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Reprinted from Evans and Barber (1977) by permission of the publisher; copyright AAAS.

favored: (a) the acetylene reduction method and (b) incorporation of the stable isotope of nitrogen, 15N. Like many other metalloenzymes that utilize gaseous substrates, nitrogenase has a broad substrate range and will reduce many small triple-bonded molecules, including acetylene. In the 1960s, it was discovered that the enzyme nitrogenase reduced acetylene to ethylene (CH2 = CCH2), which can be easily measured by gas chromatography. This method was much easier to use than the traditional method of quantifying the incorporation of 15N2 into bacterial or plant biomass. Although the advent of acetylene reduction methodology accelerated progress in our understanding of BNF, acetylene reduction is limited in its ability to accurately quantify N2 fixed in the field over a growing season, and the stoichiometry of C2H2:N2 reduced varies with environmental conditions. Indeed, most soil microbiologists and field scientists generally favor the use of 15N for quantifying N2 fixation under field conditions (Weaver and Danso, 1994). Because 15N2 is expensive and awkward to handle under field conditions, the isotope dilution approach is preferred.

The N in biological systems is composed predominantly of two stable isotopes, 15N and 14N, which constitute about 0.3663 and 99.6337%, respectively, of the N in the atmosphere. A N2-fixing organism will incorporate 14N and 15N from an N source in proportion to the concentrations of the two isotopes in that source. If one of these N sources is atmospheric N2, and because atmospheric N2 generally has a lower percentage of 15N than other sources (soil or fertilizer N, for example), the proportion of soil or fertilizer 15N assimilated by the N2 fixer will be "diluted" by the relative contribution of N2 to the cell N budget. In general, the "isotope dilution" approach to measuring BNF under soil conditions involves the addition of a small amount of 15N-enriched inorganic N to the soil to increase the 15N/14N ratio of available soil N. In principle, the 15N/14N ratio of organisms that assimilate soil N will be the same as the 15N/14N ratio of the available soil N. In the case of an organism that is fixing atmospheric N2, and assimilating soil N, its 15N/14N ratio will be lowered proportional to the amount of N2 being fixed; i.e., the 15N content of the biomass will be "diluted" as a result of BNF. The percentage of N derived from the atmosphere (%Ndfa) is represented by the following equation:

atom%15N excess in N2 -fixing plant %Ndfa = 1 ----2-—-X 100.

atom%15N excess in nonfixing plant free-living n2-fixing bacteria

Diazotrophic prokaryotes can be divided into those that carry out BNF in a symbiotic or commensalistic relationship with a eukaryote and those that fix N2 in a free-living state. Because the major factor usually limiting BNF is the C/energy supply, it is reasonable to predict that free-living photosynthetic diazotrophs would fix greater amounts of N2 under some soil conditions than free-living heterotrophs and that the latter would fix measurable amounts of N2 only in the presence of readily available plant-derived C, e.g., labile C in the rhizosphere zone of an actively growing plant, or during the decomposition of herbaceous and woody plant residues of high C:N ratio. Facultative and obligately anaerobic diazotrophic bacteria are often found in decaying wood, where it is assumed that cellulolytic and ligninolytic fungi depolymerize the sugars and phenolics necessary to support diazotrophs (Sylvester and Musgrave, 1991). A similar situation exists in agricultural straw residues, in which it has been shown that additions of both cellulolytic and dia-zotrophic bacteria enhance BNF and accelerate the decomposition of the N-deficient straw. It remains extremely difficult to quantify the contribution of BNF by free-living diazotrophs to an ecosystem, even in environments in which BNF can be detected by the acetylene reduction method. Estimates of free-living BNF in soil and plant residue-enriched environments usually range between < 1 and 10 kg N ha-1 year-1, with many natural systems occurring in the lower range of these estimates.

associative n2-fixing bacteria

Root secretions and other rhizodepositions are a major source of plant C input to soil, and diazotrophic bacteria are associated with the roots of plants (Reinhold-Hurek and Hurek, 1998). During the last third of the 20th century, many studies demonstrated that a variety of diazotrophic bacteria associate with the roots of tropical grasses, notably Paspalum and Digitaria species, where they fix measurable amounts of N2. These bacteria belong to the genera Azospirillum, Herbaspirillum, and Burkholderia. They are found both in the rhizosphere and in the intercellular spaces of the root cortex. It has been claimed these bacteria can provide between 5 and 30% of the total N accumulated by the plants. Using the 15N isotope dilution technique, Urquiaga et al. (1992) showed that some Brazilian sugarcane cultivars could derive >60% of plant N from BNF. A diazotrophic bacterium, Gluconace-tobacter diazotrophicus, was isolated from sugarcane, found to occupy internal tissues at high cell densities (106 to 107 cells g-1) (Fig. 14.3), and could grow on high sucrose concentrations (10%) and fix N2 optimally at pH 5.5. Kennedy et al. (2000) showed that a cultivar of sugarcane (SP70-1143) inoculated with a wildtype strain of Glu. diazotrophicus and grown under N-limiting conditions produced a higher biomass and contained more total N than plants inoculated with a nif- mutant (cannot fix N2) or that were not inoculated (Table 14.6). When N was not limiting, however, both the wild-type and the nif- mutant stimulated greater growth and total N content of the plants. Several questions remain unanswered. Although 15N2 was incorporated into plants inoculated with wild-type Glu. dia-zotrophicus, it is unclear what portions of the fixed N remain associated with the bacteria versus being transferred to plant tissues. In addition, it still remains unclear to what extent sugarcane growth enhancement can be attributed to BNF or to improved sequestration of soil N brought about by production of plant growth hormone-like compounds by Glu. diazotrophicus.

FIGURE 14.3 Scanning electron micrograph of Herbaspirillum seropedicae within the metaxylem of a sugarcane stem. Note that the bacteria are associated closely with the walls of the vessel. Courtesy of F. Olivares; used by permission.
TABLE 14.6 Plant Dry Weight and Total N Content of Sugarcane Plants Inoculated with Gluconacetobacter PA15 and a Nondiazotrophic Mutant, Mad3A

Treatment

Total plant dry weight (g)

Total N content (mg)

Minus fertilizer N

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