مطالعه کمپوست .... Study of Organic Matter Evolution in Citrus Compost By Isoelectrofocusing Techniq
Study of Organic Matter Evolution in Citrus Compost By Isoelectrofocusing Technique
By Trinchera, Alessandra Tittarelli, Fabio; Intrigliolo, Francesco
Three composts from citrus-processing industry wastes, sampled at prefixed times during the composting process, were studied with the aim to follow the organic matter evolution by using the isoelectrofocusing (IEF) technique. Results indicated that IEF qualitative analyses allowed evaluation of the organic matter transformation during composting process, showing a decrease of IEF peaks focused at lower values of pH (less stabilized organic matter) and a corresponding increase of peaks focused at higher value of pH (more humified material). The parameter A^sub s^%, defined as the areas sum of IEF peaks focused at pH>4.7, could be considered particularly effective as a "threshold value" to evaluate the level of organic matter evolution for the considered composts. Introduction
The quality of a compost as organic amendment in agriculture is strictly connected to its level of stability and maturity. The meaning of "compost stability" and "compost maturity" is not always clear, since sometimes "stability", which should be mainly intended as biochemical stabilization of compost organic matter (corresponding to higher amount of slowly degradable compounds) (Kirchmann 1991; Chica et al. 2003), is confused with "maturity", which instead should be associated with compost's beneficial effect on plant growth (Chen & Aviad 1990). In any case, generally, organic matter stabilization during composting, being associated with the humification process, goes in parallel with the decrease in phytotoxicity, since the production of organic substances characterized by higher molecular complexity also reduces the risk of fermentation phenomena (Hue & Liu 1995; Cooperband et al. 2003).
During the composting process, extremely different organic compounds can be formed, depending on starting materials, operative conditions, microbiological components and so on. Many authors (Sequi 1995; Zach 2002) demonstrated that amendment properties and compost stability should be searched in the humic-like fraction of compost organic matter. On the other hand, it was suggested that humic-like substances are mainly derived from the "concentration effect" of the native ones, as a consequence of the biochemical degradation of most labile organic fraction (Adani et al. 1997; Chen et al. 1997). For this reason, it is necessary to take into account the chemical characteristics of organic substances which constitute the starting compost materials and then follow their transformation during composting (Sequi 1995). This strategy should avoid erroneous evaluation, since sometimes humification index values of raw starting matrices used for compost production are very high, because of the presence of proteinaceus or glico-peptidic compounds that can interfere with the chemical determination of humified organic fraction (Ciavatta & Govi 1993). Different methods can be used to purify humic substances from composts (Ciavatta et al. 1993; Govi et al. 1994; Chefetz et al. 1996; Chen et al. 1997), allowing quantitative information to be obtained, though related to the different chemical treatments considered in the analytical procedures.
Instrumental techniques used to follow organic matter evolution during the composting process could constitute a valid contribution for improving knowledge on its humification level. Among them, the isoelectrofocusing (IEF), which is an isoelectrophoretic technique able to separate different organic compounds on the basis of their isoelectric point (pI), generally utilized in DNA separation and proteomic studies, was effectively and widely applied also to study humic matter in soils (Ciavatta & Govi 1993; Alianiello 1999; Trinchera et al. 2001; Dell'Abate et al. 2002), organic fertilizers and composts. In particular, IEF was utilized for characterizing organic matter in compost obtained from different matrices (green residues, olive oil mill wastewater, poultry manure, agro- industrial wastes, slurries, organic fraction of municipal solid wastes and so on) (Ciavatta et al. 1997; Canali et al. 1998, Alianiello et al. 1999; Tittarelli et al. 2002; Cavani et al. 2003; Trinchera et al. 2003; Alianiello & Baroccio 2004).
As reported in the literature (De Nobili et al. 1989; Govi et al. 1994; Govi et al. 1995), the more humified the organic matter is, the less acidic and higher in molecular weight are the organic compounds which compose it. This information, which is valid for soil humic matter as well as for humic-like compounds, allows use of the IEF technique to separate organic matter extracted from different materials. Recently, IEF was successfully used to qualitatively characterize compost organic matter in different phases of the composting process (Canali et al, 1998). The use of this technique for following the evolution of organic substrate from the first thermophilic phase to the last curing phase of the composting process can put in evidence the actual chemical transformation of compost organic matter, also taking into account the chemical characteristics of original starting materials.
