Photosensitivity in the Ciliated Protozoon Loxodes: Pigment Granules,Absorption and Action Spectra,Blue Light Perception,and Ecological Significance1

ABSTRACT. The ciliate Loxodes has a faint yellow-brown coloration due to large numbers of electron-dense granules located in the pellicle. Absorption and action spectra both indicate the presence of a blue-light receptor, which may be a flavin. Absorbance is minimal at wavelengths greater than 500 nm; there is a major peak at 360 nm and a pronounced shoulder at 435 nm. An action spectrum based on light-induced escape from oxic water shows a peak at 435 nm and a peak or shoulder at 360 nm. The pigment will generate superoxide when illuminated in the presence of oxygen. Loxodes living in an oxygen gradient in a spectrophotometer cell swims into and remains in anoxic water at light levels ≤10 Wm^-2. Loxodes can be exposed to light levels of 2–20 Wm^-2 in stratified lakes so its photobehavior can explain its periodic absence from oxic water. Photosensitivity in Loxodes may function as part of a predator-avoidance strategy.     

Oxidation of nitric oxide by a new heterotrophic Pseudomonas sp.

A new bacterial strain isolated from soil consumed nitric oxide (NO) under oxic conditions by oxidation to nitrate. Phenotypic and phylogenetic characterization of the new strain PS88 showed that it represents a previously unknown species of the genus Pseudomonas, closely related to Pseudomonas fluorescens and Pseudomonas putida. The heterotrophic, obligately aerobic strain PS88 was not able to denitrify or nitrify; however, strain PS88 oxidized NO to nitrate. NO was not reduced to nitrous oxide (N₂O). Nitrogen dioxide (NO₂) and nitrite (NO₂ ^–) as possible intermediates of NO oxidation to nitrate (NO₃ ^–) could not be detected. NO oxidation was inhibited under anoxic conditions and by high osmolarity, but not by nitrite. NO oxidation activity was inhibited by addition of formaldehyde, HgCl₂, and antimycin, and by autoclaving or disintegrating the cells, indicating that the process was enzyme-mediated. However, the mechanism remains unclear. A stepwise oxidation at a metalloenzyme and a radical mechanism are discussed. NO oxidation in strain PS88 seems to be a detoxification or a co-oxidation mechanism, rather than an energy-yielding process. Received: 15 November 1995 / Accepted: 24 February 1996     

Co-occurrence of denitrification and nitrogen fixation in a meromictic lake, Lake Cadagno (Switzerland)

The nitrogen cycling of Lake Cadagno was investigated by using a combination of biogeochemical and molecular ecological techniques. In the upper oxic freshwater zone inorganic nitrogen concentrations were low (up to ～3.4 μM nitrate at the base of the oxic zone), while in the lower anoxic zone there were high concentrations of ammonium (up to 40 μM). Between these zones, a narrow zone was characterized by no measurable inorganic nitrogen, but high microbial biomass (up to 4 × 10⁷ cells ml^?1). Incubation experiments with ¹⁵N‐nitrite revealed nitrogen loss occurring in the chemocline through denitrification (～3 nM N h^?1). At the same depth, incubations experiments with ¹⁵N₂‐ and ¹³C_DIC‐labelled bicarbonate, indicated substantial N₂ fixation (31.7–42.1 pM h^?1) and inorganic carbon assimilation (40–85 nM h^?1). Catalysed reporter deposition fluorescence in situ hybridization (CARD‐FISH) and sequencing of 16S rRNA genes showed that the microbial community at the chemocline was dominated by the phototrophic green sulfur bacterium Chlorobium clathratiforme. Phylogenetic analyses of the nifH genes expressed as mRNA revealed a high diversity of N₂ fixers, with the highest expression levels right at the chemocline. The majority of N₂ fixers were related to Chlorobium tepidum/C. phaeobacteroides. By using Halogen In Situ Hybridization‐Secondary Ion Mass Spectroscopy (HISH‐SIMS), we could for the first time directly link Chlorobium to N₂ fixation in the environment. Moreover, our results show that N₂ fixation could partly compensate for the N loss and that both processes occur at the same locale at the same time as suggested for the ancient Ocean.     

