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ENVIRONMENTAL MICROBIOLOGY

Terrestrial Environment Neeru Narula and Manjula Vasudeva
Department of Microbiology CCS Haryana Agricultural University Hisar – 125 004 20-Apr-2006 (Revised 06-Mar-2007)

CONTENTS Introduction Rhizosphere Phyllosphere Brief account of microbial interactions Competition Rumen microbiology Biofertilizers Biological N2 fixation Nitrogenase enzyme The nif genes Symbiotic N2 fixation Rhizobium Frankia Non-symbiotic N2 fixation Azotobacter and Azospirillum Mycorrhizae

Keywords
Terrestrial Environment, Rhizosphere, Phyllosphere, Brief account of microbial interactions, Competition, Rumen microbiology, Biofertilizers, Biological N2 fixation, Nitrogenase enzyme, The nif genes, Symbiotic N2 fixation, Rhizobium, Frankia, Non-symbiotic N2 fixation, Azotobacter and Azospirilum, Mycorrhizae.

Introduction Terrestrial environment is the process occurring within the soil and on or near the plants that influence the functioning of the ecosystem. The process of soil development involves complex interactions among the parent material (rock, sand, glacial drift etc.), topography, climate and living organisms. The term soil refers to the outer, loose material of the earth’s surface, a layer distinctly different from the underlying bedrock. Agriculturally, it is the region supporting plant life and from which plants obtain their mechanical support and many of their nutrients. Chemically, the soil contains a multitude of organic substances not present in the underlying strata. For microbiologists, the soil environment is unique in many ways. It contains a vast population of bacteria, actinomycetes, fungi, algae and protozoa. It is one of the most dynamic sites of biological interactions in nature; and it is the region where occur many of the biochemical reactions concerned in the destruction of organic matter, in the weathering of rocks and in the nutrition of agricultural crops. The physical and chemical characteristics of soil determine the nature of the environment in which microorganisms are found. These environmental characteristics in turn affect the composition of the microscopic population both qualitatively and quantitatively. It is from the soil that the water, air and the inorganic and organic nutrients are obtained. The soil serves as a buffer to the drastic changes that occur above the ground. The organisms like algae, lichens or mosses remain dormant on the dry rock and grow when moisture is present. They are phototrophic and produce organic matter which supports the growth of chemoorganotrophic bacteria and fungi. The number of chemoorganotrophs increase directly with the degree of plant cover of the rocks. CO2 produced during respiration by chemoorganotrophs is converted to carbonic acid (CO2 + H2O ------ H2CO3), which is involved in the dissolution of lime stone rocks. Many chemoorganotrophs excrete organic acids which further promote dissolution of rocks into smaller particles. Freezing and thawing also cause cracks in the rocks. In these crevices, raw soil forms and pioneering higher plants can develop. Plant roots penetrate into crevices and increase the fragmentation of the rock and hence develop rhizosphere (soil that surrounds plant roots) microflora. When the plants die, their remains are added to the soil and become nutrients for microbial development. Minerals are further rendered soluble and as water percolates, it carries some of these chemical substances deeper. As weathering proceeds, the soil increases in depth, thus permitting the development of large plants and trees. Soil animals play an important role in keeping the upper layers of the soil mixed and aerated. The movement of materials downwards results in the formation of many layers known as the soil profile which is dependent on climatic and other factors and takes hundreds of years to be formed. The soil is a complex habitat with numerous microenvironments and niches. Microorganisms are present in the soil primarily attached to soil particles. The most important factor influencing microbial activity in surface soil is the availability of water, whereas in deep soil nutrient availability plays a major role. The organic fraction of soil, often termed humus, contains the organic carbon and nitrogen which is needed for microbial development, is the dominant food reservoir. The greatest microbial activity is in the organic-rich surface layers, especially in and around the rhizosphere. The numbers and activity of soil microorganisms depend to a great extent on the balance of nutrients present. In some soils carbon is not the limiting nutrient, but instead the availability of inorganic nutrients such as phosphorous and nitrogen limit microbial productivity. 2

a diverse array of bacteria have been found including anaerobes such as sulphate reducing bacteria. are present in most deep underground soils. primarily bacteria. methanogens and homoacetogens and various aerobes and facultative aerobes. In samples collected aseptically from bore holes drilled down to 300 m. the biogeochemical significance of deep subsurface microorganisms may thus be minimal. which can extend for several hundred meters below the soil surface. Compared to microorganisms in the upper layers of soil. there is evidence that the metabolic activities of these buried microorganisms may over very long periods be responsible for some mineralization of organic compounds and release of products into the ground water (Fig.The deep soil surface. Microorganisms in the deep subsurface presumably have access to nutrients because groundwater flows through their habitats. 1: Profile of mature soil (Biology of Microorganisms) 3 . but activity measurements suggest that metabolic rates of these bacteria are rather low in their natural habitats. 1). Fig. However. A variety of microorganisms. is not a biological wasteland.

Hiltner in 1904 observed the zone of intense microbial activity around the roots and named it as rhizosphere. light and wind velocity which also interact with the leaf surface community in various ways. But as they get established. have antagonistic action against fungal parasites. These nutrients allow the dormant spores to germinate. Leaf surface carries a heterogenous population of microbes. 10 organic acids. 4 . decomposes plant material after leaf fall. It is a zone where microbial activity is usually high. mucilage and other substances together with sloughed-off root cap and other cells and exerts influence on microbial colonization. and under conditions of high humidity. The physiological and biochemical status of the leaf greatly influences the population and composition of the microflora. They may immediately grow utilizing the fresh supply of substrates present on the leaf surface or lie dormant and inactive until the leaf becomes senescent. degrade plant surface waxes and cuticles. hormones and vitamins. This is because roots excrete significant amounts of sugars. The leaf surface medium comprises exudates. The age and position of a leaf on the plant is an important factor for microbial colonization of its surface. some of them are known to fix atmospheric nitrogen for the benefit of higher plants. reaching the maximum population only on leaves which start yellowing at maturity.Rhizosphere Rhizosphere is the region immediately outside the root. produce plant hormones. they receive a potential supply of surface nutrients. relative humidity. 10 sugars. The influence of the root on soil microorganisms starts immediately after seed germination which increases as the plant grows and reaches a maximum when plants have reached the peak of their vegetative growth. For instance. Leaves at the seedling stage of plants usually harbour the least number of microbes which increases as the plants age. as in wet forests in tropical and temperate zones. which grow. activate plants to produce phytoalexins. act as a source of allerginic air-borne spores and influence the growth behaviour and root exudation of plants. reproduce and multiply on leaves in dynamic equilibrium with the existing micro. The earliest colonizers on newly formed leaves have to face almost no competition as they are devoid of any microbes. The leaf surface microbes are important in several ways. amino acids. chemical compounds resulting from biological activity of various microbes including nitrogen fixers and components resulting from atmospheric pollution. Phyllosphere Phyllosphere is the surface of the plant leaf. Many of the bacteria on leaves fix nitrogen and nitrogen fixation presumably aids these organisms in growing with the predominantly carbohydrate nutrients provided by leaves. Roots initially have little or nomicrobial colonization but as the plants grow in the soil. The structure and the chemistry of the leaf surface influences the occurrence of the plant surface colonizers apart from physical factors like temperature. have toxic effects on cattle. The bacterial count is almost always higher in the rhizosphere than it is in region of the soil devoid of roots. the microbial flora of leaves may be quite high. The rhizosphere microorganisms influence plant growth by controlling the availability and uptake of nutrients. and in fact. they face a relatively hostile environment because of widely fluctuating temperatures and the incidence of UV radiations.and macro-environment. which promote such an extensive growth of bacteria and fungi that these organisms often form microcolonies on the root surface. often many times higher. the root exudates composed of a mixture of nearly 18 amino acids.

