Olive-mill wastewaters: a promising substrate for microbial lipase production

The present study investigated the valorization of olive-mill wastewater (OMW) by its use as a possible growth medium for the microbial production of extra-cellular lipase. To this end, strains of Geotrichum candidum (NRRL Y-552 and Y-553), Rhizopus arrhizus (NRRL 2286 and ISRIM 383), Rhizopus oryzae (NRRL 6431), Aspergillus oryzae (NRRL 1988 and 495), Aspergillus niger (NRRL 334), Candida cylindracea (NRRL Y-17506) and Penicillium citrinum (NRRL 1841 and 3754, ISRIM 118) were screened. All strains were able to grow on the undiluted OMW, producing extra-cellular lipase activity. C. cylindracea NRRL Y-17506 showed the highest lipase activity on all the typologies of OMW used. Its lipase production on OMW was markedly affected by the type of nitrogen source and was induced by the addition of olive oil. The highest activity (9.23 IU ml(-1)) of the yeast was obtained on OMW supplemented with NH(4)Cl (2.4 g l(-1)) and olive oil (3.0 g l(-1)).     

Activity and elution profile of laccase during biological decolorization and dephenolization of olive mill wastewater

The performance and enzymatic strategy exhibited by basidiomycete Euc-1, a laccase producing strain, was investigated during the biodegradation of olive mill wastewater (OMW). This strain yielded better decolorization of solidified OMW than Phanerochaete chrysosporium and removed 90% of phenols (initial concentration=800 mg l(-1)), 73% of color (initial A465=4.4), and 45% of chemical oxygen demand in batch cultures containing OMW. Since partial phenol removal occurred before the detection of enzymatic activity, no plausible correlation could be established between them. In contrast, decolorization occurred only after the detection of laccase activity and coincided with its production over time. Two laccase fractions (Lac1 and Lac2) were separated by chromatography. OMW strongly induced Lac2 that was almost absent in defined liquid medium. Furthermore, Lac2 was the main laccase fraction in the presence of OMW. This study pointed out that basidiomycete Euc-1 and its ligninolytic system could be a useful tool for the bioremediation of wastewater generated in the process of olive oil extraction.     

Plasma membrane from Candida tropicalis grown on glucose or hexadecane. II. Biochemical properties and substrate-induced alterations.

Isolated plasma membranes from the yeast Candida tropicalis grown on two different carbon sources (glucose or hexadecane), had similar contents of protein (60% of total dry weight), lipid (21-24%) and carbohydrates (16-21%). Sodium dodecyl sulphate gel electrophoresis of the membrane proteins revealed 17 and 19 protein bands, respectively, for glucose and hexadecane grown cells. There were marked differences in RF values and relative peak heights between the two gels. Sterols and free fatty acids were the major components of the plasma membrane lipids. Phospholipid content was less than 2% of total plasma membrane lipids. Membrane microviscosity, as determined by fluorescence polarization, was very high (16.6 P). Fatty acid determination of membrane lipids by gas chromatography showed a significant increase of C16 fatty acids in plasma membranes of cells grown on hexadecane. Reduced-oxidized difference spectra demonstrated the presence of a b-type cytochrome in both Saccharomyces cerevisiae and C. tropicalis plasma membranes. Its concentration in C. tropicalis plasma membranes was three-fold greater in cells grown on hexadecane than in glucose grown cells.     

