Simultaneous oxidation of ammonium and p-cresol linked to nitrite reduction by denitrifying sludge

The metabolic capability of denitrifying sludge to oxidize ammonium and p-cresol was evaluated in batch cultures. Ammonium oxidation was studied in presence of nitrite and/or p-cresol by 55 h. At 50 mg/L NH₄⁺-N and 76 mg/L NO₂⁻-N, the substrates were consumed at 100% and 95%, respectively, being N₂ the product. At 50 mg/L NH₄⁺-N and 133 mg/L NO₂⁻-N, the consumption efficiencies decreased to 96% and 70%, respectively. The increase in nitrite concentration affected the ammonium oxidation rate. Nonetheless, the N₂ production rate did not change. In organotrophic denitrification, the p-cresol oxidation rate was slower than ammonium oxidation. In litho-organotrophic cultures, the p-cresol and ammonium oxidation rates were affected at 133 mg/L NO₂⁻-N. Nonetheless, at 76 mg/L NO₂⁻-N the denitrifying sludge oxidized ammonium and p-cresol, but at different rate. Finally, this is the first work reporting the simultaneous oxidation of ammonium and p-cresol with the production of N₂ from denitrifying sludge.     

Enrichment and granulation of Anammox biomass started up with methanogenic granular sludge

A sequencing batch reactor (SBR) seeded with methanogenic granular sludge was started up to enrich Anammox (Anaerobic Ammonium Oxidation) bacteria and to investigate the feasibility of granulation of Anammox biomass. Research results showed that hydraulic retention time (HRT) was an important factor to enrich Anammox bacteria. When the HRT was controlled at 30 days during the initial cultivation, the SBR reactor presented Anammox activity at t = 58 days. Simultaneously, the methanogenic granular sludge changed gradually from dust black to brown colour and its diameter became smaller. At t = 90 days, the Anammox activity was further improved. NH₄⁺-N and NO₂⁻N were removed simultaneously with higher speed and the maximum removal rates reached 14.6 g NH₄⁺-N /(m³ _reactor·day) and 6.67 g NO₂⁻-N /(m³ _reactor·day), respectively. Between t = 110 days and t = 161 days, the nitrogen load was increased to a HRT of 5 days (70 mg/l NH₄⁺ and 70 mg/l NO₂), the removal rates of ammonium and nitrite were 60.6% and 62.5% respectively. The sludge changed to red and formed Anammox granulation with high nitrogen removal activity.     

Anammox-zeolite system acting as buffer to achieve stable effluent nitrogen values

For a successful nitrogen removal, Anammox process needs to be established in line with a stable partial nitritation pretreatment unit since wastewater influent is mostly unsuitable for direct treatment by Anammox. Partial nitritation is, however, a critical bottleneck for the nitrogen removal since it is often difficult to maintain the right proportions of NO₂-N and NH₄-N during long periods of time for Anammox process. This study investigated the potential of Anammox-zeolite biofilter to buffer inequalities in nitrite and ammonium nitrogen in the influent feed. Anammox-zeolite biofilter combines the ion-exchange property of zeolite with the biological removal by Anammox process. Continuous-flow biofilter was operated for 570 days to test the response of Anammox-zeolite system for irregular ammonium and nitrite nitrogen entries. The reactor demonstrated stable and high nitrogen removal efficiencies (approximately 95 %) even when the influent NO₂-N to NH₄-N ratios were far from the stoichiometric ratio for Anammox reaction (i.e. NO₂-N to NH₄-N ranging from 0 to infinity). This is achieved by the sorption of surplus NH₄-N by zeolite particles in case ammonium rich influent came in excess with respect to Anammox stoichiometry. Similarly, when ammonium-poor influent is fed to the reactor, ammonium desorption took place due to shifts in ion-exchange equilibrium and deficient amount were supplied by previously sorbed NH₄-N. Here, zeolite acted as a preserving reservoir of ammonium where both sorption and desorption took place when needed and this caused the Anammox-zeolite system to act as a buffer system to generate a stable effluent.     

