Activity-density of <Emphasis Type="Italic">Pardosa cribata</Emphasis> in Spanish citrus orchards and its predatory capacity on <Emphasis Type="Italic">Ceratitis capitata</Emphasis> and <Emphasis Type="Italic">Myzus persicae</Emphasis>

The wolf spider Pardosa cribata Simon is the most abundant ground-dwelling spider inhabiting citrus orchards in eastern Spain. However, little is known about its activity-density and its predatory role in the citrus agrosystem. Here we report on the activity-density of P. cribata monitored by pitfall traps, and on its capacity to prey on two citrus pests that appear both in the citrus canopy and the ground cover, Ceratitis capitata (Wiedemman) and Myzus persicae (Sulzer), respectively. Pardosa cribata was present in citrus orchards throughout the year, with a peak in spring and a higher peak in summer. Pardosa cribata preyed on adults and third-instar larvae but not on pupae of C. capitata. A type II functional response was obtained for teneral-like adults, with an estimated attack rate (a′) of 0.771 ± 0.213 days⁻¹ and a handling time (T _h) of 0.051 ± 0.013 days. Pardosa cribata also preyed efficiently on M. persicae, giving a type II functional response with an estimated attack rate and handling time of 2.833 ± 0.578 days⁻¹ and 0.031 ± 0.001 days, respectively. The data reported here indicate that this wolf spider could play an important role in regulating both these pests, and therefore might contribute to developing conservation biological control strategies for citrus pests. Handling Editor: Arne Jenssen.     

Thermodynamic study of Cu2+ binding to the DAHK and GHK peptides by isothermal titration calorimetry (ITC) with the weaker competitor glycine

The peptides Asp-Ala-His-Lys (DAHK) and Gly-His-Lys (GHK) are naturally occurring Cu(II)-chelating motifs in human serum and cerebrospinal fluid. Here, the sensitive thermodynamic technique isothermal titration calorimetry was used to study the energetics of Cu(II) binding to DAHK and GHK peptides in the presence of the weaker ligand glycine as a competitor. DAHK and GHK bind Cu(II) predominantly in a 1:1 stoichiometry with conditional dissociation constants [i.e., at pH 7.4, in the absence of the competing chelators glycine and 2-(4-(2-hydroxyethyl)-1-piperazinyl)ethanesulfonic acid buffer] of 2.6 ± 0.4 × 10⁻¹⁴ M and 7.0 ± 1.0 × 10⁻¹⁴ M, respectively. Furthermore, the apparent ΔH values were measured and the number of protons released upon Cu(II) binding was determined by performing experiments in different buffers. This allowed us to determine the conditional ΔG, ΔH, and ΔS, i.e., corrected for the contributions of the weaker ligand glycine and the buffer at pH 7.4. We found that the entropic and enthalpic contributions to the Cu(II) binding to GHK and DAHK are distinct, with a enthalpic contribution for GHK. The thermodynamic parameters obtained correspond well to those in the literature obtained by other techniques, suggesting that the use of the weaker ligand glycine as a competitor in isothermal titration calorimetry provides accurate data for Cu(II) binding to high-affinity peptides, which cannot be accurately determined without the use of a competitor ligand.     

In Vitro Sensitivity of <Emphasis Type="Italic">Rhizobium</Emphasis> and Phosphate Solubilising Bacteria to Herbicides

Nitrogen fixing bacteria, rhizobia and phosphate solubilizing bacteria (PSB) are the commonly applied microbial inoculants in grain legumes (Pulses). It is important to apply herbicides to control weeds in order to augment yield of the crop. The herbicides may however, be incompatible with the microbial inoculants. This study compared the effect of the recommended pre-plant incorporated herbicide, fluchloralin (20.25 × 10⁴ ppm) and pre-emergence herbicide, pendimethalin in two doses (9 × 10⁴ and 15 × 10⁴ ppm) on the growth and survival of mungbean Rhizobium and PSB, under laboratory conditions. These herbicides were also used under field conditions in conjunction with biofertilizers (R, PSB) to improve grain yield of mungbean. It was found that fluchloralin (20.25 × 10⁴ ppm) and the lower dose of pendimethalin (9 × 10⁴ ppm) had no adverse effect on growth of Rhizobium and PSB. The higher dose of pendimethalin (15 × 10⁴ ppm) was safe on PSB but it imposed a retarding effect on the growth of Rhizobium.     

