The presence of O2 tends to suppress the reduction of NO3.

Under anaerobic conditions, the chemosynthetic autotroph, Thiobacillus denitrificans can obtain energy only by the oxidation of elementary sulphur or other sulphur com­pounds at the expense of reducing NO3, i.e., using NO3 in place of free O2.

When denitrification takes place in absence of oxygen, and with the help of nitrate ion in presence of sulphur and chalk, the equation can be written thus:

A scheme for the probable pathways of denitrification by micro-organisms is given below though direct evidences about some of the intermediates shown in the scheme are lacking. The liberation of N2 may also be achieved without the participation of enzymes, since an amino compound coming in contact with NO2 – may liberate N2.

The gaseous products such as N2O, N2 or NO2 formed by denitrifying organisms are responsible for much troublesome odour and consequent loss in certain indus­tries. NO3 – and NO2 – are commonly used for curing and preserving meat products and their decomposition is often due to unwelcome activities of denitrifying bacteria.

It is possible that denitrifies can convert appreciable quantities of fertilisers, such as (NH4)2SO4 and KNO2, added to the soil, into gaseous N2 or oxides of N2 which escape into atmosphere, thereby significantly decreasing nitrogen available for plant growth.

Ecological Aspects of N2-Fixing Organisms and Nitrogen Economy of Nature:

In arctic and subarctic conditions, the bacterial activity would necessarily be slow and consequently the soil has relatively small supply of nitrates. The plants in these areas must either be adapted to a low nitrogen intake or have some means of utilising atmospheric nitrogen.

Species of Alnus which are prominent in such areas are definitely known to form root nodules infected by actinomycetes and this symbiotic relationship may help Alnus to fix N2 and thrive in those freezing cold areas. Legumes seem very poor­ly represented.

In extreme arctic conditions lack of carbohydrate due to very short growing season appears more critical than nitrogen shortage and as a result legumes are completely absent from those areas.

In very sandy leached soils, however, a character­istic flora develops in which legumes and species of Casuarina are prominent. It is interest­ing to note that spp. of Casuarina are also known to form nitrogen-fixing nodules.

Blue-green algae and the photosynthetic bacteria are commonly the first plants to colonise bared areas of rocks and soils. The best known example of this is seen’ in the recolonisation of Krakatoa group of islands in the Pacific where a volcanic explosion in 1883 denuded the island of all visible plant life.

It is probable of course that photosynthetic and the thermophilic bacteria, Desulphovibrio, must have survived this drastic treatment for, at least in the case of some varieties of Desulphovibrio, it is known to thrive in a temp, of 80°C.!

Blue-green algae and most probably photosynthetic bac­teria were the first plants to appear in quantity on the pumice and volcanic ash a few years after the eruption—photosynthetic bacteria which use inorganic sulphur com­pounds in photosynthesis can be imagined as relishing such an environment more than any other organism, except perhaps anaerobic Desulphovibrio.

In part, the success of these organisms as colonisers is, no doubt, due to a marked ability to withstand adverse conditions such as desiccation, high temperature and high concentration of salts, particularly SO4 – , but a capacity for nitrogen fixation cannot be discounted as a possible factor determining their presence on such unpromising environment.

Mem­bers of the blue-green algae are equally important pioneers under less impressive cir­cumstances:

(i) A community of blue-green algae forms the initial stage in plant succes­sion on certain eroded soil and

(ii) They play a conspicuous part in colonisation and stabilisation of salt marshes. It is perhaps significant that majority of species occurring in the list of algae as pioneer plant communities belong to the family Nostocaceae.

Besides free-living forms there are many blue-green algae that occur in more or less intimate association with other plants, for example, with liverworts, with water-fern Azolla, with Cycads and with the flowering plant Gunnera (Fam. Haloragidaceae).

Azolla contains an Anabaena and is able to grow in a medium free of combined nitrogen, NO3 or NH4 salts. A species of Nostoc, isolated from Cycads, has been found to be scarcely capable of autotrophic nutrition and needs to be supplied with carbohydrate for growth and nitrogen fixation.

The host Cycad can grow quite well without the algal partner and it is not clear how much benefit is derived from the N2 fixed by the algae or how successful the symbiotic association is. Blue-green algae form symbiotic associations with fungi the algal partner in many lichens is often a blue-green alga.

In the lichen Collema, the alga Nostoc has definitely been shown to be nitrogen-fixing. Nitrogen fixation by the algal partner may well be the most important factor in enabling these composite orga­nisms to live on bare rocks and other similar inhospitable situations.

It has been found that some lichens contain the nitrogen-fixing bacteria Azotobacter. It seems, therefore, that many lichens are triple alliance of organisms and are capable of assimilating elementary N2 even if the blue-green algal partner fails in its duty and even if the algal partner is not a blue-green alga at all!

Agricultural land is continually losing nitrogen in various ways. The largest pro­portion is undoubtedly lost by the removal of crops and by animals grazing on the land. Very little of the combined nitrogen contained in these is returned to the soil due to the modern method of unforeseeing sewage disposal.

As a refuse, it ultimately finds its way to the sea and becomes largely unavailable to man. Comparatively large amounts are also lost by being carried away to the sea as a result of leaching or washing away of soluble nitrogenous compounds from the soil and also by the removal of soil itself by erosion.

Again by the activity of denitrifying bacteria, flourishing in a plentiful supply of organic matter and bad aeration of the soil as a result of water logging, the combined nitrogen is being continually reduced to elementary form, which escapes into atmos­phere.

That there must be a compensatory process for the maintenance of life on earth is evident from the observation that soils from which crops have been taken out are not totally depleted of combined N even if no manures are applied to the soil, since lands left without cropping rapidly gain in nitrogen.

Thus biological nitrogen fixation is undoubtedly of extreme importance for the continued existence of life on earth and it must have played an equally important part in the past. With certain minor exceptions, combined N does not occur at the present time on earth except as a result of the activity of living micro-organisms in the soil.

The natural Chilean nitrate deposits have probably resulted from the bacterial action in past. It seems possible that under conditions which prevailed upon earth when life first appeared, combined nitrogen in the form of NH3 occurred in substantial amounts in the primitive atmosphere of the earth.

Thus the necessity for nitrogen fixation did not arise for hundreds of million years. This must have provided the only source of N2 for the first living organisms. However, a stock of combined nitrogen such as NH3 could not last indefinitely, since oxidation to nitrate and the subsequent reduction of nitrates to elementary N2 would soon reduce the stock to zero.

It has actually been estimated that it would only take about 60,000 years for the total earth’s stock of combined nitrogen to disappear without any compensatory scheme of bringing back the elementary nitrogen into combined form again.

If life first appeared on the earth, say, about 3000 million years ago it is clear that organisms, capable of transforming elementary N2 back into combined state, must have appeared at a very early stage of evolution of life or life would have soon become extinct.

Actually the ancestry of sulphur bacteria which include nitrogen fixers, such as photosynthetic bacteria and Desulphovibrio, has been traced back to nearly 800,000,000 years, which indicate that the gaseous nitrogen-fixing mechanism has been in existence since—at least that time!

How young and arrogant man seems with his ancestry going back perhaps to a meagre 100,000 years.

A scheme representing the cycle of stages of nitrogen metabolism in soil and in air—nitrogen cycle—is shown.