Effect of influent substrate ratio on anammox granular sludge: performance and kinetics

Effect of influent substrate ratio on anammox process was studied in sequencing batch reactor. Operating temperature was fixed at 35 ± 1 °C. Influent pH and hydraulic retention time were 7.5 and 6 h, respectively. When influent NO₂ ^?-N/NH₄ ⁺-N was no more than 2.0, total nitrogen removal rate (TNRR) increased whereas NH₄ ⁺-N removal rate stabilized at 0.32 kg/(m³ d). ΔNO₂ ^?-N/ΔNH₄ ⁺-N increased with enhancing NO₂ ^?-N/NH₄ ⁺-N. When NO₂ ^?-N/NH₄ ⁺-N was 4.5, ΔNO₂ ^?-N/ΔNH₄ ⁺-N was 1.98, which was much higher than theoretical value (1.32). The IC₅₀ of NO₂ ^?-N was 289 mg/L and anammox activity was inhibited at high NO₂ ^?-N/NH₄ ⁺-N ratio. With regard to influent NH₄ ⁺-N/NO₂ ^?-N, the maximum NH₄ ⁺-N removal rate was 0.36 kg/(m³ d), which occurred at the ratio of 4.0. Anammox activity was inhibited when influent NH₄ ⁺-N/NO₂ ^?-N was higher than 5.0. With influent NO₃ ^?-N/NH₄ ⁺-N of 2.5–6.5, NH₄ ⁺-N removal rate and NRR were stabilized at 0.33 and 0.40 kg/(m³ d), respectively. When the ratio was higher than 6.5, nitrogen removal would be worsened. The inhibitory threshold concentration of NO₂ ^?-N was lower than NH₄ ⁺-N and NO₃ ^?-N. Anammox bacteria were more sensitive to NO₂ ^?-N than NH₄ ⁺-N and NO₃ ^?-N. TNRR would be enhanced with increasing nitrogen loading rate, but sludge floatation occurred at high nitrogen loading shock. The Han-Levenspiel could be applied to simulate nitrogen removal resulting from NO₂ ^?-N inhibition.     

Nitrogen nutrition of Salvinia natans: Effects of inorganic nitrogen form on growth,morphology, nitrate reductase activity and uptake kinetics of ammonium and nitrate

In this study we assessed the growth, morphological responses, and N uptake kinetics of Salvinia natans when supplied with nitrogen as NO₃⁻, NH₄⁺, or both at equimolar concentrations (500 μM). Plants supplied with only NO₃⁻ had lower growth rates (0.17 ± 0.01 g g⁻¹ d⁻¹), shorter roots, smaller leaves with less chlorophyll than plants supplied with NH₄⁺ alone or in combination with NO₃⁻ (RGR = 0.28 ± 0.01 g g⁻¹ d⁻¹). Ammonium was the preferred form of N taken up. The maximal rate of NH₄⁺ uptake (V_max) was 6–14 times higher than the maximal uptake rate of NO₃⁻ and the minimum concentration for uptake (C_min) was lower for NH₄⁺ than for NO₃⁻. Plants supplied with NO₃⁻ had elevated nitrate reductase activity (NRA) particularly in the roots showing that NO₃⁻ was primarily reduced in the roots, but NRA levels were generally low (<4 μmol NO₂⁻ g⁻¹ DW h⁻¹). Under natural growth conditions NH₄⁺ is probably the main N source for S. natans, but plants probably also exploit NO₃⁻ when NH₄⁺ concentrations are low. This is suggested based on the observation that the plants maintain high NRA in the roots at relatively high NH₄⁺ levels in the water, even though the uptake capacity for NO₃⁻ is reduced under these conditions.     

Rates, controls and potential adverse effects of nitrate removal in a denitrification bed

