Biodegradation of Methyl Orange by alginate-immobilized Aeromonas sp. in a packed bed reactor: external mass transfer modeling

Azo dyes are recalcitrant and xenobiotic nature makes these compounds a challenging task for continuous biodegradation up to satisfactorily levels in large-scale. In the present report, the biodegradation efficiency of alginate immobilized indigenous Aeromonas sp. MNK1 on Methyl Orange (MO) in a packed bed reactor was explored. The experimental results were used to determine the external mass transfer model. Complete MO degradation and COD removal were observed at 0.20 cm bead size and 120 ml/h flow rate at 300 mg/l of initial dye concentration. The degradation of MO decreased with increasing bead sizes and flow rates, which may be attributed to the decrease in surface of the beads and higher flux of MO, respectively. The experimental rate constants (k _ps) for various beads sizes and flow rates were calculated and compared with theoretically obtained rate constants using external film diffusion models. From the experimental data, the external mass transfer effect was correlated with a model J _D = K Re ^?(1 ? n). The model was tested with K value (5.7) and the Colburn factor correlation model for 0.20, 0.40 and 0.60 bead sizes were J _D = 5.7 Re ^?0.15, J _D = 5.7 Re ^?0.36 and J _D = 5.7 Re ^?0.48, respectively. Based on the results, the Colburn factor correlation models were found to predict the experimental data accurately. The proposed model was constructive to design and direct industrial applications in packed bed reactors within acceptable limits.     

Chromium uptake,retention and reduction in photosynthetic <Emphasis Type="Italic">Euglena gracilis</Emphasis>

Photosynthetic Euglena gracilis grown with different K₂CrO₄ concentrations was analyzed for its ability to take up, retain and reduce Cr(VI). For comparison, cells were also exposed to CrCl₃. Cellular Cr(VI) uptake at pH 7.2 showed a hyperbolic saturation pattern with K _m of 1.1 mM, V _m of 16 nmol (h × 10⁷ cells)⁻¹, and K _i sulfate of 0.4 mM. Kinetic parameters for sulfate uptake were similar, K _m = 0.83 mM, V _m = 15.9 nmol (h × 10⁷cells)⁻¹ and K _i chromate = 0.3 mM. The capacity to accumulate chromium depended on the ionic species, external concentration and pH of the incubation medium. Cr(VI) or Cr(III) accumulation was negligible in the acidic (pH 3.5) culture medium, in which Cr(VI) was abiotically reduced to Cr(III). At pH 7.2 Cr(VI) was fully stable and high accumulation (>170 nmol/1 × 10⁷ cells at 1 mM K₂CrO₄) was achieved; surprisingly, Cr(III) accumulation was also significant (>35 nmol/1 × 10⁷ cells at 1 mM CrCl₃). Cr(VI) was reduced by cells at pH 7.2, suggesting the presence of an external reductive activity. Cr(VI) induced an increased cysteine and glutathione content, but not in phytochelatins suggesting that chromium accumulation was mediated by monothiol compounds.     

Determination of Cr(VI) and Cr(III) species in parenteral solutions using a nanostructured material packed-microcolumn and electrothermal atomic absorption spectrometry

A sequential on-line preconcentration and separation system for Cr(VI) and Cr(III) species determination was developed in this work. For this purpose, a microcolumn filled with nanostructured α-alumina was used for on-line retention of Cr species in a flow-injection system. The method involves the selective elution of Cr(VI) with concentrated ammonia and Cr(III) with 1 mol L⁻¹ nitric acid for sequential injection into an electrothermal atomic absorption spectrometer (ETAAS).Analytical parameters including pH, eluent type, flow rates of sample and eluent, interfering effects, etc., were optimized. The preconcentration factors for Cr(VI) and Cr(III) were 41 and 18, respectively. The limit of detection (LOD) was 1.9 ng L⁻¹ for Cr(VI) and 6.1 ng L⁻¹ for Cr(III). The calibration graph was linear with a correlation coefficient of 0.999. The relative standard deviation (RSD) was 8.6% for Cr(VI) and 6.1% for Cr(III) (c=10 μg L⁻¹, n=10, sample volume=25 mL). Verification of the accuracy was carried out by analysis of a standard reference material (NIST SRM 1643e “Trace elements in natural water”) with a reported Cr content of 20.40±0.24 μg L⁻¹. Using the proposed methodology the total Cr content, computed as sum of Cr(III) and Cr(VI), in this SRM was 20.26±0.96 μg L⁻¹. The method was successfully applied to the determination of Cr(VI) and Cr(III) species in parenteral solutions. Concentration of Cr(III) species was found to be in the range of 0.29–3.62 μg L⁻¹, while Cr(VI) species was not detected in the samples under study.     

Evaluation of chromium(VI) removal behaviour by two isolates of Synechocystis sp. in terms of exopolysaccharide (EPS) production and monomer composition

Chromium(VI) removal and its association with exopolysaccharide (EPS) production in cyanobacteria were investigated. Synechocystis sp. BASO670 produced higher EPS (548 mg L⁻¹) than Synechocystis sp. BASO672 (356 mg L⁻¹). While the EC₅₀ of the Cr(VI) for Synechocystis sp. BASO670 and Synechocystis sp. BASO672 were determined as 11.5 mg L⁻¹, and 2.0 mg L⁻¹, respectively, there was no relation between Cr(VI) removal and EPS production. Synechocystis sp. BASO672, which has higher EPS value, removed (33%) more Cr(VI) than Synechocystis sp. BASO670. Monomer compositions of EPS of each of the isolates were determined differently. Synechocystis sp. BASO672 which removed higher Cr(VI), had higher values of uronic acid and glucuronic acid (192 μg/mg and 89%, respectively). Our results showed that EPS might play a role in Cr(VI) tolerance. Monomer composition, especially uronic acid and glucuronic acid content of EPS may have enhanced Cr(VI) removal.     

Long-term exposure of the freshwater shrimp Macrobrachium olfersii to elevated salinity: Effects on gill (Na,K)-ATPase α-subunit expression and K-phosphatase activity

The kinetic properties of a microsomal gill (Na⁺,K⁺)-ATPase from the freshwater shrimp, Macrobrachium olfersii, acclimated to 21‰ salinity for 10 days were investigated using the substrate p-nitrophenylphosphate. The enzyme hydrolyzed this substrate obeying cooperative kinetics at a rate of 123.6 ± 4.9 U mg^− 1 and K_0.5 = 1.31 ± 0.05 mmol L^− 1. Stimulation of K⁺-phosphatase activity by magnesium (V_max = 125.3 ± 7.5 U mg^− 1; K_0.5 = 2.09 ± 0.06 mmol L^− 1), potassium (V_max = 134.2 ± 6.7 U mg^− 1; K_0.5 = 1.33 ± 0.06 mmol L^− 1) and ammonium ions (V_max = 130.1 ± 5.9 U mg^− 1; K_0.5 = 11.4 ± 0.5 mmol L^− 1) was also cooperative. While orthovanadate abolished p-nitrophenylphosphatase activity, ouabain inhibition reached 80% (K_I = 304.9 ± 18.3 μmol L^− 1). The kinetic parameters estimated differ significantly from those for freshwater-acclimated shrimps, suggesting expression of different isoenzymes during salinity adaptation. Despite the ≈2-fold reduction in K⁺-phosphatase specific activity, Western blotting analysis revealed similar α-subunit expression in gill tissue from shrimps acclimated to 21‰ salinity or fresh water, although expression of phosphate-hydrolyzing enzymes other than (Na⁺,K⁺)-ATPase was stimulated by high salinity acclimation.     

