Detoxification of olive mill wastewaters by Moroccan yeast isolates

A total of 105 yeast strains were isolated from Moroccan olive oil production plants and evaluated for their ability to grow in olive oil mill wastewaters (OMW). The 9 isolates that grew best on OMW were selected for further study to evaluate their effect on removal of organic pollutants and OMW phytotoxicity (barley seed germination test). The results showed that at least four yeast isolates effectively lowered the toxicity of this effluent in addition to providing very useful materials in terms of both yeast biomass (6 g/l DW) and an irrigation fluid. This group of yeast isolates significantly reduced the concentration of total phenols (44% removal) and Chemical Oxygen Demand, COD (63% removal). The best germination rate of 80% for undiluted OMW was obtained for strain Candida holstii that also increased the pH from 4.76 to 6.75. Principal component analysis of the results obtained for the best yeast strains confirmed the importance of COD and total phenol reduction along with increase of organic nitrogen and final pH for the improvement of germination rates and phytotoxic reduction. This study has highlighted the potential of indigenous yeasts in detoxification of olive mill wastewaters.     

Transgenic tobacco plants expressing a fungal laccase are able to reduce phenol content from olive mill wastewaters

A biotechnological approach was applied to reduce phenol content in olive mill wastewaters by transgenic tobacco plants. The cDNA laccase of poxC gene from Pleurotus ostreatus, carrying its own signal peptide for extracellular secretion, was transferred into the Nicotiana tabacum genome. Transgenic tobacco plants were obtained and the recombinant enzyme was secreted into the rhizosphere by the plant root apparatus, confirming the ability of the plant machinery to recognize the fungal POXC peptide signal leader appropriately as secretory tag. Total laccase activity assayed by ABTS in transgenic lines increased sharply compared to control plants. Moreover, plants cultivated in a hydroponic solution with the addition of olive mill wastewaters were able to reduce the total phenol content up to 70%.     

Reduction of phenolics content and COD in olive oil mill wastewaters by indigenous yeasts and fungi

Olive oil mill wastewaters (OOMW) cause a recurrent environmental pollution problem. The large concentration of phenolic compounds in the organic fraction of OOMW is principally responsible for the phytotoxicity and microbial growth inhibitory effects of the effluent. Candida boidinii, Geotrichum candidum, a Penicillium sp. and Aspergillus niger HA37 were isolated from OOMW. When cultivated directly on an undiluted OOMW-based medium containing 82 g l⁻¹ COD, these strains removed only 4–8% of chemical oxygen demand (COD) and phenolics. In contrast, reduction values attaining respectively 40–73% for phenolics and 45–78% for COD removal in the undiluted OOMW-based medium were obtained when using the strains gradually acclimated to high concentration of OOMW by successive stepwise transfer from media containing COD of 20.5 up to 82 g l⁻¹. Possibly, a sufficient production level of degradation and/or detoxification enzymes has to be attained to overcome the toxic effects of the phenolic fraction of concentrated OOMW. The present investigation calls attention to the necessity of acclimation for certain fungal and yeasts strains potentially useful for treating highly polluted effluents.     

Plant growth hormone production from olive oil mill and alcohol factory wastewaters by white rot fungi

In this study, olive oil mill and alcohol factory wastewaters have been tested as growth media for the production of plant growth hormones. Funalia trogii ATCC 200800 and Trametes versicolor ATCC 200801 have been tested. Gibberellic acid (GA₃), abscisic acid (ABA), indole acetic acid (IAA), and cytokinin were determined in the culture media of these fungi. Both organisms produced enhanced levels of all three hormones in the presence of either of the wastewaters.     

