Ca requirement for aerobic nitrogen fixation by heterocystous blue-green algae

The requirement of Ca²⁺ for growth and nitrogen fixation has been investigated in two strains of heterocystous blue-green algae (Anabaena sp. and Anabaena ATCC 33047). With combined nitrogen (nitrate or ammonium) or with N₂ under microaerobic conditions, Ca²⁺ was not required for growth, at least in concentrations greater than traces. In contrast, Ca²⁺ was required as a macronutrient for growth and nitrogen fixation with air as the nitrogen source. Addition of Ca²⁺ to an aerobic culture without Ca²⁺ promoted, after a lag of several hours, development of nitrogenase activity and cell growth. Provision of air to a microaerobic culture in the absence of Ca²⁺ promoted a drastic drop in nitrogenase activity, which rapidly recovered its initial level upon restoration of microaerobic conditions. Development of nitrogenase activity in response to either Ca²⁺ or low oxygen tension was dependent on de novo protein synthesis. The role of Ca²⁺ seems to be related to protection of nitrogenase from inactivation, by conferring heterocysts resistance to oxygen.     

Heterotrophic nitrogen removal by <Emphasis Type="Italic">Providencia rettgeri</Emphasis> strain YL

Providencia rettgeri strain YL was found to be efficient in heterotrophic nitrogen removal under aerobic conditions. Maximum removal of NH₄ ⁺–N occurred under the conditions of pH 7 and supplemented with glucose as the carbon source. Inorganic ions such as Mg²⁺, Mn²⁺, and Zn²⁺ largely influenced the growth and nitrogen removal efficiency. A quantitative detection of nitrogen gas by gas chromatography was conducted to evaluate the nitrogen removal by strain YL. From the nitrogen balance during heterotrophic growth with 180 mg/l of NH₄ ⁺–N, 44.5% of NH₄ ⁺–N was in the form of N₂ and 49.7% was found in biomass, with only a trace amount of either nitrite or nitrate. The utilization of nitrite and nitrate during the ammonium removal process demonstrated that the nitrogen removal pathway by strain YL was heterotrophic nitrification-aerobic denitrification. A further enzyme assay of nitrate reductase and nitrite reductase activity under the aerobic condition confirmed this nitrogen removal pathway.     

Nickel and the metabolism of urea by Lemna paucicostata Hegelm. 6746

With urea as sole nitrogen source, the addition of 5×10^-5 M nickel sulfate to axenic cultures of Lemna paucicostata 6746 approximately doubles the rate of vegetative growth. Under a standard light-dark schedule, Ni²⁺ changes the daily pattern of respiratory CO₂ output on urea from one having a single daily peak to one with two daily peaks which resembles that on ammonium or nitrate as sole nitrogen source. It also increases CO₂ output by as much as 3-fold on a fresh-weight basis. These data represent the first confirmation in an intact higher plant of proposals, based on enzymology and tissue culture responses, for a role of Ni²⁺ in urea metabolism. Further, they indicate the possible existence of two distinct pathways of urea utilization.     

Growth of moderately thermophilic iron-oxidizing bacterium strain TI-1 in synthetic medium

The moderately thermophilic iron-oxidizing bacterium strain TI-1, which lacks enzyme systems involved in CO₂ fixation, grows at 45°C in Fe²⁺ medium supplemented with yeast extract to give a maximum cell growth of 1.0 × 10⁸ cells per ml, but does not grow in Fe²⁺ medium without yeast extract. To elucidate the physiology of the strain, a synthetic medium was developed. It was found that the best synthetic medium was Fe²⁺-6AA, containing Fe²⁺, salts, and the following six l-amino acids: alanine, aspartic acid, glutamic acid, arginine, serine, and histidine. In this medium, strain TI-1 showed a maximum cell growth of 10 × 10⁸ cells/ml. The six amino acids in the Fe²⁺-6AA medium were used not only as a carbon source but also as a source of nitrogen. Inorganic nitrogen sources, such as ammonium ion, hydrazine, hydroxylamine, nitrite, and nitrate, were not used as a sole source of nitrogen, but rather strongly inhibited the utilization of the six amino acids at 1 mM. In the Fe²⁺ (10 mM)-6AA medium supplemented with 21 mM Fe³⁺, reduction of Fe³⁺ to Fe²⁺ that was dependent on the added amino acids was observed, suggesting another role of the amino acids in the growth of strain TI-1. Washed, intact cells of strain TI-1 had the activity to reduce Fe³⁺ to Fe²⁺.     