which may act as signals which cause the partner to modify itself or which may themselves cause modifications. in which one species is suppressed while the second is not affected. or other common requirements f) Amensalism. space. the shedding of leaves. and the structure and physiology of both host and microorganism are adapted for the aerobic fixation of nitrogen. often the result of toxin production g) Parasitism and Predation. TMV and cucumber mosaic virus together cause a more severe disease than either of them alone. interactions with growing plants extend no further than the colonization of the surfaces of stems. They provide nutrients indirectly from plant exudates. the plant benefits is usually positively related to the amount of root that becomes invaded and fairly high levels of infection being most beneficial. a plant and microorganism. modifications to the host enable the partners to form the symbiosis. Associations with plants can range from those that are extremely detrimental to the plant. Odum has proposed the following relations: a) Neutralism in which the two microorganisms behave entirely independently b) Symbiosis. a condition in which there is a suppression of one organism as the two species struggle for limiting quantities of nutrients. to beneficial ones such as those formed with mycorrhizal fungi or nitrogen fixing bacteria. For symbionts. Symbiosis is the result of interaction of the two partners and both partners are modified in some way to achieve this. This must arise by the interchange of molecules between the partners. pollen. the direct attack of one organism upon another. Two members are required for the association. A number of possible interactions may occur between two species. In the symbiosis.Brief account of Microbial Interactions Plants are exposed to very large numbers of microorganisms that are present in the soil and are deposited on leaves and stems. leaves and roots because these are regions where exudates are available. Plants are the prime source of nutrients for microorganisms because they are the main source of organic matter in the environment. an association of mutual benefit to the two species but without the cooperation being obligatory for their existence or for their performance of some reaction d) Commensalisms. etc. the two symbionts relying upon one another and both benefiting the relationship c) Protocooperation. For most microorganisms. h) Synergism. in field situations is the possible synergistic effect in the plant between inducing virus and other non related viruses which could be brought to those plants from outside sources e. and also from dead plant matter. such as those with virulent pathogens.g. O2. nutrients are provided directly to microorganisms that form close associations with plants. Symbionts have developed methods that permit them to enter the host and obtain direct access to nutrients. In some cases. through interactions which do not appear to influence plant growth. It would thus seem unwise to introduce TMV into field grown tomatoes where the aphid-borne cucumber mosaic virus might be present in surrounding areas and eventually be transmitted to the tomato plants. in which only one species derives benefit while the other is unaffected e) Competition. such as Rhizobium and mycorrhizal fungi. The classical example of such a symbiosis is between leguminous plants and bacteria of the genus 5 .

Rhizobia are able to nodulate only a small proportion of the very large number of species in the family Leguminosae and one non legume. coli. the commensal. it can survive without being provided some factor or factors of host origin e. borrowed or reciprocal) defines the relationship in which some reciprocal benefit accrues to both partners.) which in turn are not usually nodulated by other rhizobia. Pisum and Lathyrus. Symbiosis is the living together in close physical association of two or more different organisms. These relationships can be very complex. the common. coli lives in the human clone. coli but E. Under nitrogen-limiting conditions legumes nodulated with active N2-fixing strains of Rhizobium benefit from the interaction and the growth of the legume plants stimulates the growth of rhizobia and other microorganisms in the soil. Parasponia. In this case. The actinomycetes Frankia nodulates a range of dicotyledonous plants. as does the cyanobacterium Nostoc on the cycad Macrozamia. In each case there is a degree of specificity. having species such as Trifolium. However. Rhozibia grow readily in culture media containing a carbon source such as mannitol or glucose. benefits while the other (sometimes called the host) is neither harmed or helped.g. Symbiosis involves intimate interactions based on mutual benefit. Lotus. the most important plant group concerned in symbiotic N2 fixation. The seat of the symbiosis is within the nodules that appear on the plant roots. ammonium or nitrate to supply the required nitrogen and several inorganic salts. When O2 is used up by the facultative anaerobic E. or not at all and such strains are either of little benefit to plant growth or are detrimental because they are utilizing the plant’s energy without providing reciprocal benefit. E. obligate anerobes such as Bacteroids are able to grow in the colon. The anaerobes benefit from their association with the host and E. There are three types of symbiotic relationships: Commensalism: (Latin com. Symbioses involving Rhizobium are only one example of the type of interaction between plants and N2 fixing microorganisms than can occur. trifolii nodulates clovers (Trifolium spp. strains of Rhizobium exist that fix N2 inefficiently. nonpathogenic strain of E.g. The spatial proximity of the two partners permits the commensal to feed on substances captured or ingested by the host. Vigna. which implies that there are mutual recognition systems. Vicia. The commensal is not directly dependent on the host metabolically and causes it no particular harm. The commensal also obtains shelter by living either on or in the host. Medicago. Mutualism: (Latin mutus. Dalea.Rhizobium. Legumes. the fixation reaction is thus the result of a true symbiosis as neither symbiont can carry out the process alone. Within this range of host plants. the commensal E. Often both the host and the commensal “eat at the same table”. together and mensa table) is a relationship in which one symbiont. specificity in the ability of particular plant and Rhizobium species to form effective symbioses is observed e. In this relationship the mutualist and the host are metabolically dependent on each other. When the commensal is separated from its host experimentally. but also grows quite well outside the host and this is a typical commensal. The range of plants with which cyanobacteria can form a symbiosis is 6 . R. coli contributes to the welfare of other symbionts. Melilotus. coli flourishes in the colon. Phaseolus. are dicotyledonous plants of the family Lesuminosae. Of particular importance to the development of the symbiotic relationship is the presence of a large population of rhizobia. coli derives no obvious benefit from the anaerobes. which is a good definition of mutualism. Crotalaria. None of the bacteria in culture solution utilize N2. The interaction between rhizobia and leguminous plants has been studied in great detail for many years.

It encompasses diatoms. citrus and almost any other plant species that can form mycorrhizal associations of the VA type. and this symbiosis is of interest as it is the only known cyanobacterial association with an angiosperm. ferns. but in one species of cycad Macrozamia communis. there are morphological adaptations of the host and also physiological adaptations to cater for the special demands of N2 fixation. They are wide spread and show little sign of specificity. As with other symbioses. Nostoc is contained in glands at the base of leaves. ranging from the lichens where the cyanobacterial partner photosynthesizes and provides the host with both carbon and nitrogen. mosses. Mycorrhizal fungi can be so important that some species of host plant are almost dependent upon them to be able to grow in soils low in PO4. many microorganisms occur in close proximity and they interact in a unique way that is in marked contrast to the behaviour of pure cultures studied by the microbiologists in the laboratory. Mycorrhizae are thus something of an enigma. ascomycetes and basidiomycetes with algae. thus resulting in a greater proportion of heterocysts in symbiotic systems. In the symbiosis where the products of N2 fixation are excreted for use by the host. Azolla and most cycad systems. Mycorrhizae are symbiotic because the plant provides the fungus with organic nutrients and in return fungus facilitates the uptake of mineral nutrients and in particular phosphate. In Azolla the cyanobacteria (Anabaena) exist in pockets within the leaf. By far the highest proportion of lichen species are associations of fungi with green algae. fungi. to the situation in cycads and in Gunnera where the endophyte is dependent wholly upon the host for carbon. The three beneficial relationships. and in Gunnera. Lichens are symbioses of fungi. protocooperation and commensalism are found to operate among the soil inhibitor. N2 is fixed by the heterocysts and diffuses into the vegetative cells. the cyanobacteria have been found inside the cells. cycads and angiosperms. These symbioses have evolved to enable the fungus to invade roots and routinely colonize from 10 to 90% of the root length with no obvious harmful effect on the plant. Studies on the differentiation of heterocysts in the filaments has shown that this is determined by the nitrogen status. Both are examples of containment that has resulted from the development of a complex interaction. but at the same time they exert detrimental influences so that both beneficial and harmful effects are evident. The cyanobacterial symbioses have a wide range. In the lichens. beans. Microorganisms in time develop certain relations that are 7 . citrus and cassava are particularly good examples. but about 25 genera have cyanobacteria as the ‘algal’ symbiont. and from a consideration of some of the associations it can be seen that there is a great diversity of interactions. grasses. the cyanobacteria exist outside the host’s cells. symbiosis. The proportion of heterocysts to vegetative cells is much higher in the symbiotic form than in the free-living cyanobacteria. N2 has to be given up to the host if the symbiosis is to be mutualistic and the high proportion of heterocysts in all these associations show that it is. In soil.very wide indeed. Mycorrhizae represent particularly interesting plant-microbe interactions because they are so wide spread and grown on lettuce can infect maize. Members of the microflora rely upon one another for certain growth substances. liverworts. through the association with Azolla where photosynthesis takes place but carbon is supplemented by the host. The cyanobacterial symbiont is Nostoc. yet they involve complex interactions. This is similar to the symbiosis with Gunnera. combined nitrogenlevels along the filament become depleted sooner than in free-fixing forms. and possibly most plants are partially dependent upon them for their PO4 nutrition.

