*2.1. Theoretical Background*

Household food waste measurement is an important step in the global efforts to reduce food losses [37]. However, a sound theoretical background, experience in practical organization, and comparable research data are scarce in this field. From the 1990s, a methodological evolution can be observed in this field, which allowed us to apply a standardized research methodology in 2016 and in our actual study (Figure 1).

**Figure 1.** Theoretical framework and organization of the study.

### 2.1.1. Diverse Methods (1990s to 2014)

Despite the growing number of studies carried out to investigate the quantity of household food waste, the diversity of methodological approaches that different researchers applied limited the comparability of most of the findings, dating back to the 1990s [18], which might have resulted in different interpretations of the research data. The following methods were used most frequently [38]:

Besides the basic methodological approach itself, a further significant bias resulted from small differences in the considerations applied by the research teams. For instance, the recorded material streams and their classification were also divergent. Typical differences found in the literature:

### 2.1.2. Standardized Method (FUSIONS, 2014)

Considering these barriers, the FUSIONS project, funded by the European Commission recognized the importance of establishing a standard for measurement methodology. Between 2012 and 2016, FUSIONS consulted researchers, industrial stakeholders, and governments about the harmonization of food waste monitoring. One of the most significant deliverables of FUSIONS was a well-balanced, widely accepted standardized methodology (that we refer to as FUSIONS methodology) to measure food waste [38], which provides the opportunity to compare country-level data. The most important considerations for household food waste measurement studies, according to the FUSIONS methodology, are the following:

### 2.1.3. First National-Level Study in Hungary Based on the FUSIONS Methodology

In 2015 the National Food Chain Safety Office of Hungary (Nébih) started a public awareness campaign on household food waste prevention, called "Wasteless", which received support in 2016 from the LIFE Framework program of the EU. Influential European food waste prevention projects were: the EU-FP7 funded FUSIONS and Horizon 2020 funded "Refresh", the "Love Food Hate Waste" campaign by WRAP, and the "Every Crumb Counts" campaign organized by different entities from the industry, academics, and NGOs. Wasteless aimed to decrease avoidable food waste in Hungarian households. In order to measure the efficiency of the communication efforts, a baseline was set based on the FUSIONS recommendations. This has been the first empirical study carried out with this methodology in the Central and Eastern European region [24]. Focus group interviews and a preliminary, representative quantitative consumer survey covering 1000 respondents were conducted to receive research insights before the first household study.

### 2.1.4. Discussion of the Experiences of the First Study

The results of the survey were published in national and international academic journals and discussed at national and international platforms (including the EU Platform on Food Losses and Food Waste, operated by the Directorate-General for Health and Food Safety of the EU Commission) and scientific conferences [24,37,44–47]. Public dissemination activities through the Wasteless communication campaign were also conducted, presenting the results to consumers. Until 2018 the campaign managed to achieve a reach of almost 50 million via di fferent media platforms [37], and the research results are widely cited by journalists even today.

### 2.1.5. Second National-Level Study in Hungary Based on the FUSIONS Methodology

A second study for household food waste measurement using the FUSIONS methodology took place in 2019. The research was organized to deliver comparable data to monitor changes in food waste quantities and composition. According to the analysis of the literature, this study has most probably been the first one to repeat a previous measurement based on the FUSIONS methodology. Although the research was not conducted as a validation action, its experiences are still valuable to indicate the replicability of the methodology.

The research was advertised in a media campaign in October 2019. The households were selected on the basis of voluntary registration. The advertisement included radio interviews (6); television interviews (3); press releases (7); online press releases (45). In total, 200 households applied to participate in the research. The sampling was conducted in November and December of 2019. The sampling period avoided all national and religious holidays.

During the measurement period, households received a kitchen scale (accuracy in grams) and a measuring glass from the research team. The measuring glass served for liquid wastes. Participants were also provided with a manual which described the scope of the research and explained clearly the di fference between avoidable and unavoidable food waste. The following definitions were used for avoidable, unavoidable, and potentially avoidable food waste [11]:

For the whole duration of the investigation, e-mail and telephone support was provided to the participants. Attention was paid to reduce bias by explaining participants that the data collection was conducted anonymously, which impeded the identification of the household. The data was administered into a unified waste log. Participating households could use an online platform or a printed sheet for this purpose. The weight and exact type of each unit of food waste had to be recorded for one week (seven days). This time frame is assumed to be long enough to reduce influences caused by the participants' compulsion to conform, originating from the fact of observation. Data on solid and liquid waste were documented in mass (grams). Weighing had to be performed prior to disposal.

A supporting unit was set up to help participants of the household measurement study taking care of:

A total of 200 households applied for participation in the survey during the media campaign, but only 165 households provided a reliable and complete data set for the analysis. The most common reasons for excluding households were interrupted communication, logs not returned by the participants, an inappropriate or non-consecutive data recording period, missing data in the logs, or inadequate details on food types that made the classification impossible.

