Environ. Sci. Technol. 2008, 42, 4896–4901
Reduced Toxicity of Olive Mill Waste Waters by Oxidative Coupling with Biomimetic Catalysis GIUSEPPE CELANO,‡ ´ ,† DANIELA ŠMEJKALOVA RICCARDO SPACCINI,† AND A L E S S A N D R O P I C C O L O * ,†,§ Dipartimento di Scienze del Suolo, della Pianta, dell’Ambiente e delle Produzioni Animali, Universita` degli Studi di Napoli “Federico II”, Via Universita` 100, 80055 Portici, Italy, Dipartimento di Produzione Vegetale, Universita` degli Studi della Basilicata, Via Nazario Sauro 85, 85100 Potenza, and Centro Interdipartimentale di Ricerca NMR-CERMANU, Universita` degli Studi di Napoli “Federico II”, Via Universita` 100, 80055 Portici, Italy
Received January 09, 2008. Revised manuscript received April 23, 2008. Accepted April 29, 2008.
Large quantities of environmentally toxic olive mill waste waters (OMWW) result from olive oil production worldwide. A synthetic water-soluble meso-tetra(2,6-dichloro-3-sulfonatophenyl)porphyrinate of iron(III) chloride (FePha) was used as biomimetic catalyst to oxidatively couple toxic phenols in OMWW fractions obtained by micro-, ultra-, and nanofiltration, and reverse osmosis. The occurrence of oxidative coupling in different OMWW size-fractions was assessed by high performance size exclusion chromatography (HPSEC), before and after conformational disruption with acetic acid, and measurements of proton spin-lattice relaxation time in the rotating frame (T1FH) through 13C-CPMAS-NMR spectroscopy. The concurrent reduction in toxicity of OMWW size-fractions brought about by the FePha treatment was monitored by an algal bioassay. HPSEC chromatograms of OMWW samples subjected to catalyzed coupling showed apparent weight-average molecular weight (Mwa) values varying from 18 to 185% larger than for control. Moreover, when such FePha-treated fractions were added to acetic acid prior to HPSEC, the Mwa values still ranged from 14 to 162% larger than for control fractions similarly treated with acetic acid. This evidence of polymerization among toxic phenols was confirmed by T1F(H) values which were significantly enhanced by the FePha treatment, thereby indicating an increased conformational rigidity of OMWW materials. These molecular changes were reflected in a significantly reduced toxicity exerted on microalgae by the OMWW size-fractions subjected to catalyzed oxidative couplings. Our results suggest that OMWW can be effectively treated with a biomimetic catalyst to induce oxidative phenol polymerization and reduce their toxicity before amendments to soils or other disposal means. * Corresponding author phone: +39 081 2539160; fax: +39 081 2539186; e-mail:
[email protected]. ‡ Dipartimento di Scienze del Suolo, della Pianta, dell’Ambiente e delle Produzioni Animali, Universita` degli Studi della Basilicata. † Universita` degli Studi di Napoli “Federico II”. § Centro Interdipartimentale di Ricerca NMR-CERMANU, Universita` degli Studi di Napoli “Federico II”. 4896
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ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 42, NO. 13, 2008
Introduction Olive (Olea europaea) mill wastewaters (OMWW) are generated during mechanical olive oil extraction. The OMWW world annual abundance reaches 3.0 × 107 m3, as mostly produced by small- and medium-size mills in Mediterranean countries (1). OMWW represent a serious threat of fresh waters contamination when accidentally or deliberately disposed into sewage systems without prior decontamination treatments (2, 3). Major components of OMWW are phenolic compounds, such as catechol, 4-methylcatechol, tyrosol, hydroxytyrosol and hydroxycinnamic acids (4), whose concentration may reach 5-10 g · L-1, depending on cultivar, harvesting season, and extraction process (5–7). The toxicity as well as the antibacterial activity of OMWW are associated with such phenolic components (8, 9). To reduce phenolic toxicity, the OMWW are commonly spread on soils, but their impact on the biosphere is unpredictable (10, 11). Tests on selected bacteria (12) and yeasts or fungi (13) suggested that OMWW have negative impact on soil microbial metabolism and inhibit their growth, thereby calling for technologies which reduce OMWW toxicity before disposal on soils. Removal of phenol toxicity from OMWW was attempted biotechnologically by either enzymatically active microorganisms such as lignin-degrading (14, 15) and white-rot fungi (16), or direct use of phenol-oxidative enzymes, such as laccase (17). The detoxification action of phenoloxidase enzymes, either within or outside microbial cells, is believed to occur through phenols coupling (18). Similarly, an increase of molecular size of humic substances under oxidative catalysis by peroxidase was explained with polymerization of humic phenolic monomers (19) or reconfiguration of fulvic constituents in natural background organic matter (20). However, unless encapsulated or immobilized, natural peroxidases are hardly recommendable for OMWW toxicity reduction because of their susceptibility to denaturation, microbial degradation, and instability in the presence of excess oxidants (21). These disadvantages may be overcome by using synthetic biomimetic alternatives, like metal-porphyrin rings, which are sterically stabilized by bulky substituents, such as 2,6dichloro sulfonatophenyl groups (22, 23). Metal-porphyrins, such as iron-porphyrin, are believed to couple phenolic compounds by a free radical mechanism. They undergo oxidation giving highly reactive iron(IV)porphyrin cation radical species, which can catalyze the oxidation of phenolic moieties into free radicals (24, 25). These are then stabilized by spontaneous mutual coupling (26), thereby resulting in oligomers or polymers of larger molecular weights, low solubility, and increased stability against further biological degradation. Iron-porphyrin was proved to catalyze the oxidative polymerization of phenolic compounds such as dihydroxybenzenes and hydroxyl-substituted cinnamic acids (27, 28), which are known OMWW components. Due to the capacity of iron-porphyrin to catalyze the oxidative coupling of natural phenols when mixed together or as components of humic matter (23, 27, 28), this work aims to verify whether the biomimetically catalyzed oxidative polymerization occurred also for phenols contained in OMWW fractions separated by size and assess the consequent reduced fractions toxicity by bioassays.
Materials and Methods Biomimetic Catalysts. Synthesis of meso-tetra-(2,6-dichloro3-sulfonatophenyl)porphyrinate of Fe(III) [Fe(TDCPPS)Cl], 10.1021/es8000745 CCC: $40.75
2008 American Chemical Society
Published on Web 05/30/2008
otherwise referred to as Fe-Pha, was previously described (23). Fe-Pha (15 mg) was dissolved in 100 mL of MilliQWater and its UV/vis spectrum recorded by using a PerkinElmer Lambda 3B Spectrophotometer from 800 to 240 nm before and after leaving the sample aging for 10 months in laboratory conditions (Figure S1 in the Supporting Information (SI)). OMWW Fractions and Treatment with Fe-Pha catalyst. OMWW were obtained from a three-phase continuous extraction plant located near Genova (Liguria, Italy) and immediately stored at -20 °C until use. They showed the following characteristics: pH 5.3, suspended solids 13 g L-1, chemical oxygen demand (COD) 38 000 mg L-1, total sugars 4.9 g L-1, total phenols 3.9 g L-1, colorimetric units 23 000. OMWW were fractionated by micro-, ultra-, and nanofiltration using membranes (Sepa Desal) with different molecular weight cutoff, and reverse osmosis (29). The fractions were filtered (F)