The capability of the algaeChlorella vulgaris andScenedesmus obliquus of utilizing amino acids as the sole nitrogen source

The capability of utilizing 20 amino acids and 2 amides as the sole nitrogen source for growth was studied in two green algae (Chlorophyceae). A comparison was made of the growth rate of algae in a mineral nutrient solution containing nitrate as the nitrogen source, with that in the same solution in which nitrogen in the form of nitrate was substituted by an equivalent nitrogen amount in the form of various amino acids. In addition to this, another series of experiments was carried out in whioh both culture media were supplied with glucose. The results show that both algae utilize a series of amino acids in dependence of their structure (mostly 3-carbon amino acids). The growth rate ofChlorella in the presence of these sources is the same as in nitrate, that ofScenedesmus even much higher. In the cultures containing glucose both algal species exhibit a higher growth rate in the media with the nitrate nitrogen source than in those with amino acids (with the exception of glycine inScenedesmus).     

Uptake and degradation of EDTA by Escherichia coli

It was found that Escherichia coli exhibited a growth by utilization of Fe(III)EDTA as a sole nitrogen source. No significant growth was detected when Fe(III)EDTA was replaced by EDTA complexes with other metal ions such as Ca²⁺, Co²⁺, Cu²⁺, Mg²⁺, Mn²⁺, and Zn²⁺. When EDTA uptake was measured in the presence of various ions, it was remarkable only when Fe³⁺ was present. The cell extract of E. coli exhibited a significant degradation of EDTA only in the presence of Fe³⁺. It is likely that the capability of E. coli for the growth by utilization of Fe(III)EDTA results from the Fe³⁺-dependent uptake and degradation of EDTA.     

Effects of nitrate on intracellular nitrite and growth of Microcystis aeruginosa

Although nitrate is a macronutrient and can serve as good nitrogen source for many species of phytoplankton, high nitrate concentrations do not benefit the growth of phytoplankton. We hypothesise that algae cultured under high nitrate concentrations can accumulate intracellular nitrite, which is produced by nitrate reductase (NR) and can inhibit the growth of algae. To assess the validity of this hypothesis, Microcystis aeruginosa was grown under different nitrate concentrations from 3.57 to 21.43 mM in low CO₂ and high CO₂ conditions for 15 days. We observed that, with increasing nitrate concentrations, the intracellular nitrite concentrations of the alga increased and the growth rates and photosynthesis declined. When grown under high CO₂ conditions, M. aeruginosa showed lower intracellular nitrite concentrations and higher growth rates and \textP_\textm^\textchla {\text{P}}_{\text{m}}^{{\text{chl}}a} , \textR_\textd^\textchla {\text{R}}_{\text{d}}^{{\text{chl}}a} , α^chla than under low CO₂ conditions. These results suggest that the accumulation of intracellular nitrite could be the cause of inhibition of algal growth under high nitrate concentrations.     

Identification of nitrogen sources and transformations within karst springs using isotope tracers of nitrogen

Isotope analyses of nitrate and algae were used to gain better understanding of sources of nitrate to Florida’s karst springs and processes affecting nitrate in the Floridan aquifer at multiple scales. In wet years, δ¹⁵N and δ¹⁸O of nitrate ranged from +3 to +9‰ in headwater springs in north Florida, indicating nitrification of soil ammonium as the dominant source. With below normal rainfall, the δ¹⁵N and δ¹⁸O of nitrate were higher in almost all springs (reaching +20.2 and +15.3‰, respectively) and were negatively correlated with dissolved oxygen. In springs with values of δ¹⁵N-NO₃ and δ¹⁸O-NO₃ greater than +10‰, nitrate concentrations declined 40–50% in dry years and variations in the δ¹⁵N and δ¹⁸O of nitrate were consistent with the effects of denitrification. Modeling of the aquifer as a closed system yielded in situ fractionation caused by denitrification of 9 and 18‰ for Δ¹⁸O and Δ¹⁵N, respectively. We observed no strong evidence for local sources of nitrate along spring runs; concentrations declined downstream (0.42–3.3?μmol-NO₃ L^?1 per km) and the isotopic dynamics of algae and nitrate indicated a closed system. Correlation between the δ¹⁵N composition of nitrate and algae was observed at regional and spring-run scales, but the relationship was complicated by varying isotopic fractionation factors associated with nitrate uptake (Δ ranged from 2 to 13‰). Our study demonstrates that nitrate inputs to Florida’s springs are derived predominantly from non-point sources and that denitrification is detectable in aquifer waters with relatively long residence time (i.e., matrix flow).     