Structural and functional studies on a variant of cystatin purified from brain of Capra hircus

Cystatins, known for their ubiquitous presence in mammalian system are thiol protease inhibitors serving important physiological functions. Here, we present a variant of cystatin isolated from brain of Capra hircus (goat) which is glycosylated but lacks disulphide bonds. Caprine brain cystatin (CBC) was isolated using alkaline treatment, ammonium sulphate fractionation (40–60%) and gel filtration chromatography on Sephacryl S-100HR column with an overall yield of 26.29% and 322-fold purification. The inhibitor gave a molecular mass of ~44 kDa as determined by SDS-PAGE and gel filtration behaviour. The Stokes radius and diffusion coefficient of CBC were 27.14 Å and 8.18 × 10^?7 cm² s^?1, respectively. Kinetic data revealed that CBC inhibited thiol proteases reversibly and competitively, with the highest inhibition towards papain (K_i = 4.10 nM) followed by ficin and bromelain. CBC possessed 34.7% α-helical content as observed by CD spectroscopy. UV, fluorescence, CD and FTIR spectroscopy revealed significant conformational change upon CBC-papain complex formation. Isothermal titration calorimetry (ITC) was used to measure the thermodynamic parameters – ΔH, ΔS, ΔG along with N (binding stoichiometry) for CBC-papain complex formation. Binding stoichiometry (N = .97 ± .07 sites) for the CBC-papain complex indicates that cystatin is surrounded by nearly one papain molecule. Negative ΔH (?5.78 kcal mol^?1) and positive ΔS (11.01 cal mol^?1 deg^?1) values suggest that the interaction between CBC and papain is enthalpically as well as entropically favoured process. The overall negative ΔG (?9.19 kcal mol^?1) value implies a spontaneous CBC-papain interaction.     

Effects of cpxR on the growth characteristics and antibiotic production of Xenorhabdus nematophila

CpxR is a global response regulator that negatively influences the antimicrobial activities of Xenorhabdus nematophila. Herein, the wildtype and ΔcpxR mutant of X. nematophila were cultured in a 5-l and 70-l bioreactor. The kinetic analysis showed that ΔcpxR significantly increased the cell biomass and antibiotic activity. The maximum dry cell weight (DCW) and antibiotic activity of ΔcpxR were 20.77 ± 1.56 g L⁻¹ and 492.0 ± 31.2 U ml⁻¹ and increased by 17.28 and 97.33% compared to the wildtype respectively. Xenocoumacin 1 (Xcn1), a major antimicrobial compound, was increased 3.07-fold, but nematophin was decreased by 48.7%. In 70-l bioreactor, DCW was increased by 18.97%, while antibiotic activity and Xcn1 were decreased by 27.71% and 11.0% compared to that in 5-l bioreactor respectively. Notably, pH had remarkable effects on the cell biomass and antibiotic activity of ΔcpxR, where ΔcpxR was sensitive to alkaline pH conditions. The optimal cell growth and antibiotic activity of ΔcpxR occurred at pH 7.0, while Xcn1 was increased 5.45- and 3.87-fold relative to that at pH 5.5 and 8.5 respectively. These findings confirmed that ΔcpxR considerably increased the biomass of X. nematophila at a late stage of fermentation. In addition, ΔcpxR significantly promoted the biosynthesis of Xcns but decreased the production of nematophin.     

