Identification and study of growth and nitrogenase activity of nitrogen fixing cyanobacteria from tropical soil

Identification of cyanobacteria species has been performed on samples coming from two different harvest areas. The most important fixing belongs to Scytonema genus. The other genus identified are Nostoc and Lyngbia. Moreover, these cells are living closely with non-fixing cyanobacteria as well as with bacteria. The growth of cells as well as nitrogenase activity has been studied on a semi-axenic strain of Scytonema, a nitrogen fixing cyanobacterium, isolated from soil crusts. The cell growth is relatively show in liquid medium depleted in combined nitrogen. The growth rate increases when nitrates are supplied to cells. A release of ammonium is observed in medium during cell culture. This release exhibits several maxima and minima during cell growth. The heterocyst cells disappear within four days when filaments are growing in nitrates supplied medium. On the contrary, the heterocyst frequency increases up to more 5% in a nitrogen depleted medium. The heterocyst frequency reaches a maxima after 4 days of culture, then decreases later on. Nitrogenase activity changes during cells growth too. The maximum activity is observed after 5 to 6 days of culture to decrease after even though the cells are still in their exponential phase of growth. Nitrogenase activity increases with light intensity, what indicate a possible relation between photosynthetic and nitrogenase activities.     

Stimulation of nitrogenase activity and photosynthesis in some cyanobacteria by glyoxylate

Abstract The effect og glyoxylate on nitrogenase activity (C₂H₂ reduction) and photosynthesis (H¹⁴CO₃ fixation and O₂ evolution) was in vestigated in the three heterocystous cyanobacteria Anabaena cylindrica, A. variabiltis and N. muscorum. Glyoxylate had virtually no effect on the rate of dark respiration and was unable to sustain photoheterotrophic growth, though some slight stimulation (= 30%) of photorophic growth was noted. A considerable stimulation of both nitrogenase and photosynthetic activities was observed in presence of glyoxylate. In the light the stimulation increased with time up to about 15-25 h after adding optimal concentrations of 4–6 mM glyoxylate. Placing glyoxylate treated samples in the dark or adding DCMU (30 μM) in the light, showed that glyoxylate initially supported significantly higher nitrogenase activity than did samples in absence of glyoxylate. However, after a prolonged incubation in the dark or in presence of DCMU glyoxylate is unable to relieve the adverse effects of such conditions. The stimulation of the nitrogenase activity was even more pronounced when the glyoxylate was added to cells preincubated in the dark (“carbon starved”) than for cells kept constantly in light. The results suggest that glyoxylate, or a metabolite, may act as an inhibitor of cyanobacterial photorespiration and this hypothesis is discussed.     

Heterotrophic metabolism and regulation of uptake hydrogenase activity in symbiotic cyanobacteria

The H₂ uptake activity of three cyanobionts isolated fromCycas revoluta, C. circinalis andazolla filiculoides was shown to be related primarily to the growth rate and independent of the main mode of carbon nutrition. Significant H₂ uptake was found in the coralloid roots ofCycas revoluta andZamia furfuracea (3 and 22 times higher than the respective C₂H₂ reduction activities). The results attained allow us to conclude that in cyanobacteria, in contrast to most nitrogen-fixing heterotrophs, uptake hydrogenase activity is not repressed by carbon substrates and that cyanobacteria in association seem to be endowed with sufficient H₂ uptake capacity to recover all of the H₂ released during the process of N₂-fixation.     

Effect of nitrogen compounds on nitrogenase activity in Azospirillum brasilense

Abstract The effect of certain nitrogen compounds on nitrogenase activity was studied in cells of Azospirillum brasilense strain Sp6, grown under microaerophilic conditions with nitrogenase fully derepressed. 0.5 mM NH₄Cl, 0.5 mM glutamine, 1.0 mM KNO₃ and 0.1 mM KNO₂ completely blocked nitrogenase activity. 1.0 mM asparagine, 1.0 mM aspartate, 1.0 mM histidine and 1.0 mM adenine did not caused no inhibition of nitrogenase; indeed asparagine, aspartate and histidine showed a slight stimulatory effect on N₂ fixation. The addition of 10 mM dl -methionine- dl -sulphoximine prevented the inhibitory effect of NH₄Cl and glutamine but did not counteract the effect of KNO₂. Rifampicin and chloramphenicol did not prevent the inhibition of nitrogenase by NH₄Cl.     

Respiration supported nitrogenase activity of isolated Rhizobium meliloti bacteroids

Bacteroids having a high level of respiration-supported nitrogenase activity were isolated from nitrogen-fixing alfalfa root nodules. Gentle maceration under anaerobic conditions in the presence of sodium succinate and a fatty acid scavenging agent were employed in this method. A large proportion of isolated bacteroids retained a triple membrane structure as shown by transmission electron microscopy. Dicarboxylic acids of the TCA cycle (malate, fumarate, succinate), but not glutamate or aspartate, supported sufficient respiratory activity to supply the nitrogenase system with ATP and reducing equivalents and to protect the nitrogenase system from inactivation by 4% oxygen over a period of 20-30 min. Sugars did not support nitrogenase activity in intact bacteroids. The properties of the isolated bacteroids were ascribed to minimal damage to the cytoplasmic membrane and peribacteroidal membrane during isolation. With succinate as substrate and oxygen as terminal electron acceptor, initial nitrogenase activity was determined at 4% oxygen in the gas phase of the assay system employed. At this oxygen concentration, the sustained rate of acetylene reduction by respiring bacteroids was linear up to 30 min. Bacteroid activity declined rapidly with time of exposure to oxygen above 4% in the gas phase. The optimum temperature range for this activity was 10-20 degrees C. Nitrogenase activity was measurable at incubation temperatures below 10 degrees C under 4% oxygen. Functionally intact bacteroids had little nitrogenase activity under anaerobic conditions in the presence of an external source of ATP and reductant. Treatment of the bacteroids with chlorpromazine eliminated respiration-supported activity and rendered the bacteroid cell membrane permeable to external ATP. Bacteroids treated with chlorpromazine had high acetylene reducing activity with external ATP and dithionite in the absence of oxygen.     

Effect of brassionosteriods on nodulation and nitrogenase activity in groundnut (Arachis hypogaea L.)

The effect of brassinolide, 24-epibrassinolide and 28-homobrassinolide on nodulation and nitrogenase activity of groundnut was studied. The tested brassinosteroids substantially increased both nodulation and nitrogenase activity.     

Immobilized cyanobacteria as a biofertilizer for rice crops; Intl. Conference on Applied Algology, Knysna, South Africa, April 1996.

N₂-fixing cyanobacteria (Anabaena azollae, symbiont strains) were immobilized in polyurethane foam and ammonia production by the cyanobacteria was investigated in the laboratory and rice field. The cyanobacterial symbiont, A. azollae - MPK-SK-AM-24 showed the highest growth rate and biomass production amongst the 5 isolates examined while A. azollae-AS-DS showed the highest nitrogenase activity followed by A. variabilis - SA₀ (wild type, non-symbiotic). Treatment of the foam-immobilized cyanobacteria with the systemic fungicide Bavistin stimulated nitrogenase activity while inhibiting glutamine synthetase (GS) activity. Free-living A. azollae-MPK-SK-AF-38, A. azollae - MPK-SK-AM-24 and A. azollae-MPK-SK-AM-27 excreted the highest amounts of ammonia into the growth medium; under foam - immobilized conditions the ammonia production increased further. Treatment of the foam - immobilized cyanobacteria with the fungicides Bavistin and Vitavax resulted in ammonia production at significantly higher rates. Rice seedlings (var. ADT 36) grown in the laboratory in conjunction with foam - immobilized A. azollae showed increased growth. A field experiment with paddy rice and foam - immobilized A. azollae strains indicated that the cyanobacteria excreted significant amounts of ammonia into the flood water in the rice fields resulting in increased chlorophyll content of the plants and increased the rice grain and straw yields. A combination of fertilizer nitrogen and inoculation with foam - immobilized cyanobacteria also significantly increased the rice grain and straw yield. Additionally, both A. azollae and A. variabilis were immobilized in sugarcane waste (bagasse), added to rice paddy and resulted in increased rice grain yield. This revised version was published online in September 2006 with corrections to the Cover Date.     

Characterization of cyanobacteria isolated from biofilms on stone monuments at Santiniketan,India

Cyanobacterial biofilms occurring on the exterior of three stone monuments at Santiniketan, India were analyzed. Species of Scytonema and Tolypothrix were the major components of these biofilms. Identification was obtained by morphometric procedures and 16S rRNA gene sequencing. Biofilms cultured for prolonged periods revealed the presence of several other cyanobacteria belonging to 14 different genera. Cyanobacteria on stone in the tropical environment of India formed a distinct cluster that was quite different from that of cyanobacteria reported for a similar substratum in temperate regions. Absorption spectra of the organisms from Santiniketan showed a high quantity of scytonemin, mycosporine-like amino acids, and carotenoids. All of the organisms survived in a desiccated state and rapidly revived after wetting. The organisms were heterocystous and nitrogenase activity was reactivated within 24?h of wetting by which time heterocysts in their filaments had also appeared.     

Relationship between microbial diversity and nitrogenase activity of Stipagrostis pennata rhizosheath

Nitrogen is the key factor for plant survival and growth, especially in the desert. Stipagrostis pennata, a sand born drought-resistant plant, could colonize pioneerly in Gurbantunggut Desert during revegetation. One strategy for their environment adaptation was the rhizosheath formatted by root-hair, mucilaginous exudates, microbial components, and soil particles, for which not only provides a favorable living microenvironment but also supplies essential nutrients. To understand the relationship between microorganisms living in rhizosheaths and the nitrogen nutrition supply, the microbial diversity and nitrogenase activity was estimated during the growth of S. pennata. Five samples of the rhizosheath, which based on the development periods of the plant, regreen, flowering, filling, seed maturating, and withering period, were collected. The nitrogenase activity was estimated by acetylene reduction and the microbial diversity was analyzed by 16S rRNA high-throughput sequencing. The results showed that the nitrogenase activity was increased slowly during regreen to flowering, while reached a peak rapidly at filling sample and then decreased gradually. A total of 274 operational taxonomic units (OTUs) were identified and significant differences in community structure and composition at each growth period. Among them, the main phyla included Actinobacteria and Proteus, which were the most abundant phyla in all periods. In addition, the microbial diversity in the grain filling period was higher than other periods in view of the analysis of alpha diversity and beta diversity. Furthermore, principal component analysis (PCA) analysis showed that the microbial communities in the filling period was low in similarity with other periods. Most importantly, the OTUs associated with nitrogen fixation is the most during the filling period, involving Phagecidae and Fucoraceae. Overall, the study not only revealed the differences in nitrogenase activity among different developmental periods in S. pennata, but also explored the potential bridges between it and community structure and diversity of bacteria.     

The effect of some terrestrial oligochaeta on nitrogenase activity in the soil

Nitrogenase activity, assayed by the acetylene reduction technique, was estimated on control soils and soils which contained several species of earthworms. The worms and casts were separately assayed for nitrogenase activity. The results indicated that nitrogenase activity was associated with gut content or body surface of worms and also their casts. Nitrogen fixation was also enhanced in soils enriched with enchytracids in the presence of leaf litter.     

The nodulation and nitrogenase activity of natural stands of mangrove legumes in a Nigerian swamp

Nodule characteristics and nitrogenase activity of Dalbergia ecastophyllum (L.) Taub, Drepanocarpus (Machearum) lunatus (L.) Mey, Pterocarpus sontelinoides (L.) and the characteristics of nodules of Baphia pubescens (Hook. f) were studied in natural mangrove stands in Nigeria. Nodules were generally spherical but occasionally elongate in Pterocarpus and Baphia; a few were also lobate in Baphia. Nodule sizes, numbers and weights varied widely between vegetations, and between sites of the same vegetation. Total nodule fresh-weight (g·m⁻²) ranged from 0.11 to 9.80, represented by the extreme values for Drepanocarpus. The plants' habitats had pH and salinity respectively in the ranges 4.8–5.49 and 0.1–25‰. Nodule acetylene reduction rates decreased in the order Pterocarpus, Drepanocarpus, Dalbergia with their corresponding aerobic rates (n moles C₂H₄ g⁻¹ freshweight) respectively as follows: 312, 23.39 and 16.07. These rates were higher than in anaerobic incubation by between 4.95 and 100%.     

Effects of fertilization rate and crop rotation on diazotrophic cyanobacteria in paddy field

Application of chemical fertilizers at the recommended level (medium fertility) or lower stimulated growth of the diazotrophic cyanobacterial population and nitrogenase activity in a paddy field. High fertilizer levels proved to be inhibitory to nitrogen-fixing cyanobacteria indicating that indiscriminate use of chemical fertilizers for a longer period drastically disturbed the natural ecological balance. The rice–mustard–moong (RMM) crop rotation was observed to be more suitable for cyanobacterial nitrogen fixation than rice–wheat–maize rotation. The cropped plots had higher nitrogenase activity than fallow plots. The low fertility coupled with RMM rotation were found to be best suited for promoting nitrogen fixation by cyanobacteria to supply the rice plants. A top dressing of chemical nitrogenous fertilizer drastically suppressed the cyanobacterial nitrogenase activity (ARA) within 12 h; the magnitude of inhibition varied with respect to the cropping system. The inhibition was overcome by the 10th day and the ARA value reached the preapplication value or even higher in the case of low fertility and medium fertility level plots. A regression equation was established to predict nitrogen fixation in a given soil ecosystem.     

O2 regulation and O2‐limitation of nitrogenase activity in root nodules of pea and lupin

The gas exchange characteristics of intact attached nodulated roots of pea (Pisum sativum cv. Finale X) and lupin (Lupinus albus cv. Ultra) were studied under a number of environmental conditions to determine whether or not the nodules regulate resistance to oxygen diffusion. Nitrogenase activity (H₂ evolution) in both species was inhibited by an increase in rhizosphere pO₂ from 20% to 30%, but recovered within 30 min without a significant increase in nodulated root respiration (CO₂ evolution). These data suggest that the nodules possess a variable barrier to O₂ diffusion. Also, nitrogenase activity in both species declined when the roots were either exposed to an atmosphere of Ar:O₂ or when the shoots of the plants were excised. These declines could be reversed by elevating rhizosphere pO₂, indicating that the inhibition of nitrogenase activity resulted from an increase in gas diffusion resistance and consequent O₂-limitation of nitrogenase-linked respiration. These results indicate that nodules of pea and lupin regulate their internal O₂ concentration in a manner similar to nodules of soybean, despite the distinct morphological and biochemical differences that exist between the nodules of the 3 species. Experiments in which total nitrogenase activity (TNA = H₂ production in Ar:O₂) in pea and lupin nodules was monitored while rhizosphere pO₂ was increased gradually to 100%, showed that the resistance of the nodules to O₂ diffusion maintains nitrogenase activity at about 80% of its potential activity (PNA) under normal atmospheric conditions. The O₂-limitation coefficient of nitrogenase (OLC_N= TNA/PNA) declined significantly with prolonged exposure to Ar:O₂ or with shoot excision. Together, these results indicate a significant degree of O₂-limitation of nitrogenase activity in pea and lupin nodules, and that yields may be increased by realizing full potential activity.     

Analysis of factors affectingin situ nitrogenase (C2H2) activity ofGalega orientalis,Trifolium pratense andMedicago sativa in temperate conditions

Summary A practical fibreglass cylinder-plastic bag system has been designed for making acetylene reduction assays in the field. Thein situ assay was used to determine seasonal patterns of nitrogenase activity for the perennial forage legumesGalega orientalis, Trifolium pratense andMedicago sativa grown under stadard management in southern Filand (60° north). Nitrogenase activity was still detected in the field plots in November, when soil temperature was 1.5°C and air temperature 0.5°C. The acetylene reduction data from weekly measurements were analyzed for correlation with plant growth rate and short-term fluctuations of environmental factors. Generally, there was a good correlation between nitrogenase activity and plant growth rate. Residual fluctuations in activity were only correlated with environmental factors in one case. The nitrogenase activity ofM. sativa was dependent on air temperature in addition to growth rate. Thus, the nitrogen fixing systems in these forage legumes seem to be an integrated part of the plants, being fairly insensitive to short-term environmental changes.Dedicated to Prof. Helge Gyllenberg on the occasion of his 60th birthday.     

黄土高原不同侵蚀类型区生物结皮固氮活性及对水热因子的响应

在野外调查的基础上,采集不同侵蚀类型区内发育至稳定阶段的生物结皮,分析水分和温度变化对生物结皮固氮活性的影响.结果表明:水蚀区、水蚀风蚀交错区、风蚀区生物结皮固氮活性表现为水蚀区(127.7μmol.m-2.h-1)>水蚀风蚀交错区(34.6μmol.m-2.h-1)>风蚀区(6.0μmol.m-2.h-1);3个侵蚀类型区生物结皮固氮的最适温度分别为35、25和15℃.在最适温度条件下,水蚀区及水蚀风蚀交错区生物结皮固氮活性在100%～40%田间持水量时差异不显著;风蚀区生物结皮固氮活性对水分变化较为敏感,当含水量降至80%田间持水量时固氮活性开始显著降低,降至20%田间持水量时,生物结皮固氮作用停止.3个侵蚀类型区生物结皮固氮活性及其对水分与温度变化响应的差异与不同侵蚀类型区的气候、环境及生物结皮物种组成有关.     

What triggers the regulation of nitrogenase activity in forage legume nodules after defoliation?

The dramatic decrease in nitrogenase activity after the defoliation of forage legumes has been recognized for a long time; however, the underlying mechanisms are not understood yet. The impact of current photosynthesis can be excluded. The precise role of carbohydrate availability is still unclear and remains to be established. From current knowledge we can conclude that, after defoliation, nitrogenase activity in legume nodules is down-regulated by a variable oxygen diffusion resistance. The triggering elements are not known; there is, however, increasing evidence that the plant's demand for symbiotically fixed nitrogen plays an important role. The possibility is here discussed that, after defoliation, a nitrogen feedback mechanism regulates nitrogenase activity through a variable oxygen diffusion resistance in the nodules.