Acetylene Reduction Test Research Articles

Pigment distribution and nitrogen fixation inAnabaena azollae

The symbiotic heterocystous cyanobacteriumAnabaena azollae present in the leaf cavities of the water fernAzolla spp. was studied. The cyanobacteria extracted from the leaf cavities showed differences in pigment composition in three species ofAzolla, i.e A.pinnata var.pinnata, A.caroliniana and A.filiculoides, as observed by pigment absorption and epifluorescence tests. These differences suggest that of these species the cyanobiont ofA. pinnata is the most actively nitrogenfixing form. This has been confirmed by nitrogen fixation (acetylene reduction) tests. Heterocysts of the symbiont ofA. pinnata were characterized by high chlorophylla and low phycocyanin content, a low fluorescence yield of chlorophyll in the heterocysts compared to vegetative cells and a gradient of phycocyanin concentration in the vegetative cells adjacent to heterocysts. This indicates that only photosystem I is present in the heterocyst. In the two otherAzolla species quantitative shifts in the pigment composition occurred suggesting a lower nitrogen fixation activity. In the cyanobiontAnabaena azollae the heterocyst frequency could reach a value of 44–45%. It is argued that there are two generations of heterocysts in a matureAzolla plant, which are concomitant with two peaks of nitrogen fixation activity correlated with leaf age,i.e. leaf number along the main axis of the plant. At both peaks of maximal N2-ase activity, only 20–25% of the heterocysts present are metabolically active as demonstrated by the reduction of Neotetrazolium chloride (NTC) in the heterocysts and darkening of nuclear emulsions by silver salt reduction. Vegetative cells of the cyanobiont reduce Neotetrazolium chloride (NTC) to formazan more rapidly than has been observed in the free-living heterocystous cyanobacteriumAnabaena cylindrica tested in parallel experiments. This feature may be due to a more permeable cell wall of the vegetative cells of the cyanobiont compared to the free-living form, since the vegetative cells of the symbiont play a role in cross-feeding of the host (Azolla). Evidence is obtained that only the heterocysts of the cyanobiont ofAzolla are involved in the nitrogen fixation process as in free-living heterocystous cyanobacterium species. This situation is different from other cyanobacterial symbioses such as inGunnera, Blasia andAnthoceros, where physiological modifications are reported in the symbiosis with another photosynthetic partner such as the absence of O2 evolution and the absence of photo-fixation of CO2 in the cyanobionts. Pigment composition and N2-ase activity in the symbiotic cyanobacteria of three Azolla species have indicated the superiority of theA. pinnata symbiont.A. pinnata var.pinnata is a semidomesticated form used in S.E. Asia for agricultural purposes (irrigated rice culture) to increase soil fertility. It is suggested that by selection (domestication) more efficient strains (clones) can be obtained, and further that with more advanced techniques such as gene mutation and genetic manipulation even more efficient and for agriculture more beneficial clones can be obtained.

Transfer of nitrogen fixation genes into Pseudomonas putida isolated from Finnish tundra soil.

The plasmid pRD1 containing the nif genes from Klebsiella pneumoniae was transferred by conjugation from Escherichia coli to Pseudomonas putida isolated from the tundra soil. 6-Cyanopurine, acetylene reduction and immunological tests showed that the nif genes were not expressed in P. putida. Existence of the nif genes in P. putida transconjugants was detected by transferring them to E. coli C600, which does not fix nitrogen. Existence of the nif genes in E. coli C600 transconjugants was detected immunologically and by acetylene reduction tests.

Nodulation of legumes, nitrogenase activity of roots and occurrence of nitrogen-fixing Azospirillum ssp. in representative soils of central Amazonia

Leguminosae do not predominate in the Brazilian Amazon rain forest, although they are among the five best represented families. Plant roots from various soils were examined for the presence of nodules, acetylene-reducing activity and N 2-fixing Azospirillum spp. Abundant nodulation was found in black earth (“terra preta dos índios”) and in one case on sandy soil under campinarana vegetation along the tributary of the upper Rio Negro. Nodules were not found on plants from many other soils and sites in this area, nor in primary or secondary forest on clayey latosol near Manaus. In sandy latosol some nodules occured in secondary forest and fewer in primary forest. Legumes in distributed clayey or sandy latosol showed more frequent nodulation. Primary forest on alluvial (“várzea”) soil, and in Bahia coastal rain forest on sandy latosol and Erythrina glauca used for shading cacao plantation were abundantly nodulated. Acetylene reduction assays showed no, or very little, nitrogenase activity of roots from forest on sandy latosol showed acetylene-reducing activity. High rates of acetylene reduction were observed in nodulated roots of primary on alluvial “várzea” soil. Root samples showed ethylene absorption in controls without acetylene which migh interface with the results of acetylene reduction tests. The incidence of Azospirillum was also higher in black earth than the other soils examined, and in soils with higher pH. The hypothesis that Azospitillum is associated with Trema micrantha roots was refuted. Roots and soils collected under cultivated grasses showed a higher incidence of Azospirillum when fertilized with phosphorus and line. Results indicate the nitrogen fixation did occur in association with roots in some soils, but not with roots of primary or secondary forest on clayey latosol in the vicinity of Manaus, which is the most common soil in Central Amazonia. The possible reasons for this are discussed.