Enzymatic synthesis of farnesyl laurate in organic solvent: initial water activity, kinetics mechanism, optimization of continuous operation using packed bed reactor and mass transfer studies

The influence of water activity and water content was investigated with farnesyl laurate synthesis catalyzed by Lipozyme RM IM. Lipozyme RM IM activity depended strongly on initial water activity value. The best results were achieved for a reaction medium with an initial water activity of 0.11 since it gives the best conversion value of 96.80%. The rate constants obtained in the kinetics study using Ping-Pong-Bi-Bi and Ordered-Bi-Bi mechanisms with dead-end complex inhibition of lauric acid were compared. The corresponding parameters were found to obey the Ordered-Bi-Bi mechanism with dead-end complex inhibition of lauric acid. Kinetic parameters were calculated based on this model as follows: V _max = 5.80 mmol l⁻¹ min⁻¹ g enzyme⁻¹, K _m,A = 0.70 mmol l⁻¹ g enzyme⁻¹, K _m,B = 115.48 mmol l⁻¹ g enzyme⁻¹, K _i = 11.25 mmol l⁻¹ g enzyme⁻¹. The optimum conditions for the esterification of farnesol with lauric acid in a continuous packed bed reactor were found as the following: 18.18 cm packed bed height and 0.9 ml/min substrate flow rate. The optimum molar conversion of lauric acid to farnesyl laurate was 98.07±0.82%. The effect of mass transfer in the packed bed reactor has also been studied using two models for cases of reaction limited and mass transfer limited. A very good agreement between the mass transfer limited model and the experimental data obtained indicating that the esterification in a packed bed reactor was mass transfer limited.     

Kinetic analyses for bindings of concanavalin A to dispersed and condensed mannose surfaces on a quartz crystal microbalance

A biotinylated mannotriose (Man3-bio) was dispersively immobilized in the matrix of biotinylated lactose (Gal-Glc-bio) on a streptavidin-covered, 27-MHz quartz crystal microbalance (QCM), and binding kinetics of concanavalin A (Con A) to Man3-bio in the Gal-Glc-bio matrix could be obtained from frequency decreases (mass increases) of the QCM. Association constants (K_a) and binding and dissociation rate constants (k_on and k_off) could be determined separately as the 1:1 and 1:2 bindings of Con A to Man3-bio on the surface. When Man3-bio was immobilized with content of 1 to 5 mol% in the matrix, the 1:1 binding of Con A to Man3-bio was obtained as K_a = (4 ± 1) × 10⁶ M⁻¹, k_on = (4 ± 1) × 10⁴ M⁻¹ s⁻¹, and k_off = (12 ± 2) × 10^–3 s⁻¹. On the contrary, when Man3-bio was immobilized with content of 20 to 100 mol% in the matrix, the 1:2 binding of Con A to Man3-bio was obtained as K_a = (14 ± 2) × 10⁶ M⁻¹, k_on = (14 ± 2) × 10⁴ M⁻¹ s⁻¹, and k_off = (7 ± 2) × 10^–3 s⁻¹. Thus, K_a for the 1:2 binding was 10 times larger than that for the 1:1 binding, with a three times larger binding rate constant (k_on) and a three times smaller dissociation rate constant (k_off). This is the first example to obtain separate kinetic parameters for the 1:1 and 1:2 bindings of lectins to carbohydrates on the surface.     

Surface-enhanced Raman spectroscopy combined with atomic force microscopy for ultrasensitive detection of thrombin

