**4. Microbial biotechnology applications in olive oil industry**

Microbial biotechnology applications in olive oil industry, mainly attempts to obtain addedvalue products from OMWs are summarised in Fig. 1. OMWs could be used as renewable low-cost substrate for industrial and agricultural microbial biotechnology as well as for the production of energy.

The chemical oxygen demand (COD) and biological oxygen demand (BOD) reduction of OMWs with a concomitant production of biotechnologically valuable products such as enzymes (lipases, ligninolytic enzymes), organic acids, biopolymers and biodegradable plastics, biofuels (bioethanol, biodiesel, biogas and biohydrogen), biofertilizers and amendments will be review.

#### **4.1 Olive mill wastes biological treatment**

Ironically, while olive oil itself provides health during its consumption, its by-products represent a serious environmental threat, especially in the Mediterranean, region that accounts for approximately 95% of worldwide olive oil production.

Olives (1 ton) Oil (∼200 kg)

Wash water (0.1-0.12 m3) Solid waste (∼400 kg)

Wash water (0.1-0.12 m3) Solid waste (500-600 kg) Fresh water for decanter (0.5-1.0 m3) Wastewater (800-950 kg)

Wash water (0.1-0.12 m3) Solid waste (800 kg)

Wastewater (∼600 kg)

Wastewater (250 kg)

Production process Inputs Outputs

Three-phase process Olives (1 ton) Oil (200 kg)

Two-phase process Olives (1 ton) Oil (200 kg)

**4. Microbial biotechnology applications in olive oil industry** 

accounts for approximately 95% of worldwide olive oil production.

Water to polish the impure oil (10 kg) - Energy (90-117 kWh) -

Energy (90-117 kWh) - Table 1. Inputs and outputs from olive oil industry (Adapted from Azbar et al. 2004)

The average amount of OMWs produced during the milling process is approximately 1000 kg per ton of olives (Azbar et al. 2004). 19.3 million tons of olive are produced annually worldwide, 15% of them used to produce olive oil (FAOSTAT 2009). As an example of the scale of the environmental impact of OMWW, it should be noted that 10 million m3 per year of liquid effluent from three-phase systems corresponds to an equivalent load of the wastewater generated from about 20 million people. Furthermore, the fact that most olive oil is produced in countries that are deficient in water and energy resources makes the need for effective treatment and reuse of OMWW (McNamara et al. 2008). Overall, about 30 million tons of OMWs per year are produced in the world that could be used as renewable

Microbial biotechnology applications in olive oil industry, mainly attempts to obtain addedvalue products from OMWs are summarised in Fig. 1. OMWs could be used as renewable low-cost substrate for industrial and agricultural microbial biotechnology as well as for the

The chemical oxygen demand (COD) and biological oxygen demand (BOD) reduction of OMWs with a concomitant production of biotechnologically valuable products such as enzymes (lipases, ligninolytic enzymes), organic acids, biopolymers and biodegradable plastics, biofuels (bioethanol, biodiesel, biogas and biohydrogen), biofertilizers and

Ironically, while olive oil itself provides health during its consumption, its by-products represent a serious environmental threat, especially in the Mediterranean, region that

Energy (40-63 kWh) -

Traditional process

negative or low-cost substrates.

production of energy.

amendments will be review.

**4.1 Olive mill wastes biological treatment** 

(pressing)

Fig. 1. Potential uses of olive mill wastes in microbial biotechnology.

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 OMWs. The management of wastes from olive oil extraction is an industrial activity submitted to three main problems: the generation of waste is seasonal, the amount of waste is enormous and there are various types of olive oil waste (Giannoutsou et al. 2004).

OMWs have the following properties: dark brown to black colour, acidic smell, a high organic load and high C/N ratio (chemical oxygen demand or COD) values up to 200 g per litre, a chemical oxygen demand/biological oxygen demand (COD/BOD5) ratio ranging from 2.5 to 5.0, indicating low biodegrability, an acidic pH of between 4 and 6, high concentration of phenolic substances 0.5–25 g per litre with more than 30 different phenolic compounds and high content of solid matter. The organic fraction contains large amounts of proteins (6.7–7.2%), lipids (3.76–18%) and polysaccharides (9.6–19.3%), and also phytotoxic components that inhibit microbial growth as well as the germination and vegetative growth of plants (Roig et al. 2006; McNamara et al. 2008).

