**2. Olive oil microbiology**

In applied microbiology, specific microorganisms employed to remove environmental pollutants or industrial productions have often been isolated from specific sites. For example, when attempting to isolate an organism that can degrade or detoxify a specific target compound like OMW, sites may be sampled that are known to be contaminated by

Overall, these findings show that yeasts are able to contribute in a positive or negative way to the organoleptic characteristics of the olive oil. Necessary microbiological research carried out so far on olive oil is still needed. From the available scientific data up to now, it is not possible to establish that other species of microorganisms are useful and harmful in stabilizing the oil quality. In particular, it is not known if the yeasts in the freshly produced olive oil can modify some parameters responsible for the quality of virgin olive oil. Further microbiological studies on olive oil proffer to isolation of new microorganisms with biotechnological potential. The OMWs due to their particular characteristics, in addition to fat and triglycerides, sugars, phosphate, polyphenols, polyalcohols, pectins and metals, could provide microorganisms with biotechnological potential and low-cost fermentation substrates. For example, the exopolysaccharideproducing bacterium *Paenibacillus jamilae* (Aguilera et al. 2001) and the obligate alkaliphilic *Alkalibacterium olivoapovliticus* (Ntougias

According to the last report of Food and Agriculture Organisation of the United Nations (FAOSTAT 2009), 2.9 million tons of olive oil are produced annually worldwide, 75.2% of which are produced in Europe, with Spain (41.2%), Italy (20.1%) and Greece (11.4%) being the highest olive oil producers. Other olive oil producers are Asia (12.4%), Africa (11.2%), America (1.0%) and Oceania (0.2%). Olive oil production is a very important economic activity, particularly for Spain, Italy and Greece; worldwide, there has been an increase in

Multiple methods are used in the production of olive oil, resulting in different waste products. The environmental impact of olive oil production is considerable, due to the large amounts of wastewater (OMWW) mainly from the three-phase systems and solid waste. The three-phase system, introduced in the 1970s to improve extraction yield, produces three streams: pure olive oil, OMWW and a solid cake-like by-product called olive cake or *orujo*. The olive cake, which is composed of a mixture of olive pulp and olive stones, is transferred to central seed oil extraction plants where the residual olive oil can be extracted. The twophase centrifugation system was introduced in the 1990s in Spain as an ecological approach for olive oil production since it drastically reduces the water consumption during the process. This system generates olive oil plus a semi-solid waste, known as the two-phase olive-mill waste (TPOMW) or *alpeorujo* (Alburquerque et al. 2004; McNamara et al. 2008;

The olive oil industry is characterized by its great environmental impact due to the production of a highly polluted wastewater and/or a solid residue, olive skin and stone (olive husk), depending on the olive oil extraction process (Table 1) (Azbar et al. 2004).

Pressure and three-phase centrifugation systems produce substantially more OMWW than two-phase centrifugation, which significantly reduces liquid waste yet produces large amounts of semi-solid or slurry waste commonly referred to as TPOMW. The resulting solid waste is about 800 kg per ton of processed olives. This ''alpeorujo'' still contains 2.5–3.5% residual oil and about 60% water in the two-phase decanter system (Giannoutsou et al.

and Russell 2001) were isolated from olive mill wastes.

**3. Olive mill waste as renewable low-cost substrates** 

production of about 30% in the last 10 years (FAOSTAT 2009).

Morillo et al. 2009).

2004).

this material. These environments provide suitable conditions to metabolize this compound by microorganisms.

Recent microbiological research has demonstrated the presence of a rich microflora in the suspended fraction of freshly produced olive oil. The microorganisms found in the oil derive from the olives' carposphere which, during the crushing of the olives, migrate into the oil together with the solid particles of the fruit and micro-drops of vegetation water. Having made their way to the new habitat, some microbic forms succumb in a brief period of time whereas others, depending on the chemical composition of the oil, reproduce in a selective way and the typical microflora of each oil (Zullo et al. 2010).

Newly produced olive oil contains numerous solid particles and micro-drops of olive vegetation water containing, trapped within, a high number of microorganisms that remain during the entire period of olive oil preservation. The microbiological analyses highlighted the presence of yeasts, but not of bacteria and moulds (Ciafardini and Zullo 2002). Several isolated genus of yeasts were identified as *Saccharomyces*, *Candida* and *Williopsis* (Ciafardini et al. 2006).

Some types of newly produced oil are very bitter since they are rich in the bitter-tasting secoiridoid compound known as oleuropein, whereas after a few months preservation, the bitter taste completely disappears following the hydrolysis of the oleuropein. In fact, the taste and the antioxidant capacity of the oil can be improved by the β-glucosidaseproducing yeasts, capable of hydrolysing the oleuropein into simpler and less bitter compounds characterized by a high antioxidant activity. Oleuropein present in olive oil can be hydrolysed by β-glucosidase from the yeasts *Saccharomyces cerevisiae* and *Candida wickerhamii.* The absence of lipases in the isolated *S. cerevisiae* and *C. wickerhamii* examined that the yeasts contribute in a positive way to the improvement of the organoleptic characteristics of the oil without altering the composition of the triglycerides (Ciafardini and Zullo 2002).