Cloning,sequencing, and characterization of a gene (narT) encoding a transport protein involved in dissimilatory nitrate reduction inStaphylococcus carnosus

A Tn917 mutant ofStaphylococcus carnosus TM300, nrIII, was isolated and characterized. Mutant nrIII did not take up nitrate or accumulate nitrite when grown in B-medium supplemented with up to 10 mM nitrate under anoxic conditions; however, it displayed wild-type levels of benzyl viologen-linked nitrate reductase activity. Cultivated in B-medium with nitrate under oxic conditions, mutant nrIII accumulated fivefold less nitrite than the wild-type. The mutation inS. carnosus nrIII could be complemented with a 2-kb chromosomalEcoRI-HpaI fragment from the wild-type. The gene affected by transposon insertion in mutant nrIII was cloned and sequenced. Analysis of the deduced amino acid sequence revealed that this gene, designatednarT, encodes a highly hydrophobic 42-kDa transmembrane protein of 388 amino acids and shows similarities to transport proteins that play a role in nitrate import or nitrite export. The inability of nrIII to take up nitrate under anoxic conditions and its ability to take up and accumulate nitrite in the presence of benzyl viologen, a nitrate ionophore, under the same conditions suggest that NarT represents a transport protein required for nitrate uptake under anoxic conditions inS. carnosus.     

Root contribution to nitrate reduction in barley seedlings (Hordeum vulgare L.)

Summary The leaf and root nitrate reductase activities were measured in 7 day-old barley seedlings by anoxic nitrite accumulation in darkness, during 48h after the transfer from a N-starved medium to a 1.5 mM K¹⁵NO₃ medium. Thisin situ nitrate reduction was compared with the¹⁵N incorporation in the reduced N fraction of the whole seedlings.The nitrate reduction integrated fromin situ measurements was lower than the reduced¹⁵N accumulation. The rootin situ nitrate reductase activity seemed to account for only the third of the real root nitrate reduction, which may have been responsible for the overall underestimation. This discrepancy was partly explained by the ability of the root to reduce nitrite in an anoxic environment.These results suggest that, after correction of thein situ estimation of the nitrate reduction. the roots contribute to about 50% of the total assimilation.     

The denitrification capacity of sediment from a hypereutrophic lake

SUMMARY.

• 1 In sediment from Wintergreen Lake, Michigan, denitrification was not detectable by the acetylene inhibition method at in situ nitrate concentrations. When nitrate was added to sediment slurries, denitrification capacities up to 18.8μg N g^-1 h^-1 were measured. The denitrification capacities decreased with increasing sediment depth and distance from shore.
• 2 The high denitrification capacities in these sediments which under natural conditions had no supply of nitrate and oxygen suggested that denitrifies with alternative mechanisms for anaerobic energy conversion were present. Nitrous oxide was a significant portion of the N-gas produced immediately after the nitrate addition. Small amounts (4–5% of the total N-gas production) of nitric oxide accumulated in the early phase of nitrate reduction. Presumably after depletion of nitrate and nitrite both N₂O and NO were further reduced to N₂.
• 3 About 70%r of the added nitrate was denitrified, and the remainder was assumed to have been reduced to ammonium.

     

Lithotrophic growth ofSulfurospirillum deleyianum with sulfide as electron donor coupled to respiratory reduction of nitrate to ammonia

Sulfurospirillum deleyianum grew in batch culture under anoxic conditions with sulfide (up to 5 mM) as electron donor, nitrate as electron acceptor, and acetate as carbon source. Nitrate was reduced to ammonia via nitrite, a quantitatively liberated intermediate. Four moles of sulfide were oxidized to elemental sulfur per mole nitrate converted to ammonia. The molar growth yield per mole sulfide consumed, Y_m, was 1.5 ± 0.2 g mol^–1 for the reduction of nitrate to ammonia. By this type of metabolism, S. deleyianum connected the biogeochemical cycles of sulfur and nitrogen. The sulfur reductase activity in S. deleyianum was inducible, as the activity depended on the presence of sulfide or elemental sulfur during cultivation with nitrate or fumarate as electron acceptor. Hydrogenase activity was always high, indicating that the enzyme is constitutively expressed. The ammonia-forming nitrite reductase was an inducible enzyme, expressed when cells were cultivated with nitrate, nitrite, or elemental sulfur, but repressed after cultivation with fumarate. Received: 13 March 1995 / Accepted: 29 May 1995     

Microbial processes of the carbon and sulfur cycles in an ice‐covered,iron‐rich meromictic lake Svetloe (Arkhangelsk region,Russia)

Biogeochemical, isotope geochemical and microbiological investigation of Lake Svetloe (White Sea basin), a meromictic freshwater was carried out in April 2014, when ice thickness was ～0.5 m, and the ice‐covered water column contained oxygen to 23 m depth. Below, the anoxic water column contained ferrous iron (up to 240 μμM), manganese (60 μM), sulfide (up to 2 μM) and dissolved methane (960 μM). The highest abundance of microbial cells revealed by epifluorescence microscopy was found in the chemocline (redox zone) at 23–24.5 m. Oxygenic photosynthesis exhibited two peaks: the major one (0.43 μmol C L^?1 day^?1) below the ice and the minor one in the chemocline zone, where cyanobacteria related to Synechococcus rubescens were detected. The maximum of anoxygenic photosynthesis (0.69 μmol C L^?1 day^?1) at the oxic/anoxic interface, for which green sulfur bacteria Chlorobium phaeoclathratiforme were probably responsible, exceeded the value for oxygenic photosynthesis. Bacterial sulfate reduction peaked (1.5 μmol S L^?1 day^?1) below the chemocline zone. The rates of methane oxidation were as high as 1.8 μmol CH₄ L^?1 day^?1 at the oxi/anoxic interface and much lower in the oxic zone. Small phycoerythrin‐containing Synechococcus‐related cyanobacteria were probably involved in accumulation of metal oxides in the redox zone.     

Intracellular nitrite accumulation: The cause of growth inhibition of Microcystis aeruginosa exposure to high nitrite level

The aim of the present study is to test the role of intracellular nitrite in external nitrite suppressing algal growth. We examined the growth of Microcystis aeruginosa at different nitrite levels under high nitrate conditions and without nitrate conditions. There were higher intracellular nitrite and lower P_m^chla, R_d ^chla, α^chl, maximum cell density and specific growth rate in high nitrate group than nitrate absence group at 5 mg NO₂^?‐N L^?1. At 10 and 15 mg NO₂^?‐N L^?1, P_m^chla, R_d ^chla, α^chl, maximum cell densities and specific growth rates in the high nitrate group became higher than those of the nitrate absence group, while a lower intracellular nitrite in the high nitrate group than nitrate absence group was observed. In addition, the intracellular nitrite and the growth of M. aeruginosa in the high nitrate group did not change from 5 to 10 mg NO₂^?‐N L^?1. In the nitrite uptake experiment, with nitrite concentration increasing from 5 to 15 mg NO₂^?‐N L^?1, maximum nitrite uptake rate of alga increased, and half‐saturation constant of alga decreased. These results indicate that external nitrite inhibited algal growth through stimulating intracellular nitrite rise, which resulted from overexpression of nitrite transporter.     

Nitrate and nitrite in interstitial waters of eelgrass beds in relation to the rhizosphere

The distribution of nitrate and nitrite in the interstitial water of the sediment of eelgrass (Zostera marina) bed of Izembek Lagoon, Alaska, were investigated. Their concentrations were relatively high (0 to 9.8 μg-at.N·1^?1, average 4.8 for nitrate; 0 to 4.0 μ-at.N·1^?1, average 1.9 for nitrite) although the sediments were anoxic and contained hydrogen sulphide. The rates of bacterial denitrification measured by ¹⁵N tracer technique ranged from 0.49×10^?10 to 1.2 × 10^?9 g-atN·g^?1·h^?1. When a steady state is maintained, the loss of nitrate and nitrite must be balanced by their production by bacterial nitrification. Experimentally determined rate of nitrification in the sediment was of the same order. A model experiment demonstrated that oxygen is transported from leaves to rhizomes and roots of eelgrass and released into the sediment. The oxygen is used for nitrification in the rhizosphere in anoxic sediments.     

Gaseous NO2 as a regulator for ammonia oxidation of Nitrosomonas eutropha

Cells of Nitrosomonas eutropha strain N904 that were denitrifying under anoxic conditions with hydrogen as electron donor and nitrite as electron acceptor were unable to utilize ammonium (ammonia) as an energy source. The recovery of ammonia oxidation activity was dependent on the presence of NO₂. Anaerobic ammonia oxidation activity was observed in a helium atmosphere supplemented with 25 ppm NO₂ after 20 h. Ammonia oxidation activity was detected after 2–3 days using an oxic atmosphere with 25 ppm NO₂. In contrast, ammonia consumption started after 8–9 days under oxic conditions without the addition of NO₂; in this case, small amounts of NO and NO₂ were detected and their concentrations increased with increasing ammonia oxidation activities. Hardly any ammonia oxidation was detected when nitrogen oxides were removed by intensive aeration. It would seem, therefore, that NO₂ is the master regulatory signal for ammonia oxidation in Nitrosomonas eutropha. Anaerobic ammonia oxidation activity was inhibited by the addition of NO. This inhibition was partly compensated by either increasing the NO₂ concentration or by using 2,3-dimercapto-1-propane-sulfonic acid as a NO binding substrate. DMPS was inhibitory to nitrification under oxic conditions, while increased amounts of NO or NO₂ led to increased oxidation activities.     

Spatial heterogeneity and short‐term oxygen dynamics in the rhizosphere of Vallisneria spiralis: Implications for nutrient cycling

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Nitrite Uptake Patterns in Wheat Seedlings as Influenced by Nitrate and Ammonium

Nitrite and nitrate uptake by wheat (Triticum vulgare) from 0.5 mM potassium solutions both showed an apparent induction pattern characterized by a slow initial rate followed by an accelerated rate. The accelerated phase was more rapid for nitrate uptake, was initiated earlier, and was seriously restricted by the presence of equimolar nitrite. The accelerated phase of nitrite uptake was restricted by nitrate to a lesser extent. The two anions seem not to be absorbed by identical mechanisms. Ammonium pretreatments or prior growth with ammonium had relatively little influence on the pattern of nitrite uptake. However, prior growth with nitrate eliminated the slow initial phase and induced development of the accelerated phase of nitrite uptake. A beneficial effect was noted after 3 h nitrate pretreatment and full development had occurred by 12 h nitrate pretreatment. The evidence suggests that a small amount of tissue nitrite, which could be supplied either by absorption or by nitrate reduction, was specifically required for induction of the accelerated phase of nitrite uptake. Cycloheximide (2 μg ml^?1) seriously restricted development of the accelerated phase of nitrite uptake, but its effect was not as severe when it was added after the accelerated phase had been induced by prior exposure to nitrite or nitrate. However, translocation of ¹⁵N from the absorbed nitrite was sharply decreased under the latter conditions, indicating a difference in sensitivity of the uptake and translocation processes to cycloheximide. Potassium uptake was greater from KNO₃ than from KNO₂ and in both instances it was enhanced during the early stages of the accelerated phase of anion uptake. Moreover, addition of NaNO₃ to KNO₂ substantially increased potassium uptake. A coupling between anion and potassium uptake was therefore evident, but the coupling was not obligatory because the accelerated phase of nitrite uptake could occur in absence of rapid potassium uptake.     

Mass Spectrometric Studies of the Effect of pH on the Accumulation of Intermediates in Denitrification by Paracoccus denitrificans

We have used a quadrupole mass spectrometer with a gas-permeable membrane inlet for continuous measurements of the production of N₂O and N₂ from nitrate or nitrite by cell suspensions of Paracoccus denitrificans. The use of nitrate and nitrite labeled with ¹⁵N was shown to simplify the interpretation of the results when these gases were measured. This approach was used to study the effect of pH on the production of denitrification intermediates from nitrate and nitrite under anoxic conditions. The kinetic patterns observed were quite different at acidic and alkaline pH values. At pH 5.5, first nitrate was converted to nitrite, then nitrite was converted to N₂O, and finally N₂O was converted to N₂. At pH 8.5, nitrate was converted directly to N₂, and the intermediates accumulated to only low steady-state concentrations. The sequential usage of nitrate, nitrite, and nitrous oxide observed at pH 5.5 was simulated by using a kinetic model of a branched electron transport chain in which alternative terminal reductases compete for a common reductant.     

Effects of gaseous NO2 on cells of Nitrosomonas eutropha previously incapable of using ammonia as an energy source

Cells of Nitrosomonas eutropha grown under anoxic conditions with hydrogen as electron donor and nitrite as electron acceptor were initially unable to oxidize ammonia (ammonium) and hydroxylamine when transferred to oxic conditions. Recovery of ammonia and hydroxylamine oxidation activity was dependent on the presence of NO₂. Under oxic conditions, without addition of NO₂, ammonia consumption started after 8 – 9 days, and small amounts of NO and NO₂ were detectable in the gas atmosphere. Removing these nitrogen oxides by intensive aeration, ammonia oxidation activity decreased and broke off after 15 days. Addition of gaseous NO₂ (25 ppm) led to a fast recovery of ammonia oxidation (3 days). Simultaneously, the arrangement of intracytoplasmic membranes (ICM) changed from circular to flattened vesicles, the protein pattern revealed an increase in the concentration of a 27 and a 30 kDa polypeptide, and the cytochrome c content increased significantly.     

Effect of internal recycle on the nitrogen removal efficiency of an anaerobic/anoxic/oxic (A2/O) wastewater treatment plant (WWTP)

Internal recycle ratio is an important parameter in anaerobic/anoxic/oxic (A²/O) wastewater treatment plant (WWTP) operation. An increase in this ratio decreases nitrate and nitrite concentration in the effluent and hence improves the nitrogen removal efficiency, even though the economical cost increases simultaneously. Determining the most favourable recycle ratio taking into account both considerations is an important item in A²/O WWTP operational optimisation. In this work, the effect of recycle ratio on nitrogen removal when using different influent nitrogen loads was tested in a pilot A²/O WWTP. Experimental results obtained show how increasing the internal recycle ratio from 0 to 5 produced a 12% increase in nitrogen removal. This increase was achieved by improving N–NO_x removal by 9% with an increase in N–NH₄⁺ removal of 3%.     

Effect of transient oxic conditions on the composition of the nitrate-reducing community from the rhizosphere of Typha angustifolia

Within a nitrate-reducing bacterial community, a niche differentiation between denitrifying and nitrate ammonifying bacteria may be determinated by a complex of environmental parameters, such as the availability of carbon, nitrate, and oxygen. Hence, oxygen- and carbon-releasing aerenchymatous plants may affect the composition of the nitrate-reducing community in waterlogged sediment. The composition of the nitrate-reducing community in the rhizosphere of the aerenchymatous plant species Typha angustifolia was compared with the community in nonrhizospheric sediment. All three functional groups (NO₂ ^– accumulators, N₂O producers, and presumed NH₄ ⁺ producers) were present at both sites with an ratio of 36:45:12 and 43:22:18 for nonrhizospheric and rhizospheric sediments, respectively. Most of the isolated were gram-negative, and approximately 50% of these strains demonstrated an obligatory oxidative metabolism.In the absence of nitrate, Enterobacteriaceae (belonging to the NO₂ ^– accumulating group) became dominant during enrichment of bacteria from the rhizosphere of T. angustifolia in a chemostat with glycerol (20 mM) as substrate, both under strictly anoxic and transient oxic conditions. Addition of nitrate to the chemostats led to the predominance of denitrifying pseudomonads, irrespective of the presence or absence of oxygen. However, in the presence of nitrate under anoxic conditions, enterobacteria persisted in the medium together with pseudomonads.It was concluded that oxidative bacteria such as pseudomonads are the better competitors for limiting amounts of glycerol, provided oxygen or nitrate is present. In the absence of these electron acceptors, fermentative bacteria become dominant.     

Estimation of the microbial biomass in tidal flat sediment by fumigation-extraction

A transect of ten profiles was laid out in 20 m intervals on a tidal sand flat approximately 100 m from the east shore of Sylt until the next tideway was reached. Sediment samples were taken from 0–2 cm depth (oxic layer) and 2–4 cm depth (anoxic layer). The average content of organic carbon (C) was 2.41 mg g⁻¹ in the oxic layer and 1.86 mg g⁻¹ in the anoxic layer. The organic C content correlated positively with non-biomass C, 0.5M K₂SO₄ extractable C, total nitrogen (N), cation exchange capacity (CEC), and the textural classes <200 μm, and negatively correlated with the coarse sand fraction. The average total C:N ratio was 7.0 in the oxic layer and 6.7 in the anoxic layer, indicating that the C input comes entirely from the microflora. CHCl₃-labile C was measured by the fumigation-extraction method and was converted to microbial biomass C (values in brackets). The average content of CHCl₃-labile C was 407 μg g⁻¹ (903 μg g⁻¹) in the oxic layer and 214 μg g⁻¹ (476 μg g⁻¹) in the anoxic layer. CHCl₃-labile C did not correlate with CEC and the textural classes <200 μm, indicating that conditions other than the physical environment determine this fraction (C input, grazing).