multiply and assume dominance. cellulolytic fungi produce from cellulose a number of organic acids that serve as carbon sources for noncellulolytic bacteria and fungi. or by the autotrophic formation of nitric and sulphuric acid which affects the proliferation of acid-sensitive microorganisms. In mixed cultures of several microorganisms in laboratory media or in partially sterilized soil. but commensal relationships are quite frequent. These growth factors are synthesized by certain microorganisms. (a) the rivalry for limiting nutrients. Microbiological competition for available carbon. A second beneficial association arises from the need of many microorganisms for accessory growth substances. Competition Microbial Competition The categories of deleterious interactions are summarized by the terms competition. 8 . The disappearance itself is probably the result of competitive effects since specific toxic substances active against the alien bacteria are difficult to demonstrate. is probably one of the more important interactions between organisms. Aerobes may permit the growth of obligate anaerobes by consuming the O2 in the environment. its growth rate. (b) the release by one species of products toxic to its neighbours and (c) the direct feeding of one organism upon a second. and its nutritional complexity. The habitat is foreign and the invader fails to find a niche. As a first approximation. parasitism and predation. the attack on nematodes by predacious fungi. e. particularly those concerned with N2 fixation. Alteration of the environment to the detriment of certain microbial species may occur through the synthesis of metabolic products that are bacterostatic or bactericidal by the utilization of oxygen which leads to the suppression of obligate aerobes. In competition. Because the supply of nutrients in soil is perennially inadequate. when large populations of alien bacteria are added to soil. the ability of an organism to compete is probably governed by its capacity to utilize the carbonaceous substrates found in soil. the digestion of fungal hyphae by bacteria and the lysis of bacteria and actinomycetes by bacteriophages. minerals or oxygen is quite common. In addition to these instances of commensalism and protocooperation. several well documented example of true symbiosis are in evidence. as they can develop at the expense of growth factors obtained from the environment. some species are suppressed while others survive. competition for carbon. space or oxygen.beneficial and others that are detrimental. but the decomposition results in the formation of products utilized by the second. Predatory and parasitic activities likewise are not rare. and their excretion permits the proliferation of nutritionally complex soil inhabitants. however. one of which can attack a substrate not available to the second organism. It is likely that the role of an element in modifying the biological equilibrium is determined by the demand of the microflora and the supply in the soil. A simple nutrition could be advantageous. amensalism. One of the more important beneficial associations is that involving two species. but the presence in soil of growth factors suggests the effective competitors need not be nutritionally independent. This type of commensalism is not infrequent in nature and it is the way many polysaccharides are transformed to nutrients supporting non-specialized microorganisms. The microbial decomposition of biologically produced inhibitors that prevent the proliferation of sensitive species is another instance of a beneficial relationship. Predation and parasitism are observed in the feeding upon bacteria by protozoa and myxobacteria. i. Sometimes the benefit is mutual. the invaders do not establish and soon die out. Therefore. The usual cause of this phenomenon is the competition for nutrients. certain microorganisms dominate through their capacity to make most effective use of the limiting factors in the environment.e.g.

Essentially all types of predators or parasites are present in the soil ecosystems. the daily increases in bacteria and protozoa seem to be inversely related. The virulence of individual fungi varies greatly even in a single species. thereby reducing iron availability to competing organisms. Most commonly studied siderophore-synthesizing microbes from the view of controlling plant pathogens are members of the fluorescent pseudomonad group. Myxobacteria and myxomycetes also affect the true bacteria by feeding directly upon them. Bacteria. protozoa. Parasitism and Predation Predators and parasites. The growth-controlling impact of the former compounds in soil has been reasonably well accepted. In well-mannered fields. 9 . because the substances can be quantified easily in soil samples and their interactions with soil microbial populations can be shown. or extra cellular enzymes that lyse other bacteria. These substances selectively complex ferric ion with a high affinity. as is commonly described for protozoa. organisms that feed upon living biomass. Siderophores are extra cellular.Amensalism It is the suppression of the growth of one organism by the products of growth of a second organism. they can be classified as antibiotics. The feeding activity of predators and infectivity of parasites maintains a younger. capturing and consuming nematodes or amoebae. Another group of biologically synthesized compounds that appear to be useful in reducing plant disease through antagonism of pathogens are siderophores. bacteriophages. fungi or algae may be produced. Protozoa. Certain fungi are predacious. bolellovibrios. Two major types of biological inhibitors or toxins are produced by soil microbes: those effective at high concentrations (organic acids. The role of antibiotics within the soil ecosystem is more problematic. but when they result in suppression of microbial growth at low concentrations. a change in either group will bring about a qualitative and quantitative change in the other. Because the pH may reach values as low as 2. The presence of a nutrient supply in the form of bacteria is essential for the development of soil protozoa and large numbers of bacteria must be ingested for one protozoan cell division. commonly reduces soil pH through the oxidation of sulphide to sulfate. as well as nematodes are all active in soil ecosystems. This may result from a situation as simple as the alteration of the soil pH or the production of the growth-inhibiting or lethal biological product. play a key role in the soil ecosystem. more active. probably reducing the abundance of edible cells and serving as a biological antagonist in maintaining the equilibrium. and the parasitized species is thereby often eliminated. Parasites and predators maintain the soil bacterial and fungal populations in an active state and enhance nutrient movement between soil reservoirs through consumption of microbial biomass. Fungi are capable of parasitizing one another. chelators) and those that are effective at low concentrations (antibiotics). therefore are undoubtedly a key factor in limiting the size of the bacterial population. These substances appear to be active at higher concentrations than is characteristic of antibiotics. soil microbial population. lowmolecular weight (500 to 1000 dalltons) iron-transporting compounds synthesized by a variety of microorganisms growing under low iron conditions. These organisms may ingest their nutrients by consuming intact cells (holozoic feeding). the growth of any pH sensitive microbes is inhibited. The parasitism may entail a penetration into the host’s mycelium or a coiling around the host’s hyphae. Thiobacillus spp. and the study of the nematode-trapping fungi may prove of practical value in the control of plant diseases caused by nematodes. In the predator prey relationship between protozoa and bacteria. one group increases as the other decreases. A key consideration in evaluating predator or parasite behaviour in any ecosystem parasitic relates to the observation that both the host and parasites or prey and predators coexist in the same ecosystem. which prey on other bacteria.

