Evaluation of olive oil mill wastewater toxicity on the mitochondrial bioenergetics after treatment with Candida oleophila

Abstract

In a previous work the ability of Candida oleophila to use phenolic compounds as sole carbon and energy source at high concentrations without an additional carbon source was reported. C. oleophila grown in bioreactor batch cultures in a diluted and sterilized olive oil mill wastewater (OMW) caused a significant decrease in the total tannins content but no significant alteration was observed in phenolic acid and fatty acid content. Both treated and untreated OMWs were tested to evaluate the capacity in interfering with mitochondrial bioenergetics. Mitochondrial respiration was not affected by treated OMW on the range of used concentrations, contrary to the untreated OMW. Furthermore, mitochondrial membrane potential and respiratory complexes were always significantly less affected by treated OMW in comparison with untreated OMW. However, supplementary treatment should be applied before OMW could be considered non-toxic.

Introduction

The growing consumer demand for olive oil, as a consequence of its proved benefits for human health (Tuck and Hayball, 2002), has become a positive factor for higher productions of this natural fat. Olive oil extraction industries are mainly located in Mediterranean countries, producing seasonally large amounts of black olive wastewaters generally referred as olive mill wastewaters (OMWs). The amount of OMW produced is dependent on the extraction process used. In general, for each ton of olives processed, 1.3 m³ of black waters are produced (Vitolo et al., 1999). This black liquid wastewater is highly pollutant, since it presents high biological oxygen demand (BOD) values (50–100 gO₂/L) as well as chemical oxygen demand (COD) values (80–200 gO₂/L) (Khoufi et al., 2006). These are 200–400 times higher than those of a typical municipal sewage (Cossu et al., 1993). Besides its high organic loading, the presence of polyphenols and tannins, high content of suspended solids, and acidity make these waters highly recalcitrant to conventional water treatment making the management and disposal of olive oil mill effluents a serious environmental problem (Paixão et al., 1999).

The research on OMW valorization has been focused on the degradation/elimination of phenolic compounds, since their breakdown is considered as the limiting step on the biotreatment of OMW (Fountoulakis et al., 2002). Therefore many different methods have already been proposed to decompose OMW (Aggelis et al., 2003; Benitez et al., 1999, Benitez et al., 1997; Fadil et al., 2003; Gotsi et al., 2005; Pinto et al., 2002). In several of these works, microorganisms like filamentous fungi have been used to pre-treat OMW, since many are able to reduce polyphenol contents, making these waters suitable for secondary conventional treatment (Fadil et al., 2003).

Several investigations have already been carried out to evaluate OMW toxicity on microorganisms (Paixão et al., 1999). These clearly show that OMWs are highly toxicant not only to microorganisms but also to microcrustaceans as Daphnia magna or Tamnocephalus platyurus although the parameter responsible for this toxicity was not clear.

Furthermore, Sert et al. (1998) have shown that ferulic acid, a phenolic acid frequent in OMW, carries out an interference with both l-malate dehydrogenase and malic enzyme. Our previous studies have shown that mitochondrial bioenergetics is strongly disturbed by OMW (Martins et al., 2007). Interference with mitochondrial bioenergetics is known to be a part of the process of cell injury by assorted agents and by a variety of mechanisms (Wallace et al., 1997). Mitochondrial dysfunction promoted by any toxicant can lead to apoptotic cell death and to some neuronal degenerative diseases (Eckert et al., 2003). Furthermore, mitochondrion has proved itself to be a good model to study the action of many xenobiotics on cell toxicity, since data obtained from such studies are generally well correlated with cytotoxicity parameters reported in cell cultures and whole organisms (Knobeloch et al., 1990).

Filamentous fungi like Geotrichum sp., Aspergillus sp., Pleurotus sp., or Phanerochaete sp. have proved to be efficient in eliminating some organic loading and phenolic fractions of OMW (Borja et al., 1995a, Borja et al., 1995b; Gharsallah et al., 1999). Yeasts are mainly unicellular fungi, which when used in full-scale wastewater plants do not present the bulking problems associated with filament formation. Besides, in previous works (Amaral et al., 2005), yeasts isolated by our working group, belonging to genus Candida, have shown the ability to use phenolic compounds as sole carbon and energy source at high concentrations (1000 mg/L). Based on these previous results, and considering the problem of technology application at large scale, the aim of this study was to evaluate the ability of Candida oleophila to remove phenolic and other toxic compounds from a real olive mill effluent, and analyse the effect of this biodegradation on the mitochondrial bioenergetics using isolated mitochondria from rat liver.