On the other hand, the presence of some lipase-producing yeast can worsen oil quality through triglycerides hydrolysis. Two lipase-producing yeast strains *Saccharomyces cerevisiae* 1525 and *Williopsis californica* 1639 were found to be able to hydrolyse olive oil triglycerides. The lipase activity in *S. cerevisiae* 1525 was confined to the whole cells as cell-bound lipase, whereas in *W. californica* 1639, it was detected as extracellular lipase. Furthermore, the free fatty acids of olive oil proved to be good inducers of lipase activity in both yeasts. The microbiological analysis carried out on commercial extra virgin olive oil demonstrated that the presence of lipase-producing yeast varied from zero to 56% of the total yeasts detected (Ciafardini et al. 2006).

Some dimorphic species can also be found among the unwanted yeasts present in the olive oil, considered to be opportunistic pathogens to man as they have often been isolated from immunocompromised hospital patients. Recent studies demonstrate that the presence of dimorphic yeast forms in 26% of the commercial extra virgin olive oil originating from different geographical areas, where the dimorphic yeasts are represented by 3-99.5% of the total yeasts. The classified isolates belonged to the opportunistic pathogen species *Candida parapsilosis* and *Candida guilliermondii*, while among the dimorphic yeasts considered not pathogenic to man, the *Candida diddensiae* species (Koidis et al. 2008; Zullo and Ciafardini 2008; Zullo et al. 2010).

this material. These environments provide suitable conditions to metabolize this compound

Recent microbiological research has demonstrated the presence of a rich microflora in the suspended fraction of freshly produced olive oil. The microorganisms found in the oil derive from the olives' carposphere which, during the crushing of the olives, migrate into the oil together with the solid particles of the fruit and micro-drops of vegetation water. Having made their way to the new habitat, some microbic forms succumb in a brief period of time whereas others, depending on the chemical composition of the oil, reproduce in a

Newly produced olive oil contains numerous solid particles and micro-drops of olive vegetation water containing, trapped within, a high number of microorganisms that remain during the entire period of olive oil preservation. The microbiological analyses highlighted the presence of yeasts, but not of bacteria and moulds (Ciafardini and Zullo 2002). Several isolated genus of yeasts were identified as *Saccharomyces*, *Candida* and *Williopsis* (Ciafardini

Some types of newly produced oil are very bitter since they are rich in the bitter-tasting secoiridoid compound known as oleuropein, whereas after a few months preservation, the bitter taste completely disappears following the hydrolysis of the oleuropein. In fact, the taste and the antioxidant capacity of the oil can be improved by the β-glucosidaseproducing yeasts, capable of hydrolysing the oleuropein into simpler and less bitter compounds characterized by a high antioxidant activity. Oleuropein present in olive oil can be hydrolysed by β-glucosidase from the yeasts *Saccharomyces cerevisiae* and *Candida wickerhamii.* The absence of lipases in the isolated *S. cerevisiae* and *C. wickerhamii* examined that the yeasts contribute in a positive way to the improvement of the organoleptic characteristics of the oil without altering the composition of the triglycerides (Ciafardini and

On the other hand, the presence of some lipase-producing yeast can worsen oil quality through triglycerides hydrolysis. Two lipase-producing yeast strains *Saccharomyces cerevisiae* 1525 and *Williopsis californica* 1639 were found to be able to hydrolyse olive oil triglycerides. The lipase activity in *S. cerevisiae* 1525 was confined to the whole cells as cell-bound lipase, whereas in *W. californica* 1639, it was detected as extracellular lipase. Furthermore, the free fatty acids of olive oil proved to be good inducers of lipase activity in both yeasts. The microbiological analysis carried out on commercial extra virgin olive oil demonstrated that the presence of lipase-producing yeast varied from zero to 56% of the total yeasts detected

Some dimorphic species can also be found among the unwanted yeasts present in the olive oil, considered to be opportunistic pathogens to man as they have often been isolated from immunocompromised hospital patients. Recent studies demonstrate that the presence of dimorphic yeast forms in 26% of the commercial extra virgin olive oil originating from different geographical areas, where the dimorphic yeasts are represented by 3-99.5% of the total yeasts. The classified isolates belonged to the opportunistic pathogen species *Candida parapsilosis* and *Candida guilliermondii*, while among the dimorphic yeasts considered not pathogenic to man, the *Candida diddensiae* species (Koidis et al. 2008; Zullo and Ciafardini

selective way and the typical microflora of each oil (Zullo et al. 2010).

by microorganisms.

et al. 2006).

Zullo 2002).

(Ciafardini et al. 2006).

2008; Zullo et al. 2010).

Overall, these findings show that yeasts are able to contribute in a positive or negative way to the organoleptic characteristics of the olive oil. Necessary microbiological research carried out so far on olive oil is still needed. From the available scientific data up to now, it is not possible to establish that other species of microorganisms are useful and harmful in stabilizing the oil quality. In particular, it is not known if the yeasts in the freshly produced olive oil can modify some parameters responsible for the quality of virgin olive oil. Further microbiological studies on olive oil proffer to isolation of new microorganisms with biotechnological potential. The OMWs due to their particular characteristics, in addition to fat and triglycerides, sugars, phosphate, polyphenols, polyalcohols, pectins and metals, could provide microorganisms with biotechnological potential and low-cost fermentation substrates. For example, the exopolysaccharideproducing bacterium *Paenibacillus jamilae* (Aguilera et al. 2001) and the obligate alkaliphilic *Alkalibacterium olivoapovliticus* (Ntougias and Russell 2001) were isolated from olive mill wastes.
