Denitrification of PVA-immobilized denitrifying photosynthetic bacterium,Rhodobacter sphaeroides

A newly isolated denitrifying strain, Rhodobacter sphaeroides NII2 was immobilized in polyvinyl alcohol (PVA) gel, and the properties of the cells in the gel were examined. The immobilized cells had low or almost no denitrification activity, but the cells were activated by incubation in light with culture medium for denitrification containing 0.5% nitrate and no other nitrogen source. Cells grown in the dark were activated by incubation at an earlier stage and to a higher rate than the light-grown cells. The activation was markedly enhanced in the PVA gel with a low cell concentration. The immobilized cells consumed nitrate with a temporary accumulation of NO₂ and evolved nitrogen gas. The immobilized cells could use various organic compounds as electron donors for denitrification. Thus, the immobilized cells were applied to a continuous treatment of synthetic wastewater using an aparatus devised by this laboratory. The results showed an efficient removal of NO₃-N from the test water.     

Growth and nutrient removal properties of a freshwater microalga Scenedesmus sp. LX1 under different kinds of nitrogen sources

Microalgae have received much attention for the inorganic nutrient removal in tertiary treatment of domestic wastewater. Effect of different kinds of nitrogen sources on the growth and nitrogen/phosphorus removal properties of a newly isolated freshwater microalga, Scenedesmus sp. LX1, from a low-nutrient environment condition was studied and reported in this paper. The order of specific growth rate of the microalga with different nitrogen sources was NH₄-N > urea-N > NO₃-N. With nitrate or urea as nitrogen source, the microalga could grow well and remove both nitrogen and phosphorus efficiently (90% nitrogen and nearly 100% phosphorus were removed). However, with ammonium as the nitrogen source, the maximum algal density was relatively low, and the nitrogen and phosphorus removal efficiencies were as low as 31.1% and 76.4%, respectively. This was caused by the inhibitory effect of algal culture's acid pH due to H⁺ releasing from NH₄⁺ during algal cultivation process.     

Dilution of 15N in Dunaliella tertiolecta by uptake of an unidentified compound following nitrate exhaustion

Nitrate (about 20 μM) was added as ¹⁵NO₃ to a nitrate-limited continuous culture of Dunaliella tertiolecta at steady-state. Nitrate uptake was then estimated from the decrease in nitrate in the medium, the incorporation of ¹⁵N into cells, and the increase in cellular nitrogen. Although the overall nitrogen budget over 5 h was balanced, there were large differences in estimates (up to a factor of five) of nitrate assimilation by the three methods on shorter time scale. After nitrate was exhausted from the medium, cellular nitrogen continued to increase while the ¹⁵N content of the particulate matter decreased over the next 1.5 h. This indicated that an unidentified, unlabelled nitrogen form, which was neither nitrite, ammonium nor dissolved free amino acids, was being taken up by the cells, at rates comparable to those of nitrate. This phenomenon leads to an underestimation of new biomass production when assessed through ¹⁵N incorporation into cells.     

A method for the laboratory culture of the planktonic rotifer Keratella cochlearis (Gosse)

Procedures for the continuous laboratory culture of Keratella cochlearis in a defined medium and upon an algal food are described. Culturing success appears to be a function of food availability as well as composition. This availability requirement is satisfied by the use of test tubes and inverted titration plate concavities as culture vessels. The satisfactory culture medium contains an ammonia compound as a nitrogen source.     

Influence of Carbon Source on Nitrate Removal by Nitrate-Tolerant Klebsiella oxytoca CECT 4460 in Batch and Chemostat Cultures