Although there have been several studies on the topic with similar or even smaller sample sizes ([15] n = 101; [16] n = 192; [19] n = 61; [41] n = 61; [48] n = 13), a post-hoc calculation was performed to determine the power of the test, for which data from the previous Hungarian study [24] served as basis. The power of the test proved to be 87.5%, which meets the criterion of being above 80% [49–51].

Based on the household data, a detailed classification was made by the research team (e.g., meals, bakery products, fresh vegetables, etc.). Each item had to be recorded by the participants as accurately as possible (e.g., not 'bread' but 'bread crust', not 'chicken' but 'chicken skin', etc.). Avoidable and unavoidable food waste units were recorded separately by the households. Subsequently to the measurement, the research team conducted a post-hoc validation of the categorization performed by the consumers.

After data cleaning, the analysis was conducted for each household. Data on food waste per person were calculated in each food waste category. The results were extrapolated to one year by multiplying the results of the one week by 52.

### 2.1.6. Afterlife of Research Results

Planning the afterlife of the research data has been a part of the theoretical framework. This inspection is considered to be an element in a time series study, with the objective of providing information to policy makers on a regular basis and also helping to optimize public awareness-raising activities. Since the reporting on the food waste situation became a compulsory activity of all EU member states [35], the study will also serve to deliver national level data to the EU Commission with respect to Hungarian households. Experiences with the FUSIONS methodology are still scarcely available, and therefore sharing the findings of this research may help other research teams to start their own activities in this field.


**4.3. SNPStream (Beckman coulter)**

This method involves a single-base extension assay and tag array technology. It starts with a multiplexed SNP-specific PCR followed by a primer extension reaction using tagged primers and fluorescent-labeled nucleotide terminators, i.e., ddNTPs. The products are captured on a tag array, which is then scanned to detect the hybridized extension primers and produce calls. It allows the processing of up to three million genotypes in 384 samples at a time. This genotyping system combines solid-phase primer extension assay and universal tags for SNP genotyping. The instrument allows processing of 4,600–3,000,000 genotypes per day [167].


**6. Affine Formulation**

In the usual formulation of General Relativity, one defines a covariant derivative ∇, which is a map among tensors. Then, one can define curvature and torsion and eventually get the field equations for ECSK theory or General Relativity by setting torsion to zero.

One may wonder if this latter formulation is equivalent to the one we have been implementing through principal bundles and principal connection.

The answer is positive and is given in the next two sections.

16 Where we have introduced the notation Ω*k* (*M*, *P* ×Ad g) := Ω*k* (*M*, ad*P*).


**4. Conclusions**

Density functional theory calculation using DFT/B3LYP/6-31 + G (d) level of theory has been performed for four histamine H2 receptor antagonists, cimetidine, famotidine, nizatidine, and ranitidine with the help of Gaussian software. Vibrational analysis (IR) of four histamine H2 receptor antagonists has been generated by using DFT. Generated vibrational results of nizatidine were compared with the experimental result and the computed vibrational results found to be in good agreement with the experimental result. From thermochemical parameters of histamine H2 receptor antagonist, it was found that famotidine is having superior thermodynamics parameters among the four with high free energy, zero-point energy, enthalpy and low entropy. Comparing the global descriptive parameters of histamine H2 receptor antagonist, nizatidine is found to be having higher softness, ionization potential, electron affinity, chemical potential and lower hardness, which shows that it is more stable and chemically reactive. Higher value of electrophilicity index indicates its high biological activity. Nizatidine is found to be having lower energy gap, which shows that nizatidine is more chemically reactive. In case of stability, reactivity and biological activity ranitidine comes next to nizatidine and among them, cimetidine is less stable and having lower biological activity. At the same time, the Gibb's free energy revealed that the solubility of nizatidine is not sufficient to have adequate bioavailability. Finally, we could amorphousize nizatidine by quench cooling technique and found that it stable in amorphous state without showing any recrystallization tendency during super cooling and the subsequent heating in the metastable state. Nizatidine has a glass transition temperature around 282.1 K and was found to be stable over the measured temperature range up to 307 K.


**1. Introduction**

166 New Insights into the Prevention and Treatment of Bulimia Nervosa

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and clinical practice (pp.142- 153). New York, London: The Guilford Press 2002 [32] Tiggemann, M. Media Influences on Body Image Development. In: T.F.Cash, T.

[33] Tiggemann, M. Media exposure, body dissatisfaction and disordered eating: television and magazines are not the same. European Eating Disorders Review, 200311, 418-430. [34] Garner, D.M., Olmsted, M.P., Bohr, Y., Garfinkel, P.E. *The Eating Attitudes Test:*

[35] Cash, T. R., Pruzinsky, T. Future challenges for body image theory, research and clinical

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Physical activity and exercise has a widely known positive effect on various physiological and psychological variables, and lack of physical activity has been shown as an independent factor for obesity, type 2 diabetes, hypertension, certain types of cancer and other diseases (Pedersen & Saltin, 2006). However, in the Diagnostic and Statistical Manual for Mental Disorders version four (DSM-IV) (APA, 1994) excessive amounts of exercise is listed as one possible weight compensatory behavior among patients with bulimia nervosa (BN). In this chapter I will describe the effects of physical activity, and the motives for physical activity among patients with BN. Furthermore, the two-edged sword aspect of physical activity among patients with BN will be explored. This duality comes to show on one hand because of the excessiveness and the abuse of physical activity, and on the other hand the beneficial and therapeutic effects of correctly dosed physical activity in treatment of BN.


**4. Tea phytochemicals as Nutraceuticals in different diseases**

Tea is beneficial in protection and prevention of numerous critical diseases such as different cancers, diabetes, neurodegenerative and cardiovascular diseases [29–31]. The polyphenols, the major phytochemicals present in tea are known for their antioxidant properties due to their abilities to scavenge free radical species such as hydroxyl radical (OH), hydrogen peroxide (H2O2) and superoxide anion (O2.) and thus their consumption confer them as potential cancer chemopreventive agents as well as other free radical induced diseases. The polyphenol extract of tea has been effectively used in cancer prevention, protection cardiovascular diseases, reduce weight-loss, anti-allergic, prebiotics, osteoarthritis protection etc. [32, 33]. Green tea extract comprises of nearly 40% of polyphenols of which nearly 25% accounts for different catechins and their gallates of which EGCG is the highest in quantity (about 11%), the most studied polyphenols in therapeutic properties. Black tea polyphenols theaflavins, theaflavin gallates (which are dimers of two different catechins and their gallates) and thearubigins with 2-6% in quantity in addition to 3-10% of catechins gain attention in different diseases [34].

Herein, we wish to discuss the health benefits of tea in common and major diseases such as diabetes, cardiovascular diseases, obesity, cancers, as well as others minor diseases related to microbial and inflammatory types. The role of major phtochemicals of tea in different diseases is outlined in **Table 3**.


**3. Cyclodextrins covalently attached to polymeric chains**

This section aims to summarize new research that has emerged in the past few years on materials composed of synthetic or natural polymers to which CD derivatives have been chemically attached. The polymers discussed here were chosen considering the extensive investigations and applications in pharmaceutics and biomedicine as drug excipients, biocompatible alternative materials in tissue engineering, contrast enhancers, molecular recognition models, etc. To achieve novel and/or enhanced properties of these classes of compounds, the functionalization with different CD units is employed. To covalently attach CDs onto polymer chains, multiple types of crosslinking agents (citric acid, epichlorohydrin, aldehydes, carbodiimides, and amines) can be used. In recent years, polymers functionalized with CD units have been studied for the development of a variety of polymeric networks [41, 42]. Moreover, the polymeric material based on CD units can be modulated in such a way to form nano/micro/macroparticles, gels, micelles, coating films, or fibers [43–45]. Many studies in this regard are performed using chitosan, mostly due to its remarkable biological properties, including antimicrobial activity, nontoxicity, biocompatibility, and biodegradability, and at the same time to the possibility to functionalize it [46, 47].

For instance, Campos *et al.* took this advantage and appended β-CD on chitosan nanoparticles in the presence of N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide (EDC) and N-hydroxysuccinimide (NHS), thus developing a potential carrier for botanical pesticides [48]. A green-assembly strategy to obtain β-CD-chitosan functionalized graphene oxide hydrogels in the presence of sodium ascorbate has been reported, with application as recyclable decontaminants in wastewater treatment [49]. Both simple polymers and polymer mixtures are favorable to the attachment of CD units, as demonstrated by the research of Hardy *et al.* regarding the complex formed between chitosan and alginate for the release of piroxicam [50]. The research revealed that CD appending on chitosan generates a decrease in the number of amino groups, thus modulating the alginate complexation. In a recent study, alginate has also been functionalized with either host units (n-alkyl amine CD derivatives) or guest units (adamantane) [42]. In this particular case, the grafting procedure was

performed in aqueous media in the presence of EDC and N-hydroxysulfosuccinimide (NHSS). The properties of the gel obtained by mixing the two types of alginates in the presence of Ca2+ ions were influenced by the host-guest interaction and the length of the alkyl chain of the β-CD derivatives. Thus, by mixing the decorated alginates with CD and adamantyl units, materials suitable for encapsulating both large molecules and small species can be obtained. In addition, the functionalization of alginate with units of β-CD derivatives by using Cu(I)-catalyzed azide-alkyne cycloaddition click reaction led to materials with good drug release properties [51]. The same procedure was used in another study, showing that the grafting degree of β-CD on alginate can be controlled and modulates the release/uptake of the model molecule methyl orange [52]. In addition, a composite based on sodium alginate grafted with β-CD using epichlorohydrin and NaOH was successfully obtained for the first time and used as a matrix in the immobilization of *Arthrobacter simplex* cells for cortisone acetate biotransformation [53].

Not only natural polymers were grafted with CD derivatives, but also synthetic ones. In this regard, the polyacrylic acid was modified by attaching a CD derivative (2-aminoethyl)amino-deoxy-β-CD) [54]. The study targeted to obtain the selfassembly of β-CD and adamantyl moieties covalently linked to polyacrylate networks for application in controlled complexation and release of ethyl orange, methyl orange, and methyl red. Another study developed a multistimulus responsive supramolecular hydrogel based on host-guest and electrostatic interactions between β-CD dimer and methoxy-azobenzene molecules grafted on polyacrylic acid [55]. The obtained materials showed thermo-, photo-, and pH-responsive behavior determining a reversible sol-gel transition.

Chabalala *et al*. described the grafting of β-CD molecules on polyacrylonitrile using citric and sulfuric acids as crosslinkers [56]. The nanofiber membranes produced by the electrospinning method were used for the adsorption of bromophenol blue and atrazine. Mono-(6-ethylenediamine-6-deoxy)-β-CD was appended in the presence of EDC/NHS crosslinking agents onto the external surface of a plasma separation membrane based on polyvinylidene fluoride [57].


*Supporting open minds since 2005*

Postharvest Technology - Recent Advances, New Perspectives and Applications http://dx.doi.org/10.5772/intechopen.95208 Edited by Md Ahiduzzaman

#### Contributors

Theophilus M. Maduabuchukwu Ikegwu, Clement C. Ezegbe, Chioke A. Okolo, Chigozie E. Ofoedu, Mahesh Prasad Thakur, Chandra Shekhar Shukla, Harvinder K Singh, Atma Nand Tripathi, P. Sudha, P. Rajkumar, A. Astina Joice, I.P. Sudagar, R. Arulmaru, Kofi Owusu-Akyaw Oduro, Shepard Ndlela, Nelson L. Mwando, Samira A. Mohamed, Trina Adhikary, Ch. Durga Hemanth Kumar, Zhongli Pan, Chang Chen, Ryusuke Oishi, Barkat Hussain, Rayees Ahmad, Shafiya Hassan, Showkat Ahmad, Syed Nighat, Yendrambamb K. Devi, Kounser Javeed, Salma Usmani, Mohd Javid Ansari, Sait Erturk, Mustafa Alkan, Federico Félix Hahn Schlam

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**2. Processes affecting the distribution of heat around the surface of the Earth**

The Sun is far enough away that the solar radiation arrives at the surface of the Earth as a beam. Since the Earth is round, the bulk of the energy arrives within the Tropics, 23.5° north and south of the Equator. Under present-day conditions, the Sun does not rise for at least a day in winter at latitudes higher than 66.7° north and south due to the tilt of the axis of the Earth.

**Figure 2** shows the typical fate of insolation in summer entering the atmosphere of the Earth at about 50°N latitude. In practice, the amount of insolation reaching the ground will also depend on the sum of the effects of numerous climatic cycles pulling in multiple directions [14, 18, 19]. There are a very large number of these interacting with one another, so they normally largely cancel one another out. However, they are also largely responsible for the continual noise in the climatic signal that makes it advisable to have a 30-year average of climatic data where possible.

There is one special group of cycles that are critical in determining the amount of solar radiation arriving at a given location on the surface of the Earth over long periods of time, *viz.*, the Milankovitch cycles first thought of by Adhemar (Croll [20, 21]). Milankovitch [22, 23] refined the calculations of the effect of the three types of Earth orbital movements that can alter the Sun's incoming radiation by up to 25% in the zone subtropical zones (30–60° north and south of the equator. They are the shape of the Earth's orbit (eccentricity, a 100,000 year cycle), the

#### **Figure 2.**

*Typical fate of the solar energy reaching the surface of the atmosphere and proceeding down towards the ground.*

angle of tilt of the Earth's axis relative to the Earth's orbital plane (obliquity, from 21.1–24.5°in a 41,000 year cycle), and the direction of the Earth's axis of rotation is pointing (precession, in a 23,000 year cycle).

Milankovich calculated that cold events might occur approximately every 41,000 years and subsequent research confirms that they did occur at 41,000-year intervals between one and three million years ago. About 800,000 years ago, the cycle of the Ice Ages lengthened to 100,000 years, matching Earth's eccentricity cycle [24]. However, the cycles should not affect the data for the last two decades.


**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.

 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 <IP_ADDRESS>) 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 <IP_ADDRESS>) 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).