Determination of aerobic biodegradation kinetics of olive oil mill wastewater

Aerobic biological treatment was conducted for the treatment of high strength olive oil mill wastewater (OMW). Two different approaches were used for kinetic modeling of OMW biodegradation. TOC removal and CO₂–C evolution were monitored in an open and a closed bioreactor systems, respectively. Gompertz, Refractory organics plus first-order (RFO) and Chen–Hashimoto equations were applied to estimate the kinetic parameters by using the data from bioreactors. Furthermore, change in oxidation stage of carbon was monitored and temperature dependency of OMW biodegradation was investigated based on activation energy. At room temperature, 64% of TOC was removed in the open bioreactor while cumulative CO₂–C evolution was 6.32 g L⁻¹ in closed the bioreactor. Higher biodegradation efficiency and kinetic parameters were obtained at 25 °C rather than 10 °C. Gompertz and RFO equations provided better fitting with CO₂–C and TOC data, respectively. Experimental and kinetic estimations indicated that OMW constituted of approximately 30% refractory organics. The comparison of two different modeling approaches showed that kinetic modeling based on CO₂–C provided better correlation with the experimental data. Temperature coefficient indicated that biological degradation of OMW is slightly dependent on temperature.     

Bioremediation of olive oil mill wastewater and production of microbial biomass

Olive oil mill wastewater (OMWW) was used as a substrate for the culture of a mixture of edible fungi in order to obtain a potentially useful microbial biomass and to induce a partial bioremediation of this fastidious waste. Before fermentation, the OMWW underwent an alkaline-oxidative treatment with the aim of decreasing the polyphenolic content which is the main cause of its toxicity. The fungal mixture grew fairly well in the treated OMWW and reached a maximum of biomass production within about 14 days of fermentation at room temperature. Up to 150–160 g of wet biomass was obtained per liter of OMWW. Analysis of the partially dehydrated biomass revealed a protein content of about 13 g% and 6 g% of row fiber. A relevant presence of unsaturated fatty acids was found, as well as the presence of significant amounts of vitamins A and E, nicotinic acid, calcium, potassium and iron. The possibility of using the microbial biomass produced from OMWW as an additive to animal feed is discussed.     

Codigestion of olive oil mill wastewaters with manure, household waste or sewage sludge

Combined anaerobic digestion of oil mill effluent(OME) together with manure, household waste (HHW) orsewage sludge was investigated. In batch experimentsit was shown that OME could be degraded into biogaswhen codigested with manure. In codigestion with HHWor sewage sludge, OME dilution with water (1:5) wasrequired in order to degrade it. Using continuouslystirred lab-scale reactors it was shown thatcodigestion of OME with manure (50:50 and 75:25 OMEto manure ratios) was successful with a theoreticalOME utilization of 75% and with approx. 87%reduction of the lipids content in OME. An OMEutilization of approx. 55%, and lipid reduction of73% was reached in codigestion with HHW (50:50 and75:25 OME to HHW ratios). The results showed thatthe high buffering capacity contained in manure,together with the content of several essentialnutrients, make it possible to degrade OME withoutprevious dilution, without addition of externalalkalinity and without addition of external nitrogensource.     

Characterisation of Paenibacillus jamilae strains that produce exopolysaccharide during growth on and detoxification of olive mill wastewaters

A total of 10 bacterial strains were isolated from a compost of corn treated with olive mill wastewaters (OMW) and selected by their capacity to synthesize exopolysaccharides (EPS). Morphological, physiological, biochemical and nutritional tests were used for a phenotypic study. A numerical analysis showed that all strains were 90% similar to each other. A DNA–DNA hybridization assay confirmed that all the strains belonged to Paenibacillus jamilae species. All the characterized strains were able to produce EPS growing on OMW batch cultures. The strain which was able to produce the highest EPS yield was chosen to perform an assay for testing its putative detoxifying activity, and it showed to reduce more than half the toxic capacity of the OMW. The results presented in this study, indicated the possible perspectives for using these bacterial strains to produce EPS and contribute to the bioremediation of the waste waters that are produced in the olive oil elaboration process.     