The rumen temperature is about 39-40°C in the cow due to the (exothermic) microbial fermentation. Food remains in the rumen for about 9-12 hrs. The acidity of the fermentation products is counteracted by the buffering action of the ruminant’s saliva – which is produced in copious amounts and contains sodium bicarbonate and sodium hydrogen phosphate. The host animal absorbs the fatty acids from the rumen and from the omasum and abomasums and eliminates the gases by erutation (the ruminant uses fatty acids rather than glucose as primary sources of energy and carbon). propionic and butyric and the gases CO2 and methane. the rumen contents contain approximately 1010 cells bacteria ml-1 rumen fluid. The number and type of microorganisms depend upon the nature of animal’s diet and on the period of time since the last intake of food. buffalo. Here chemical digestive processes begin that continue in the small and large intestine. the rumen and reticulum are not fully developed and ingested food passes from the oesophagus via the oesophageal groove to the omasum and abomasum – thus by passing the rumen). such as gases (Fig. but this time the material passes to the omasum. primarily acetic. vitamins and assimilable carbon and energy yielding substrates. During this period. Many microbial cells formed in the rumen are digested in the gastrointestinal tract and serve as a major source of proteins and vitamins for the animal. Food enters the rumen mixed with saliva containing bicarbonate and is churned in a rotary motion during which the microbial fermentation occurs. Rumen is a special organ within which the digestion of cellulose and other plant polysaccharides occurs through the activity of special microbial populations. Now finely divided solids. A ruminant is thus nutritionally superior to a non-ruminant when subsisting on foods that are deficient in protein. 6 litres in a sheep) and its position in the alimentary tract as the organ where ingested food goes first. Several different bacteria hydrolyze 10 . goat. well mixed with saliva. Rumen bacteria Biochemical reactions taking place in rumen are complex and hence involve a large number of microorganisms where anaerobic bacteria dominate. camel etc. small honey comb like. cellulolytic bacteria and protozoa hydrolyze cellulose to the disaccharide cellobiose and to free glucose units. Omasum and abomasums (the stomach). 2a & b). Since many of the microorganisms of the rumen are able to grow on urea as a sole nitrogen source. and the wild animals such as deer. The bulk of this protein ends up in the animal itself. Released glucose then undergoes bacterial fermentation with the production of volatile fatty acids (VFAs). Their digestive system includes four compartments. an organ more like a true (acidic) stomach. finally ending in the abomasum. This peristallic action grinds the cellulose into a fine suspension. Mammals lack the enzymes needed to digest cellulose.5. (In the suckling animal. break down plant material ingested by the host animal and provide the animal with protein. which assists in microbial attachment. pH varies with diet but generally it is between 6-6. The rumen contents are anaerobic. giraffe are the ruminants. which are regurgitated into the mouth where they are chewed again.Rumen Microbiology Ruminants are the herbivorous mammals. the large pouch. The rumen has a large size (100-150 litres in a cow. so the rumen containing a large number of microorganisms (mainly bacteria and protozoa) which play an essential role in ruminant nutrition. The food mass then passes gradually into the reticulum where it is formed into small clumps called cuds. it is often supplied in cattle feed in order to promote microbial protein synthesis. Domestic animals such as cows. sheep. The reduction potential in rumen is –30 mV. are swallowed again.

then starch digesting bacteria Ruminobacter amylophilus and Succinomonas amylolytica develop. (a) (b) Fig. succinate is converted to propionate and CO2 and lactate is fermented to acetic and other acids by Selenomonas and Megaphaera. which is high in pectin. then pectin digesting bacterium Lachnospira multiparus is in the rumen flora. In the rumen 65% CO2 and 35% CH4 are present and which leave the rumen by belching. hemicellulose and pectins. H2 quickly reduces CO2 to CH4 by methanogens. If a ruminant is gradually switched from cellulose to a diet of high in starch (grains). If an animal is fed legume hay. Rumen fungi also degrade plant polysaccharides as well as partially degrades lignin.cellulose to sugars and ferment sugars to VFAs. Fibrobacter succinogens and Ruminococcus albus are cellulolytic anaerobes. rumen also has protozoal fauna (about 106/ml) which are obligate anaerobes as well as anaerobic fungi that ferment cellulose to VFAs. (b) Biochemical reactions in the rumen. In addition to prokaryotes. In fermentation process. A number of rumen bacteria produce ethanol which is fermented to acetate + H2. (Biology of Microorganisms) 11 .2: (a) Schematic diagram of the rumen and gastrointestinal system of a cow.

different P solubilizing bacteria and mycorrhizae are of significance. Biofertilizers have an important role to play in improving the nutrient supplies to crop plants as well as trees in Indian agriculture as an alternate source of soil fertility building through renewable sources. reindeer. ferments starch to lactate which acidifies the rumen which is called acidosis. Other animals Buffalo. acidophilic Lactobacillus spp.e.g. These can help in increasing the biologically fixed atmospheric N or increase the native P availability to crop plants. phosphorus solubilization or mobilization. industrial production of biofertilizers has come to help farmers to economize on chemical fertilizer inputs. death of the animal e. c) They produce certain antimicrobial agents and suppress the incidence of pathogens. an explosive growth of Streptococcus bovis from normal growth of 107 cell/ml to 1010 cells/ml takes place. They are cheap.Sometimes changes in the microbial composition of the rumen is fatal i. blue green algae and Azolla are important. predominate and cause a further fall in pH. b) They produce and liberate various plant growth promoting substances. They contain multichambered stomach whose fore stomach is similar to rumen and show abundant volatile fatty acids similar to cattle. They are products containing living microorganisms which have the ability to mobilize nutritionally important elements from nonusable to usable form through biological process. vitamins and help in better and quick growth.e. Among those associated with increased P availability. S. caribon and elk are also ruminants. Thus. Among the biofertilizers useful in increasing N supply. Normally. Azospirillum. the microorganisms are evolved after intensive researches and are included in certain carriers such as charcoal. N2 fixing bacteria Rhizobium. Biofertilizers are known to make a number of positive contributions in soil thereby improving soil health in general and crop health in particular by various mechanisms as under: a) They fix atmospheric nitrogen and provide to various crops. lignite or peat. Grains contain high level of starch whereas grasses contain cellulose. killing off normal rumen flora. if a cow is changed abruptly from forage to grain diet. d) They solubilize or mobilize phosphorus in soil. e) They improve soil physical. Beleen whales also have a rumen like fermentation. Biofertilizers can be defined as preparations containing live efficient microbes performing various functions like nitrogen fixation. Azotobacter. bovis is a lactic acid bacterium. Such carbohydrates lead to a proliferation of acid producing bacterium which cause a fall in pH and consequent loss of protozoans and many species of bacteria. Biofertilizers are also called microbial inoculants. Following are the various groups of biofertilizers for different crops which are being popularly used: 12 . Biofertilizers Biofertilizers can be defined as a microbial preparation containing N2 fixing or PO4solubilizing or celluoylic or such other useful microorganisms which by virtue of special biochemical processes can increase the availability of a certain important nutrients in the vicinity of the root system leading to better plant growth and crop productivity. deer. chemical and biological health of soil. economical sources of nutrients and are ecofriendly i. nonpolluting in nature. As a result.

Cellomonas. They fix atmospheric nitrogen and produce plant growth promoting substances and Vitamins. Nitrogenase is a functional enzyme which reduces N2 to ammonia and depends on energy source from ATP. 2. metalcontaining enzyme found only in certain prokaryotic organisms. 3). Actinoplane Biological N2 fixation The utilization of atmospheric N2 gas as a source of nitrogen is called nitrogen fixation. IAA. These bacteria are benefited by root exudates of plants and help the plants by fixing atmospheric nitrogen and producing plant growth promoting substances. Phosphate solubilizing biofertilizer: Many times phosphate becomes a imiting factor for plant growth because much of it in the soil is bound as insoluble calcium. Azospirillum Biofertilizers: Azospirillum form a loose symbiosis with nonleguminous crops and are known as associative symbionts. Popularly used PGPR are Pseudomonas. Predomonas. designated as Mo-Fe protein (nitrogenase) and the other iron protein (nitrogenase reductase). Plant growth promoting rhizobacteria (PGPR): PGPR are also being used a biofertilizer as they are able to produce various phytohormones like. 3. The nitrogenase has two components: one containing Mo-Fe. Azotobacter biofertilizers are used for non leguminous crop like cereals millets and oil seed. Bacteria provide nitrogen to plants and plants in turn provide carbon sources and other nutrients to bacteria e. Rhizobial Biofertilizers: Fix atmospheric nitrogen in symbiosis with leguminous crops. 4. Certain artificial substrates that are structurally similar to N2. polymyxa.Nitrogen fixing biofertilizers 1. iron or aluminium phosphates. Nitrogenase and associated regulatory proteins are encoded by the nif regulation. Mycorrhiza are commonly used phosphate solubilizing biofertilizers. Biological N2 fixation is brought about by free-living bacteria or blue-green algae. Agrobacterium. Alcaligenes. Aspergillus. the reduction of N2 to NH3 involves a complex enzyme system called nitrogenase. Most nitrogenase contain molybdenum or vanadium and iron as metal cofactors and the process of N2 fixation is highly energy-demanding. Bacillus mregaterium. Bacillus. Both the components are essential for nitrogenase activity. which consists of dinitrogenase and dinitrogenase reductase. B. Arthrobacter. Prokaryotes both anaerobic and aerobic fix N2. N2 fixation. Cyanobacterial Biofertilizers or BGA Biofertilizers: These are useful in rice fields. which make use of N2 by non-symbiotic means and by symbiotic associations composed of a microorganisms and a higher plant. 6. No eukaryotic organisms fix N2. They also produce certain antimicrobial agents which keep away pathogens.g. Cytokinin and gibberellins etc which are important for plant growth and productivity. 13 . Produce unspecified plant growth promoting substances thereby induce profuse root and shoot growth. Rhizobium. Azotobacter Biofertilizers: Fix atmospheric nitrogen a symbiotically in soil. such as acetylene and cyanide are also reduced by nitrogenase (Fig. The availability of phosphates therefore depends on the degree of solubilization of insoluble phosphates by various organic and iorganic acids produced by the microorganisms thereby solubilize insoluble phosphates and make it available to the plant. 5. There are some bacteria called symbiotic fix N2 only in association with certain plants.

enabling it to reduce the MoFe protein. The electrons come from ferredoxin that has been reduced in a variety of ways: i) by photosynthesis in cyanobacteria. for every two electron transfer by nitrogenase. ATP is hydrolyzed 14 . whereas the aerobic Azotobacter uses electrons from NADPH to reduce ferredoxin. the Fe protein has 4 iron atoms.+ 16 ATP Æ 2NH3 + H2 + 16 ADP + 16 P.g. Therefore. ATP binding changes the conformation of the Fe protein and lowers its reduction potential. Fig. 3: Pathway of nitrogen fixation (Agricultural Microbiology) Nitrogenase enzyme The reduction of N2 to ammonia is catalysed by the enzyme nitrogenase. Fe protein is first reduced by ferredoxin. Clostridium pasteurianum (an anaerobic bacterium) reduces ferredoxin during pyruvate oxidation. iii) Fermentations in anaerobic bacteria e. The MoFe protein contains 2 atoms of molybdenum and 28 to 32 atoms of iron. N2 reduction requires at least 8 electrons and 16 ATP moles. 4 ATPs per pair of electrons.ATP N2 + 3H2 Æ 2 NH3 For this reaction. ii) Respiratory processes in aerobic N2 fixers. then it binds ATP. Nitrogenase is a complex system consisting of two major protein components a MoFe protein joined with one or two Fe proteins. Reaction has a high activation energy because molecular N2 is an unreactive gas with a triple bond between the two N2 atoms. four ATP moles are required. N2 + 8H+ + 8e.

reduced MOFe protein donates electrons to atomic nitrogen. The H2 reacts with diimine (HN=NH) to form N2 and H2 (Fig 4).when this electron transfer occurs.g. cyanide and azide) 15 . Nitrogenase is quite sensitive to O2 and must be protected from O2 inactivation within the cell.Harley-Klein) Nitrogenase can reduce a variety of molecules containing triple bonds (e. 4: Mechanism of Nitrogenase Action (Prescott. The reduction of N2 to NH3 occurs in 3 steps. Finally. each of which requires an electron pair. The overall process actually requires at least 8 electrons and 16 ATPs because nitrogenase also reduces protons to H2. Fig. acetylene. Six electron transfers take place and this requires a total 12 ATPs per N2 reduced.

called nif genes are found in both symbiotic and free living systems. Once molecular N2 has been reduced to ammonia. FeMoCo As for B As for B As for B Codes for a flavodoxin Codes for a pyruvate: flavodoxin oxidoreductase Codes for an activator mole for the other nif genes Codes for a repressor molecule for the other nif genes Possibly concerned with molybdenum uptake Possibly concerned with processing the FeMo protein As S Unknown Unknown 16 . The genes that code for these proteins are all adjacent to one another on the Klebsiella chromosomes. Proteins have been identified and in some cases functions for these genes are known. The nif-genes have been investigated most thoroughly in Klebsiella. The nif genes The genes for nitrogen fixation. one gene will code for this protein. The symbiotic activation of nif-genes in the Rhizobium is dependent on low oxygen concentration. then several genes will be necessary to code for the nitrogen-fixing system. each of which will require one gene. Work with Klebsiella pneumoniae has shown that there are 17 nif-genes. If one gene codes for the synthesis of one polypeptide. The Fe protein is composed of two sub-units but. which in turn is regulated by another set of genes called fix-genes which are common for both symbiotic and free living nitrogen fixation systems.Æ H2C = CH2 The rate of reduction of acetylene to ethylene is even used to estimate nitrogenase activity. the ammonia can be incorporated into organic compounds.HC = CH + 2H+ + 2e. The MoFe protein has two different sub-units. but in the Rhizobium-legume system. The molybdenum cofactor will require a gene and further genes will be necessary to code for any special electron donors in the system. the nif genes are distinct. as each of these is the same. Functions of the nif genes of Klebsiella pneumonia Gene nif H nif D nif K nif M nif B nif N nif E nif V nif F nif J nif A nif L nif Q nif S nif U nif X nif Y Function of the gene or gene product Codes fore the sub-unit of the Fe protein Codes for the sub-unit of the FeMo protein Codes for the ß-sub-unit of the FeMo protein Activation of the Fe protein Involved in the synthesis and insertion of the iron molybdenum cofactor.

Mutations in the nif M gene results in an inactive Fe protein. mutations in several genes affect the activity of the MoFe protein. or alteration of the particular proteins in mutants. If this is true. which does not inhibit hydrogen evaluation from normal nitrogenase. When FeMoCo was obtained from the nif V mutant protein and was combined with protein of a nif B mutant. Carbon monoxide. as the provision of electrons depends upon the metabolism of pyruvate. sodium dithionite. so that the product of the nif M gene must be involved in modifying the protein in some way. 5a & 5b). The nif J product has been shown to be the enzyme pyruvate : flavodoxin oxidoreductase. the reduction of nitrogenase and then acetylene can be achieved. B. The genes which control the synthesis of the nitrogenase proteins will be present in all the species that fix nitrogen. The purpose of these genes is to control the expression of the other genes in the nif region (Fig.mutants can be rendered active by providing Azotobacter flavodoxin. which catalyses the oxidation of pyruvate to produce reduced flavodoxin: Pyruvate + CoA + flavodoxin ----. which demonstrates that there is some specificity for the reductant and that flavodoxins from different species may differ. However. nif B.The nif H. then nine genes are needed to produce the complete active MoFe protein. when the flavodoxins from Azotobacter is used. From studies of nif V. inhibits hydrogen evaluation from nif V mutants. Thus it was concluded that the nif V product modifies FeMoCo in order to produce effective nitrogenase. The genes nif X and nif Y have been identified by means of their polypeptide products from cloned fragments of the nif region. Two genes are concerned with electron transport to nitrogenase: nif F and J. E and V are connected with the synthesis of the molybdenum cofactor. perhaps incorporating the Fe-S cluster. N and E have been identified with the synthesis of FeMoCo and the nif Q product’s action is thought to be the acquisition of molybdenum. N. the activity is one third of that with the flavodoxin from Klebsiella. The products of the genes nif S and U are thought to modify the MoFe protein. the genes concerned with electron transport will differ.acetyl CoA + flavodoxin + CO2 ox red With flavodoxin and the pure enzyme in the reaction mixture. Mutations of nif V give an altered substrate specificity. the nif V-phenotype was obtained. Three other genes. These mutants are unable to reduce N2 but can reduce acetylene. The function of these genes has however not been established. 17 . Extracts of mutants of both of these genes can fix nitrogen if they are provided with the artificial electron donor.mutants it has been concluded that FeMoCo contains the binding site for N2 and CO. D and K genes code for the polypeptides of the Fe and MoFe proteins. It is interesting to note that the genes nif F and J for electron transport to nitrogenase are transcribed in the opposite direction to the other nif genes. These genes are readily identified by the lack of. Extracts of nif F. a protein from which FeMoCo is absent. However. The remaining two genes are nif A and nif L. It is thus assumed that the product of nif F is a flavodoxin. Thus five of the genes nif Q. although there is no hard evidence for this as yet. Similarly. when FeMoCo from a normal protein was added to the nif B mutant protein a normal protein resulted.