Indole-3-acetic acid (IAA) amide conjugates play an important role in balancing levels of free IAA in plant cells. The GH3 family of proteins conjugates free IAA with various amino acids. For example, auxin levels modulate expression of the Oryza sativa (rice) GH3-8 protein, which acts to prevent IAA accumulation by coupling the hormone to aspartate. To examine the kinetic properties of the enzyme, we developed a liquid chromatography–tandem mass spectrometry (LC–MS/MS) assay system. Bacterially expressed OsGH3-8 was purified to homogeneity and used to establish the assay system. Monitoring of the reaction confirms the reaction product as IAA–Asp and demonstrates that production of the conjugate increases proportionally with both time and enzyme amount. Steady-state kinetic analysis using the LC–MS/MS-based assay yields the following parameters: V/E_t^IAA = 20.3 min⁻¹, K_m^IAA = 123 μM, V/E_t^ATP = 14.1 min⁻¹, K_m^ATP = 50 μM, V/E_t^Asp = 28.8 min⁻¹, K_m^Asp = 1580 μM. This is the first assignment of kinetic values for any IAA–amido synthetase from plants. Compared with previously described LC- and thin-layer chromatography (TLC)-based assays, this LC–MS/MS method provides a robust and sensitive means for performing direct kinetic studies on a range of IAA-conjugating enzymes.     

Interaction of cryptogein with its binding sites in tobacco plasma membrane studied using the piezoelectric biosensor

Elicitins are low-molecular-weight proteins representing the elicitor family secreted by many species of the oomycete Phytophthora. Elicitins induce a hypersensitive reaction in tobacco, a process that is triggered by binding of elicitin to the high-affinity site on the plasma membrane. Specific interaction of cryptogein with the binding sites on tobacco plasma membranes was studied using the piezoelectric biosensor in real time in a flow-through mode. Cryptogeins (wild-type and mutant forms) were covalently immobilized on the sensing surface, and membrane vesicles containing receptors were in solution. Kinetic characterization of the interaction provided values of kinetic rate association (k_a) = 5.74 · 10⁶ M¹ s⁻¹ and kinetic rate dissociation (k_d) = 6.87 10⁻⁴ s⁻¹ constants, respectively. The kinetic equilibrium dissociation constant was calculated as K_D = 12.0 nM. The piezoelectric biosensor appeared to be a convenient tool for studying interactions of receptors embedded in membrane vesicles.     

Modelling and simulation of steady-state phenol degradation in a pulsed plate bioreactor with immobilised cells of Nocardia hydrocarbonoxydans

A novel bioreactor called pulsed plate bioreactor (PPBR) with cell immobilised glass particles in the interplate spaces was used for continuous aerobic biodegradation of phenol present in wastewater. A mathematical model consisting of mass balance equations and accounting for simultaneous external film mass transfer, internal diffusion and reaction is presented to describe the steady-state degradation of phenol by Nocardia hydrocarbonoxydans (Nch.) in this bioreactor. The growth of Nch. on phenol was found to follow Haldane substrate inhibition model. The biokinetic parameters at a temperature of 30 ± 1 °C and pH at 7.0 ± 0.1 are μ _m = 0.5397 h⁻¹, K _S = 6.445 mg/L and K _I = 855.7 mg/L. The mathematical model was able to predict the reactor performance, with a maximum error of 2% between the predicted and experimental percentage degradations of phenol. The biofilm internal diffusion rate was found to be the slowest step in biodegradation of phenol in a PPBR.     

Gill (Na+,K+)-ATPase from the blue crab Callinectes danae: modulation of K+-phosphatase activity by potassium and ammonium ions

