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Chapter 4 Nitrogen Fixation Air is four-fifths nitrogen, yet it is the absence of this particular element that most commonly limits food production. Neither man, animals, nor high- er plants can use elemental nitrogen; it must first be "fixed," that is, com- bined with other elements such as hydrogen, carbon, or oxygen before it can be assimilated. Certain bacteria and algae have the ability to utilize (fix) gaseous nitrogen from the air. Some microorganisms work symbiotically in nodules on the roots of plants, with the plant providing food and energy for the bacteria, which, in turn, fix nitrogen from the air for their host. Other kinds of bac- teria and algae work independently and fix nitrogen for their own use, but these are often limited in their activity because of the lack of a dependable energy supply. Bacteria that fix nitrogen in nodules on the roots of leguminous plants are called rhizobia (Figure 4.1~. Other microorganisms that produce nodules on certain nonleguminous plants are classified as Franks spp. and are actino- mycetes. Freshly crushed nodules from the same plant species also will induce nodulation on these plants. Recently Callaham et al. (1978) have induced nodulation in shrubs of sweet fern (so mptonia peregrine with a pure culture of Frankia. Leguminous plants have been known for centuries to enrich soils, but the reason was not understood until 1886 when two Ghan scientists, Hellriegel and Wilfarth, found that the bacteria in the nodules on the leguminous root brought about nitrogen fixation. Nitrogen-fixing microorganisms fix an estimated 175 million t of nitrogen annually, or about 70 percent of our total supply. The remainder is produced in chemical fertilizer factories. With the rising world population and the declining supply of fossil fuels required to manufacture chemical nitrogen fertilizer, it may be necessary to rely more on microorganisms to satisfy plant needs for nitrogen. Some of the nitrogen-fixing systems involving micro- organisms are described in the following sections. 59
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6Q MICROBIAL PROCESSES S~BE ANS WITH NODUL E 5 F1X A1R NlTROGEN \w ~~ ~~ o # ~ ~ -_ f~1 / FICURE 4.1 RhizobisofthepIopeIkindappUedtolegum~ousseedsbe~Ieplan1~g caninducenodules OI nitrogen bctoIiestofonm ontheIOOtS.lhesepIovidetheplant with usablenKIogen.ThepIocessof~pply~gtheIb~obiatoseed~caDed Elocution. (PhotogIaphcouI1esyofJoeC.BuIton)
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NITROGEN FIXATION Symbiotic Systems Rhizobium- Leguminous Plant Associations 61 Of all the systems of biological nitrogen fixation, the Rhizobium-legumi- nous plant association has been the most reliable. Legumes in association with nodule bacteria fix at least 35 million t of nitrogen annually valued at several billion U.S. dollars. Yet this beneficial association of nodule bacteria with legumes has only been partially explored. Of the 13,000 known species of legumes, only about 100 are grown commercially. Further, much of the seed is planted without inoculations with the nodule bacteria. Modulation, if it oc- curs under these conditions, is by native soil rhizobia, which are often either ineffective or too few in number to bring about effective nitrogen fixation. Plant Groups and Rhizobium Species. Many soils do not contain the proper nodule bacteria to bring about nitrogen fixation and successful growth of legumes. In many cases when the bacteria are present, they are ineffec- tive—that is, they produce nodules that provide little or no nitrogen. Farmers can enhance nitrogen fixation by adding the proper nitrogen-fixing bacteria to leguminous seeds before they are planted. Less than a kilogram of high- quality inoculant, properly applied to legume seeds, can replace more than 100 kilograms of fertilizer nitrogen per hectare. Certain groups of leguminous plants are nodulated by a single kind of Rhizobium. The bacteria that nodulate each of these groups are often con- sidered a species. All plants susceptible to nodulation by a Rhizobium species constitute a "cross-inoculation" group. The Rhizobium species and their cor- responding plant or cross-inoculation groups are given in Table 4.1. Effective nitrogen-fixing nodules on some common legumes are shown in Figure 4.2. These nodules are usually large and are often concentrated on the primary root. In contrast, ineffective nodules are small, numerous, and scat- tered over the root system (Figure 4.3~. Rhizoblum- H ost I nteractions. Strains of rhizobia cannot be described as effective or ineffective without specifying the exact species of legume host. Strains of rhizobia that are good nitrogen-f~xers in association with one host are often worthless on another. There is voluminous literature on this point, but so many of the leguminous plants cultured in the tropics and subtropics are nodulated by the cowpea rhizobia that special mention is justified. The cowpea cross-inoculation group encompasses numerous genera and species of plants. Rhizobium strains effective on a wide spectrum of plants within this group are scarce. Specific inocula containing two or three strains known to be highly effective on the particular legume may be needed to assure good yields.