CHEMOSENSORY RESPONSES OF THE SYMBIOTIC DINOFLAGELLATE SYMBIODINIUM MICROADRIATICUM (DINOPHYCEAE)1

Motile Symbiodinium microadriaticum (Freudenthal 1962) were attracted to a variety of nitrogen-containing compounds, including ammonium, nitrate, urea and some amino acids. No chemosensory response to phosphate, sulphate, vitamins, trace metals or sugars was evident. Motile algae responded to concentrations of ammonium, nitrate, and urea at least as low as 10^?6 M. High concentrations (≥ 10^?2 M) of ammonium appeared to inhibit attraction of motile algae. Calculations using ammonium release rates from various aposymbiotic hosts suggest that motile S. microadriaticum can respond to released ammonium ca. 1 cm from the source. Cultured algae were not attracted to combined nitrogen cues for at least 2 days after inoculation into seawater with dissolved combined low nitrogen. Algae freshly isolated from starved animals were normally motile the day following isolation and attracted to ammonium and nitrate when maintained in seawater containing < 1 μM ammonium and nitrate. The algae lost their ability to orient to nitrogen attractants the day after incubation into culture medium containing high levels of ammonium and nitrate. These results suggest that chemosensory behavior is suppressed when nutrients are present in the ambient medium or are stored by the alga. There were few differences in chemosensory abilities in different strains of S. microadriaticum to the attractants assayed, suggesting that selection for a particular strain by a host species may not be due to differential chemosensory ability or cues. However, the absence of chemical attraction of motile S. microadriaticum to infected hosts may act to preserve strain selection occurring at other steps in the infection process of aposymbiotic hosts.     

Proton stoichiometric imbalance during algae photosynthetic growth on various nitrogen sources: toward metabolic pH control

The use of algae as a potential platform for fuels or biochemical production requires process design and control that can be implemented at agronomic scales. Toward achieving pH control in large unmixed systems, we present a rigorous set of direct measurements of non-buffered proton uptake and efflux during growth on ammonium and nitrate, observing nearly unit molar proton imbalance H⁺/OH^? respectively for these nitrogen sources. This proton imbalance can be shown to be consistent with the initial assimilation steps of nitrogen from glutamate to peptide bonds which indicates that the remainder of metabolism is largely net proton balanced. These results are refined by demonstrating pH balance for growth with incrementally fed nitric acid and ammonium hydroxide. In contrast to the typical assumption of simple charge balance, each displays a slight proton uptake (around 10 % excess) that is considerably lower than urea, which displayed a molar H⁺ uptake per N assimilated of up to 33 %. This work illustrates details of proton imbalance that have been largely obscured in laboratory work due to use of elevated CO₂ and its associated carbonate equilibrium. Combined with the recent demonstration of preferential, mutually exclusive assimilation of ammonium over nitrate in Chlorella and Chlamydomonas, these results provide the stoichiometry and dynamics of photosynthetic algae growth needed to implement large-scale pH control in the absence of buffering.     

Stimulatory effects of certain amino acids,sugars, organic acids,nucleic acids and metals on nitrate utilization by alkalophilic Bacillus

The growth of alkalophilic Bacillus no. A-40-2 with nitrate as the nitrogen source was highly stimulated by the addition of 0.1% of certain amino acids, sugars, organic acids, nucleic acids, or Fe²⁺ or Mn²⁺ at concentrations of 10 mM or more to the medium, resulting in maximum growth after 24 h. Other alkalophilic Bacillus strains also showed the same results. A decrease in the amount of nitrate in the medium was observed. The optimum pH of nitrate reductase was 7.5.     

Growth of benthic freshwater algae on dairy manures

A potential alternative to land application of livestock manures for cropproduction is the production of algae to recover the nitrogen andphosphorus present in the manure. Compared to terrestrial plants,filamentous algae have exceedingly high growth and nutrient uptake rates. Moreover, they are capable of year-round growth in temperate climates,can be harvested on adapted farm-scale equipment, and yield a biomassthat should be valuable as an animal feed supplement. The objective of thisresearch was to evaluate algal turf scrubber (ATS) technology to removenitrogen, phosphorus and chemical oxygen demand from raw andanaerobically digested dairy manure. Laboratory-scale ATS units wereoperated by continuously recycling wastewater and adding manure effluentsdaily. ATS units were seeded with algal consortia from a nearby streamand grown using dairy manures from two different dairy farms. Algalbiomass was harvested weekly and dried prior to analysis for total Kjeldahlnitrogen, total phosphorus, and inorganic constituents. Wastewater sampleswere analyzed for total Kjeldahl nitrogen, ammonium, nitrate,orthophosphate, conductivity and chemical oxygen demand. Using atypical manure input containing 0.6–0.96 g total nitrogen day^-1,the dried algal yield was approximately 5 g m^-2 day^-1. Thedried algae contained approximately 1.5–2% phosphorus and 5–7%nitrogen. Algal nitrogen and phosphorus accounted for 42–100% ofinput ammonium-nitrogen (33–42% of total nitrogen) and 58–100%of input total phosphorus, respectively.     

