Olive Mill Waste Application To Soil Biology Essay

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The organic matter is a soil fraction regulating the biological activity of soils, so a satisfactory content in organic material, dominates the soil fertility, Economou et al. (1), Chouliaras et al. (2), Gougoulias et al. (3).

The very important amounts of olive mill waste produced in olive cultivation areas, ranging between 1,75x106 and 2, 25 x106 tons/year of water-waste for Greece, Kyriazopoulos (4) then the profitable use of these organic materials as soil amendments are beneficial both to soil improvement and environmental protection.

Numerous methods are used for the treatments of olive mill waste, then the respective products added to the soil, act various effects on soil properties and plant growth, Ouzounidou Georgia et al. (5). The waste could be applicable as composted material while the raw waste could have beneficial effects concerning time and cost, Lopez-Pineiro et al. (6).

The beneficial effects of these amendments are related to soil organic matter increase, Carbonnell et al. (7) then, consequently, to soil chemical and physical property improvement, Ntoulas et al. (8). In according to Lopez-Pineiro et al. (9), successive applications of the de-oiled two-phase olive mill waste on soil as amendment, may be an effective management practice for controlling their ability to increase P-availability. The two-phase olive mill waste application to olive grove soil, increased organic carbon, total N, available P & K, aggregated stability and, in general, increased olive production, Lopez-Pineiro et al. (10).

The effect of this waste material on soil microbial activity was also assessed, Kotsou et al. (11), Hameed et al. (12), Saadi et al. (13). Moreover, the antimicrobial effect, probably due to polyphenolic compounds, occurring in olive mill waste water against fungi, was demonstrated by Vagelas et al. (14) and Vagelas et al. (15). Further, more according to Abid et al. (16), water thermophilic bacteria as well as actinomycetes dominated over eumycetes, during composting of the olive mill waste.

The aim of this work was to examine the effects of biological and chemical properties on soil, caused by the rate and the nature of the solid-waste or water soluble-waste of olive mill, in vitro.


The experiment lasted for two periods of incubation. During the 1st period, an amount od wast tested either in liquid or solid form was added to the soil. During the 2nd period different mixtures of both form materials were tested.

1st experiment - separate application of each form waste to soil

In this study, 13.51, 27.03 and 40.54 g of air dried solid waste material containing 2.5, 5.0 and 7.5 g of organic matter, were added to 50 g of air dried, light textured soil respectively, which was poor in organic matter. All these materials â€" soil and waste â€" were obtained from the region of Larissa (Greece, Table 1). Into 50 g of the same soil 41.7, 83.3 an 125.0 g of liquid waste was also added, containing 2.5, 5.0 and 7.5 g of organic matter, respectively. In the middle of the incubation period, all treatments of the 1st experiment, were leached with distilled water (1soil: 5H2O) and all water extracts were collected to be analysed, in order to approach the ecological effect of rainfall in natural leaching conditions.

2nd experiment - application of both from waste to soil, in mixture

This experiment was established based on the data of the 1st experiment, then an adequate mixture of the two form waste was chosen, in order to be tested.

In this study, 24.39 g of solid waste +8.13 g of liquid waste, (3S:1L), 20.4 g of solid waste+ 20.4 g of liquid waste, (1S:1L), and 13.7 g of solid waste+41.1 g of liquid waste, (1S:3L), were added to 50 g of the same soil, respectively. Each mixture contained 5.0 g of organic matter.

In the incubator, the treatments were prepared in triplicates and kept at 28 oC for a period of 15 weeks. During the first three weeks of the incubation period, the moisture was maintained at two-thirds of field capacity, but for the next three weeks the soils were left to dry. This process was repeated until the end of the incubation period. According to Wu and Brookes (17), the alternation of drying and rewetting soil samples enhances mineralization of both soil biomass organic matter and non-biomass organic matter.

At the end of the incubation period, soil samples were analysed using the following methods which are referred by Page et al. (18).

Organic carbon was analysed by chemical oxidation with 1 mol L-1 K2Cr2O7 and titration of the remaining reagent with 0.5 mol L-1 FeSO4.

Both ammonium and nitrate nitrogen were extracted with 0,5 mol L-1 CaCl2, and estimated by distillation in presence of MgO and Dewarda alloy, respectively.

Organic phosphorus was measured after mineralization by combustion of the soil sample and subtraction of the mineral phosphorus amounts, which had previously been estimated in the laboratory. The mineral amounts were extracted with 1 mol L-1 H2SO4 and all forms were measured by spectroscopy.

Available P forms (P-Olsen) were extracted with 0.5 mol L-1 NaHCO3 and measured by spectroscopy.

Exchangeable forms of potassium were extracted with 1 mol L-1 CH3COONH4 and measured by flame photometer.

Available forms of Mn, Zn and Cu were extracted with DTPA (Diethylene Triamine Penta Acetic Acid 0.005 mol L-1 + CaCl2 0.01 mol L-1 + Triethanolamine 0.1 mol L-1) and measured by atomic absorption.

Total humus compounds (Humic+Fulvic acids) were extracted at pH:12 with NaOH, and Humic acids were precipitated at pH: 2 with HCl, according to fractionation of soil organic matter protocol, proposed by Chouliaras et al. (19).

The soil organic carbon contents were transformed in organic matter contents by multiplying by 1.724, which is an experimental factor, reported by Hesse (20).