*Bacillus pumilus* OMWW Culture in OMWW 50% phenol reduction (Ramos-Cormenzana

*Candida holstii* OMWW Culture in OMWW 39% phenol reduction (Ben Sassi et al. 2008)

Method Results Reference

47% colour removal 22.7%

57.7% phenol reduction (Giannoutsou

Tannins content reduction (Peixoto et al. 2008)

60% COD reduction (Asses et al. 2009)

57% phenol reduction (Giannoutsou

61% phenol reduction (Giannoutsou

et al. 1996)

et al. 2004)

2004)

(Aouidi et al. 2009)

(Kachouri and Hamdi

(Di Gioia et al. 2001)

(Gonçalves et al. 2009)

(Gonçalves et al. 2009)

(Fadil et al. 2003)

et al. 2004)

et al. 2004)

(Chtourou et al. 2004)

(Lanciotti et al. 2005)

(García García et al. 2000)

(García García et al. 2000)

(Sampedro et al. 2007a)

(Fadil et al. 2003)

(Robles et al. 2000)

OMWW Culture in OMWW 70% COD reduction (Piperidou et al. 2000)

phenol reduction

polyphenols content

compounds and COD

compounds and COD

51.7% phenol reduction

< 30% phenol reduction

polyphenols

OMWW Culture in OMWW 67-82% COD reduction (Wu et al. 2009)

phenol reduction

phenol reduction

phenol reduction

reduction

44.3% phenol reduction

Biodegradation of aromatic compounds

Microorganism Waste

Bacteria *Azotobacter vinelandii* 

*Lactobacillus paracasei* 

*Lactobacillus plantarum* 

Yeasts

*Geotrichum candidum*

*Geotrichum candidum*

*Trichosporon cutaneum* 

*Yarrowia lipolytica* W29

Molds

*Fusarium oxysporum* 

*Pseudomonas putida*  and *Ralstonia* spp.

*Candida boidinii* TPOMW Fed-batch

*Saccharomyces* spp. TPOMW Fed-batch

*Candida oleophila* OMWW Bioreactor batch

type

OMWW Culture in OMWW

OMWW Culture two strains in OMWW

microcosm

*Candida cylindracea* OMWW Culture in OMWW reduction of phenolic

culture with OMWW

*Candida rugosa* OMWW Culture in OMWW reduction of phenolic

*Candida tropicalis* OMWW Culture in OMWW 62.8% COD reduction

bioreactors with OMWW

microcosm

microcosm

*Yarrowia lipolytica* OMWW Culture in OMWW 20-40% COD reduction

*Aspergillus niger* OMWW Culture in OMWW 73% COD reduction 76%

*Aspergillus terreus* OMWW Culture in OMWW 63% COD reduction 64%

*Penicillium* spp. OMWW Culture in OMWW 38% COD reduction 45%

*Aspergillus* spp. OMWW Culture in OMWW 52.5% COD reduction

OMWW Culture in OMWW removal of mono- and

DOR Culture in DOR 16-71% phytotoxicity

OMWW Culture in

TPOMW Fed-batch

with cheese whey's

OMWW Culture in OMWW Increase of simple

OMWs treatment processes tested employ physical, chemical, biological and combined technologies. Several disposal methods have been proposed to treat OMWs, such as traditional decantation, thermal processes (combustion and pyrolysis), agronomic applications (e.g. land spreading), animal-breeding methods (e.g. direct utilisation as animal feed or following protein enrichment), physico-chemical treatments (e.g. precipitation/flocculation, ultrafiltration and reverse osmosis, adsorption, chemical oxidation processes and ion exchange), extraction of valuable compounds (e.g. antioxidants, residual oil, sugars), and biological treatments (Morillo et al. 2009).

Among the different options, biological treatments or bioremediation are considered the most environmentally compatible and the least expensive (Mantzavinos and Kalogerakis 2005). Bioremediation is a treatment process employing naturally microorganisms (bacteria and fungi like yeasts, molds and mushrooms) to break down, or degrade, hazardous substances into less toxic or non-toxic substances. Bioremediation technologies can be classified as *in-situ* (bioaugmentation, bioventing, biosparging) or *ex-situ* (bioreactors, landfarming, composting and biopiles). *In-situ* bioremediation treats the contaminated water or soil where it was found, whereas *ex-situ* bioremediation processes involve removal of the contaminated soil or water to another location prior to treatment (Arvanitoyannis et al. 2008).

Bioremediation occurs either under aerobic or anaerobic conditions. Many aerobic biological processes, technologies and microorganisms have been tested for the treatment of OMWs, aimed at reducing organic load, dark colour and toxicity of the effluents (Table 2). 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 OMWs (McNamara et al. 2008). A number of different species of bacteria, yeasts, molds and mushrooms have been tested in aerobic processes to treat OMWs that are listed (Table 2).

A number of studies have utilized bacterial consortia for bioremediation of OMWW. Bioremediation of OMWW using aerobic consortia has been quite successful in these studies, achieving significant reductions in COD (up to 80%) and the concentration of phytotoxic compounds, and complete removal of some simple phenolics (Zouari and Ellouz 1996; Benitez et al. 1997). A combined bacterial–yeast system of *Pseudomonas putida* and *Yarrowia lipolytica* were used to degrade OMWW (De Felice et al. 1997).

Anaerobic bioremediation of OMWs has employed, almost exclusively, uncharacterized microbial consortia derived from municipal and other waste facilities. This technique presents a number of advantages in comparison to the classical aerobic processes: (a) a high degree of purification with high-organic-load feeds can be achieved; (b) low nutrient requirements are necessary; (c) small quantities of excess sludge are usually produced; and (d) a combustible biogas is generated (Dalis et al. 1996; Zouari and Ellouz 1996; Borja et al. 2006). Combined aerobic–anaerobic systems have also been used effectively in the bioremediation of OMWs (Hamdi and Garcia 1991; Borja et al. 1995). Aerobic processes are applied waste streams of OMWs with low organic loads, whereas anaerobic processes are applied waste streams with high organic loads.

314 Olive Oil – Constituents, Quality, Health Properties and Bioconversions

OMWs treatment processes tested employ physical, chemical, biological and combined technologies. Several disposal methods have been proposed to treat OMWs, such as traditional decantation, thermal processes (combustion and pyrolysis), agronomic applications (e.g. land spreading), animal-breeding methods (e.g. direct utilisation as animal feed or following protein enrichment), physico-chemical treatments (e.g. precipitation/flocculation, ultrafiltration and reverse osmosis, adsorption, chemical oxidation processes and ion exchange), extraction of valuable compounds (e.g. antioxidants,

Among the different options, biological treatments or bioremediation are considered the most environmentally compatible and the least expensive (Mantzavinos and Kalogerakis 2005). Bioremediation is a treatment process employing naturally microorganisms (bacteria and fungi like yeasts, molds and mushrooms) to break down, or degrade, hazardous substances into less toxic or non-toxic substances. Bioremediation technologies can be classified as *in-situ* (bioaugmentation, bioventing, biosparging) or *ex-situ* (bioreactors, landfarming, composting and biopiles). *In-situ* bioremediation treats the contaminated water or soil where it was found, whereas *ex-situ* bioremediation processes involve removal of the contaminated soil or water to another location prior to treatment (Arvanitoyannis et

Bioremediation occurs either under aerobic or anaerobic conditions. Many aerobic biological processes, technologies and microorganisms have been tested for the treatment of OMWs, aimed at reducing organic load, dark colour and toxicity of the effluents (Table 2). 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 OMWs (McNamara et al. 2008). A number of different species of bacteria, yeasts, molds and mushrooms have been tested in aerobic

A number of studies have utilized bacterial consortia for bioremediation of OMWW. Bioremediation of OMWW using aerobic consortia has been quite successful in these studies, achieving significant reductions in COD (up to 80%) and the concentration of phytotoxic compounds, and complete removal of some simple phenolics (Zouari and Ellouz 1996; Benitez et al. 1997). A combined bacterial–yeast system of *Pseudomonas putida* and