The kinetic properties of a microsomal gill (Na⁺,K⁺)-ATPase from the blue crab Callinectes danae were analyzed using the substrate p-nitrophenylphosphate. The (Na⁺,K⁺)-ATPase hydrolyzed PNPP obeying cooperative kinetics (n=1.5) at a rate of V=125.4±7.5 U mg⁻¹ with K_0.5=1.2±0.1 mmol l⁻¹; stimulation by potassium (V=121.0±6.1 U mg⁻¹; K_0.5=2.1±0.1 mmol l⁻¹) and magnesium ions (V=125.3±6.3 U mg⁻¹; K_0.5=1.0±0.1 mmol l⁻¹) was cooperative. Ammonium ions also stimulated the enzyme through site–site interactions (n_H=2.7) to a rate of V=126.1±4.8 U mg⁻¹ with K_0.5=13.7±0.5 mmol l⁻¹. However, K⁺-phosphatase activity was not stimulated further by K⁺ plus NH₄⁺ ions. Sodium ions (K_I=36.7±1.7 mmol l⁻¹), ouabain (K_I=830.3±42.5 μmol l⁻¹) and orthovanadate (K_I=34.0±1.4 nmol l⁻¹) completely inhibited K⁺-phosphatase activity. The competitive inhibition by ATP (K_I=57.2±2.6 μmol l⁻¹) of PNPPase activity suggests that both substrates are hydrolyzed at the same site on the enzyme. These data reveal that the K⁺-phosphatase activity corresponds strictly to a (Na⁺,K⁺)-ATPase in C. danae gill tissue. This is the first known kinetic characterization of K⁺-phosphatase activity in the portunid crab C. danae and should provide a useful tool for comparative studies.     

Performance and kinetic evaluation of anaerobic moving bed biofilm reactor for treating milk permeate from dairy industry

High strength milk permeate derived from ultra-filtration based cheese making process was treated in an anaerobic moving bed biofilm reactor (AMBBR) under mesophilic (35 °C) condition. Total chemical oxygen demand (TCOD) removal efficiencies of 86.3–73.2% were achieved at organic loading rates (OLR) of 2.0–20.0 g TCOD L⁻¹ d⁻¹. A mass balance model gave values of methane yield coefficient (Y_G/S) and cell maintenance coefficient (k_m) of 0.341 L CH₄ g⁻¹ TCOD_removed and 0.1808 g TCOD_removed g⁻¹ VSS d⁻¹, respectively. The maximum substrate utilization rate U_max was determined as 89.3 g TCOD L⁻¹ d⁻¹ by a modified Stover–Kincannon model. Volumetric methane production rates (VMPR) were shown to correlate with the biodegradable TCOD concentration through a Michaelis–Menten type equation. Moreover, based on VMPR and OLR removed from the reactor, the sludge production yield was determined as 0.0794 g VSS g⁻¹ TCOD_removed.     

Tryptophan residue is essential for immunoreactivity of a diagnostically relevant peptide epitope of A. fumigatus

The role of tryptophan (Trp¹⁷) in immunoreactivity of P1, the diagnostically relevant peptide from a major allergen/antigen of Aspergillus fumigatus, was evaluated by chemically modifying tryptophanyl residue of P1. In BIAcore kinetic studies, unmodified P1 showed a 100-fold higher binding with ABPA (Allergic Bronchopulmonary Aspergillosis) patients’ IgG [K_D (equilibrium dissociation constant) = 2.74 e⁻⁸ ± 0.13 M] than the controls’ IgG (K_D = 2.97 e⁻⁶± 0.14 M), whereas chemically-modified P1 showed similar binding [K_D patients’ IgG = 3.25 e⁻⁷± 0.16 M, K_D controls’ IgG = 3.86 e⁻⁷± 0.19 M] indicating loss of specific immunoreactivity of P1 on tryptophan modification. Modified P1 showed loss of specific binding to IgE and IgG antibodies of ABPA patients in ELISA (Enzyme-Linked Immunosorbent Assay). The study infers that tryptophan residue (Trp¹⁷) is essential for immunoreactivity of P1.     