Denitrification beds are a simple approach for removing nitrate (NO₃⁻) from a range of point sources prior to discharge into receiving waters. These beds are large containers filled with woodchips that act as an energy source for microorganisms to convert NO₃⁻ to nitrogen (N) gases (N₂O, N₂) through denitrification. This study investigated the biological mechanism of NO₃⁻ removal, its controlling factors and its adverse effects in a large denitrification bed (176 m × 5 m × 1.5 m) receiving effluent with a high NO₃⁻ concentration (>100 g N m⁻³) from a hydroponic glasshouse (Karaka, Auckland, New Zealand). Samples of woodchips and water were collected from 12 sites along the bed every two months for one year, along with measurements of gas fluxes from the bed surface. Denitrifying enzyme activity (DEA), factors limiting denitrification (availability of carbon, dissolved organic carbon (DOC), dissolved oxygen (DO), temperature, pH, and concentrations of NO₃⁻, nitrite (NO₂⁻) and sulfide (S²⁻)), greenhouse gas (GHG) production - as nitrous oxide (N₂O), methane (CH₄), carbon dioxide (CO₂) - and carbon (C) loss were determined. NO₃⁻-N concentration declined along the bed with total NO₃⁻-N removal rates of 10.1 kg N d⁻¹ for the whole bed or 7.6 g N m⁻³ d⁻¹. NO₃⁻-N removal rates increased with temperature (Q₁₀ = 2.0). In laboratory incubations, denitrification was always limited by C availability rather than by NO₃⁻. DO levels were above 0.5 mg L⁻¹ at the inlet but did not limit NO₃⁻-N removal. pH increased steadily from about 6 to 7 along the length of the bed. Dissolved inorganic carbon (C-CO₂) increased in average about 27.8 mg L⁻¹, whereas DOC decreased slightly by about 0.2 mg L⁻¹ along the length of the bed. The bed surface emitted on average 78.58 μg m⁻² min⁻¹ N₂O-N (reflecting 1% of the removed NO₃⁻-N), 0.238 μg m⁻² min⁻¹ CH₄ and 12.6 mg m⁻² min⁻¹ CO₂. Dissolved N₂O-N increased along the length of the bed and the bed released on average 362 g dissolved N₂O-N per day coupled with N₂O emission at the surface about 4.3% of the removed NO₃⁻-N as N₂O. Mechanisms to reduce the production of this GHG need to be investigated if denitrification beds are commonly used. Dissolved CH₄ concentrations showed no trends along the length of the bed, ranging from 5.28 μg L⁻¹ to 34.24 μg L⁻¹. Sulfate (SO₄²⁻) concentrations declined along the length of the bed on three of six samplings; however, declines in SO₄²⁻ did not appear to be due to SO₄²⁻ reduction because S²⁻ concentrations were generally undetectable. Ammonium (NH₄⁺) (range: <0.0007 mg L⁻¹ to 2.12 mg L⁻¹) and NO₂⁻ concentrations (range: 0.0018 mg L⁻¹ to 0.95 mg L⁻¹) were always very low suggesting that anammox was an unlikely mechanism for NO₃⁻ removal in the bed. C longevity was calculated from surface emission rates of CO₂ and release of dissolved carbon (DC) and suggested that there would be ample C available to support denitrification for up to 39 years.This study showed that denitrification beds can be an efficient tool for reducing high NO₃⁻ concentrations in effluents but did produce some GHGs. Over the course of a year NO₃⁻ removal rates were always limited by C and temperature and not by NO₃⁻ or DO concentration.     

Stimulating denitrification in a stream mesocosm with elemental sulfur as an electron donor

The heavy use of fertilizers in agricultural lands can result in significant nitrate (NO₃⁻) loadings to the aquatic environment. We hypothesized that biological denitrification in agricultural ditches and streams could be enhanced by adding elemental sulfur (S^o) to the sediment layer, where it could act as a biofilm support and electron donor. Using a bench-scale stream mesocosm with a bed of S^o granules, we explored NO₃⁻ removal fluxes as a function of the effluent NO₃⁻ concentrations. With effluent NO₃⁻ ranging from 0.5 mg N L⁻¹ to 4.1 mg N L⁻¹, NO₃⁻ removal fluxes ranged from 228 mg N m⁻² d⁻¹ to 708 mg N m⁻² d⁻¹. This is as much as 100 times higher than for agricultural drainage streams. Sulfate (SO₄²⁻) production was high due to aerobic sulfur oxidation. Molecular studies demonstrated that the S^o amendment selected for Thiobacillus species, and that no special inoculum was required for establishing a S^o-based autotrophic denitrifying community. Modeling studies suggested that denitrification was diffusion limited, and advective flow through the bed would greatly enhance NO₃⁻ removal fluxes. Our results indicate that amendment with S^o is an effective means to stimulate denitrification in a stream environment. To minimize SO₄²⁻ production, it may be better to place S^o deeper in the sediment layer.     

Short term effects of hypoxia and bioturbation on solute fluxes, denitrification and buffering capacity in a shallow dystrophic pond

