**1. Introduction**

[39] Tchobanoglous, G. and Burton, F.L., (1997) Wastewater Engineering: Treatment, Dis‐ posal, Reuse, Metcalf and Eddy Inc., McGraw-Hill Publication, New Delhi.

[40] Thakur Ravindra Y and Patil Yogesh B (2009) Management of thiocyanate pollution using a novel low cost natural waste biomass.*South Asian Journal of Management Re‐*

[41] Van Zyl, Andries W., Harrison, Susan T.L., van Hille, Robert P. (2011) Biodegrada‐ tion of thiocyanate by a mixed microbial population. In: Mine Water – Managing the

[42] Westley, J. (1981) *Cyanide and sulfane sulfur*. In: Vennesland, B., ed., Cyanide in Biolo‐

[43] Wood, A.P., Kelly, D.P., McDonald, I.R., Jordan, S.L., Morgan, T.D., Khan, S., Murell, J.C. and Borodina, E., (1998) A novel pink-pigmented facultative methylotroph, *Methylobacterium thiocyanatum* sp. nov., capable of growth on thiocyanate as sole ni‐

[44] Wood, J.L. (1975) Biochemistry, In: Newmann, A.A., ed., Thiocyanic Acid and its De‐

[45] Zargury, G.J., Oudjehani, K. and Deschenes, L. (2004) Characterisation and availabili‐ ty of cyanide in solid mine tailings from gold extraction plants. *Science of the Total En‐*

*search* 1(2): 85-96.

50 Applied Bioremediation - Active and Passive Approaches

*vironment* 320: 211-224.

Challenges (IMWA 2011), Aachen, Germany.

gy, Academic Press, London, pp. 201-212.

trogen source. *Arch. Microbiol.*, 169, 148-158.

rivatives, Academic Press, London, United Kingdom.

The cultivation of olive trees and the production and use of olive oil has been a well-known and established practice in the Mediterranean region for more than 7000 years [1].

Olive is the most extensively cultivated fruit crop in the world, counting 9,2 million hectares of area harvested in 2009 and its cultivation area has tripled in the past 50 years [2].

Over the last decade, olive oil production has increased about 40% worldwide and Europe obtained an increase of 45% in production [3], due to its high dietetic and nutritional value (the high smoke point-210 °C- and an excellent lipid profile as the proportion of saturated, mono-unsaturated and poly-unsaturated fatty acids is 14:77:9) [1]. It is generally accepted that olive oil consumption brings benefits to human health, such as reduction of risk factors of coronary heart disease, prevention of several types of cancers, and modifications of immune and inflammatory responses [4].

Mediterranean Countries produce more than 98% of the world's olive oil, which is estimated at over 2.5 million metric tons *per* year. Three quarters of the annual production in the world comes from European Union, in particular Spain (36% of the worldwide production), Italy (24% of the world's total), and Greece (17% of the global production) [3].

These data reflect the importance of olive oil sector in the Mediterranean area and consequently the magnitude of the problems related with the disposal of large amounts of olive mill wastewaters (OMW). Many studies report that OMW is a major pollutant to surface and

© 2013 Bevilacqua et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. ground water resources in the Mediterranean basin [5]. Moreover, olive oil production is no longer restricted to the Mediterranean basin, and new producers such as Australia, USA and South America will also have to face the environmental problems posed by OMW [6].

tion/flocculation, ultrafiltration and reverse osmosis, adsorption, chemical oxidation processes and ion exchange), extraction of valuable compounds (e.g. antioxidants, residual oil, sugars), agronomic applications (e.g. land spreading), animal-breeding methods (e.g. direct utilisation as animal feed or following protein enrichment) and biological treatments [8]. Among the different options, biological treatments are considered the most environmentally compatible

Bioremediation of Olive Mill Wastewater by Yeasts – A Review of the Criteria for the Selection of Promising Strains

http://dx.doi.org/10.5772/56916

53

Two different approaches have been developed for OMW biological treatment: aerobic and anaerobic processes [16]. Some drawbacks of OMW bioremediation under anaerobic condi‐ tions are the difficulties to remove high-molecular weight phenols [16], the need for a long period for the adaptation of microorganisms, the high costs for the storage [17]; on the other

Early studies focused on the use of specific bacterial species, including *Bacillus pumilus* [18], *Arthrobacter* sp. [19], *Azotobacter vinelandii* [20], *Azotobacter chroococcum* [21], *Pseudomonas putida* and *Ralstonia* sp. [22] and various bacterial consortia [23-25]. In general, aerobic bacteria appeared to be very effective against some low-molecular-mass phenolic compounds but are relatively ineffective against the more complex polyphenolics responsible for the dark