This study was carried out on three composts from citrus- processing industry wastes which had been previously analyzed for stability (by measurements of CO2 evolution of amended soil), genotoxicity and phytotoxicity (De Simone et al. 2001; Tittarelli et al. 2003). Obtained results demonstrated their stability, maturity and absence of any genotoxic effect.
The objective of this work was to verify the effectiveness of isoelectrofocusing technique for monitoring organic matter evolution in composts from agroindustrial citrus waste, in order to widen its application field.
Materials and Methods
The considered composts were prepared by mixing agro-industrial wastes, coming from the citrusprocessing industry. The starting materials were pastazzo (a mixture of citrus pulp and skins, which represents the main residue of citrus-processing squeezing treatment) and sludge (citrus-processing industry effluents, which undergo purification treatment), together with green residues, used as bulking agent. All composting trials were performed in the Azienda Sperimentale "Palazzelli" (Lentini) of the C.R.A. - Istituto Sperimentale per l'AgrumicolturaAcireale (CT, Italy) (Rapisarda et al. 1998; Intrigliolo et al. 2001). Table 1 reports the main chemical-physical parameters of the starting matrices.
Three heaps were prepared on concrete platforms, taking into account the chemical analysis of pastazzo and sludge and the C/N ratio of green residues (which was 77). Other chemical parameters of green residues were not considered useful for heap preparation, being their contribution to composting, apart from C/N ratio, mainly as the bulking agent. Mix ratios were as follows:
TABLE 1.
Main chemical physical parameters of pastazzo and sludge.
Compost C1: pastazzo 40% - sludge 20% - green residues 40%. Composting time: February - June 1999 (winter-spring season); Compost C2: pastazzo 40% - sludge 20% - green residues 40%. Composting time: May - September 1999 (spring-summer season); Compost C3: pastazzo 60% - green residues 40%. Composting time: May - September 1999 (spring-summer season).
The first and the second composts were characterized by the same starting mixture, but they differed for the seasonal period during which the composting process was carried out (cold climate during C1 thermophilic phase and hot and dry climate in case of compost C2 and C3).
Pastazzo and green residues utilized for C1, C2 and C3 were collected in advance and adequately stored in a protected area, in order to guarantee the same chemical-physical characteristics of starting materials. The third compost differed from Cl and C2 for the lack of sludge in the starting mixture (with the corresponding increase of pastazzo percentage); the C3 composting process was carried out in the same period of compost C2. All piles were protected by a Gore-tex(R) cover in order to avoid excessive moisture due to rainfall.
The temperature of the three heaps was measured during the whole composting period and moisture maintained at the optimum values (5060%). Piles were turned more frequently during the thermophilic phase and less frequently during the mesophilic composting phase. Samples of composts were taken from each pile at prefixed time: mixing time (T0), after 29 (T1), 67 (T2), 89 (T3), 130 (T4) and 165 days (T5). Each sample was constituted by six sub-samples taken after turning the heap, then mixed until homogenization. The samples were oven dried at 50[degrees]C, ground and sieved at 1 mm and finally stored for subsequent analyses. All analyses were performed in four replicates.
In order to assess the level of organic matter evolution in composts samples (T0-T5), the following widely accepted parameters were determined: pH, C/N ratio, total extractable C, humic and fulvic acid C and humification indeces (official parameters requested by national law on fertilizer). Nitrogen determinations were performed by dry combustion (Dumas method).