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62 TABLE 4.1 Rhizobium Speaes and Plants Nodulated MICROBIAL PROCESSES Rhizobium Species Plants Nodulated Designated R. mel~loti Medicago sativa (alfalfa) Melilotus sp. (sweet clover) Medicago sp. (burr and barrel medic annuals) Trigonella foenum graecum (fenugreek) R. trifolii Trifolium sp. (clovers) R. leguminosarum Pisum sativam (garden and field peas) Vicia faba (broad bean) Lens esculenta (lentils) Lathyrus sp. (peavine) R. phaseoli Phaseolus vulgaris (common, field, haricot, kidney, pinto, snap beans, etc.) P. coccineus (runner bean, scarlet runner) R. Iupini Lupinus sp. (all lupine) Ornithopus sativus (serradella) R. japonicum Glycine max (soybean) Undesignated Rhizobium sp. Vigna unf~iculata (cowpea) (Cowpea type) Arachis hypogaea (peanut, groundnut) Vigna radiata (mung bean) Phaseolus lunatus (lima bean) P. acutifolius (tepary bean) Psophocarpus tetragonolobus (winged bean) Sphenostylis sp. (African yam bean) Pachyrhizas sp. (iicamus) Centrosema sp. (centro) Mucuna deeringiana (velvet bean) Canavalia ensiformis ( ack bean) Lablab purpureus (hyacinth bean) Phaseolus aconitifolius (moth bean) Cyamopsis tetragonoloba (guar) Voandzeia subterranea (Bambara groundnut) Cajanus cajan (pigeon pea) Desmodillm sp. Cassia sp. Lespedeza sp. Indigofera sp. Crotalaria sp. Pueraria sp. Rhizobium sp. Cicer arietinum (chick-pea, garbanzo) Coronilla varia (crownvetch) Onobrychis vicisefolia (sainfoin) Leucaena leucocephala (ipil-ipil) Petalostemum sp. (prairie clover) Albizzia julibrissin Lotus sp. (t~efoils) Anthyllis vulneraria (kidney vetch) Sesbania sp. Sources: R. E. Buchanan, and N. E. Gibbons, eds. 1974. Bergey~s Manual of Determina- tive Bacteriology. 8th edition. Baltimore: The Williams and Wilkins Co. E. B. Fred; I. L. Baldwin; and E. McCoy. 1932. Root-Nodule Bactena and LeguminousPlants Madison: University of Wisconsin Press.
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NITROGEN FIXATION . .. ... .... .. . ::::::;:.:.;::..~.~:TRGGEN:::::::::::::~::XINS:::::::::::::N:0006::ES::::: .............. . . .. ... .... . . . . . . ...... .. . . . . .... .. ... ........ .. . .. .. ... . .................................................................... .............................. ................................ WIN~h ~ : . n ,. . ~ . ..~. ..~ ~ ..~ ., , y ... ... ...... ... .~ .. .. . ~ hi. ~ ~ ~ .............. ... -: ......... to $~|C$~@f ~166¢ BEAN FIGURE 4.2 Nodules or nitrogen factories on the roots of important food legumes: winged bean, Psophocarpus tetragonolobus; peanut, Arachis hypogaea; chickpea, Cicer arietinum; and field bean, Phaseolus Yulgaris (Photographs courtesy of Joe C. Burton) 63
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64 MICROBIAL PROCESSES ~~ s s saws s s .s s~sis~ ~ EA~N~!NOOULE~S~ ~~ s FIGURE 4.3 Rhizobia vary ~ then nUIoqen~x~g Maiden Some aIe ~e~c1~e; they produce nodules and use Mod that the plant provides, but Ox 1~1~ OI no nitrogen. Ethers me fictive; these produce large nodules and Ox appreciable amounts of nitrogen (Photographs courtesy of Joe C Burton)
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NITROGEN FIXATION 65 Nitrogen Fixed by Leguminous Plants. The amount of nitrogen fixed in Rhizobium-leguminous plant associations varies with both the bacteria and legumes as well as environmental factors. Forage legumes usually fix more nitrogen than do grain legumes because carbohydrate requirements resulting from seed development are small, whereas with grain legumes the developing large seeds impose a large demand on the carbohydrate supply. The amounts of nitrogen fixed are uncertain, because of the methods of measurement, but relative quantities as related to host species give valid comparisons (Table 4.21. Rhizobium species in association with leguminous vegetables, in addition to increasing plant protein, make the plants richer in vitamins and mineral content by assisting general growth. Healthy, well-fed plants are more palat- aole and nutritious than plants suffering from lack of nitrogen. Limitations A major limitation to culture of leguminous crops is the lack of viable, effective inocula for many of the legumes. Leguminous crops and soil condi- tions vary with each location. Rhizobium strains should be selected for partic- ular legumes and soil and climatic conditions. However, Rhizobium inocu- lar~ts are highly perishable and often lose viability before reaching their destination. TABLE 4.2 Nitrogen Fixed by Various Rhizobium-Legume Associations Plant Approximate Ranges of Nitrogen Fixed (kg/ha/yr) Alfalfa, Medicago saliva Sweet Clover, Melilotus sp. Clover, Trifolium sp. Cowpea, Vigna unguiculata Faba bean, Vicia faba Lentils, Lens sp. Lupines, Lupinus sp. Peanuts, Arachis hypogaea Soybeans, Glycine max Mung bean, Vigna radiate Velvet bean, Mucuna pruriens Pasture legumes, Desmodium sp., Lespedeza sp. 100-300 125 1 00-150 85 240-325 100 1 5 0-200 50 60-80 55 115 1 00-400 Sources: Adapted from R. C. Burns, and R. W. F. Hardy, 1975. Nitrogen fixation in bacteria and higher plants Berlin: Springer-Verlag; and W. S. Silver, and R. W. F. Hardy, 1976. Biological nitrogen fixation in forage and livestock systems. American Society of Agronomy Special Publication No. 28. pp. 1-34.
