Suitability of some local agro-industrial wastes as carrier materials for cyanobacterial inoculant

Survival and nitrogenase efficiency ofNostoc commune andN. austinii were evaluated monthly in four carrier materials (sugarcane bagasse, wheat straw, wheat bran and peat) at 10, 30 and 40 °C. Survival, as well as nitrogenase activity, of both species was much better in peat, followed by wheat bran, sugarcane bagasse than in wheat straw at 10 and 30 °C up to three months, the activity ofN. commune being better thanN. austinii. None of the materials tested was found to be superior to peat as carrier ofNostoc species but the results indicated that wheat bran and sugarcane bagasse can be used as inoculant carriers with relative success. Storage of inoculants in these carriers is feasible at 30 °C up to three months.     

PHYSIOLOGICAL ECOLOGY OF NITROGEN FIXING BLUE-GREEN ALGAL CRUSTS IN THE UPPER-SUBALPINE LIFE ZONE1

Blue-green algal (cyanobacterial) crusts composed of nitrogen fixing Nostoc commune Voucher ex Born. et Flah. and Tolypothrix conglutinata var. colorata Ghose were studied in the upper-subalpine life zone, Mission Mountain Wilderness, Montana. Rates of ethylene production were highest in the submerged shoreline crusts, lower for exposed crusts pioneering rocky shorelines and lowest in the Carex meadow. Nitrogenase activity (acetylene reduction technique) was constant between 200–285% crust moisture content (wet/dry weight) and then rapidly declined to 0 between 200–140%. Optimal temperatures for ethylene production by illuminated cells was between 20–30° C for T. conglutinata, 20° C for N. commune and about 25° C in darkness for both species. Nitrogenase activity by T. conglutinata in culture was unaffected by repeated freeze-thaw treatments whereas N. commune was severely inhibited. In contrast, N₂-ase activity of these two species in an intact crust was unaffected by repeated freeze-thaw treatments. Application of nitrogen-free growth medium to intact crusts increased nitrogenase activity by 3.7 times implying that these were mineral deficient under field conditions. Photosynthesis was light saturated at 125 μmol-m^?2.s^?1 whereas nitrogenase activity was light independent for cells with carbohydrate reserves. When carbohydrate reserves were reduced by 8 h incubation in darkness, between 1–3 h of illumination were required to restore nitrogenase activity to 80% of the maximum rate. Biochemical pathway inhibitor studies employing DCMU, MFA, and CCCP showed that oxidative metabolism was the source of reductant for acetylene reduction. Tetrazolium precipitation in heterocysts paralleled acetylene reduction activity in the inhibitor treated cells.     

Effect of soil temperature on nitrogen fixation on roots of rice and reed

Summary The relation of nitrogenase activity (ethylene evolution) to soil temperature or incubation temperature of roots was determined on two genera of swamp plants, namely rice (Oryza sativa) cultivated in tropical climate and reed (Phragmites communis) grown in temperate regions. For both intact rice plants and excised rice roots the optimum temperature was 35°C. On excised roots nitrogenase activity responded more sensitivity to changes in temperature. In contrast to intact rice plants no ethylene evolution occurred on excised roots at 17 and 44°C. On reed roots temperature optimum was between 26 and 30°C which is clearly lower than on rice (35°C). The temperature range in which nitrogen fixation occurred was, however, similar to that of rice, although on a lower level. The results suggest a higher potential of the tropics for associative N₂ fixation, while in cooler climates the lower temperatures appear to be a major limiting factor.     