Strategies for dephenolization of raw olive mill wastewater by means of <Emphasis Type="Italic">Pleurotus ostreatus</Emphasis>

The reduction of polyphenols content in olive mill wastewater (OMW) is a major issue in olive oil manufacturing. Although researchers have pointed out the potential of white-rot fungus in dephenolizing OMW, the results available in the literature mainly concern pretreated (sterilized) OMW. This paper deals with the reduction of polyphenols content in untreated OMW by means of a white-rot fungus, Pleurotus ostreatus. Dephenolization was performed both in an airlift bioreactor and in aerated flasks. The process was carried out under controlled non-sterile conditions, with different operating configurations (batch, continuous, biomass recycling) representative of potential industrial operations. Total organic carbon, polyphenols concentration, phenol oxidase activity, dissolved oxygen concentration, oxygen consumption rate, and pH were measured during every run. Tests were carried out with or without added nutrients (potato starch and potato dextrose) and laccases inducers (i.e., CuSO₄). OMW endogenous microorganisms were competing with P. ostreatus for oxygen during simultaneous fermentation. Dephenolization of raw OMW by P. ostreatus under single batch was as large as 70%. Dephenolization was still extensive even when biomass was recycled up to six times. OMW pre-aeration had to be provided under continuous operation to avoid oxygen consumption by endogenous microorganisms that might spoil the process. The role of laccases in the dephenolization process has been discussed. Dephenolization under batch conditions with biomass recycling and added nutrients proved to be the most effective configuration for OMW polyphenols reduction in industrial plants (42–68% for five cycles).     

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.     

Phenolic removal in olive oil mill wastewater using loofah-immobilized Phanerochaete chrysosporium

Summary Olive oil mill wastewater (OMW) has a high organic load, and this is a serious concern of the olive industry. Conventional biological wastewater treatments, despite their simplicity and suitable performance are ineffective for OMW treatment since phenolics possess antimicrobial activity. In order to carry out a proper treatment of OMW, use of a microorganism able to degrade the phenolics is thus necessary. In this study the ability of Phanerochaete chrysosporium to degrade the phenolic compounds of OMW and to decrease the chemical oxygen demand (COD) using cells immobilized on loofah was examined. The basal mineral salt solution along with glucose, ammonium sulfate and yeast extract was used to dilute the OMW appropriately. The fungus did not grow on the concentrated OMW. The extent of removal in this bio-treatment, of total phenols (TP) and the COD were 90 and 50%, respectively, while the color and aromaticity decreased by 60 and 95%, respectively. The kinetic behavior of the loofah-immobilized fungus was found to follow the Monod equation. The maximum growth rate μ_max was 0.045 h⁻¹ while the Monod constant based on the consumed TP and COD were (mg/l) 370 and 6900, respectively.     

Olive oil mill waste waters decoloration and detoxification in a bioreactor by the white rot fungus Phanerochaete flavido-alba

Olive oil mill wastewater (OMW) is produced as waste in olive oil extraction. With the purpose of treating this highly polluting waste, a number of experiments were conducted in a laboratory-scale bioreactor with the white rot fungus Phanerochaete flavido-alba (P. flavido-alba). It is known that this fungus is capable of decolorizing OMW in static or semistatic cultures at Erlenmeyer scale and at 30 degrees C. The objective of this work was to prove that P. flavido-alba could decolorize OMW in submerged cultures and that it is capable of reducing OMW toxicity at room temperature (25 degrees C) and in a laboratory-scale bioreactor. In the experiments conducted, manganese peroxidase (MnP) and laccase enzymes were detected; however, unlike other studies, lignin peroxidase was not found to be present. Decoloration obtained after treatment was 70%. The reduction of aromatic compounds obtained was 51%, and the toxicity of the culture medium was reduced by up to 70%. We can therefore state that P. flavido-alba is capable of reducing important environmental parameters of industrial effluents and that prospects are positive for the use of this process at a larger scale, even when working at room temperature.     

Reuse of microbially treated olive mill wastewater as fertiliser for wheat (Triticum durum Desf.)

