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Olive orchard amended with olive mill wastewater: Effects on olive fruit and olive oil quality
Journal of Hazardous Materials 172 (2009) 1544–1550
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Olive orchard amended with olive mill wastewater: Effects on olive fruit and olive oil quality B. Mechri a,b,∗ , M. Issaoui a , A. Echbili a , H. Chehab b , F.B. Mariem b , M. Braham b , M. Hammami a a b
Laboratoire de Biochimie, USCR Spectrométrie de Masse, UR Nutrition et Désordres Métaboliquesm, Faculté de Médecine, Monastir, Tunisia Laboratoire d’Ecophysiologie, Institut de L’Olivier de Sousse, Tunisia
a r t i c l e
i n f o
Article history: Received 3 May 2009 Received in revised form 6 August 2009 Accepted 6 August 2009 Available online 13 August 2009 Keywords: Olive mill wastewater Olive oil Polyphenols Soluble carbohydrate
a b s t r a c t The aim of this work was to study the effects of agronomic application of olive mill wastewater (OMW) in a field of olive trees on olive fruit and olive oil quality. Agronomic application of OMW increased significantly the fungal:bacteria ratio, whereas the root colonisation and the photosynthetic rates decreased significantly. Consequently, the oil content expressed as a percentage of dry weight, decreased significantly after agronomic application of OMW. Land spreading of OMW altered the relative proportion of individual olive fruit sugar and decreased significantly the nitrogen (N) and phosphorus (P) of the fruit. A significant increase was observed in total phenol content of oil after agronomic application of OMW. ␣-Tocopherol content, on the contrary, decreased with OMW application. The fatty acid composition of the oil was not affected by the treatments. To our knowledge, this is the first report of change in the olive fruit and olive oil quality following agronomic application of OMW. © 2009 Elsevier B.V. All rights reserved.
1. Introduction In the Mediterranean area, which has gained world recognition as a centre of olive growing, limiting energy is not, generally speaking, a factor and nutritive elements can be supplied by means of adequate fertilisation, while scarce water availability is the main factor which could negatively affect production. A number of studies have revealed that in 2025, in most parts of the Mediterranean countries, per capita water availability will be decreased by 60% compared to 1990 resulting in the reduction of irrigated surfaces and the gradual deterioration in water quality [1]. Therefore, the development of irrigation strategies for optimising olive management under conditions of limited water resources is of crucial importance for improvement of the long-term profitability within the concept of sustainable olive growing. The large quantities of olive oil mill wastewater (OMW) produced during olive oil processing may represent a low cost source of water. Thus, the recycling of the OMW and their use as water for irrigation in the agriculture is an attractive perspective for the Mediterranean countries. The organic fraction of the OMW contains a complex consortium of phenolic substances, some nitrogenous compounds (especially amino acids), organic acids, sugars, tan-
∗ Corresponding author at: Laboratoire de Biochimie, USCR Spectrométrie de Masse, UR Nutrition et Désordres Métaboliques, Faculté de Médecine, Monastir, Tunisia. Tel.: +216 73 462 200; fax: +216 73 460 737. E-mail address: [email protected] (B. Mechri). 0304-3894/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.jhazmat.2009.08.026
nins, pectins, carotenoids, and almost all of the water soluble constituents of the olives [2,3]. The inorganic fraction contains chloride, sulphate, and phosphoric salts of potassium as well as calcium, iron, magnesium, sodium, copper, and other trace elements in various chemical forms. The inorganic constituents at the concentration levels found in OMW are not toxic; quite the reverse, they may potentially serve as good sources of plant nutrients and renders this effluent potentially suitable for recycling as a soil amendment [4]. In countries on the South bank of the Mediterranean with severe water deficient environments and with soils usually characterized by a scarcity of organic matter, the use of OMW for soil fertilisation could be doubly beneficial. For these reasons, increasing attention has been given to find the best methods to spread OMW in the field of olive trees and to recycle both the organic matter and the nutritive elements in the soil crop system. Recent papers have been published about the effects of spreading OMW on the soil cultivated with cereals and other annual crops [5], grapevine [6] and with olive trees [7,8]. The aim of such research is to increase production and reduce costs, while maintaining product quality and protecting the environment. Many factors may affect olive quality, including cultural practices such as fertilisation [9], the irrigation management [11] and agronomic practices adopted in the field [10]. Conflicting data have also been reported on the effect of irrigation on the olive fruit and fatty acid composition [11–13], as well as on the polyphenols rates in the oils [14]. However, no research has been published on the effect of irrigation with OMW on olive fruit or oils quality.
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The present work was aimed at evaluating, in natural situations (field of olive trees), the effects of agronomic application of OMW on some compounds in olive fruit (specifically, sugar, nitrogen and phosphorus) and olive oil quality. 2. Materials and methods 2.1. Characteristics of the OMW used for irrigation The original OMW used in the present study was obtained from an olive oils production plant located in the city of Ouled Jaballah, Tunisia, which uses discontinuous process for extraction of olive oils. The main characteristics of the OMW were pH: 5.1; electrical conductivity (EC): 9.1 ds m−1 ; salinity: 6.37 g l−1 ; COD: 93 g l−1 ; N: 1340 mg l−1 ; P: 720 mg l−1 ; K: 6200 mg l−1 ; phenols: 8400 mg l−1 ; and glucose 1200 mg l−1 . 2.2. Field site and sampling Tree located at Ouled Jaballah, Tunisia, North latitude 35◦ 12 , East longitude, 10◦ 59 , spaced 12 m × 12 m apart were selected in 2004 for the experiments [7,8]. The climate of this region is typical Mediterranean, semiarid to arid, with an average rainfall of 200 mm year−1 and an average annual temperature of 18–20 ◦ C. Physico-chemical characteristics of the soil at this site were as follows: pH: 8.53; EC: 0.44 ds m−1 ; sand: 78.1%; clay: 12.85%; silt: 5.1%; organic C: 0.37%; N: 0.042%; Olsen P: 20.86 mg kg−1 ; Exchangeable K: 0.43 meq 100 g−1 . The experiment included three levels of OMW application (TC: 0 m3 ha−1 , T1: 30 m3 ha−1 , T2: 60 m3 ha−1 and T3: 150 m3 ha−1 ). Three plots (576 m2 each: 24 m × 24 m) were designed for each treatment. This amendment was realised in December 2004 in one application [7,8]. All sampling events included the collection of soil samples (0–20 cm deep) from five random locations in each plot at harvest (February 2006). Roots and olive fruits samples were collected at this time. A composite root sample was collected per plot, for determination of arbuscular mycorrhizal (AM) fungi colonisation. From each plot, approximately 2 kg of fruit was taken to determine olive fruits and olive oils quality. 2.3. Soil fatty acid methyl ester (FAME) analysis Lipids were extracted from the soil samples (3 g) as well as the root samples (30 mg) using the EL-FAME method [15]. Briefly, a mild alkaline hydrolysis (0.2 M KOH in methanol) was used to extract whole cell fatty acids (FAs). This included FAs from phospholipids, glycolipids and neutral lipids. The FAME extraction residue was dissolved in hexane. The hexane layer was transferred to a clean tube, and the hexane was evaporated off, after which FAMEs were resuspended in 0.5 ml of hexane–methyl tert-butyl ether (1:1) for analysis. Individual FAMEs were analyzed on a Hewlett-Packard 5890 gas chromatograph equipped with a flame ionisation detector and a HP-5MS column (95% dimethyl–5% diphenyl polysiloxane, length 30 m × 0.25 mm). The temperature was programmed to increase from 170 to 270 ◦ C at a rate of 5 ◦ C min−1 . The temperature was increased to 270 ◦ C for 2 min between samples in order to clean the column. Identification of FAMEs was by retention time and confirmed by gas chromatography–mass spectrometry (GC–MS). The following FAMEs were designated as bacterial: i15:0, a15:0, 15:0, 16:19, i17:0, a17:0, 17:0, 17:18, cy17:0 and cy19:0. Soil FAME 18:19 and 18:26 were used as indicators of saprophytic fungi. Soil FAME 16:15 was used to indicate (AM) fungi abundance. Root FAME 16:15 analysis was used as index for the development of AM colonisation in the olive trees roots [7].