Screening for mutants of Rhizobium japonicum with defects in nitrogen fixing ability.

Mutants of Rhizobium japonicum with reduced ex planta nitrogenase activity could be isolated with high frequency by direct screening of ultra-violet mutagenized bacteria growing as spots on the surface of an appropriate agar medium permitting derepression of nitrogenase synthesis. Small glass chambers fitted with a serum cap were pushed into the agar around each spot of growth, forming a small enclosed gas space which was made to 10% acetylene, permitting assessment of nitrogenase activity by the acetylene reduction test. Four mutants were isolated from a total of 305 screened spots. Two mutants had almost no ex planta activity, one of which had no symbiotic activity despite normal nodulation (ineffective), the other had only somewhat reduced activity symbiotically. Two other mutants with less than half wild-type activity ex planta were normal in symbiosis.

Enrichment and isolation of new nitrogen-fixing hydrogen bacteria

An enrichment method for nitrogen fixing hydrogen bacteria is described. The procedure invariably resulted in the isolation of yellow-pigmented coryneform bacterial strains assigned to Corynebacterium autotrophicum. The procedure included a serial transfer in an ammonium-free mineral liquid medium under an atmosphere of 10% hydrogen, 5% oxygen, 10% carbon dioxide and 75% nitrogen, followed by a short alkali treatment and by streaking on nutrient broth-succinate agar. The ability to fix nitrogen was confirmed by the acetylene reduction test and by 15N2 incorporation.

Nitrogen‐fixing Bacteria Associated with Graminaceous Roots with Special Reference to Spirillum lipoferum Beijerinck

AbstractMethods and results for field estimation of nitrogen fixation in the rhizosphere of grasses and cereals and the problem of choosing a reliable method for estimates based on acetylene reduction tests are reviewed. Growth and nitrogen fixation of Azotobacter spp. and of Spirillum lipoferum in plant rhizospheres are discussed on the basis of their physiology. Special consideration is given to the oxygen sensitivity of the nitrogenase reaction and adaptation to microaerophilic conditions, plant root exudates, plant specificity and genotype, mineral nitrogen and to microbial synergism, antagonism and competition. A better understanding of factors essential for a successful saprophytic competition of S. lipoferum in the root environment could allow utilization of its nitrogen fixing potential in crop production. Examples of growth promoting and ineffective associations of this organism with various graminaceous host plants are given.

Nitrogenase Activity of Rhizobium japonicum Growing on Agar Surfaces in Relation to Slime Production, Growth and Survival

A reproducible, simple method for assessing nitrogenase activity by the acetylene reduction test on agar plates is described. The effects of phosphate, succinate and arabinose on nitrogenase activity and slime production by R. japonicum strain 61-a-101, growing on a solid medium with leucine or glutamine as sole source of fixed nitrogen, were investigated. Slime production varied with different media but there was no simple correlation between slime production and high nitrogenase activity. Arabinose greatly enhanced nitrogenase activity of bacteria growing on glutamine as sole nitrogen source, especially if the amino acid concentration was 1 mm or less. Without arabinose, the optimum concentration of glutamine for nitrogenase activity was 2 to 2·5 mm, but 1 mm-glutamine plus 50 mm-arabinose gave twice the activity [8 to 10 nmol ethylene produced h–1 (mg protein)–1].

LIGHT‐DEPENDENT AND LIGHT‐INDEPENDENT PRODUCTION OF HYDROGEN GAS BY PHOTOSYNTHESIZING RHODOSPIRILLUM RUBRUM MUTANT C

Abstract. Rhodospirillum rubrum mutant C grew photosynthetically in the light and produced copious amounts of H2. During light‐growth mutant C produced 7.9mmol of H2 in medium with 9mmol of Na‐pyruvate per mg protein. When parent strain R. rubrum S1 was grown similarly, these cells only produced a trace amount of H2. Light‐grown mutant C evolved H2 by H2‐nitrogenase and formic hydrogenlyase. Although both hydrogenases were previously detected in R. rubrum S1, the activities of the reactions in light‐grown mutant C were higher and they operated under different conditions. In the parent strain S1, the production of nitrogenase was strongly repressed during growth in medium enriched with organic nitrogen and the cells only reduced 0.06 pmol of acetylene per mg protein after 30 min in the light. Under similar conditions, nitrogenase activity measured by the acetylene reduction test in mutant C was 10‐fold greater. In addition to nitrogenase, mutant C also produced large amounts of H2 with formate as an intermediate when the cells were grown with Na‐pyruvate. Formic hydrogenlyase in mutant C operated equally well in anaerobic light or dark conditions. The analogous formate oxidation reaction in parent strain S1 only functioned in the dark. These data, compared with results with R. rubrum S1 suggested that C was a regulatory mutant. Additional observations suggested that formic hydrogenlyase occurred constitutively in R. rubrum. Pyruvate formate‐lyase, however, which produced formate for formic hydrogenlyase, was only detected in the cells after growth in media with Na‐pyruvate. The reaction was not formed when R. rubrum was grown in the light in media with dl‐malate as the sole carbon substrate.