The nitrate-tolerant organism Klebsiella oxytoca CECT 4460 tolerates nitrate at concentrations up to 1 M and is used to treat wastewater with high nitrate loads in industrial wastewater treatment plants. We studied the influence of the C source (glycerol or sucrose or both) on the growth rate and the efficiency of nitrate removal under laboratory conditions. With sucrose as the sole C source the maximum specific growth rate was 0.3 h⁻¹, whereas with glycerol it was 0.45 h⁻¹. In batch cultures K. oxytoca cells grown on sucrose or glycerol were able to immediately use sucrose as a sole C source, suggesting that sucrose uptake and metabolism were constitutive. In contrast, glycerol uptake occurred preferentially in glycerol-grown cells. Independent of the preculture conditions, when sucrose and glycerol were added simultaneously to batch cultures, the sucrose was used first, and once the supply of sucrose was exhausted, the glycerol was consumed. Utilization of nitrate as an N source occurred without nitrite or ammonium accumulation when glycerol was used, but nitrite accumulated when sucrose was used. In chemostat cultures K. oxytoca CECT 4460 efficiently removed nitrate without accumulation of nitrate or ammonium when sucrose, glycerol, or mixtures of these two C sources were used. The growth yields and the efficiencies of C and N utilization were determined at different growth rates in chemostat cultures. Regardless of the C source, yield carbon (Y_C) ranged between 1.3 and 1.0 g (dry weight) per g of sucrose C or glycerol C consumed. Regardless of the specific growth rate and the C source, yield nitrogen (Y_N) ranged from 17.2 to 12.5 g (dry weight) per g of nitrate N consumed. In contrast to batch cultures, in continuous cultures glycerol and sucrose were utilized simultaneously, although the specific rate of sucrose consumption was higher than the specific rate of glycerol consumption. In continuous cultures double-nutrient-limited growth appeared with respect to the C/N ratio of the feed medium and the dilution rate, so that for a C/N ratio between 10 and 30 and a growth rate of 0.1 h⁻¹ the process led to simultaneous and efficient removal of the C and N sources used. At a growth rate of 0.2 h⁻¹ the zone of double limitation was between 8 and 11. This suggests that the regimen of double limitation is influenced by the C/N ratio and the growth rate. The results of these experiments were validated by pulse assays.     

Effects of atrazine on the assimilation of inorganic nitrogen in cereals

The inclusion of sub-lethal amounts ofthe herbicide atrazine [2-chloro-4-(ethylamino)-6-(isopropylamino)-s-triazine] in the nutrient solution supplied to maize and barley increased the growth of the root and shoot and the uptake of nitrate. The activities of nitrate and nitrite reductases, glutamine synthetase and glutamate synthase were enhanced and the amino acid and nitrate contents of the xylem sap increased. All these effects of atrazine were found only in plants grown with nitrate as the nitrogen source. The uptake of ¹⁵NO₃^? and its incorporation into protein in the root and shoot of maize and barley seedlings was significantly greater in the atrazine treated plants. However, a stimulation in the incorporation of leucine-[¹⁴C] into TCA-precipitable protein of detached leaves from 7-day-old barley seedlings was obtained only in the absence of a supply of combined nitrogen either in the culture medium or in the in vitro incubation mixture containing the labelled amino acid.     

Interrelationships between carbohydrate metabolism and nitrogen assimilation in cultured plant cells

Summary The effect of the nature and concentration of the nitrogen source on respiratory activity and removal of carbohydrate from the medium in suspension cultured sycamore (Acer pseudoplatanus L.) cells was determined. Comparison was also made of the rates of uptake of the two alternative nitrogen sources, nitrate and glutamate, at differing initial nitrogen concentrations within the range 7–14 mM. The initial pH of the culture medium before inoculation was 5.2; after inoculation the pH of both nitrate and glutamate cultures rose to reach an eventual level in the range 7.0–7.1. Glutamate was removed from the medium more slowly than nitrate. Under the particular conditions of culture used the growth of the cells was nitrogen limited. Sugar uptake from the medium continued for some time after the nitrogen in the medium was depleted. The data show that although cell division and protein content are nitrogen-limited, dry weight and fresh weight yields may also be determined in a complex interaction through carbohydrate availability. There were no obvious differences in respiratory activity between cultures grown on nitrate or glutamate.     

