Citric acid production by Yarrowia lipolytica cultivated on olive-mill wastewater-based media

Abstract

Yarrowia lipolytica ACA-DC 50109 cultivated on olive-mill wastewater (O.M.W.)-based media, enriched with commercial-industrial glucose, presented an efficient cell growth. Parameters of growth were unaffected by the presence of O.M.Ws in the growth medium. In diluted O.M.Ws enriched with high glucose amounts (initial sugar concentration, 65 g l⁻¹), a notable quantity of total citric acid was produced (28.9 g l⁻¹). O.M.W.-based media had a noteworthy stimulating effect on the production of citric acid, since both final citric acid concentration and conversion yield of citric acid produced per unit of sugar consumed were higher when compared with the respective parameters obtained from trials without added O.M.W. Adaptation of the strain in O.M.W.-based media favoured the biosynthesis of cellular unsaturated fatty acids (principally of oleic and palmitoleic acids). Additionally, a non-negligible decrease of the phenolic compounds in the growth medium [up to 15% (wt/wt)], a slight decrease of the phyto-toxicity, and a remarkable decolourisation of the O.M.W. were observed. All these results suggest the potentiality of O.M.Ws utilisation in the fermentation process of citric acid production.

Introduction

The manufacturing process of olive oil production yields a liquid fraction, which is called olive-mill wastewater (O.M.W.). This important residue of the olive oil industry is one of the most difficult to treat wastes because of its high content in phenolic compounds (Gharsallah et al., 1999, Garcia Garcia et al., 2000, Aggelis et al., 2003, Ammar et al., 2005). Besides the presence of phenolic substances, O.M.Ws cause serious environmental problems due to their potentially high concentration in sugars, tannins, pigments and emulsified oil that result in increased C.O.D. values, and their black colour that stains indelibly the soil (Scioli and Vollaro, 1997, Tsioulpas et al., 2002, Fadil et al., 2003). To reduce pollution in O.M.Ws, chemical or biological processes have been used (Mantzavinos and Kalogerakis, 2005, Crognale et al., 2006). One principal strategy involves the use of moulds (e.g. Phanerochaete spp., Pleurotus spp., Panus tigrinus, Geotrichum spp., Lentinula edodes, Trametes versicolor or Aspergillus spp.), which have been found capable of reducing C.O.D. values and breaking down phenolic compounds of the O.M.Ws treated (Sayadi and Ellouz, 1992, Sayadi and Ellouz, 1995, Gharsallah et al., 1999, Garcia Garcia et al., 2000, Tsioulpas et al., 2002, Aggelis et al., 2003, Fenice et al., 2003, D’Annibale et al., 2004, Ayed et al., 2005, Dhouib et al., 2006). Likewise, prokaryotic microorganisms (e.g. Pseudomonas putida, Klebsiella oxytoca, Lactobacillus plantarum, Citrobacter diversus) have the capability to degrade phenolic compounds, in the presence or absence of sugars in the growth media (Seker et al., 1997, Lamia and Moktar, 2003, Ammar et al., 2005). In some cases, yeasts or yeast-like species have been used in the valorisation of O.M.Ws in order to produce single-cell protein (S.C.P.) together with other metabolites (e.g. alcohol or enzymes). In most of the cases, the yeast strains employed belonged to the genera Torulopsis (Bambalov et al., 1989), Cryptococcus (C. albidus) (Federici et al., 1988), Saccharomyces (S. norbensis, S. oleaceus, S. chevalierie, S. rouxii) (Bambalov et al., 1989, Gharsallah, 1993), Yarrowia (Y. lipolytica) (De Felice et al., 1997, Scioli and Vollaro, 1997, Lanciotti et al., 2005) and Candida (C. krusei, C. cylindricae, C. tropicalis) (Gharsallah, 1993, Fadil et al., 2003, Ettayebi et al., 2003, D’Annibale et al., 2006).

In recent investigations, Yarrowia lipolytica strain ACA-DC 50109 has been cultivated on raw glycerol utilised as the sole substrate in nitrogen-limited cultures, and produced remarkable quantities of citric acid (Papanikolaou et al., 2002). Moreover, this process has been successfully simulated with the aid of various numerical models (Papanikolaou and Aggelis, 2003). The aim of the present investigation was to valorise O.M.Ws by producing citric acid in batch cultures using the aforementioned microorganism. The study had a double orientation, to both valorise and detoxify the effluent.