Nitrogen fixation associated withEichhornia crassipes

Azotobacter associated withEichhornia crassipes were found to be concentrated in the slime formed around petiole bases. ‘Massive’ colonies formed where raw slime was applied to nitrogen-free medium inoculated with Azotobacter. Slime in which Azotobacter had been killed did not stimulate the formation of massive colonies unless live Azotobacter cells were mixed into it. Acetylene reduction tests indicated that Azotobacter associated with Eichhornia did not fix nitrogen unless the plant was suffering extreme nitrogen deficiency, and that the maximum rate of fixation was inadequate for rapid plant growth.

Bacterial degradation of ethylene and the acetylene reduction test

The basic assumption that ethylene is not degraded during the acetylene reduction test was found to be valid for the systems tested. Acetylene inhibited ethylene degradation by both pure cultures and soil, while acetylene-utilizing bacteria did not co-oxidize ethylene.

Enrichment and isolation of nitrogen fixing hydrogen bacteria.

An enrichment method for nitrogen fixing hydrogen bacteria is described. The procedure invariably resulted in the isolation of yellow-pigmented coryneform bacterial strains assigned to Corynebacterium autotrophicum. The procedure included a serial transfer in an ammonium-free mineral liquid medium under an atmosphere of 10% hydrogen, 5% oxygen, 10% carbon dioxide and 75% nitrogen, followed by a short alkali treatment and by streaking on nutrient broth-succinate agar. The ability to fix nitrogen was confirmed by the acetylene reduction test and by 15N2 incorporation.

Nitrogen fixation by nodulated species of Pavetta and Psychotria

Summary Leaves bearing bacteria-filled nodules from seven Pavetta and two Psychotria species were tested for nitrogenase activity by the acetylene reduction method for up to 24 hours with negative results. Four of the Pavetta species and the two Psychotria species used in the acetylene reduction tests were also grown in a nitrogen-free culture solution for 75 days. The plants developed severe symptoms of nitrogen deficiency and no increase in their nitrogen content could be detected during the experimental period.

Environmental effects on the growth of nitrogen‐fixing bacteria

Studies with Azotobacter chroococcum in chemostats have clarified several aspects of the physiology of nitrogen fixation by this obligate aerobe. Sensitivity to excessive oxygenation was specific to nitrogen-fixing populations and was not shown by those assimilating ammonia; C- or P-limited populations were hypersensitive. Oxygen sensitivity was attributable to the known ease with which the enzyme nitrogenase is damaged chemically by oxygen and it represented a response by the organism to prevent such damage to the enzyme in vivo . In the presence of excess of all other nutrients, N 2 fixation was regulated intracellularly and chemostat populations showed the properties normally associated with N-limited populations (inverse relation of biomass to growth rate), complicated by a selection of fast-growing variants at dilution rates approaching wash-out. Populations could become adapted to high oxygen tensions and showed augmented respiration rates and increased contents of cytochrome oxidase. The acetylene reduction test indicated that the nitrogenase content was unchanged but that the optimal P 0 2 value for activity had increased. Thus respiration appeared to perform a protective function; when respiratory protection was inadequate, the organisms were able to inactivate their enzyme rapidly and reversibly. This process has been called “conformational protection” because evidence exists that the enzyme is then physically orientated, possibly by association with subcellular membranes, in a manner in which the oxygen-sensitive components are inaccessible to 0 2 . In this state it would be inactive towards N 2 .

Nitrogen fixation in rumen contents indicated by the acetylene reduction test.

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The presence of N2-fixing bacteria in the intestines of man and animals.

SUMMARY: Cultures of N2-fixing bacteria have been isolated from the intestinal contents of humans, pigs and guinea-pigs. Acetylene-reduction tests indicate that N2-fixation may be occurring in the intestines. Best N2-fixation was by cultures provisionally identified as Klebsiella aerogenes, but other genera were also involved. All cultures fixed more N2 anaerobically than aerobically but some fixation occurred when 20% O2 was present.

Use of Pankhurst tubes to assay acetylene reduction by facultative and anaerobic nitrogen-fixing bacteria.

Pankhurst tubes are simple and inexpensive vessels in which to screen large numbers of bacterial isolates by the acetylene reduction test for nitrogen fixation. A modification is described which allows, in addition, optical density measurements of growth. The method is well suited to most probable number estimations of facultative and anaerobic nitrogen-fixing bacteria in soils and on root surfaces.