Transepithelial Taurine Transport in Caco-2 Cell Monolayers

Here we characterized transepithelial taurine transport in monolayers of cultured human intestinal Caco-2 cells by analyzing kinetic apical and basolateral uptake and efflux parameters. Basolateral uptake was Na⁺- and Cl⁻- dependent and was inhibited by β-amino acids. Uptake by this membrane showed properties similar to those of the apical TauT system. In both membranes, taurine uptake fitted a model consisting of a non-saturable plus a saturable component, with a higher half-saturation constant and transport capacity at the apical membrane (K_m, 17.1 μmol/L; V_max, 28.4 pmol·cm⁻²·5 min⁻¹) than in the basolateral domain (K_m, 9.46 μmol/L; V_max, 5.59 pmol·cm⁻²·5 min⁻¹). The non-saturable influx component, estimated in the absence of Na⁺ and Cl⁻, showed no significant differences between apical and basolateral membranes (K_D, 89.2 and 114.7 nL·cm⁻² · 5 min⁻¹, respectively). Taurine efflux from the cells is a diffusive process, as shown in experiments using preloaded cells and in trans-stimulation studies (apical K_D,72.7 and basolateral K_D, 50.1 nL·cm⁻²·5 min⁻¹). Basolateral efflux rates were significantly lower than passive influx rates. We conclude that basolateral taurine uptake in Caco-2 cells is mediated by a transport mechanism that shares some properties with the apical system TauT. Moreover, calculation of unidirectional and transepithelial taurine fluxes reveals that apical influx of this amino acid is higher than basolateral efflux rates, thereby enabling epithelial cells to accumulate taurine against a concentration gradient.     

Bacterial Cr(VI) Reduction Concurrently Improves Sunflower (<Emphasis Type="Italic">Helianthus Annuus</Emphasis> L.) Growth

Four Cr(VI)-reducing bacterial strains (Ochrobactrum intermedium, CrT-2, CrT-3 and CrT-4) previously isolated from chromium-contaminated sites were inoculated on to seeds of sunflower (Helianthus annuus var SF-187), which were germinated and grown along with non-inoculated controls with chromate salts (300 μg CrCl₃ or K₂CrO₄ ml⁻¹). Severe reduction (20%) in seed germination was observed in Cr(VI) stress. Plant height decreased (36%) with Cr(VI) when compared with chromium-free control, while O. intermedium inoculation resulted a 20% increment in this parameter as compared to non-inoculated chromium-free control. CrT-3 inoculation resulted a 69% increment in auxin content as compared to non-inoculated control. O. intermedium caused 30% decrease in chromium uptake in sunflower plant roots under Cr(VI) stress as compared to chromium-free control plants.     

Comparison of oxygen mass transfer coefficient in simple and extractive fermentation systems

Aeration and agitation are important variables to ensure effective oxygen transfer rate during aerobic bioprocesses; therefore, the knowledge of the volumetric mass transfer coefficient (k_La) is required. In view of selecting the optimum oxygen requirements for extractive fermentation in aqueous two-phase system (ATPS), the k_La values in a typical ATPS medium were compared in this work with those in distilled water and in a simple fermentation medium, in the absence of biomass. Aeration and agitation were selected as the independent variables using a 2² full factorial design. Both variables showed statistically significant effects on k_La, and the highest values of this parameter in both media for simple fermentation (241 s⁻¹) and extractive fermentation with ATPS (70.3 s⁻¹) were observed at the highest levels of aeration (5 vvm) and agitation (1200 rpm). The k_La values were then used to establish mathematical correlations of this response as a function of the process variables. The exponents of the power number (N³D²) and superficial gas velocity (V_s) determined in distilled water (α = 0.39 and β = 0.47, respectively) were in reasonable agreement with the ones reported in the literature for several aqueous systems and close to those determined for a simple fermentation medium (α = 0.38 and β = 0.41). On the other hand, as expected by the increased viscosity in the presence of polyethylene glycol, their values were remarkably higher in a typical medium for extractive fermentation (α = 0.50 and β = 1.0). A reasonable agreement was found between the experimental data of k_La for the three selected systems and the values predicted by the theoretical models, under a wide range of operational conditions.     

