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High grade purified hydroxytyrosol was produced in small scale by recovery from three-phase olive mill wastewaters, combining a soxhlet continuous liquid–liquid extraction procedure with two chromatographic steps, which were consistent with silica gel middle pressure and percolation on C-18 phase chromatography. The highly pure orthodiphenol was characterized by HPLC, EI-MS and 1 H-NMR methods. The convenience of this procedure and the utilization of the compound for laboratory uses are briefly discussed.

Dihydrogenarsenate [H(2)AsO(4)(-), As(V)] or dichromate [Cr(2)O(7)(2-), Cr(VI)] at pH=4.0 showed to be sorbed on a Fe(OH)(x)-polymerin complex and ferrihydrite to a greater extent than on polymerin, the organic polymeric fraction of olive oil mill wastewater (OMW). In particular, the maximum amount (x(m)) of arsenate sorbed on Fe(OH)(x)-polymerin complex was similar to that on ferrihydrite (880.26 and 743.02 mmol kg(-1), respectively), and was much greater than that sorbed on polymerin (384.25 mmol kg(-1)). The sorption of dichromate was to a comparable extent on Fe(OH)(x)-polymerin complex and ferrihydrite (205.90 and 254.88 mmol kg(-1), respectively). Cr(III), a less toxic chromium form, mainly, and Cr(V) were indeed the effective forms sorbed on polymerin (200 mmol kg(-1)), as a consequence of the redox reaction of the strongly toxic Cr(VI) with the CH(2)OH groups of the polysaccharide moiety of this bio-sorbent, according to the data deriving from XPS and DRIFT analyses. The potential exploitation of the selected sorbents for the removal of As(V) or Cr(VI) from aqueous effluents is briefly discussed.

A comparative study was performed on the sorption capacity of the phenoxy acid herbicide cyhalofop on polymerin (from olive oil mill effluents), ferrihydrite, and a ferrihydrite-polymerin complex, by using a batch equilibrium method. The most efficient sorbent showed to be ferrihydrite followed by the ferrihydrite-polymerin complex and polymerin. Cyhalofop acid bound to ferrihydrite by a combination of ionic and ion-dipole bonding, whereas the same herbicide bound to the ferrihydrite-polymerin complex by ionic bonding and to polymerin by hydrogen bonding. Simulated wastewaters contaminated with cyhalofop acid were completely purified by two sorption cycles on ferrihydrite and five cycles on the ferrihydrite-polymerin complex, whereas the same wastewaters maintained a constant residue even after five sorption cycles on polymerin. For the first time, the possible use of a mineral (ferrihydrite) and an organo-mineral complex (ferrihydrite-polymerin) as a filter for the control of the herbicide contamination in point sources is proposed and briefly discussed.

This study investigated the sorption of paraquat and 2,4-D on polymerin, the humic acid-like fraction of olive mill wastewater. Effects of pH, contact time, initial concentration and sorbent dosage on the sorption of both herbicides were studied. The sorption mechanism of paraquat on polymerin was consistent with the ion exchange of this herbicide with Ca, Mg and K natively occurring in the sorbent; in contrast, 2,4-D was bound to polymerin by hydrogen bonding. Simulated wastewaters contaminated with paraquat were purified after three sorption cycles on polymerin renewed at each cycle, at a solid/liquid ratio of 0.5, whereas those containing 2,4-D showed a maximal residue removal of 44% after two sorption cycles at the same ratio. The possible application of this model to other water-soluble herbicides, as well as the possible exploitation of polymerin as a bio-filter for the decontamination of pollution point sources is briefly discussed.

Three α-elicitins, named hibernalin1, hibernalin2 and hibernalin3 (hib1, hib2 and hib3, respectively), were isolated by reverse phase-low-pressure liquid chromatography from culture filtrates of Phytophthora hibernalis Carne 1925, the causal agent of citrus lemon brown rot. Hib1 proved to be identical to syringicin previously isolated from culture filtrates of Phytophthora syringae. Hib2 and hib3 shared the same primary structure with hib1, but contained, at position 50, Met sulphoxide or sulphone, respectively. By SDS-PAGE, the three proteins showed the same electrophoretic mobility, corresponding to about 10 kDa. Exact Mr values were obtained by MALDI-TOF-MS (10,194.82 for hib1, 10,209.33 for hib2 and 10,223.80 for hib3), while by ESI-MS an M r value of 10,194.90 was found for hib1 and no results for hib2 and hib3. The hibernalin forms showed a high propensity to self-association, after exposure to acetonitrile. Hib1 showed to be active in both the hypersensitivity response and electrolytes leakage assays; the sample containing hib1 and hib2 was only weakly active in the first assay and inactive in the second assay, while the sample containing all three hibernalin forms proved to be inactive in both tests. It is proposed that the different activities of the three hibernalin samples could be very likely attributed to both Met50 oxidation and aggregation. © 2007 The Japanese Biochemical Society.

High-performance anion exchange chromatography coupled with pulsed amperometric detection was used for the quantitative determination of total and free sugars in olive oil mill waste waters (OMWW). Automated amino acid ion chromatography was employed to analyse total and free amino acids in the same OMWW. Sugars were analysed in samples pre-purified by means of a three-step purification procedure involving: (i) methanol precipitation of OMWW; (ii) dialysis of the obtained solid and liquid fractions; and (iii) chromatographic purification on RP18 phase followed by Amberlite resin. The amino acids were determined directly in samples obtained from the first two steps performed for sugar analysis. The analysis carried out with the reported methodologies allowed the quantitative determination of total sugars and amino acids and the differentiation between their free and bound forms. The sugars determined were arabinose, fructose, galactose, glucose, rhamnose, xylose, galacturonic and glucuronic acids, and the amino acids were Asp, Glu, Thr, Ser, Pro, Gly, Ala, Val, Met, Ile, Leu, Tyr, Phe, Lys, His, Arg and Cys. Asn, Gln, and Trp were not detected. The technological, biotechnological and environmental advantages arising from this analytical methodology applied to OMWW are briefly discussed. Copyright © 2003 John Wiley & Sons, Ltd.

