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

The selection of yeasts intended as functional starter for the bioremediation of OMW is a quite complex process, involving different steps; figure 1 proposes a possible scheme.

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 of promising yeasts.

#### **2.1. Isolation**

information is now available on the indigenous yeasts present in the OMW and their possible

**Yeasts Method Results Reference**

*Candida boidinii* Fed-batch microcosm 42.2% - [36]

*C. cylindracea* Culture in OMW 27% 45.8-70.2% [38]

*C. diddensiae* Culture in OMW 32.14-43.56% 55.40-64.84% [41]

*C. ernobii* Culture in OMW 34.09-35.23% 51.85-62.65 [41] *C. holstii* Culture in OMW 39% 57.93% [41]

*C. rugosa* Culture in OMW 12.2-20.4% 20.4-62.2% [38]

*C. tropicalis* Culture in OMW 51.7% 62.8% [44]

*Geotrichum* sp. Culture in OMW 46.6% 55% [44]

**Phenol Reduction COD Reduction**

12.8-31.3% 27.4-55.9% [39]

51% 62% [47]

Culture in OMW 40% 45% [37]

Culture in OMW 36.2% 48.4% [40]

Culture in OMW 10-72% - [42]

culture with OMW 20.3% (tannins) - [43] Culture in OMW 83% 55% [12]

Culture in OMW 15.3% 31.1-62.2% [38]

OMW - 12.4-62% [11]

OMW - 25-65% [16]

Culture in OMW 20-41% 25-56% [49] Culture in OMW 46% 51% [37]

Fed-batch microcosm 42.9% - [36] OMW from industrial mills 25-31% 20-23% [45] Culture in OMW 47% 77% [48]

OMW from industrial mills 25% 18% [45] Culture in OMW 12-36.5% 39.4-69.7% [46]

use for performing biodegradation of the waste.

54 Applied Bioremediation - Active and Passive Approaches

*C. oleophila* Bioreactor batch

*G. candidum* Culture in bioreactors with

Bioreactor batch culture with OMW

Culture in bioreactors with a mixture of OMW (75%) and pig slurry (25% v/v)

Culture in bioreactors with

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

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 samples are serially diluted and plated on opportune media, like acidified Malt Extract Agar [55], Potato Dextrose Agar and Yeast Malt Agar [56], YEPD agar (Yeast Extract Potato Dextrose) supplemented with 50 μg/mL of ampicillin [45]. Then, yeasts are selected on the basis of colony morphology.

**2.3. Enzymatic traits**

**2.4. Identification**

Spain).

protocols to assess enzymatic traits.

**2.5. Functional characterization**

decrease of the pollutant impact of OMW.

the microbial toxicity towards *Bacillus cereus*.

**2.6. Selection of promising strains and validation**

absorbance measurement at 390 nm.

through some commercial miniaturized kits [61].

Yeasts intended for bioremediation should be assessed for different enzymatic traits; some of them (pectinolytic, lipolytic and protease activities) rely on the ability to persist in a stressful environment, whereas other traits are strongly related with the ability to remove phenols. For example, Taccari and Ciani [60] reported that ligninolytic enzymes lignin peroxidase (LiP), manganese peroxidase (MnP) and laccase, characterized by a low substrate specificity, are involved in the degradation of polyphenols in OMW. Reference [61] reports the most common

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

57

For long time yeasts have been identified through the fermentation/assimilation profiles of sugars; a good profile should include the assays for the following sugars: D-glucose, Dgalactose, maltose, α-methyl-D-glucoside, sucrose, trehalose, melibiose, lactose, cellobiose, melezitose, raffinose, inulin, starch and D-xylose. Nowadays, these assays are usually run

It is well known that the phenotypic identification shows some limits and drawbacks, therefore yeast identification should be performed through genotypic method. One of the most used approach is PCR amplification of the region spanning ITS1 and ITS2 and the 5.8S rRNA gene (5.8S–ITS region) and subsequent restriction analysis, following the protocols by the references [62, 63]; the results of amplification and restriction are used as input data for an analysis through a specific database (for example Yeast-id database, CECT, University of Valencia,

For yeasts intended for bioremediation, phenol removal, the decrease of BOD/COD and OMW decolorization could be referred to as the functional traits, as they are strictly related to the

Focusing on phenol removal, yeasts should be inoculated onto aliquots of OMW under laboratory conditions (static temperature and agitation) for some days [55]. Thereafter, the amount of residual phenols can be assessed through HPLC equipments or simply using the method by Folin-Ciocalteau [64]. Other authors [12,65] evaluated indirectly phenol removal through toxicity attenuation, thus they studied the phytoxicity of OMW towards seeds and