Fig. the best studied nitrogen fixing organism. (b) The genetic structure of the nif regulation in Klebsiella pneumoniae. (Biology of Microorganisms) 18 . 5: The nitrogenase system (a) Steps in nitrogen fixation: reduction of N2 to 2 NH3.

sometimes with the accumulation of acid but never of gas. Continued plant and bacterial division and formation of the mature root nodule. the root hair curls as a result of the action of substances excreted by the bacterium called Nod factors and the bacteria enter the root hair and induce formation by the plant of a cellulosic tube. Rhizobium cells penetrate into the root hair via the root hair tip. Root cells adjacent to the root hairs subsequently become infected by rhizobia and Nod factors stimulate plant cell division. Rhizobium bacteria stimulate leguminous plants to develop root nodules. Formation of deformed bacterial cells. which the bacteria infect and inhabit. the most important plant group concerned in symbiotic N2 fixation. The energy requirement for biological N2 fixation appears to be high and this becomes the limiting factor in the quantity of N2 fixed in different legumeRhizobium combinations. The seat of the symbiosis is within the nodules that appear on the plant roots.0 µ long. The bacteria multiply rapidly within the plant cells and are transformed into swollen. Atmospheric N2 gets 19 . The classical example of such a symbiosis is that between leguminous plants and bacteria of the genus Rhizobium. Invasion of the root hair by the bacterial formation of an infection thread. There are two main types of rhizobia. The roots of leguminous plants secrete a variety of organic materials that stimulate the growth of a rhizosphere microflora. non-spore forming. misshapen and branch forms called bacteroids. The bacteria are Gram negative. within the plant cells and development of the nitrogen-fixing state. The nodule fixes N2 only for a short duration when it is in the highest symbiotic relationship with the plant. Following binding. they grow in the rhizosphere and build up to high population densities. Bacteroids become surrounded simply or in small groups by portions of the plant cell membrane. 6). Rhizobium The rhizobia are soil organisms that inhabit the rhizosphere of legumes and other plants. Only after the formation of bacteroids does nitrogen fixation begins. are put in R. For successful symbiotic N2 fixation. those bacteria which nodulate clovers. are dicotyledonous plants of the family Leguminosae. Legumes.2 µ to 3. called the infection thread.9 µ wide and 1. Fifty percent of natural nitrogen fixation is accomplished by the Rhizobium legume association. a plant and a microorganism. “fast growers” and “slow growers”. trifolii and those that nodulate peas and vetches are put in R. If there are rhizobia in the soil. The nodules develop in a complex series of steps (Fig. They are typically motile and utilize several carbohydrates. N2 fixation takes place in these nodules and the effective nodules are pink in color. a healthy plant growing in sufficient light and an effective nodule forming bacterium are required. (i) (ii) (iii) (iv) (v) Recognition of the correct partner on the part of both plant and bacterium and attachment of the bacterium to root hairs. aerobic rods. Rhizobium non legumes. 0. leguminosarum. frankia and Angiosperm and cyanobacterial associations. bacteroids. Different symbiotic associations are Rhizobium-legumes.5 to 0. Travel to the main root via the infection thread. A specific adhesion protein called rhicadhesin which is present on the surface of Rhizobium is a calcium binding protein and binds calcium complexes on the root hair surface. The division of Rhizobium into species is based on the interaction with plants. which spreads down the root hair. eventually leading to formation of the nodule. Attachment of bacterium to plant in the legume – Rhizobium symbiosis is the first step in the formation of nodules.Symbiotic N2 fixation Two members are required for the association.

micronutrients and interaction with other soil organisms. light. phosphorus. Legume-Rhizobium symbiosis is influenced by a variety of factors like host. temperature. bacterial strains.reduced to NH3 by the nitrogenase system and then to amino acids in the root nodules. Fig. combined N2. 6: Steps in the formation of a root nodule in legume infected by Rhizobium (Biology of Microorganisms) 20 . soil pH.

The plant-derived peribacteroid membrane (PBM) forms the envelop for the bacteroids. streptomycete like. although when assayed in cell extracts the nitrogenase of Frankia is sensitive to molecular O2. Intact cells of Frankia fix N2 at full O2 tensions. This is because Frankia protects its nitrogenase by localizing it in terminal swellings on the cells called Vesicles. This root nodule symbiosis has been reported in at least 8 families of plants. The members of this genus are actinomycetes : most of these bacteria at sometime in their life cycle have a filamentous habit which often superficially bears some morphological resemblance to the fungi. since presence of O2 inhibits nitrogenase enzyme of the bacterium. Like Alder. N2 fixing organism called Frankia. The bacteria in the nodule undergo limited DNA replication and division and then transform into bacteroids. 7). They are however. There is specific gene-controlled interaction taking place between the bacteroids and the PBM. 21 . many of which show no evolutionary relationships to one another. Some of the nod genes induce the host plant to react by producing nodulins which are flavanoids. the nif genes are distinct.Symbiotic process is controlled by a number of nod genes of which some are host specific and the other are common nod genes. which is required in large quantities for N2 fixation. the bacterium through infective. The plant supplies carbon compounds. prokaryotes with hyphae of smaller dimensions – in Frankia typically less than 2 µm diameter-than fungi. should ultimately come under the control of the host (Fig. The symbiotic activation of nif-genes in the Rhizobium is dependent on low O2 concentration. but in the Rhizobium-legume system. The vesicles contain thick walls of laminated structure that act as a barrier to O2 diffusion. accompanied by the genetic transcription and translation through DNA.e. The Rhizobium bacterial cells and the host cells cooperate intimately in respect of cellular metabolism with the required energy and growth regulation. The leghaemoglobin (Lb) present in the nodule binds O2 so as to facilitate N2 fixation by the bacterium. which helps the bacteria to produce ATP. which in turn is regulated by another set of genes called fix genes which are common for both symbiotic and free-living N2 fixation systems. Frankia vesicles resemble the heterocysts produced by some filamentous cyanobacteria as localized sites of N2 fixation. The genes for N2 fixation. Frankia The Alder tree (genus alnus) has N2-fixing root nodules that harbor a filamentous. derived from photosynthesis in the shoot. Frankia nodulates a number of other small woody plants. An important feature of Frankia is that many strains can fix N2 at normal O2 concentration at rates sufficient to support growth in culture. thus maintaining the O2 tension within vesicles at levels compatible with nitrogenase activity. are found in both symbiotic and free living systems. Some nod genes are required for root-hair curling and for cell division. The exchange of carbon sources with that of the nitrogenous substances is balanced as to make the bacterium a symbiont instead of a pathogen i. 6). like intact cells of Azotobacter. This suggests that the nodulation process in the Frankia symbiosis is more of a generalized phenomenon than the highly specific process observed in the Rhizobium-legume symbiosis and this holds promise for experimental attempts to expand the Frankia symbiosis to agriculturally important plants (Fig. N2 fixation in such cultures is inhibited by the addition of combined N2. called nif genes.