Metal cations were quantitatively detected by atomic absorption spectrometry in samples of olive oil mill waste waters obtained by a pressure process (omww(1)) (K, 17.1; Mg, 2.72; Ca, 2.24; Na, 0.40; Fe, 0.123; Zn, 0.0630; Mn, 0.0147; Cu, 0.00860 g L(-)(1)) and a centrifugation process (omww(2)) (K, 9.80; Mg, 1.65; Ca, 1.35; Na, 0. 162; Fe, 0.0330; Zn, 0.0301; Mn, 0.00910; Cu, 0.00980 g L(-)(1)). The inorganic anions, determined in the same samples by ion chromatography, proved to be Cl(-), H(2)PO(4)(-), F(-), SO(4)(2)(-), and NO(3)(-) (1.61, 1.05, 0.66, 0.52, and 0.023 g L(-)(1), respectively, in omww(1) and 0.61, 0.40, 0.25, 0.20, and 0.0090 g L(-)(1), respectively, in omww(2)). Most of the metal cations were revealed to be bound to the omww organic polymeric fraction (opf), composed of polysaccharides, phenol polymers, and proteins. Opf relative molecular weight was substantially estimated in the range between 1000 and 30000 Da for approximately 75% and in the range from 30000 to 100000 Da for approximately 25%. The free residual cations pool proved to be neutralized by the inorganic counteranions. Finally, the possible exploitation of this material in agriculture and in environmental biotechnology processes is also discussed in the light of its chemical and biochemical oxygen demand parameters.

Electron ionization and fast atom bombardment mass spectrometry (EI- and FAB-MS) are the spectroscopic methods most extensively used for analysing the main polyphenols naturally occurring in olive oil mill waste waters (omww) and in dropped olive leaves. EI-MS is very suitable for analysing catechol, 4-methylcatechol, tyrosol and hydroxytyrosol, the main polyphenols identified in omww. The EI method has also proved to be suitable for analysing their acetyl derivatives, but does not allow the molecular ion of diacetyltyrosol and its isomers to be detected. FAB-MS is the only adequate ionization method for detecting the molecular ion of diacetyltyrosol and is more suitable than EI-MS for analysing triacetylhydroxytyrosol. The mechanisms of fragmentation of the acetyl derivatives of tyrosol and hydroxytyrosol were determined using the EI and FAB ionization methods. In addition, FAB-MS, which was performed in both positive and negative ion modes, was shown to be the only adequate ionization method for analysing oleuropein, a phenol glucoside which occurs naturally in olive leaves. The positive ion FAB-MS was shown to be much more suitable than EI-MS for analysing the aglycone obtained following the hydrolysis of oleuropein by β-glucosidase. These results confirmed the versatility of FAB-MS for analysing low molecular weight compounds, for which EI-MS proved to be an unsatisfactory method. Copyright © 1999 John Wiley & Sons, Ltd.

Putaminoxin and pinolidoxin, two structurally related nonenolides, isolated respectively from organic extracts of Phoma putaminum and Aschochyta pinodes cultures, together with some of their natural analogs and synthetic derivatives, were used in a structure-activity relationship study. Their phytotoxic, antifungal and zootoxic activities were assayed with the aim to find compounds with potential herbicidal properties. The strongest phytotoxic compounds proved to be putaminoxin and pinolidoxin, whose activity appeared to be correlated to the integrity of the nonenolide ring and to the presence of both the hydroxy groups and the unmodified propyl side chain. None of the assayed nonenolides showed antifungal activity, whereas pinolidoxin analogs and derivatives showed high to weak zootoxicity.

A new unusual phytotoxic bis-amino acid N-glucoside, named ascaulitoxin, was isolated from the culture filtrate of Ascochyta caulina, the causal agent of leaf and stem necrosis of Chenopodium album, a promising mycoherbicide for the biological control of this common noxious weed. Ascaulitoxin, characterized by extensive use of NMR techniques and chemical methods as N 2 - (2,4,7-triamino-5-hydroxy)-octanedioyl-β- d -glucopyranoside , showed phytotoxic activity against host and non-host plants.

A phytotoxic macrolide and a hexaketide, putaminoxins B and C, respectively, were isolated together with putaminoxin, the main phytotoxin, from liquid culture filtrates of Phoma putaminum, the causal agent of leaf necrosis of Erigeron annuus, a common weed of fields and pastures. Putaminoxins B and C were characterized, using spectroscopic methods, as a new disubstituted nonenolide and a new disubstituted cyclononendione, respectively. Assayed on leaves of weedy and cultivated host plants, putaminoxins C showed toxic effects similar to those produced by putaminoxin. Copyright (C) 1997 Elsevier Science Ltd.

The activity of cytochalasin B was tested on 8 Phytophthora species, while the same toxin, some of its derivatives and natural analogues, namely cytochalasin F and deoxaphomin, were assayed at 2 x 10(-5) - 2 x 10(-4) M on the most sensitive species, P. cactorum. A significant inhibitory activity on P. cactorum was shown by cytochalasin B, its 7-monoacetyl derivative, and deoxaphomin. The hydroxy group at C-20 and the conformational freedom of the macrocyclic ring proved to be important structural features for this activity. The 7-hydroxy group at C-7 appeared to have no influence on this toxicity, while a size reduction associated with the carbocyclic nature of the macrocycle seems to lightly increase the activity. The 7-O-acetylcytochalasin B showed selective toxic activity on P. cactorum at the tested concentration, thus suggesting a possible use as a fungicide for this compound.