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2.4. Photosynthesis measurements Photosynthetic rates (A) were measured in the field by using a LI-6400 gas exchange system (Li-Cor, Lincoln, NE, USA) from 11:00 to 13:00 in February 2006 [7]. Net photosynthesis rate (A in mol CO2 m−2 s−1 ), was measured at fixed CO2 concentration (CO2 at 400 mol mol−1 ), air temperature (25 ◦ C), relative humidity (60%) and a photosynthetic photon flux density (1500 mol m−2 s−1 ). 2.5. Olive analyses 2.5.1. Soluble carbohydrate determination The olive soluble carbohydrates were extracted according to the method described by Bartolozzi et al. [16]. Briefly the soluble carbohydrates from composite olive samples were extracted twice in 80% ethanol at 70 ◦ C. Extracts were dried and converted into trimethylsilyl ethers with a silylation mixture made up of pyridine, hexamethyldisilazane and trimethylchlorosilane. Soluble sugars were analyzed using a Hewlett-Packard 5890 series II gas chromatograph equipped with a flame ionisation detection (FID) system and a HP-5MS column (30 m × 0.25 mm) as described by Bartolozzi et al. [16]. Identification of individual carbohydrate was achieved by use of the relative retention times, i.e., in comparison to that of the standard. These were compared to those identified earlier by gas chromatography–mass spectrometry. 2.5.2. Olive N and P determination Olive nitrogen (N) content was determined by the Kjeldahl method [17]. The analysis of phosphorus (P) was carried out following the official method of the AOAC no. 970-39 Phosphorus in Fruits and Fruit Products (spectrophotometric molybdovanadate method) [18]. The method is based on the capacity of P (in phosphoric acid form) in an acid solution and in the presence of V5+ and Mo6+ to form a yellow phosphomolybdovanadate complex whose absorbance can be measured at 400 nm. 2.6. Olive oil analyses 2.6.1. Determination of oil content Oil content was determined by extracting dry material with 40–60 ◦ C petroleum ether using a Soxhlet apparatus. The extract was dried at 70 ◦ C and weighed. 2.6.2. Fatty acid determination The fatty acid composition of the oil was determined by gas chromatography (GC) as fatty acid methyl esters (FAMEs). Methyl esters were prepared from olive oil by vigorous shaking of a solution of oil in hexane (0.2 g in 3 ml) with 0.4 ml of 2N methanolic potassium hydroxide. Individual FAMEs were separated and quantified by gas chromatography with a FID detector. The temperature was programmed to increase from 170 to 270 ◦ C at a rate of 5 ◦ C min−1 . Nitrogen was used as a carrier gas. Fatty acids were identified by comparing retention times with standard compounds. 2.6.3. Total phenol content determination Total phenols and amounts were quantified colorimetrically [19]. Phenolic compounds were isolated by triple extraction of a solution of oil in hexane with a water–methanol mixture (60:40, v/v). The Folin–Ciocalteau reagent (Merck Schuchardt OHG, Hohenbrunn, Germany) was added to a suitable aliquot of the combined extracts, and the absorption of the solution at 725 nm was measured. Values are given as mg of caffeic acid per kilogram of oil.
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2.6.4. ˛-Tocopherol determination ␣-Tocopherol was evaluated by high-performance liquid chromatography (HPLC) with direct injection of an oil in hexane solution [20]. Separation by HPLC was carried out using a Hewlett-Packard liquid chromatographic system (Waldbronn, Germany) with an HP-1100 pump system and a Rheodyne Model 7125 injector (Cotati, CA, USA) with a final volume loop of 20 l. The detector was an HP-1040 M photodiode-array detection (DAD) system. The data were stored and processed by an HPLC Chemstation (Dos Series) (Hewlett-Packard). The column was a Supelco ODS-2 (150 mm × 4.6 mm I.D., 5 mm particle size). The mobile phase consisted of methanol/water (96:4, v/v) at a flow rate of 2 ml min−1 . ␣-Tocopherol was quantified by the internal standard method. Results are given as mg of ␣-Tocopherol per kilogram of oil. 2.7. Statistical analyses All statistical values (mean values, standard deviation) were calculated using the SPSS statistic program. Pearson’s correlation coefficients were used when calculating correlations between mannitol and oil content. In addition, Duncan’s multiple range tests were used to determine significant differences among data. Olive fruits compositions (olive N, olive P and soluble carbohydrates) were evaluated with the XLSTAT 2006 Version 2006.06 using principal components analysis (PCA) in order to compare the effects of OMW on olive fruits quality. 3. Results and discussion 3.1. Soil microbial community, AM fungus colonisation and photosynthesis after agronomic application of OMW The abundance of the saprophytic fungi as indicated by the sum of FAME 18:19 and 18:26 was significantly higher in the OMW amended soil than that in the control soil (Table 1), indicating that OMW constitutes an adequate substrate for saprotrophic fungi [7,8]. Agronomic application of OMW increased significantly the fungal:bacteria ratio from 0.23 in the control soil to 1.11 in the soil amended with 150 m3 ha−1 OMW. The modify values of fungi:bacteria was an agreement with the change in the soil microbial community following agronomic application of OMW [7,8]. The abundance of AM fungi and the root colonisation decreased significantly in the olive trees irrigated with OMW as compared to the control. The decreased root colonisation might have decreased the photosynthetic rate, as photosynthesis is under some control by sink demand [21]. It is possible that the sink strength of the roots in the OMW amended soil is lower, due to the reduced root colonisation after agronomic application of OMW. Wright et al. [21] demonstrated that AM fungal colonisation stimulated the rate of
Fig. 1. Glucose, fructose, mannitol and galactose content in olive fruits following agronomic application of OMW (TC: control, T1: 30 m3 ha−1 OMW, T2: 60 m3 ha−1 OMW and T3: 150 m3 ha−1 OMW). Different letters indicate significantly different values at P ≤ 0.05 according to Duncan test.
photosynthesis sufficiently to compensate for the carbon requirement of the fungus. In view of the above background, the following questions were addressed: (i) is the specific environment created after agronomic application of OMW (increase of saprophytic fungi, decrease of bacteria, AM fungi and root colonisation) beneficial or detrimental to the growth of olive fruits? and (ii) If land spreading of OMW has an effect (positive effect or negative effect) on the development of olive fruits, does the olive oil quality change? 3.2. Olive fruits soluble carbohydrate after agronomic application of OMW Sugars are the main soluble components in olive tissues and play an important role, providing energy for metabolic changes. They are important components of the cell-wall, related to the textural properties [22]. Results of the GC determinations are given in Fig. 1. The major sugars in control and OMW irrigated olive-pulp were glucose, fructose and galactose. Differences between TC, T1, T2 and T3 treatments were significant for glucose and fructose and nonsignificant for galactose. Glucose was the predominant component in the TC and the T1 treatments, while fructose was the predominant component in the T2 and T3 treatments. Generally glucose accounted for 57.58%, fructose for 17.32% and galactose for 4.89% of the total sugars in the control, while glucose accounted for 42.24%, 37.19%, 24.92%, fructose for 40.8%, 48.18%, 59.11% and galactose for 2.46%, 2.51%, 4.% of the total sugars in the T1, T2 and T3 treatments respec-
Table 1 Influence of OMW treatments on soils microbial community, roots colonisation and olive trees photosynthesis.
a
Fungi (% total FAME) Bacteriab (% total FAME) Fungi:bacteria AM fungic (% total FAME) Root colonisationd (%) Photosynthesis (mol m−2 s−1 )
Control
Agronomic application of OMW (m3 ha−1 )
TC
T1 (30)
4.56 20.05 0.22 1.47 14.3 8.67
± ± ± ± ± ±
0.76d 1.41a 0.04d 0.4a 0.95a 0.79a
9.39 20.22 0.46 1.4 12.5 8.57
± ± ± ± ± ±
T2 (60) 1.73c 1.21a 0.03c 0.3a 0.9ab 0.65a
11.63 19.42 0.59 1.24 10.5 8.38
± ± ± ± ± ±
T4 (150) 1.71b 1.64a 0.01b 0.16b 0.73bc 0.52ab
18.11 16.38 1.1 0.82 9.1 5.68
± ± ± ± ± ±
0.26a 0.53b 0.03a 0.02c 1.82c 1.62c
The effect of OMW treatment was tested with one-way ANOVA (mean value ± SE, n = 3 for fungi, n = 3 for bacteria, n = 3 for colonisation, n = 6 for photosynthesis), and mean values in individual line followed by the same letter are not significantly different at P < 0.05 (Duncan test). a 18:26 and 18:19. b i 15:0, a15:0, 15:0, 16:19, 16:1 7, i17:0, a17:0, 17:0, cy17:0, 18:17 and cy19:0. c Estimated as the soils FAME 16:15 analysis. d Estimated as the roots FAME 16:15 analysis.
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tively. Mannitol was also found, with a content ranging between 17.67% in the control to 13.08%, 10.04% and 8.66% in the T1, T2 and T3 treatments respectively. Such significant differences in the relative proportion of sugar probably reflect the metabolic behaviour of each olive tree in relation to different specific environment conditions created in soil following land spreading of OMW. Marsilio et al. [23] reported that the differences in the individual sugars in olive fruits reflect the metabolic behaviour of each cultivar in relation to the genotype and to different climatic and environmental conditions. 3.3. Olive P and N following irrigation with OMW Agronomic application of OMW decreased significantly the olive fruit content of N and P (Fig. 2). The role of AM fungi in the stimulation of growth and nutrient uptake of many host plants is well documented [24]. Olive plants are known to form AM fungi [7,25], and can be colonized by different species, including Glomus mosseae, Glomus clarum, Glomus caledonium, Glomus monosporum, Glomus intraradices and Glomus viscosum [25,26]. In this study, agronomic application of OMW decreased significantly the root FAME 16:15. The fatty acids 16:15 have been used to estimate the extraradical biomass in pot cultures and the amount of these fatty acids were well correlated with the AM hyphal length [27]. OMW, that have negative effect on the AM hyphal length, might consequently affect the mycorrhizal effectiveness on nutrient uptake. In principle, AM plants having a developed extraradical mycelium could potentially absorb more micronutrients than AM plants with less developed extraradical hyphae. The higher nutrient uptake in mycorrhizal plants might be attributed to the contribution of
Fig. 3. Oil accumulation in the olive fruits after agronomic application of OMW. Different letters indicate significantly different values at P ≤ 0.05 according to Duncan test.
external hyphae which explore a large volume of soil and thus absorb more N, P and certain others nutrients [28,29]. However, the negative impact of agronomic application of OMW on the olive fruit N and P found in the present study can be explained by this mechanism. This implies that the differences in the olive N and P concentration we observed after agronomic application of OMW were due to factors in the OMW amended soil that inhibit the AM fungal root colonisation and thus the development of AM fungi [7]. It has been reported that AM fungi enhance plant nutrient acquisition in alkaline soil [30], and alleviate cultural and environmental stresses [24], through greater effective root area and the activation and excretion of various enzymes by AM fungi roots and/or hyphae [31]. 3.4. Oil accumulation in the olive fruits after agronomic application of OMW The oil accumulation behaviour is illustrated in Fig. 3. The oil content expressed as a percentage of dry weight decreases after agronomic application of OMW. Nergiza and Engez [32] found a positive correlation between the oil accumulation and sugar content. Marsilio et al. [23] indicated that the relative amount of mannitol in the fruit might indicate the potential of a cultivar for oil biosynthesis. A good correlation coefficient (r = 0.848; P < 0.001) was established in this study between mannitol and oil content (Fig. 4). Thus, the mannitol in the olive, as well as other polyols in many other higher plants, might be of specific importance in the metabolic transformation and synthesis of the fruit storage material [33]. Jiménez et al. [22] reported that sugars are the main
Fig. 2. Effect of agronomic application of OMW on the olive fruits nitrogen (A) and phosphorus (B) (TC: control, T1: 30 m3 ha−1 OMW, T2: 60 m3 ha−1 OMW and T3: 150 m3 ha−1 OMW). Different letters indicate significantly different values at P ≤ 0.05 according to Duncan test.
Fig. 4. Relationship between the olive oil content and the olive fruits mannitol (n = 12).
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Table 2 Fatty acids composition (%) in olive oil following agronomic application of OMW. Fatty acids (%)
Control
Agronomic application of OMW (m3 ha−1 )
TC (0)
T1 (30)
T2 (60)
T3 (150)
Saturated Palmitic C16:0 Margaric C17:0 Stearic C18:0 Arachidic C20:0
20.12 0.09 2.55 0.46
Monounsaturated Oleic C18:1 Decenoic C17:1 Palmitoleic C16:1
55.49 ± 0.1 0.1 ± 0.01 2.6 ± 0.43
55.09 ± 0.9 0.15 ± 0.03 3 ± 0.21
54.29 ± 0.33 0.11 ± 0.06 2.85 ± 0.22
55.47 ± 0.36 0.1 ± 0.04 2.39 ± 0.18
Polyunsaturated Linoleic C18:2 ␣-Linolenic C18:3
21.29 ± 0.5 0.76 ± 0.08
21.83 ± 0.3 0.67 ± 0.01
21.33 ± 0.3 0.86 ± 0.07
21.65 ± 0.6 0.88 ± 0.01
± ± ± ±
0.4 0.03 0.3 0.05
20.55 0.14 2.38 0.44
soluble components in olive tissues and play an important role, providing energy for metabolic changes. They are important components of the cell-wall, related to the textural properties of the fruit and act as precursors for olive oil biosynthesis. Based on these results, it can be said that the change in the individual olive fruit carbohydrate after agronomic application of OMW can be considered as a potential factor modifying oil biosynthesis. 3.5. Fatty acid composition following agronomic application of OMW Agronomic application of OMW did not cause significant variations in fatty acids composition (Table 2). Fatty acids were palmitic (16:0), palmitoleic (16:1), heptadecanoic (17:0), heptadecenoic (17:1), stearic (18:0), oleic (18:1), linoleic (18:2), linolenic (18:3) and eicosanoic (20:0). The percentages of saturated (palmitic + heptadecanoic + stearic + eicosanoic), monounsaturated (palmitoleic + heptadecenoic + oleic) and polyunsaturated (linoleic + linolenic) fatty acids of lipid classes and the unsaturated/saturated, monounsaturated/polyunsaturated and oleic/linoleic acid ratios did not appear to be significantly influenced by irrigation with OMW.
± ± ± ±
0.36 0.04 0.1 0.04
20.7 0.08 2.59 0.49
± ± ± ±
0.2 0.01 0.33 0.07
20.07 0.07 2.73 0.46
± ± ± ±
0.26 0.01 0.1 0.07
cially for oil obtained from olive trees irrigated with 150 m3 ha−1 , making this hypothesis more plausible to explain these results. Cinquanta et al. [37] have noted that qualitative and quantitative compositions of olive oil’s phenols are strongly affected by the agronomic and technological conditions of its production. Among agronomic parameters are cultivar, pedoclimatic conditions of production and some agronomic techniques such as the irrigation [38]. We found in this study that agronomic application of OMW appeared capable of influencing the quantitative composition of olive oil phenols. The effect of agronomic application of OMW on the qualitative composition of olive oil phenols should be examined in future studies.
3.6. Olive oil polyphenol and ˛-Tocopherol content after agronomic application of OMW Polyphenols content, a component of great importance in oil quality because of its antioxidant effects, increased significantly following agronomic application of OMW (Fig. 5A). To our knowledge land spreading of OMW creates a stress situation that induces the production of phenolics. In fact, the decline in the root colonisation might to consequently affect the mycorrhizal effectiveness on nutrient uptake which, in turn, could have declined the olive nutrient content. These changes in soil properties and soil microbial community observed following agronomic application of OMW can generate a stress situation that induces high oil phenol content. This makes it rather unlikely that the high oil phenol content observed after agronomic application of OMW can be related to the significant decrease in the olive N and P content. In the case of low nutrient availability, as occurs in our experiments (especially in T3 treatment), phenylalanine preferentially flows in to phenylpropanoids synthesis via phenylalanine ammonia lyase rather than toward synthesis of protein [34]. As observed in other species, phenylalanine ammonia lyase enzyme could play a key role in olive. After phenylalanine deamination, trans-cinnamic acid is formed which is the precursor of polyphenolic substances [35]. In our study, trees fruits protein content, estimated from its N content [36], was lower whereas polyphenol content in olive oil was higher, espe-
Fig. 5. Effect of agronomic application of OMW on the olive oil polyphenol (A) and ␣Tocopherol (B) content from different OMW treatments (TC: control, T1: 30 m3 ha−1 OMW, T2: 60 m3 ha−1 OMW and T3: 150 m3 ha−1 OMW). Different letters indicate significantly different values at P ≤ 0.05 according to Duncan test.
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and the whole plant carbon balance, and consequently, on chemical characteristics of oil. Several works have shown that several factors such as environmental and agronomic factors have an effect on plant physiologic behaviour and, consequently, on oil chemical properties [40,41]. We found that agronomic application of OMW not only modify the soil properties, the soil microbial community and the olive trees key physiological parameters, but also appeared capable of influencing the olive fruits characteristics and oil quality. Our results support the hypothesis that the change in the functioning of arbuscular mycorrhizas following agronomic application OMW should be considered as a potential factor influencing olive and oil quality. Acknowledgements The authors wish to thank M. Imed cheraief for his help in GC/MS analysis. This study was supported by the Ministère de l’Enseignement Supérieur, Ministère de la Recherche Scientifique de la technologie et de développement des compétences (UR03/ES08 Nutrition et Désordres Métaboliques) and Institut de l’Olivier de Sousse. References
Fig. 6. Effects of agronomic application of OMW on the olive quality. (A) PCA of olive after agronomic application of OMW. (B) PCA showing loading values for olive N, olive P, fructose, galactose, mannitol and glucose of the treatments plots given in (A). The variance explained by the each principal component axis is shown in parentheses (() control; () 30 m3 ha−1 OMW; () 60 m3 ha−1 OMW; () 150 m3 ha−1 OMW).