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66 MICROBIAL PROCESSES A limitation to the legume symbiosis system, particularly with the grain legumes, is the relatively short season of active nitrogen fixation in the nodule, especially with plants such as field beans (Phaseolus vulgaris). Soy- beans fix nitrogen a little longer. Nitrogen fixation with the soybean could be doubled if the period of active nitrogen fixation in the nodule could be increased by as little as 10 days (Hardy and Havelka, 1970~. A limitation to the symbiotic system in leguminous plants is the in- adequacy of inoculants and inoculation methods to ensure a greater propor- tion of nodules from the applied rhizobia when seeds are planted in soils infested with highly infective rhizobia of poor nitro~en-fixing properties. Research Needs · Rhizobium strains should be selected for the specific legume crops being grown in each country. Strains should be selected for their nitrogen- fixing ability and competitiveness under the prevailing soil and climatic con- ditions. · Small-scale inoculant production methods should be studied. Effective use of leguminous plants will depend both on effective Rhizobium strains and a dependable delivery system that will help ensure a high production of nodules by the inoculum rhizobia. · In devising delivery systems, consideration should be given to overcoming an aggressive native population of both infective rhizobia and other micro- organisms. The latter group of plant pathogens—as well as insects—may neces- sitate separate application of inoculants and protective chemicals to the seeds. · New genera and species of legumes should be studied. Leguminous plants such as the winged bean, Psophocarpus tetragonolobus, the climbing lima, Phaseolus lunatus, the yam bean, Sphenostylis stenocarpa, and the hyacinth bean, Lablab purpureus, all of which are climbers, can be very productive under humid tropical conditions. Further, they will fix nitrogen for months, providing the pods are gathered regularly and not allowed to mature on the vine. Frankia-Nonleguminous Plant Associations Numerous nonleguminous trees and woody shrubs form root nodules and fix atmospheric nitrogen under natural conditions. The organism responsible, found in the nodules (called an endophyte), is an actinomycete (Frankia sp.) and infection and nodule initiation have recently been achieved with the cultured organism (Callaham et al, 1978~. Crushed nodules from a growing plant of the same species readily induce nodulation in most cases. Now that the endophyte from one nonleguminous nodulating plant has been isolated and cultivated, this can be done with others, thereby greatly facilitating cul- ture of these nodulating nonleguminous plants. The same organism has now
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NITROGEN FIXATION been shown to nodulate Alnus, Myrica, and Comptonia. ~ . . . . ~ 67 Noniegum~nous trees and woody shrubs capable of fixing nitrogen com- prise 145 species in 15 different ger~era of 7 plant families. Within such a large group of plants, there is adaptability to a range of diverse soil and climatic conditions. These plants often thrive in nitrogen-deficient eroded areas and on sand dunes, barren slopes, and even arid soils. Certain species are pioneers. They are the first to grow after glaciers have receded. Others make an early appearance after volcanic eruptions and help develop soils from lava. The hardiness of this group of plants is partially attributable to their ability to fix nitrogen in association with Frankia sp. Many species also bene- fit from association with external or ectotrophic mycorrhizae, and can thus survive minimum phosphorus levels in the soil. The economic impact of the nodulating nonlegume application is mainly in forestry rather than agricul- tural crops. Trees of the birch family, especially Alnus sp., provide wood for timber in many countries; the red alder (Alnus rubra Bong) (Figure 4.4) can fix as much as 300 kg of nitrogen per ha per year. ............ .. FIGURE 4.4 Nodules on red alder, Alnus rubra Bong. Red alder can fix as much as 300 kg nitrogen per ha per year (Table 4.3) when effectively nodulated. (Photograph courtesy of H. J. Evans)
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68 MICROBIAL PROCESSES The nitrogen-fixing abilities of these nonleguminous trees and shrubs when they are well nodulated are almost comparable to those of the Rhizobium- leguminous plant associations, providing the stands are kept for several years. Silvester (1977) has tabulated reports on nitrogen fixed by various species (Table 4.3~. The unique abilities of these nodulating nonleguminous trees and shrubs to pioneer in new soils, increase fertility, and enrich the soil for growth of economic crops should not be overlooked. Such plants can provide the base for expansion of areas for food production, arid they can help to restore soils disrupted by the removal of coal and minerals. Limitations The nodulating nonleguminous trees and shrubs are long-term crops, mak- ing them unsuitable when annual harvests of farm crops must be made for subsistence. Recently a new plant (Da tisca cannabina) was discovered, which nodulates like Alnus, is not woody, and is propagated by seed. There may be many others of this type. Most known valuable species grow best in cool or temperate climates or at him altitudes in the tropics. With further study, species well adapted to the lowland tropics may also be discovered. Seeds are very limited in availability. TABLE 4.3 Nitrogen Fixed by Various Genera and Species of Nodulating Non- leguminous Trees and Shrubs Species Age-Years Nitrogen kg/ha/yr* Alnus crispa 0-5 362 15-20 1 15 1 0-60 40 Alnus glutinosa 0-8 125 20 56-1 30 Alnus ru bra 2-15 325 Gasuarina equisetifolia 0-13 58 Ceanothussp. - 60 Coriaria arborea 14-25 129-192 Dryas drummondii 0-25 12 Hippophae rhamnoides 10-15 15 13-16 179 Myrica gale 3 9 *These are values reported in the literature; fixation rates vary widely with conditions and should be treated as indicative estimates only, not as definitive rates. Source: W. B. Silvester, 1977. Dinitrogen fixation by plantassociationsexcludingle- gumes. In A treatise on dinitrogen fixation, R. W. F. Hardy and A. H. Gibson, eds. New York: John Wiley and Sons.