Influences of Lead (II) Chloride on the Nitrogen Metabolism of Spinach

Lead (Pb²⁺) is a well-known highly toxic element. The mechanisms of the Pb²⁺ toxicity are not well understood for nitrogen metabolism of higher plants. In this paper, we studied the effects of various concentrations of PbCl₂ on the nitrogen metabolism of growing spinach. The experimental results showed that Pb²⁺ treatments significantly decreased the nitrate nitrogen absorption and inhibited the activities of nitrate reductase, glutamate dehydrogenase, glutamine synthase, and glutamic–pyruvic transaminase of spinach, and inhibited the synthesis of organic nitrogen compounds such as protein and chlorophyll. However, Pb²⁺ treatments increased the accumulation of ammonium nitrogen in spinach cell. It implied that Pb²⁺ could inhibit inorganic nitrogen to be translated into organic nitrogen in spinach, thus led to the reduction in spinach growth.     

Growth, nitrogen utilization and biodiesel potential for two chlorophytes grown on ammonium, nitrate or urea

Nitrogen removal from wastewater by algae provides the potential benefit of producing lipids for biodiesel and biomass for anaerobic digestion. Further, ammonium is the renewable form of nitrogen produced during anaerobic digestion and one of the main nitrogen sources associated with wastewater. The wastewater isolates Scenedesmus sp. 131 and Monoraphidium sp. 92 were grown with ammonium, nitrate, or urea in the presence of 5 % CO₂, and ammonium and nitrate in the presence of air to optimize the growth and biofuel production of these chlorophytes. Results showed that growth on ammonium, in both 5 % CO₂ and air, caused a significant decrease in pH during the exponential phase causing growth inhibition due to the low buffering capacity of the medium. Therefore, biological buffers and pH controllers were utilized to prevent a decrease in pH. Growth on ammonium with pH control (synthetic buffers or KOH dosing) demonstrated that growth (rate and yield), biodiesel production, and ammonium utilization, similar to nitrate- and urea-amended treatments, can be achieved if sufficient CO₂ is available. Since the use of buffers is economically limited to laboratory-scale experiments, chemical pH control could bridge the gap encountered in the scale-up to industrial processes.     

Combined Effect and Mechanism of Acidity and Lead Ion on Soybean Biomass

Heavy metal pollution and soil acidification are serious global environmental issues. The combined pollution from acidification and heavy metal has become a new environmental issue in regions where the two issues simultaneously occur. However, studies on combined pollution are still limited. In the current study, we investigated the combined effect and mechanism of acidity and heavy metal [lead ion (Pb²⁺)] on soybean biomass as well as on growth, nitrogen nutrition, and antioxidant system in soybean roots. Results showed that the combined treatment with acidity and Pb²⁺ decreased the soybean biomass. At pH 4.5, the soybean biomass in the combined treatment with acidity and 0.9 mmol L^?1 Pb²⁺ was lower than that in the combined treatment with acidity and Pb²⁺ at 0.3 or 1.5 mmol L^?1. This result was also observed at pH 3.5 and 3.0. The combined treatment with acidity and Pb²⁺ also resulted in the following consequences: root growth inhibition; decrease in nitrate, ammonium, and malondialdehyde contents; increase in nitrite reductase activity; and decrease in peroxidase activity. The extent at which the test indexes decreased/increased in the combined treatment was higher than that in the single acidity treatment. The correlation analysis results indicated that the decrease in the soybean biomass in the combined treatment with acidity and Pb²⁺ resulted from the decrease in the root growth, nitrate–nitrogen assimilation, and peroxidase activity.     

Exogenous Ca<Superscript>2+</Superscript> alleviates nitrogen and water deficit,and improves growth of wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis>) seedlings exposed to high temperature

This study was designed to examine whether exogenous Ca²⁺ would improve nitrogen nutrition, water status and growth of high temperature (HT)-stressed wheat (Triticum aestivum) seedlings. Wheat plants were exposed to 35/30 and 25/20°C as temperature control. Some of HT-stressed plants were simultaneously treated with 4 mM Ca²⁺. External Ca²⁺ could obviously improve growth of HT-exposed wheat seedlings indicated by the biomass. Compared with Ca²⁺-untreated plants, total nitrogen content showed a significant increase in Ca²⁺-treated plants under HT stress, this primarily resulted from enhanced nitrate reductase activity and depressed loss of ammonium through photorespiration. External Ca²⁺ application could also increase leaf relative water content and alleviate osmotic stress via increased K⁺ ion and water-soluble carbohydrates in HT-stressed plants. Whereas free proline content showed remarkable decline in Ca²⁺-treated plants at HT stress.     