Two different strategies for the removal of trivalent chromium (Cr(3+)) from contaminated water are reported. The first one is based on the sorption process on an organo-mineral complex, named LDH-HP, obtained in turn by sorption of polymerin, the humic acid-like fraction occurring in olive oil mill wastewater, on a layered double hydroxide (LDH) of magnesium and aluminium with carbonate in the interlayer. This sorption process is preliminary developed on simulated wastewater (SW) as theoretical model and successively applied on tannery process wastewaters (TPWs) natively containing Cr(3+). The removal of Cr(3+) from TPW is lower than that observed for SW, because of the large compositional variability of TPW. The second one is based on the direct production of a LDH of magnesium and chromium (Cr(3+)), using as starting material TPW, naming LDH-TPW the complex produced. This process allows the complete removal of Cr(3+) from TPW and also the abatement of chemical oxygen demand, indicating to be a very promising purification process for an industrial application.

The ‘crude’ filtrate (CF) of Phytophthora cactorum containing phytotoxin(s), having some properties similar to the toxins isolated from other Phytophthora species, was processed by three steps (acetone precipitation, dialysis and gel filtration chromatography). The CF fractions corresponding to the progressive steps of purification were tested for phytotoxicity on tomato seedlings and for activity on cell trans-membrane electrical potential (Em) of susceptible and resistant apple rootstocks (Malus domestica). The fractions (F4), obtained from chromatography on Sephadex G50 fine and eluted in the zone corresponding to a molecular weight of 15 ± 2 kD, induced a specific alteration on susceptible apple cell membranes. These metabolites, even though incompletely purified, are able to induce a high and specific activity on susceptible apple cell Em only, not on resistant ones. As a consequence, they may be of potential use in screening for insensitive cells. Zusammenfassung Einflus von Partiell Gelauterten Phytotoxinen von Phytophthora cactorum auf Plasmamembranen von Apfelzellen In Rohfiltraten (CF) von Phytophthora cactorum wurden Phytotoxine mit ahnlichen Eigenschaften von Toxinen anderer Phytophthora-Arten nachgewiesen. Die Filtrate wurden in einem Drei-Phasen-Prozes fraktioniert (Azentonniederschlag, Dialyse und Gelfiltration) und bezuglich Phytotoxizitat an Tomatensamlingen und Beeinflussung des elektrischen Potentials (Em) auf Zellmembranen von anfalligen und resistenten Apfelwurzelstocken (Malus domesticus) gepruft. Die Gelfitrationsfraktion (F4) mit einem Molekulargewicht von 15 ± 2 kD fuhrte zu einer spezifischen Veranderung bei Zellmembranen der anfalligen Apfelvarietat. Die in der Fraktion enthaltenen P. cactorum-Metaboliten induzieren nur bei Zellmembranen von anfalligen, nicht aber von resistenten, Apfelvarietaten eine heftige Veranderung des elektrischen Potentials. Sie konnten deshalb auch zur Selektion von resistenten Apfelvarietaten verwendet werden.

Among the main polyphenols occurring in olive oil vegetation waters (VW), catechol showed the most deterrent action on the oviposition of Bactrocera oleae (Gmelin); 4-methylcatechol was less active, whereas hydroxytyrosol and tyrosol were inactive. In contrast, synthetic o-quinone was found to be stimulant at 7.5 x 10(-2) M. Two other synthetic derivatives of catechol, diacetylcatechol and guaiacol, were also deterrent, suggesting these compounds undergo a biochemical transformation into catechol by means of the bacterial symbionts of B. oleae. VW and their phenolic extracts showed deterrence only when highly concentrated, while natural olive juice was strongly deterrent. Experiments carried out to evaluate the effect of olive juice and catechol on the fecundity of B. oleae showed that they strongly reduce this function. Moreover, the possible utilization of VW and their bioactive polyphenols in protection of olives against Batrocera oleae is discussed.

Ascochyta pisi var. pisi . a phytopathogenic fungus responsible for pea anthracnose, produced toxic metabolites when grown on autociaved wheat kernels. The main phytotoxin, called ascosalitoxin, was isolated and characterized as 2,4-dihydroxy-3-methyl-6-(1,3-di-methyl-2-oxopentyl)benzaldehyde by spectroscopic and chemical analysis of both the toxin and its acetyl derivative. Ascosalitoxin displayed phytotoxic activity on pea and bean leaves and pods, and on tomato seedlings. Assayed on brine shrimps ( A. salina ) and on Geotrichum candidum the toxin showed no toxicity.

Three new pinolidoxins called epi-, dihydro-, and epoxy-pinolidoxin (2, 4, and 5, respectively) were isolated from Ascochyta pinodes grown on wheat, and their structures were determined using spectroscopic and chemical methods. They were characterized as 7-epi-, 5,6-dihydro-, and 5,6-epoxy-pinolidoxin, respectively. Assayed on pea and bean leaves, epipinolidoxin and dihydropinolidoxin caused necrotic lesions, whereas epoxypinolidoxin was inactive. Only epipinolidoxin and epoxypinolidoxin were active using the brine shrimp assay

Polymerin is a humic acid-like polymer, which we previously recovered for the first time from olive oil mill waste waters (OMWW) only, and chemically and physicochemically characterized. We also previously investigated its versatile sorption capacity for toxic inorganic and organic compounds. Therefore, a review is presented on the removal, from simulated polluted waters, of cationic heavy metals [Cu(II), Zn, Cr(III)] and anionic ones [Cr(VI)) and As(V)] by sorption on this natural organic sorbent in comparison with its synthetic derivatives, K-polymerin, a ferrihydrite-polymerin complex and with ferrihydrite. An overview is also performed of the removal of ionic herbicides (2,4-D, paraquat, MCPA, simazine, and cyhalofop) by sorption on polymerin, ferrihydrite, and their complex and of the removal of phenanthrene, as a representative of polycyclic aromatic hydrocarbons, by sorption on this sorbent and its complexes with micro- or nanoparticles of aluminum oxide, pointing out the employment of all these sorbents in biobed systems, which might allow the remediation of water and protection of surface and groundwater. In addition, a short review is also given on the removal of Cu(II) and Zn from simulated contaminated waters, by sorption on the humic acid-like organic fraction, named lignimerin, which we previously isolated for the first time, in collaboration with a Chilean group, from cellulose mill Kraft waste waters (KCMWW) only. More specifically, the production methods and the characterization of the two natural sorbents (polymerin and lignimerin) and their derivatives (K-polymerin ferrihydrite-polymerin, polymerin-microAl(2)O(3) and -nanoAl(2)O(3), and H-lignimerin, respectively) as well as their sorption data and mechanism are reviewed. Published and original results obtained by the cyclic sorption on all of the considered sorbents for the removal of the above-mentioned toxic compounds from simulated waste waters are also reported. Moreover, sorption capacity and mechanism of the considered compounds on polymerins and lignimerins are evaluated in comparison with other known natural sorbents, especially of humic acid nature and other organic matter. Some of their technical aspects and relative costs are also considered. Finally, the possible large-scale application of the considered sorption systems for water remediation is briefly discussed.

Two additional cytochalasins were isolated from liquid culture filtrates of Phoma exiqua var. heteromorpha together with some already known cytochalasins. The two new metabolites, named cytochalasin U and V, represent the first natural occurrence of a 25,26-dioxa[16] and 25-oxa[15]cytochalasan, respectively, both bearing a formyl group on the macrocyclic ring. Cytochalasins U and V showed respectively significant and weak activity in the brine shrimp assav.

Fusaric and 9,10-dehydrofusaric acids and their corresponding methyl esters were isolated from the culture filtrates of Fusarium nygamai. The methyl esters were characterized by chemical and spectroscopic methods and reported here for the first time as naturally occurring products. When assayed on tomato leaves and seedlings at 2.7 × 10−3 and 2 × 10−4 M, respectively, fusaric and 9,10-dehydrofusaric acids and their methyl esters showed wide chlorosis rapidly evolving into necrosis as well as a strong inhibition of root elongation, respectively. When assayed at 10−4 M on brine shrimps (Artemia salina), fusaric and 9,10-dehydrofusaric acids did not prove to be toxic, while their methyl esters showed a toxicity level of 50%, expressed as mortality.

Mg-Al layered double hydroxides (LDHs) containing chloride (LDH-Cl) or carbonate (LDH-CO(3)) in the interlayer were obtained at room temperature and after calcitation at 450 degrees C (LDH-Cl-450 and LDH-CO(3)-450) and were characterized by X-ray diffraction and Fourier transform infrared analyses. Sorption isotherms of a humic acidlike fraction naturally occurring in olive oil mill waste waters, named polymerin, on these LDH minerals were carried out. Because LDH-CO(3) showed the highest capacity to sorb polymerin among the four LDH minerals synthesized, two organo-mineral complexes named LDH-CO(3)-LP (low polymerin) and LDH-CO(3)-HP (high polymerin) were prepared by coprecipitation (LDH-CO(3)-LP) and sorption onto a preformed LDH-CO(3) mineral (LDH-CO(3)-HP). These complexes were characterized chemically and physicochemically, and their stability to pH and after thermal treatment at 80 degrees C were evaluated. The diffuse reflectance infrared Fourier transform and X-ray analysis of the complexes indicated that polymerin was sorbed only on the external surfaces of LDH-CO(3) and no intercalation occurred. The LDH-polymerin complexes appeared to be more stable than LDH-CO(3) at pH 4.0 and showed that they were able to sorb both As(V) and Zn. Because the waste waters are usually contaminated with mixtures of pollutants in cationic and anionic forms, the LDH-polymerin complexes appear more suitable than the sorbents in a potential water remediation process.

Catechol, 4-methylcatechol, tyrosol and hydroxytyrosol were isolated and characterized as the main polyphenols from olive oil mill waste waters. In addition, the corresponding acetates were prepared. In phytotoxicity assays carried out on tomato (Lycopersicon esculentum) and vegetable marrow (Cucurbita pepo) plants, the compounds were selectively toxic, except for 4-methylcatechol and its acetate. The vegetation waters remained phytotoxic even after total extraction of the polyphenols, suggesting that other chemical products contribute to the overall phytotoxicity.

Two additional cytochalasins were isolated from liquid culture filtrates of Phoma exiqua var. heteromorpha together with some already known cytochalasins. The two new metabolites, named cytochalasin U and V, represent the first natural occurrence of a 25,26-dioxa[16] and 25-oxa[15]cytochalasan, respectively, both bearing a formyl group on the macrocyclic ring. Cytochalasins U and V showed respectively significant and weak activity in the brine shrimp assav.

High performance liquid chromatography and high performance thin layer chromatography were used in combination to check the occurrence of the bioactive cytochalasins A, B, C, D, E, H and J in cultures of some phytopathogenic species of Ascochyta, isolated from infected plants. Cytochalasins A and B were readily detected and estimated in the culture filtrates of Ascochyta heteromorpha Sch. and Sacc., the causal agent of foliar disease of oleander (Nerium oleander L.). The two compounds were also detected in oleander leaves naturally infected by A. heteromorpha suggesting that these compounds play a role in the disease of the ornamental plant. None of the above mentioned cytochalasins were detected in the culture filtrates of the other fungi tested.

Intent of this study was to explore the potential application of polymerin, the polymeric, dissolved organic matter fraction from olive oil wastewaters, in technologies aimed at remediating hydrophobic organic compounds (HOCs) point-source pollution. Phenanthrene binding with polymerin was investigated. Moreover, the effect of addition of micro and nanoscale aluminum oxides (Al2O3) was studied, as well as sorption of polymerin on the oxides. Phenanthrene binding capacity by polymerin was notably higher than the sorption capacities for both types of Al2O3 particles. Polymerin sorption on nanoparticles was nearly 100 times higher than microparticles. In a three-phase system, using microparticles, higher phenanthrene sorption was found by adding into water polymerin, oxides and phenanthrene simultaneously. In contrast, using nanoparticles, a considerable enhancement of phenanthrene sorption was shown by adding phenanthrene to a pre-formed and dried polymerin–oxide complex. These findings support the application of polymerin, especially associated with Al2O3 nanoparticles, in remediation of water contaminated with HOCs. This work highlights the significant role of nanoparticles.