␣-Tocopherol decrease with increasing dose of OMW (Fig. 5B), showing an inverse trend than polyphenols. ␣-Tocopherol seems to become effective when the activity of the polar phenolic fraction is reduced and primary products of autoxidation reach a critical concentration [39]. 3.7. Chemometric analysis Principal component analyses of olive fruits compositions (olive N, olive P and sugars) accounted for 74.65% of the variance on the first component, while the second component accounted for 18.35%. This explained 93.01% of the total variance (Fig. 6). PCA of the samples revealed separation of the four treatments into four distinct clusters. The first cluster (TC), the second cluster (T1), the third cluster (T2) and the fourth cluster (T3), where the first cluster (control plots) and the second cluster (olive trees irrigated with 30 m3 ha−1 ) were positively associated with PC1 whereas the third cluster (olive trees irrigated with 60 m3 ha−1 ) and the fourth cluster (olive trees irrigated with 150 m3 ha−1 ) were negatively associated with PC1 (Fig. 6A). This indicated that clear differentiation exists between olives obtained from TC and T1 treatments and those obtained from T2 and T3 treatments. This was also illustrated by the fact that olives N, olives P, glucose and mannitol are more common to the right of PC1 (TC and T1 treatments), whereas fructose is more common to the left on PC 1 (T2 and T3 treatments) (Fig. 6B). 4. Conclusion Agronomic applications of OMW were demonstrated by this study to have a major impact on the growth and establishment of AM fungi. Conversely, the AM fungi have considerable effects on plant physiology apparently through changes to the mineral status
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Journal of Hazardous Materials journal homepage: www.elsevier.com/locate/jhazmat
Olive orchard amended with olive mill wastewater: Effects on olive fruit and olive oil quality B. Mechri a,b,∗ , M. Issaoui a , A. Echbili a , H. Chehab b , F.B. Mariem b , M. Braham b , M. Hammami a a b
Laboratoire de Biochimie, USCR Spectrométrie de Masse, UR Nutrition et Désordres Métaboliquesm, Faculté de Médecine, Monastir, Tunisia Laboratoire d’Ecophysiologie, Institut de L’Olivier de Sousse, Tunisia
a r t i c l e
i n f o
Article history: Received 3 May 2009 Received in revised form 6 August 2009 Accepted 6 August 2009 Available online 13 August 2009 Keywords: Olive mill wastewater Olive oil Polyphenols Soluble carbohydrate
a b s t r a c t The aim of this work was to study the effects of agronomic application of olive mill wastewater (OMW) in a field of olive trees on olive fruit and olive oil quality. Agronomic application of OMW increased significantly the fungal:bacteria ratio, whereas the root colonisation and the photosynthetic rates decreased significantly. Consequently, the oil content expressed as a percentage of dry weight, decreased significantly after agronomic application of OMW. Land spreading of OMW altered the relative proportion of individual olive fruit sugar and decreased significantly the nitrogen (N) and phosphorus (P) of the fruit. A significant increase was observed in total phenol content of oil after agronomic application of OMW. ␣-Tocopherol content, on the contrary, decreased with OMW application. The fatty acid composition of the oil was not affected by the treatments. To our knowledge, this is the first report of change in the olive fruit and olive oil quality following agronomic application of OMW. © 2009 Elsevier B.V. All rights reserved.
1. Introduction In the Mediterranean area, which has gained world recognition as a centre of olive growing, limiting energy is not, generally speaking, a factor and nutritive elements can be supplied by means of adequate fertilisation, while scarce water availability is the main factor which could negatively affect production. A number of studies have revealed that in 2025, in most parts of the Mediterranean countries, per capita water availability will be decreased by 60% compared to 1990 resulting in the reduction of irrigated surfaces and the gradual deterioration in water quality [1]. Therefore, the development of irrigation strategies for optimising olive management under conditions of limited water resources is of crucial importance for improvement of the long-term profitability within the concept of sustainable olive growing. The large quantities of olive oil mill wastewater (OMW) produced during olive oil processing may represent a low cost source of water. Thus, the recycling of the OMW and their use as water for irrigation in the agriculture is an attractive perspective for the Mediterranean countries. The organic fraction of the OMW contains a complex consortium of phenolic substances, some nitrogenous compounds (especially amino acids), organic acids, sugars, tan-
∗ Corresponding author at: Laboratoire de Biochimie, USCR Spectrométrie de Masse, UR Nutrition et Désordres Métaboliques, Faculté de Médecine, Monastir, Tunisia. Tel.: +216 73 462 200; fax: +216 73 460 737. E-mail address: [email protected] (B. Mechri). 0304-3894/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.jhazmat.2009.08.026
nins, pectins, carotenoids, and almost all of the water soluble constituents of the olives [2,3]. The inorganic fraction contains chloride, sulphate, and phosphoric salts of potassium as well as calcium, iron, magnesium, sodium, copper, and other trace elements in various chemical forms. The inorganic constituents at the concentration levels found in OMW are not toxic; quite the reverse, they may potentially serve as good sources of plant nutrients and renders this effluent potentially suitable for recycling as a soil amendment [4]. In countries on the South bank of the Mediterranean with severe water deficient environments and with soils usually characterized by a scarcity of organic matter, the use of OMW for soil fertilisation could be doubly beneficial. For these reasons, increasing attention has been given to find the best methods to spread OMW in the field of olive trees and to recycle both the organic matter and the nutritive elements in the soil crop system. Recent papers have been published about the effects of spreading OMW on the soil cultivated with cereals and other annual crops [5], grapevine [6] and with olive trees [7,8]. The aim of such research is to increase production and reduce costs, while maintaining product quality and protecting the environment. Many factors may affect olive quality, including cultural practices such as fertilisation [9], the irrigation management [11] and agronomic practices adopted in the field [10]. Conflicting data have also been reported on the effect of irrigation on the olive fruit and fatty acid composition [11–13], as well as on the polyphenols rates in the oils [14]. However, no research has been published on the effect of irrigation with OMW on olive fruit or oils quality.
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The present work was aimed at evaluating, in natural situations (field of olive trees), the effects of agronomic application of OMW on some compounds in olive fruit (specifically, sugar, nitrogen and phosphorus) and olive oil quality. 2. Materials and methods 2.1. Characteristics of the OMW used for irrigation The original OMW used in the present study was obtained from an olive oils production plant located in the city of Ouled Jaballah, Tunisia, which uses discontinuous process for extraction of olive oils. The main characteristics of the OMW were pH: 5.1; electrical conductivity (EC): 9.1 ds m−1 ; salinity: 6.37 g l−1 ; COD: 93 g l−1 ; N: 1340 mg l−1 ; P: 720 mg l−1 ; K: 6200 mg l−1 ; phenols: 8400 mg l−1 ; and glucose 1200 mg l−1 . 2.2. Field site and sampling Tree located at Ouled Jaballah, Tunisia, North latitude 35◦ 12 , East longitude, 10◦ 59 , spaced 12 m × 12 m apart were selected in 2004 for the experiments [7,8]. The climate of this region is typical Mediterranean, semiarid to arid, with an average rainfall of 200 mm year−1 and an average annual temperature of 18–20 ◦ C. Physico-chemical characteristics of the soil at this site were as follows: pH: 8.53; EC: 0.44 ds m−1 ; sand: 78.1%; clay: 12.85%; silt: 5.1%; organic C: 0.37%; N: 0.042%; Olsen P: 20.86 mg kg−1 ; Exchangeable K: 0.43 meq 100 g−1 . The experiment included three levels of OMW application (TC: 0 m3 ha−1 , T1: 30 m3 ha−1 , T2: 60 m3 ha−1 and T3: 150 m3 ha−1 ). Three plots (576 m2 each: 24 m × 24 m) were designed for each treatment. This amendment was realised in December 2004 in one application [7,8]. All sampling events included the collection of soil samples (0–20 cm deep) from five random locations in each plot at harvest (February 2006). Roots and olive fruits samples were collected at this time. A composite root sample was collected per plot, for determination of arbuscular mycorrhizal (AM) fungi colonisation. From each plot, approximately 2 kg of fruit was taken to determine olive fruits and olive oils quality. 2.3. Soil fatty acid methyl ester (FAME) analysis Lipids were extracted from the soil samples (3 g) as well as the root samples (30 mg) using the EL-FAME method [15]. Briefly, a mild alkaline hydrolysis (0.2 M KOH in methanol) was used to extract whole cell fatty acids (FAs). This included FAs from phospholipids, glycolipids and neutral lipids. The FAME extraction residue was dissolved in hexane. The hexane layer was transferred to a clean tube, and the hexane was evaporated off, after which FAMEs were resuspended in 0.5 ml of hexane–methyl tert-butyl ether (1:1) for analysis. Individual FAMEs were analyzed on a Hewlett-Packard 5890 gas chromatograph equipped with a flame ionisation detector and a HP-5MS column (95% dimethyl–5% diphenyl polysiloxane, length 30 m × 0.25 mm). The temperature was programmed to increase from 170 to 270 ◦ C at a rate of 5 ◦ C min−1 . The temperature was increased to 270 ◦ C for 2 min between samples in order to clean the column. Identification of FAMEs was by retention time and confirmed by gas chromatography–mass spectrometry (GC–MS). The following FAMEs were designated as bacterial: i15:0, a15:0, 15:0, 16:19, i17:0, a17:0, 17:0, 17:18, cy17:0 and cy19:0. Soil FAME 18:19 and 18:26 were used as indicators of saprophytic fungi. Soil FAME 16:15 was used to indicate (AM) fungi abundance. Root FAME 16:15 analysis was used as index for the development of AM colonisation in the olive trees roots [7].
