Biochemical studies on the effect of various carbon sources on growth,nitrogen fixtion,and main cellular constituents of azotobacter vinelandii,strain IV grown under various cultivation conditions

The accessibility of different carbon compounds to Azotobacter vinelandii and the productivity of nitrogen fixation were studied under static and shaking culture conditions. The nature of the carbon source applied was found to affect the yield of bacterial mass and nitrogen metabolism of the tested organism. On the basis of the efficiency of dinitrogen fixation and the yield efficiency ratio it was obvious that (sucrose + mannitol) as a source of carbon is optimum for both growth and dinitrogen fixation by A. vinelandii grown under static and shaking culture conditions. Furthermore, it was found that the highest crude protein efficiency ratio (14.6) and total carbohydrates efficiency ratio (4.3) were obtained with (sucrose + mannitol) as energy source for this organism under shaking culture condition. The experimental organism is able to convert the soluble nitrogenus substances present in molasses into more complex protein as well as to utilize the molasses as a source of energy for the fixtion of atmospheric nitrogen. The tested organism was unable to utilize sodium salicylate as the sole source of carbon.     

Effects of photoperiodicity and light irradiance on phosphate-limited Oscillatoria agardhii in chemostat cultures

Oscillatoria sp. strain 23 is a filamentous, non-heterocystous cyanobacterium that fixes nitrogen aerobically. Although, in this organism nitrogenase is inactivated by oxygen a high tolerance is observed. Up to a pO₂ of 0.15 atm, oxygen does not have any measurable effects on acetylene reduction. Higher concentrations of oxygen inhibited the activity to a relatively high degree. Evidence for two mechanisms of oxygen protection of nitrogenase in this cyanobacterium was obtained. A high rate of synthesis of nitrogenase may allow the organism to maintain a certain amount of active enzyme under aerobic conditions. Secondly, a switch off/on mechanism may reversibly convert the active enzyme into a non-active form which is insensitive to oxygen inactivation after a sudden and short-term exposure to high oxygen concentrations. It is conceived that these mechanisms in addition to a temporal separation of nitrogen fixation from oxygenic photosynthesis sufficiently explain the regulation process of aerobic nitrogen fixation in this organism.Abbreviations DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - CAP chloramphenicol     

Effect of dissolved oxygen on nitrogen fixation by A. vinelandii. I. Free cell cultures

As part of an investigation into the use of biological nitrogen fixation for fertilizer ammonia production, continuous culture studies of respiration and nitrogen fixation in the aerobic bacteria Azotobacter vinelandii under oxygen-limited conditions were conducted. Respiration and growth rates followed Monod forms with respect to dissolved oxygen concentration. However, specific nitrogen fixation rate and nitrogenase activity exhibited maximum values at dissolved oxygen concentrations of ca. 0.02 mM (10% of air saturation). These results suggest careful control of oxygen in the environment is necessary to optimize fixed nitrogen production by this organism.     

Nitrate levels affect the development of the black locust-Rhizobium symbiosis

Summary Experiments with black locust (Robinia pseudoacacia L.) seedlings grown under strictly controlled laboratory conditions indicated that the availability of nitrate has a marked impact on nitrogen fixation. When nitrate concentrations were very low, both nodulation and seedling growth were impaired, whereas nitrate concentrations high enough to promote plant growth strongly inhibited symbiotic nitrogen fixation. When nitrate was added to the growth medium after infection, nodulation and nitrogen fixation of the seedlings decreased. This effect was even more marked when nitrate was applied before infection with rhizobia. Higher nitrogen concentrations also reduced nodule number and nodule mass when applied simultaneously with the infecting bacteria. The contribution of symbiotic nitrogen fixation to black locust shoot mass by far exceeded its effects on shoot length and root mass. When nitrate availability was very low, specific nitrogen fixation (i. e. nitrogenase activity per nodule wet weight) was improved with increasing nitrogen supply, but rapidly decreased with higher nitrogen concentrations.     