Free cells of Aspergillus niger were grown on olive mill wastewater (OMW) supplemented with rock phosphate (RP) in an air-lift bioreactor in batch and repeated-batch processes. The fungus grew well and reduced the chemical oxygen demand of the waste by 35% and 64% in the batch and repeated-batch (fourth batch) processes, respectively. Total sugar content was consistently reduced (ca. 60%) in both processes while reduction of total phenols was minimal. RP was solubilised and maximum soluble P was 0.63 and 0.75 gl(-1) in the batch and repeated-batch (third batch), respectively. Several types of OMW+/-RP, microbially-treated or not, were tested in a greenhouse for their fertilising ability on a soil-wheat (Triticum durum Desf.) model system. Beneficial effects were highest using OMW treated by the repeated-batch process. The treated plants showed an increase in seed biomass, spike number, and kernel weight. Harvest index was highest (0.49+/-0.04) after treatment with OMW from the repeated-batch process.     

Biological treatment of olive mill wastewater by non-conventional yeasts

The ability of lipolytic yeasts to grow on olive mill wastewater (OMW)-based medium and to produce high-value compounds while degrading this waste, was tested. OMW collected from three-phase olive mills from the North region of Portugal were characterized and used. OMW with COD ranging from 100 g L⁻¹ to 200 g L⁻¹ were supplemented with yeast extract and ammonium chloride. Studies of OMW consumption were carried out in batch cultures of Candida rugosa, Candida cylindracea and Yarrowia lipolytica. All strains were able to grow in the OMW-based media, without dilution, to consume reducing sugars and to reduce COD. C. cylindracea was the best strain concerning the lipase production and the reduction of phenolic compounds and COD. For all strains, the phenols degradation was quite difficult, mostly when more easily degradable carbon source is still present in the medium. Among the phenolic compounds tested catechol is the most inhibitory to the cells.     

Aerobic biodegradation and detoxification of wastewaters from the olive oil industry

Growth and polyphenol biodegradation by three microorganisms namely Geotrichum sp., Aspergillus sp. and Candida tropicalis were studied on olive mill wastewater (OMW). These three microorganisms were selected for their tolerance to the polyphenols. The biodegradation process of OMW was investigated in batch regime by conducting experiments where the initial concentration of chemical oxygen demand (COD) was varied. Furthermore, some tests were performed to determine the most important nutrients necessary for aerobic degradation of OMW. Average COD removals were 55.0%, 52.5% and 62.8% in wastewaters fermented with Geotrichum sp., Aspergillus sp. and C. tropicalis, respectively. The maximum removal of polyphenols was 46.6% (Geotrichum sp.), 44.3% (Aspergillus sp.) and 51.7% (C. tropicalis). In addition, significant decolorization was evident.     

Decolourisation and dephenolisation potential of selected <Emphasis Type="Italic">Aspergillus</Emphasis> section <Emphasis Type="Italic">Nigri</Emphasis> strains – <Emphasis Type="Italic">Aspergillus tubingensis</Emphasis> in olive mill wastewater

Aspergillus section Nigri strains Aspergillus aculeatus Ege-K 258, A. foeditus var. pallidus Ege-K156, A. niger Ege-K 4 and A. tubingensis Ege-K 265 were used to treat olive mill wastewater (OMW) in an investigation aimed at exploring their dephenolisation and decolourisation ability and, consequently, the economic feasibility of using any or all of these strains in a pre-treatment step in the processing of OMW. Of these strains A. tubingensis Ege-K 265 resulted in an 80% decolourisation of twofold-diluted OMW and a 30% decolourisation of undiluted OMW; in addition, it was able to remove approximately 30% of all phenolic compounds in both twofold-diluted and undiluted OMW. We conclude that A. tubingensis Ege-K 265 could be effectively used in the pre-treatment step of a combined aerobic-anaerobic process to solve the environmental problems caused by OMW in Mediterranean countries.     