Comparative study of olive oil mill wastewater treatment using free and immobilized Coriolopsis polyzona and Pycnoporus coccineus

The efficiency of the two white-rot fungi Pycnoporus coccineus and Coriolopsis polyzona in the Olive Oil Mill Wastewater (OOMW) treatment was investigated. Both fungi were active in the decolourisation and COD removal of OOMW at 50 g/L COD, but only the first fungus remains effective on the crude effluent (COD=100 g/L). Moreover P. coccineus was less affected by oxygen supplementation and exhibited a high tolerance to agitation in comparison to C. polyzona. However, it required a nitrogen supplementation to obtain faster and higher COD removal. To overcome the negative effect of agitation on fungi growth and efficiency, immobilisation of C. polyzona and P. coccineus in polyurethane foam was applied. The immobilized system showed better COD decreases during three consecutive batches without remarkable loss of performances. The results obtained in this study suggested that immobilized C. polyzona and especially immobilized P. coccineus might be applicable to a large scale for the removal colour and COD of OOMW.     

Development and characterization of edible chitosan/olive oil emulsion films

The properties of plasticized chitosan-olive oil emulsion films prepared with increasing oil concentrations were investigated. Emulsifying nature of chitosan was enough to stabilize olive oil droplets in the film forming emulsions; hence homogeneous, thin and translucent films were obtained in all cases. The homogeneity of the lipid globules distribution in the films was confirmed by contact angle measurements and optical microscopy. All the tensile properties (Young Modulus, strength and maximum elongation) increased with olive oil concentration and were explained considering the interactions developed between lipid and carbohydrate phases in addition to the lubricant characteristics of the oil. Moisture sorption, water vapor permeation through the films and effective diffusion coefficients decreased as oil concentration increases, as a result of the non-polar nature of the lipid. Total soluble matter measurements were used to confirm the development of strong associations between chitosan and olive oil.     

Future prospects and constraints of olive mill wastewaters use and treatment: A review

There has been an increasing number of biotechnical processes for use or treatment of olive mill wastewaters (OMW) over the last twenty years, both at laboratory size and on pilot scale. This paper reviews the methods described in the literature emphasizing the most important features and constraints of each of these processes.     

Activity and location of olive oil phenolic antioxidants in liposomes

The antioxidant activity of hydroxytyrosol, hydroxytyrosol acetate, oleuropein, 3,4-dihydroxyphenylelenolic acid (3,4-DHPEA-EA) and 3,4-dihydroxyphenylelenolic acid dialdehyde (3,4-DHPEA-EDA) towards oxidation initiated by 2,2'-azobis(2-amidinopropane) hydrochloride in a soybean phospholipid liposome system was studied. The antioxidant activity of these olive oil phenols was similar and the duration of the lag phase was almost twice that of alpha-tocopherol. Trolox, a water-soluble analogue of alpha-tocopherol, showed the worst antioxidant activity. However, oxidation before the end of the lag phase was inhibited less effectively by the olive oil phenols than by alpha-tocopherol and Trolox. Synergistic effects (11-20% increase in lag phase) were observed in the antioxidant activity of combinations of alpha-tocopherol with olive oil phenols both with and without ascorbic acid. Fluorescence anisotropy of probes and fluorescence quenching studies showed that the olive oil phenols did not penetrate into the membrane, but their effectiveness as antioxidants showed they were associated with the surface of the phospholipid bilayer.     

Production of added‐value metabolites by Yarrowia lipolytica growing in olive mill wastewater‐based media under aseptic and non‐aseptic conditions