Organic matter extraction was carried out on 2 g of each sample of compost with 100 mL of a solution NaOH/Na^sub 4^P^sub 2^O^sub 2^ 0.1N for 48 hours at 65[degrees]C. After centrifugation and ftracts were stored at 4[degrees]C under nitrogen atmosphere. Isoelectric focusing (IEF) of extracted organic matter for composts C1, C2 and C3 (from T0 to T5 samples) were carried out in a Multiphore II, LKB electrophoretic cell, according to Govi et al. (1994). The same IEF separation was performed on the starting matrices: pastazzo, sludge and green residues. Ten milliliters of NaOH/Na^sub 4^P^sub 2^O^sub 7^ extracts were dialysed in 6,000-8,000 Dalton membranes, lyophilized and then electrofocused in a pH range 3.5-8.0, on a polyacrylamide (acrylamide/bis-acrylamide: 37.5/1) slab gel, using 1 mL of a mixture of carrier ampholytes (Pharmacia Biotech) constituted by: 25 units of Ampholine pH 3.5-5.0; 10 units of Ampholine pH 5.0-7.0; 5 units of Ampholine pH 6.0-8.0. A prerun (2h 30'; 1200V; 21mA; 8W; 1[degrees]C) was performed and the pH gradient formed in the slab gel was checked by a specific surface electrode. The electrophoretic run (2h; 1200V; 21mA; 8W; 1[degrees]C) was carried out loading the same C amount of water-resolubilized extracts (1 mg C x 50 [mu]L^sup -1^ x sample^sup -1^). The bands obtained were stained with an aqueous solution of Basic Blue 3 (30%) for 18 h and then scanned by an Ultrascan-XL Densitometer(R) (Amersham -Pharmacia).
In order to compare the isoelectric focusing data among the different matrices and the samples (T0-T5) of C1, C2 and C3, the same operative conditions were applied for all IEF separations. Each isoelectrophoretic run was performed two times to test the repeatability of IEF profiles.
The same IEF procedure was applied to standard IHSS humic acids, extracted from soil and peat. About 3.5 mg of lyophilized standard humic acids (HS) were solubilized in 100 [mu]L of bidistilled water, in order to obtain the same carbon concentration used for soil samples (1 mg C x 50 [mu]L^sup -1^ x sample^sup -1^). The IEF run was thus carried out by using the same operative conditions described above.
Peaks detected from each electrophoretic profile of composts were sequentially numbered and their areas were integrated, considering 100% of the area of the entire profile. This elaboration allowed the relative peak areas to be independent from the organic matter content of the analyzed sample. Regression analysis was performed by single linear regression analysis routine (Statwin Program).
Results and Discussion
The evaluation of the composting processes, starting from the analysis of the process parameters, can give, roughly, an indication of the residual amount of fermentiscible organic matter.
In particular, C1 pile was characterized by a high average value of temperature (50-60[degrees]C) (Figure 1), which was maintained for more than three months during the composting period, decreasing to 40[degrees]C only after four months. This trend suggested a slackening in organic matter evolution and in compost maturation, probably due to the winter environmental conditions (open-air piles). On the contrary, the trends of temperature for C2 and C3 piles (Figure 1) showed a rapid increase (up to 60 - 65 [degrees]C) during the first weeks, slowly decreasing in the following months. After 5 months of composting, temperature reached 35-38 [degrees]C and, even when optimal moisture condition were maintained, did not increase significantly.
The trends reported above represent the typical self-heating pattern expected for a regular composting process characterized by a thermophilic phase, followed by mesophilic and curing phases (Miller 1993).
High acidity of pastazzo was differently managed by several authors working on citrus waste compost (Correia Guerrero et al. 1995; Van Heeerden et al. 2002). In our experiment, we monitored pH evolution over time. In any case, pH value of starting mixtures ranged from 6.0 to 7.0 and increased over time, until reaching 8.0 - 8.5 at the end of the composting processes, as reported in Figure 2. On the basis of what is reported above, chemical influence of citrus waste acidity on extractability of compost organic matter, and in particular, of humic-like substances, was not expected. Moreover the initial pH, ranging between 6.0-7.0, guaranteed optimal conditions for all microbial groups involved from the beginning of composting process (Miller 1993).
FIGURE 1. Temperature trends for C1, C2 and C3 composts.
FIGURE 2. pH values of composts from T0 to T5 samples.
FIGURE 3. C/N ratio of composts from T0 to T5 samples.
In order to have more detailed information on organic matter evolution, C/N ratio (Figure 3) total organic carbon (C^sub tot^%), extractable carbon (C^sub extr^%), humic and fulvic acids carbon (C %), not humified carbon (C^sub NH^%), humincation index (HI) (Table 2) and humification rate (Figure 4) for composts C1, C2 and C3 during the composting processes were determined. The application of several parameters, such as temperature of the composting process, C/ N ratio, humification parameters and qualitative characterization of organic matter, could constitute a basic data-set which contribute to the characterization of compost organic matter during the stabilization process. Results obtained suggested that the three composts underwent high organic matter stabilization confirmed also by other biochemical parameters (Tittarelli et al. 2002). Effectively, C/N ratio decreased notably during composting for all the three composts, from the initial 29-35 to the final 12-15 value, indicating that more than 25% of organic C was mineralized during composting.