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NITROGEN FIXATION Research Needs 69 · Improved technology is needed for seed collection, production, and handling for the more promising species. · Dependable laboratory-produced cultures of the endophytes specific to all important species would be very helpful. · Surveys should be conducted to determine if nitrogen-~xing species of these nodulating nonleguminous plants grow in the lowland tropics. · Plants should be evaluated for nitrogen-fixing ability as well as for their value as human and animal food. Azolla-Anabacna Associations A small floating freshwater fern, Azolla pinnate invades lowland rice fields in Indonesia, southern China, Vietnam, and other tropical areas. The upper lobe of the Azolla leaflet contains a large leaf cavity inhabited by the blue- green alga, Anabaena azolla. The symbiotic nature of the association is evi- denced by two findings: 1) the algae in the leaf cavity have 15-20 percent nitrogen-f~xing cells (heterocysts) as compared to 5 percent in free-living Anabaena species, and 2) the algae grow very poorly when taken from the leaf cavity and placed on an inorganic medium with no combined nitrogen. Growth is obtained in some cultures when the growth medium is supple- mented with an organic compound in the form of 0.5 percent sugar (fruc- tose). But in subsequent studies, nitrogenase activity of this isolated algae has been only about half that of algae growing symbiotically in the leaf. The Azolla-Anabaena association is literally a live floating nitrogen fac- tory, using energy from photosynthesis to fix atmospheric nitrogen. Under Indonesian environmental conditions, the Azolla-Anabaena association can fix from 100 to 150 kg of nitrogen per ha per year in approximately 40-60 t of biomass. It is important not to let the fern cover the water completely in the rice paddies. Because the rice can be damaged *om excessive shading of the paddy water, 50 percent coverage is ideal. Nitrogen fixation occurs at night as well as during the day, but the rate of fixation is lower in the dark. Azolla pinnate plants floating on the water surface of irrigated rice paddies are shown in Figure 4.5. Azolla has been used extensively in Asia as a forage crop for duck and pig feed, but its greatest potential appears to be as a green manure. On a dry- weight basis it contains about 23.8 percent crude protein, 4.4 percent fat, 6.4 percent starch, and 9.5 percent fiber. Vietnam and Thailand have used Azolla for years in their system of rice culture. Stocks of Azolla are kept during the hot season for multiplication and distribution when cooler weather comes. The stocks are then used to seed other paddies fertilized with ashes, urine, and rotted manure. Azolla vegetation can double approximately every 5 days under favorable conditions.
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7Q MICROBIAL PROCESSES FIGURE 4.S Smog Doming nitrogen ~ctoI~s on ~ Hooded tics paddy. The small fern Czar p~m hubris blue~Ieen algae, ~~e~ razor, which ~ nitrogen Together they provide nitrogen HI ~ Intuit crop. (Photograph courtesy of J. U. Backing)
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NITROGEN FIXATION 71 Nitrogen fixation by Anabaena azolla is apparently not adversely affected by the level of combined nitrogen in the water because the organisms are ac- tually sheltered in the leaflet cavity. The Azolla must die off and the nitrogen must be mineralized before it becomes available to the plant. This happens when the temperature rises (to as much as 40°-45° C in Indonesia), and the Azolla dies and settles to the bottom of the paddy. Nitrogen is released from the decomposing cells and becomes available to the rice plants. The rice plants then turn green and filtering (the production of multiple shoots from the same plant) increases. Limitations Use of the Azolla-Anabaena association for food production is limited to agricultural soils that can be flooded, and it is best adapted to rice culture. Azolla could possibly be used, however, as a nitrogen source for other aquatic plants such as tare (Colocasia esculenta) or water chestnut (Eleocharis dulcis). In addition, the association may be beneficial in removing nutrients from sewage treatment lagoons. Water, plenty of sunshine, and a temperature regime that favors rhythmic self-destruction of the fern seem to be the most important requirements for success. Research Neecis · More information is needed about nitrogen-fixation efficiencies of dif- ferent Azolla-Anabaena strain combinations. It is possible that more efficient nitrogen-fixing combinations can be discovered. · Good husbandry should be developed for using Azolla-Anabaena in rice culture systems, as has been done in Indonesia and Vietnam. Technology for using Azolla under different soil and climate conditions is needed, particu- larly for temperate areas. · Experiments should be made to determine how best to grow Azolla with rice to increase both nitrogen fixation and rice yields. Asymbiotic Fixation Blue-Green Algae The blue-green algae are perhaps our most widespread group of nitrogen fixers because they are present almost everywhere on land, in fresh water, and in the sea. Regardless of environmental conditions (except at low pH), there are almost always present some forms that fix atmospheric nitrogen. Blue-