Yarrowia lipolytica ACA‐YC 5033 was grown on glucose‐based media in which high amounts of olive mill wastewaters (OMWs) had been added. Besides shake‐flask aseptic cultures, trials were also performed in previously pasteurized media while batch bioreactor experiments were also done. Significant decolorization (～58%) and remarkable removal of phenolic compounds (～51% w/w) occurred, with the latter being amongst the highest ones reported in the international literature, as far as yeasts were concerned during their growth on phenol‐containing media. In nitrogen‐limited flask fermentations the microorganism produced maximum citric acid quantity ≈19.0 g/L [simultaneous yield of citric acid produced per unit of glucose consumed (Y_Cit/Glc)≈0.74 g/g]. Dry cell weight (DCW) values decreased at high phenol‐containing media, but, on the other hand, the addition of OMWs induced reserve lipid accumulation. Maximum citric acid concentration achieved (≈52.0 g/L; Y_Cit/Glc≈0.64 g/g) occurred in OMW‐based high sugar content media (initial glucose added at ≈80.0 g/L). The bioprocess was successfully simulated by a modified logistic growth equation. A satisfactory fitting on the experimental data occurred while the optimized parameter values were found to be similar to those experimentally measured. Finally, a non‐aseptic (previously pasteurized) trial was performed and its comparison with the equivalent aseptic experiment revealed no significant differences. Yarrowia lipolytica hence can be considered as a satisfactory candidate for simultaneous OMWs bioremediation and the production of added‐value compounds useful for the food industry.     

Biodegradation of olive oil mill wastewaterby Coriolus versicolor and Funalia trogii:effects of agitation, initial COD concentration, inoculum size and immobilization

The biodegradation of olive oil mill wastewater (OOMW) by Coriolus versicolor and Funalia trogii was investigated. Initial COD concentration, agitation and inoculum size were all found to be significant for biodegradation. Adding glucose, sulphate or nitrogen had no effect on biodegradation. During growth in optimum conditions, C.versicolor removed approximately 63% COD, 90% phenol and 65% colour within 6 days and F. trogii removed approximately 70% COD, 93% phenol and 81% colour of the OOMW used. The fungi also excreted large amounts of extracellular laccase into the medium. High biodegradation yields were also obtained by fungi immobilized in calcium alginate gels.     

Optimisation of biodegradation conditions for the treatment of olive mill wastewater

The aim of the present paper was to optimise the conditions of aerobic treatment of olive mill wastewater. To do so, the waste was treated following the experimental optimal design methodology studying the set of factors susceptible to influence the treatment (pH, C/N ratio, aeration and temperature). The results of a first series of experiments showed a strong correlation between the reduction in the levels of polyphenols and three of the parameters studied, i.e. the C/N ratio, aeration and temperature. Optimised conditions led to a 94% drop in polyphenols.

Then, for a finer study of the conditions, just two parameters were varied, the pH and the C/N ratio. The results showed that the conditions of pH modification (addition of lime or sodium hydroxide) and the C/N ratio (urea or ammonium nitrate) allowed the microbiological activity to be very significantly improved. This led to polyphenol reductions of 51% and 76%.     

Influence of olive oil and its components on mesenchymal stem cell biology

Extra virgin olive oil is characterized by its high content of unsaturated fatty acid residues in triglycerides, mainly oleic acid, and the presence of bioactive and antioxidant compounds. Its consumption is associated with lower risk of suffering chronic diseases and unwanted processes linked to aging, due to the antioxidant capacity and capability of its components to modulate cellular signaling pathways. Consumption of olive oil can alter the physiology of mesenchymal stem cells(MSCs). This may explain part of the healthy effects of olive oil consumption, such as prevention of unwanted aging processes. To date,there are no specific studies on the action of olive oil on MSCs, but effects of many components of such food on cell viability and differentiation have been evaluated. The objective of this article is to review existing literature on how different compounds of extra virgin olive oil, including residues of fatty acids,vitamins, squalene, triterpenes, pigments and phenols, affect MSC maintenance and differentiation, in order to provide a better understanding of the healthy effects of this food. Interestingly, most studies have shown a positive effect of these compounds on MSCs. The collective findings support the hypothesis that at least part of the beneficial effects of extra virgin olive oil consumption on health may be mediated by its effects on MSCs.     