For all the three composts, organic matter transformation took place, since an increase of humified fraction (C^sub HA+FA^), with a corresponding decrease of not humified fraction (C^sub NH^), was verified. This behavior is more accentuated in compost C3, produced without sludge (the final value of HI for C3 was 0.11, against 0.32 and 0.36 of composts C1 and C2, respectively).
TABLE 2.
C^sub extr^ C^sub HA+EA,^ C^sub NH^ and HI for C1, C2 and C3 composts from T0 to T5 samples (all data reported are mean values, calculated on dry matter).
FIGURE 4. Humification rate (HR %) of composts from T0 to T5 samples.
In order to verify whether this increase in humic fraction was due to an actual transformation of organic matter into more chemically stable compounds or to a "concentration effect" of the original humic-like substances formerly present in the raw matrices, a qualitative organic matter characterization of starting materials and composts during the stabilization process was carried out by using the isoelectrofocusing technique (IEF). In Figure 5, the IEF profiles for pastazzo, sludge and green residues are reported.
FIGURE 5. Isoelectric focusing profiles of NaOH/Na^sub 4^P^sub 2^O^sub 7^ extracts of starting matrices for composts C1, C2 and C3 (only few significant peaks numbers, corresponding to the related pI in Table 3, are reported.)
The IEF profiles of the matrices showed, for sludge, a very simple pattern, characterized by a first band focused at pH 3.5 (peak 1), a second band focused at pH 3.85 (peak 4) and two groups of three and two large bands in the range between 4.15-4.4, respectively. The IEF profiles of pastazzo and green residues were not resolved, with an intense peak 1 and a series of large peaks focused between pH 3.9-4.6. These configurations attested the low level of humification of the extracted organic compounds from raw matrices, firstly for the absence of a complete separation among the groups of substances during the IEF run and secondly for the pH range in which these compounds focused (not higher then 4.6).
As reported in literature, the more humified organic substances concentrate in the last and less acidic portion of the IEF spectra (De Nobili et al. 1989; Govi et al. 1994; Canali et al. 1998; Cavani & Ciavatta 2003). Actually, during humification process, the fulvic acids, characterized by lower molecular weight and higher acidity, condense to form more stable humic acids with higher molecular weight due to the presence of polymerized structures, which undergo from aliphatic to more aromatic character (Zach 2002). With respect to fulvic acids, humic acids are chemically different (lower O and higher C content, lower acidity) for the condensation of acidic functional groups (ethers and esters formations). Therefore, humic acids are characterized by the decrease of free phenolic and carboxylic groups (Stevenson 1994), together with the increased dispersion of negative charge through the polymerized structures. This determines an increase of the isoelectric point of humic acids, with a corresponding decrease of their electrophoretic mobility into the IEF gel in which the pH gradient was created. In order to have an IEF reference for humified organic matter, in Figure 6, the IEF profiles of purified IHSS standard humic acids from soil and peat are reported. These IEF profiles, obtained from humified materials, are well resolved, with weak peaks focused at pH values 3.5-4.3 and a group of intense peaks between 4.4-5.2 characterized by high intensity, especially at pH 4.7 (peak 12).
In Figure 7, the IEF profiles of samples T0-T5 for composts C1, C2 and C3 are reported. For C1 and C2 composts, characterized by the same starting composition (pastazzo+sludge+green residues), a notable transformation of the original organic matter during the composting process took place. In C1T0 and C2T0, the IEF profiles showed a sharp peak 1 at pH 3.5 and a group of not well resolved peaks focused between 3.85 and 4.7, very similar to those of sludge IEF profile. For the subsequent composts samples, a little decrease in the intensity of peak 1 and a strong increase of the bands focused between 4.4 and 4.75 was noticed. In particular, the last band at pH 4.7 - 4.8, seemed to be an index of compost stabilization, since it became preponderant at the end of composting process.