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72 MICROBIAL PROCESSES green algae fix nitrogen in Antarctic waters as well as in hot springs. They operate over the range of 0°-60°C. One reason for their wide range of adaptability is that they include many species, each of which may fix nitrogen under varying conditions. Nitrogen- fixing species occur in the genera Anabaena, Aulosira, Cylindrospermum, Gloeotricha, Tolypothrix, Calothrix, Nostoc, Haplosiphon, and others. In rice culture, the blue-green algae can be depended on to provide nitro- gen consistently. In long-term soil fertility experiments at the International Rice Research Institute (IRRI) in the Philippines, 23 consecutive rice crops have been harvested from soils unfertilized with nitrogen, without any ap- parent decline in soil nitrogen. The algae replaced the nitrogen removed by the rice crops. Studies at the Agricultural Research Center in Giza, Egypt, have shown that two blue-green algae, Tolypothr~c tenuis and Aulosira fertilissima, fix more nitrogen than other forms in that region. In a rotation with rice every third year, it has been found advantageous to grow the effective algae and inoculate the rice fields shortly after planting. Of the rice cultured, 10 percent is now inoculated with a dried algal preparation of the two effective cultures and this percentage is expected to increase rapidly in the future. Inocula are also supplied to farmers in India by the Agricultural Research Institute in New Delhi. The importance of blue-green algae in rice paddies has long been recog- nized. But these microorganisms also operate very well in desert regions; they use the moisture of night dews during the early morning hours and fix nitro- gen during this period of temporary activity. In the western United States blue-green algae in crusts on the soil surface fix considerable amounts of nitrogen per hectare per year. Nitrogenase activity (the activity of the enzyme that splits molecular N2) ceases when the crusts become dry, but it is measur- ably reactivated within 2 hours after crusts are moistened. The importance of the role of the blue-green algae in fixing nitrogen was not appreciated until the acetylene-reduction technique of measuring nitro- gen fixation was developed. Now it is possible to study algal fixation in streams and lakes and under various soil conditions, and the significance of the blue-green algae in our world food production is becoming more evident. Their main assets are 1) the wide range of adaptation to temperature and moisture, and 2) their ability to respond quickly when environmental condi- tions are suitable and to grow rapidly in paddy fields. The blue-green algae fill ecological niches left by other systems of biological nitrogen fixation. Limitations From a management standpoint, knowledge of how to use the blue-green algae effectively is meager. Nitrogen fixed by these microorganisms is not
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NITROGEN FIXATION 73 readily available to food crops—the algal cells must decompose and the nitro- gen must be mineralized. Although this may not be a problem in continuous rice culture, it could be a major obstacle in other planting systems. Algal growth in freshwater lakes is usually undesirable because they cause the water to become stagnant. Technology on use of algal tissue as feed for livestock or food for human consumption is needed. At present, its use is chiefly as a green manure. Research bleeds · A survey should be made to determine the occurrence of good nitrogen- . . . wing species. · Strains should be selected for environmental adaptation as well as nitro- gen-fixing potential. · Technology related to husbandry and how to prepare, store, and dis- tribute inoculants is needed. · Methods of culturing starter cultures and distributing dependable in- ocula should be developed; biological and chemical methods of control will result in better husbandry and more efficient handling of effective algae. . Information is needed on how to encourage the growth of desirable organisms and discourage the growth of undesirable ones. The role of pred- ators in reducing algal nitrogen fixation should be investigated and the use of algae adapted to local soils. · Studies should be conducted on the use of algae for human food and animal feed. The large biomass of algae could possibly provide high-quality edible protein for animal consumption. Free-Living Nitrogen-Fixing Bacteria Free-living nitrogen fixers from at least 25 genera and many taxonomic groups of bacteria are known. These organisms occur in diverse habitats; their requirements for oxygen, a specific energy source, electron acceptors, and other factors vary widely. Fixation of significant amounts of nitrogen is dependent upon a suitable carbon and energy supply. An adequate source of energy is one of the most critical limiting factors to nitrogen fixation by free-living organisms. One of three systems of obtaining energy may be utilized: · Energy may be obtained through breakdown of plant residues in soil. Only rich, fertile soils harbor organic residues in amounts sufficient to pro- vide significant energy. Clostridium, Klebsiella, and most Azotobacter species rely on this source.