Effect of nitrogen-mediated changes in alkalinity on pH control and CO(2) supply in intensive microalgal cultures

The freshwater alga Scenedesmus obliquus was grown in continuous culture at a fixed dilution rate of 0.5/day, but at varying pH in the range 4.17-10.67. The pH was regulated in the range 4.17-7.67 by continuously bubbling 1% CO(2)-enriched air into the cultures and by varying the source of nitrogen (NO(3) (-), NH(4) (+), or urea) in the growth medium, which, in turn, led to changes in culture alkalinity. Culture alkalinity and P(CO(2) ) were the sole determinants of pH. A pH-stat system, together with NO(3) (-) in the medium, was used to regulate the pH in the range 7.92-10.67. Maximum productivity, which occurred at pH 6.6, was dependent on N source only to the extent that culture alkalinity was a function of nitrogen uptake. The results demonstrate that the choice of N is a critical factor in controlling the pH of large-scale algal cultures. NH(4) (+) is a poor source of N because it leads to destruction of culture alkalinity and concomitant growth-inhibiting reductions in pH, whereas NO(3) (-) has an opposite effect, although pH is not so severely affected in this case. Urea is, by far, the most suitable N source for maximizing algal yield when it is supplied in combination with the proper amounts of HCO(3) (-) alkalinity in the growth medium and percent CO(2) in the bubbled gas that will lead to an equilibrium pH near the optimum pH.     

Removal of Nitrate from Groundwater by Cyanobacteria: Quantitative Assessment of Factors Influencing Nitrate Uptake

The feasibility of biologically removing nitrate from groundwater was tested by using cyanobacterial cultures in batch mode under laboratory conditions. Results demonstrated that nitrate-contaminated groundwater, when supplemented with phosphate and some trace elements, can be used as growth medium supporting vigorous growth of several strains of cyanobacteria. As cyanobacteria grew, nitrate was removed from the water. Of three species tested, Synechococcus sp. strain PCC 7942 displayed the highest nitrate uptake rate, but all species showed rapid removal of nitrate from groundwater. The nitrate uptake rate increased proportionally with increasing light intensity up to 100 μmol of photons m⁻² s⁻¹, which parallels photosynthetic activity. The nitrate uptake rate was affected by inoculum size (i.e., cell density), fixed-nitrogen level in the cells in the inoculum, and aeration rate, with vigorously aerated, nitrate-sufficient cells in mid-logarithmic phase having the highest long-term nitrate uptake rate. Average nitrate uptake rates up to 0.05 mM NO₃⁻ h⁻¹ could be achieved at a culture optical density at 730 nm of 0.5 to 1.0 over a 2-day culture period. This result compares favorably with those reported for nitrate removal by other cyanobacteria and algae, and therefore effective nitrate removal from groundwater using this organism could be anticipated on large-scale operations.     

Enhancement of nitric oxide solubility using Fe(II)EDTA and its removal by green algae <Emphasis Type="Italic">Scenedesmus</Emphasis> sp.

A photoautotrophic cultivation of green algae Scenedesmus cells was used for the removal of nitric oxide (NO) from a model flue gas mixture. In an attempt to improve the solubility of NO in the culture broth, the addition of Fe(II)EDTA to the cultivation was investigated. The addition of Fe(II)EDTA greatly enhanced NO-dissolution in the culture broth and subsequently increased the algal-uptake of NO. NO was assimilated as a source of nitrogen for the growth of Scenedesmus cells since there was a steady increase in cell density with no other nitrogen source in the culture except the incoming NO. 40–45% of NO removal was maintained for more than 12 days with the addition of 5 mM Fe(II)EDTA in a 1-L air-lift type photobioreactor system fed with 300 ppm of NO gas at a rate of 0.3 wm. However, the NO-dissolution-enhancing capacity of Fe(II)EDTA did not reach its full potential due to its oxidation to Fe(III)EDTA, possibly induced by molecular oxygen that evolved from algal photosynthesis, and subsequent loss of chelating capabilities.     

Nitrate reductase activity in Paul's scarlet rose suspension cultures and the differential role of nitrate and molybdenum in induction

Induction of nitrate reductase (EC 1.6.6.1) activity was measured in Paul's Scarlet rose cell suspensions cultured in media containing nitrate (NO ₃ ^- ) or urea (U) as nitrogen source, and with (+Mo) or without molybdenum (-Mo). There was a lag of 30 min during induction by NO ₃ ^- in +Mo cultures but no lag occurred during induction after adding Mo to NO ₃ ^- -Mo or to U-Mo cultures preincubated with NO ₃ ^- . Actinomycin D, cycloheximide, and puromycin completely blocked induction by NO ₃ ^- , but had no effect on the initial rate of induction by Mo. Cycloheximide and puromycin blocked induction by NO ₃ ^- more quickly than actinomycin D. Induction by NO ₃ ^- appeared to involve mRNA-dependent synthesis of apoprotein followed by rapid activation with molybdenum in intact cells independently of protein synthesis. Nitrate-induced apoprotein appeared less stable than the holoenzyme. When induced by NO ₃ ^- in the absence of Mo, apoprotein concentration was about half the amount of maximally induced nitrate reductase. Cycloheximide stabilised preformed nitrate reductase which disappeared steadily in the presence of puromycin. Apoprotein was not stabilised by either antimetabolite.Abbreviations Mo molybdenum - NO ₃ ^- +Mo standard, MX₁ culture medium - NO ₃ ^- -Mo MX₁ medium purified of Mo - NR nitrate reductase - PSR Paul's Scarlet rose - U urea - U+Mo MX₁ medium with NO ₃ ^- replaced by urea - U-Mo MX₁ medium with NO ₃ ^- replaced by urea and also purified of Mo     

Removal of inorganic nitrogen sources from water by the algal biofilm of the aerial microalga Trentepohlia aurea

Removal of inorganic nitrogen sources by cells of the aerial microalga Trentepohlia aurea grown on the surface of substrate, such as filter paper, has been investigated in a batch system. When the alga grew on the paper dampened with medium, it actively ingested inorganic nitrogenous compounds in the medium. Immobilized cells on the filter papers were called algal biofilm in this study. When the algal biofilms were soaked in modified Bold's Basal medium (using 1 g NH₄Cl l^–1 as a N source), the removal rate was 4.25 mg ammonium-N l^–1 day^–1 in 40 days. In modified medium with added 26 mg nitrite-N, the removal rate of the total inorganic N ion by the biofilms reached 5.11 mg N l^–1 day^–1. This removal rate of total N ion was higher than that in the medium by addition of 26 mg nitrate-N. In addition, we tried to examine simultaneous removal of ammonium, nitrate, and nitrite ions and growth inhibition of cyanobacteria in the medium by using the algal biofilms. Consequently, it was demonstrated that the algal biofilms of T. aurea could be utilized as a biofunctional material for the purification of wastewater.     

Production and Consumption of Nitric Oxide by Three Methanotrophic Bacteria

We studied nitrogen oxide production and consumption by methanotrophs Methylobacter luteus (group I), Methylosinus trichosporium OB3b (group II), and an isolate from a hardwood swamp soil, here identified by 16S ribosomal DNA sequencing as Methylobacter sp. strain T20 (group I). All could consume nitric oxide (nitrogen monoxide, NO), and produce small amounts of nitrous oxide (N₂O). Only Methylobacter strain T20 produced large amounts of NO (>250 parts per million by volume [ppmv] in the headspace) at specific activities of up to 2.0 × 10⁻¹⁷ mol of NO cell⁻¹ day⁻¹, mostly after a culture became O₂ limited. Production of NO by strain T20 occurred mostly in nitrate-containing medium under anaerobic or nearly anaerobic conditions, was inhibited by chlorate, tungstate, and O₂, and required CH₄. Denitrification (methanol-supported N₂O production from nitrate in the presence of acetylene) could not be detected and thus did not appear to be involved in the production of NO. Furthermore, cd₁ and Cu nitrite reductases, NO reductase, and N₂O reductase could not be detected by PCR amplification of the nirS, nirK, norB, and nosZ genes, respectively. M. luteus and M. trichosporium produced some NO in ammonium-containing medium under aerobic conditions, likely as a result of methanotrophic nitrification and chemical decomposition of nitrite. For Methylobacter strain T20, arginine did not stimulate NO production under aerobiosis, suggesting that NO synthase was not involved. We conclude that strain T20 causes assimilatory reduction of nitrate to nitrite, which then decomposes chemically to NO. The production of NO by methanotrophs such as Methylobacter strain T20 could be of ecological significance in habitats near aerobic-anaerobic interfaces where fluctuating O₂ and nitrate availability occur.     

Effects of different nitrogen species on sensitivity and photosynthetic stress of three common freshwater diatoms

Different sources of nitrogen pose diverse effects to algal community, but the mechanism of inhibitory effects of nitrogen sources on freshwater diatoms is not fully understood. The purpose of this study was to compare biomass, photosynthetic activity, and morphological structure of three common freshwater diatoms (Cyclotella meneghiniana, Nitzschia sp., and Gomphonema parvulum) under different nitrogen sources (NO₃ ^? or NH₄ ⁺). The sorption characteristic of each diatom was investigated, and chlorophyll a (Chl-a) content and oxygen evolution rate were analyzed to investigate stress of different nitrogen sources on each diatom in the batch experiments. Ammonium lowered the growth rate of C. meneghiniana and Nitzschia sp. when it was supplied in addition to growth-saturating nitrate concentrations, suggesting a combined effect of inhibition of nitrate uptake and direct ammonium stress. Oxygen evolution rate of Nitzschia sp. showed that the direct ammonium stress on the photosynthetic activity can be alleviated by coexistence of nitrate in the nitrogen enriched treatment, but not for C. meneghiniana and G. parvulum, which may be caused by a different nitrate transporter system within algal cells. Transmission electron microscopy was used to assess the toxicity of ammonium on ultrastructural chloroplast of each diatom. Ultrastructural changes in chloroplasts showed undefined electron-dense granules and lipid droplets, but the membrane integrity of cell was maintained, suggesting an adaptation to adjustment to ammonia stress. Results showed that Cyclotella meneghiniana and Nitzschia sp. were more sensitive to ammonium stress than Gomphonema parvulum on growth, but the mechanism remains unclear.     

Urea uptake by the scleractinian coral Stylophora pistillata

Urea can be one of the major sources of nitrogen for phytoplankton, but little is known about its importance for corals. Experiments were therefore designed to assess the uptake rates of urea by the scleractinian coral Stylophora pistillata; ¹⁵N-urea was used to follow the incorporation of nitrogen into the zooxanthellae and animal tissue. The uptake kinetics of urea in the tissue of S. pistillata showed that there is a concentration-dependent uptake of urea. The transport of urea was composed of a linear component (diffusion) at concentrations higher than 6 μmol N-urea l^− 1 and an active carrier-mediated component, at lower concentrations. The value of the carrier affinity (K_m = 1.05 μmol urea l^− 1) indicates a good adaptation of the corals to low levels of urea in seawater. At the in situ concentration of ca. 0.2 μmol N-urea l^− 1, the uptake rate was equal to 0.1 nmol N h^− 1 cm^− 2. Urea uptake was at least four times higher in the animal than in the algal fraction, and five times higher when corals were incubated in the light than in the dark. These results could be explained by the involvement of urea in the calcification process, which is also enhanced by light. Comparison of urea uptake rates with nitrate or ammonium uptake rates for the same S. pistillata species, at in situ concentrations, showed that urea is preferred to nitrate and may therefore be an important source of nitrogen for scleractinian corals.     

Culture of Scenedesmus sp. LX1 in the modified effluent of a wastewater treatment plant of an electric factory by photo-membrane bioreactor

To investigate the coupled technology for advanced wastewater treatment and microalgal biomass production, a photo-membrane bioreactor was constructed. The microalga Scenedesmus sp. LX1 was cultured in the bioreactor using liquor prepared from the effluent of an electronic device factory. The algal cell growth, nitrate nitrogen removal, orthophosphate phosphorus removal were investigated. When cultured with batch operation, the average specific growth rate was about 0.09 d⁻¹, and low nitrogen (N), phosphorus (P) concentrations in the liquor were achieved. However, under continuous operation with an inflow of 60 L h⁻¹, the average specific growth rate was only 0.02 d⁻¹, and removal rates of 100% for orthophosphate P and 46% for nitrate N were achieved. With the inflow of 120 L h⁻¹, the accumulated metal ions in the bioreactor adversely affected the algal cells. The algal cells were much easier to settle, and the removal efficiency for N and P decreased.     