Organic matter humification in olive oil mill wastewater by abiotic catalysis with manganese(IV) oxide

The chemical changes occurring in an olive oil mill wastewater (OMW) sample digested catalytically with MnO(2) for 30 and 60 days were evaluated comparatively with those occurring in the same OMW left standing for the same time in an open-air lagoon. Both treatments increased the pH and electrical conductivity and decreased the contents of dry matter, total organic C and total N, and C/N ratio of OMW. The humic acid (HA)-like fraction isolated from the fresh OMW was characterized by a marked aliphatic character, small O and acidic functional group contents, marked presence of proteinaceous materials, partially modified lignin moieties and polysaccharides-like structures, extended molecular heterogeneity, and small degrees of aromatic ring polycondensation, polymerization and humification. With increasing the time of either lagooning or catalytic digestion, a loss of aliphatic materials and an increase of extraction yield, oxygenation, acidic functional groups, carbohydrates and aromaticity occurred in the HA-like fractions. The more evident changes measured for the HA-like fractions from catalytically-digested OMW, with respect to those from lagooned OMW, indicated that MnO(2) was able to catalyze organic matter humification in OMW.     

Anaerobic co-digestion of olive mill wastewater with olive mill solid waste in a tubular digester at mesophilic temperature

Anaerobic co-digestion is a well established process for treating many types of organic wastes, both solid and liquid. In this study we have investigated, on a laboratory scale, the anaerobic co-digestion of olive mill wastewater (OMW) with olive mill solid waste (OMSW) using semi-continuous, feeding, tubular digesters operated at mesophilic temperatures. Each digester was fed with an influent, composed of OMW and OMSW, at an organic loading rate (OLR) varying between 0.67 and 6.67 g COD/l/d. The hydraulic retention times (HRT) were 12, 24 and 36 days. The TCOD concentrations of OMW used as the main substrate were 24, 56 and 80 g COD/l; the amount of the dry OMSW used as a co-substrate was fixed to approximately 56 g/l of OMW. The results indicated that the best methane production was about 0.95 l/l/day obtained at an OLR = 4.67 g COD/l/d, corresponding to influent TCOD = 56 g COD/l at an HRT = 12d. In contrast, the maximum TCOD removal efficiency (89%) was achieved at an OLR = 0.67 g COD/l/d, corresponding to influent TCOD = 24 g COD/l at an HRT = 36 d. Moreover, the inhibition of biogas production was observed at the highest OLR studied.     

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.     

Influence of salts and phenolic compounds on olive mill wastewater detoxification using superabsorbent polymers

For a selection of nine commercially available superabsorbent polymers, the absorption capacity was evaluated for the principal absorption-inhibition constituent of OMW, mineral salts and for phytotoxic-components, the phenolic compounds. A double exponential model was established for electrical conductivities ranging 4.2-25,000 microS cm(-1). For solutions of phenolic compounds ranging 0-0.5 g l(-1), a distribution coefficient near unit was achieved, while for OMW, the phenolic compounds were concentrated inside the gel as the distribution coefficient was 1.4. Correction of OMW pH towards neutrality was found to increase the absorption capacity by up to 35%. The phytotoxicity was assessed by the germination of Lepidium sativum. Inhibition in plant growth occurred for all OMW dilutions without superabsorbent polymers application. For 5% of OMW (COD 5 gl(-1) and 200 ppm of phenolic compounds) immobilised in PNa2 (1 gl(-1)), plant growth was promoted being observed a 120% growth germination, thus indicating that olive mill wastewater detoxification occurred.     

Phenolic removal in olive oil mill wastewater by strains of Pleurotus spp. in respect to their phenol oxidase (laccase) activity

The ability of several Pleurotus spp. strains to remove phenolic compounds from an olive oil mill wastewater (OMW) was studied. All strains tested in this work were able to grow in OMW without any addition of nutrients and any pre-treatment, except sterilization. High laccase activity was measured in the growth medium, while 69-76% of the initial phenolic compounds were removed. The black color of OMW became yellow-brown and brighter as the strains grew. The lowest phenolic concentrations were reached after 12/15 days. A decrease of the phytotoxicity, as described by the parameter Germination Index, was noticed in the OMW treated with some Pleurotus spp strains, although this decrease was not proportional to the phenolic removal. A new parameter, namely Phenol-toxicity Index, was considered in the present paper. Using this parameter it was found that the remaining phenolics and/or some of the oxidation products of the laccase reaction in the treated OMW were more toxic than the original phenolic compounds.     