Characterisation of acidic sites of Pseudomonas biomass capable of binding protons and cadmium and removal of cadmium via biosorption

Summary The ability of Pseudomonas aeruginosa to accumulate Cd(II) ions from wastewater industries was experimentally investigated and mathematically modelled. From the potentiometric titration and non-ideal competitive analysis (NICA) model, it was found that the biomass contains three acidic sites. The values of proton binding (pK _i =1.66±3.26×10⁻³, 1.92±1.63×10⁻⁴ and 2.16±3.79×10⁻⁴) and binding constant of cadmium metal ions (pK _M1=1.99±2.45×10⁻³ and pK _M2=1.67±4.08×10⁻³) on the whole surface of biomass showed that protonated functional groups and biosorption of Cd(II) ions could be attributed to a monodentate binding to one acidic site, mainly the carboxylic group. From the isothermal sorption experimental data and Langmuir model, it was also found that the value of Langmuir equilibrium (pK _f) constant is 2.04±2.1×10⁻⁵ suggesting that the carboxyl group is the main active binding site. In addition, results showed that the maximum cadmium capacity (q _max) and affinity of biomass towards cadmium metal ions (b) at pH 5.1 and 20 min were 96.5±0.06 mg/g and 3.40×10⁻³± 2.10×10⁻³, respectively. Finally, interfering metal ions such as Pb(II), Cu(II), Cr(III), Zn(II), Fe(II), Mn(II), Ca(II) and Mg(II) inhibited Cd(II) uptake. Comparing the biosorption of Cd(II) by various Pseudomonas isolates from contaminated environment samples (soil and sewage treatment plant) showed that maximum capacities and equilibrium times were different, indicating that there was a discrepancy in the chemical composition between biomasses of different strains.     

Evaluation of Staphylococcus aureus DNA aptamer by enzyme‐linked aptamer assay and isothermal titration calorimetry

To monitor the specificity of Staphylococcus aureus aptamer (SA‐31) against its target cell, we used enzyme‐linked aptamer assay. In the presence of target cell, horseradish peroxidase–conjugated streptavidin bound to biotin‐labeled SA‐31 showed specific binding to S   aureus among 3 different bacteria with limit of detection of 10³ colony‐forming unit per milliliter. The apparent K _a was 1.39 μM⁻¹ ± 0.3 μM⁻¹. The binding of SA‐31 to membrane proteins extracted from cell surface was characterized using isothermal titration calorimetry, and the effect of changes in binding temperature and salt concentrations of binding buffer was evaluated based on thermodynamic parameters (K _a, ΔH , and ΔG ). Since binding of aptamer to its targets solely depends on its 3‐dimensional structure under experimental conditions used in selection process, the change in temperature and ion concentration changed the affinity of SA‐31 to its target on surface of bacteria. At 4°C, SA‐31 did not show an affinity to its target with poor heat change upon injection of membrane fraction to aptamer solution. However, the apparent association constants of SA‐31 slightly varied from K _a = 1.56 μM⁻¹ ± 0.69 μM⁻¹ at 25°C to K _a = 1.03 μM⁻¹ ± 0.9 μM⁻¹ at 37°C. At spontaneously occurring exothermic binding reactions, affinities of S  aureus aptamer to its target were also 9.44 μM⁻¹ ± 0.38 μM⁻¹ at 50mM, 1.60 μM⁻¹ ± 0.11 μM⁻¹ at 137mM, and 3.28 μM⁻¹ ± 0.46 μM⁻¹ at 200 mM of salt concentration. In this study, it was demonstrated that enzyme‐linked aptamer assay and isothermal titration calorimetry were useful tools for studying the fundamental binding mechanism between a DNA aptamer and its target on the outer surface of S  aureus .     