The comparison among the IEF profiles for compost C1 and C2 allowed to detect dsting. In C1, the increase of peak 13 at pH 4.75 was gradual, since at the end of the thermophuic phase (60 days - T2), it did not reach its maximum intensity. Only at the end of the curing phase (T5) was this peak the highest of the entire profile. For compost C2, after 90 days, the T3 configuration pattern was comparable to the T5 one, since it did not change significantly during the last curing phase (T4-T5). This difference in organic matter transformation could be due to the different evolution of the process, as revealed by compost temperature trends (Figure 1-C1/ C2), and probably caused by different air temperature. Pile C1 was prepared in winter and probably the cold atmospheric conditions determined a slackening in microbial activity in the heap. On the contrary, for C2 pile, which was prepared in spring, the hot climate favored a fast increase of temperature during the first 60 days of composting, probably accelerating the process of mineralization/ humification of compost organic matter. It is important to point out the strong similarity of the starting mixtures' IEFs (C1T0 and C2T0) and the difference between the less acidic part of the IEF profiles of the last compost samples (C1T5 and C2T5), which attests to the role of environmental conditions in open air composting system. FIGURE 6. Isoelectric focusing profiles of NaOH/Na^sub 4^P^sub 2^O^sub 7^ extracts of IHSS standards humic acids from soil and peat (peak numbers, corresponding to the related pI in Table 3, are reported).
FIGURE 7. Isoelectric focusing profiles of NaOH/Na^sub 4^P^sub 2^O extracts of C1, C2 and C3 composts (T0-T5) (only few significant peaks numbers are reported).
The most important difference among the first sample C3T0 and the other two composts samples collected at T0 time is the clear IEF configuration pattern found in correspondence with the starting mixture (Figure 7). A strong band at pH 3.5 (peak 1), a little band at pH 3.85 (peak 4) and two groups of bands, with increasing intensity between pH 4.15-4.3 (peaks 6, 7 and 8) and a decreasing one between 4.4-4.7 (peaks 9-12) are detected. In C3T1 IEF profile, there is a significant decrement of the first band at pH 3.5 (less humified compounds, with more acidic pI) and a corresponding change in the pH region between 4.15-4.7, with the increase of the last peak at pH 4.7 (more stable compounds, with higher pI). Besides, a group of not well resolved bands between 4.8 and 5.3 appeared in the C3T1 profile (peaks 13-18). The following samples C3T2-C3T3 seemed to maintain the same characteristics, while IEF profiles of C3T4 and C3T5 showed a splitting of the last band in two not well separated peaks 12 and 13 at pH 4.7 and 4.75, respectively. The little bands focused in the pH range 4.8-5.3 did not show significant modifications from C3T1 and C3T5 samples. In this compost, the increase of the last bands at pH 4.7-4.75 was extremely evident, as the quite complete disappearance of peak 1 at pH 3.5, attesting the high level of stabilization of C3T5 organic matter. Moreover, the presence of the bands between 4.8-5.3, which were completely absent in C1 and C2 composts, indicated a high level of humification, probably linked to the difference in the C3 starting mixture.
FIGURE 8. Rings diagrams of sequential IEF peaks areas for C1, C2 and C3. Each compost sample (T0-T5) is represented by a ring, while each colour corresponds to an IEF peak. More internal rings correspond to T0 sample and more external ones to the T5 samples for each compost.
A certain similarity was found among IEF of humic matter obtained from studied composts and IHSS standard humic acids from soil and peat. As indicated by the comparison of IEF profiles derived from IHSS standard humic acids from peat (commonly defined as a strongly humified organic material) and those of T2-T5 samples of composts, the appearance or the increase of peaks focused at pH >/= 4.7 (not present in starting mixtures) showed the qualitative transformation of organic matter and the high level of humification reached by the considered composted material.
In order to better analyze the IEF profiles of the composts, the area sum of IEF peaks focused at pH >/= 4.7 was calculated for each composts samples (A^sub s^ %). In Table 3, the percentage area of each numbered peak and the corresponding A^sub s^ % value for composts C1, C2 and C3 (T0-T5) were reported.