Some saprobe fungi (Phlebia radiata, Trametes versicolor, Coriolopsis rigida, Pycnoporus cinnabarinus, Fomes sclerodermus or Pleurotus pulmonarius) were able to bioconvert the ethyl acetate fraction (DEAF) and the corresponding aqueous exhausted fraction (EAF) of dry olive mill residue (DOR), reducing their phytotoxicity on Lepidium sativum seeds. Large amount of hydroxytyrosol together with other eight monomeric phenols were found in the native DEAF fraction, which represents a good source of antioxidants. P. radiata, T. versicolor and F. sclerodermus caused an effective phytotoxicity reduction of EAF in the concentration range of 25–3 g l 1 . In particular, in the range between 12.5 and 3 g l 1 , the EAF samples inoculated with P. radiata and F. sclerodermus surprisingly stimulated the germinability of L. sativum, suggesting their use as a potential biofertilizer. This is the first report which showed the bioconversion of the above fractions in shorter time with respect to the previous findings concerning DOR. The possible implications of laccase in the decrease of DEAF and EAF phytotoxicity was also discussed. � 2006 Elsevier Ltd. All rights reserved.

Dry olive mill residue (DOR) from the olive oil production by two phase centrifugation system was fractionated by a consecutive continuous solid–liquid extraction obtaining the EAF, PF, MF and WF fractions with ethyl acetate, n-propanol, methanol and water, respectively. The chemical, chromatographic and mass spectrometric analyses showed EAF, PF and MF to be mainly composed of simple phenols, phenolic acids, flavonoids and glycosilated phenols (glycosides of phenols, secoiridoids and flavonoids), whereas WF was mainly consisting of polymerin, the metal organic polymeric mixture previously identified in olive oil mill waste waters and composed of carbohydrates, melanin, proteins and metals (K, Na, Ca, Mg and Fe). The identification in DOR of oleoside, 6 0 -b-glucopyranosyloleoside and 6 0 -b-rhamnopyranosyl-oleoside, and of its organic polymeric component, known as polymerin, are reported for the first time in this paper. The inoculation of the previously mentioned fractions with saprobe fungi Coriolopsis rigida, Pycnoporus cynnabarinus or Trametes versicolor indicated these fungi to be able to metabolize both the phenols and glycosilated phenols, but not polymerin. In correspondence, EAF, PF, MF and WF, which proved to be toxic on Lepidium sativum, decreased their toxicity after incubation with the selected fungi, WF showing to be also able to stimulate the growth of the selected seeds. The phytotoxicity appeared mainly correlated to the monomeric phenols and, to a lesser extent, to the glycosilated phenols, whereas polymerin proved to be non toxic. However, the laccase activity was not associated with the decrease of phytotoxicity. The valorization of DOR as a producer of high added value substances of industrial and agricultural interest in native form and after their bioremediation for a final objective of the total DOR recycling is also discussed. � 2007 Elsevier Ltd. All rights reserved.

A dark and complex salified organic polymeric mixture, named lignimerin, was for the first time recovered from Kraft cellulose mill wastewater (KCMW) and characterized by chemical, spectroscopic and relative molecular weight (RMW) analysis. Lignimerin proved to be composed of polyphenols (57.00%), carbohydrates (22.26%) and proteins (7.42%). It also contained metals (6.93%), mainly Ca and, to much lower extent, Mg, Na, Al, Fe, K, Mn, Zn and Cu, bound to the carboxylate and phenate groups. The distribution of lignimerin RMW was assessed to be approximately between 1000 and 8600 Da, as well as to consist of lignin and tannin, protein and polysaccharide moieties, strongly aggregated each other. H-lignimerin, its acid derivative, revealed a chemical composition and a RMW distribution very close to that of lignimerin, but a marked metal cations decreasing (1.60%) with respect to lignimerin (6.93%). The humic acid-like nature of both the polymers was assessed. Their potential use as bio-adsorbents of heavy metals is briefly discussed.

A hydroxytyrosol triacetyl derivative was very efficiently produced as a highly pure stabilized antioxidant compound by a short treatment of olive mill waste water (OMWW) organic extracts, rich in hydroxytyrosol, with an acetylating mixture composed of HClO4-SiO2 and Ac2O (Chakborti and Gulhane reaction), in mild and safe conditions. A successive single step of middle pressure liquid chromatography (MPLC) purification of the reaction product was performed, with an overall yield of 35.6%. (This process, including both the Chakborti and Gulhane reaction and the MPLC purification, is protected by an international patent under PCT/IT2005/000781.) The o-diphenol triacetyl derivative was also produced by direct reaction of hydroxytyrosol, previously purified by MPLC, with HClO4-SiO2 and Ac2O, with an overall yield of 29.5%. A further procedure for the production of the hydroxytyrosol triacetyl derivative was consistent with the direct treatment of raw OMWW with the acetylating agent and a single step of MPLC purification, with an overall yield of 27.6%. The purified natural triacetylhydroxytyrosol confirmed the same strong protective effects against the oxidative stress in human cells as the corresponding synthetic compound, likely because of the biochemical activation of the acetyl derivative into the active parent hydroxytyrosol by esterases. We therefore propose the utilization of OMWW for recovering hydroxytyrosol as a natural antioxidant in a chemically stabilized form, with a good yield, which can be potentially used as a nontoxic functional component in nutritional, pharmaceutical, and cosmetic preparations.