Transport mechanism and metabolism of olive oil hydroxytyrosol in Caco-2 cells

3,4-dihydroxyphenylethanol (hydroxytyrosol; DPE) is the major phenolic antioxidant present in extra virgin olive oil, either in a free or esterified form. Despite its relevant biological effects, no data are available on its bioavailability and metabolism. The aim of the present study is to examine the molecular mechanism of DPE intestinal transport, using differentiated Caco-2 cell monolayers as the model system. The kinetic data demonstrate that [(14)C]DPE transport occurs via a passive diffusion mechanism and is bidirectional; the calculated apparent permeability coefficient indicates that the molecule is quantitatively absorbed at the intestinal level. The only labelled DPE metabolite detectable in the culture medium by HPLC (10% conversion) is 3-hydroxy-4-methoxyphenylethanol, the product of catechol-O-methyltransferase; when DPE is assayed in vitro with the purified enzyme a K(m) value of 40 microM has been calculated.     

Entrapment of lipase into K-carrageenan beads and its use in hydrolysis of olive oil in biphasic system

The porcine pancrease lipase was immobilized by entrapment in the beads of K-carrageenan and cured by treatment with polyethyleneimine (PEI) in the phosphate buffer. The retention of hydrolytic activity of lipase and compressive strength of the beads were examined. The activity of free and immobilized lipase was assessed by using olive oil as the substrate. The immobilized enzyme exhibited a little shift towards acidic pH for its optimal activity and retained 50% of its activity after 5 cycles. When the enzyme concentration was kept constant and substrate concentration was varied the K_m and V_max were observed to be 0.18 × 10⁻² and 0.10, and 0.10 × 10⁻² and 0.09 respectively, for free and for entrapped enzymes. When the substrate concentration was kept constant and enzyme concentration was varied, the values of K_m and V_max were observed to be 0.19 × 10⁻⁷ and 0.41, and 0.18 × 10⁻⁷ and 0.41 for free and entrapped enzymes. Though this indicates that there is no conformational change during immobilization, it also shows that the reaction velocity depends on the concentration. Immobilized enzyme showed improved thermal and storage stability. Hydrolysis of olive oil in organic–aqueous two-phase system using fixed bed reactor was carried out and conditions were optimized. The enzyme in reactor retained 30% of its initial activity after 480 min (12 cycles).     

The fate of olive oil polyphenols in the gastrointestinal tract: Implications of gastric and colonic microflora-dependent biotransformation

We have conducted a detailed investigation into the absorption, metabolism and microflora-dependent transformation of hydroxytyrosol (HT), tyrosol (TYR) and their conjugated forms, such as oleuropein (OL). Conjugated forms underwent rapid hydrolysis under gastric conditions, resulting in significant increases in the amount of free HT and TYR entering the small intestine. Both HT and TYR transferred across human Caco-2 cell monolayers and rat segments of jejunum and ileum and were subject to classic phase I/II biotransformation. The major metabolites identified were an O-methylated derivative of HT, glucuronides of HT and TYR and a novel glutathionylated conjugate of HT. In contrast, there was no absorption of OL in either model. However, OL was rapidly degraded by the colonic microflora resulting in the formation of HT. Our study provides additional information regarding the breakdown of complex olive oil polyphenols in the GI tract, in particular the stomach and the large intestine.     

Selection of non-conventional yeasts and their use in immobilized form for the bioremediation of olive oil mill wastewaters

The yeast population dynamics in olive wastewaters (OMW), sampled in five mills from Salento (Apulia, Southern Italy), were investigated. Three hundred yeasts were isolated in five industrial mills and identified by molecular analysis. Strains belonging to Geotrichum, Saccharomyces, Pichia, Rhodotorula and Candida were detected. Five G. candidum strains were able to grow in OMW as the sole carbon source and to reduce phenolics, chemical oxygen demand (COD) and antimicrobial compounds. One G. candidum isolate was selected for whole-cell immobilization in calcium alginate gel. The COD and phenolic reduction obtained with immobilized cells showed a 2.2- and 2-fold increase compared to the removal obtained with free cells, respectively. The immobilization system enhanced yeast oxidative activity by avoiding the presence of microbial protease in treated OMW. To our knowledge, this is the first report on G. candidum whole-cell immobilization for OMW bioremediation.