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2.4. Photosynthesis measurements Photosynthetic rates (A) were measured in the field by using a LI-6400 gas exchange system (Li-Cor, Lincoln, NE, USA) from 11:00 to 13:00 in February 2006 [7]. Net photosynthesis rate (A in mol CO2 m−2 s−1 ), was measured at fixed CO2 concentration (CO2 at 400 mol mol−1 ), air temperature (25 ◦ C), relative humidity (60%) and a photosynthetic photon flux density (1500 mol m−2 s−1 ). 2.5. Olive analyses 2.5.1. Soluble carbohydrate determination The olive soluble carbohydrates were extracted according to the method described by Bartolozzi et al. [16]. Briefly the soluble carbohydrates from composite olive samples were extracted twice in 80% ethanol at 70 ◦ C. Extracts were dried and converted into trimethylsilyl ethers with a silylation mixture made up of pyridine, hexamethyldisilazane and trimethylchlorosilane. Soluble sugars were analyzed using a Hewlett-Packard 5890 series II gas chromatograph equipped with a flame ionisation detection (FID) system and a HP-5MS column (30 m × 0.25 mm) as described by Bartolozzi et al. [16]. Identification of individual carbohydrate was achieved by use of the relative retention times, i.e., in comparison to that of the standard. These were compared to those identified earlier by gas chromatography–mass spectrometry. 2.5.2. Olive N and P determination Olive nitrogen (N) content was determined by the Kjeldahl method [17]. The analysis of phosphorus (P) was carried out following the official method of the AOAC no. 970-39 Phosphorus in Fruits and Fruit Products (spectrophotometric molybdovanadate method) [18]. The method is based on the capacity of P (in phosphoric acid form) in an acid solution and in the presence of V5+ and Mo6+ to form a yellow phosphomolybdovanadate complex whose absorbance can be measured at 400 nm. 2.6. Olive oil analyses 2.6.1. Determination of oil content Oil content was determined by extracting dry material with 40–60 ◦ C petroleum ether using a Soxhlet apparatus. The extract was dried at 70 ◦ C and weighed. 2.6.2. Fatty acid determination The fatty acid composition of the oil was determined by gas chromatography (GC) as fatty acid methyl esters (FAMEs). Methyl esters were prepared from olive oil by vigorous shaking of a solution of oil in hexane (0.2 g in 3 ml) with 0.4 ml of 2N methanolic potassium hydroxide. Individual FAMEs were separated and quantified by gas chromatography with a FID detector. The temperature was programmed to increase from 170 to 270 ◦ C at a rate of 5 ◦ C min−1 . Nitrogen was used as a carrier gas. Fatty acids were identified by comparing retention times with standard compounds. 2.6.3. Total phenol content determination Total phenols and amounts were quantified colorimetrically [19]. Phenolic compounds were isolated by triple extraction of a solution of oil in hexane with a water–methanol mixture (60:40, v/v). The Folin–Ciocalteau reagent (Merck Schuchardt OHG, Hohenbrunn, Germany) was added to a suitable aliquot of the combined extracts, and the absorption of the solution at 725 nm was measured. Values are given as mg of caffeic acid per kilogram of oil.
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2.6.4. ˛-Tocopherol determination ␣-Tocopherol was evaluated by high-performance liquid chromatography (HPLC) with direct injection of an oil in hexane solution [20]. Separation by HPLC was carried out using a Hewlett-Packard liquid chromatographic system (Waldbronn, Germany) with an HP-1100 pump system and a Rheodyne Model 7125 injector (Cotati, CA, USA) with a final volume loop of 20 l. The detector was an HP-1040 M photodiode-array detection (DAD) system. The data were stored and processed by an HPLC Chemstation (Dos Series) (Hewlett-Packard). The column was a Supelco ODS-2 (150 mm × 4.6 mm I.D., 5 mm particle size). The mobile phase consisted of methanol/water (96:4, v/v) at a flow rate of 2 ml min−1 . ␣-Tocopherol was quantified by the internal standard method. Results are given as mg of ␣-Tocopherol per kilogram of oil. 2.7. Statistical analyses All statistical values (mean values, standard deviation) were calculated using the SPSS statistic program. Pearson’s correlation coefficients were used when calculating correlations between mannitol and oil content. In addition, Duncan’s multiple range tests were used to determine significant differences among data. Olive fruits compositions (olive N, olive P and soluble carbohydrates) were evaluated with the XLSTAT 2006 Version 2006.06 using principal components analysis (PCA) in order to compare the effects of OMW on olive fruits quality. 3. Results and discussion 3.1. Soil microbial community, AM fungus colonisation and photosynthesis after agronomic application of OMW The abundance of the saprophytic fungi as indicated by the sum of FAME 18:19 and 18:26 was significantly higher in the OMW amended soil than that in the control soil (Table 1), indicating that OMW constitutes an adequate substrate for saprotrophic fungi [7,8]. Agronomic application of OMW increased significantly the fungal:bacteria ratio from 0.23 in the control soil to 1.11 in the soil amended with 150 m3 ha−1 OMW. The modify values of fungi:bacteria was an agreement with the change in the soil microbial community following agronomic application of OMW [7,8]. The abundance of AM fungi and the root colonisation decreased significantly in the olive trees irrigated with OMW as compared to the control. The decreased root colonisation might have decreased the photosynthetic rate, as photosynthesis is under some control by sink demand [21]. It is possible that the sink strength of the roots in the OMW amended soil is lower, due to the reduced root colonisation after agronomic application of OMW. Wright et al. [21] demonstrated that AM fungal colonisation stimulated the rate of
Fig. 1. Glucose, fructose, mannitol and galactose content in olive fruits following agronomic application of OMW (TC: control, T1: 30 m3 ha−1 OMW, T2: 60 m3 ha−1 OMW and T3: 150 m3 ha−1 OMW). Different letters indicate significantly different values at P ≤ 0.05 according to Duncan test.