Several strains of filamentous fungi have revealed interesting capacities for the removal of problematic OMW compounds [26]. A variety of white-rot fungi have been used including *Phanerochaete chrysosporium* [27], *Trametes versicolor* [28], *Pleurotus* spp. [29], *Funalia trogii* [30-31], *Lentinus edodes* [3]. Although Garcia et al. [32] studied the ability of *Aspergillus niger* and *Aspergillus terreus*to remove phenol compounds from OMW, the use of *Aspergillus* spp. is

According to a recent review, fungi - including white rot fungi - are more effective than bacteria for the degradation of the phenols of OMW [6]. The high efficiency of fungi relies upon the structure of the aromatic compounds present in OMW; they are analogous to those of many lignin monomers, and only a few microorganisms, mainly white rot fungi, are able to efficiently degrade lignin by producing ligninolytic enzymes such as lignin peroxidases, manganese peroxidases and laccases [6]. However, there is usually a need to employ a heat pre-treatment to facilitate establishment of introduced fungi [26, 33]. Starter cultures for bioremediation usually requires aeration, and the duration of treatment is ca. 8-24 days, depending on some process variables such as degree of dilution, aeration and supplementation [6]. In addition, only some white-rot fungi were reported as able to perform decolorization and COD reduction in OMW when the active COD is >50 g/L [34]. Finally, the application of fungi for OMW treatment on a large scale was limited by the difficulty of achieving continuous culture because

To overcome this limitation, the use of yeasts could be a promising way. In fact, among the mentioned microbiota, yeasts are the more adapted and resistant to high concentrations of phenols and low pH values of mill wastes, allowing them to dominate this environment [35]. Some genera have already been tested successfully to detoxify and/or decolourise OMW, including *Candida*, *Geotrichum*, *Pichia*, *Saccharomyces*, *Trichosporon* and *Yarrowia* (table 1). Little

and the least expensive methods [9].

colouration of OMW [3].

hand, the aerobic protocols do not show these limits.

not so common as the application of white rot fungi.

of the formation of filamentous pellets and mycelia [16].

OMW (*acque re*fl*ue* in Italy; *alpechin* in Spain; *katsigaros* in Greece; *zebar* in Arab countries) is a dark red to black-coloured, mildly acidic liquid of high conductivity, obtained from mechanical olive processing during olive oil production [7]. Only in the Mediterranean area, OMW generation varies between 10 × 106 and 30 × 106 m3 [3]. In general, the quality and quantity of OMW, and consequently the environmental impact, depends on many fac‐ tors, such as the type of olives, the type of soil, the cultivation system and the produc‐ tion process [8]. The traditional cold press method typically generates about 50% of OMW relative to the initial weight of the olives, while the continuous centrifugation process generates 80–110% of OMW due to the continuous washing of the olive paste with warm water prior to oil separation from the paste [9].

The problems connected with OMW depend on their high chemical oxygen demand (COD) (up to 100 g/L), biological oxygen demand (BOD) (13-46 g/L), low pH (4–5), and other recalcitrant organic compounds, such as water-soluble phenols (hydroxytyrosol, tyrosol, catechol, methylcatechol, caffeic acid, vanillic acid, *p*-coumaric acid, etc.) and polyphenols originating from the olives [1]; the conductivity of OMW is around 18.0 mmhos/cm, while the average value of TSS (total suspended solids) and VSS (volatile suspended solids) are respec‐ tively 40-60 g/L and 30-50 g/L, with a TOC (total organic matter) of 10-30 g/L and TN (total nitrogen) of 0.6-1.4 g/L [1]. OMW contains also other mineral elements (P2O5, K2O, Na, Mg, Fe, Cu etc.), but the amount of these compounds is greatly variable.

OMW is one of the most complex agro-industrial effluent [10]. Most of the problems associated with OMW pollution can be attributed to the phenolic fraction [11]. Monomeric phenols of OMW have been associated with the phytotoxic and antimicrobial properties of these waste‐ waters, while the dark brownish color of OMW, which is particularly recalcitrant to decolor‐ ization, has been attributed to the polymerization of tannins and low molecular weight phenolic compounds [12].

OMW are often poured into the soil (up to 50 m3 *per* hectar in Italy) or disposed of in sewage, causing soil and water pollution. In fact untreated OMW are able to change the microbial composition of the soil through their antibacterial activity and produce phytopathogenic effects due to their high toxicity [13] (i.e. 1 m3 of OMW is equivalent to 100-200 m3 of domestic sewage) [1]. Due to the high organic load of OMW, it may contribute significantly to eutro‐ phication of recipients in which fluid exchange rates are low (closed gulfs, estuaries, lakes, etc.). An additional adverse impact of OMW on the environment is the aesthetic degradation caused by its strong odour and dark coloration [14]. Furthermore, environmental regulations and enforcements have become more and more strict [15], thus there is the need of new guidelines to manage these wastes; in fact the most olive oil is produced in Countries that are deficient in water and energy [3].

For these reasons, in recent years, several disposal methods have been proposed such as thermal processes (combustion and pyrolysis), physico-chemical treatments (e.g. precipita‐ tion/flocculation, ultrafiltration and reverse osmosis, adsorption, chemical oxidation processes and ion exchange), extraction of valuable compounds (e.g. antioxidants, residual oil, sugars), agronomic applications (e.g. land spreading), animal-breeding methods (e.g. direct utilisation as animal feed or following protein enrichment) and biological treatments [8]. Among the different options, biological treatments are considered the most environmentally compatible and the least expensive methods [9].

ground water resources in the Mediterranean basin [5]. Moreover, olive oil production is no longer restricted to the Mediterranean basin, and new producers such as Australia, USA and South America will also have to face the environmental problems posed by OMW [6].

is a dark red to black-coloured, mildly acidic liquid of high conductivity, obtained from mechanical olive processing during olive oil production [7]. Only in the Mediterranean area, OMW generation varies between 10 × 106 and 30 × 106 m3 [3]. In general, the quality and quantity of OMW, and consequently the environmental impact, depends on many fac‐ tors, such as the type of olives, the type of soil, the cultivation system and the produc‐ tion process [8]. The traditional cold press method typically generates about 50% of OMW relative to the initial weight of the olives, while the continuous centrifugation process generates 80–110% of OMW due to the continuous washing of the olive paste with warm

The problems connected with OMW depend on their high chemical oxygen demand (COD) (up to 100 g/L), biological oxygen demand (BOD) (13-46 g/L), low pH (4–5), and other recalcitrant organic compounds, such as water-soluble phenols (hydroxytyrosol, tyrosol, catechol, methylcatechol, caffeic acid, vanillic acid, *p*-coumaric acid, etc.) and polyphenols originating from the olives [1]; the conductivity of OMW is around 18.0 mmhos/cm, while the average value of TSS (total suspended solids) and VSS (volatile suspended solids) are respec‐ tively 40-60 g/L and 30-50 g/L, with a TOC (total organic matter) of 10-30 g/L and TN (total nitrogen) of 0.6-1.4 g/L [1]. OMW contains also other mineral elements (P2O5, K2O, Na, Mg, Fe,

OMW is one of the most complex agro-industrial effluent [10]. Most of the problems associated with OMW pollution can be attributed to the phenolic fraction [11]. Monomeric phenols of OMW have been associated with the phytotoxic and antimicrobial properties of these waste‐ waters, while the dark brownish color of OMW, which is particularly recalcitrant to decolor‐ ization, has been attributed to the polymerization of tannins and low molecular weight

OMW are often poured into the soil (up to 50 m3 *per* hectar in Italy) or disposed of in sewage, causing soil and water pollution. In fact untreated OMW are able to change the microbial composition of the soil through their antibacterial activity and produce phytopathogenic

sewage) [1]. Due to the high organic load of OMW, it may contribute significantly to eutro‐ phication of recipients in which fluid exchange rates are low (closed gulfs, estuaries, lakes, etc.). An additional adverse impact of OMW on the environment is the aesthetic degradation caused by its strong odour and dark coloration [14]. Furthermore, environmental regulations and enforcements have become more and more strict [15], thus there is the need of new guidelines to manage these wastes; in fact the most olive oil is produced in Countries that are deficient

For these reasons, in recent years, several disposal methods have been proposed such as thermal processes (combustion and pyrolysis), physico-chemical treatments (e.g. precipita‐

of domestic

effects due to their high toxicity [13] (i.e. 1 m3 of OMW is equivalent to 100-200 m3

*ue* in Italy; *alpechin* in Spain; *katsigaros* in Greece; *zebar* in Arab countries)

OMW (*acque re*

fl

52 Applied Bioremediation - Active and Passive Approaches

phenolic compounds [12].

in water and energy [3].

water prior to oil separation from the paste [9].

Cu etc.), but the amount of these compounds is greatly variable.

Two different approaches have been developed for OMW biological treatment: aerobic and anaerobic processes [16]. Some drawbacks of OMW bioremediation under anaerobic condi‐ tions are the difficulties to remove high-molecular weight phenols [16], the need for a long period for the adaptation of microorganisms, the high costs for the storage [17]; on the other hand, the aerobic protocols do not show these limits.

Early studies focused on the use of specific bacterial species, including *Bacillus pumilus* [18], *Arthrobacter* sp. [19], *Azotobacter vinelandii* [20], *Azotobacter chroococcum* [21], *Pseudomonas putida* and *Ralstonia* sp. [22] and various bacterial consortia [23-25]. In general, aerobic bacteria appeared to be very effective against some low-molecular-mass phenolic compounds but are relatively ineffective against the more complex polyphenolics responsible for the dark colouration of OMW [3].