Biomolecules released by plants and microorganisms have an important effect on the mobility of trace elements in soil environments. We have found that in the presence of low-molecular-mass organic ligands (e.g. oxalate (OX) and citrate), the adsorption of heavy metals on short-range-ordered oxides increased with an increase in the initial organic ligand/Me molar ratio (rL) up to 10, whereas on well-crystallized Al- or Fe-oxides, the adsorption initially increased up to 2.5 (goethite) or 5 (bayerite) and then decreased. The rL value, the nature of the organic ligands and heavy metals, and the surface properties of the sorbents are critical in determining whether metal adsorption is enhanced or inhibited. Organic ligands inhibit the adsorption of trace elements in anionic form (arsenate (As), arsenite, and selenite). The adsorption of heavy metals and metalloids on variable-charge minerals is influenced not only by the presence, nature, and concentration of organic ligands but also by the sequence of addition of trace elements on the sorbents. Trace elements in cationic and anionic form show a different adsorption capacity onto metal oxides, organic matter, and organomineral complexes. Heavy metals also compete for sorption sites on these soil components. However, we found that Cu inhibits the sorption of Zn more on ferrihydrite than on an organomineral complex or a humic acid-like material.

A study on the individual sorption of Cr(III), Cu, and Zn on polymerin, the humic-acid-like fraction of olive mill wastewater, and its derivatives, K-polymerin and an Fe(OH)x-polymerin complex, showed that these heavy metals were strongly sorbed on polymerin and K-polymerin in the order Cr(III)CuZn. The sorption on Fe(OH)x-polymerin was to a lower extent compared with that of the other two sorbents, but to a higher extent compared with ferrihydride [Fe(OH)x]. Combined atomic absorption spectrometry and diffuse reflectance infrared Fourier transform spectroscopy analyses showed that the selected heavy metals were individually sorbed on polymerin by means of a cation exchange mechanism, which was consistent with the replacement of Ca, Mg, K, and H bound to the carboxylate groups of the biosorbent and the concomitant chelation of the heavy metals by the OH groups of polymerin polysaccharide component. In binary combination and equimolar ratio, Cu was sorbed by polymerin more selectively than Zn. In ternary combination and equimolar ratio, Cr(III), Cu, and Zn were sorbed by polymerin in the order Cr(III)CuZn. The sorbing capacity of Zn and Cu was strongly influenced by Cr(III), whereas the sorbing capacity of Cr(III) was not affected bythe presence of the other two metals. The overall sorbing capacity of the binary and ternary mixtures of the three metals on polymerin proved to be considerable and much greater than that on Fe(OH)x-polymerin. Simulated wastewaters contaminated with Cu and Zn were purified after three sorption cycles by polymerin renewed at each cycle, whereas those containing a mixture of Cr(III), Cu, and Zn showed residues of Zn after five cycles. We briefly discuss environmental and industrial advantages for a possible exploitation of polymerin.

A dark and complex metal polymeric organic mixture, named polymerin, was recovered from olive oil mill wastewaters (OMWW) and characterized by chemical analysis, diffuse reflectance infrared Fourier transform spectroscopy (DRIFTS), and atomic absorption spectrometry (AAS). Polymerin proved to be composed of carbohydrates (52.40 mg 100(-1), w/w), melanin (26.14 mg 100(-1)), and proteins (10.40 mg 100(-1)), and the respective composition of monosaccharides, phenols, and amino acids was determined. It also contained metals (11.06 mg 100(-1)), mainly K(+) and, to a lesser extent, Na(+), Ca(2+), Mg(2+), Zn(2+), Fe(3+), and Cu(2+), which were naturally bound and chelated through carboxylate anions and other characteristic nucleophilic functional groups naturally occurring in polymerin. The distribution of polymerin relative molecular size was assessed to be approximately between 500.0 and 2.0 kDa by calibrated molecular weight gel filtration chromatography, indicating also that a fraction consisted of protein, melanin, and polysaccharide, strongly aggregated to each other in a supramolecular status by a combination of covalent and hydrogen bonds and CH/Pi interactions, and another fraction of only free polysaccharide. Polymerin was transformed into a potassium salt deglycosylated derivative, named KSDpolymerin, which was also characterized by chemical analysis, DRIFTS, and AAS. KSDpolymerin consisted of carbohydrates (6.00 mg 100(-1)), melanin (52.49 mg 100(-1)), and proteins (35.40 mg 100(-1)), and the composition of monosaccharides, phenols, and amino acids was determined. It also contained metals (6.11 mg 100(-1)), mainly K(+) and to a lesser extent Na(+), Ca(2+), Mg(2+), Zn(2+) and Fe(3+), bound as in polymerin. All the organic components were strongly linked in a supramolecular aggregate status and the relative average molecular size proved to be 6.3 kDa. Finally, we briefly discuss the possible use of such polymerins in agriculture as bioamendments and macro- and microelement biointegrators and as a biofilter for toxic metal removal, in light of their similarity with humic acids.

The dark polymeric organic fraction rich in potassium recovered from olive oil mill waste waters (OMWW) and named polymerin and the potassium salified deglycosylated polymerin derivative (K-SDpolymerin) were easily transformed into their metal derivatives by saturation with various metals, including Na, Cu, Zn, Mn, Fe, and Al. Saturated metal polymerins were characterized by diffuse reflectance infrared Fourier transform spectroscopy and atomic absorption spectrometry. Tests on tomato plants of the various polymerins showed that only the soluble polymerin, K-SDpolymerin, and the insoluble Mn-SDpolymerin were significantly toxic. The toxic effects of OMWW on tomato at the original concentration and diluted 1:10 were much stronger than those of any polymerin. The possible exploitation of polymerins as bioamendments and/or metal biointegrators as a functon of their phytotoxic effects, their humic acid-like nature, and their richness in macro- and micronutrient metals is also discussed.

