A quantitative proteomic approach to highlight Phragmites sp. adaptation mechanisms to chemical stress induced by a textile dyeing pollutant.

Phragmites sp. is present worldwide in treatment wetlands though the mechanisms involved in the phytoremediation remain unclear. In this study a quantitative proteomic approach was used to study the prompt response and adaptation of Phragmites to the textile dyeing pollutant, Acid Orange 7 (AO7). Previously, it was demonstrated that AO7 could be successfully removed from wastewater and mineralized in a constructed wetland planted with Phragmites sp. This azo dye is readily taken up by roots and transported to the plant aerial part by the xylem. Phragmites leaf samples were collected from a pilot scale vertical flow constructed wetland after 0.25, 3.25 and 24.25h exposure to AO7 (400mgL-1) immediately after a watering cycle used as control. Leaf soluble protein extraction yielded an average of 1560 proteins in a broad pI range (pH3-10) by two-dimensional gel electrophoresis. A time course comparative analysis of leaf proteome revealed that 40 proteins had a differential abundance compared to control (p<0.05) within a 3.25h period. After 24.25h in contact with AO7, leaf proteome was similar to control. Adaptation to AO7 involved proteins related with cellular signalling (calreticulin, Ras-related protein Rab11D and 20S proteasome), energy production and conversion (adenosine triphosphate synthase beta subunit) carbohydrate transport and metabolism (phosphoglucose isomerase, fructose-bisphosphate aldolase, monodehydroascorbate reductase, frutockinase-1 and Hypothetical protein POPTR_0003s12000g and the Uncharacterized protein LOC100272772) and photosynthesis (sedoheptulose-1,7-bisphosphatase and ferredoxin-NADP+ reductase). Therefore, the quantitative proteomic approach used in this work indicates that mechanisms associated with stress cell signalling, energy production, carbohydrate transport and metabolism as well as proteins related with photosynthesis are key players in the initial chemical stress response in the phytoremediation process of AO7.

Purification of plant complex protein extracts in non-denaturing conditions by in-solution isoelectric focusing.

An alternative approach for plant complex protein extracts pre-purification by in-solution isoelectric focusing in non-denaturing conditions is presented. The separation of biologically active proteins, in narrow ranges of isoelectric point (pI) was obtained by a modified OFFGEL electrophoresis. Two different water-soluble protein extracts from Phragmites leaves were fractionated into 24 fractions within a 3-10 pI range at 10 °C in the absence of denaturing/reducing agents. One-dimensional electrophoretic analysis revealed different protein distribution patterns and the effective fractionation of both protein extracts. Peroxidase activity of each fraction confirmed that proteins remained active and pre-purification occurred. Biological triplicates assured the needed reproducibility.

Constructed wetland treatment system in textile industry and sustainable development.

This study focuses on the evaluation of the adequacy and sustainability of a constructed wetland (CW), with vertical flow (VF) design to treat a strongly coloured textile wastewater.Secondly an accidental AO7 overloaded discharge (700 mg l(-1)) was studied. A set of three similar VFCW beds (3x1 m2), operating in series, allowed also the efficient treatment of the AO7 heavy loaded wastewaters. The treated effluent quality enables water reuse for irrigation purposes or within the process.

Phragmites australis peroxidases role in the degradation of an azo dye.

Phragmites australis are commonly used in constructed wetlands either for domestic sewage or industrial effluents treatment. The aerobic mineralization mechanisms of Acid Orange 7, AO7, in a Vertical Flow Constructed Wetland (VFCW) planted with P. australis suggest that AO7 degradation pathway may involve enzymes like peroxidases (POD), known to degrade some recalcitrant pollutants. In this context, the aim of this study was to evaluate the role of POD extracted from the VFCW P. australis leaves in the decolourization of AO7, which belongs to the very restricted group of bio-degradable azo dyes and is widely used in the textile industry. Leaves' crude extract (CE) was purified by protein fractioning with ammonium sulphate (20-80%). AO7 (0.14 mM) decolourization rate of each CE fraction was determined using hydrogen peroxide (0.2 mM) as a co-substrate. A maximum specific activity of 6.8 x 10(-3) micromol QNNM min(-1) mg protein(-1) was obtained for the 40-60% fraction. The results obtained suggest that P. australis may be a good candidate for the treatment of AO7 contaminated effluents in a VFCW, as very high removal efficiencies were achieved at pilot scale and in vitro studies leading to the decolourization of the dye, suggesting a positive and active role of P. australis in the removal mechanisms within the VFCW. Moreover, some questions were put forward regarding the participation of other important plant enzymes in the degradation process.

Vertical flow constructed wetland for textile effluent treatment.

A pulse feed vertical flow constructed wetland (VFCW) proved to be efficient in the treatment of a textile effluent being able to buffer, dilute and treat an Acid Orange (AO7) accidental discharge. The influence of the flooding level (FL) and pulse feed (PF) duration on the removal efficiencies of a VFCW was examined. Average AO7 removal efficiencies of 70% were achieved for an AO7 Inlet concentration of 700 mgl(-1) applied during 15 min cycle(-1) (every three hours) at a hydraulic load of 13 lm(-2) cycle(-1) and an FL of 21%. The VFCW was modelled by analogy with a combination of ideal reactors. The simplest combination that best reproduced the experimental results was an association of 2 reactors in series plus 1 reactor accounting the dead volumes. The model parameters helped to understand the hydrological and kinetic processes occurring in VFCW. Through the model simulation it was shown that 3 VFCW in series were enough to efficiently treat an organic mass load of 76 gAO7 m(-2) day(-1) in 9 hours and fulfil the discharge legislation. In this work it was possible to establish that the overall degradation kinetics was of first order.

Aerobic degradation of acid orange 7 in a vertical-flow constructed wetland.

Biological, aerobic degradation of an azo dye and of the resultant, recalcitrant, aromatic amines in a constructed wetland (CW) was demonstrated for the first time. A vertical-flow CW, planted with Phragmites sp. was fed with 127 mgl(-1) of acid orange 7 (AO7) at hydraulic loads of 28, 40, 53 and 108 l m(-2) day(-1). Color removal efficiencies of up to 99% clearly demonstrate cleavage of the azo bond, also confirmed by the similar AO7 removal and SO(4)(2-) release rates revealing that adsorption onto the matrix was constant. The positive redox potential at the outlet demonstrates that aerobic conditions were present. Chemical oxygen demand and total organic carbon removal efficiencies of up to 93% were also indicative of AO7mineralization. The degradation of sulfanilic acid was confirmed by the presence of NO(3)(-), SO(4)(2-) and secondary metabolites, which suggest at least two degradation pathways leading to a common compound, 3-oxoadipate.

Influence of salts and phenolic compounds on olive mill wastewater detoxification using superabsorbent polymers.

For a selection of nine commercially available superabsorbent polymers, the absorption capacity was evaluated for the principal absorption-inhibition constituent of OMW, mineral salts and for phytotoxic-components, the phenolic compounds. A double exponential model was established for electrical conductivities ranging 4.2-25,000 microS cm(-1). For solutions of phenolic compounds ranging 0-0.5 g l(-1), a distribution coefficient near unit was achieved, while for OMW, the phenolic compounds were concentrated inside the gel as the distribution coefficient was 1.4. Correction of OMW pH towards neutrality was found to increase the absorption capacity by up to 35%. The phytotoxicity was assessed by the germination of Lepidium sativum. Inhibition in plant growth occurred for all OMW dilutions without superabsorbent polymers application. For 5% of OMW (COD 5 gl(-1) and 200 ppm of phenolic compounds) immobilised in PNa2 (1 gl(-1)), plant growth was promoted being observed a 120% growth germination, thus indicating that olive mill wastewater detoxification occurred.

Detoxification of olive mill wastewater using superabsorbent polymers.

The detoxification of agro-industrial effluents using superabsorbent polymers is a new and innovative process. Olive mill wastewater constitutes a major environmental problem in Mediterranean countries due to the large volumes generated, the seasonality of the industry, and the high content of polyphenols and organic matter. The application of superabsorbent polymers allows olive mill wastewater to be used as a fertilizer, as it is immobilized, increasing the biological activity that decreases its phytotoxicity, thus making its water, organic matter and mineral content usable for plant nutrition. Various parameters that characterise olive mill wastewater were evaluated after absorption in 2 different superabsorbent polymers (SAP1 and SAP2). The organic matter was equally distributed in both phases, while there was a concentration of protein and sodium in solution. The K:Na ratio decreased from 70:1 to 2:1. The polyphenol desorption from the gel into solution was found to follow Fick's law. The mass transfer coefficients were 0.147 min(-1) and 0.0085 min(-1) for SAP1 and SAP2, respectively. Phytotoxicity tests were carried out with SAP2. Olive mill wastewater in SAP2 with polyphenol concentrations up to 200 mg l(-1) revealed no phytotoxicity, and even stimulated Lepidium sativum growth, while olive mill wastewater without superabsorbent polymer revealed growth inhibition for all concentrations tested. Caffeic acid degradation by the immobilised biomass followed zero order kinetics. Degradation constants of 0.087 mg l(-1) min(-1) gSAP2(-1) and 1.156 mg l(-1) min(-1) gSAP2(-1) were found. Fungi that developed in the plant growth medium were identified as Aspergillus sp. and Penicillium sp.