15N natural abundance of plant and soil components of a Banksia woodland ecosystem in relation to nitrate utilization, life form, mycorrhizal status and N2-fixing abilities of component species

Studies of the variation in δ¹⁵N values for plants from a fire-prone Banksia woodland in South West Australia showed that pioneer herbaceous, non-mycorrhizal species which were active in nitrate reduction and storage, had the highest values (1.81%c). A detailed study of one such species Ptilotus polystachus demonstrated a close correspondence between the δ¹⁵N values of soil nitrate, xylem nitrate and leaf total nitrogen, suggesting an exclusive reliance on nitrate ions as nitrogen source. These pioneer species also showed a preponderance of the chloroplastic isoform of glutamine synthetase while woody species generally had higher activity associated with the cytosolic isoform. The group comprising monocotyledonous hemicryptophytes and geophytes contained species with slightly positive δ¹⁵N values and moderately active in nitrate reduction and storage. Nitrogen-fixing species had the lowest δ¹⁵N values (–0.36‰), irrespective of their apparent utilisation of nitrate. However, woody resprouter species which had low levels of nitrate reduction and storage had δ¹⁵N values which fell within the range of values obtained for the miscellaneous assemblage of N₂-fixing species. Consequently, ¹⁵N abundance values failed to distinguish N₂ fixing from non-fixing woody species, and therefore, could not be used in the ecosystem to determine the dependence of putative nitrogen fixing species on N₂ fixation. The study demonstrated complex patterns of nitrogen utilization in the ecosystem in which exploitation of different nitrogen resources related to plant life form and the physiological attributes of nitrogen assimilation by component species.     

SEASONAL FLUCTUATIONS IN TISSUE NITROGEN FOR FIVE SPECIES OF PERENNIAL MACROALGAE IN RHODE ISLAND SOUND1

Tissue nitrogen was assessed monthly for 16 months in five species of perennial macroalgae representing three phyla at one location in Rhode Island Sound. The species showed a remarkable similarity in their pattern of seasonal fluctuation in both nitrate and total nitrogen. The period of greatest accumulation (January through March) coincided with the period of highest concentration of inorganic nitrogen in the water, and for most of these algae it was also the time of-least growth. Conversely, the period of lowest tissue nitrogen (50% of the winter value, May through July) coincided with the period of lowest inorganic nitrogen in the water and highest algal growth. The greatest accumulation of nitrate was found in Laminaria saccharina (L.) Lamour. (80 μmol·g dry wt.^?1), four times as much as that measured simultaneously in the other species and 560 times the ambient concentration. By April the concentration of internal nitrate had dropped to nearly undetectable levels, but in August it began to accumulate again—a pattern that was repeated in Chondrus crispus Stackh. In Ascophyllum nodosum (L.) Le Jolis, Fucus vesiculosus L. and Codium fragile subsp. tomentosoides (Van Goor) Silva, the period of negligible internal nitrate level extended from March to December. The greatest concentration of total tissue nitrogen was measured in C. crispus (4.8% dry wt.), double the maximum in L. saccharina (2.3% dry wt.).     

The utilization of organic nitrogen for growth of algae: physiological aspects

The ability of 27 algae belonging to 11 taxonomic divisions to grow at the expense of organic nitrogen was tested in axenic culture. Experiments were carried out in buffered media: morpholine propanesulphonic acid (MOPS) was used successfully for all fresh water strains except diatoms. It was not used as a nitrogen source by any strain examined but served as a good source of sulphur for four strains. The range of substrates used was extensive and included amino acids, urea, acetamide, urate and some nucleosides. Growth rates varied widely but growth yields were generally comparable to those attained with nitrate or ammonia except for substrates containing more than one utilizable nitrogen atom. Limited experiments were carried out in the dark, and it was found that a given substrate was equally suitable for both dark heterotrophic and photolithotrophic growth. Levels of chlorophyll a were measured during growth of two algae with nitrate, glycine and urate. With nitrate, levels of specific chlorophyll a declined some ten-fold during growth: with poor substrates supporting only slow growth, levels were more uniform but were only 10–15% of those attained during growth with good substrates. One strain grown with MOPS as sole source of sulphur produced cultures with levels of chlorophyll a about half those found in sulphate-grown cultures. Tolerance of sodium chloride was examined in a few strains and even putatively fresh water strains were found to be appreciably tolerant.