photosynthesis sufficiently to compensate for the carbon requirement of the fungus. In view of the above background, the following questions were addressed: (i) is the specific environment created after agronomic application of OMW (increase of saprophytic fungi, decrease of bacteria, AM fungi and root colonisation) beneficial or detrimental to the growth of olive fruits? and (ii) If land spreading of OMW has an effect (positive effect or negative effect) on the development of olive fruits, does the olive oil quality change? 3.2. Olive fruits soluble carbohydrate after agronomic application of OMW Sugars are the main soluble components in olive tissues and play an important role, providing energy for metabolic changes. They are important components of the cell-wall, related to the textural properties [22]. Results of the GC determinations are given in Fig. 1. The major sugars in control and OMW irrigated olive-pulp were glucose, fructose and galactose. Differences between TC, T1, T2 and T3 treatments were significant for glucose and fructose and nonsignificant for galactose. Glucose was the predominant component in the TC and the T1 treatments, while fructose was the predominant component in the T2 and T3 treatments. Generally glucose accounted for 57.58%, fructose for 17.32% and galactose for 4.89% of the total sugars in the control, while glucose accounted for 42.24%, 37.19%, 24.92%, fructose for 40.8%, 48.18%, 59.11% and galactose for 2.46%, 2.51%, 4.% of the total sugars in the T1, T2 and T3 treatments respec-
Table 1 Influence of OMW treatments on soils microbial community, roots colonisation and olive trees photosynthesis.
a
Fungi (% total FAME) Bacteriab (% total FAME) Fungi:bacteria AM fungic (% total FAME) Root colonisationd (%) Photosynthesis (mol m−2 s−1 )
Control
Agronomic application of OMW (m3 ha−1 )
TC
T1 (30)
4.56 20.05 0.22 1.47 14.3 8.67
± ± ± ± ± ±
0.76d 1.41a 0.04d 0.4a 0.95a 0.79a
9.39 20.22 0.46 1.4 12.5 8.57
± ± ± ± ± ±
T2 (60) 1.73c 1.21a 0.03c 0.3a 0.9ab 0.65a
11.63 19.42 0.59 1.24 10.5 8.38
± ± ± ± ± ±
T4 (150) 1.71b 1.64a 0.01b 0.16b 0.73bc 0.52ab
18.11 16.38 1.1 0.82 9.1 5.68
± ± ± ± ± ±
0.26a 0.53b 0.03a 0.02c 1.82c 1.62c
The effect of OMW treatment was tested with one-way ANOVA (mean value ± SE, n = 3 for fungi, n = 3 for bacteria, n = 3 for colonisation, n = 6 for photosynthesis), and mean values in individual line followed by the same letter are not significantly different at P < 0.05 (Duncan test). a 18:26 and 18:19. b i 15:0, a15:0, 15:0, 16:19, 16:1 7, i17:0, a17:0, 17:0, cy17:0, 18:17 and cy19:0. c Estimated as the soils FAME 16:15 analysis. d Estimated as the roots FAME 16:15 analysis.
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tively. Mannitol was also found, with a content ranging between 17.67% in the control to 13.08%, 10.04% and 8.66% in the T1, T2 and T3 treatments respectively. Such significant differences in the relative proportion of sugar probably reflect the metabolic behaviour of each olive tree in relation to different specific environment conditions created in soil following land spreading of OMW. Marsilio et al. [23] reported that the differences in the individual sugars in olive fruits reflect the metabolic behaviour of each cultivar in relation to the genotype and to different climatic and environmental conditions. 3.3. Olive P and N following irrigation with OMW Agronomic application of OMW decreased significantly the olive fruit content of N and P (Fig. 2). The role of AM fungi in the stimulation of growth and nutrient uptake of many host plants is well documented [24]. Olive plants are known to form AM fungi [7,25], and can be colonized by different species, including Glomus mosseae, Glomus clarum, Glomus caledonium, Glomus monosporum, Glomus intraradices and Glomus viscosum [25,26]. In this study, agronomic application of OMW decreased significantly the root FAME 16:15. The fatty acids 16:15 have been used to estimate the extraradical biomass in pot cultures and the amount of these fatty acids were well correlated with the AM hyphal length [27]. OMW, that have negative effect on the AM hyphal length, might consequently affect the mycorrhizal effectiveness on nutrient uptake. In principle, AM plants having a developed extraradical mycelium could potentially absorb more micronutrients than AM plants with less developed extraradical hyphae. The higher nutrient uptake in mycorrhizal plants might be attributed to the contribution of
Fig. 3. Oil accumulation in the olive fruits after agronomic application of OMW. Different letters indicate significantly different values at P ≤ 0.05 according to Duncan test.
external hyphae which explore a large volume of soil and thus absorb more N, P and certain others nutrients [28,29]. However, the negative impact of agronomic application of OMW on the olive fruit N and P found in the present study can be explained by this mechanism. This implies that the differences in the olive N and P concentration we observed after agronomic application of OMW were due to factors in the OMW amended soil that inhibit the AM fungal root colonisation and thus the development of AM fungi [7]. It has been reported that AM fungi enhance plant nutrient acquisition in alkaline soil [30], and alleviate cultural and environmental stresses [24], through greater effective root area and the activation and excretion of various enzymes by AM fungi roots and/or hyphae [31]. 3.4. Oil accumulation in the olive fruits after agronomic application of OMW The oil accumulation behaviour is illustrated in Fig. 3. The oil content expressed as a percentage of dry weight decreases after agronomic application of OMW. Nergiza and Engez [32] found a positive correlation between the oil accumulation and sugar content. Marsilio et al. [23] indicated that the relative amount of mannitol in the fruit might indicate the potential of a cultivar for oil biosynthesis. A good correlation coefficient (r = 0.848; P < 0.001) was established in this study between mannitol and oil content (Fig. 4). Thus, the mannitol in the olive, as well as other polyols in many other higher plants, might be of specific importance in the metabolic transformation and synthesis of the fruit storage material [33]. Jiménez et al. [22] reported that sugars are the main
Fig. 2. Effect of agronomic application of OMW on the olive fruits nitrogen (A) and phosphorus (B) (TC: control, T1: 30 m3 ha−1 OMW, T2: 60 m3 ha−1 OMW and T3: 150 m3 ha−1 OMW). Different letters indicate significantly different values at P ≤ 0.05 according to Duncan test.
Fig. 4. Relationship between the olive oil content and the olive fruits mannitol (n = 12).