The primary structure of syringicin (syr), a new acidic α-elicitin, isolated from culture filtrates of Phytophthora syringae, causal agent of citrus fruit rot, has been determined using a combined approach based on Edman degradation and MALDI-MS (TTCTT TQQTA AYVAL VSILS DSSFN QCATD SGYSM LTATA LPTTA QYKLM CASTA CKTMI TKIVS LNAPD CELTV PTSGL VLNVY SYANG FSSTC ASL). Syr has 98 amino acids with a Mr of 10194.6±0.2, which was determined by electrospray ionisation-mass spectrometry (ES-MS) and in agreement with three disulphide bridges, located between Cys3-Cys71, Cys27-Cys56 and Cys51-Cys95. Syr induces a hypersensitive response and electrolyte leakage in tobacco. These are characteristic elicitor properties of the group and in agreement with the molecular mechanism recently proposed for this kind of protein. Finally, its possible applications in biological agriculture and biomedicine are briefly discussed. © 2001 Elsevier Science Ltd. All rights reserved.

Elicitin 172, an acid protein with elicitor activity, has been isolated in true form from culture filtrates of Phytophthora nicotianae , the causal agent of crown and root rot of tomato ( Lycopersicon esculentum ). The M r (10,349±1) of the purified protein, determined by ES–MS, is identical to that calculated for parasiticein using the mean isotopic composition and assuming the occurrence of three disulfide bridges. The primary structure of elicitin 172, determined using also MALDI–MS experiments, shows complete identity with parasiticein, with elicitin 310 and a cloned elicitin gene from P. parasitica (= P. nicotianae ), confirming conservation of the elicitin sequence within a single species. The protein induces necrosis (hypersensitive reaction) on tobacco, but no symptoms on tomato, when applied on the leaves. Tomato pretreated with elicitin 172 was affected by P. nicotianae , as well as by the phytotoxic aggregates, naturally occurring with the elicitin in the non permeated dialysis fraction of culture filtrates. Finally, the elicitin induce protection of capsicum ( Capsicum annuum) and vegetable marrow ( Cucurbita pepo ) from P. capsici .

Hydroxytyrosol, a polyphenol with very interesting antioxidant properties, which naturally occurs in virgin olive oil and mainly in olive oil mill waste waters, was synthesized by reducing 3, 4-dihydroxyphenylacetic acid with LiAlH(4) in tetrahydrofuran under refluxing for 2 h. The yield of reaction was 82.8%. The spectroscopic and HPLC data of the synthesized compound proved to coincide fully with those of a pure sample obtained by the chromatographic recovery from olive oil mill waste waters (yield = 91 mg/L). This synthetic method appears to be the most convenient compared with those reported in the literature and is more convenient than the chromatographic recovery. The tri- and diacetyl derivatives of the synthetic compound were also prepared for structure-bioactivity relationship studies. A brief discussion is given on the economical and ecological aspects regarding the production of hydroxytyrosol.

Pure-grade oleuropein, a bitter, hypotensive, phenolic glucoside, was obtained from organic extracts of olive plant leaves by two chromatographic steps. The purified compound was characterized by spectroscopic NMR and FAB-MS methods. The glucoside underwent chemical and enzymatic hydrolysis. Aglycone was characterized by spectroscopic methods (H-1-NMR and FAB-MS). Glucose was measured by enzymatic methods. The enzymatic hydrolysis of oleuropein was carried out by a soluble beta-glucosidase. The reaction was characterized in terms of kinetic parameters, optimal pH value, activation energy, inhibition constant by glucose, and thermal stability. Preliminary experiments were also performed in a continuous-flow ultrafiltration membrane reactor. The cut-off of the membrane was lower than the molecular-weight of the enzyme, thus determining beta-glucosidase confinement within the reactor. Under these conditions, beta-glucosidase had a good long-term stability. This is an encouraging results in view of possible industrial applications.

p ]Among the main polyphenols isolated from waste waters of olive (Olea europea) mill, catechol, 4-methylcatechol and hydroxytyrosol were toxic for Pseudomonas syringae subsp. savastanoi, the causai agent of olive knot disease. In particular the most toxic was 4-methylcatechol, Among some synthetic derivatives of catechol, as o-quinone, guaiachol, veratrole and diacetylcatechol, which were tested on P. savastanoi and on Corynebacterium michiganense (Gram+), o-quinone was strongly toxic for both bacteria, whereas veratroie and diacetylcatechol were toxic only for P. savastanoi. From fruits and leaves of olive was isolated oieuropein, a well known phenolic glucoside, from which was prepared its agtycone, by using a commercial β-giucosidase and hydroxytyrosol by chemical hydroiysis. The oieuropein was significantly toxic on P. savastanoi at a concentration of 10-3 and 10-4 M, while its aglycone was toxic in the range between 10-2 and 10-4 M and hydroxytyrosot at 10-2 and 10-3 M.

A further cytochalasin was isolated from liquid culture filtrates of Phoma exigua var. heteromorpha together with cytochalasins U and V and other well-known cytochalasins. The metabolite, named cytochalasin W, was characterized by spectroscopic and chemical methods as a new 24-oxa[14]cytochalasan, bearing a formyl group on the macrocyclic ring. Cytochalasin W showed toxic activity in the brine shrimp assay.

Olive oil vegetation waters (VW) were highly toxic to both phytopathogenic Pseudomonas syringae (Smith, Yung et al.) pv. savastanoi (Gram-negative) and Corynebacterium michiganense (Gram-positive) and showed bactericidal activity in their original concentration (in raw form). Among the main polyphenols, present in the waste waters, methylcatechol proved to be the most toxic to Ps. savastanoi at 10(-4) mol l-1, and also demonstrated bactericidal activity, while on Coryne. michiganense it was only slightly active; catechol and hydroxytyrosol were less active on Ps. savastanoi, but inactive on Coryne. michiganense; tyrosol and its synthetic isomers 1,2- and 1,3-tyrosol were completely inactive on both bacteria. Among the derivatives of VW polyphenols considered, acetylcatechol and guaiacol were selectively toxic for Ps. savastanoi, while o-quinone was strongly toxic for both bacteria. The minor carboxylic polyphenols of VW at 10(-4) mol l-1 were all inactive on the bacteria. VW, catechol, 4-methylcatechol and the less abundant carboxylic polyphenols proved to be toxic on Hep2 human cells. Finally the possibility of using the active polyphenols in agriculture in an integrated pest management program for the protection of the olive plant is discussed.