For investigating the effect of added material on the soil microflora (bacterial and fungal communities), a small amount of soil was spread onto Potato Dextrose Agar (PDA) plates and incubated for two days at 25 oC in darkness. After the incubation period the number of bacterial colonies formed were counted. The same plates were further incubated as above for another six days. After the incubation period the emerged fungi per plate and per treatment were recognised under the microscope.

Statistical analyses

The experiment was repeated and the completely randomized design with four replications was used. Tukey’s procedures were used to detect and separate the mean treatment differences at P = 0.05. Statistical analyses were performed by the statistical program MINITAB (21).


1st experiment - separate application of each form waste to soil

The water extracts, taken by leaching the soil treatments during the incubation period, showed a significant increase of soil salinity and alkalinity in all the samples amended with water soluble waste; in these water extract treatments, a significant elevation in P and K water soluble forms was also attested in comparison with control, (Fig-1).

The organic contents of the treatments at the end of the incubation period showed a high rate of organic matter biodegradation, about >30% for all the samples amended with soluble waste. On the contrary, a strong resistance to biodegradation has been shown by solid waste; a significant increase of salinity and alkalinity was also reveled for the two upper rates of liquid applications, and available forms of P and K were also found significantly increased in these treatments with water soluble waste, (Fig-2).

The NO3- content of all treatments at the end of the incubation period is higher in comparison with NH4+ , but that (N-NO3-/ N-NH4+) ratio is lower in the case of water leaching extracts taken during the incubation time, which could be explained by a necessary nitrification process, achieved for all over the incubation period. A significant increase for P-organic synthesis was also reveled for all treatments.

Despite the elevation in available forms for Cu, Mn, Zn in comparison to the control for all treatments, an insignificant elevation of total contents for these metals was attested, and the increase of exchangeable Na did not cause any sodicity risk for soil, (Fig-3).

The humic acid contents are always predominating in comparison with fulvic acid contents for treatments with solid waste, (Fig-2).

According to our results, soil bacterial numbers increased significantly by L3 (mill wastewater) treatment where as L1 treatment inhibited soil bacterial growth (Table 2). Changes in total soil bacteria number treated with S1 and S3 are supported by the fact that a higher concentration of available Zn was found at S3 treatment compared with S1 (Figure 3).

2nd experiment - application of both from waste to soil, in mixture

The mixture of these two sorts of waste added to soil, produced a well-decomposed material during incubation, concerning organic matter for both solid or water soluble waste; the organic matter of these mixture is decomposed about >40%, then organic matter of solid waste is better decomposed under the effect of the water soluble waste, in comparison to the results of the 1st experiment where solid waste is separately added to soil. The salinity was further increased in the case of the mixture 1S:3L, but soil pH was slightly affected in all treatments. At the end of the incubation period, relative high amounts of N-NH4+ forms yet existed, while P-organic content increased significantly in all treatments. Available forms of P, K, and Mn were increased especially at the mixture 1S:3L, also the available forms of Cu and Zn increased for all treatments. The increase of exchangeable Na did not cause any sodicity risk for soil by these mixtures either, (Fig-4).

Moreover, figure 4 showed that, when the available Zn increased, total soil bacterial numbers decreased (Table 3, treatment 1S: 3L), suggesting that 1S: 3L treatment contains high available Zn accumulation with negative impacts on soil bacterial community. Furthermore, it seems that olive mill wastewater has a significant impact on soil borne fungi belonging to genera Rhizopus (Table 2 and 3) and it might have allowed the growth of some fungi species of Aspergillus and Fusarium (Table 2 and 3).


The organic content of the treatments at the end of the incubation period showed a high rate of organic matter biodegradation for samples amended with water soluble waste. However, a strong resistance to biodegradation has been proved by solid waste, when the two kinds of waste are added separately; the organic matter of solid waste is better decomposed under the effect of the water soluble waste, when the two waste sorts are added together; a probable explanation for that effect could be the higher content in fulvic acids of water soluble waste, and these more labile compounds are estimated as more biodegradable by soil micro flora, Jacquin & Chouliaras (22). The salinity elevation is more affected by liquid waste application, but it is important to remark the increased presence of available forms of P, K, Zn, Mn and Cu in the soils amended with the mixture of solid +water soluble waste, in the ratio (1S:3L).

According to our findings the soil amended separately and combining with the solid (S) or liquid (L) form of olive mill waste, reduced bacterial growth significantly duo to the increase of heavy metals, such as Zn and Cu. Toxicity of these heavy metals (Zn, Cu) to the organism (soil bacterial community) is well known, Frostegard et al. (23), Giller et al. (24), Novac et al. (25). According to our results, soil amended combining with solid (S) and liquid (L) form of olive mill waste in 1S:3L showed significantly fewer total soil bacteria numbers and a much bigger amount of Zn and Cu compared to all other treatments which suggested that soil treated with this mixture conducted toxic. Moreover, this study showed that the soil amended with 1S:3L mixture increased the growth of fungi belonging to the subdivision Deuteromicotina, class Deuteromycetes (particularly Aspergillus and Fusarium) and the fungus belonging to genus Mucor (class Zygomycetes) concluded that a) these soil fungi are resistant to toxic metals, such as Zn and Cu and b) it is seems that in the present study these fungi species increased the exchangeable K in soil. According to literature microorganisms like fungi species Aspergillus and Fusarium are known for their resistance to heavy metals, Kapoor et al. (26), Ahmad et al. (27), Bishnoi and Garima (28). Based on this, we can conclude that soil amended with 1S:3L olive mill waste mixture could be an alternative way to chemical control.