Plasma Levels of Zinc,Copper, and Ceruloplasmin in Patients after Undergoing Laparoscopic Adjustable Gastric Banding

Laparoscopic adjustable gastric banding (LAGB) causes significant weight loss in morbidly obese adults. However, its consequences on nutritional status still remain unclear. There are a few studies determining the nutritional status after LAGB and none have focused on the serum levels of zinc (Zn), copper (Cu), and ceruloplasmin (CP). We aimed to investigate the effects of LAGB surgery on plasma Zn, Cu, and CP levels. Thirty patients with LAGB with morbid obesity were included. Blood samples were collected preooperatively and in the postoperative third month to determine plasma Zn, Cu, and CP levels. The mean preoperative and postoperative body mass indexes (BMI) were 44.9 ± 7.4 kg/m² and 44.1 ± 6.5 kg/m², respectively. The mean weight loss was 12.9 ± 3.3 kg at the postoperative third month. The postoperative Zn (500 ± 130 ng/ml), Cu (280 ± 80 ng/ml), and CP (23.9 ± 8.8 mg/dl) values were statistically significantly lower than the preooperative Zn (740 ± 230 ng/ml), Cu (370 ± 80 ng/ml) and CP (33.3 ± 15.7 mg/dl) levels (p < 0.05). Decreases in the plasma levels of Zn, Cu, and CP were seen postoperatively following LAGB surgery. The nutritional status of LAGB-applied patients should be monitored and mineral supplementation may be considered.     

Sorption of Cu(II) and Cd(II) by extracellular polymeric substances (EPS) from Aspergillus fumigatus

Sorption of Cu(II) and Cd(II) onto the extracellular polymeric substances (EPS) produced by Aspergillus fumigatus was investigated for the initial pH of the solution, EPS concentrations, contact time, NaCl concentration, initial metal ion concentration and the presence of other ions in the solution. The results showed that the adsorption of metal ions was significantly affected by pH, EPS concentrations, initial metal concentration, NaCl concentration and co-ions. The sorption of Cu(II) and Cd(II) increased with increasing pH and initial metal ion concentration but decreased with an increase in the NaCl concentration. The maximum sorption capacities of A. fumigatus EPS calculated from the Langmuir model were 40 mg g⁻¹ EPS and 85.5 mg g⁻¹ EPS for Cu(II) and Cd(II), respectively. The binary metal sorption experiments showed a selective metal binding affinity in the order of Cu(II) > Pb(II) > Cd(II). Both the Freundlich and Langmuir adsorption models described the sorption of Cu(II) and Cd(II) by the EPS of A. fumigatus adequately. Fourier transform infrared spectroscopy (FTIR) analysis revealed that carboxyl, amide and hydroxyl functional groups were mainly correlated with the sorption of Cu(II) and Cd(II). Energy dispersive X-ray (EDX) system analysis revealed that the ion-exchange was an important mechanism involved in the Cu(II) and Cd(II) sorption process taking place on EPS.     

Biosorption of heavy metals by lyophilized cells of <Emphasis Type="Italic">Pseudomonas stutzeri</Emphasis>

Biosorptive capacity of Pb(II), Cd(II) and Cu(II) by lyophilized cells of Pseudomonas stutzeri was investigated based on Langmuir and Freundlich isotherms. Biosorptive capacity for Pb(II), Cd(II) and Cu(II) decreased with an increase of metal concentration, reaching 142, 43.5 and 36.2 mg/g at initial concentration of 300 mg/l, respectively. Biosorption capacity for metal ions increased with increasing pH. The optimum pH for biosorption rate of Cd(II) and Cu(II) were 5.0, and 6.0 for Pb(II) biosorption. The experimental data showed a better fit with the Langmuir model over the Freundlich model for metal ions throughout the range of initial concentrations. The maximum sorptive capacity (q _max) obtained from the Langmuir equation for Pb(II), Cd(II) and Cu(II) were 153.3 (r ²  = 0.998), 43.86 (r ²  = 0.995), and 33.16 (r ²  = 0.997) for metal ions, respectively. The selectivity order for metal ions towards the biomass of P. stutzeri was Pb(II) > Cd(II) > Cu(II) for a given initial metal ions concentration. The interactions between heavy metals and functional groups on the cell wall surface of bacterial biomass were confirmed by FTIR analysis. The results of this study indicate the possible removal of heavy metals from the environment by using lyophilized cells of P. stutzeri.     

Muscle fiber type characteristics in females with chronic obstructive pulmonary disease. A preliminary study

Chronic obstructive pulmonary disease (COPD) is known to elicit intrinsic abnormalities in male skeletal muscle. However, it is unclear to what extent these changes occur in women and whether they are fiber-type specific. We investigated fiber-type specific differences in selected histochemical properties in muscle obtained from women with moderate to severe COPD compared to healthy control (CON) women. Tissue was obtained from the vastus lateralis in five COPD patients (age 66.9 ± 2.6 years; FEV₁ = 43 ± 7%) and eight CON (age 68 ± 4.9 years; FEV₁ = 113 ± 4.2%). Compared to CON, the distribution (30.6 ± 5.2 vs. 57.9 ± 4.6%) and cross sectional area of type I (CSA, 5660 ± 329 vs. 3586 ± 257 μm²) and type IIA (2770 ± 302 vs. 2099 ± 206 μm²) were lower (P < 0.05) and higher (P < 0.05), respectively, in COPD. Disease state did not alter either the distribution or CSA of the IIA, IIAX or type X subtypes. Although differences were found between fiber types in the number of capillary contacts (n) (I > IIAX, IIX; IIA > IIX) and the capillaries per CSA (μm²10⁻³) (I < IIA, IIAX, IIX), no differences were found between CON and COPD. Succinic dehydrogenase activity and sarcoplasmic reticulum (SR) Ca²⁺-ATPase activity, measured photometrically (OD units), were higher (P < 0.05), and lower (P < 0.05), respectively, in type I compared to the type II fiber subtypes. These properties were not altered with COPD. COPD in females is accompanied by a higher percent of type II fibers, a larger CSA of type I and type IIA fibers, both of which occur in the absence of differences in oxidative potential and the potential for SR Ca²⁺-sequestration.     

Flow injection assays for NADH and ethanol using photosensitized rose bengal and luminol–copper (II) chemiluminescence system

The online photoreaction of the rose bengal photosensitized luminol–copper (II) chemiluminescence (CL) system was used for the determination of β-nicotinamide adenine dinucleotide (NADH) and ethanol (EtOH) in pharmaceutical formulations combined with a flow injection technique. NADH can significantly enhance the CL emission of the reaction. For EtOH, alcohol dehydrogenase in soluble form was utilized in the presence of nicotinamide adenine dinucleotide resulting in NADH production. The limit of detection (3σ blank, 𝑛 = 3) of 4.0 × 10⁻⁸ and 2.17 × 10⁻⁵ M, and linear range 1.3 × 10⁻⁷ to 2.5 × 10⁻⁵ M (R² = 0.9998, n = 6) and 0.11–2.17 × 10⁻³ M (R² = 0.9996, n = 6) were obtained for NADH and EtOH respectively. The injection rate was 100 h⁻¹ with a relative standard deviation (n = 3) of 1.5–4.8% in the range studied for both analytes. The procedure was satisfactorily applied to pharmaceutical formulations with recoveries in the range 91.6 ± 3.0% to 110 ± 2.0% for NADH and 88 ± 3.0% to 95.4 ± 4.0% for EtOH. The results obtained were very consistent and did not differ considerably from the reported approaches at a 95% confidence limit. The possible mechanism of the CL reaction is also explained briefly.     

Human serum albumin coordinates Cu(II) at its N-terminal binding site with 1 pM affinity

The conditional stability constant at pH 7.4 for Cu(II) binding at the N-terminal site (NTS) of human serum albumin (HSA) was determined directly by competitive UV–vis spectroscopy titrations using nitrilotriacetic acid (NTA) as the competitor in 100 mM NaCl and 100 mM N-(2-hydroxyethyl)piperazine-N′-ethanesulfonic acid (Hepes). The log K _NTS^c value of 12.0 ± 0.1 was determined for HSA dissolved in 100 mM NaCl. A false log log K _NTS^c value of 11.4 ± 0.1 was obtained in the 100 mM Hepes buffer, owing to the formation of a ternary Cu(NTA)(Hepes) complex. The impact of the picomolar affinity of HSA for Cu(II) on the availability of these ions in neurodegenerative disorders is briefly discussed.     

Heavy metal pollution monitoring using the brown seaweed Padina durvillaei in the coastal zone of the Santa Rosalía mining region, Baja California Peninsula, Mexico

The coastal marine sediments near Santa Rosalía on the eastern coast of the Baja California Peninsula (Mexico) are heavily polluted by metals due to copper mining and smelting over the past century (up to 1984). The present study compares the accumulation of metals in the brown seaweed Padina durvillaei from the central segment of the coast of Santa Rosalía (polluted by Co, Cu, Mn and Zn) and from two segments north and south of the known “hot spot”. The seaweed samples were collected in May and August 2004 and June 2005 at 13 stations located along the Santa Rosalía mining region. Heavy metal concentrations were measured by instrumental neutron activation analysis (Co and Fe) directly in dried homogenized subsamples or by flame atomic absorption spectrophotometry (Cd, Cu, Mn, Ni, Pb and Zn) after complete strong acid digestion of sub-samples. The means and standard deviations of the concentrations in dry tissues of Padina durvillaei specimens for all the studied metals and stations lie in the following sequence: Cd (3.6 ± 1.6 mg kg⁻¹) < Co (6.5 ± 6.1 mg kg⁻¹) < Pb (7.8 ± 6.2) < Ni (9.96 ± 5.28 mg kg⁻¹) < Cu (53 ± 38 mg kg⁻¹) < Zn (63 ± 43 mg kg⁻¹) < Mn (295 ± 269 mg kg⁻¹) < Fe (2243 ± 2325 mg kg⁻¹). This increase of the average concentrations was statistically significant. Principal Component Analysis showed that Factor 1 (36.46%) displays high positive loadings for Co, Cu, Mn and Zn, reflecting the influence of local anthropogenic pollution on the seaweed composition, while Factor 2 (26.91%) is important for Co, Fe and Ni and probably corresponds to the adsorption or accumulation of terrigenous elements of the regional origin, while Factor 3, with a high positive loading for Pb and a high negative loading for Cd, is probably controlled by local and regional anthropogenic input of Pb and episodic supply of Cd by local upwellings. The results of ANOVA for each element do not show any significant differences between the average concentrations for Cd, Fe, Ni and Pb in the seaweed of the three segments, or between the central and southern segments for Cu, Mn and Zn. Cobalt contents in the seaweed from the northern and central segments are, however, significantly different from the southern segment. This indicates that element concentrations in Padina durvillaei generally do not follow the drastic increases and gradients of Cu, Co, Mn and Zn contents detected in surface sediments. The apparent contradiction could be explained by a low geochemical mobility of these metals in the polluted sediments, composed mainly of stable residues of smelter wastes, with very low content of organic matter usually driving diagenetic mobilization of some metals into interstitial waters and then to the overlying water.     

Energetics of Acidianus ambivalens growth in response to oxygen availability

All life requires energy to drive metabolic reactions such as growth and cell maintenance; therefore, fluctuations in energy availability can alter microbial activity. There is a gap in our knowledge concerning how energy availability affects the growth of extreme chemolithoautotrophs. Toward this end, we investigated the growth of thermoacidophile Acidianus ambivalens during sulfur oxidation under aerobic to microaerophilic conditions. Calorimetry was used to measure enthalpy (ΔH_inc) of microbial activity, and chemical changes in growth media were measured to calculate Gibbs energy change (ΔG_inc) during incubation. In all experiments, Gibbs energy was primarily dissipated through the release of heat, which suggests enthalpy‐driven growth. In microaerophilic conditions, growth was significantly more efficient in terms of biomass yield (defined as C‐mol biomass per mole sulfur consumed) and resulted in lower ΔG_inc and ΔH_inc. ΔG_inc in oxygen‐limited (OL) and oxygen‐ and CO₂‐limited (OCL) microaerophilic growth conditions resulted in averages of ?1.44 × 10³ kJ/C‐mol and ?7.56 × 10² kJ/C‐mol, respectively, and average ΔH_inc values of ?1.11 × 10⁵ kJ/C‐mol and ?4.43 × 10⁴ kJ/C‐mol, respectively. High‐oxygen experiments resulted in lower biomass yield values, an increase in ΔG_inc to ?1.71 × 10⁴ kJ/C‐mol, and more exothermic ΔH_inc values of ?4.71 × 10⁵ kJ/C‐mol. The observed inefficiency in high‐oxygen conditions may suggest larger maintenance energy demands due to oxidative stresses and a preference for growth in microaerophilic environments.     

Bioaccumulation and biosorption efficacy of Trichoderma isolate SP2F1 in removing copper (Cu(II)) from aqueous solutions

In our study, we isolated the isolate Trichoderma SP2F1 from sediment samples from the Penchala River, heavily contaminated with effluents from nearby industrial areas. Qualitative and quantitative screening using plate and broth assay, respectively, supplemented with various concentrations of Cu(II) showed the isolate was able to tolerate 6 mM CuSO₄, although growth was also detected in broths with 10 mM CuSO₄. Trichoderma spp. was able to remove Cu(II) in aqueous solutions in both viable and non-viable cell forms. Bioaccumulation capacity of viable SP2F1 cells removed 19.60 mg g⁻¹ of Cu(II) after 168 h incubation, while the maximum Cu(II) biosorption capacity for non-viable SP2F1 cells was 28.75 mg g⁻¹ of Cu(II). Results here showed that Trichoderma spp isolate SP2F1 has good potential for application in Cu(II) removal, can be used to treat sewage waste by applying either in viable or non-viable cell forms.     

The effects of copper on the photosynthetic response of <Emphasis Type="Italic">Phaeocystis cordata</Emphasis>

We investigated the effects of limiting (1.96 × 10⁻⁹ mol l⁻¹ total Cu, corresponding to pCu 14.8; where pCu = −log [Cu²⁺]) and toxic Cu concentrations up to 8.0 × 10⁻⁵ mol l⁻¹ total Cu (equivalent to pCu 9.5) on growth rates and photosynthetic activity of exponentially grown Phaeocystis cordata, using batch and semi-continuous cultures. With pulse amplitude modulated (PAM) fluorometry, we determined the photochemical response of P. cordata to the various Cu levels, and showed contrasting results for the batch and semi-continuous cultures. Although maximum photosystem II (PSII) quantum yield (Φ_M) was optimal and constant in the semi-continuous P. cordata, the batch cultures showed a significant decrease in Φ_M with culture age (0–72 h). The EC50 for the batch cultures was higher (2.0 × 10⁻¹⁰ mol l⁻¹, pCu9.7), than that for the semi-continuous cultures (6.3 × 10⁻¹¹ mol l⁻¹, pCu10.2). The semi-continuous cultures exhibited a systematic and linear decrease in Φ_M as Cu levels increased (for [Cu²⁺] < 1.0 × 10⁻¹² mol l⁻¹, pCu12.0), however, no effect of high Cu was observed on their operational PSII quantum yield (Φ′_M). Similarly, semi-continuous cultures exhibited a significant decrease in Φ_M, but not in Φ′_M, because of low-Cu levels. Thus, Cu toxicity and Cu limitation damage the PSII reaction centers, but not the processes downstream of PSII. Quenching mechanisms (NPQ and Q _n) were lower under high Cu relative to the controls, suggesting that toxic Cu impairs photo-protective mechanisms. PAM fluorometry is a sensitive tool for detecting minor physiological variations. However, culturing techniques (batch vs. semi-continuous) and sampling time might account for literature discrepancies on the effects of Cu on PSII. Semi-continuous culturing might be the most adequate technique to investigate Cu effects on PSII photochemistry.     

Effects of heavy metals on aerobic denitrification by strain Pseudomonas stutzeri PCN-1

Effects of heavy metals on aerobic denitrification have been poorly understood compared with their impacts on anaerobic denitrification. This paper presented effects of four heavy metals (Cd(II), Cu(II), Ni(II), and Zn(II)) on aerobic denitrification by a novel aerobic denitrifying strain Pseudomonas stutzeri PCN-1. Results indicated that aerobic denitrifying activity decreased with increasing heavy metal concentrations due to their corresponding inhibition on the denitrifying gene expression characterized by a time lapse between the expression of the nosZ gene and that of the cnorB gene by PCN-1, which led to lower nitrate removal rate (1.67∼6.67 mg L⁻¹ h⁻¹), higher nitrite accumulation (47.3∼99.8 mg L⁻¹), and higher N₂O emission ratios (5∼283 mg L⁻¹/mg L⁻¹). Specially, promotion of the nosZ gene expression by increasing Cu(II) concentrations (0∼0.05 mg L⁻¹) was found, and the absence of Cu resulted in massive N₂O emission due to poor synthesis of N₂O reductase. The inhibition effect for both aerobic denitrifying activity and denitrifying gene expression was as follows from strongest to least: Cd(II) (0.5∼2.5 mg L⁻¹) > Cu(II) (0.5∼5 mg L⁻¹) > Ni(II) (2∼10 mg L⁻¹) > Zn(II) (25∼50 mg L⁻¹). Furthermore, sensitivity of denitrifying gene to heavy metals was similar in order of nosZ > nirS ≈ cnorB > napA. This study is of significance in understanding the potential application of aerobic denitrifying bacteria in practical wastewater treatment.

     

Subcellular fractionation of Cu exposed oysters,Crassostrea virginica,and Cu accumulation from a biologically incorporated Cu rich oyster diet in Fundulus heteroclitus in fresh and sea water

In order to examine the effect of salinity on Cu accumulation from a naturally incorporated diet, oysters (Crassostrea virginica) were exposed in sea water for 96 days to four waterborne [Cu]: 2.9 ± 0.7 (control), 4.3 ± 0.6, 5.4 ± 0.5, and 10.7 ± 1.0 µg L^? 1. After 96 days, the control whole body [Cu] increased from 2.1 ± 0.5 to 9.1 ± 1.1 µg g^? 1 w.w. and the highest [Cu] was 163.4 ± 27.1 µg g^? 1 w.w. in the oysters. Despite large differences in tissue [Cu], there was no effect on the fraction of trophically available metal in the oyster suggesting that trophic transfer will correlate well with tissue [Cu]. The control and highest [Cu] oysters became diet for killifish (Fundulus heteroclitus) in fresh and seawater for 40 days. The two diets contained 84.7 ± 5.1 and 850.5 ± 8.8 µg Cu g^? 1 d.w. Fish were fed a combined diet of oyster and a pellet supplement (20.5 ± 1.0 µg Cu g^? 1 d.w.) both at 5% body mass day^? 1. In killifish, Cu increased ~ 7% in gills and 100% in intestines after 6 weeks of exposure to the high Cu diet. No other tissues accumulated Cu above control levels. An 11-fold difference free Cu²⁺ concentrations was predicted in intestinal fluid between fresh and sea water, but there was no corresponding effect of salinity on intestinal Cu accumulation suggesting that Cu is not accumulated as the free ion.