Several strains of filamentous fungi have revealed interesting capacities for the removal of problematic OMW compounds [26]. A variety of white-rot fungi have been used including *Phanerochaete chrysosporium* [27], *Trametes versicolor* [28], *Pleurotus* spp. [29], *Funalia trogii* [30-31], *Lentinus edodes* [3]. Although Garcia et al. [32] studied the ability of *Aspergillus niger* and *Aspergillus terreus*to remove phenol compounds from OMW, the use of *Aspergillus* spp. is not so common as the application of white rot fungi.

According to a recent review, fungi - including white rot fungi - are more effective than bacteria for the degradation of the phenols of OMW [6]. The high efficiency of fungi relies upon the structure of the aromatic compounds present in OMW; they are analogous to those of many lignin monomers, and only a few microorganisms, mainly white rot fungi, are able to efficiently degrade lignin by producing ligninolytic enzymes such as lignin peroxidases, manganese peroxidases and laccases [6]. However, there is usually a need to employ a heat pre-treatment to facilitate establishment of introduced fungi [26, 33]. Starter cultures for bioremediation usually requires aeration, and the duration of treatment is ca. 8-24 days, depending on some process variables such as degree of dilution, aeration and supplementation [6]. In addition, only some white-rot fungi were reported as able to perform decolorization and COD reduction in OMW when the active COD is >50 g/L [34]. Finally, the application of fungi for OMW treatment on a large scale was limited by the difficulty of achieving continuous culture because of the formation of filamentous pellets and mycelia [16].

To overcome this limitation, the use of yeasts could be a promising way. In fact, among the mentioned microbiota, yeasts are the more adapted and resistant to high concentrations of phenols and low pH values of mill wastes, allowing them to dominate this environment [35].

Some genera have already been tested successfully to detoxify and/or decolourise OMW, including *Candida*, *Geotrichum*, *Pichia*, *Saccharomyces*, *Trichosporon* and *Yarrowia* (table 1). Little information is now available on the indigenous yeasts present in the OMW and their possible use for performing biodegradation of the waste.

**Yeasts Method Results Reference**

Bioremediation of Olive Mill Wastewater by Yeasts – A Review of the Criteria for the Selection of Promising Strains

*Pichia guilliermondii* Culture in OMW 25.09-33.52% 34.47-53.21% [41] *Pichia* sp. Culture in OMW 40% 41.04% [41] *P. fermentans* OMW from industrial mills 26% 18% [45] *P. holstii* OMW from industrial mills 17% 15% [45] *Saccharomyces* sp. Fed-batch microcosm 38.8% - [36] *Trichosporon cutaneum* Culture in OMW > 80% >80% [50]

*Yarrowia lipolytica* Culture in OMW ≤78.2% 1.47-41.22% [51]

**Table 1.** Phenol removal and COD decrease in OMW by yeasts. A review of the literature. -, data not available.

complex process, involving different steps; figure 1 proposes a possible scheme.

The selection of yeasts intended as functional starter for the bioremediation of OMW is a quite

Namely, after strain isolation from OMW, yeasts should be characterized (step 1) and identi‐ fied (2); then, some promising isolates could be studied in relation to their functional properties (phenol removal and COD/BOD decrease). Finally, a multivariate approach could be used to choose the best strains for the final validation under laboratory and factory-scale conditions. In the following sections, there are some details on the most important assays for the selection

This is a critical step as it important to recover yeasts and many times they are not able to grow

Generally, OMW are stored under controlled conditions (for example at 25 °C) and let to ferment; for example, authors of reference [55] analyzed OMW for 90 days. Periodically, the

Culture in bioreactors with

**2. Yeast selection: A step-by step protocol**

of promising yeasts.

under laboratory conditions.

**2.1. Isolation**

**Phenol Reduction COD Reduction**

http://dx.doi.org/10.5772/56916

55

Culture in OMW 64% 88% [48]

Culture in OMW - 67-82% [52]

Culture in OMW 19.2-31.3% 21.6-52.6% [38] Culture in OMW 25.3% 23.5-51.3% [38] Culture in OMW 20% 23.1-50.9% [38] Culture in OMW 43-72% 54-79% [54] Culture in OMW 39-68% 75-80% [54]

OMW - 80% [53]


Bioremediation of Olive Mill Wastewater by Yeasts – A Review of the Criteria for the Selection of Promising Strains http://dx.doi.org/10.5772/56916 55


**Table 1.** Phenol removal and COD decrease in OMW by yeasts. A review of the literature. -, data not available.