The effects of short term hypoxia on bioturbation activity and inherent solute fluxes are scarcely investigated even if increasing number of coastal areas are subjected to transient oxygen deficits. In this work dark fluxes of oxygen (O₂), dissolved inorganic carbon (TCO₂) and nutrients across the sediment-water interface, as well as rates of denitrification (isotope pairing), were measured in intact sediment cores collected from the dystrophic pond of Sali e Pauli (Sardinia, Italy). Sediments were incubated at 100, 70, 40 and 10% of O₂ saturation in the overlying water, with both natural benthic communities, dominated by the polychaete Polydora ciliata (11.100 ± 2.500  ind. m^− 2), and after the addition of individuals of the deep-burrower polychaete Hediste diversicolor. Below an uppermost oxic layer of ~ 1 mm, sediments were highly reduced, with up to 6 mM of S²⁻ in the 5 mm layer. Flux of S²⁻ and O₂ calculated from pore water gradients were 8.61 ± 1.12 and − 2.27 ± 0.56 mmol m^− 2 h^− 1, respectively. However, sediment oxygen demand (SOD) calculated from core incubation was − 10.52 ± 0.33 mmol m^− 2 h^− 1, suggesting a major contribution of P. ciliata to O₂-mediated sulphide oxidation. P. ciliata also strongly stimulated NH₄⁺ and PO₄³⁻ fluxes, with rates ~ 15 and ~ 30 folds higher, respectively, than those estimated from pore water gradients. P. ciliata activity was significantly reduced at 10% O₂ saturation, coupled to decreased rates of solutes transfer. The addition of H. diversicolor further stimulated SOD, NH₄⁺ efflux and SiO₂ mobilisation. Similarly to P. ciliata, the degree of stimulation of SOD and NH₄⁺ flux by H. diversicolor depended on the level of oxygen saturation. TCO₂ regeneration, respiratory quotients, PO₄³⁻ fluxes and denitrification of added ¹⁵NO₃⁻ were not affected by the addition of H. diversicolor, but depended upon the O₂ levels in the water column. Denitrification rates supported by water column ¹⁴NO₃⁻ and sedimentary nitrification were both negligible (< 0.5 µmol m^− 2 h^− 1). They were not significantly affected by oxygen saturation nor by bioturbation, probably due to the limited availability of NO₃⁻ in the water column (< 3 µM) and O₂ in the sediments. This study demonstrates for the first time the integrated short term effect of transient hypoxia and bioturbation on solute fluxes across the sediment-water interface within a simplified lagoonal benthic community.     

Morphological and physiological adaptive traits of Mediterranean narrow endemic plants: The case of Centaurea gymnocarpa (Capraia Island,Italy)

A case study on Centaurea gymnocarpa Moris & De Not., a narrow endemic species, was carried out by analyzing its morphological, anatomical, and physiological traits in response to natural habitat stress factors under Mediterranean climate conditions. The results underline that the species is particularly adapted to the environment where it naturally grows. At the plant level, the above-ground/below-ground dry mass (1.73 ± 0.60) shows its investment predominately in the above-ground structure with a resulting total leaf area per plant of 1399 ± 94 cm². The senescent attached leaves at the base of the plant contribute to limit leaf transpiration by shading soil around the plant. Moreover, the dense C. gymnocarpa leaf pubescence, leaf rolling, the relatively high leaf mass area (LMA = 12.3 ± 1.3 mg cm⁻²) and leaf tissue density (LTD = 427 ± 44 mg cm⁻³) contribute to limit leaf transpiration, also postponing leaf death under dry conditions. At the physiological level, a relatively low respiration/photosynthesis ratio (R/P_N) in spring results from high R [2.26 ± 0.59 μmol (CO₂) m⁻² s⁻¹] and P_N [12.3 ± 1.5 μmol (CO₂) m⁻² s⁻¹]. The high photosynthetic nitrogen use efficiency [PNUE = 15.5 ± 0.4 μmol (CO₂) g⁻¹ (N) s⁻¹] shows the large amount of nitrogen (N) invested in the photosynthetic machinery of new leaves, associated to a high chlorophyll content (Chl = 35 ± 5 SPAD units). On the contrary, the highest R/P_N ratio (1.75 ± 0.19) in summer is due to a significant P_N decrease and increase of R in response to drought. The low PNUE [1.5 ± 0.2 μmol (CO₂) g⁻¹ (N) s⁻¹] in this season is indicative of a greater N investment in leaf cell walls which may contribute to limit transpiration. On the contrary, the low R/P_N ratio (0.05 ± 0.02) in winter is resulting from the limited enzyme activity of the respiratory apparatus [R = 0.23 ± 0.08 μmol (CO₂) m⁻² s⁻¹] while the low PNUE [3.5 ± 0.2 μmol (CO₂) g⁻¹ (N) s⁻¹] suggests that low temperatures additionally limit plant production. The experiment of the imposed water stress confirms that the C. gymnocarpa growth capability is in conformity with the severe conditions of its natural habitat, likewise as it may be the case with others narrow endemic species that have occupied niches with similar extreme conditions.     