(Biology of Microorganism) Non-symbiotic N2 fixation Biological N2 fixation is brought about by free-living bacteria or blue-green algae. the bacteria use the N2 salt rather than N2 from the atmosphere. 7(a&b): Frankia nodules and Frankia cells. A number of environmental factors govern the rate and magnitude of non-symbiotic N2 fixation and the transformation is markedly affected by the physical and chemical characteristics of their habitat: • Microorganisms that assimilate N2 have the ability to utilize ammonium and sometimes nitrate and other combined forms of nitrogen. which make use of N2 by non-symbiotic means. 22 . ammonium salts are used preferentially and often at a greater rate than molecular N2 so that the presence of ammonium. ineffect. i.e. inhibits the fixation.a b Fig. (a) Root nodules of the common alder Alnus glutiosa (b) Frankia culture purified from nodules of Componia pergrina. In fact.

Their absence is associated directly with pH. the bacteria generally. production of plant growth hormones. Blue-green algae bacteria. The bacterium not only provides the nitrogen but produces a variety of growth promoting substances. but the need for calcium can sometimes be replaced by strontium. As a rule. H2PO4-. These strains are better competitors than the non excreting strains. the nitrogenase is more susceptible to O2. calcium and iron are critical for the fixation reaction. Similarly. Rb++ and Fe++. environments more acid than pH 6. Where there is sufficient substrate it is likely that the O2 23 . vitamin-B and anti fungal substances. but microorganisms do not use nitrate unless molybdate is present although the molybdenum requirement for nitrate utilization is less than for N2 fixation. The benefits of Azotobacter inoculation are: enhanced branching of roots. A requirement for Ca has been demonstrated during N2 assimilation by blue-green algae and some Azotobacter spp. Algae. About 1 mg of phosphorus must be assimilated by Azotobacter for each 5 to 10 mg of N2 fixed.• • • • • • • • • Many inorganic nutrients are necessary for the development of the microorganisms. Clostridium. Aerobacter and Achromobacter. as when it is grown on a medium low in carbon.0 whereas the acid tolerance of Clostridium falls between Azotobacter and Bcijerinckia. Azotobacter is characteristically sensitive to high hydrogen ion concentrations.. Azotobacter and Azospirillum Azotobacter is a free living heterotrophic nitrogen fixing bacterium encountered in neutral to alkaline soil conditions. grows better at O2 concentration lower than atmospheric when fixing N2. Molybdenum. K+.0 are free of the organism or contain very few Azotobacter cells. will neither grow nor fix N2 in culture media having a pH below 6. Beijerinckia spp. Another important characteristic of Azotobacter associated crop improvement is excretion of ammonia in the rhizosphere in the presence of root exudates and help in modification of nutrient uptake by the plants. application of organic carbon containing sources to the soil improves the asymbiotic N2 fixation capacity by the diazotroph. increased nitrate reductase activity and antifungal compounds. Azotobacter has a very high rate of respiration and when the organism is deprived of respirable substrate. gibberellic acid (GA). enhancement of uptake of NO3. for growth upon fixed compounds of N2. however. develop poorly in media and are sparse in soils more acid than approximately pH 6. but the specific requirement for N2 metabolism is often difficult to establish because Fe is required. N2 fixation therefore occurs in an aerobic environment and there must be a mechanism to prevent the access of O2 to the O2-sensitive proteins. Molybdenum is required for the metabolism of N2. The genus Azotobacter is highly versatile in utilizing carbon sources therefore. Azotobacter therefore. In like manner. Fe salts are implicated in the N2 metabolism of Azotobacter. to a lesser extent. improved water status of plants. NH4+.0. There is some evidence that the occurrence of Azotobacter is also related to the available PO4 content of soils. These include indole acetic acid (IAA). do not possess the acid sensitivity like Azotobacters and they develop and fix N2 from pH 3 to 9. The distribution of blue-green algae in wet paddy fields is likewise associated with the PO4 content of the soil. Members of this genus are strict aerobes: O2 is required for metabolism and also to fix N2. N2 fixation is inhibited when the organism is suddenly exposed to an O2 concentration higher than that in which it has been grown.

It has a wide host range. It can fix atmospheric N2 in pure culture and under microaerophilic conditions. The tropical soils have much higher Azotobacter populations than those found under temperate climates. These organisms use root exudates for their carbon and energy source while fixing N2. In addition to N2 fixation. In soil. Azospirillum is a mesophyllic bacterium and is reported to occur in association with crops grown in acidic to alkaline pH range. The genus Azotobacter comprises large. Inoculation of soil or seed with Azotobacter is effective in increasing yields of crops in well manured soil with high organic matter content. These microorganism contribute to plant functioning in natural environments. have been isolated from the rhizosphere of a large number of monocotyledons and a few dicotyledon plants.concentration will be lower than that of atmospheric because of the respiration of both Azotobacter and of other microorganisms in the vicinity. Mycorrhizae Mycorrhizae are fungus root associations. The word mycorrhizae comes from the Greek words meaning fungus and roots. (iii) orchid and (iv) 24 . Its useful characters include high N2 fixation capacity. Crops grown to pre-treated seed give increased yields up to about 10 to 30%. hormonal effects have also been shown to be responsible for at least part of yield increase following inoculation with Azospirillum. It is a tropical bacterium and has a high optimum temperature so that it does not occur to any great extents in temperature latitude. low energy requirement and tolerance to high soil temperature for its suitability under tropical conditions. Gram-negative. Azospirillum participates in all steps of the N2 cycle except nitrification. first discovered by Albert Bernhard Frank in 1885. obligately aerobic rods capable of fixing N2 non-symbiotically. Application of nitrogenous fertilizers drastically reduces the Azotobacter population in soil. Azospirillum lipoferum has been observed to fix atmospheric N2 in the cortical cells of the roots of maize. Substantial increases in yield were reported following the inoculation of sorghum and pearl millet with Azospirillum brasilense under several agro-climatic conditions in India. their numbers vary from a few to a few hundred per gm of soil. Five types of mycorrhizae can be recognized: (i) Ectomycorrhizae which form a sheath around roots but lack intracellular penetration of the cortical cells. Azospirilla are metabolically versatile and can grow vigorously in the presence of nitrogenous compounds present in the soil but as soon as the external N2 supply is exhausted the bacteria shift to diazotrophy. increase the number of root hairs and root hormone exudation. It has also been shown that Azospirillum and Azotobacter. Azospirillum spp. commonly found in association with roots of cereals and grasses has received great interest as a biofertilizer. agriculture and reclamation. three types of Endomycorrhizae: (ii) ericoid. Azospirillum: an associative micro aerophilic N2 fixer. The roots of 95% of all kinds of vascular plants are normally involved in symbiotic associations with mycorrhizae (Fig 8). besides enhancing N2 uptake by plants. This genus of spirally curved Gram negative bacteria is interesting as its members not only live in the rhizosphere of grasses but can also enter the root cortex. Azospirillum is a O2-sensitive and can fix N2 only at low O2 concentrations. Use of Azospirillum inoculum under saline alkaline conditions is possible because strains adapted to these stress conditions maintained high N2 activity.