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74 MICROBIAL PROCESSES · Certain bacteria are favored by root exudates of some plant genotypes that are very efficient photosynthesizers. Exudates from the roots are selec- tive energy sources for particular bacteria. This relationship is often referred to as an "associative symbiosis"; a recent international symposium on nitro- gen fixation recommended that it should be termed "biocoenosis." Azoto- bacter paspali, Beijerinkia sp., and Spirillum lipoferum fit into this category. (Recent work has suggested that Spinllum lipoferum should be reclassified as two species of Azospirillum: A. Iipoferum and A. brasilense iKrieg and Tarrand, 19771.) · Other bacteria carry out photosynthesis themselves. But requirements for growth are so restrictive that these microorganisms are not considered highly important agronomically. Rhodospirillum rubrum and other photo- synthetic bacteria are in this group. The presence of nitrogen-fixing bacteria in the root zone does not assure that they are actively fixing nitrogen; it does indicate the capability for nitrogen fixation if there is sufficient energy and other conditions are present for growth. If ammonia or nitrates are present in the soil, the organisms will use these to produce new cells and will conserve energy in preference to fixing nitrogen. Energy used for nitrogen fixation cannot be used in repro- duction. The real nitrogen contribution of free-living nitrogen fixers to the soil is uncertain. With a mixed population under natural conditions, it is difficult to assess the contributions of single species. Clostridium, Klebsiella, and several Enterobacter species are credited with substantial nitrogen fixation when energy-rich soils are flooded, but the iden- tities of the major aerobic genera are uncertain. Azotobacter species, on the other hand, prefer a moist, aerated environment, but they, too, are dependent upon an adequate source of carbon. In both cases, large amounts of energy- rich materials are required if significant amounts of nitrogen are to be fixed. Nitrogen fixation efficiency is low. It takes the equivalent of about 50 kg of sucrose for Azotobacter species to fix 1 kg of nitrogen at the oxygen concen- trations found in air. The inoculation of soils and seeds of nonleguminous plants with prepara- tions of Azotobacter chroococcum has been practiced in Russia and India for many years. Some types of Azotobacter have been credited with increasing crop yields as a result of the nitrogen they fixed, but the low concentration of cells in the soil could not have fixed appreciable amounts of nitrogen. With the new highly sensitive techniques for measuring nitrogen fixation, some doubt has been cast on the real contribution of free-living bacteria to soil nitrogen. Bacterial inoculation experiments rarely show yield increases as great as the 10 percent level required to attribute statistical significance to the results. The greatest increases are on very fertile soil. There is little proba-
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NITROGEN FIXATION 75 bility Mat any sound inoculation practice for free-living bacteria will be devel- oped soon, and many of the reports of alleged growth stimulation are now considered dubious. In highly fertile soils, Azotobacter species are sometimes believed to pro- duce growth factors and vitamins that are beneficial to vegetables. Benefits from Azotobacter inoculation often are attributed to these growth factors rather than to nitrogen fixation. The effect is manifested only in the highly fertile garden soils used in vegetable production. The results of these studies are still equivocal. The discovery that the association of Bahia grass (Paspalum notatum) with Azotobacter paspali in tropical soils resulted in nitrogen fixation stimu- lated new interest in this field. Interest was heightened by the subsequent finding by Dobereiner in Brazil that an associative symbiotic relationship between Digitaria decumbens, cultivar "transvala," and the microorganism Azospirillum lip oferum also brought about nitrogen fixation. It was reasoned that tropical grasses with their more efficient 4-carbon photosynthetic cycle could indeed provide the abundance of energy needed for fixation that was lacking in other systems. The enthusiasm has been dampened somewhat by the great variability that has characterized field studies, and more study is needed to identify the limiting factors and devise agronomic practices to bypass them. Large vari- ations in growth cycles are observed with cereals, and they appear to fix nitrogen only during the reproductive phase. Interactions of nitrogen fixa- tion, nitrate assimilation, and denitrification raise the question whether the nitrogen is being lost as rapidly as it is being fixed. On the other hand, nitrate reductase-negative mutants of Azospirillum species are now available that fix nitrogen in the presence of high levels of nitrate. Much more study is needed to identify the factors important for vigorous fixation and to reduce the high degree of variability. Limitations Too little is known of the physiology of this unique associative symbiosis for it to be used effectively. In some cases, for instance, the organism may enter the root cortex, but fail to proliferate enough to effect significant nitrogen fixation. We need to know the reason for this. To date, firm data have not been published to establish that Azospirillum and free-living nitrogen fixers contribute substantial amounts of nitrogen and increase crop yields under field conditions. The conditions required for good inoculation trials are difficult to attain. These conditions are: 1) low numbers of microorganisms already present in the root zone with dinitrogen fixing capability; 2) inoculum able to compete