Anaerobic bioremediation of OMWs has employed, almost exclusively, uncharacterized microbial consortia derived from municipal and other waste facilities. This technique presents a number of advantages in comparison to the classical aerobic processes: (a) a high degree of purification with high-organic-load feeds can be achieved; (b) low nutrient requirements are necessary; (c) small quantities of excess sludge are usually produced; and (d) a combustible biogas is generated (Dalis et al. 1996; Zouari and Ellouz 1996; Borja et al. 2006). Combined aerobic–anaerobic systems have also been used effectively in the bioremediation of OMWs (Hamdi and Garcia 1991; Borja et al. 1995). Aerobic processes are applied waste streams of OMWs with low organic loads, whereas anaerobic processes are

*Yarrowia lipolytica* were used to degrade OMWW (De Felice et al. 1997).

residual oil, sugars), and biological treatments (Morillo et al. 2009).

processes to treat OMWs that are listed (Table 2).

applied waste streams with high organic loads.

al. 2008).


In general, available scientific information shows that fungi are more effective than bacteria at degrading both simple phenols and the more complex phenolic compounds present in olive-mill wastes. For example, several species of the genus *Pleurotus* were found to be very effective in the degradation of the phenolic substances present in OMWs (Hattaka 1994). For OMWs biotreatment in large-scale, the use of filamentous fungi have considerable problems because of the formation of fungal pellets and other aggregations. The use of yeast in

In recent years, many researchers have utilized OMWs as growth substrates for microorganisms, obtaining a reduction of the COD level, together with enzyme production. The addition of nutrients can modify the pattern of degrading enzymes production by

Lipases (EC 3.1.1.3) are among the most important classes of industrial enzymes (Darvishi et al. 2009). Many microorganisms are known as potential producers of lipases including bacteria, yeast, and fungi. Several reviews have been published on microbial lipases

Lipolytic fungal species, such as *Aspergillus oryzae*, *Aspergillus niger*, *Candida cylindracea*, *Geotrichum candidum*, *Penicillium citrinum*, *Rhizopus arrhizus* and *Rhizopus oryzae* were preliminarily screened for their ability to grow on undiluted OMWW and to produce extracellular lipase. A promising potential for lipase production was found by *C. cylindracea* 

Among the different yeasts tested, the *Y. lipolytica* most adapted to grow on OMW. the *Y. lipolytica* strains were produced 16-1041 U/L of lipase on OMWs and also reduced 1.5-97% COD, 80% BOD and 0-72% phenolic compounds of OMWs (Fickers et al. 2011). The yeasts *Saccharomyces cerevisiae* and *Candida wickerhamii* produce β-glucosidase enzyme to hydrolyse

Olive oil cake (OOC) used as a substrate for phytase production in solid-state fermentation using three strains of fungus *Rhizopus* spp. OOC of initial moisture 50% was fermented at 30°C for 72 hours and inoculated with *Rhizopus oligosporus* NRRL 5905, *Rhizopus oryzae* NRRL 1891 and *R. oryzae* NRRL 3562. The results indicated that all three *Rhizopus* strains

Tannase could be utilized as an inhibitor of foam in tea production, clarifying agent in beer and fruit juices production, in the pharmaceutical industry and for the treatment of tannery effluents. *Aspergillus niger* strain HA37, isolated from OMW, was incubated on a synthetic medium containing tannic acid and on diluted OMW on a rotary shaker at 30°C. On the medium containing tannic acid, tannase production was 0.6, 0.9 and 1.5 U/ml at 0.2%, 0.5%

Extracellular ligninolytic enzymes such as lignin peroxidase (LiP), manganese peroxidase (MnP) and laccase (Lac) were produced by the white rot fungus *Phanerochaete flavido-alba* with a concomitant decoloration and decrease in phenolic content and toxicity of OMWW. Laccase was the sole ligninolytic enzyme detected in cultures containing monomeric aromatic compounds. Laccase and an acidic manganese-dependent peroxidase (MnPA, pI

bioreactors could be a way forward to overcome this limitation.

specific microorganisms from OMWs. (De la Rubia et al. 2008).