Robust biological nitrogen fixation in a model grass–bacterial association

Nitrogen‐fixing rhizobacteria can promote plant growth; however, it is controversial whether biological nitrogen fixation (BNF) from associative interaction contributes to growth promotion. The roots of Setaria viridis, a model C₄ grass, were effectively colonized by bacterial inoculants resulting in a significant enhancement of growth. Nitrogen‐13 tracer studies provided direct evidence for tracer uptake by the host plant and incorporation into protein. Indeed, plants showed robust growth under nitrogen‐limiting conditions when inoculated with an ammonium‐excreting strain of Azospirillum brasilense. ¹¹C‐labeling experiments showed that patterns in central carbon metabolism and resource allocation exhibited by nitrogen‐starved plants were largely reversed by bacterial inoculation, such that they resembled plants grown under nitrogen‐sufficient conditions. Adoption of S. viridis as a model should promote research into the mechanisms of associative nitrogen fixation with the ultimate goal of greater adoption of BNF for sustainable crop production.     

Relationship between growth,nitrogen fixation and assimilation in a legume (Medicago sativa L.)

Summary Symbiotic N₂ fixation, NO ₃ ⁻ assimilation and protein accumulation in the shoots were measured simultaneously in alfalfa (Medicago sativa L.) grown in the field or in pots, in order to study how the balance between the two modes of nitrogen nutrition could be influenced by agronomic factors, such as harvest, mineral nitrogen supply and drought stress. During periods of rapid growth, fixation and assimilation may function simultaneously; they are antagonistic at the beginning and at the end of the growth cycle, when the nitrogen requirement of the plant is lower. When nitrogen nutrition does not limit growth, mineral nitrogen supply favours assimilation at the expense of fixation, but does not modify the amount of nitrogen accumulated, which is adjusted to the growth capacity of the plant. After cutting, nitrate assimilation compensated for the decrease in fixation and supplied the plant with the nitrogen required by the regrowth, the proliferation of which determined the fixation recovery. Drought stress decreased N₂ fixation much more than NO ₃ ⁻ assimilation. The latter made growth recovery possible when water supply conditions became normal again. These results suggested the existence of an optimum level of nitrate assimilation, which differed depending on the age of the plants and allowed both maximum growth and fixing activity.     

Influence of Different Ecological Groups of Earthworms on the Intensity of Nitrogen Fixation

The results of studying nitrogen fixation in organic substrates processed by different ecological groups of earthworms suggest that the earthworms Aporrectodea caliginosa actively stimulate nonsymbiotic nitrogen fixation. In this respect, they exceed the manure worms Eizenia foetida tens of times due to the formation in the organic substrate of conditions favorable for the nitrogen fixing bacteria, and namely: low content of nitrogen easily accessible for microorganisms, changes in the structure of the microbial community of the substrate in favor of non-spore forms of bacteria, and suppression of the growth of saprophyte bacilli, the main competitors of nitrogen fixing bacteria for carbon nourishment sources.     

<Emphasis Type="Italic">Bd</Emphasis> oxidase homologue of photosynthetic purple sulfur bacterium <Emphasis Type="Italic">Allochromatium vinosum</Emphasis> is co-transcribed with a nitrogen fixation related gene

Purple sulfur bacteria, which are known to be the most ancient among anoxygenic phototrophs, play an important role in the global sulfur cycle. Allochromatium vinosum oxidizes reduced sulfur compounds such as hydrogen sulfide, elemental sulfur and thiosulfide. At low oxygen concentrations, A. vinosum can grow chemotrophically using oxygen as the terminal electron acceptor. Being also a nitrogen fixer, A. vinosum is faced with the paradox of co-existence of aerobic metabolism and nitrogen fixation. Due to growth difficulties, only a few studies have dealt with the aerobic metabolism of the organism and, until now, there has been no information about the genes involved in the respiratory metabolism of purple sulfur bacteria. In this article we show the first terminal oxidase gene for A. vinosum. The presence of a Bd type of quinol oxidase is necessary to protect nitrogenases against the inhibitory effects of oxygen. In this case, a nitrogen fixation related gene is part of the cyd operon and this gene is co-transcribed with cydAB genes. Bd oxidase of A. vinosum may be the earliest form of oxidase where the function of the enzyme is to scavenge the contaminant oxygen during nitrogen fixation. This may be an important clue about the early evolution of oxygenic photosynthesis, perhaps as a protective mechanism for nitrogen fixation.     

Non-symbiotic nitrogen fixation in soil

Summary 1. The fixation of N¹⁵-labeled nitrogen in small vessels of California soil under various conditions of pH, substrate level, oxygen tension, and other soil conditions was observed.2. Nitrate concentrations greater than 1.0–1.5 microequivalents per gram soil were found to suppress nitrogen fixation but not the growth ofAzotobacter.3. Large amounts of nitrogen were fixed when soluble organic substrates (e.g. glucose or sucrose) were added to the soil.4. Moderate fixation also resulted from the inversion of a disc of sod.5. Fixed nitrogen appeared largely in the nitrate and ammonia-amide fractions with that in the nitrate fraction probably representing nitrification of more reduced initial products of fixation.6. Under conditions of these experiments growing grass did not enhance fixation. At higher light intensities, however, such an enhancement might be observed.7. The incorporation of grass cuttings, straw or alfalfa meal into the soil caused only a slight increase in fixation.8. The inoculation of soils with large populations ofAzotobacter did not result in increased fixation.This investigation was supported in part by a contract with the United States Atomic Energy Commission.     

Phytotoxin resistance ofRhizobium meliloti mutants

The efficiency of twoRhizobium meliloti mutants for nodulation and nitrogen fixation (acetylene reduction) under three concentrations ofp-coumaric and ferulic acids, was evaluated using a sand-culture medium. The effect of the test concentrations of both acids on the growth of alfalfa was also determined. The results revealed that the test mutants showed considerable resistance to the higher concentration of the test phytotoxins in terms of nodulation and nitrogen fixation, whereas seedling growth was inhibited by increased phytotoxin concentrations. The possible advantages of mutants resistance to the phytotoxins under field conditions are briefly discussed.     

Ammonium-nitrogen metabolism and nitrogen fixation in azotobacter vinelandii

The probable effect of increasing levels of ammonium nitrogen on the growth, efficiency of nitrogen fixation, and main cellular constituents of Azotobacter vinelandii was studied under shaking and static culture conditions. The presence of NH₄⁺-N up to 50 mgl^-1 level has no harmful effect on the multiplication as well as the yield efficiency ratio of the tested organism. A. vinelandii was able to fix dinitrogen in the presence of NH₄⁺-N when both nitrogen sources were available in the culturing medium. The efficiency of nitrogen fixation was affected by the initial presence of NH₄⁺-N in the medium, it was quite low at the highest level. The crude protein efficiency ratio was correlated inversely with the initial NH₄⁺-N concentration, whereas the total carbohydrate efficiency ratio as well as the total lipid efficiency ratio were positively correlated with the NH₄⁺-N concentration. The presence of NH₄⁺-N in the culturing medium has no essential influence on the qualitative composition of the amino acids in the Azotobacter cells.     

Field measurements of nitrogen-fixing activity of intact saplings ofAlnus maximowiczii in the subalpine zone of Mt Fuji

Nitrogen-fixing (acetylene-reducing) activity of intact saplings ofAlnus maximowiczii was measured under natural conditions in the subalpine zone of Mt Fuji. The nitrogen-fixing activity was detected from the middle of June when expansion of leaves had just begun to the end of October when the shedding of leaves was almost completed. Diurnal changes in the activity were almost parallel with those of ground temperature. The measured nitrogen-fixing activity was related to ground temperature and total leaf area. Using this relation, annual nitrogen fixation was estimated from the data of ground temperature and leaf area measured in the field. The amount of annual nitrogen fixation was almost the same as that of nitrogen used for annual growth. It was concluded that nitrogen fixation by nodules made a considerable contribution to the nitrogen economy in the saplings ofA. maximowiczii.     

The Fixation of Nitrogen associated with the Root Nodules of Myrica gale L., with Special Reference to its pH Relation and Ecological Significance

The paper deals with the fixation of nitrogen by nodulated plantsof Myrica gale under experimental conditions, the fixation being,in the first year of development, of a magnitude comparableto that of legumes under similar conditions. The effect of acidityon the fixation has been investigated. The Myrica organism ismarkedly adapted to acid conditions. The observations are correlatedwith field data in Britain and help to explain the distributionand habits of the species.     

Influence of host cultivars and Bradyrhizobium strains on the growth and symbiotic performance of soybean under salt stress

This paper examines the importance of salt tolerance of host cultivars, Bradyrhizobium strains, and host-Bradyrhizobium combinations on the symbiotic nitrogen fixation potential of soybean under NaCl and KCl salt stress. Plants were grown in a soil medium, and the experiments were conducted under controlled environment growth room conditions. Bradyrhizobium growth was examined in yeast-mannitol broth andB. japonicum strains tolerant of NaCl and KCl (80 mM) stress were identified. Soybean cultivar Williams, which was sensitive to salt stress, performed poorly both in growth and symbiotic nitrogen fixation, irrespective of whether it was matched with a tolerant or sensitive Bradyrhizobium strain. Tolerant cultivar Manchu sustained nodulation and nitrogen fixation, irrespective of whether it was matched with a tolerant or sensitive Bradyrhizobium strain. Evidence presented here suggests a need, first to select soybean cultivars that are tolerant to salt stress, and then to match them with tolerant and effective Bradyrhizobium strains.     

Physiological and biochemical studies on nitrogen fixing blue-green algae

Summary Experiments were made to investigate the influence exerted by calcium, strontium, cobalt and molybdenum on the growth and nitrogen fixation of a local strain of Nostoc commune.Calcium, cobalt and molybdenum at least in micro amounts were found to be required for the achievement of best growth and highest nitrogen fixation.Calcium seems to be irreplaceable by strontium, the latter element exerted a strong inhibitory action on both growth and nitrogen fixation processes.     

Geosiphon pyriforme,an Endosymbiotic Consortium of a Fungus and a Cyanobacterium (Nostoc), Fixes Nitrogen

The bladders of Geosiphon pyriforme, an endosymbiotic consortium of a fungus and the cyanobacterium Nostoc punctiforme, show nitrogenase activity. This suggests that the organism is capable of nitrogen fixation.     

Some characteristics of Methylococcus mobilis sp. nov.

A methane-assimilating coccus was isolated from a continuous culture. Under certain conditions, in pure culture the cells grew in sarcina-like, refractile clusters.That these clusters resembled multiple-bodied cysts was confirmed by electron microscopy. In addition, tube-like structures, not previously reported in methane-oxidizing bacteria, were found. Motile cells, with one or, rarely, two flagella were occasionally observed. Methane and methanol were exclusively assimilated as the sole source of carbon and energy. Formaldehyde, formate, lower alkanes, alcohols and aldehydes, with the exception of acetaldehyde, were oxidized by cell suspensions. The presence of a number of key enzymes in cell-free extracts suggested that the ribulose-monophosphate pathway of carbon assimilation is active. This agreed with the internal membrane structure found (type I organism).Nitrate, ammonia, peptone, yeast extract, asparagine, aspartic acid and glutamic acid were assimilated as sources of nitrogen. No nitrogen fixation could be demonstrated.The cells contained 9.1–9.7% nitrogen and 7–10% lipid, dependent on the growth conditions. Poly--hydroxybutyric acid could not be detected. The guanine+cytosine (G+C) content of the DNA was found to be 56.3%. As a name for the organism Methylococcus mobilis sp. nov. is proposed.Abbreviations used G+C Guanine+Cytosine     

Nodulation and nitrogen fixation in extreme environments

Biological nitrogen fixation is a phenomenon occurring in all known ecosystems. Symbiotic nitrogen fixation is dependent on the host plant genotype, theRhizobium strain, and the interaction of these symbionts with the pedoclimatic factors and the environmental conditions. Extremes of pH affect nodulation by reducing the colonization of soil and the legume rhizosphere by rhizobia. Highly acidic soils (pH<4.0) frequently have low levels of phosphorus, calcium, and molybdenum and high concentrations of aluminium and manganese which are often toxic for both partners; nodulation is more affected than host-plant growth and nitrogen fixation. Highly alkaline soils (pH>8.0) tend to be high in sodium chloride, bicarbonate, and borate, and are often associated with high salinity which reduce nitrogen fixation. Nodulation and N-fixation are observed under a wide range of temperatures with optima between 20–30°C. Elevated temperatures may delay nodule initiation and development, and interfere with nodule structure and functioning in temperate Iegumes, whereas in tropical legumes nitrogen fixation efficiency is mainly affected. Furthermore, temperature changes affect the competitive ability ofRhizobium strains. Low temperatures reduce nodule formation and nitrogen fixation in temperate legumes; however, in the extreme environment of the high arctic, native legumes can nodulate and fix nitrogen at rates comparable to those observed with legumes in temperate climates, indicating that both the plants and their rhizobia have successfully adapted to arctic conditions. In addition to low temperatures, arctic legumes are exposed to a short growing season, a long photoperiod, low precipitation and low soil nitrogen levels. In this review, we present results on a number of structural and physiological characteristics which allow arctic legumes to function in extreme environments.