A^sub s^ % parameter showed a strong increase of its value during the three composting processes, even if in C3T5 the percentage of peak areas focused at pH>/=4.7 resulted about two times higher with respect to those of C1T5 and C2T5. This seemed to be in relation with the different starting materials used in the three piles, attesting the better stabilization of compost C3 compared to C1 and C2, as confirmed also by the humification indexes reported above (Table 2). Ring diagrams of the sequential peaks areas for each sample of composts C1, C2 and C3 are reported in Figure 8. This represervation enhances differences among the considered IEF profiles, underlining the different organic matter evolution in composts C1, C2 and C3. The comparison between C1 and C2 showed that the transformation followed by organic matter during the composting process was different. The number of bands focused in the range of pH 3.5-4.75 were the same (1-13), but the configuration pattern of C2 was extremely simple and similar from T1 to T5, while, for C1, the organic matter evolution was slower and gradual from T0 to T5. It is worth noting the size increase of peak 13 in C1 compared to C2. The situation of C3 had to be considered apart from the other two. The most evident characteristic of the C3 rings diagram was the high number (18) of IEF peaks at the end of the composting process, which suggested the high heterogeneity of organic compounds formed during the stabilization process, especially in the pH range from 4.7 to 5.5.
TABLE 3.
IEF peaks areas (normalised to 100%) and As % parameter (pH>/ =4.7) for compost C1, C2 and C3 (from T0 to T5). Sequence numbers represent the corresponding sequence of IEF peaks for each sample profile. Reported values are the means of four replicates.
This finding seems to indicate that the presence of citrus- processing sludge in the starting mixture reduces the percentage of lignocellulosic material inducing the formation of few and less humified products while, in C3, the microbial community degraded mainly the ligno-cellulosic fraction from pastazzo and green residues, which constituted an excellent substrate to synthesize humic-like matter.
It was, then, considered possible to use the A^sub s^ % value as a quality parameter for evaluating the level of compost organic matter stability (Canali et al. 1998; Trinchera et al. 1999). A^sub s^ data were in accordance to those obtained by quantitative determination of composts' humified organic matter (HI). For C1, C2 and C3 composts, a strong correlation among A^sub s^ % and the corresponding HI was found, with a value of R^sup 2^ = 0.85 (Figure 9).
FIGURE 9. Correlation between As % and HI for C1, C2 and C3 during composting (T0-T5).
Conclusion
The isoelectrofocusing technique applied to the considered composts furnished useful information on the organic matter transformation during the citrus composting process. Results obtained indicated that IEF qualitative analyses made it possible to follow the organic matter evolution during composting, showing a decrease of peaks focused at lower values of pH and a corresponding increase of peaks focused at higher values. The IEF configuration patterns seemed to confirm the hypothesis of increase in humic, more stable substances, since the electrophoretic profiles obtained from the end products looked extremely different with respect to the original starting materials and similar to those of humic acids extracted from soil and peat.
The different IEF profiles of C1 and C2 composts give some information on the role of composting conditions in organic matter transformation. As reported by Trautmann and Olynciw (2000), the longer the thermophilic phase, the more diverse is the microbial community it supported, so that the organic matter transformation results are more complex. In our study, during the curing phase (90- 165 days), C1 compost seems to sustain further rearrangements of organic compounds until reaching the final level of evolution (C1T5). For compost C2, a comparable level of organic matter complexity was reached after 90 days, with the consequent higher homogeneity of the produced organic compounds. For the C3 compost, the absence of sludge in the starting mixture seemed to be a positive factor, since the level of organic matter humification shown at the end of the composting process was higher than those of C1 and C2, as attested by both the IEF profiles and the other considered parameters. It can be supposed that the absence of sludge in the C3 starting mixture determined different metabolic pathways and, consequently, a higher level of heterogeneity of organic compounds produced during composting.
The use of A^sub s^% to quantify previously considered qualitative information of compost organic matter seemed to be effective for citrus composts. In order to consider A^sub s^% value as a compost quality parameter, it should be tested on different typology of composted materials, to individuate for each compost (from green residues, agro-industrial wastes, municipal solid wastes, and so on) a "A^sub s^ threshold value" corresponding to a defined level or organic matter evolution.
Acknowledgements
This work was carried out in the framework of MiPAF Project "Ricerche e Sperimentazioni nel settore dell'Agrumicoltura italiana", paper No 183.
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Alessandra Trinchera1, Fabio Tittarelli1 and Francesco Intrigliolo2
1. CRA - Istituto Sperimentale per la Nutrizione delle Piante, Rome, Italy
2. CRA - Istituto Sperimentale per l'Agrumicoltura, Acireale (CT)