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Table 2 Fatty acids composition (%) in olive oil following agronomic application of OMW. Fatty acids (%)
Control
Agronomic application of OMW (m3 ha−1 )
TC (0)
T1 (30)
T2 (60)
T3 (150)
Saturated Palmitic C16:0 Margaric C17:0 Stearic C18:0 Arachidic C20:0
20.12 0.09 2.55 0.46
Monounsaturated Oleic C18:1 Decenoic C17:1 Palmitoleic C16:1
55.49 ± 0.1 0.1 ± 0.01 2.6 ± 0.43
55.09 ± 0.9 0.15 ± 0.03 3 ± 0.21
54.29 ± 0.33 0.11 ± 0.06 2.85 ± 0.22
55.47 ± 0.36 0.1 ± 0.04 2.39 ± 0.18
Polyunsaturated Linoleic C18:2 ␣-Linolenic C18:3
21.29 ± 0.5 0.76 ± 0.08
21.83 ± 0.3 0.67 ± 0.01
21.33 ± 0.3 0.86 ± 0.07
21.65 ± 0.6 0.88 ± 0.01
± ± ± ±
0.4 0.03 0.3 0.05
20.55 0.14 2.38 0.44
soluble components in olive tissues and play an important role, providing energy for metabolic changes. They are important components of the cell-wall, related to the textural properties of the fruit and act as precursors for olive oil biosynthesis. Based on these results, it can be said that the change in the individual olive fruit carbohydrate after agronomic application of OMW can be considered as a potential factor modifying oil biosynthesis. 3.5. Fatty acid composition following agronomic application of OMW Agronomic application of OMW did not cause significant variations in fatty acids composition (Table 2). Fatty acids were palmitic (16:0), palmitoleic (16:1), heptadecanoic (17:0), heptadecenoic (17:1), stearic (18:0), oleic (18:1), linoleic (18:2), linolenic (18:3) and eicosanoic (20:0). The percentages of saturated (palmitic + heptadecanoic + stearic + eicosanoic), monounsaturated (palmitoleic + heptadecenoic + oleic) and polyunsaturated (linoleic + linolenic) fatty acids of lipid classes and the unsaturated/saturated, monounsaturated/polyunsaturated and oleic/linoleic acid ratios did not appear to be significantly influenced by irrigation with OMW.
± ± ± ±
0.36 0.04 0.1 0.04
20.7 0.08 2.59 0.49
± ± ± ±
0.2 0.01 0.33 0.07
20.07 0.07 2.73 0.46
± ± ± ±
0.26 0.01 0.1 0.07
cially for oil obtained from olive trees irrigated with 150 m3 ha−1 , making this hypothesis more plausible to explain these results. Cinquanta et al. [37] have noted that qualitative and quantitative compositions of olive oil’s phenols are strongly affected by the agronomic and technological conditions of its production. Among agronomic parameters are cultivar, pedoclimatic conditions of production and some agronomic techniques such as the irrigation [38]. We found in this study that agronomic application of OMW appeared capable of influencing the quantitative composition of olive oil phenols. The effect of agronomic application of OMW on the qualitative composition of olive oil phenols should be examined in future studies.
3.6. Olive oil polyphenol and ˛-Tocopherol content after agronomic application of OMW Polyphenols content, a component of great importance in oil quality because of its antioxidant effects, increased significantly following agronomic application of OMW (Fig. 5A). To our knowledge land spreading of OMW creates a stress situation that induces the production of phenolics. In fact, the decline in the root colonisation might to consequently affect the mycorrhizal effectiveness on nutrient uptake which, in turn, could have declined the olive nutrient content. These changes in soil properties and soil microbial community observed following agronomic application of OMW can generate a stress situation that induces high oil phenol content. This makes it rather unlikely that the high oil phenol content observed after agronomic application of OMW can be related to the significant decrease in the olive N and P content. In the case of low nutrient availability, as occurs in our experiments (especially in T3 treatment), phenylalanine preferentially flows in to phenylpropanoids synthesis via phenylalanine ammonia lyase rather than toward synthesis of protein [34]. As observed in other species, phenylalanine ammonia lyase enzyme could play a key role in olive. After phenylalanine deamination, trans-cinnamic acid is formed which is the precursor of polyphenolic substances [35]. In our study, trees fruits protein content, estimated from its N content [36], was lower whereas polyphenol content in olive oil was higher, espe-
Fig. 5. Effect of agronomic application of OMW on the olive oil polyphenol (A) and ␣Tocopherol (B) content from different OMW treatments (TC: control, T1: 30 m3 ha−1 OMW, T2: 60 m3 ha−1 OMW and T3: 150 m3 ha−1 OMW). Different letters indicate significantly different values at P ≤ 0.05 according to Duncan test.
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and the whole plant carbon balance, and consequently, on chemical characteristics of oil. Several works have shown that several factors such as environmental and agronomic factors have an effect on plant physiologic behaviour and, consequently, on oil chemical properties [40,41]. We found that agronomic application of OMW not only modify the soil properties, the soil microbial community and the olive trees key physiological parameters, but also appeared capable of influencing the olive fruits characteristics and oil quality. Our results support the hypothesis that the change in the functioning of arbuscular mycorrhizas following agronomic application OMW should be considered as a potential factor influencing olive and oil quality. Acknowledgements The authors wish to thank M. Imed cheraief for his help in GC/MS analysis. This study was supported by the Ministère de l’Enseignement Supérieur, Ministère de la Recherche Scientifique de la technologie et de développement des compétences (UR03/ES08 Nutrition et Désordres Métaboliques) and Institut de l’Olivier de Sousse. References
Fig. 6. Effects of agronomic application of OMW on the olive quality. (A) PCA of olive after agronomic application of OMW. (B) PCA showing loading values for olive N, olive P, fructose, galactose, mannitol and glucose of the treatments plots given in (A). The variance explained by the each principal component axis is shown in parentheses (() control; () 30 m3 ha−1 OMW; () 60 m3 ha−1 OMW; () 150 m3 ha−1 OMW).
␣-Tocopherol decrease with increasing dose of OMW (Fig. 5B), showing an inverse trend than polyphenols. ␣-Tocopherol seems to become effective when the activity of the polar phenolic fraction is reduced and primary products of autoxidation reach a critical concentration [39]. 3.7. Chemometric analysis Principal component analyses of olive fruits compositions (olive N, olive P and sugars) accounted for 74.65% of the variance on the first component, while the second component accounted for 18.35%. This explained 93.01% of the total variance (Fig. 6). PCA of the samples revealed separation of the four treatments into four distinct clusters. The first cluster (TC), the second cluster (T1), the third cluster (T2) and the fourth cluster (T3), where the first cluster (control plots) and the second cluster (olive trees irrigated with 30 m3 ha−1 ) were positively associated with PC1 whereas the third cluster (olive trees irrigated with 60 m3 ha−1 ) and the fourth cluster (olive trees irrigated with 150 m3 ha−1 ) were negatively associated with PC1 (Fig. 6A). This indicated that clear differentiation exists between olives obtained from TC and T1 treatments and those obtained from T2 and T3 treatments. This was also illustrated by the fact that olives N, olives P, glucose and mannitol are more common to the right of PC1 (TC and T1 treatments), whereas fructose is more common to the left on PC 1 (T2 and T3 treatments) (Fig. 6B). 4. Conclusion Agronomic applications of OMW were demonstrated by this study to have a major impact on the growth and establishment of AM fungi. Conversely, the AM fungi have considerable effects on plant physiology apparently through changes to the mineral status
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