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76 MICROBIAL PROCESSES with established root-zone bacteria; 3) soil conditions that will support pro- liferation of the organism to produce a large biomass of cells; 4) low levels of available combined nitrogen in the soil; and 5) adequate substrate or plant exudate to supply the energy required for fixation. Research Needs · More knowledge is needed on the physiology of free-living, nitrogen- fixing bacteria. Drily a few strains of the microorganisms, and even fewer genotypes of the host plants, have been studied. A report on nitrogen fixation in wheat, Triticum aest~vam, is of particular interest. Roots from lines con- taining the S-D chromosome were covered with a gelatinous material (prob- ably a polysaccharide) which favored proliferation and nitrogen fixation by gram-positive bacteria within the gelatinous layer on the roots in contrast to wheat lines without this chromosome. O Mass screening for nitrogen-fixing activity of numerous grass genotypes and strains of microorganisms should prove rewarding. · Greater emphasis should be placed on field studies. Acetylene-reduction tests should be used when needed, but in field studies, increased yields and higher quality are of greater significance than the amount of nitrogen fixed. · Attempts Would be made to modify the rhizosphere bacterial com- munity to allow the inoculum strain to become established. References and Suggested Reading Rhizobium-Leguminous Plant Associations Brill, W. J. 1977. Biological nitrogen fixation. Scientific American 236:68-74. Buchanan, R. E., and Gibbons, N. E., eds. 1974. Bergey's manual of determinative bacteriology. 8th edition. Baltimore: The Williams and Wilkins Co. Burns, R. C., and Hardy, R. W. F. 1975. Nitrogen fixation in bacteria and higher plants. Berlin: Springer-Verlag. Burris, R. H. 1975. The acetylene reduction technique. In Nitrogen fixation by free- living microorganisms: International Biological Programme 6, pp. 249-257. Cam- bridge, England: Cambridge University Press. Burton, J. C. 1967. Rhizobium culture and use. In Microbial technology, H. J. Peppler, ea., pp. 1-33. Huntington, New York: Robert E. Krieger Publishing Co. Evans, H. J. 1969. How legumes fix nitrogen. In Crops grown-a century later, Agricul- tural Experiment Station Bulletin No. 708, pp. 110-127. New Haven: Connecticut Agricultural Experiment Station. 1975. Enhancing biological nitrogen fixation: proceedings of a workshop held on June 6, 1974. Sponsored by Energy Related Research and the Division of Biological and Medical Sciences of the National Science Foundation. Washington, D.C.: U.S. National Science Foundation. Fred, E. B.; Baldwin, I. L.; and McCoy, E. 1932. Root-nodule bacteria and leguminous plants. Madison: University of Wisconsin Press. Hardy, R. W. F., and Havelka, U. D. 1970. Nitrogen fixation research, a key to world food. Science 188:633-643. Silver, W. S., and Hardy, R. W. F. 1976. Biological nitrogen fixation in forage and livestock systems. American Society of Agronomy Special Publication No. 28, pp. 1-34. Madison, Wisconsin: American Society of Agronomy.
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NITROGEN FIXATION 77 Skinner, K. J. 1976. Nitrogen fixat~on-key to a brighter future for agriculture. Chemical and Engineering News 54:22-35. Frankia-Non leguminous Plant Associations Allen, E. K., and Allen, O. N. 1964. Non-leguminous plant symbiosis. In Microbiology and soil fertility, 25th Annual Biology Colloquium, C. M. Gilmour and O. N. Allen, eds., pp. 77-106. Corvallis: Oregon State University Press. Becking, J. H. 1977. Dinitrogen-fixing associations in higher plants other than legumes. In A treatise on dinitrogen Taxation, R. W. F. Hardy and W. Silver, eds., Section III: Biology, pp. 185-276. New York: John Wiley and Sons. Bond, G. 1974. Root-nodule symbiosis with actinomycete-like organisms. In The biology of nitrogen fixation, A. Quispel, ea., pp. 342-378. Amsterdam: North-Holland Pub- l~ishing Co. Callaham, D.; Tredici, P. D.; and Torrey, J. G. 1978. Isolation and cultivation in vitro of the actinomycete causing root nodulation in Comptonza. Science 199:899-902. Silvester, W. B. 1977. Dinitrogen fixation by plant associations excluding legumes. In A treatise on dinitrogen fixation, R. W. F. Hardy and A. H. Gibson, eds., Section IV: Agronomy and ecology, pp. 141-190. New York: John Wiley and Sons. Torrey, J. G. 1978. Nitrogen fixation by actinomycete-nodulated angiosperms. Bio- Science 28:586-592. Azolta-Anabaena Associations Becking, J. H. 1975. Contribution of plant-algae associations. In Proceedings of the International Symposium on Nitrogen Fixation, pp. 556-580. Pullman: Washington State University Press. Mague, T. H. 1977. Ecological aspects of dinitrogen fixation by blue-green algae. In Treatise on dinitrogen fixation, R. W. F. Hardy and A. H. Gibson, eds., Section IV: Agronomy and ecology, pp. 85-140. New York: John Wiley and Sons. Moore, A. W. 1969. Azolla: biology and agronomic significance. Biological Review 35: 35-37. Peters, G. A. 1975. Studies on the Azolla: Anabaena symbiosis. In Proceedings of the International Symposium on Nitrogen Fixation, W. E. Newton and C. J. Nyman, eds., pp. 592-610. Pullman: Washington State University Press. 1978. Blue-green algae and algal associations. BioScience 28:580-585 Blue-Green Algae Burris, R. H. 1975. The acetylene-reduction technique. In Nitrogen fixation by free- living microorganisms. International Biological Programme 6, pp. 249-257. Cam- bridge, England: Cambridge University Press. Dart, P. J., and Day, J. M. 1977. Non-symbiotic nitrogen fixation in soil. In Soil micro- biology, N. Walker, ea., pp. 225-252. New York: John Wiley and Sons. Fogg, G. E. 1971. Nitrogen fixation in lakes. In Plant and soil special volume: biological nitrogen fixation in natural and agricultural habitats. Proceedings of the Technical Meetings on Biological Nitrogen Fixation of the International Biological Program (Section PP-N), Prague and Wageningen, 1970, T. A. Lie and E. G. Mulder, eds., pp. 393-401. The Hague: Martinus Nijhoff. Hendrikkson, E. 1971. Algae nitrogen fixation in temperate regions. In Plant and soil special volume: biological nitrogen fixation in natural and agricultural habitats. Pro- ceedings of the Technical Meetings on Biological Nitrogen Fixation of the Inter- national Biological Program (Section PP-N), Prague and Wageningen, 1970, T. A. Lie and E. G. Mulder, eds., pp. 415-419. The Hague: Martinus Nijhoff. Rinaudo, G.; Balandreau, J.; and Dommergues, Y. 1971. Algae and bacterial non- symbiotic nitrogen fixation in paddy soils. In Plant and soil special volume: biological nitrogen fixation in natural and agricultural habitats. Proceedings of the Technical Meetings on Biological Nitrogen Fixation of the International Biological Program (Section PP-N), Prague and Wageningen, 1970, T. A. Lie and E. G. Mulder, eds., pp. 471-479. The Hague: Martinus Nijhoff.