Effects of incubation temperature and oxygen tension on nitrogenase activity of legume root nodules

Summary Acetylene reduction and H₂ evolution by legume root nodules from several plant species depended on incubation temperature; some nodules were active from 2 to 40°C. Acetylene reduction rates differed between plant species, with maximum activity at temperatures between 20 and 30°C forVicia faba, V. sativa, Trifolium pratense, T. subterraneum, Medicago truncatula and soybean, at 35°C forM. sativa and at 40°C for cowpea. OnlyM. sativa and cowpea reduced substantial amounts at 37.5°C. Temperatures from 2 to 10°C only slightly lessened activity ofT. subterraneum andV. sativa nodules. Nitrogenase functioned at temperatures which prevent establishment of other aspects of the symbiosis. The rate of acetylene reduction was constant for several hours at temperatures below 15°C, and activity continued for several days at 2°C for some species, but declined with time at warmer temperatures. Some nitrogenase was denatured at warmer temperatures, but the O₂ tension in the assay vial also affected activity. In closed assay vessels nodule respiration decreased the pO₂ and reduced nitrogenase activity. Activity was restored by adding O₂ or regassing assay vials with air or Ar/O₂. When the pO₂ was maintained, acetylene reduction and H₂ evolution by detached soybean nodules continued unchanged for 6 h.     

Comparative growth and metabolism of gelatinous colonies of three cyanobacteria,Nostoc commune,Nostoc pruniforme and Nostoc zetterstedtii,at different temperatures

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Rewetting of drought-resistant blue-green algae: Time course of water uptake and reappearance of respiration,photosynthesis, and nitrogen fixation

Summary The response of the terrestrial blue-green algae Nostoc flagelliforme, Nostoc commune, and Nostoc spec. to water uptake has been investigated after a drought period of approximately 2 years. Rapid half-times of rewetting (0.6, 3.3, and 15.5 min, respectively) are found. The surfaceto-mass ratio of the three species is inversely correlated to the speed of water uptake and loss. The ecological relevance of these different time courses is discussed.Respiration starts immediately after a 30-min rewetting period, whereas photosynthetic oxygen evolution reaches its maximum activity after 6 and 8 h with N. commune and N. flagelliforme, respectively. In the dark, recovery of oxygen uptake by N. commune is somewhat impaired, while slightly stimulated with N. flagelliforme. With both species, recovery of photosynthesis is inhibited by darkness.Using colonies kept dry for two years, nitrogenase activity of N. commune attains its maximum 120 to 150 h after rewetting, while only 50 h were needed with algal mats kept dry for two days.Thus, after a 2-year drought period, the physiological sequence of reactivation is respiration—photosynthesis—nitrogen fixation. Respiration and photosynthesis precede growth and are exhibited by existing vegetative cells, whereas recovery of nitrogen fixation is dependent on newly differentiated heterocysts.     

Use of membrane filters for soil algal bioassays

A culture technique for eucaryotic and procaryotic soil algae involving the use of membrane filters superimposed on the surface of inorganic nutrient agar is described. Nostoc commune grew exponentially when cultured in this manner. No significant differences in biomass production or nitrogenase activity were detected among culture subsets within replicate experiments run under standard conditions. Estimates of daily growth rates (0.340), culture doubling time (48.9 h), and nitrogenase activity (14.54 nM C₂H₂ reduced μg⁻¹ chl a h⁻¹) were consistent with laboratory and field estimates reported for several planktonic species of Anabaena and strains of Nostoc commune isolated from diverse terrestrial habitats. Therefore, the filter-culture technique is an alternative which may be superior to traditional liquid culture methodology for studies involving certain soil procaryotic and eucaryotic algae.     

EFFECT OF TEMPERATURE ON THE SENSITIVITY OF NITROGENASE TO OXYGEN IN TWO HETEROCYSTOUS CYANOBACTERIA1

The effect of temperature and oxygen on nitrogenase activity in two heterocystous cyanobacteria, Anabaena variabilis Kütz. ATCC29413 and Nostoc sp. PCC7120, was investigated. The cyanobacteria were grown under a 12:12 light:dark (L:D) cycle at 27°C and were subsequently exposed to different temperatures (27, 36, 39, and 42°C) at different steady‐state O₂ concentrations (20, 10, 5, 0%). Light response curves of nitrogenase activity were recorded under each of these conditions using an online acetylene reduction assay combined with a sensitive laser photoacoustic ethylene detection method. The light response curves were fitted with the rectangular hyperbola model from which the model parameters N_m, N_d, and α were derived. In both strains, nitrogenase activity (N_tot = N_m + N_d) was the highest at 39°C–42°C and at 0% O₂. The ratio N_tot/N_d was 4.1 and 3.1 for Anabaena and Nostoc, respectively, indicating that respectively 25% and 33% of nitrogenase activity was supported by respiration (N_d). N_tot/N_d increased with decreasing O₂ concentration and with increasing temperature. Hence, each of these factors caused a relative increase in the light‐driven nitrogenase activity (N_m). These results demonstrate that photosynthesis and respiration both contribute to nitrogenase activity in Anabaena and Nostoc and that their individual contributions depend on both O₂ concentration and temperature as the latter may dynamically alter the flux of O₂ into the heterocyst.     

Annual carbon fixation in terrestrial populations of Nostoc commune (Cyanobacteria) from an Antarctic dry valley is driven by temperature regime

Nostoc commune Vaucher (a cyanobacterium) is a very conspicuous terrestrial primary producer in Victoria Land, continental Antarctica. Because polar ecosystems are considered to be especially sensitive to environmental changes, understanding the environmental constraints on net carbon (C) fixation by N. commune is necessary to determine the effects of environmental changes on the ecological functioning of ice‐free areas of the continent. A model describing net C fixation in terrestrial populations of N. commune in an Antarctic dry valley was constructed using field and laboratory measurements in which N. commune colonies were exposed to different combinations of incident irradiance (400–700 nm), temperature, and degree of desiccation. For desiccated N. commune mats with water content ≤ 30% saturation, net C fixation was highly variable between replicates and could not be modelled. However, for colonies at > 30% saturation, rates of net C fixation and dark respiration depended strongly on irradiance and temperature. Net C fixation reached a maximum rate of 21.6 μg C m^{− 2} s^{− 1} at irradiance of approximately 250 μmol m^{− 2} s^{− 1} and the optimum temperature of 20.5 °C. Agreement between predicted short‐term net C fixation and field and laboratory measurements allowed estimation of total seasonal fixation, using previously published environmental data. Annual net C fixation was estimated in the range 14.5–21.0 g C fixed m^{− 2}Nostoc mat, depending on year/season. Estimates for different seasons correlated with thermal time (accumulated hours above 0 °C during the year) rather than irradiance, in contrast to communities in local lacustrine environments, where irradiance is the main driver of primary productivity. In the terrestrial habitat, N. commune appears to compromise between an ability to capitalize on short periods of higher temperature and efficient utilization of lower irradiance at low temperature. The relationship between thermal time and net annual C fixation by N. commune is strongly linear.     

Effects of high temperature on nodulation and nitrogen fixation by Phaseolus vulgaris L.

Screening of Rhizobium leguminosarum bv. phaseoli strains showed some that were able to nodulate common beans (Phaseolus vulgaris L.) at high temperatures (35 and 38°C/8 h/day). The nodulation ability was not related to the capability to grow or produce melanin-like pigment in culture media at high temperatures. However, nodules formed at high temperatures were ineffective and plants did not accumulate N in shoots. Two thermal shocks of 40°C/8 h/day at flowering time drastically decreased nitrogenase activity and nodule relative efficiency of plants otherwise grown at 28°C. Recovery of nitrogenase activity began only after seven days, when new nodules formed; total incorporation of N in tops did not recover for 2 weeks. Non-inoculated beans receiving mineral N were not affected by the thermal shock, and when growing continuously at 35 or 38°C had total N accumulated in shoots reduced by only 18%.     

Effects of low temperatures on nitrogenase activity in sainfoin (Onobrychis viciifolia) nodulated by arctic rhizobia

The temperate forage legume sainfoin (Onobrychis viciifolia) is readly nodulated by rhizobia isolated from arctic legumes (Astragalus and Oxytropis species). We have investigated the effects of low temperatures on nitrogenase activity in sainfoin nodulated by arctic and temperate (homologous) rhizobia. At low temperatures, nitrogenase activity of arctic rhizobia measured either with detached nodules or with whole plants, was higher than that of temperate rhizobia. At 5°C and 10°C, nitrogenase activity values of arctic rhizobia represented 12% and 33% of those measured at 20°C, while lower values of 3.7% and 22.4% were observed with temperate rhizobia. This cold adaptation was also reflected on bacterial growth where, at 5°C and 10°C, arctic rhizobia showed a shorter doubling time and synthesized more protein than temperate rhizobia.     

COMPARATIVE STUDY ON THE PHOTOSYNTHETIC PROPERTIES OF PRASIOLA (CHLOROPHYCEAE) AND NOSTOC (CYANOPHYCEAE) FROM ANTARCTIC AND NON‐ANTARCTIC SITES1

The terrestrial cyanobacterium Nostoc commune Vaucher ex Bornet et Flahault occurs worldwide, including in Japan and on the Antarctic continent. The terrestrial green alga Prasiola crispa (Lightf.) Kütz. is also distributed in Antarctica. These two species need to acclimate to the severe Antarctic climate including low ambient temperature and desiccation under strong light conditions. To clarify this acclimation process, the physiological characteristics of the photosynthetic systems of these two Antarctic terrestrial organisms were assessed. The relative rate of photosynthetic electron flow in N. commune collected in Japan and in Antarctica reached maxima at 900 and 1,100 μmol photons · m^?2 · s^?1, respectively. The difference seemed to reflect the presence of high amounts of UV‐absorbing substances within the Antarctic cyanobacterium. On the other hand, the optimal temperatures for photosynthesis at the two locations were 30°C–35°C and 20°C–25°C, respectively. This finding suggested a decreased photosynthetic thermotolerance in the Antarctic strain. P. crispa exhibited desiccation tolerance and dehydration‐induced quenching of PSII fluorescence. Re‐reduction of the photooxidized PSI reaction center, P700, was also inhibited at fully dry states. Photosynthetic electron flow in P. crispa reached a maximum at 20°C–25°C and at a light intensity of 700 μmol photons ? m^?2 ? s^?1. Interestingly, the osmolarity of P. crispa cells suggested that photosynthesis is performed using water absorbed in a liquid form rather than water absorbed from the air. Overall, these data suggest that these two species have acclimated to optimally photosynthesize under conditions of the highest light intensity and the highest temperature for their habitat in Antarctica.     

Desiccation independence of terrestrialNostoc commune ecotypes (cyanobacteria)

The subspeciesNostoc commune var.flagelliforme andN. commune var.commune are found in China (Ningxia Province, Inner Mongolia) as two morphologically different ecotypes of the desiccation-independent cyanobacteriumN. commune. The first ecotype, but not the second, colonizes arid areas. Various biochemical parameters and water dependence of photosynthesis and nitrogen fixation were compared for both ecotypes. Different patterns of water stress proteins were found in the two ecotypes. Repeated desiccation resulted in an enhanced desiccation independence for photosynthesis and, in the case of the ecotypecommune, for nitrogen fixation. The different response of nitrogenase of both ecotypes towards repeated cycles of rewetting and desiccation under conditions simulating the natural environment is discussed in terms of the energy balance of the colonies that are adapted to different environmental conditions.     

Changes in Water Soluble Protein Profile of Antarctic Cyanobacterium Nostoc Commune under Temperature Downshift from 25° to 5 °C

The cyanobacterium Nostoc commune (Nostocales) is an isolate from the Schirmacher Oasis Antarctica. The cyanobacterium is psychrotropic in nature; and maintained in laboratory at 25?°C temperature, in unialgal form. Here, we studied the change in protein profile of water soluble proteins from exponentially growing N. commune upon downshift from its optimum growth temperature (25?°C) to a low temperature (5?°C). Experimental set up used to analyze the proteome were- a sudden shift to low temperature (i.e., cold shock), after short- (8?days) and long-term acclimation (7?weeks) to low temperature (5?°C). Cold-shock resulted in an increase in Low molecular weight proteins (LMWPs) with clouding of diffused proteins. Further increase in the duration of incubation period (short- and long-term acclimation) caused dissociation of proteins, indicated by NaCl (50–600mM) induced dissociation of proteins. That is, high molecular weight proteins (HMWPs) dissociated into LMWPs resulting in an increased number of protein bands. This was further confirmed by addition of LMWPs (≤10KDa) resulting in re-association of proteins into HMWPs. Hence, we report that the cold-induced synthesis of LMWPs (≤10kDa) is a strategy adopted by the N. commune to survive at low temperature of Antarctica.     

Sensitivity of the common bean (Phaseolus vulgaris L.) symbiosis to high soil temperature

Summary Experiments were done to test whether N fixation is more sensitive to high soil temperatures in common bean than in cowpea or soybean. Greenhouse experiments compared nodulation, nitrogenase activity, growth and nitrogen accumulation of several host/strain combinations of common bean with the other grain legumes and with N-fertilization, at various root temperatures. Field experiments compared relative N-accumulation (in symbiotic relative to N-fertilized plants) of common bean with cowpea under different soil thermal regimes. N-fertilized beans were unaffected by the higher temperatures, but nitrogen accumulation by symbiotic beans was always more sensitive to high root temperatures (33°C, 33/28°C, 34/28°C compared with 28°C) than were cowpea and soybean symbiosis. Healthy bean nodules that had developed at low temperatures functioned normally in acetylene reduction tests done at 35°C. High temperatures caused little or no suppression of nodule number. However, bean nodules produced at high temperatures were small and had low specific activity. ForP. vulgaris some tolerance to high temperature was observed among rhizobium strains (e.g., CIAT 899 was tolerant) but not among host cultivars. Heat tolerance ofP. acutifolius andP. lunatus symbioses was similar to that of cowpea and soybean. In the field, high surface soil temperatures did not reduce N accumulation in symbiotic beans more than in cowpea, probably because of compensatory nodulation in the deeper and cooler parts of the soil.     

Accumulation of trehalose in response to desiccation and salt stress in the terrestrial cyanobacterium Nostoc commune

Changes in photosynthetic activity and trehalose levels in field‐isolated, natural colonies of the terrestrial cyanobacterium Nostoc commune responding to desiccation and salt stress were investigated. As the water content decreased in N. commune colonies during desiccation, photosynthetic O₂‐evolving activity decreased and no activity was detected in desiccated colonies. A high level of O₂ evolution was restored in the colonies as they absorbed atmospheric moisture, indicating that only a small amount of water is required for reactivation of photosynthesis. No detectable trehalose was found in fully hydrated N. commune colonies; however, trehalose accumulation occurred in response to water loss during desiccation and high levels of trehalose were detected in the air‐dried colonies. Moreover, a 0.2 M NaCl treatment also induced trehalose accumulation to a level equivalent to that by desiccation. Photosynthetic O₂ evolution was inhibited by 0.2 M NaCl, indicating that N. commune can tolerate only low levels of salt. These results suggest that cessation of photosynthesis and trehalose accumulation occur in response to both matric water stress (desiccation) and osmotic water stress (high salt concentration), and that while trehalose may be a less effective osmoprotective compound than others, it is important for the extreme tolerance to desiccation observed in terrestrial cyanobacterium.     

Four chemotypes of the terrestrial cyanobacterium Nostoc commune characterized by differences in the mycosporine‐like amino acids

The cyanobacterium Nostoc commune is adapted to terrestrial environments and has a cosmopolitan distribution. Four genotypes of N. commune can be identified based on differences in their 16S rRNA genes, and these genotypes are distributed throughout Japan without regional specificity. Mycosporine‐like amino acids (MAAs) are UV‐absorbing pigments, and novel glycosylated MAA derivatives with radical scavenging activities have been identified in N. commune. In this study, we investigated the consistency of the relationship between MAA compositions and N. commune genotypes. The MAA compositions were different in a genotype‐specific manner, suggesting that the types of MAA derivatives can feasibly be used as chemotaxonomic markers to characterize N. commune. The novel 756‐Da MAA, which was identified as an aglycone of the 1050‐Da MAA and named nostoc‐756, occurred in genotype C of N. commune. Nostoc‐756 functioned as a radical scavenger in vitro. In conclusion, N. commune is classified into four groups representing genetically different chemotypes, namely, the arabinose‐bound porphyra‐334 producer (chemotype A), the glycosylated nostoc‐756 producer (chemotype B), the nostoc‐756 producer (chemotype C) and the glycosylated palythine‐threonine producer (chemotype D). Either the molecular taxonomical method or chemical analysis of a characteristic secondary metabolite is sufficient to identify the types of N. commune; however, there are no obvious ecophysiological differences that allow us to distinguish them.     

Does oxygen limit nitrogenase activity in soybean exposed to elevated CO2?

Soybean (Glycine max L. Merr) plants grown under control (360 µmol mol^?1) or elevated CO₂ concentration (800 µmol mol^?1) from 33 to 42 d after sowing were assayed for various components of in vivo nitrogenase activity to test the hypothesis that increasing carbohydrate supply to nodules would increase the potential (i.e. O₂ saturated) nitrogenase activity and impose a more severe O₂ limitation on both nodule metabolism and total nitrogenase activity. Within 51 h of elevated CO₂ treatment, significant increases relative to control plants were seen in total nitrogenase activity expressed per plant. After 6 d of elevated CO₂, the total nitrogenase activity per plant was 18% higher than that in control. This was attributed to an initial increase in nodule size, and a subsequent increase in nodule number following plant exposure to elevated CO₂. However, after 9 d of elevated CO₂, the potential and total nitrogenase activities per gram nodule dry weight were lower, not higher than corresponding values in plants in the control treatment. These results did not support the hypothesis. It was concluded that the metabolic capacity of the control nodules were not limited by carbohydrate supply, at least at the assay temperatures employed here.     

Physiological studies on antarctic Prasiola crispa and Nostoc commune at low temperatures

Summary Photosynthetic gas exchange measurements and ¹⁴CO₂-fixation experiments were performed with Antarctic Prasiola crispa and Nostoc commune at low temperatures. In the case of Prasiola photosynthetic activity was found as low as-15°C, wile with Nostoc photosynthesis was suppressed below-5°C. At decreasing temperatures the metabolism of Prasiola is modified to enhance sugar phosphate synthesis, which might serve as a protective agent against freezing. The fixation pattern of Nostoc did not change near the freezing point; the total sugar phosphates amounted to approximately 50% at all temperatures tested. The differences observed may be explained by the different environments of the two algae.     

DEPRESSION OF MASTIGOCLADUS LAMINOSUS (CYANOPHYTA) NITROGENASE ACTIVITY UNDER NORMAL SUNLIGHT INTENSITIES1

The effect of light intensity (PAR) on the nitrogenase activity of Mastigocladus laminosus Cohn was studied by the acetylene reduction technique. Benthic mat from a thermal stream, Hot River, in Yellowstone National Park was used in both experimental and in situ incubations. This hot spring maintained a mean pH of 7.0, was essentially isothermal (ca. 50°C), and had virtually no upstream to downstream physicochemical gradients (P > 0.05). Two surveys of the stream showed that nitrogenase of the M. laminosus mat was significantly more active (P > 0.02) under low light intensities than under high intensities, 252 and 712 μE · m^?2· s^?1, respectively. Maximum activity of Hot River Mastigocladus (268 nmol C₂H₄· mg Chl a^?1· h^?1) occurred at 50% full midday light intensities; the rates at low light (mean = 247 nmol C₂H₄· mg Chl a^?1· h^?1) were significantly (P > 0.001) greater than those at high light (mean = 106). The results indicate that M. laminosus nitrogenase activity is low light adapted and suggest that the temporal pattern for nitrogen fixation might be significantly different from that of thermophilic Calothrix.