Performance evaluation of various bioreactors for the removal of Cr(VI) and organic matter from industrial effluent

Performances of various bioreactors under different operating conditions were evaluated with respect to hexavalent chromium (Cr(VI)) reduction and COD removal. Continuous reactor studies were carried out with (i) aerobic suspended growth system, (ii) aerobic attached growth system, and (iii) anoxic attached growth system, using both synthetic and actual industrial wastewater. Arthrobacter rhombi-RE (MTCC7048), a Cr(VI) reducing strain enriched and isolated from chromium contaminated soil, was used in all the bioreactors for Cr(VI) biotransformation and COD removal. Aerobic and anoxic batch experiments were conducted to evaluate the bio-kinetic parameters. The bio-kinetic parameters for aerobic system were: μ_max = 2.34/d, K_s = 190 mg/L (as COD), K_i = 3.8 mg/L of Cr(VI), and Y_T = 0.377. These parameters for anoxic conditions were: μ_max = 0.57/d, K_s = 710 mg/L (as COD), K_i = 8.77 mg/L of Cr(VI), and Y_T = 0.13. Aerobic attached growth system, operated at a hydraulic retention time (HRT) of 24 h and an organic loading rate (OLR) of 3 kg/m³/d, performed better than aerobic suspended and the anoxic attached growth systems operated under identical conditions, while treating synthetic wastewater as well as industrial effluent.     

Nitrogen utilization by the raphidophyte Heterosigma akashiwo: Growth and uptake kinetics in laboratory cultures

The nitrogen uptake and growth capabilities of the potentially harmful, raphidophycean flagellate Heterosigma akashiwo (Hada) Sournia were examined in unialgal batch cultures (strain CCMP 1912). Growth rates as a function of three nitrogen substrates (ammonium, nitrate and urea) were determined at saturating and sub-saturating photosynthetic photon flux densities (PPFDs). At saturating PPFD (110 μE m⁻² s⁻¹), the growth rate of H. akashiwo was slightly greater for cells grown on NH₄⁺ (0.89 d⁻¹) compared to cells grown on NO₃⁻ or urea, which had identical growth rates (0.82 d⁻¹). At sub-saturating PPFD (40 μE m⁻² s⁻¹), both urea- and NH₄⁺-grown cells grew faster than NO₃⁻-grown cells (0.61, 0.57 and 0.46 d⁻¹, respectively). The N uptake kinetic parameters were investigated using exponentially growing batch cultures of H. akashiwo and the ¹⁵N-tracer technique. Maximum specific uptake rates (V_max) for unialgal cultures grown at 15 °C and saturating PPFD (110 μE m⁻² s⁻¹) were 28.0, 18.0 and 2.89 × 10⁻³ h⁻¹ for NH₄⁺, NO₃⁻ and urea, respectively. The traditional measure of nutrient affinity—the half saturation constants (K_s) were similar for NH₄⁺ and NO₃⁻ (1.44 and 1.47 μg-at N L⁻¹), but substantially lower for urea (0.42 μg-at N L⁻¹). Whereas the α parameter (α = V_max/K_s), which is considered a more robust indicator for substrate affinity when substrate concentrations are low (<K_s), were 19.4, 12.2 and 6.88 × 10⁻³ h⁻¹/(μg-at N L⁻¹) for NH₄⁺, NO₃⁻ and urea, respectively. These laboratory results demonstrate that at both saturating and sub-saturating N concentrations, N uptake preference follows the order: NH₄⁺ > NO₃⁻ > urea, and suggests that natural blooms of H. akashiwo may be initiated or maintained by any of the three nitrogen substrates examined.     

In situ measurement of fine root water absorption in three temperate tree species—Temporal variability and control by soil and atmospheric factors