Ectomycorrhizal fungi penetrate intra cellularly and partially replaces the middle lamellae cortical cells of feeder roots. Betulaceae and Fugaceae. especially conifers. In a forest. almost every root of every tree is mycorrhizal. Example of plant spp. 8: Components of the mycorrhizal symbiosis. Salicaceae. forming ectomycorrhizal associations are spp.vesicular-arbuscular mycorrhiza which colonize the root cortical cells intracellularly and (v) Ectendomycorrhizae which form sheath and produce intracellular penetrations (Fig 9). beeches and oaks and are most highly developed in temperate forests. they obtain their nutrients from root secretions. show the typical fungal sheath. This fungal mantle varies from one to two hyphal diameters to as many as 30 or 40 depending upon the fungal associate. These fungi produce plant growth substances that induce morphological alterations in the roots. a single species of pine can form mycorrhizae with over 40 species of fungi. Most ectomycorrhizal fungi are 25 . The short roots. causing characteristically short dichotomously branched mycorrhizal roots to be formed. Fig. Most mycorrhizal fungi do not attack cellulose and leaf litter but instead use simple carbohydrates for growth and usually have one or more vitamin requirements. The mycorrhizal fungi are never found in nature except in association with roots and hence can be considered obligate symbionts. there is little species specificity involved. Despite the close relationship between fungus and root. These fungi form a dense mycelial net around and between the plant cells termed Hartig net. The root system of a mycorrhizal tree is composed of both long and short roots. Phosphate enters the plant. both directly from the soil and through the fungus. which are characteristically dichotomously branched. Ectomycorrhizal associations are also characterized by a dense. the host and the environmental conditions. (Advances in Agricultural Microbiology) Ectomycorrhizae: are found mainly in forest-trees. generally continuous hyphal network over the feeder root surface called a fungal mantle. in the families Pinaceae. along with other mineral nutrients. whereas long roots are usually uninfected.

The VAM fungi penetrates the outermost cortex region. swellings. corn. Currently. Vesicles appear to be organelles for the storage of lipid and energy reserves and arbuscules. horticultural and ornamental crops. All orchids are infected at some stage in their life cycle by the Orchidaceous mycorrhizal fungi. as well as some forest tree spp. These fungi do not form dense fungal mantles. which are not infected by endomycorrhizal fungi. Cortinariaceae. Tricholomataceae. Rhizopogonaceae and Sclerodermataceae). Russulaceae. VAM colonization originates from hyphae arising from soil borne propagules. The ectomycorrhizal fungi help in the phosphorus nutrition of plants through increased surface area of absorption. the mycorrhizal fungi are reported to increase the availability of water to plants. but they do develop a loose. VA mycorrhizal fungus grown on lettuce can infect maize. which resemble the haustoria of rust fungi and mildews are complex ramifications of small branches of the fungus that provide sites for nutrient exchange. or minute branches. Azotobacter and phosphate solubilizers in the rhizosphere and suppress the growth of root pathogenic fungi and nematodes. Boletaceae. that can form mycorrhizal associations of the VA type. are phycomyces many of which are in the genus Endogone. which would otherwise be unavailable to the plant. In addition. 26 . without the plant. Mycorrhizal hyphae have been found to be very efficient in the uptake of phosphorus from the soil. Endotrophic mycorrhizae are found in wheat. is the most common fungal symbiont. as blueberry is an important cash crop. beans. several laboratories are studying this association. offer protection against some of the soil-borne plant pathogens and enhance rooting and survival of cuttings through production of growth hormones. Mycorrhizal fungi have been observed to improve plant growth through better uptake of P and Zn from soil. intermittent arrangement of mycelium on the root surface. Recent studies show that plant flavonoids may stimulate spore germination and this could lead to the development of plant-free cultures of these mycorrhizae.basidomycetes (primarily of the families Amanitaceae. where they grow intracellularly and form coils. grasses. On reaching the cortex. A recent study has shown that artificial inoculation of orchids with the mycorrhizal fungus is not necessary as the fungus is abundantly present in nature. Pezizella ericae. resulting in more vigorous growth under drought conditions. usually members of the zygomycetes. oranges and many other commercial crops. but in phosphorus deficient plants they penetrate deep into the cortex and help the plant to obtain the nutrient from the soil. as well as most pasture and rangeland grasses. citrus and almost any other plant spp. when the plant is well supplied with phosphorus. hyphae grow into cells by tree like dichotomous branching to give arbuscules. apples. Ectomycorrhizal fungi have more limited host ranges and there are plant spp. oval structures called vesicles may form which have storage functions. In this association the fungal hyphae penetrate the outer cortical cells of the plant root. The ericoid mycorrhiza is seen in members of heath family like blueberry and Erica. beans. When colonization is well established. Endomycorrhizae: are distinguished by the fact that the fungus penetrates the cortical cells of feeder roots and may form large vesicles and arbuscles (hence the term vesicular-arbuscular mycorrhizae (VAM). Endomycorrhizae are of particular interest. Endomycorrhizae are formed by most agronomic. Recent studies have shown that they stimulate beneficial organisms like Rhizobium. which can be cultured. as it has not been possible to grow these fungi. tomatoes. that do not form ectomycorrhizae. The fungal spp. an ascomycete.

These benefits have distinct energy costs for the plant in the form of photosynthate required to support the plant’s “mycorrhizal habit”. Many tropical soils are so phosphorus deficient that they cannot respond to N2 until this deficiency is corrected.Ectodomycorrhizae: These mycorrhizae resemble ectomycorrhizae in forming a Hartig net and a fungal mantle. Jones. Rangaswamy and D. It is here that endomycorrhiza takes on added significance. where nutrients do not limit plant functioning to the same degree. trees that were artificially inoculated at the time of planting grow much more rapidly than uninoculated trees. especially phosphorus. Suggested Reading 1. Microbiology (1999) by Lansing M. where trees that are mycorrhizal. Harley & Donald A. Under certain conditions the plant is apparently willing to trade photosynthate produced with the increased water acquisition for H2O. T. The formation of mycorrhizae is particularly pronounced in land low in phosphorus and N2 and high nutrient levels are correlated with poor mycorrhizal development. Mycorrhizae are thus something of an enigma.D. 6.R. mycorrhizae can increase a plant’s competitiveness.T. Nitrogen Fixation in Plants by (1986) R. Avances in Agricultural Microbiology (1982) by Subba Rao. 5. the mycorrhizae aid in water uptake.G. John M. Dixon & C. These symbioses have evolved to enable the fungus to invade roots and routinely colonize from 10% to 90% of the root length with no obvious harmful effect on the plant. They are wide spread and show little sign of specificity. Gray & J. Fletcher. yet they involve complex interactions and possibly most plants are partially dependent upon them for their PO4 nutrition. N2 fixing microorganisms can increase soil N2 while mycorrhizal fungi effectively augment the absorbing surface of the roots. It is well established that the mycorrhizal plant is able to absorb nutrients from its environment more efficiently than does a nonmycorrhizal one. Mycorrhizal fungi can be so important that some species of host plant are almost dependent upon them to be able to grow in soils low in PO4. Martinko. A resemblance with the endomycorrhizae is associated with their penetration of cortical cells. as it enhances the absorption of phosphorus.G. 4. This improved nutrient absorption is probably due to the greater surface area provided by the fungal mycelium. allowing increased transpiration rates in comparison with nonmycorrhizal plants. 7. Prescott. Madigan. Brock Biology of Microorganisms (1997) by Michael T. In wet environments they increase the availability of nutrients. 2.J. In arid environments. citrus and cassava are particularly good example.O. Depending on the environment of the plant. The beneficial effect on the plant of the mycorrhizal fungus is best observed in poor soils. This mycorrhizal grouping is the least studied and the nature of the fungal symbionts has not been totally elucidated. Ecology of Microbial Communities (1987) by M. John P. Agricultural Microbiology (1996) by G. 3. Bagayaraj. Jack Parker. thrive but non mycorrhizal one do not. Mycorrhizae are symbiotic because the plant provides the fungus with organic nutrients and the fungus facilitates the uptake of mineral nutrients and in particular PO4. 27 . which ordinarily lack a suitable fungal inoculum. When trees are planted in prairie soils. A mycorrhizae is a mutualistic symbiosis between plant and fungus localized in a root or root-like structure in which energy moves primarily from plant to fungus and inorganic resources move from fungus to plant. Introduction to Soil Microbiology (1961) by Martin Alexander. Wheeler. Klein.

9. 28 . Soil Microbiology by (1994) Robert L.8.L. Tate III.Dogra. Plant-Microbe Interactions. 1 by Tsune Kosnge & Eugene W.C. Nester. Molecular & Genetic Perspectives (1984) Vol. Plant Microbe Interaction in Sustainable Agriculture (1995) by R. A. Khurana & R. 10.K. Behl.