Other traits are the reduction of COD and BOD [45, 66], as well as waste decolorization; for this last assay, OMW should be diluted with distilled water and then analyzed through

Choosing the most promising strains is the final step for a starter selection; as reported elsewhere [59], the management of a such large amount of data (many strains, each of them

An interesting approach was proposed by other authors [57]; they optimized the protocol for the isolation of bacteria able to remove phenols from wastewater and slurry, but their method, with some modifications, could be used successfully for yeasts. OMW should be added to a mineral salt medium (MSM) containing (g/L): Na2HPO4, 1.6; KH2PO4, 0.4; NH4NO3, 0.5; MgSO4\*7H2O, 0.2; CaCl2, 0.025; FeCl3, 0.0025 with and without 1% glucose (w/v) as an additional carbon source.

Different concentrations of phenol (100, 200, 300, 500, 700, and 900 mg/L) should be added to the medium; after adjusting the pH to 7.0, the samples can be stored at 25°C for at least 5 days and then plated onto MSM agar plates, with and without glucose.

#### **2.2. Technological characterization**

The technological characterization of yeasts deals with both the taxonomic assays and the technological traits (growth requirements and enzymatic traits).

The most important trait is the effect of phenols on yeast growth/survival; this characteristic includes both the ability to use phenols as carbon sources and the growth/survival in OMW. Phenol assimilation can be assessed on Yeast Nitrogen Base (a laboratory medium without carbon source), added with either caffeic, vanillic or *p*-coumaric acid [55]. Another way to assess the suitability of yeasts for bioremediation is the evaluation of growth in OMW or in solid/liquid media containing OMW [58].

A modification of this last assay was proposed by Aissam et al. [37], who cultured yeasts into lab media containing increasing amounts of OMW (from 50 to 100%) to induce yeast adaptation to such a stressful environment.

Although the assimilation of phenols and the growth in OMW are the most important traits for the selection of promising yeasts for bioremediation, some other interesting characteristics are the thermal profile (*i.e.* the minimal and maximum temperatures of growth, the optimal temperature), the effect of pH, and nitrogen assimilation. The effects of temperature and pH can be evaluated through a spectrophotometric measurement, followed by the calculation of Growth Index, as proposed for yeasts intended as starters for table olives [59].

On the other hand, nitrogen assimilation should be assessed in a poor medium, containing a single nitrogen source (for example KNO3 or ethylamine) [55]; this assay, as well as spore production, has also a taxonomic potential: for example *Saccharomyces cerevisiae* is not able to use nitrate as the only nitrogen source, whereas other yeasts do it.

### **2.3. Enzymatic traits**

samples are serially diluted and plated on opportune media, like acidified Malt Extract Agar [55], Potato Dextrose Agar and Yeast Malt Agar [56], YEPD agar (Yeast Extract Potato Dextrose) supplemented with 50 μg/mL of ampicillin [45]. Then, yeasts are selected on the basis of colony

An interesting approach was proposed by other authors [57]; they optimized the protocol for the isolation of bacteria able to remove phenols from wastewater and slurry, but their method, with some modifications, could be used successfully for yeasts. OMW should be added to a mineral salt medium (MSM) containing (g/L): Na2HPO4, 1.6; KH2PO4, 0.4; NH4NO3, 0.5; MgSO4\*7H2O, 0.2; CaCl2, 0.025; FeCl3, 0.0025 with and without 1% glucose (w/v) as an

Different concentrations of phenol (100, 200, 300, 500, 700, and 900 mg/L) should be added to the medium; after adjusting the pH to 7.0, the samples can be stored at 25°C for at least 5 days

The technological characterization of yeasts deals with both the taxonomic assays and the

The most important trait is the effect of phenols on yeast growth/survival; this characteristic includes both the ability to use phenols as carbon sources and the growth/survival in OMW. Phenol assimilation can be assessed on Yeast Nitrogen Base (a laboratory medium without carbon source), added with either caffeic, vanillic or *p*-coumaric acid [55]. Another way to assess the suitability of yeasts for bioremediation is the evaluation of growth in OMW or in

A modification of this last assay was proposed by Aissam et al. [37], who cultured yeasts into lab media containing increasing amounts of OMW (from 50 to 100%) to induce yeast adaptation

Although the assimilation of phenols and the growth in OMW are the most important traits for the selection of promising yeasts for bioremediation, some other interesting characteristics are the thermal profile (*i.e.* the minimal and maximum temperatures of growth, the optimal temperature), the effect of pH, and nitrogen assimilation. The effects of temperature and pH can be evaluated through a spectrophotometric measurement, followed by the calculation of