Different sensitivity of Phragmites australis and Glyceria maxima to high availability of ammonium-N

The ability to cope with NH₄⁺-N was studied in the littoral helophytes Phragmites australis and Glyceria maxima, species commonly occupying fertile habitats rich in NH₄⁺ and often used in artificial wetlands. In the present study, Glyceria growth rate was reduced by 16% at 179 μM NH₄⁺-N, and the biomass production was reduced by 47% at 3700 μM NH₄⁺-N compared to NO₃⁻-N. Similar responses were not found in Phragmites. The amounts (mg g⁻¹ dry wt) of starch and total non-structural carbohydrates (TNC) in rhizomes were significantly lower in NH₄⁺ (8.9; 12.2 starch; 20.1; 41.9 TNC) compared to NO₃⁻ treated plants (28.0; 15.6 starch; 58.5; 56.3 TNC) in Phragmites and Glyceria, respectively. In addition, Glyceria showed lower amounts (mg g⁻¹ dry wt) of soluble sugars, TNC, K⁺, and Mg²⁺ in roots under NH₄⁺ (5.6; 14.3; 20.6; 1.9) compared to NO₃⁻ nutrition (11.6; 19.9; 37.9; 2.9, for soluble sugars, TNC, K⁺, and Mg²⁺, respectively), while root internal levels of NH₄⁺ and Ca²⁺ (0.29; 4.6 mg g⁻¹ dry wt, mean of both treatments) were only slightly affected. In Phragmites, no changes in soluble sugars, TNC, Ca²⁺, K⁺, and Mg²⁺ contents of roots (7.3; 14.9; 5.1; 17.3; 2.6 mg g⁻¹ dry wt, means of both treatments) were found in response to treatments. The results, therefore, indicate a more pronounced tolerance towards high NH₄⁺ supply in Phragmites compared to Glyceria, although the former may be susceptible to starch exhaustion in NH₄⁺-N nutrition. In contrast, Glyceria's ability to colonize fertile habitats rich in NH₄⁺ is probably related to the avoidance strategy due to shallow rooting or to the previously described ability to cope with high NH₄⁺ levels when P availability is high and NO₃⁻ is also provided.     

Bicarbonate use in three aquatic plants

Our study aimed to test the ability of aquatic plants to use bicarbonate when acclimated to three different bicarbonate concentrations. To this end, we performed experiments with the three species Ceratophyllum demersum, Egeria densa, Lagarosiphon major to determine photosynthetic rates under varying bicarbonate concentrations. We measured bicarbonate use efficiency, photosynthetic performance and respiration. For all species, our results revealed that photosynthetic rates were highest in replicates grown at low alkalinity. Thus, E. densa had approx. five times higher rates at low (264 ± 15 μmol O₂ g⁻¹ DW h⁻¹) than at high alkalinity (50 ± 27 μmol O₂ g⁻¹ DW h⁻¹), C. demersum had three times higher rates (336 ± 95 and 120 ± 31 μmol O₂ g⁻¹ DW h⁻¹), and L. major doubled its rates at low alkalinity (634 ± 114 and 322 ± 119 μmol O₂ g⁻¹ DW h⁻¹). Similar results were obtained for bicarbonate use efficiency by E. densa (136 ± 44 and 43 ± 10 μmol O₂ mequiv. L⁻¹ g⁻¹ DW h⁻¹) and L. major (244 ± 29 and 82 ± 24 μmol O₂ mequiv. L⁻¹ g⁻¹ DW h⁻¹). As to C. demersum, efficiency was high but unaffected by alkalinity, indicating high adaptation ability to varied alkalinities. A pH drift experiment supported these results. Overall, our results suggest that the three globally widespread worldwide species of our study adapt to low inorganic carbon availability by increasing their efficiency of bicarbonate use.     

Reevaluation of the nutrient mineralization process by infaunal bivalves (Ruditapes philippinarum) in a shallow lagoon in Hokkaido, Japan