NRRL Y-17506 on OMWW (D'Annibale et al. 2006).

oleuropein present in olive oil (Ciafardini and Zullo 2002).

produced very low titers of enzyme on OOC (Ramachandran et al. 2005).

and 1% initial tannic acid concentration, respectively (Aissam et al. 2005).

(Arpigny and Jaeger 1999; Vakhlu and Kour 2006; Treichel et al. 2010).

**4.2 Enzymes** 


OMWW: olive oil wastewater, TPOMW: two-phase olive-mill waste, COD: chemical oxygen demand, TOC: Total organic carbon, DOR: olive-mill dry residue.

Table 2. Aerobic treatment of OMWs by microorganisms

In general, available scientific information shows that fungi are more effective than bacteria at degrading both simple phenols and the more complex phenolic compounds present in olive-mill wastes. For example, several species of the genus *Pleurotus* were found to be very effective in the degradation of the phenolic substances present in OMWs (Hattaka 1994). For OMWs biotreatment in large-scale, the use of filamentous fungi have considerable problems because of the formation of fungal pellets and other aggregations. The use of yeast in bioreactors could be a way forward to overcome this limitation.

#### **4.2 Enzymes**

316 Olive Oil – Constituents, Quality, Health Properties and Bioconversions

TPOMW Culture in TPOMW 9.2% TOC reduction 14.5%

*Coriolopsis polyzona* OMWW Culture in OMWW 75% colour removal (Jaouani et al. 2003)

*Lentinus tigrinus* OMWW Culture in OMWW Effective in decolorization (Jaouani et al. 2003) *Pleurotus eryngii* OMWW Culture in OMWW > 90% phenol reduction (Sanjust et al. 1991) *Pleurotus floridae* OMWW Culture in OMWW > 90% phenol reduction (Sanjust et al. 1991) *Pleurotus ostreatus* OMWW Culture in OMWW 100% phenol reduction (Tomati et al. 1991)

*Pleurotus ostreatus* OMWW Solid-state culture 67% phenol reduction (Fountoulakis

TPOMW Culture in TPOMW 9.7% TOC reduction 66.2%

*Pleurotus sajor-caju* OMWW Culture in OMWW > 90% phenol reduction (Sanjust et al. 1991) *Pleurotus spp.* OMWW Culture in OMWW 76% phenol reduction (Tsioulpas et al. 2002)

TPOMW Culture in TPOMW 7.6% TOC reduction 88.7%

OMWW: olive oil wastewater, TPOMW: two-phase olive-mill waste, COD: chemical oxygen demand,

Method Results Reference

(García García et al. 2000)

(Sampedro et al.

2007b)

(Sampedro et al. 2007b)

(Yesilada et al. 1997)

(Yesilada et al. 1997)

(D'Annibale et al.

(Aggelis et al. 2003)

(Saavedra et al. 2006)

(Sampedro et al.

(Sampedro et al.

(Sampedro et al.

(Sampedro et al.

et al. 2002)

2007b)

2007b)

2007b)

2007b)

2004)

52% phenol reduction (Blánquez et al. 2002)

75% COD reduction 92% phenol reduction

phenol reduction

TPOMW Solid-state culture 70% phenol reduction (Linares et al. 2003)

89% phenol reduction

phenol reduction

phenol reduction

phenol reduction

complete

phenol reduction

phenol reduction

phenol reduction

phenol reduction

OMWW Culture in OMWW Effective in decolorization (Jaouani et al. 2003)

72.3% phenol reduction

Phenol reduction nearly

Microorganism Waste

*Phanerochaete chrysosporium* 

*Phanerochaete chrysosporium* 

*Phanerochaete flavido-alba*

*Phanerochaete flavido-alba* 

Mushrooms

*Pleurotus pulmonarius* 

*Pycnoporus cinnabarinus* 

*Pycnoporous coccineus* 

type

*Pleurotus ostreatus* OMWW Culture in

OMWW Culture in

OMWW Culture in

bioreactors with OMWW

bioreactors with OMWW

*Coriolopsis rigida* TPOMW Culture in OMWW 9% TOC reduction

*Coriolus versicolor* OMWW Culture in OMWW 65% COD reduction 90%

*Funalia trogii* OMWW Culture in OMWW 70% COD reduction 93%

*Lentinula edodes* OMWW Culture in OMWW 65% COD reduction 88%

bioreactors with OMWW

*Pleurotus ostreatus* TPOMW Plastic bag 22% TOC reduction 90%

*Phlebia radiata* TPOMW Culture in TPOMW 13% TOC reduction 95.7%

*Poria subvermispora* TPOMW Culture in TPOMW 13.2% TOC reduction

TOC: Total organic carbon, DOR: olive-mill dry residue.