Miniature heat balance-sap flow gauges were used to measure water flows in small-diameter roots (3–4 mm) in the undisturbed soil of a mature beech–oak–spruce mixed stand. By relating sap flow to the surface area of all branch fine roots distal to the gauge, we were able to calculate real time water uptake rates per root surface area (J_s) for individual fine root systems of 0.5–1.0 m in length. Study aims were (i) to quantify root water uptake of mature trees under field conditions with respect to average rates, and diurnal and seasonal changes of J_s, and (ii) to investigate the relationship between uptake and soil moisture θ, atmospheric saturation deficit D, and radiation I. On most days, water uptake followed the diurnal course of D with a mid-day peak and low night flow. Neighbouring roots of the same species differed up to 10-fold in their daily totals of J_s (<100–2000 g m⁻² d⁻¹) indicating a large spatial heterogeneity in uptake. Beech, oak and spruce roots revealed different seasonal patterns of water uptake although they were extracting water from the same soil volume. Multiple regression analyses on the influence of D, I and θ on root water uptake showed that D was the single most influential environmental factor in beech and oak (variable selection in 77% and 79% of the investigated roots), whereas D was less important in spruce roots (50% variable selection). A comparison of root water uptake with synchronous leaf transpiration (porometer data) indicated that average water fluxes per surface area in the beech and oak trees were about 2.5 and 5.5 times smaller on the uptake side (roots) than on the loss side (leaves) given that all branch roots <2 mm were equally participating in uptake. Beech fine roots showed maximal uptake rates on mid-summer days in the range of 48–205 g m⁻² h⁻¹ (i.e. 0.7–3.2 mmol m⁻² s⁻¹), oak of 12–160 g m⁻² h⁻¹ (0.2–2.5 mmol m⁻² s⁻¹). Maximal transpiration rates ranged from 3 to 5 and from 5 to 6 mmol m⁻² s⁻¹ for sun canopy leaves of beech and oak, respectively. We conclude that instantaneous rates of root water uptake in beech, oak and spruce trees are above all controlled by atmospheric factors. The effects of different root conductivities, soil moisture, and soil hydraulic properties become increasingly important if time spans longer than a week are considered.     

Ion and acid–base regulation in the freshwater mummichog (Fundulus heteroclitus): a departure from the standard model for freshwater teleosts

Ion and acid–base balance were examined in the freshwater-adapted mummichog (Fundulus heteroclitus) using a series of treatments designed to perturb the coupling mechanisms. Unidirectional Cl⁻ uptake (J^Cl_in) was extremely low whereas J^Na_in was substantial (three- to sixfold higher); comparable differences occurred in unidirectional efflux rates (J^Cl_out, J^Na_out). J^Cl_in was refractory to all treatments, suggesting that Cl⁻/base exchange was unimportant or absent. Indeed, no base excretion or modulation of ion fluxes occurred for acid–base balance for up to 8 h after NaHCO₃⁻ loading (injections of 1000 or 3000 nequiv.·g⁻¹). Acute environmental low pH (4.5) and amiloride (10⁻⁴ M) treatments caused concurrent inhibition of J^Na_in and net H⁺ excretion (J^H+_net), indicating the presence of Na⁺/H⁺ exchange. J^Na_in was elevated and J^H+_net restored during recovery from both treatments, but this exchange did not appear to be dynamically adjusted for acid–base homeostasis. High external ammonia exposure (1 mmol·l⁻¹) initially blocked ammonia excretion (J^Amm_net) but had no effect on J^Na_in, whereas high pH (9.4) reduced both J^Amm_net and J^Na_in. Inhibition of J^Na_in by the low pH and amiloride treatments had no effect on J^Amm_net. These results indicate that ammonia excretion is entirely diffusive and independent of both Na⁺uptake and the protons that are transported via the Na⁺/H⁺ coupling. In addition, ureagenesis served as a compensatory mechanism during high external ammonia exposure, as a marked elevation in urea excretion partially replaced the inhibited J^Amm_net. In all treatments, changes in the Na⁺–Cl⁻ net flux differential were consistent with changes in J^H+_net measured by traditional water titration techniques, indicating that the former can be used as an estimate of the acid–base status of the fish. Overall, the results demonstrate that the freshwater-adapted F. heteroclitus does not conform to the ion/acid–base relationships described in the standard model based on commonly studied species such as trout, goldfish, and catfish.