Respiration profiles in monitoring the composting of by-products from the olive oil agro-industry

The composting of olive press cake (OPC) repeatedly mixed either with olive mill wastewater (OPC+OMW) or with tap water (OPC+W) was studied using the thermogradient respirometer, an apparatus that determines the respiration rates from a substrate over a wide range of different temperatures (respiratory profile). The composting processes took place over a period of five months during which nine moistenings of the OPC were performed with the respective liquids. The composting resulted in detoxification of the materials used in both treatments, as indicated by seed germination tests. However, the repeated applications of OMW resulted in recurring thermophilic phases (following each application) and in greater pH and conductivity increases in the final product, as compared to water applications. Respiration measurements performed at 35 degrees C were good indicators of the mean metabolic potential in the compost piles (the mean respiration derived from the whole respiration profile over a wide range of environmental temperatures). However, respiration measurements at higher temperatures (48.5 degrees C) were better indicators of the respiration activity occurring in situ. Following the initial thermophilic phase, the respiration potential of the composts at high temperatures (42-63 degrees C) increased drastically compared to their respiration potential at lower temperatures (17-42 degrees C) indicating the establishment of a thermophilic microflora. Subsequently, only the periodic new substrate-C applications in the form of OMW resulted in increased ratios of low temperature-to-high temperature respiration potential. These ratios decreased again following the respective thermophilic phase that each new OMW application had induced.     

Activity and substrate specificity of the alcohol dehydrogenases of n-alkane oxidizing yeasts

The activity and substrate specificity of alcohol dehydrogenases (ADH) in the fractions of cytosol and membrane particles were compared in the yeasts Torulopsis candida, Candida lipolytica and Candida tropicalis grown in media with glucose and hexadecane. In all studied yeast cultures growing in the medium with hexadecane, NAD-dependent ADH specifically dehydrogenating only medium and higher alcohols are induced in the membrane structures of the cells. Soluble ADH are found in the cytosol of the cultures grown either on glucose or on hexadecane. These ADH oxidize all alcohols with the carbon chain length from C2 to C16. As was found by electrophoresis in polyacrylamide gel, the number of ADH molecular forms in the cytosol fraction of the cultures depends on the carbon growth substrate being used and the peculiarities of yeast culture.     

Use of Geotrichum candidum for olive mill wastewater treatment in submerged and static culture

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 olive mill wastewater (OMW). Optimisation of different factors showed that dilution, carbon and ammonium concentrations significantly affected decolourisation and activities of ligniolytic peroxidases (LiP and MnP) on OMW. Moreover, addition of olive oil and agitation improved the lipase production. Batch and continuous OMW treatments in settler or bubble column bioreactors showed high COD and colour removal efficiencies of 60% and 50%, respectively. Lipolytic activity was greater in the batch bubble column whereas, LiP and MnP productions were improved in the settler. The performance of the continuous processes decreased with the decrease of hydraulic retention time (HRT). It has been shown that decolourisation and biodegradation decreased with an average of 40% and 45%, respectively, by decreasing the HRT from 4 d to 1.7 d.     

Olive oil history,production and by-product management

In this review paper, the history of the olive tree and the development of the main olive oil extraction systems employed in the past are presented. Furthermore, the management techniques employed from antiquity until today for the treatment of olive mill wastewater (OMW), which constitutes the most important by-product of olive oil production, are addressed and compared. Reference is also made to global olive oil production, the environmental impacts of the uncontrolled disposal of OMW, and to the legal framework concerning its management in the main olive oil producing countries. Other by-products of olive processing are also discussed.