冬小麦等4种作物对铵,硝态氮的吸收能力

采用水培试验探讨了冬小麦、大豆、油菜和莴笋４种作物对硝、铵态氮的相对吸收能力以及这两种氮源对它们生长发育的影响。试验表明：（１）不同氮源对供试作物的生长发育影响极大。供给硝态氮,这些作物生长发育良好,供给等量的ＮＯ＾－３和ＮＨ＾－４（１：１）时,蔬菜作物莴笋生长量下降幅度最大;供给铵态氨,莴笋和大豆极为敏感,供给ＮＯ＾－３时莴笋吸氮量显著高于供给等氮量ＮＯ＾－３和ＮＨ＾＋４,莴上麦供给等量ＮＯ＾－     

Hydrogen uptake by the nitrogen-starved cyanobacterium Anabaena cylindrica

The role of the oxyhydrogen reaction in the nitrogen metabolism of Anabaena cylin-drica, particularly under conditions of dinitrogen starvation, was investigated. It was shown that although this reaction supports nitrogenase activity in the dark, when the cells are deprived of nitrogen the rate of hydrogen uptake is little changed. Measurements of ammonia excretion into the medium in the presence of methionine sulfoximine under such conditions indicated that hydrogen uptake supported the turnover of cell protein as an alternative source of nitrogen. In the absence of H₂ and O₂ in the dark, nitrogenase activity was negligible but protein turnover continued. In their presence nitrogenase activity was greatly stimulated; turnover was also stimulated but to a greater extent in the absence of nitrogenase substrates. The oxyhydrogen reaction also stimulated uptake of ammonium ions by intact filaments in argon in the dark. Only at very low hydrogen tensions can net hydrogen formation be obtained in argon/CO₂ in the light, casting considerable doubt on the suitability of hydrogenase-containing organisms for biophotolytic hydrogen formation. Addition of exogenous ammonia to the cultures incubated in argon resulted in a pronounced stimulation of H₂ uptake; nitrate and its derivatives had no such effect, nor did various amino acid derivatives of ammonia.     

Increase in Nitrate Reductase Activity with Ammonium Chloride in Bacillus licheniformis by Shaking Culture

In shaking culture, nitrate reductase activity in the cell-free extracts of Bacillus licheniformis increased with the addition of NH₄Cl to the medium containing NaNO₃ as a single nitrogen source, where amounts of nitrogen sources were sufficient for cell growth. This increase of nitrate reductase activity therefore suggests that the activity is not for nitrate assimilation but for other physiological functions containing a dissimilatory nitrate reduction.     

Accumulation,mobilization and turn-over of glycogen in the blue-green bacterium Anacystis nidulans

1. Accumulation of glycogen up to a constant amount per cell was observed during the post-exponential phase of growth, in the presence of an excess of a utilizable carbon source. Cell multiplication was reproducibly controlled by growth of the organism in a nitrogen-limiting medium under photoautotrophic conditions (presence of light, air plus CO₂).
2. Temporary starvation, i.e. by removal of light or by the addition to an illuminated culture of DCMU, 3-(3′,4′-dichlorophenyl)-1,1′-dimethylurea, a specific inhibitor of photosystem II, lead to a mobilization of glycogen in the cell. Furthermore, Anacystis nidulans, having accumulated glycogen by virtue of preculture under nitrogen-limiting conditions, will resume cell division when the culture medium is complemented with a nitrogen source. The ability of the organism to use glycogen as an endogenous carbon source for growth was observed by addition of a nitrogen source to nitrogen-starving cells and simultaneous removal of CO₂.
3. During the period of constant amount of glycogen per cell the reserve polysaccharide was subject to turnover as demonstrated with a pulse chase-labelling technique. The demonstration of a turnover—for the first time with a bacterial species—indicated a strict balance in the relative rate of synthesis and degradation.