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FP5

NEWTECHOMW Sintesi della relazione

Project ID: ICA3-CT-2002-10033
Finanziato nell'ambito di: FP5-INCO 2
Paese: Italy

Detoxification of OMW by biotic and abiotic catalysts

The efficiency of a pure commercial laccase and a synthesized birnessite in the detoxification of OMW was evaluated. Experiments were performed on the whole OMW and on the two fractions deriving from the Soxhlet extraction procedure, used for the isolation and preparation of hydroytyrosol. This procedure produces a phenolic extract rich in lipophylic monomeric phenols and an exhausted fraction, rich of all the other OMW components including polymeric phenols, amino acids, proteins and carbohydrates. The phytotoxicity of samples before and after laccase action was assessed by germination test using L. esculentum (tomato) and L. sativum seeds. The bacterial toxicity was determined with B. megaterium. The results clearly indicate that a 48 h treatment with laccase was most efficient in reducing phenol content on crude OMW than on its phenolic extract. A corresponding decrease of phytotoxicity was observed which was much more evident when G.I./phenol index was considered. By contrast no decrease of bacterial toxicity was observed. These results seem to suggest that either some OMW components improve or enhance the activity of laccase or that some phenols, probably present in the phenolic extract at higher concentrations inhibit laccase activity and than other OMW components rather than monomeric phenols are responsible of OMW toxicity.

To deeply understand these behaviour further experiments were performed on the catalytic action of laccase on single phenolic compounds tested alone or in combination of two, three or more phenolic mixtures. According to the obtained results, the phenols were classified in three classes: consistently transformed by laccase, recalcitrant to laccase action and showing UV-Vis spectra with either changed shapes or appearance of new absorbance peaks.

Further comparative experiments performed with three different laccases: a plant laccase from R. vernicifera, and two fungal laccases from C. polyzona and from T. versicolor indicated that the three enzymes had a different kinetic behaviour towards the substrates and were differently affected by their concentrations. Therefore, the choice of the most convenient enzymatic catalyst to apply for the transformation of a such complex phenolic mixture as OMW is a very difficult task, being the responses of the different phenols, presumably present in OMW, so differentiate to the different enzymatic catalysts.

Investigations carried out with birnessite showed a completely different behaviour as compared to laccase activity. The abiotic catalyst was more efficient than laccase in the phenol transformation reactions. Treated samples were also analysed about their antibacterial activity and the obtained results showed a complete disappearance of antibacterial activity after 2 h treatment. Previous experiments indicated that the OMW phytotoxicity was only partially reduced and not completely eliminated by the treatment with laccase or birnessite although the complete removal of monomeric phenols occurred.

The indication that other OMW components are responsible of their phytotoxicity may be drawn. Phytotoxicity tests, performed with crude OMW, OMW treated with two soils differing for their chemical and physical properties for 24 h (an incubation time sufficient to eliminate completely the presence of monomeric phenols detectable to HPLC analysis and corresponding to a decrease of total phenol content of about 30-50%) and OMW-soxhlet extract and its corresponding exhausted fraction indicated a lower toxicity for the OMW after their treatment with only one of the two soils.

However, when the data were elaborated in terms of GI/phenol concentration no significant differences were evident between crude OMW and treated-OMW, containing half content of phenolics. This seems to suggest that other OMW components are responsible of their toxicity or that the used test has a low sensitivity.

The results obtained with OMW and their extracts and exhausted fractions obtained with both tomato and Lepidium sativum seeds still indicated that no a substantial difference in phytotoxicity was evidenced between crude OMW and their fractions by germination tests performed on tomato seed. In contrast, a higher sensitivity of germination tests was observed with Lepidium sativum. Indeed with these seeds a significant difference of toxicity appeared between the extract and exhausted fractions, that was not visible with tomato seeds. Furthermore, it was evident that at the same phenol concentration the exhausted fraction exhibited a lower toxicity than the extract one and the effect was evident also when the GI were normalized by the phenols contained in the two fractions.

Informazioni correlate

Contatto

Liliana GIANFREDA, (Full Professor of Agricultural Biochemistry)
Tel.: +39-081-25392179
Fax: +39-081-2539186
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