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78 MICROBIAL PROCESSES Stewart, W. D. P. 1966. Nitrogen fixation by free-living organisms. In Nitrogen fixation in plants, pp. 68-83. London: Athlone Press. Distributed in the United States by Humanities Press, Atlantic Highlands, New Jersey. , ed. 1976. Blue-green algae. Nitrogen-fxation by free-living micro-organisms. Inter- national Biological Programme Series 6, pp. 129-229. Cambridge, England: Cambridge University Press. Free-Living Nitrogen-Fixing Bacteria Barber, L. E.; Tjepkema, J. D.; Fussell, S. A.; and Evans, H. J. 1976. Acetylene reduction (nitrogen fixation) associated with corn inoculated with Spirillum. Applied and Envi- ronmental Microbiology 32:108-113. Burris, R. H.; Albrecht, S. L.; and Okon, Y. 1978. Physiology and biochemistry of Spirillum lipoferurrt In Limitations and potentials for biological nitrogen fixation in the tropics, Vol. 10, Basic Life Sciences, Proceedings of a Conference on Limitations and Potentials of Biological Nitrogen Fixation in the Tropics, Brasilia, Brazil Johanna Dobereiner, Robert H. Burris, Alexander Hollaender, Avilio A. Franco Carlos A. Neyra, and David Barry Scott, eds., pp. 303-315. New York: Plenum Press. Dart, P. J., and Day, J. M. 1975. Nitrogen fixation in the field other than by nodules. In Soil microbiology: a critical view, Norman Walker, eden pp. 225-252. London: Butter- worth's Scientific Publications. Knowles, R. 1977. The significance of asymbiotic dinitrogen fixation by bacteria. In A treatise on dinitrogen fixation, R. W. F. Hardy and A. H. Gibson, eds., Section IV: Agronomy and ecology, pp. 33-84. New York: John Wiley and Sons. Krieg, N. R., and Tarrand, J. J. 1977. Taxonomy of the root-associated nitrogen fixing bacterium Spirillum lipoferum. In Limitations and potentials for biological nitrogen fixation in the tropics, Vol. 10, Basic Life Sciences, Proceedings of a Conference on Limitations and Potentials of Biological Nitrogen Fixation in the Tropics. Brasilia, Brazil. Johanna Dobereiner, Robert H. 3urris, Alexander Hollacnder? Avilio A. Franco, Carlos A. Neyra and David Barry Scott, eds., pp. 317-333. New York: Plenum Press. c, O Research Contacts Rh/zobium-Leguminous Plant Associations R. H. Burris, Department of Biochemistry, University of Wisconsin, Madison, Wisconsin 53706, U.S.A. R. A. Date, CSIRO, The Cunningham Laboratory, Mill Road, St. Lucia, Queensland, Australia, 4067. Deane F. Weber, Cell Culture and Nitrogen Fixation Laboratory, Beltsville Agricultural Research Center, U.S. Department of Agriculture, Beltsville, Maryland 20705, U.S.A. Frankia-Nonlegum~nous Plant Associations J. H. Becking, Institute for Atomic Sciences in Agriculture, 6 Keyenbergseweg, Postbus 48, Wageningen, The Netherlands. W. B. Silvester, Department of Biological Sciences, University of Waikato, Private Bag, Hamilton, New Zealand. Azolla-Anabeena Associations Alan W. Moore, CSIRO, The Cunningham Laboratory, Mill Road, St. Lucia, Queensland, Australia, 4067. D. W. Rains, Plant Growth Laboratory, University of California, Davis, California 95616, U.S.A.
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NITROGEN FIXATION 79 Blue-Green Algae T. M. Mague, Bigelow Laboratory for Ocean Sciences, McKown Point, West Boothbay Harbor, Maine 04575, U.S.A. W. D. P. Stewart, Department of Biological Sciences, University of Dundee, Dundee, DD1 4HN, Scotland. I. Watanabe, International Rice Research Institute, Los Banos, The Philippines. Free-Living Nitrogen-Fixing Bacteria Lynn Barber, Department of Microbiology, Oregon State University, Corvallis, Oregon 97331, U.S.A. R. H. Burris, Department of Biochemistry, University of Wisconsin, Madison, Wisconsin 53706, U.S.A. Johanna Dobereiner, EMBRAPA, 23460 Seropedica, Rio de Janeiro, Brazil. David H. Hubbell, Soil Science Department, University of Florida, Gainesville, Florida 32611, U.S.A.
Representative terms from entire chapter: