Toxicity and isolation of the cyanobacterium Nodularia spumigena from the southern Baltic Sea in 1986

Three water bloom samples were collected in August 1986 from the southern Baltic Sea. Acute toxicity of the samples was determined by mouse bioassay and the toxins were further studied by HPLC. The bloom samples contained equal amounts of cyanobacteria Nodularia spumigena and Aphanizomenon flos-aquae and were hepatotoxic. Two hepatotoxic Nodularia spumigena strains were isolated from the samples. The isolates produce a toxic peak indistinguishable from the bloom material in the HPLC analysis. The toxicity of the fractions was verified by mouse bioassay. Thus the toxicity of the bloom samples was in all likelihood caused by Nodularia spumigena.     

Calcium requirement in nitrogen fixation in the cyanobacterium Synechococcus RF-1

Under diurnal 16/8-h light-dark cycles, ethyleneglycol-bis-(-aminoethyl ether)-N,N,N,N-tetraacetic acid (EGTA) at 1 mM completely blocked the appearance of rhythmic N₂-fixing activity in Synechococcus RF-1. Ca²⁺ at 2 mM, when supplied either together with or several hours after the EGTA application, restored the nitrogenase activity, whereas, when Ca²⁺ was supplied several hours later, the peak of nitrogenase activity was shifted from the dark to the light period in which the activity is normally suppressed. Sr²⁺ also reversed the inhibition by EGTA, but only partially. When O₂ in the gas phase above the culture was below 1%, the inhibition of nitrogenase activity by EGTA was reduced to less than 20% of the control value without EGTA. Thus Ca²⁺ appears to be required by the cell to protect its nitrogenase from inactivation by O₂. In media without EGTA, a close correlation between nitrogenase activity and concentrations of Ca²⁺ was also observed.Abbreviation EGTA ethyleneglycol-bis-(-aminoethyl ether)-N,N,N,N-tetraacetic acid     

Growth,N2 fixation and photosynthesis in a cyanobacterium,Trichodesmium sp., under Fe stress

Trichodesmium sp., isolated from the Great Barrier Reef lagoon, was cultured in artificial seawater media containing a range of Fe concentration. Fe additions stimulated growth, N₂ fixation, cellular chlorophyll a content, light-saturated chlorophyll a-specific gross photosynthetic capacity (P_m ^chla) and the dark respiration rate (R_d ^chla). Cell yields only doubled for 9 nM Fe relative to zero added Fe, whereas N₂ fixation increased 11-fold considerably for 450 nM Fe. The results suggest that N₂ fixation of Trichodesmium is more sensitive to Fe limitation than are the cell yields.     

Production of N 2 O by Ammonia Oxidizing Bacteria at Subfreezing Temperatures as a Model for Assessing the N 2 O Anomalies in the Vostok Ice Core

It was suggested that the abnormally high N ₂ O values found in 130,000–160,000 year-old Vostok ice core samples, characterized by high δ ¹⁵ N and low δ ¹⁸ O values, resulted from in situ microbial N ₂ O production. To substantiate these observations we obtained new geochemical data from the last glacial period and showed the existence of additional small N ₂ O anomalies. To test the hypothesis that microbial metabolism could contribute to these anomalies, we developed protocols for examining the ability of Nitrosomonas cryotolerans cells to produce N ₂ O at subfreezing temperatures. Our results show that these model, frozen cultures produce N ₂ O at temperatures as low as ?32°C.     

A Fuzzy Logic Model to Describe the Cyanobacteria Nodularia spumigena Blooms in the Gulf of Finland, Baltic Sea

A fuzzy logic model to describe the seasonal evolution of Nodularia spumigena blooms in the Gulf of Finland was built and calibrated on the basis of monitoring data. The model includes three phosphate sources: excess phosphate after the annual spring bloom and parameterised phosphate transport to the upper mixed layer by turbulent mixing and upwelling events. Surface layer temperature and wind mixing form the physical conditions controlling the growth of N. spumigena. Model simulations revealed that phosphate input caused by turbulent mixing and upwelling have to be taken into account to achieve the best fit with observed data. Testing the fuzzy model for early prediction of maximum N. spumigena biomass about a month before the usual occurrence of blooms, gave good results. The potential use of the model for prediction of bloom risk at a certain location along the Estonian or Finnish coast was tested. The bloom transport velocities used in the fuzzy model were pre-calculated by a 3D numerical circulation model for different wind regimes.     

Growth physiology of a marine nitrogen-fixing cyanobacterium (Nodularia harveyana) in outdoor culture

The performance ofNodularia harveyana, a N₂-fixing cyanobacterium isolated from seawater, has been studied outdoors in two different culture systems: open pond (OP) and tubular photobioreactor (TPR). The productivity in both devices was influenced by areal density. The maximum yield obtained was 12.0 g (d.wt) m^–2 day^–1 in OP and 14.0 g (d.wt) m^–2 day^–1 in TPR in August, corresponding to the highest solar radiation received. In a month-long experiment with the cyanobacterium cultivated in TPR at high circulation speed, a net increase in productivity was obtained over that at low circulation speed. The influence of temperature on the productivity of the cultures grown in open ponds and tubular photobioreactors has been investigated. The higher productivity obtained in TPR compared to OP was attributed to its better controlled temperature conditions. In outdoor culture the maximum nitrogenase activity did not coincide with the maximum light intensity, but occurred in early afternoon. The amount of carbohydrate accumulated during the day probably influenced the rate of dark nitrogenase activity and its duration in the night.     

Biological nitrogen fixation: Investments,expectations and actual contributions to agriculture

Inputs of biologically fixed N into agricultural systems may be derived from symbiotic relationships involving legumes and Rhizobium spp., partnerships between plants and Frankia spp. or cyanobacteria, or from non-symbiotic associations between free-living diazotrophs and plant roots. It is assumed that these N₂-fixing systems will satisfy a large portion of their own N requirements from atmospheric N₂, and that additional fixed N will be contributed to soil reserves for the benefit of other crops or forage species. This paper reviews the actual levels of N₂ fixation attained by legume and non-legume associations and assesses their role as a source of N in tropical and sub-tropical agriculture. We discuss factors influencing N₂ fixation and identify possible strategies for improving the amount of N₂ fixed.     

Growth characteristics of non-heterocystous cyanobacterium Plectonema boryanum with N2 as nitrogen source

Strains of filamentous, non-heterocystous cyanobacteria from the Pasteur Culture Collection (PCC), able to synthesize nitrogenase under anaerobic test conditions, were tested for growth with N₂ as sole nitrogen source at low O₂ partial pressure (less than 0.05%). Plectonema boryanum (PCC 73110) exhibited exponential growth under these conditions. This capacity was restricted to light intensities not exceeding 500 lux. Growth rates were 0.014/h at 200 and 0.023 at 500 lux and similar to those of anaerobic and aerobic control cultures with nitrate as N-source. For N₂-fixing cultures incubated at 200 and 500 lux, acetylene reduction rates were 4–8 and 5–14 nmol C₂H₄ per mg protein per min, respectively. The ratio of phycocyanine to chlorophyll was higher (200 lux) or slightly reduced (500 lux) in N₂-fixing cultures as compared to control cultures with nitrate as N-source. On the basis of epifluorescence microscopy and microfluorimetry, no differences in pigment contents were found between individual cells or filaments of N₂-fixing cultures. Also no noteworthy differences were observed between the pycobiliprotein composition of individual cells in N₂ fixing cultures as compared to nitrate-grown controls. Thus the observed exponential growth of P. boryanum at low light intensities implies simultaneous nitrogen fixation and oxygenic photosynthesis. Additional continuous culture experiments showed that N₂-fixing exponential growth was dependent on O₂ partial pressures lower than 0.2–0.4%.The other strains tested (PCC 6412, 6602, 7403, 7104) did not grow under such conditions.Abbreviations Chl chlorophyll - PBP phycobiliproteins - PC phycocyanin - PCC Pasteur Culture Collection - OD optical density     