On the other hand, nitrogen assimilation should be assessed in a poor medium, containing a single nitrogen source (for example KNO3 or ethylamine) [55]; this assay, as well as spore production, has also a taxonomic potential: for example *Saccharomyces cerevisiae* is not able to

Growth Index, as proposed for yeasts intended as starters for table olives [59].

use nitrate as the only nitrogen source, whereas other yeasts do it.

and then plated onto MSM agar plates, with and without glucose.

technological traits (growth requirements and enzymatic traits).

morphology.

additional carbon source.

**2.2. Technological characterization**

56 Applied Bioremediation - Active and Passive Approaches

solid/liquid media containing OMW [58].

to such a stressful environment.

Yeasts intended for bioremediation should be assessed for different enzymatic traits; some of them (pectinolytic, lipolytic and protease activities) rely on the ability to persist in a stressful environment, whereas other traits are strongly related with the ability to remove phenols.

For example, Taccari and Ciani [60] reported that ligninolytic enzymes lignin peroxidase (LiP), manganese peroxidase (MnP) and laccase, characterized by a low substrate specificity, are involved in the degradation of polyphenols in OMW. Reference [61] reports the most common protocols to assess enzymatic traits.

### **2.4. Identification**

For long time yeasts have been identified through the fermentation/assimilation profiles of sugars; a good profile should include the assays for the following sugars: D-glucose, Dgalactose, maltose, α-methyl-D-glucoside, sucrose, trehalose, melibiose, lactose, cellobiose, melezitose, raffinose, inulin, starch and D-xylose. Nowadays, these assays are usually run through some commercial miniaturized kits [61].

It is well known that the phenotypic identification shows some limits and drawbacks, therefore yeast identification should be performed through genotypic method. One of the most used approach is PCR amplification of the region spanning ITS1 and ITS2 and the 5.8S rRNA gene (5.8S–ITS region) and subsequent restriction analysis, following the protocols by the references [62, 63]; the results of amplification and restriction are used as input data for an analysis through a specific database (for example Yeast-id database, CECT, University of Valencia, Spain).

### **2.5. Functional characterization**

For yeasts intended for bioremediation, phenol removal, the decrease of BOD/COD and OMW decolorization could be referred to as the functional traits, as they are strictly related to the decrease of the pollutant impact of OMW.

Focusing on phenol removal, yeasts should be inoculated onto aliquots of OMW under laboratory conditions (static temperature and agitation) for some days [55]. Thereafter, the amount of residual phenols can be assessed through HPLC equipments or simply using the method by Folin-Ciocalteau [64]. Other authors [12,65] evaluated indirectly phenol removal through toxicity attenuation, thus they studied the phytoxicity of OMW towards seeds and the microbial toxicity towards *Bacillus cereus*.

Other traits are the reduction of COD and BOD [45, 66], as well as waste decolorization; for this last assay, OMW should be diluted with distilled water and then analyzed through absorbance measurement at 390 nm.

### **2.6. Selection of promising strains and validation**

Choosing the most promising strains is the final step for a starter selection; as reported elsewhere [59], the management of a such large amount of data (many strains, each of them studied for different parameters) is quite difficult and complex. A possible solution could be the use of multivariate statistical approaches, like the Principal Component Analysis, Cluster Analysis or Multiple Correspondence Analysis or all of them in a sequence.

**2.** yeasts can be used for the aerobic and anaerobic treatment of wastes;

**5.** yeasts can be used in continuous or in batch cultures, while moulds do not;

lipases) using OMW as medium;

**Step 1**

**Step 2**

**Step 3**

**Figure 1.** Selection of yeasts for phenol removal in OMW

bacteria do not.

**3.** the yield of moulds in phenol removal is high, many times higher than for yeasts; however, micelia could absorb phenols and release them again in the case of a prolonged storage;

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

59

**4.** some yeasts could be used to produce biomass and useful metabolites (for example

**6.** some yeasts are able to remove both low and high molecular weight phenols, whereas

Yeast kinetic

Yeast isolation and technological characterization

Phenotyping

Genotyping

Functional characterization

Validation

Screening of promising strains in OMW

10

**Starter selection and validation at industrial level**

The main result of the multivariate approach is the choice of the best strains (3-10) for an *in vivo* validation; however, yeasts require a preliminary optimization and/or validation in small volumes and under controlled conditions.

Some variables that should be assessed are:

