**4.7 Pharmaceutical**

The enhancing effect of various concentrations of 18 oils and a silicon antifoam agent on erythromycin antibiotic production by *Saccharopolyspora erythraea* was evaluated in a complex medium containing soybean flour and dextrin as the main substrates. The highest titer of erythromycin was produced in medium containing 55 g/l black cherry kernel oil (4.5 g/l). The titers of erythromycin in the other media were also recorded, with this result: black cherry kernel > water melon seed > melon seed > walnut > rapeseed > soybean > (corn = sesame) > (olive = pistachio = lard = sunflower) > (hazelnut = cotton seed) > grape seed > (shark = safflower = coconut). In medium supplement with olive oil, concentration of erythromycin was 2.15±0.03 and 2.75±0.02 g/l before and after optimization, respectively (Hamedi et al. 2004).

### **4.8 Biofuels**

It is widely recognised that clean and sustainable technologies, e.g. biofuels, are only part of the solution to the impending energy crisis. Comparing the heating value of biohydrogen (121 MJ/kg), methane (50.2 MJ/kg) and bioethanol (23.4 MJ/kg), the production of hydrogen will be more attractive. Nevertheless, the use of biohydrogen is still not practical and thus there is a higher demand for methane and bioethanol because they can be used directly as biofuels with the existing technology (Duerr et al. 2007).

Ethanol production as a biofuel from OMWs with high content of organic matter is interesting (Li et al. 2007). The two main components of TPOMW (stones and olive pulp) as substrates were used to production of ethanol by a simultaneous saccharification and fermentation process (Ballesteros et al. 2001). In recent study, an enzymatic hydrolysis and subsequent glucose fermentation by baker's yeast were evaluated for ethanol production

homologs, (Rha-C10-C10) and (Rha-Rha-C10-C10), when grown in olive oil waste water or

A few edible fungi, especially species of *Pleurotus*, can also be grown using OMWs as the source of nutrients by the application of different strategies. Recently the cultivation of the

Hydroxy fatty acids (HFAs) are known to have special properties such as higher viscosity and reactivity compared to other normal fatty acids. These special properties used in a wide range of applications including resins, waxes, nylons, plastics, lubricants, cosmetics, and additives in coatings and paintings. Some HFAs are also reported as antimicrobial agents against plant pathogenic fungi and some of food-borne bacteria. Bacterium *Pseudomonas aeruginosa* PR3 produce several hydroxy fatty acids from different unsaturated fatty acids. Of those hydroxy fatty acids, 7,10-dihydroxy-8(E)-octadecenoic acid (DOD) was efficiently produced from oleic acid by strain PR3. DOD production yield from olive oil was 53.5%. Several important environmental factors were also tested. Galactose and glutamine were optimal carbon and nitrogen sources, and magnesium ion was required for DOD production

The enhancing effect of various concentrations of 18 oils and a silicon antifoam agent on erythromycin antibiotic production by *Saccharopolyspora erythraea* was evaluated in a complex medium containing soybean flour and dextrin as the main substrates. The highest titer of erythromycin was produced in medium containing 55 g/l black cherry kernel oil (4.5 g/l). The titers of erythromycin in the other media were also recorded, with this result: black cherry kernel > water melon seed > melon seed > walnut > rapeseed > soybean > (corn = sesame) > (olive = pistachio = lard = sunflower) > (hazelnut = cotton seed) > grape seed > (shark = safflower = coconut). In medium supplement with olive oil, concentration of erythromycin was 2.15±0.03 and 2.75±0.02 g/l before and after optimization, respectively

It is widely recognised that clean and sustainable technologies, e.g. biofuels, are only part of the solution to the impending energy crisis. Comparing the heating value of biohydrogen (121 MJ/kg), methane (50.2 MJ/kg) and bioethanol (23.4 MJ/kg), the production of hydrogen will be more attractive. Nevertheless, the use of biohydrogen is still not practical and thus there is a higher demand for methane and bioethanol because they can be used

Ethanol production as a biofuel from OMWs with high content of organic matter is interesting (Li et al. 2007). The two main components of TPOMW (stones and olive pulp) as substrates were used to production of ethanol by a simultaneous saccharification and fermentation process (Ballesteros et al. 2001). In recent study, an enzymatic hydrolysis and subsequent glucose fermentation by baker's yeast were evaluated for ethanol production

directly as biofuels with the existing technology (Duerr et al. 2007).

oyster mushroom *Pleurotus ostreatus* was suggested on OMWW (KalmIs et al. 2008).

in waste frying oils consisting from olive/sunflower (Pantazaki et al. 2010).

**4.6 Food and cosmetics** 

from olive oil (Suh et al. 2011).

**4.7 Pharmaceutical** 

(Hamedi et al. 2004).

**4.8 Biofuels** 

using dry matter of TPOMW. The results showed that yeasts could effectively ferment TPOMW without nutrient addition, resulting in a maximum ethanol production of 11.2 g/l and revealing the tolerance of yeast to TPOMW toxicity (Georgieva and Ahring 2007).

Anaerobic digestion is a biological process in which organic material is broken down by microorganisms. Unlike composting, the process occurs in the absence of air. Anaerobic digestion is a practical alternative for the treatment of TPOMW, which produces biogas. The TPOMW is biodegradable by anaerobic digestion at mesophilic temperatures in stirred tank reactors, with COD removal efficiencies in the range of 72–89% and an average methane yield coefficient of 0.31 dm3 CH4 per gramme COD removed. Hydrogen production was coupled with a subsequent step for methane production, giving the potential for production of 1.6 mmol H2 per gramme of TPOMW (Borja et al. 2006).

The OMW used as a sole substrate for the production of hydrogen gas with *Rhodobacter sphaeroides* O.U.001. The bacterium was grown in diluted OMW media, containing OMW concentrations between 20% and 1% in a glass column photobioreactor at 32°C. The released gas was nearly pure hydrogen, which can be utilized in electricity producing systems, such as fuel cells. The maximum hydrogen yield (145 ml) was obtained with 3% and 4% OMW concentrations. However, as well as hydrogen production, COD, BOD and phenol reduction from OMW were recorded (Eroglu et al. 2004).

Biodiesel, a fuel that can be made from renewable biological sources such as vegetable oils or animals fats, has been recognized recently as an environment friendly alternative fuel for diesel engines. Among liquid biofuels, biodiesel derived from vegetable oils is gaining ground and market share as diesel fuel in Europe and the USA. A mixture of frying olive oil and sunflower oil for the production of methyl esters that can be used as biodiesel (Encinar et al. 2005).

#### **4.9 Biofertilizers**

As far as agronomic use of the waste is concerned, the idea of re-using microbially treated OMWW as fertiliser has been also proposed. An acidogenic fungus strain *Aspergillus niger* was grown in either free or immobilised form on OMWW with rock phosphate added in order to solubilise it. It was found that at optimized process conditions (moisture 70%; corn steep liquor as a nitrogen source; inoculum size of 3-4 ml; presence of slow release phosphate), the filamentous fungal culture was able to produce 58 U phytase/g dry substrate and 31 mg soluble phosphate per flask (Vassilev et al. 1997; Vassilev et al. 2007).

#### **4.10 Biomass**

Already 50 years ago, the production of yeast biomass using OMWW in a chemostat for use in industrial applications was reported. The microbial biomass produced from OMW fermentations either as an additive to animal feed or to improve its agronomic use. For example, an intense degradation of most polluting substances of OMWW and the production of biomass could be used as an animal feed integrator using a chemical– biological method (Morillo et al. 2009).

Seven strains of *Penicillium* isolated from OMWW disposal ponds were tested for biomass production and biodegradation of undiluted OMWW. Best results were obtained by using

adjust C/N ration between 25-30. At the end of 50 days of composting using *Trichoderma harzianum* and *Phanerochaete chrysosporium*, cellulose and lignin were highly degraded. It was found that after 30 days, *P. chrysosporium* and *T. harzianum* degraded approximately 71.9%

Treated OMW may find applications as a raw material in various biotechnological processes or as animal food. The appropriate utilization of by-products in animal nutrition can improve the economy and the efficiency of agricultural, industrial and animal production. The olive pomace was alkali-treated, transferred to culture flasks and inoculated with the above fungi. After inoculation, the fermentation process was carried out at 25°C for 60 days. The results indicated that *Oxysporus* spp. degraded lignin up to 69%, whereas *Phanerochaete chrysosporium* and *Schizophyllum commune* delignified olive pomace 60% and 53%, respectively. However, the potential use of treated olive pomace as a feed for poultry is still under investigation. The fermented olive pomace can be used as a feed for the poultry

The olive oil industry generates large amounts of olive mill wastes (OMWs) as by-products that are harmful to the environment. About 30 million tons of OMWs per year are produced in the world. Thus, more research is needed on the development of new bioremediation technologies and strategies of OMWs, as well as the valorisation by microbial biotechnology. The fermentation of fatty low-value renewable carbon sources like OMWs aiming at the production of various added-value metabolites is a noticeable interest in the

Microbiological studies show that presence of yeasts, but not of bacteria and moulds in the olive oil. Some of the yeasts are considered useful as they improve the organoleptic characteristics of the oil during preservation, whereas others are considered harmful as they can damage the quality of the oil through the hydrolysis of the triglycerides. Olive oil and its by-products could provide a source of low-cost fermentation substrate and isolation of new

OMWs treatment processes that employ physical, chemical, biological and combined technologies have been tested. Among the different options, biological treatments or bioremediation are considered the most environmentally compatible and the least expensive. Bioremediation occurs either under aerobic or anaerobic conditions. Aerobic processes are applied waste streams of OMWs with low organic loads, whereas anaerobic

Microbial biotechnology strategies and methods in olive oil industry were used to reduce chemical oxygen demand (COD), biological oxygen demand (BOD) and phenolic compounds of OMWs with a concomitant production of biotechnologically valuable products such as enzymes (lipases, β-glucosidase, phytase, tannase, lignin peroxidase, manganese peroxidise, laccase and pectinases), organic acids (citric, isocitric and oxalic acids), biopolymers and biodegradable plastics (xanthan, β-glucan and polyhydroxyalkanoates), biosurfactants, food

sector of industrial microbiology and microbial biotechnology.

processes are applied waste streams with high organic loads.

microorganisms with biotechnological potentials.

of the lignin and 59.25% of the cellulose, respectively (Haddadin et al. 2009).

**4.12 Animal feed** 

**5. Conclusion** 

industry (Haddadin et al. 2002).

strain P4, which formed 21.50 g (dry weight) of biomass per litre of undiluted wastewater after 20 days of cultivation. This and other strains also carried out an outstanding reduction of the COD and the phenolic content of OMW, as well as a pH raise (Robles et al. 2000). The *Y. lipolytica* strain ATCC 20255 strain has been effective in the treatment of OMWW that yield of the biomass (single-cell protein) was 22.45 g/l (Scioli and Vollaro 1997).

Microalgal biomass is as a potential source of proteins, carbohydrates, pigments, lipids, and hydrocarbons. In addition, the biomass can be used as a low-release fertilizer. This chemical composition has great variation, depending on the species, culture medium, and the operating conditions. Microalga *Scenedesmus obliquus* was used to biomass production from rinse water (RW) from two-phase centrifugation in the olive-oil extraction industry. Maximum specific growth rate, 0.044 per hour was registered in the culture with 5% RW and reduces 67.4% BOD when operating with 25% RW. The greater specific rate of protein synthesis during the exponential phase was 3.7 mg/g h to 50% RW (Hodaifa et al. 2008).

Microbial lipid (single cell oil or SCO) production has been an object of research and industrial interest for more than 60 years. Microorganisms can store triacylglycerol (TAG) as intracellular oil droplets. *Gordonia* sp. DG accumulated more than 50% lipid with most tested wastes, while only 29, 36 and 41% was accumulated in presence of olive mill waste, hydrolyzed barely seeds and wheat bran, respectively (Gouda et al. 2008).

Carbon-limited cultures were performed on waste cooking olive oil, added in the growth medium at 15 g/l, and high biomass quantities were produced up to 18 g/l. Cellular lipids were accumulated in notable quantities in almost all cultures. *Aspergillus* sp. ATHUM 3482 accumulated lipid up to 64% (w w) in dry fungal mass. In parallel, extracellular lipase activity was quantified, and it was revealed to be strain and fermentation time dependent, with a maximum quantity of 645 U/ml being obtained by *Aspergillus niger* NRRL 363. Storage lipid content significantly decreased at the stationary growth phase (Papanikolaou et al. 2011).

#### **4.11 Compost**

Composting is the aerobic processing of biologically degradable organic waste to produce a reasonably stable, granular material and valuable plant nutrients. Composting removes the phytotoxicity of the residues within a few weeks and allows the subsequent enrichment of croplands with nutrients that were originally taken up by olive tree cultivation. Composting of OMWs requires the proper adjustment of pH, temperature, moisture, oxygenation and nutrients, thereby allowing the adequate development of the microbial populations (Arvanitoyannis and Kassaveti 2007).

Among the possible technologies for recycling the TPOMW, composting is gaining interest as a sustainable strategy to recycle this residue for agricultural purposes. Dry olive cake alone or mixed with municipal biosolids vermicomposted for 9 months in order to examine the behaviour of three specific humic substance-enzyme complexes. During the process, βglucosidase synthesis and release was observed, whereas no significant change in urease and phosphatase activity was recorded. The vermicomposted olive cake, alone or in blends with biosolids, could be effectively used as amendment due to their ability to reactivate the C, P and N-cycles in degraded soils for regeneration purposes (Benitez et al. 2005).

Olive pomace, a wet solid waste from the three-phase decanters and presses, was composted by using a reactor for a period of 50 days in four bioreactors. Urea was added to adjust C/N ration between 25-30. At the end of 50 days of composting using *Trichoderma harzianum* and *Phanerochaete chrysosporium*, cellulose and lignin were highly degraded. It was found that after 30 days, *P. chrysosporium* and *T. harzianum* degraded approximately 71.9% of the lignin and 59.25% of the cellulose, respectively (Haddadin et al. 2009).
