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.
Contents
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 15N, 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 15N with various nitrogen- fixing organisms, it is always found that the heavy 15N appears in the greatest extent in glutamic acid just as it does if 14N 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 15N, 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 15N. 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). 16N2 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:
Material:
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:
5S+6KNO3+2CaCO3
→3K2SO4 +2CaSO4 +2CO2 +3N2+660,000 cal.
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.