Effect of iron ions (Fe<Superscript>2+</Superscript>, Fe<Superscript>3+</Superscript>) on the formation and structure of aerobic granular sludge

Aerobic granulation is a promising technology for wastewater treatment, but problems regarding its formation and stability need to be solved. Divalent metal ions, especially Ca²⁺, Mg²⁺ and Mn²⁺, have been demonstrated to play an important role in the process of aerobic granulation. Here, we studied whether iron ions can affect aerobic granulation. Granular sludge formed without iron ion addition (<0.02 mg Fe²⁺ L^?1) was fluffy and had a finger-type structure and filamentous out-growth. The addition of iron ions to concentrations of 1 and 10 mg Fe²⁺ L^?1 repressed the finger-type structure and filamentous out-growth. The results show that chemical precipitation in the granules with iron ion addition was higher than that in the granules without ferrous addition. The amount of precipitates was higher inside the granules than outside. This study demonstrates that iron ions (Fe²⁺/Fe³⁺) increase the size and stability of aerobic granular sludge but do not affect the granulation time, which is the time that the first granular sludge is observed. The study shows that aerobic granular sludge technology can be confidently applied to actual wastewater containing a high concentration of iron compounds.     

Coprecipitation of Th<Superscript>4+</Superscript> and the purified extracellular polysaccharide produced by bacterium <Emphasis Type="Italic">Bradyrhizobium</Emphasis> (<Emphasis Type="Italic">Chamaecytisus</Emphasis>) BGA-1

The soil bacterium Bradyrhizobium (Chamaecytisus) strain BGA-1 produces an extracellular polymeric substance (EPS) that, in the presence of Fe³⁺, Al³⁺ or Th⁴⁺ solutions, forms a gel-like precipitate composed of polysaccharide, protein, lipopolysaccharide and the metal. Precipitation of the main component of the EPS, the extracellular polysaccharide, and thorium was studied. The precipitate was stable, but redissolved at pH values below 3.0 or in the presence of 10 mM EDTA. In the precipitate, the ratio thorium/basic repeating unit of the polysaccharide ranged from 0.4 to 0.8 mol/mol. Soluble polysaccharide–thorium complexes were not found, and larger polysaccharide molecules were precipitated in preference to smaller ones. Kinetic studies showed a non-linear dependence of the precipitate on the concentrations of both thorium and polysaccharide. The behaviors of the purified polysaccharide and of whole EPS with the thorium-containing precipitate were compared. The results suggested that EPS components other than polysaccharide are able to modify the precipitating ability of the polysaccharide. Thus, whole EPS is a better substrate than the purified polysaccharide for the removal of thorium from its solutions.     

Competitive binding of Fe<Superscript>3+</Superscript>, Cr<Superscript>3+</Superscript>, and Ni<Superscript>2+</Superscript> to transferrin

Competitive binding of Fe³⁺, Cr³⁺, and Ni²⁺ to transferrin (Tf) was investigated at various physiological iron to Tf concentration ratios. Loading percentages for these metal ions are based on a two M ⁿ⁺ to one Tf (i.e., 100% loading) stoichiometry and were determined using a particle beam/hollow cathode–optical emission spectroscopy (PB/HC-OES) method. Serum iron concentrations typically found in normal, iron-deficient, iron-deficient from chronic disease, iron-deficient from inflammation, and iron-overload conditions were used to determine the effects of iron concentration on iron loading into Tf. The PB/HC-OES method allows the monitoring of metal ions in competition with Fe³⁺ for Tf binding. Iron-overload concentrations impeded the ability of chromium (15.0 μM) or nickel (10.3 μM) to load completely into Tf. Low Fe³⁺ uptake by Tf under iron-deficient or chronic disease iron concentrations limited Ni²⁺ loading into Tf. Competitive binding kinetic studies were performed with Fe³⁺, Cr³⁺, and Ni²⁺ to determine percentages of metal ion uptake into Tf as a function of time. The initial rates of Fe³⁺ loading increased in the presence of nickel or chromium, with maximal Fe³⁺ loading into Tf in all cases reaching approximately 24%. Addition of Cr³⁺ to 50% preloaded Fe³⁺–Tf showed that excess chromium (15.0 μM) displaced roughly 13% of Fe³⁺ from Tf, resulting in 7.6 ± 1.3% Cr³⁺ loading of Tf. The PB/HC-OES method provides the ability to monitor multiple metal ions competing for Tf binding and will help to understand metal competition for Tf binding.     

Cloning,deletion, and overexpression of a glucose oxidase gene in <Emphasis Type="Italic">Aureobasidium</Emphasis> sp. P6 for Ca<Superscript>2+</Superscript>-gluconic acid overproduction

This study aimed to overexpress a glucose oxidase gene (GOD1) in Aureobasidium sp. P6 to achieve Ca²⁺-gluconic acid (GA) overproduction. The GOD1 gene was cloned, deleted, and overexpressed. A protein deduced from the GOD1 gene of Aureobasidium sp. P6 strain had 1824 bp that encoded a protein with 606 amino acids, with a conserved NADB-ROSSMAN domain and a GMC-oxred domain. Deleting the GOD1 gene made the disruptant GOK1 completely lose the ability to produce GA and GOD1 activity, whereas overexpressing the GOD1 gene rendered the transformant GOEX8 to produce considerably more Ca²⁺-GA (160.5?±?5.6 g/L) and higher GOD1 activity (1438.6?±?73.2 U/mg of protein) than its parent P6 strain (118.7?±?4.3 g/L of Ca²⁺-GA and 1100.0?±?23.6 U/mg of GOD1 protein). During a 10-L fermentation, the transformant GOEX8 grown in the medium containing 160.0 g/L of glucose produced 186.8?±?6.0 g/L of Ca²⁺-GA, the yield was 1.2 g/g of glucose, and the volumetric productivity was 1.7 g/L/h. Most of the produced GOD1 were located in the yeast cell wall. The purified product was identified to be a GA. The transformant GOEX8 overexpressing the GOD1 gene could produce considerably more Ca²⁺-GA (186.8?±?6.0 g/L) than its wild-type strain P6.     

Cd<Superscript>2+</Superscript> extrusion by P-type Cd<Superscript>2+</Superscript>-ATPase of <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> 17810R via energy-dependent Cd<Superscript>2+</Superscript>/H<Superscript>+</Superscript> exchange mechanism

Cd²⁺ is highly toxic to Staphylococcus aureus since it blocks dithiols in cytoplasmic 2-oxoglutarate dehydrogenase complex (ODHC) participating in energy conservation process. However, S. aureus 17810R is Cd²⁺-resistant due to possession of cadA-coded Cd²⁺ efflux system, recognized here as P-type Cd²⁺-ATPase. This Cd²⁺ pump utilizing cellular energy—ATP, ?μ _H ⁺ (electrochemical proton potential) and respiratory protons, extrudes Cd²⁺ from cytoplasm to protect dithiols in ODHC, but the mechanism of Cd²⁺ extrusion remains unknown. Here we propose that two Cd²⁺ taken up by strain 17810R via Mn²⁺ uniporter down membrane potential (?ψ) generated during glutamate oxidation in 100 mM phosphate buffer (high P_iB) are trapped probably by high affinity sites in cytoplasmic domain of Cd²⁺-ATPase, forming SCdS. This stops Cd²⁺ transport towards dithiols in ODHC, allowing undisturbed NADH production, its oxidation and energy conservation, while ATP could change orientation of SCdS towards facing transmembrane channel. Now, increased number of P_i-dependent protons pumped electrogenically via respiratory chain and countertransported through the channel down ?ψ, extrude two trapped cytoplasmic Cd²⁺, which move to low affinity sites, being then extruded into extracellular space via ?ψ-dependent Cd²⁺/H⁺ exchange. In 1 mM phosphate buffer (low P_iB), external Cd²⁺ competing with decreased number of P_i-dependent protons, binds to ψ_s of Cd²⁺-ATPase channel, enters cytoplasm through the channel down ?ψ via Cd²⁺/Cd²⁺ exchange and blocks dithiols in ODHC. However, Mg²⁺ pretreatment preventing external Cd²⁺ countertransport through the channel down ?ψ, allowed undisturbed NADH production, its oxidation and extrusion of two cytoplasmic Cd²⁺ via Cd²⁺/H⁺ exchange, despite low P_iB.     

Studies on the activation of isocitrate dehydrogenase kinase/phosphatase (AceK) by Mn<Superscript>2+</Superscript> and Mg<Superscript>2+</Superscript>

Isocitrate dehydrogenase kinase/phosphatase (AceK) is a bifunctional enzyme with both kinase and phosphatase activities that are activated by Mg²⁺. We have studied the interactions of Mn²⁺and Mg²⁺ with AceK using isothermal titration calorimetry (ITC) combined with molecular docking simulations and show for the first time that Mn²⁺ also activates the enzyme activities. However, Mn²⁺ and Mg²⁺ exert their effects by different mechanisms. Although they have similar binding constants (of 1.11?×?10⁵ and 0.98?×?10⁵ M^?1, respectively) for AceK and induce conformational changes of the enzyme, they do not compete for the same binding site. Instead Mn²⁺ appears to bind to the regulatory domain of AceK, and its effect is transmitted to the active site of the enzyme by the conformational change that it induces. The information in this study should be very useful for understanding the molecular mechanism underlying the interaction between AceK and metal ions, especially Mn²⁺ and Mg²⁺.     

Blood clearance and occupational exposure for <Superscript>177</Superscript>Lu-DOTATATE compared to <Superscript>177</Superscript>Lu-PSMA radionuclide therapy

The main target of this work is to examine blood clearance and external exposure for ¹⁷⁷Lu-DOTATATE compared with new emerging ¹⁷⁷Lu-PSMA therapy. Blood clearance and radiation exposure of 31 patients treated with 5.5?±?1.1 GBq ¹⁷⁷Lu-DOTATATE were compared to those of 23 patients treated with 7.4 GBq ¹⁷⁷Lu-PSMA. Dose rates were measured at several distances and time points up to 120 h after treatment. Blood samples were collected conjunctively after infusion. Caregiver’s cumulative dose was measured by means of an OSL (optically stimulated luminescence) dosimeter for 4–5 days and medical staff’s dose was also estimated using electronic personal dosimeters. Finger dose was determined via ring TLD (Thermoluminescence Dosimeter) for radiopharmacists and nurses. Dose rates due to ¹⁷⁷Lu-DOTATATE at a distance of 1 m, 4 h and 6 h after infusion, were 3.0?±?2.8 and 2?±?1.9 µSv/(h GBq), respectively, while those due to ¹⁷⁷Lu-PSMA were 3.1?±?0.8 and 2.2?±?0.9 µSv/(h GBq). Total effective dose of 17 caregivers was 100–200 µSv for ¹⁷⁷Lu-DOTATATE therapy. Mean effective doses to nurses and radiopharmacists were 5 and 4 µSv per patient, respectively, while those for physicists and physicians were 2 µSv per patient. For ¹⁷⁷Lu-DOTATATE, effective half-life in blood and early elimination phase were 0.31?±?0.13 and 4.5?±?1 h, while they were found as 0.4?±?0.1 and 5?±?1 h, respectively, for ¹⁷⁷Lu-PSMA. The first micturition time following ¹⁷⁷Lu-DOTATATE infusion was noted after 36?±?14 min, while the second and third voiding times were after 74?±?9 and 128?±?41 min, respectively. It is concluded that blood clearance and radiation exposure for ¹⁷⁷Lu-DOTATATE are very similar to those for ¹⁷⁷Lu-PSMA, and both treatment modalities are reasonably reliable for outpatient treatment, since the mean dose rate [2.1 µSv/(h GBq)] decreased below the dose rate that allows release of the patient from the hospital (20 µSv/h) after 6 h at 1 m distance.     

Effect of trace elements and optimization of their composition for the nitrification of a heterotrophic nitrifying bacterium, <Emphasis Type="Italic">Acinetobacter harbinensis</Emphasis> HITLi7<Superscript>T</Superscript>, at low temperature

The effects of trace elements on ammonium degradation performance and extracellular polymeric substances (EPS) secretion of Acinetobacter harbinensis HITLi7^T at low temperature were investigated. Response surface methodology (RSM) was applied to obtain the optimal composition of trace elements and analyze their correlation. In this study, the results indicated that the ammonium removal performance could be enhanced by the presence of 0.1 mg L^?1 Fe, Mn, or B in pure cultivation. When the concentrations of Fe and Mn were 0.2 mg L^?1, the ammonium removal rates of the novel strain HITLi7^T were 0.49 ± 0.01 mg L^?1·h^?1 and 0.58 ± 0.01 mg L^?1·h^?1, respectively, while it was the low concentration of 0.05 mg L^?1 B that showed the maximum ammonium removal rate (0.56 ± 0.02 mg L^?1·h^?1) of strain HITLi7^T. The regression model was obtained and the optimal formulation of trace elements was: B 0.064 mg L^?1, Fe 0.12 mg L^?1, and Mn 0.1 mg L^?1. Based on these values, the experimental ammonium removal rate could reach 0.59 mg L^?1·h^?1, which matched well with the predicted response. The study also found that the addition of trace elements, causing high ammonium removal rates, resulted in a high polysaccharide (PS) ratio in the EPS secreted by Acinetobacter harbinensis HITLi7^T. Especially under the optimal conditions, the PS ratio reached the highest value of 49.9%.     

Citrate analysis using capillary electrophoresis and complexation with Eu<Superscript>3+</Superscript>-tetracycline

A sensitive assay for citrate was developed. Citrate was incubated with 50 μM Eu³⁺-tetracycline and the complex separated using capillary electrophoresis utilizing post-column laser-induced luminescence detection in a sheath flow cuvette. Signal was linear with citrate concentration from 10 μM to 200 nM. Injection volumes were 320 pL. For the 200 nM sample, this corresponds to the injection of 64 amoles of citrate. Separation time was?<?90 s with a total run time of 5 min. As an application the method was used to analyze citrate in agricultural and medicinal products. The method was also used to develop an assay for the enzyme citrate synthase.     

Biologically Fe<Superscript>2+</Superscript> oxidizing fluidized bed reactor performance and controlling of Fe<Superscript>3+</Superscript> recycle during heap bioleaching: an artificial neural network-based model

The performance of a biological Fe²⁺ oxidizing fluidized bed reactor (FBR) was modeled by a popular neural network-back-propagation algorithm over a period of 220 days at 37 °C under different operational conditions. A method is proposed for modeling Fe³⁺ production in FBR and thereby managing the regeneration of Fe³⁺ for heap leaching application, based on an artificial neural network-back-propagation algorithm. Depending on output value, relevant control strategies and actions are activated, and Fe³⁺ production in FBR was considered as a critical output parameter. The modeling of effluent Fe³⁺ concentration was very successful, and an excellent match was obtained between the measured and the predicted concentrations.     

Carbon flows in eutrophic Lake Rotsee: a <Superscript>13</Superscript>C-labelling experiment

The microbial segment of food webs plays a crucial role in lacustrine food-web functioning and carbon transfer, thereby influencing carbon storage and CO₂ emission and uptake in freshwater environments. Variability in microbial carbon processing (autotrophic and heterotrophic production and respiration based on glucose) with depth was investigated in eutrophic, methane-rich Lake Rotsee, Switzerland. In June 2011, ¹³C-labelling experiments were carried out at six depth intervals in the water column under ambient light as well as dark conditions to evaluate the relative importance of (chemo)autotrophic, mixotrophic and heterotrophic production. Label incorporation rates of phospholipid-derived fatty acid (PLFA) biomarkers allowed us to differentiate between microbial producers and calculate group-specific production. We conclude that at 6 m, net primary production (NPP) rates were highest, dominated by algal photoautotrophic production. At 10 m —the base of the oxycline— a distinct low-light community was able to fix inorganic carbon, while in the hypolimnion, heterotrophic production prevailed. At 2 m depth, high label incorporation into POC could only be traced to nonspecific PLFA, which prevented definite identification, but suggests cyanobacteria as dominating organisms. There was also depth zonation in extracellular carbon release and heterotrophic bacterial growth on recently fixed carbon. Large differences were observed between concentrations and label incorporation of POC and biomarkers, with large pools of inactive biomass settling in the hypolimnion, suggesting late-/post-bloom conditions. Net primary production (115 mmol C m^?2 d^?1) reached highest values in the epilimnion and was higher than glucose-based production (3.3 mmol C m^?2 d^?1, highest rates in the hypolimnion) and respiration (5.9 mmol C m^?2 d^?1, highest rates in the epilimnion). Hence, eutrophic Lake Rotsee was net autotrophic during our experiments, potentially storing large amounts of carbon.     

Aluminum resistance in wheat involves maintenance of leaf Ca<Superscript>2+</Superscript> and Mg<Superscript>2+</Superscript> content,decreased lipid peroxidation and Al accumulation,and low photosystem II excitation pressure

The phytotoxic aluminum species (Al³⁺) is considered as the primary factor limiting crop productivity in over 40 % of world’s arable land that is acidic. We evaluated the responses of two wheat cultivars (Triticum aestivum L.) with differential Al resistance, cv. Yecora E (Al-resistant) and cv. Dio (Al-sensitive), exposed to 0, 37, 74 and 148 μM Al for 14 days in hydroponic culture at pH 4.5. With increasing Al concentration, leaf Ca²⁺ and Mg²⁺ content decreased, as well as the effective quantum yield of photosystem II (PSII) photochemistry (Φ _PSII ), while a gradual increase in leaf membrane lipid peroxidation, Al accumulation, photoinhibition (estimated as F _v /F _m ), and PSII excitation pressure (1 ? q _p ) occurred. However, the Al-resistant cultivar with lower Al accumulation, retained larger concentrations of Ca²⁺ and Mg²⁺ in the leaves and kept a larger fraction of the PSII reaction centres (RCs) in an open configuration, i.e. a higher ratio of oxidized to reduced quinone A (Q_A), than plants of the Al-sensitive cultivar. Four times higher Al concentration in the nutrient solution was required for Al-resistant plants (148 μM Al) than for Al-sensitive (37 μM Al), in order to establish the same closed RCs. Yet, the decline in photosynthetic efficiency in the cultivar Dio was not only due to closure of PSII RCs but also to a decrease in the quantum yield of the open RCs. We suggest that Al³⁺ toxicity may be mediated by nutrient deficiency and oxidative stress, and that Al-resistance of the wheat cultivar Yecora E, may be due at least partially, from the decreased Al accumulation that resulted to decreased reactive oxygen species (ROS) formation. However, under equal internal Al accumulation (exposure Al concentration: Dio 74 μM, Yecora E 148 μM) that resulted to the same oxidative stress, the reduced PSII excitation pressure and the better PSII functioning of the Al-resistant cultivar was probably due to the larger concentrations of Ca²⁺ and Mg²⁺ in the leaves. We propose that the different sensitivities of wheat cultivars to Al³⁺ toxicity can be correlated to differences in the redox state of Q_A. Thus, chlorophyll fluorescence measurements can be a promising tool for rapid screening of Al resistance in wheat cultivars.     

N-terminus of PutCAX2 from <Emphasis Type="Italic">Puccinellia tenuiflora</Emphasis> affects Ca<Superscript>2+</Superscript> and Ba<Superscript>2+</Superscript> tolerance in yeast

Cation/H⁺ exchangers (CAXs) are membrane proteins that transport Ca²⁺ and other cations using the H⁺ gradient generated by H⁺-ATPase or H⁺-pyrophosphatase. This study reports the characterization of CAX2 from Puccinellia tenuiflora with respect to molecular and functional properties. PutCAX2 was cloned from a cDNA library of P. tenuiflora seedlings. The expression of PutCAX2 in shoots and roots was induced by Ca²⁺ and Ba²⁺ treatments. A green fluorescent protein (GFP) marker revealed that PutCAX2 was located on the endoplasmic reticulum (ER) membrane. Four yeast transformants were created using GFP fusion PutCAX2 and truncated PutCAX2s, and their growth in the presence of various cations (Fe³⁺, Al³⁺, Mn²⁺, Cu²⁺, Co²⁺, Ni²⁺, Mg²⁺, Zn²⁺, Na⁺, Li⁺, Ca²⁺, and Ba²⁺) was analyzed. The N-terminally truncated PutCAX2 (GFP-ΔNPutCAX2) and the N and C-terminally truncated PutCAX2 (GFP-ΔNCPutCAX2) transformants grew well in the presence of 100 and 150 mM Ca²⁺ or 8 and 20 mM Ba²⁺, whereas the GFP-PutCAX2 and C-terminally truncated PutCAX2 (GFP-ΔCPutCAX2) transformants did not show any tolerance to Ca²⁺ or Ba²⁺. The Ba²⁺ content in whole yeast cells expressing GFP-ΔNPutCAX2 or GFP-ΔNCPutCAX2 was lower than that in other yeast transformants. Moreover, the efflux experiment showed that the Ba²⁺ efflux rate of yeast cells expressing GFP-ΔNPutCAX2 and GFP-ΔNCPutCAX2 was higher than that of other yeast cells. To our knowledge, this is the first report on the molecular and functional characterization of a novel ER-localized CAX protein from a wild halophyte plant; the results suggest that the N-terminus of PutCAX2 acts as an auto-inhibitory domain, which affects the Ca²⁺ and Ba²⁺ tolerance of yeast.     

Effect of Mn<Superscript>2+</Superscript> augmentation on reinforcing aerobic sludge granulation in a sequencing batch reactor

Two sequencing batch reactors were synchronously operated to investigate the effect of manganese (II) (Mn²⁺) augmentation on aerobic granulation. Reactor 1 (R1) was added with 10 mg/L Mn²⁺, while there was no Mn²⁺ augmentation in reactor 2 (R2). Results showed that R1 had a faster granulation process than R2 and R1 performed better in chemical oxygen demand (COD) and ammonium nitrogen (NH₄⁺–N) removal efficiencies. Moreover, the mature granules augmented with Mn²⁺ behaved better on their physical characteristics and size distributions, and they also had higher production of extracellular polymeric substances (EPS) content. The result of three-dimensional excitation and emission matrix fluorescence showed that Mn²⁺ had the function of causing organic material diversity (especially proteins diversity) in EPS fraction from granules. Polymerase chain reaction and denaturing gradient gel electrophoresis techniques were employed to analyze the microbial and genetic characteristics in mature granules. The results exhibited that Mn²⁺ augmentation was mainly responsible for the higher microbial diversity of granules from R1 compared with that from R2. Uncultured sludge bacterium A16 (AF234726) and Rhodococcus sp. WTZ-R2 (HM004214) were the major species in R1, while only uncultured sludge bacterium A16 (AF234726) in R2. Moreover, there were eight species of organisms found in both two aerobic granules, and three species were found only in aerobic granules from R1. It could be concluded that Mn²⁺ could enhance the sludge granulation process and have a key effect role on the biological properties during the sludge granulation.     

Morphological and physiological responses of root tip cells to Fe<Superscript>2+</Superscript> toxicity in rice

Two genotypes of rice (Oryza sativa L.), Azucena (iron tolerant) and IR64 (iron sensitive), were used to investigate the numbers and survival rates of root border cells (namely, in situ border cells) in plants that were exposed to excess iron (Fe²⁺). Additionally, we examined the changes in the root tip cell morphology and activities of protective enzymes in response to Fe²⁺ toxicity. The results showed that Fe²⁺ toxicity hindered the development of root border cells (RBCs) and that higher Fe²⁺ concentrations caused root cap cell walls to thicken. In the iron-sensitive rice variety, these changes lowered RBC survival rate and lead to programmed cell death. Low concentrations of Fe²⁺ were shown to facilitate the development of RBCs in the iron-tolerant rice variety and that the activities of the protective enzymes superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) were elevated in the iron-tolerant variety, thus suggesting that rice root tips could defend against Fe²⁺ toxicity by producing RBCs, root cap cells, and protective enzymes.     

Modulation of the reaction cycle of the Na<Superscript>+</Superscript>:Ca<Superscript>2+</Superscript>, K<Superscript>+</Superscript> exchanger

Ca²⁺ concentration in retinal photoreceptor rod outer segment (OS) strongly affects the generator potential kinetics and the receptor light adaptation. The response to intense light stimuli delivered in the dark produce potential changes exceeding 40 mV: since the Ca²⁺ extrusion in the OS is entirely controlled by the Na⁺:Ca²⁺, K⁺ exchanger, it is important to assess how the exchanger ion transport rate is affected by the voltage and, in general, by intracellular factors. It is indeed known that the cardiac Na⁺:Ca²⁺ exchanger is regulated by Mg-ATP via a still unknown metabolic pathway. In the present work, the Na⁺:Ca²⁺, K⁺ exchanger regulation was investigated in isolated OS, recorded in whole-cell configuration, using ionic conditions that activated maximally the exchanger in both forward and reverse mode. In all species examined (amphibia: Rana esculenta and Ambystoma mexicanum; reptilia: Gecko gecko), the forward (reverse) exchange current increased about linearly for negative (positive) voltages and exhibited outward (inward) rectification for positive (negative) voltages. Since hyperpolarisation increases Ca²⁺ extrusion rate, the recovery of the dark level of Ca²⁺ (and, in turn, of the generator potential) after intense light stimuli results accelerated. Mg-ATP increased the size of forward and reverse exchange current by a factor of ∼2.3 and ∼2.6, respectively, without modifying their voltage dependence. This indicates that Mg-ATP regulates the number of active exchanger sites and/or the exchanger turnover number, although via an unknown mechanism. Proceedings of the XVIII Congress of the Italian Society of Pure and Applied Biophysics (SIBPA), Palermo, Sicily, September 2006.     

Ca<Superscript>2+</Superscript> Dependence and Inhibitory Effects of Trifluoperazine on Plasma Membrane ATPase of <Emphasis Type="Italic">Thermoactinomyces vulgaris</Emphasis>

Ca²⁺ enhanced the plasma membrane Ca²⁺-ATPase (PMCA) specific activities in wild-type strain 1227 and mutant strains 1278, 1286, and 1261 of Thermoactinomyces vulgaris. The Ca²⁺-ATPase specific activities showed marked increase with increasing concentrations of Ca²⁺ added in the form of CaCl₂ in the culture medium and reached the optimum values at 0.6 mM in strains 1227, 1278, and 1286 and at 0.7 mM in strain 1261 of T. vulgaris. Trifluoperazine, a specific blocker of calmodulin, when added in vivo at concentrations of 2 M and 8 M along with the respective optimal concentrations of Ca²⁺, decreased the PMCA-specific activities to a low level in a dose-dependent manner. The results of the present investigation suggest the presence of a Ca²⁺-dependent protein activator (CaDPA) in the microenvironment constituting this enzyme; and such Ca²⁺-modulated protein has been assigned to play an important role in the enhancement of PMCA levels in this aerobic, spore-forming, thermophilic actinomycete.     

Direct formation of axes in new plantlets of <Emphasis Type="Italic">Kappaphycus alvarezii</Emphasis> (Doty) Doty,as influenced by the use of AMPEP K<Superscript>+</Superscript>, spindle inhibitors,and plant growth hormones

In order to induce the direct formation of erect dark brown vegetative shoots of Kappahycus alvarezii, under micro-propagation conditions, the agricultural bio-stimulant “Acadian Marine Plant Extract Powder” (or AMPEP) K⁺ (a Canadian commercial extract of the brown seaweed Ascophyllum nodosum with enhanced levels of potassium) was used singly or in combination with colchicine or oryzalin at different concentrations plus (synthetic) plant growth regulators (i.e., PGR, IAA, and kinetin) over 45 days of incubation. Amongst the treatments tested, 5 mg L^?1 AMPEP K⁺ with PGR produced the longest direct axis shoots (9.6 ± 0.33 mm), followed by 0.1 mg L^?1 AMPEP K⁺ + 1.0 mg L^?1 oryzalin with PGR (8.7 ± 0.00 mm), 10 mg L^?1 AMPEP K⁺ + 0.5 mg L^?1 colchicine with PGR (8.6 ± 1.20 mm), and 0.5 mg AMPEP K⁺ only (5.3 ± 1.8 mm). The shortest, direct axis shoots measured were the following: 8.3 ± 0.33 mm with 0.1, 1 and 10 mg L^?1 AMPEP K⁺ with PGR, 6.0 ± 0.58 mm with 0.5 mg L^?1 AMPEP K⁺ + 0.1 mg L^?1 oryzalin with PGR, 5.3 ± 0.89 mm with 0.5 mg L^?1 AMPEP K⁺ + 0.1 mg L^?1 colchicine with PGR, and finally, 2.7 ± 0.33 mm with 0.1 mg L^?1 AMPEP K⁺ only. The percentage of direct shoots formed ranged from 93 to 100 % and 87 to 100 % in AMPEP K⁺ with PGR and AMPEP K⁺ only, respectively, while 62–100 % and 87–100 % were observed in AMPEP K⁺ + colchicine with PGR and AMPEP K⁺ + oryzalin with PGR, respectively. The use of AMPEP K⁺ with the PGR resulted in the longest and highest percentage formation of direct axis shoots. These observations, taken together, suggested the efficacy of AMPEP K⁺ used as a protocol to induce direct, erect shoots for the micro-propagation of K. alvarezii plantlets under laboratory conditions.     

Subcellular distribution and chemical forms of Co<Superscript>2+</Superscript> in three barley genotypes under different Co<Superscript>2+</Superscript> levels

The toxicity of many heavy metals in plants is closely associated with its subcellular distribution and chemical forms. The subcellular distribution and chemical forms of cobalt (Co²⁺) were investigated using 3 barley genotypes differing in Co²⁺ toxicity resistance, namely Yan66 (resistant), Ea 52 (sensitive), and Humai 4 (moderate), under two Co²⁺ levels (25 and 100 µM). Higher Co²⁺ level in cultural solution significantly increased Co²⁺ accumulation in all subcellular fractions, with vacuole and cell wall having higher concentration. In comparison with 25 µM Co²⁺, 100 µM Co²⁺ treatment caused significant increase of Co²⁺ concentration in the forms of F-NaCl (extracted with 1 M NaCl), F-Ac (extracted with 2% HAc), F-HCl (extracted by 0.6 M HCl), and F-residue (residue forms) in both shoots and roots. There was a significant difference among genotypes in Co²⁺ subcellular distribution and chemical forms, with Ea52 accumulating more Co²⁺ in organelles and Yan66 accumulating more Co²⁺ in vacuole and cell wall. Moreover, the inorganic form of Co²⁺ extracted with 80% ethanol (F-ethanol) and water-soluble form (F-H₂O) were significantly increased in Ea52, while Yan66 accumulated more Co²⁺ in the forms of low-bioavailable molecules (F-NaCl, F-HAc, and F-HCl). The results suggest that the vacuolar sequestration and cell wall deposition of Co²⁺ is a key resistant mechanism for genotype Yan66.     

Strain improvement of <Emphasis Type="Italic">Trichoderma viride</Emphasis> for increased cellulase production by irradiation of electron and <Superscript>12</Superscript>C<Superscript>6+</Superscript>-ion beams

Objectives

To improve cellulase production and activity, Trichoderma viride GSICC 62010 was subjected to mutation involving irradiation with an electron beam and subsequently with a ¹²C⁶⁺-ion beam.

Results

Mutant CIT 626 was the most promising cellulase producer after preliminary and secondary screening. Soluble protein production and cellulase activities were increased mutifold. The optimum temperature, pH and culture time for the maximum cellulase production of the selected mutant were 35 °C, pH 5 and 6 days. The highest cellulase production was obtained using wheat bran. The prepared cellulases from T. viride CIT 626 had twice the hydrolytic performance with sawdust (83 %) than that from the parent strain (42.5 %). Furthermore, molecular studies demonstrated that there were some key mutation sites suggesting that some amino acid changes in the protein caused by base mutations had led to the enhanced cellulase production and activity.

Conclusions

Mutagenesis with electron and ¹²C⁶⁺-ion beams could be developed as an effective tool for improvement of cellulase producing strains.