Margarida M. Correia dos Santos

Enzymatic biotransformation of the azo dye Sudan Orange G with bacterial CotA-laccase

In the present study we show that recombinant bacterial CotA-laccase from Bacillus subtilis is able to decolourise, at alkaline pH and in the absence of redox mediators, a variety of structurally different synthetic dyes. The enzymatic biotransformation of the azo dye Sudan Orange G (SOG) was addressed in more detail following a multidisciplinary approach. Biotransformation proceeds in a broad span of temperatures (30-80 degrees C) and more than 98% of Sudan Orange G is decolourised within 7h by using 1 U mL(-1) of CotA-laccase at 37 degrees C. The bell-shape pH profile of the enzyme with an optimum at 8, is in agreement with the pH dependence of the dye oxidation imposed by its acid-basic behavior as measured by potentiometric and electrochemical experiments. Seven biotransformation products were identified using high-performance liquid chromatography and mass spectrometry and a mechanistic pathway for the azo dye conversion by CotA-laccase is proposed. The enzymatic oxidation of the Sudan Orange G results in the production of oligomers and, possibly polymers, through radical coupling reactions. A bioassay based on inhibitory effects over the growth of Saccharomyces cerevisiae shows that the enzymatic bioremediation process reduces 3-fold the toxicity of Sudan Orange G.

Speciation of Inorganic Arsenic in Natural Waters by Square-Wave Cathodic Stripping Voltammetry

A fast and sensitive voltammetric method for the determination of arsenite and total inorganic arsenic in natural water systems is described using square-wave cathodic stripping voltammetry at a hanging mercury drop electrode. All the determinations were done in 1 M HCl, in the presence of 5 ppm copper(II) and dissolved oxygen. Discrimination between both oxidation states is achieved just by varying the composition of the supporting electrolyte: reduction of As(V) to As(III) is carried out at room temperature by a mixture of potassium iodide and ascorbic acid and inorganic arsenic determined as As(III)+As(V), while As(III) is measured in the absence of the reducing agent. For an accumulation time of 60 s the detection limits were 0.7 ppb for As(III) in 1 M HCl, and 5 ppm Cu(II), 0.6 ppb for As(III)+As(V) in 1 M HCl, 5 ppm Cu(II) and 0.04 % (w/v) KI+0.02 % (w/v) ascorbic acid. The precision, expressed as relative standard deviation (RSD) at 95 % confidence level, was calculated as 9 % from ten individual measurements taken at 2 ppb standard. The method was tested by the measurement of As(V) and As(III) in real and synthetic natural water samples.

Rapid pK measurements for multibasic weak acids by gradient flow injection titration

Abstract p K values of phthalic acid, phosphoric acid and EDTA (p K 2 , p K 3 and p K 4 only) have been determined by rapid gradient flow injection titration. Key factors in experimental design have been investigated, viz. pH range of the titration, direction of titration, flow-rate and weak acid concentration. Under optimum conditions the technique is well suited to the determination of p K values with a precision of 0.01–0.05 p K and in good agreement with literature values. It is less well suited than conventional batch titration to the determination of very low or high p K values at low weak acid concentrations.

An integrated gradient chamber and potentiometric detector for flow injection analysis: Part 2. Measurement of acidity constants of weak acids

Abstract A gradient chamber with potentiometric detection is evaluated for rapid determination of the acidity constants of weak acids. The best precision was obtained when an acidic carrier solution was used. p K a values determined for acetic and hydrofluoric acids show good precision (σ=0.012 p K a ) and agreement with critical literature values is better than 0.1.

An integrated gradient chamber and potentiometric detector for flow injection analysis : Part 1. Assessment of the system

Summary An integrated gradient chamber and Potentiometric flow cell is described. Good response is demonstrated for flat membrane glass electrodes and a range of other ion-selective electrodes. Mixing in the gradient chamber is evaluated by using Potentiometrie electrodes and a conductivity detector. The values of the time constants obtained are consistent for all experiments, but not in accordance with the theoretically predicted values. Therefore, they should be considered as empirical constants.

Evaluating trace element bioavailability and potential transfer into marine food chains using immobilised diatom model species Phaeodactylum tricornutum, on King George Island, Antarctica

In order to evaluate trace element bioavailability and potential transfer into marine food chains in human impacted areas of the Fildes Peninsula (King George Island, South Shetland Islands Archipelago), element levels (Cr, Ni, Cu, Zn, Cd, and Pb) were determined in water, sediments, phytoplankton, and in diatom Phaeodactylum tricornutum Bohlin (Bacillariophyceae) cells immobilised in alginate and exposed to water and sediments, from the Bellingshausen Dome (reference site) and Ardley Cove (human impacted area), during January 2014. High element concentrations in exposed P. tricornutum indicated element mobilisation from sediments into the water. Levels in exposed cells reflected the sediment element content pattern, comparable to those found in phytoplankton, supporting phytoplankton as an important path of trace element entry into marine food chains. This study clearly shows immobilised P. tricornutum as good proxy of phytoplankton concerning element accumulation efficiency, and an effective tool to monitor trace element contamination in polar coastal ecosystems.

Determination of nickel, calcium and magnesium in xylem sap by flame atomic absorption spectrometry using a microsampling technique.

INTRODUCTION Knowledge of xylem sap chemical composition is important to the understanding of translocation, detoxification and tolerance mechanisms. However, the small amount of sample available often hampers its characterisation. Hence, low volume consumption techniques are needed for xylem sap analysis. OBJECTIVE To develop a microsampling technique for the determination of elements in xylem sap from different plants by flame atomic absorption spectrometry (FAAS). METHODOLOGY The microsampling device was optimised in terms of sample volume and integration time. The analytical characteristics of the microsampling technique (micro-FAAS) were established and compared with those of FAAS with traditional continuous nebulisation. The method was validated by means of an independent technique. RESULTS Ca, Mg and Ni were determined in a 50 microL aliquot of xylem sap solution/element that was introduced directly into the flame via the microsampling accessory. Good precision was obtained with relative standard deviations of 1.1, 0.6 and 2.3% for Ca, Mg and Ni, respectively. Matrix effects resulting from the physical characteristics of the samples and possible chemical interferences caused by phosphate and/or sulphate were ruled out. CONCLUSION A simple, rapid and reproducible microsampling technique coupled to FAAS was developed and successfully applied in the determination of Ca, Mg and Ni in xylem sap.

Improved voltammetric method for simultaneous determination of Pt and Rh using second derivative signal transformation – application to environmental samples

The determination of Platinum-group elements (PGE) in relevant environmental matrices is a challenging task. Sensitive and accurate analytical procedures for simultaneous determination of Pt and Rh are still needed. In this study, we report for the first time on the use of second derivative signal transformation to the ultra-trace simultaneous determination of Pt and Rh by Adsorptive Cathodic Stripping Voltammetry (AdCSV). With that step, the ill-defined peaks typically observed in the original voltammograms are transformed into well-shaped peaks, resulting in accurate detection. The experimental conditions were investigated and optimised: a suitable electrolyte for both elements, with less reagents consumption, (0.25M H2SO4, 0.05M HCl, 0.01M FA and 0.5mM HZ), deposition time (td) and deposition potential (Ed). For td = 120s and Ed = -0.75V, linear relationships r > 0.999 were obtained in the concentration range up to 5.8ngL-1 (27 pM) for Pt and up to 3.4ngL-1 (34 pM) for Rh. Limits of detection were 0.2ngL-1 for Pt and 0.08ngL-1 for Rh. Lower values can be achieved by increasing the deposition time. Limits of quantification, LOQ, calculated as 3 times LOD, were 0.5ngL-1 for Pt and 0.2ngL-1 for Rh. The sensitivity of Pt was affected by elevated Zn concentrations, whereas a minor effect was observed for Rh. However, Pt and Rh determinations were not influenced using the standard addition method. Precision as intermediate precision and expressed as relative standard deviation, based on Pt and Rh spiked solutions and digested road dust CRM BCR-723 was 17% and 20% for Pt and Rh, respectively. Recoveries of CRM were around 90% for both elements. The method was successfully applied in the simultaneous determination of Pt and Rh in sediments from Tagus estuary and, for the first time, dissolved Rh was determined in water samples of a waste water treatment plant. Application of this technique in a multidisciplinary approach will be a relevant contribution to the current understanding of PGE cycle and fate in the environment.

Binding of vanadium to human serum transferrin - voltammetric and spectrometric studies

Previous studies generally agree that in the blood serum vanadium is transported mainly by human serum transferrin (hTF). In this work through the combined use of electrochemical techniques, matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry and small-angle X-ray scattering (SAXS) data it is confirmed that both VIV and VV bind to apo-hTF and holo-hTF. The electrochemical behavior of solutions containing vanadate(V) solutions at pH=7.0, analyzed by using two different voltammetric techniques, with different time windows, at a mercury electrode, Differential Pulse Polarography (DPP) and Cyclic Voltammetry (CV), is consistent with a stepwise reduction of VV→VIV and VIV→VII. Globally the voltammetric data are consistent with the formation of 2:1 complexes in the case of the system VV-apo-hTF and both 1:1 and 2:1 complexes in the case of VV-holo-hTF; the corresponding conditional formation constants were estimated. MALDI-TOF mass spectrometric data carried out with samples of VIVOSO4 and apo-hTF and of NH4VVO3 with both apo-hTF and holo-hTF with V:hTF ratios of 3:1 are consistent with the binding of vanadium to the proteins. Additionally the SAXS data suggest that both VIVOSO4 and NaVVO3 can effectively interact with human apo-transferrin, but for holo-hTF no clear evidence was obtained supporting the existence or the absence of protein-ligand interactions. This latter data suggest that the conformation of holo-hTF does not change in the presence of either VIVOSO4 or NH4VVO3. Therefore, it is anticipated that VIV or VV bound to holo-hTF may be efficiently up-taken by the cells through receptor-mediated endocytosis of hTF.