Carbon metabolism and bacteroid respiration in nodules of chick-pea (Cicer arietinum L.) plants grown under saline conditions

ABSTRACT

The present work investigates the relationships between nitrogen fixation, carbon metabolism and oxygen consumption by bacteroids of Mesorhizobium ciceri in root nodules of chick-pea plants. Its aim was to establish whether some of the compounds which accumulate under salt stress may be used as respiratory substrates by bacteroids to fuel their own metabolism and nitrogenase activity. Plants were grown in a growth chamber, and salt stress was induced by adding 50 mM NaCl to the nutrient solution at sowing. The data presented here show a rise in fermentative metabolism in nodules of chick-pea plants exposed to high salinity, and suggest that proline, lactate or ethanol, may play an important role as energy-yielding substrates for bacteroids in this plant species. The bacteroids could utilize glucose as a respiratory substrate both under control and saline conditions, while malate did not appear to be the preferred substrate in the presence of salt.     

Biological nitrogen fixation associated with sugar cane

A recent¹⁵N dilution/N balance study confirmed that certain sugar cane varieties are capable of obtaining large contributions of nitrogen from plant-associated N₂ fixation. It was estimated that up to 60 to 80% of plant N could be derived from this source, and under good conditions of water and mineral nutrient supply, it may be possible to dispense with N fertilization of these varieties altogether. The recently discovered bacterium,Acetobacter diazotrophicus, apparently responsible for this N₂ fixation associated with the plants, has unique physiological properties for a diazotroph, such as tolerance to low pH, and high sugar and salt concentrations, lack of nitrate reductase, and nitrogenase activity which tolerates short-term exposure to ammonium. Furthermore, it also behaves as an endophyte, in that it is unable to infect sugar cane plants unless through damaged tissue or by means of VA mycorrhizae and is propagated via the planting material (stem pieces).     

Carbon isotopes in terrestrial ecosystem pools and CO2 fluxes

Stable carbon isotopes are used extensively to examine physiological, ecological, and biogeochemical processes related to ecosystem, regional, and global carbon cycles and provide information at a variety of temporal and spatial scales. Much is known about the processes that regulate the carbon isotopic composition (delta(13)C) of leaf, plant, and ecosystem carbon pools and of photosynthetic and respiratory carbon dioxide (CO(2)) fluxes. In this review, systematic patterns and mechanisms underlying variation in delta(13)C of plant and ecosystem carbon pools and fluxes are described. We examine the hypothesis that the delta(13)C of leaf biomass can be used as a reference point for other carbon pools and fluxes, which differ from the leaf in delta(13)C in a systematic fashion. Plant organs are typically enriched in (13)C relative to leaves, and most ecosystem pools and respiratory fluxes are enriched relative to sun leaves of dominant plants, with the notable exception of root respiration. Analysis of the chemical and isotopic composition of leaves and leaf respiration suggests that growth respiration has the potential to contribute substantially to the observed offset between the delta(13)C values of ecosystem respiration and the bulk leaf. We discuss the implications of systematic variations in delta(13)C of ecosystem pools and CO(2) fluxes for studies of carbon cycling within ecosystems, as well as for studies that use the delta(13)C of atmospheric CO(2) to diagnose changes in the terrestrial biosphere over annual to millennial time scales.     

现场模拟添加磷、铁及胶体对北部湾2007年春季生物固氮的影响

生物固氮是海洋氮循环的重要过程。固氮作用能够促进海洋初级生产力的提高,增强海洋吸收CO₂能力,对于降低大气CO₂浓度,减缓温室效应具有重要意义。2007年春季对北部湾海区进行了现场模拟添加P,Fe和胶体实验,并应用乙炔还原法（ARA,Acetylene Reducing Activity）分析其固氮速率,研究其主要影响因素。结果表明,温盐与固氮速率相关性不显著（P > 0.05）。对固氮速率来说,外加P其变化范围为-97%-545%,外加Fe其变化范围为-86%-146%,外加胶体其变化范围为-96%-1456%。对叶绿素含量而言,外加P其变化范围为-24%-50%,外加Fe其变化范围为-32%-36%,外加胶体其变化范围为-53%-41%。N可能是春季北部湾海区浮游植物生长的限制因子,固氮作用主要受到P限制,而外加胶体对固氮生物和固氮速率表现出较大的促进作用。     

Biological N2 fixation by heterotrophic and phototrophic bacteria in association with straw

Much of the crop residues, including cereal straw, that are produced worldwide are lost by burning. Plant residues, and in particular straw, contain large amounts of carbon (cellulose and hemicellulose) which can serve as substrates for the production of microbial biomass and for biological N₂ fixation by a range of free-living, diazotrophic bacteria. Microorganisms with the dual ability to utilise cellulose and fix N₂ are rate, but some strains that utilize hemicellulose and fix N₂ have been found. Generally, cellulolysis and diazotrophy are carried out by a mixed microbial community in which N₂-fixing bacteria utilise cellobiose and glucose produced from straw by cellulolytic microorganisms. N₂-fixing bacteria include heterotrophic and phototrophic organisms and the latter are apparently more prominent in flooded soils such as rice paddies than in dryland soils. The relative contributions of N₂ fixed by heterotrophic diazotrophic bacteria compared with cyanobacteria and other phototrophic bacteria depend on the availability of substrates from straw decomposition and on environmental pressures. Measurements of asymbiotic N₂ fixation are limited and variable but, in rice paddy systems, rates of 25 kg N ha^-1 over 30 days have been found, whereas in dryland systems with wheat straw, in situ measurements have indicated up to 12 kg N ha^-1 over 22 days. Straw-associated N₂ fixation is directly affected by environmental factors such as temperature, moisture, oxygen concentration, soil pH and clay content as well as farm management practices. Modification of managements and use of inoculants offer ways of improving asymbiotic N₂ fixation.In laboratory culture systems, inoculation of straws with cellulolytic and diazotrophic microorganisms has resulted in significant increases in N₂ fixation in comparison to uninoculated controls and gains of N of up to 72 mg N fixed g^-1 straw consumed have been obtained, indicating the potential of inoculation to improve N gains in composts that can then be used as biofertilisers. Soils, on the other hand, contain established, indigenous microbial populations which tend to exclude inoculant microorganisms by competition. As a consequence, improvements in straw-associated N₂ fixation in soils have been achieved mostly by specific straw-management practices which encourage microbial activity by straw-decomposing and N₂-fixing microorganisms.Further research is needed to quantify more accurately the contribution of asymbiotic N₂ fixation to cropping systems. New strains of inoculants, including those capable of both cellulolytic and N₂-fixing activity, are needed to improve the N content of biofertilisers produced from composts. Developments of management practices in farming systems may result in further improvements in N₂ fixation in the field.     

15N-Determined effect of inoculation with N2 fixing bacteria on nitrogen assimilation in Western Canadian wheats

Summary A two-year field study was undertaken using¹⁵N isotope techniques to differentiate between stimulation of N uptake and N₂ fixation in Western Canadian cultivars of spring wheat (Triticum aestivum L. emend Thell) and durum (T. turgidum L. emend Bowden) in response to inoculation with N₂-fixing bacteria. Bacterial inoculation either had no effect or lowered the % N derived from the fertilizer and the fertilizer use efficiency. Despite the depression of fertilizer uptake, inoculants did not alter the relative uptake from soil and fertilizer-N pools indicating that bacterial inoculation did not alter rooting patterns. Nitrogen-15 isotope dilution indicated that N₂ fixation did occur. In 1984, % plant N derived from the atmosphere (% Ndfa) due to inoculation with Bacillus C-11-25 averaged 23.9% while that withAzospirillum brasilense ATCC 29729 (Cd) averaged 15.5%. In 1985, higher soil N levels reduced these values by approximately one-half. Cultivar x inoculant interactions, while significant, were not consistent across years. However, these interactions did not affect cultivars ‘Cadet’ and ‘Rescue’. In agreement with previous results, ‘Cadet’ performed well with all inoculants in both years while ‘Rescue’ performed poorly. Among 1984 treatments, the N increament in inoculated plants was positively correlated with % Ndfa but no such correlation existed in 1985. N₂ fixation averaged over all cultivars and strains was 17.9 and 6.7 kg N fixed ha⁻¹ in 1984 and 1985, respectively. Highest rates of N₂ fixation were estimated at 52.4 kg N ha⁻¹ for ‘Cadet’ in 1984 and 31.3 kg N ha⁻¹ for ‘Owens’ in 1985, both inoculated with Bacillus C-11-25, an isolate from southern Alberta soils. Inoculation with either ofAzospirillum brasilense strain Cd (ATCC29729) or 245 did not result in as consistent or as high N₂ fixation, suggesting that these wheats had not evolved genetic compatability with this exogenous microorganism. These agronomically significant amounts of N₂ fixation occurred under optimally controlled experimental conditions in the field. It is yet to be determined if N₂ fixation would occur in response to bacterial inoculation under dryland conditions commonly occurring in Western Canada. Contribution from Agriculture Canada Research Station, Lethbridge, Alberta, Canada.     

Total denitrification and the ratio between N2O and N2 during the growth of spring barley

Summary Total denitrification (N₂O+N₂) and nitrous oxide emission were measured on intact soil cores using the acetylene inhibition technique.Total denitrification from the depth 0–8 cm during the growth period from April to August was 7 kg N/ha from plots supplied with 30 kg N/ha and 19 kg N/ha from plots supplied with 120 kg N/ha. The amounts of precipitation, plant growth, and N application were found to affect the denitrification rate. These factors also affected the ratio (N₂O+N₂)/N₂O, which varied from 1.0 to 7.2. Plant growth and precipitation increased the proportion of N₂ produced, whereas a high nitrate content increased the proportion of N₂O.     

Influence of carbon availability on the production of NO, N2O, N2 and CO2 by soil cores during anaerobic incubation

Net productions of permanent soil atmosphere gases (N₂, CO₂, O₂) and temporary gases (N₂O, NO) were monitored in soil cores using a non-interfering, fully automated measuring technique allowing highly time resolved measurements over prolonged periods. The influence of changes in available organic carbon on CO₂, N₂O, NO and N₂ production was studied by changing the soil carbon content through aerobic preincubations of different length, up to 21 days.The aerobic preincubation caused an increase in NO₃ ^- concentration and a decrease in available carbon content. Available carbon content dominated both CO₂ and total N gas (N₂+N₂O+NO) production during anaerobiosis. Both CO₂ and total N gas production rates decreased with increasing length of the previous aerobic preincubation, this in spite of the higher initial NO₃ ^- concentration.Total denitrification rates were closely related to the anaerobic CO₂ production rates. No relation was found between water soluble carbon content and total denitrification. The N₂O/N₂ ratio could be explained by an interaction of carbon availability, NO₃ ^- concentration and enzyme status. Net N₂O consumption was monitored. The balance between cumulative total N gas production and NO₃ ^- consumption varied according to the different treatments. Cumulative N₂O production exceeded cumulative N₂ production for 0 up to 5 days.     

Enhancement of nitrogen fixation in lentil,faba bean,and soybean by dual inoculation with Rhizobia and mycorrhizae

The combined effect of Vesicular Arbuscular Mycorrhizae (VAM) and Rhizobium on the cold season legumes, lentil and faba bean, as well as on summer legume, soybean, were studied in soils with low indeginous VA mycorrhizal spores. Inoculation of the plant with VA mycorrhizal fungi increased the level of mycorrhizal root infection of lentil, faba bean and soybean. The inoculation with Rhizobium had no significant effect on VA mycorrhizal infection percent, but VA mycorrhizal inoculation increased nodulation of the three legumes. The inoculation with Rhizobium alone significantly increased plant dry weight and N content of lentil and faba bean as well as seed yield of soybean. VA mycorrhizal inoculation also significantly increased plant dry weight and phosphorus content of the plants as did fertilization with superphosphate. Rock phosphate fertilization, however, had no significant effect on plant growth or phosphorus uptake. The addition of rock phosphate in combination with VA mycorrhizal inoculation significantly increased plant dry weight and P uptake of the plants. The dual inoculation with both rhizobia and mycorrhizae induced more significant increases in plant dry weight, N and P content of lentil and faba bean as well as seed yield of soybean than inoculation with either VA mycorrhizae or Rhizobium alone.     

Relationship between nitrogen fixation and nitrate metabolism in the Nodularia strains M1 and M2

Nitrogen fixation and nitrate-reduction activities were determined in photoautotrophic cultures of two wild-type strains of cyanobacterium Nodularia, spp. M1 and M2. Air could support growth of the two strains at a similar rate in the presence or absence of exogenous nitrate, ammonium and/or bicarbonate. Nitrogenase activity in air-grown cultures varied with culture age, and totally disappeared after 6 h of darkness. Recovery took place upon culture re-illumination. Ammonium at a concentration of 1 mM resulted in the total disappearance of nitrogenase activity and of heterocysts. In contrast, 20 mM nitrate hardly affected nitrogenase activity and heterocyst formation after ten generations. Under the same conditions, either ammonium or nitrate completely abolished nitrogenase activity and heterocyst formation in Anabaena sp. PCC 7119, a typical heterocystous strain. The inefficiency of nitrate in inhibiting nitrogen fixation in Nodularia M1 and M2 seemed to be caused by a low nitrate-reductase activity, and not by an impairment of nitrate-uptake activity. On the other hand, the presence of nitrate was not required for uptake activity to be expressed in Nodularia.Abbreviation NR nitrate reductase We thank C. Fernández-Cabrera (Consejo Superior de Investigaciones Científicas, CSIC, Madrid, Spain) for technical assistance, and Dr. G. Pérez-Silva (CSIC) for his collaboration in the Anabaena NR assays. This work was supported by grants from Spanish CI-CyT (PB 87-0204 and PB 92-0497).     

Outdoor mass culture ofAzolla spp.: yields and efficiencies of nitrogen fixation

Summary The productivity of three species of Azolla (A. pinnata, A. filiculoides andA. caroliniana) in outdoor culture has been evaluated at different planting densities. The highest yields were obtained with biomass concentration ranging from 40 to 70g d.w. m^–2. The mean productivity over a 90 days period (from May 10th to August 10th) ranged from 10g d.w. m^–2 day^–1 forA. filiculoides up to 11.5 g d.w. m^–2 day^–1 forA. caroliniana. The nitrogen content of the dried biomasses was 48.3 mg (g d.w.)^–1 forA. pinnata, 51.5mg (g d.w.)^–1 forA. filiculoides and 52.3 mg (g d.w.)^–1 forA. caroliniana. Very little variations of the nitrogen content of the ferns during the experimental period were observed.The nitrogen-fixing efficiency of the Azolla-Anabaena azollae symbiosis grown in outdoor conditions was evaluated both by direct measurement of the amount of N₂ fixed by the culture and by the C₂H₂-reduction and H₂-evolution tests in an air atmosphere. These tests were performed outdoor under the same environmental conditions as the growing cultures. For all the species the ratios of C₂H₂-reduced to N₂-fixed were unexpectedly low, ranging from 2.04 (A. pinnata) to 1.50 (A. caroliniana).The results suggest that the reliability of the C₂H₂-reduction assay, particularly when applied to complex biological N₂-fixing systems, must be re-examined.     

15N2 measurement of nitrogen fixation by legumes and actinorhizals: theory and practice

A gas-tight chamber has been constructed to calibrate the ¹⁵N isotope dilution method against direct ¹⁵N₂ measurements. The theoretical basis for such estimates is given, and the practical problems associated with the experiments are discussed.