Table 2. Aerobic treatment of OMWs by microorganisms

In recent years, many researchers have utilized OMWs as growth substrates for microorganisms, obtaining a reduction of the COD level, together with enzyme production. The addition of nutrients can modify the pattern of degrading enzymes production by specific microorganisms from OMWs. (De la Rubia et al. 2008).

Lipases (EC 3.1.1.3) are among the most important classes of industrial enzymes (Darvishi et al. 2009). Many microorganisms are known as potential producers of lipases including bacteria, yeast, and fungi. Several reviews have been published on microbial lipases (Arpigny and Jaeger 1999; Vakhlu and Kour 2006; Treichel et al. 2010).

Lipolytic fungal species, such as *Aspergillus oryzae*, *Aspergillus niger*, *Candida cylindracea*, *Geotrichum candidum*, *Penicillium citrinum*, *Rhizopus arrhizus* and *Rhizopus oryzae* were preliminarily screened for their ability to grow on undiluted OMWW and to produce extracellular lipase. A promising potential for lipase production was found by *C. cylindracea*  NRRL Y-17506 on OMWW (D'Annibale et al. 2006).

Among the different yeasts tested, the *Y. lipolytica* most adapted to grow on OMW. the *Y. lipolytica* strains were produced 16-1041 U/L of lipase on OMWs and also reduced 1.5-97% COD, 80% BOD and 0-72% phenolic compounds of OMWs (Fickers et al. 2011). The yeasts *Saccharomyces cerevisiae* and *Candida wickerhamii* produce β-glucosidase enzyme to hydrolyse oleuropein present in olive oil (Ciafardini and Zullo 2002).

Olive oil cake (OOC) used as a substrate for phytase production in solid-state fermentation using three strains of fungus *Rhizopus* spp. OOC of initial moisture 50% was fermented at 30°C for 72 hours and inoculated with *Rhizopus oligosporus* NRRL 5905, *Rhizopus oryzae* NRRL 1891 and *R. oryzae* NRRL 3562. The results indicated that all three *Rhizopus* strains produced very low titers of enzyme on OOC (Ramachandran et al. 2005).

Tannase could be utilized as an inhibitor of foam in tea production, clarifying agent in beer and fruit juices production, in the pharmaceutical industry and for the treatment of tannery effluents. *Aspergillus niger* strain HA37, isolated from OMW, was incubated on a synthetic medium containing tannic acid and on diluted OMW on a rotary shaker at 30°C. On the medium containing tannic acid, tannase production was 0.6, 0.9 and 1.5 U/ml at 0.2%, 0.5% and 1% initial tannic acid concentration, respectively (Aissam et al. 2005).

Extracellular ligninolytic enzymes such as lignin peroxidase (LiP), manganese peroxidase (MnP) and laccase (Lac) were produced by the white rot fungus *Phanerochaete flavido-alba* with a concomitant decoloration and decrease in phenolic content and toxicity of OMWW. Laccase was the sole ligninolytic enzyme detected in cultures containing monomeric aromatic compounds. Laccase and an acidic manganese-dependent peroxidase (MnPA, pI

citric acid. Identification tests showed that these isolates belonged to the species *Y. lipolytica*. M1 and M2 strains produced maximum levels of lipase on olive oil, and high levels of citric

The highest oxalic acid quantity (5 g/l) was obtained by the strain *Aspergillus* sp. ATHUM 3482 on waste cooking olive oil medium. For strain *Penicillium expansum* NRRL 973 on this medium sole organic acid detected was citric acid with maximum concentration achieved