Phoma putaminum, the causal agent of leaf necrosis of Erigeron annuus, a common weed of field and pasture, produced toxic metabolites when grown in liquid culture. The main phytotoxin, named putaminoxin, was isolated and characterized using spectroscopic and chemical methods as (5S)5-hydroxy-9-propyl-6-nonen-9-olide, a new 10-macrolide. When assayed on leaves of host and non-host plants, putaminoxin showed a wide range of toxicity, with leaves of E. annuus being most sensitive.

A comparative study was performed on the sorption capacity of the phenoxy acid herbicide (4-chloro-2-methylphenoxyacetic acid), also known as MCPA, on a single-walled carbon nanotube (SWCNT), three multi-walled carbon nanotubes with average outer diameters of 15, 30 and 50 nm (MWCNT15, MWCNT30 and MWCNT50), and nanoscale metal oxides (Al2O3, TiO2 and ZnO). The most efficient sorbent was the SWCNT, followed by the three MWCNTs. The metal oxide nanoparticles showed a much lower sorption capacity. The herbicide bound to the tested carbon nanotubes, by a combination of electron donor–acceptor (π–π EDA) interactions and hydrogen bonds. On the contrary, MCPA bound to the considered metal oxide nanoparticles by electrostatic interactions occurring between the carboxylate anions of the same herbicide and OH2+ of the sorbent. Experiments of cyclic sorption showed the pesticides to be totally removed by only two sorption cycles on the SWCNT and MWCNT15, whereas the same waste water was purified after four cycles on nano Al2O3. Finally, desorption studies carried out in ethanol showed a potential re-use of the SWCNT.

A patented process to produce and purify large amounts of cytochalasin B is described. The fungal microorganism, Phoma exigua var. heteromorpha, is grown on a solid medium, constituted by autoclaved wheat kernels. The fungal culture, obtained growing the fungus in optimized environmental conditions, is extracted by organic solvent, to obtain crystalline cytochalasin B, in more than 3 g/kg of dried wheat culture. The advantages of this process are: production higher than that obtained with other known processes; lower production expenses; lower quantities of solvents; exclusion of chromatographic purifications. Moreover, the production of large amounts of cytochalasin B allow to prepare a wide number of derivatives, with different biological activities.

Ascochyta pinodes , the causal agent of pea anthracnose, cultured on sterilized wheat, produces toxic metabolites. The main phytotoxin, named pinolidoxin, was isolated and characterized using spectral and chemical methods as 2-(2,4-hexadienoyloxy)-7,8-dihydroxy-9-propyl-5-nonen-9-olide, a new phytotoxic 10-macrolide. When assayed on host (pea) and non-host (bean) plants, pinolidoxin was highly toxic; towards brine shrimps it was only weakly toxic.

It has been discovered that 1,2,3-benzenetricarboxylic acid and some 5-substituted derivatives are specific and competitive inhibitors of the mitochondrial tricarboxylate carrier. In order to characterize these acids a study was carried out using electron ionization (EI), chemical ionization (CI) and fast atom bombardment (FAB) mass spectrometry on the free acids, the potassium salt of 1,2,3-benzenetricarboxylic acid and the corresponding methyl esters. Unimolecular decomposition processes were also studied using B/E linked scan experiments in order to correlate peaks present in the FAB mass spectra of the above compounds. The FAB ionization method, as expected, was the most suitable for analysing the very polar and thermolabile acids and the only method which could be used for the analysis of the potassium salt. EI and CI turned out to be appropriate methods for analysing the less polar and thermostable trimethyl esters.

A thin layer chromatographic (TLC) method has been developed to detect the major polyphenols (the main pollutants) in olive oil vegetation waters. The method involves reversed phase TLC (C18-TLC) and silica gel high performance TLC (Si-HPTLC) on the organic extracts of oil vegetation waters (VW), using catechol, 4-methylcatechol, tyrosol, hydroxytyrosol and oleuropein as standards. Using Si-HPTLC analysis it is possible to detect only tyrosol and hydroxytyrosol, whilst with C18-TLC analysis all of the main polyphenols present except oleuropein can be detected. Confirmation of the identities of the polyphenolic compounds was obtained by C18-TLC and Si-HPTLC analysis of the acetylated organic extracts of VW together with the more stable acetyl derivatives of the phenols as standards.

The possible presence of toxic metabolites in the culture extracts of 24Ascochyta strains, grown on autoclaved wheat, was ascertained by the use of the biological assay on brine shrimps (Artemia salina). Only in the culture extract ofA lathyri, that showed a very high toxicity, the presence of cytochalasins A and B was revealed by tlc,(1)H nmr and fab-mass spectra.SinceA heteromorpha, previously described as the firstAscochyta species to produce cytochalasins, has been reclassified asPhoma exigua varheteromorpha, this is therefore the first report on the cytochalasin production by a trueAscochyta species.

Three further cytochalasins from Phoma exigua var. heteromorpha , grown on wheat, were isolated and characterized by spectroscopic methods and by chemical correlation with cytochalasin B. They were identified as cytochalasin T, a new 24-oxa[14]cytochalasan, cytochalasin F and 7- O -acetylcytochalasin B, both isolated for the first time from this fungus. Cytochalasin F showed significant activity in the brine shrimp assay and on tomato seedling growth.