Previous estimations of nutrient mineralization in the water column by infaunal bivalves might have been overestimated because of underestimation of the uptake process by microphytobenthos in the field. We conducted field surveys of environmental conditions and quantitative sampling of Ruditapes philippinarum in a shallow lagoon system (Hichirippu Lagoon, eastern Hokkaido, Japan) in August 2006. We recorded the spatial distribution pattern and the molar ratio of dissolved inorganic nutrients to determine the limiting nutrients for microphytobenthos, to evaluate the input and output of nutrients at the entrance of the lagoon station, and to estimate potential nutrient mineralization by R. philippinarum. Our aim was to reevaluate the nutrient mineralization process by infaunal bivalve species. In this study, the mean standing stock of microphytobenthos inhabiting surface sediment (5 mm thick) on the tidal flats was 100 times higher than that of phytoplankton (1 m depth). Low N/P and high Si/N ratios (mean = 2.6 and 17.6, respectively) near the entrance of the lagoon compared to those of microphytobenthos (N:P:Si = 10.1:1:18) clearly suggest N deficiency. The flux of NH₄-N coming into the lagoon was 3.4 kmolN d^− 1, and the flux out was − 3.7 kmolN d^− 1. Thus, assuming that there would have been no phytoplankton and microphytobenthos uptake during the day, 0.3 kmolN d^− 1 of NH₄-N was produced within the lagoon. However, the NH₄-N mineralization rate of the clams has been estimated to be approximately 7.7 ± 6.8 kmolN d^− 1. Thus, 96% (7.4 kmolN d^− 1, i.e., 7.7 kmolN d^− 1 minus 0.3 kmolN d^− 1) of the NH₄-N mineralized by the clam was consumed by microphytobenthos. In contrast, if all the NH₄-N inflow (3.1 kmolN d^− 1) was consumed by the microalgae before outflow, 52% (4.0 kmolN d^− 1, i.e., 7.7 kmolN d^− 1 minus 3.7 kmolN d^− 1) of the NH₄-N mineralized by the clams should have been consumed by microphytobenthos. Microphytobenthos on the tidal flats (11.3 ± 11.8 kmolN) used all of the surplus nutrients (between 4.0 and 7.4 kmolN d^− 1), and the temporal division rate [=(NH₄-N uptake)/(standing stock of microphytobenthos)] of microphytobenthos would have to be between 0.35 and 0.65 d^− 1. Residual NH₄-N (0.3 - 3.7 kmolN d^− 1) was the water-column source and accounted for 12-148% of NH₄-N in the water column near the entrance of the lagoon (2.5 ± 1.4 kmolN) per day. This is the first field-based observation with a quantitative evaluation of nutrient mineralization by infaunal bivalves and nutrient uptake by microphytobenthos.     

Importance of the operating pH in maintaining the stability of anoxic ammonium oxidation (anammox) activity in moving bed biofilm reactors

Two bench-scale parallel moving bed biofilm reactors (MBBR) were operated to assess pH-associated anammox activity changes during long term treatment of anaerobically digested sludge centrate pre-treated in a suspended growth partial nitrification reactor. The pH was maintained at 6.5 in reactor R1, while it was allowed to vary naturally between 7.5 and 8.1 in reactor R2. At high nitrogen loads reactor R2 had a 61% lower volumetric specific nitrogen removal rate than reactor R1. The low pH and the associated low free ammonia (FA) concentrations were found to be critical to stable anammox activity in the MBBR. Nitrite enhanced the nitrogen removal rate in the conditions of low pH, all the way up to the investigated level of 50 mg NO₂-N/L. At low FA levels nitrite concentrations up to 250 mg NO₂-N/L did not cause inactivation of anammox consortia over a 2-days exposure time.     

Determination of the membrane permeability characteristics of Pacific oyster, Crassostrea gigas, oocytes and development of optimized methods to add and remove ethylene glycol

In order for cryopreservation to become a practical tool for aquaculture, optimized protocols must be developed for each species and cell type. Knowledge of a cell’s osmotic tolerance and membrane permeability characteristics can assist in optimized protocol development. In this study, these characteristics were determined for Pacific oyster oocytes and modified methods for loading and unloading ethylene glycol (EG) were tested. Oocytes were found to behave as ideal osmometers and their osmotically inactive fraction (V_b) was calculated to be 0.48. Oocytes exposed to NaCl solutions of 0.6 to 2.3 Osm fertilized at rates equivalent to oocytes left in seawater. This corresponds to volume changes of +27.3 and −38.1 ± 1.2%. The permeability of the oocytes to water (L_p) was determined to be 3.8 ± 0.4 × 10⁻², 5.7 ± 0.8 × 10⁻², and 13.2 ± 1.3 × 10⁻² μm min⁻¹ atm⁻¹, when measured at temperatures of 5, 10 and 20 °C. The respective EG permeability values (P_s) were 9.5 ± 0.1 × 10⁻⁵, 14.6 ± 1.2 × 10⁻⁵, and 41.7 ± 2.4 × 10⁻⁵ cm min⁻¹. The activation energies for L_p and P_s were determined to be 14.5 and 17.5 kcal mol⁻¹, respectively. Different models for EG loading and unloading from oocytes were developed and tested. Post-thaw fertilization did not differ significantly between a published step addition method and single step addition at 20 °C. This represents a considerable reduction in handling. The results of this study demonstrate that the cryobiological characteristics of a given cell type should be taken into account when developing cryopreservation methods.     

Nitrogen nutrition of Canna indica: Effects of ammonium versus nitrate on growth, biomass allocation, photosynthesis, nitrate reductase activity and N uptake rates

The effects of inorganic nitrogen (N) source (NH₄⁺, NO₃⁻ or both) on growth, biomass allocation, photosynthesis, N uptake rate, nitrate reductase activity and mineral composition of Canna indica were studied in hydroponic culture. The relative growth rates (0.05-0.06 g g⁻¹ d⁻¹), biomass allocation and plant morphology of C. indica were indifferent to N nutrition. However, NH₄⁺ fed plants had higher concentrations of N in the tissues, lower concentrations of mineral cations and higher contents of chlorophylls in the leaves compared to NO₃⁻ fed plants suggesting a slight advantage of NH₄⁺ nutrition. The NO₃⁻ fed plants had lower light-saturated rates of photosynthesis (22.5 μmol m⁻² s⁻¹) than NH₄⁺ and NH₄⁺/NO₃⁻ fed plants (24.4-25.6 μmol m⁻² s⁻¹) when expressed per unit leaf area, but similar rates when expressed on a chlorophyll basis. Maximum uptake rates (V_max) of NO₃⁻ did not differ between treatments (24-35 μmol N g⁻¹ root DW h⁻¹), but V_max for NH₄⁺ was highest in NH₄⁺ fed plants (81 μmol N g⁻¹ root DW h⁻¹), intermediate in the NH₄NO₃ fed plants (52 μmol N g⁻¹ root DW h⁻¹), and lowest in the NO₃⁻ fed plants (28 μmol N g⁻¹ root DW h⁻¹). Nitrate reductase activity (NRA) was highest in leaves and was induced by NO₃⁻ in the culture solutions corresponding to the pattern seen in fast growing terrestrial species. Plants fed with only NO₃⁻ had high NRA (22 and 8 μmol NO₂⁻ g⁻¹ DW h⁻¹ in leaves and roots, respectively) whereas NRA in NH₄⁺ fed plants was close to zero. Plants supplied with both forms of N had intermediate NRA suggesting that C. indica takes up and assimilate NO₃⁻ in the presence of NH₄⁺. Our results show that C. indica is relatively indifferent to inorganic N source, which together with its high growth rate contributes to explain the occurrence of this species in flooded wetland soils as well as on terrestrial soils. Furthermore, it is concluded that C. indica is suitable for use in different types of constructed wetlands.     

Effects of inorganic nitrogen forms on growth, morphology, nitrogen uptake capacity and nutrient allocation of four tropical aquatic macrophytes (Salvinia cucullata, Ipomoea aquatica, Cyperus involucratus and Vetiveria zizanioides)

This study assesses the growth and morphological responses, nitrogen uptake and nutrient allocation in four aquatic macrophytes when supplied with different inorganic nitrogen treatments (1) NH₄⁺, (2) NO₃⁻, or (3) both NH₄⁺ and NO₃⁻. Two free-floating species (Salvinia cucullata Roxb. ex Bory and Ipomoea aquatica Forssk.) and two emergent species (Cyperus involucratus Rottb. and Vetiveria zizanioides (L.) Nash ex Small) were grown with these N treatments at equimolar concentrations (500 μM). Overall, the plants responded well to NH₄⁺. Growth as RGR was highest in S. cucullata (0.12 ± 0.003 d⁻¹) followed by I. aquatica (0.035 ± 0.002 d⁻¹), C. involucratus (0.03 ± 0.002 d⁻¹) and V. zizanioides (0.02 ± 0.003 d⁻¹). The NH₄⁺ uptake rate was significantly higher than the NO₃⁻ uptake rate. The free-floating species had higher nitrogen uptake rates than the emergent species. The N-uptake rate differed between plant species and seemed to be correlated to growth rate. All species had a high NO₃⁻ uptake rate when supplied with only NO₃⁻. It seems that the NO₃⁻ transporters in the plasma membrane of the root cells and nitrate reductase activity were induced by external NO₃⁻. Tissue mineral contents varied with species and tissue, but differences between treatments were generally small. We conclude, that the free-floating S. cucullata and I. aquatica are good candidate species for use in constructed wetland systems to remove N from polluted water. The rooted emergent plants can be used in subsurface flow constructed wetland systems as they grow well on any form of nitrogen and as they can develop a deep and dense root system.     

Reactivation of effluent granular sludge from a high-rate Anammox reactor after storage

In this study, effluent sludge from a high-rate Anammox reactor was used to re-start new Anammox reactors for the reactivation of Anammox granular sludge. Different start-up strategies were evaluated in six upflow anaerobic sludge blanket (UASB) reactors (R₁–R₆) for their effect on nitrogen removal performance. Maximal nitrogen removal rates (NRRs) greater than 20 kg N/m³/day were obtained in reactors R₃–R₅, which were seeded with mixed Anammox sludge previously stored for approximately 6 months and 1 month. A modified Boltzmann model describing the evolution of the NRR fit the experimental data well. An amount of sludge added to the UASB reactor or decreasing the loading rate proved effective in relieving the substrate inhibition and increasing the NRR. The modified Stover–Kincannon model fit the nitrogen removal data in the Anammox reactors well, and the simulation results showed that the Anammox process has great nitrogen removal potential. The observed inhibition in the Anammox reactors may have been caused by high levels of free ammonia. The sludge used to seed the reactors did not settle well; sludge flotation was observed even after the reactors were operated for a long time at a floating upward velocity (F_s) of greater than 100 m/h. The settling sludge, however, exhibited good settling properties. Scanning electron microscopy showed that the Anammox granules consisted mainly of spherical and elliptical bacteria with abundant filaments on their surface. Hollows in the granules were also present, which may have contributed to sludge floatation.     

Performance of a pilot-scale sewage treatment: an up-flow anaerobic sludge blanket (UASB) and a down-flow hanging sponge (DHS) reactors combined system by sulfur-redox reaction process under low-temperature conditions

Performance of a wastewater treatment system utilizing a sulfur-redox reaction of microbes was investigated using a pilot-scale reactor that was fed with actual sewage. The system consisted of an up-flow anaerobic sludge blanket (UASB) reactor and a down-flow hanging sponge (DHS) reactor with a recirculation line. Consequently, the total COD_Cr (465 ± 147 mg L⁻¹; total BOD of 207 ± 68 mg L⁻¹) at the influent was reduced (70 ± 14 mg L⁻¹; total BOD of 9 ± 2 mg L⁻¹) at the DHS effluent under the conditions of an overall hydraulic retention time of 12 h, a recirculation ratio of 2, and a low-sewage temperature of 7.0 ± 2.8 °C. A microbial analysis revealed that sulfate-reducing bacteria contributed to the degradation of organic matter in the UASB reactor even in low temperatures. The utilized sulfur-redox reaction is applicable for low-strength wastewater treatment under low-temperature conditions.     

Process of inorganic nitrogen transformation and design of kinetics model in the biological aerated filter reactor

As one of the plug-flow reactors, biological aerated filter (BAF) reactor was divided into four sampling sectors to understand the characteristics of elemental nitrogen transformation during the reaction process, and then the different characteristics of elemental nitrogen transformation caused by different NH₃-N loadings, biological quantities and activities in each section were obtained. The results showed that the total transformation ratio in the nitrifying reactor was more than 90% in the absence of any organic carbon resource, at the same time, more than 65% NH₃-N in the influent were nitrified at the filter height of 70 cm below under the conditions of the influent runoff 9-19 L/h, the gas-water ratio 4-5:1, the dissolved oxygen 3.0-5.8 mg/L and the NH₃-N load 0.28-0.48 kg NH₃-N/m³ d. On the base of the Eckenfelder mode, the kinetics equation of the NH₃-N transformation along the reactor was S_e = S₀ exp(−0.0134D/L^1.2612).     

Estimation of nitrogen dynamics in a vertical-flow constructed wetland

The vertical-flow constructed wetland (VFCW) is a promising engineering technique for removal of excess nutrients and certain pollutants from wastewater and stormwater. The aim of this study was to develop a model using the STELLA software for estimating nitrogen (N) dynamics in an artificial VFCW (i.e., a substrate column with six zones) associated with a growing Cyperus alternifolius species under a wetting (wastewater) -to-drying ratio of 1:3. The model was calibrated by our experimental data with a reasonable agreement prior to its applications. Simulations showed that rates of NH₄⁺-N and NO₃⁻-N leaching decreased with increasing zone number (or column depth), although such a decrease was much more profound for NH₄⁺-N. Our simulations further revealed that rate of NH₄⁺-N leaching decreased with time within each zone, whereas rate of NO₃⁻-N leaching increased with time within each zone. Additionally, both the rates of NH₄⁺-N and NO₃⁻-N leaching through zones followed the water flow pattern: breakthrough during wetting period and cessation during drying period. In general, the cumulative amounts of total nitrogen (TN) were in the following order: leaching > denitrification > uptake > settlement. About 54% of the TN from the wastewater flowed out of the VFCW system, 18% of TN lost due to denitrification, 6% of TN was taken up by roots of a single plant (one hill), and the rest of 22% TN from the wastewater was removed from other mechanisms, such as volatilization, adsorption, and deposition. This study suggested that to improve the overall performance of a VFCW for N removal, prevention of N leaching loss was one of the major issues.