Exopolysaccharides (EPSs) often show clearly identified properties that form the basis for a wide range of applications in food, pharmaceuticals, petroleum, and other industries. The production of these microbial polymers using OMWW as a low-cost fermentation substrate has been proposed (Ramos-Cormenzana et al. 1995). This approach could reduce the cost of polymer production because the substrate is often the first limiting factor. Moreover, OMWW contains free sugars, organic acids, proteins and other compounds such as phenolics that could serve as the carbon source for polymer production, if the chosen

Xanthan gum, an extracellular heteropolysaccharide produced by the bacterium *Xanthomonas campestris* has been obtained from OMWW. Growth and xanthan production on dilute OMWW as a sole source of nutrients were obtained. Addition of nitrogen and/or salts led to significantly increased xanthan yields with a maximum of 7.7g/l (Lopez and

The fungus *Botryospheria rhodina* has been used for the production of β-glucan from OMWW with yield of 17.2 g/l and a partial dephenolisation of the substrate (Crognale et al. 2003). A metal-binding EPS produced by *Paenibacillus jamilae* from OMWs. Maximum EPS production (5.1 g/l) was reached in batch culture experiments with a concentration of 80%

Polyhydroxyalkanoates (PHAs) are reserve polyesters that are accumulated as intracellular granules in a variety of bacteria. Of these polymers, poly-β-hydroxybutyrate (PHB) is the most common. Since the physical properties of PHAs are similar to those of some conventional plastics, the commercial production of PHAs is of interest. However, these biodegradable and biocompatible 'plastics' are not priced competitively at the present, mainly because the sugars (i.e. glucose) used as fermentation feed-stocks are expensive. Finding a less expensive substrate is, therefore, a major need for a wide commercialisation of these products. Large amounts of biopolymers containing β-hydroxybutyrate (PHB) and copolymers containing β-hydroxyvalerate (P[HB-co-HV]) are produced by *Azotobacter chroococcum* in culture media amended with alpechin (wastewater from olive oil mills) as the

Rhamnolipids, typical biosurfactants produced by *Pseudomonas aeruginosa*, consist of either one or two rhamnose molecules, linked to one or two fatty acids of saturated or unsaturated alkyl chain between C8 and C12. The *P. aeruginosa* 47 T2 produced two main rhamnolipid

microorganism is able to metabolize these compounds (Fiorentino et al. 2004).

acid on citric acid fermentation medium (Mafakher et al. 2010).

3.5 g/l (Papanikolaou et al. 2011).

Ramos-Cormenzana 1996).

sole carbon source (Pozo et al. 2002).

**4.5 Biosurfactants** 

**4.4 Biopolymers and biodegradable plastics** 

of OMWW as fermentation substrate (Morillo et al. 2007).

62.8) were accumulated in cultures with OMWW or polymeric pigment. Furthermore, modified manganese-dependent peroxidases were observed mainly in OMWWsupplemented cultures. Laccase was more stable to the effect of OMWW toxic components and was accumulated in monomeric aromatic-supplemented cultures, suggesting a more important role than manganese-dependent peroxidases in OMWW detoxification. Alternatively, MnPA accumulated in cultures containing the polymeric pigment seemed to be more essential than laccase for degradation of this recalcitrant macromolecule by *P. flavido-alba*. (Ruiz et al. 2002).

Enzyme laccase, produced by fungus *Pycnoporus coccineus*, is responsible for OMWW decolorization and decrease COD and phenolic compounds. The highest laccase level was 100 000 U/l after 45 incubation-days. The enzyme was stable at pH 7, at room temperature and showed a half-life of 8 and 2 h at 50 and 60°C, respectively (Jaouani et al. 2005). In order to decolourise OMWW efficiently, production and differential induction of ligninolytic enzymes by the white rot *Coriolopsis polyzona*, were studied by varying growth media composition and/or inducer addition (Jaouani et al. 2006). The production of lignin peroxidase (LiP), manganese peroxidase (MnP) and lipases by *Geotrichum candidum* were performed in order to control the decolourisation and biodegradation of OMWW (Asses et al. 2009).

Sequential batch applications starting with adapted *Trametes versicolor* FPRL 28A INI and consecutive treatment with *Funalia trogii*, possible to remove significant amount of total phenolics content and higher decolorization as compared to co-culture applications. Also highest laccase and manganese peroxidase acitivities were obtained with *F. trogii* (Ergul et al. 2010).
