Biochemical activity and chemical-structural properties of soil organic matter after 17 years of amendments with olive-mill pomace co-compost

Journal of Environmental Management 147 (2015) 278e285

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Biochemical activity and chemical-structural properties of soil organic matter after 17 years of amendments with olive-mill pomace co-compost V. Aranda a, *, C. Macci b, E. Peruzzi b, G. Masciandaro b a b

University of Ja
en, Department of Geology, Campus Las Lagunillas s/n, 23071 Ja
en, Spain Consiglio Nazionale delle Ricerche (CNR), Istituto per lo Studio degli Ecosistemi (ISE), Area della Ricerca, Via Moruzzi 1, 56124 Pisa, Italy

a r t i c l e i n f o

a b s t r a c t

Article history: Received 23 April 2014 Received in revised form 21 August 2014 Accepted 28 August 2014 Available online 22 September 2014

This study evaluates soil fertility, biochemical activity and the soil's ability to stabilize organic matter after application of composted olive-mill pomace. This organic amendment was applied in two different olive groves in southern Spain having different soil typologies (carbonated and silicic). Olive grove soils after 17 years of organic management with application of olive-mill pomace co-compost were of higher quality than those with conventional management where no co-compost had been applied. The main chemical parameters studied (total organic carbon, total nitrogen, available phosphorus, exchangeable bases, cation exchange capacity, total extractable carbon (TEC), and humic-to-fulvic acids ratio), significantly increased in soils treated with the organic amendment. In particular, the more resistant pool of organic matter (TEC) enhanced by about six and eight fold in carbonated and silicic soils, respectively. Moreover, the amended silicic soils showed the most significant increases in enzyme activities linked to C and P cycles (b-glucosidase twenty-five fold higher and phosphatase seven fold higher). Organic management in both soils induced higher organic matter mineralization, as shown by the higher pyrrole/ phenol index (increasing 40% and 150% in carbonated and silicic soils, respectively), and lower furfural/ pyrrole index (decreasing 27% and 71% in carbonated and silicic soils, respectively). As a result of mineralization, organic matter incorporated was also more stable as suggested by the trend of the aliphatic/aromatic index (decreasing 36% and 30% in carbonated and silicic soils, respectively). Therefore, management system and soil type are key factors in increasing long-term C stability or sequestration in soils. Thus application of olive-oil extraction by-products to soils could lead to important mid-to -longterm agro-environmental benefits, and be a valuable alternative use for one of the most widespread polluting wastes in the Mediterranean region. © 2014 Elsevier Ltd. All rights reserved.

Keywords: Organic amendment Composted olive-mill pomace Soil enzyme activity Pyrolysis-GC

1. Introduction About 80% of Spanish olive crops are concentrated in Andalusia, the largest olive-growing area worldwide, with over 30% of the world's production of olive oil (Spanish Agency for Olive Oil database, 2009). Within Andalusia, the most important olive oil
n (southern Spain). producing areas are in the province of Jae The conventional and intensive agricultural methods used in olive orchards in this area cause soil fertility degradation, erosion and soil compaction, in addition to polluting surface waters (Castro et al., 2008). The main effect of this type of management is loss in organic matter content, which becomes especially significant in * Corresponding author. E-mail address: [email protected] (V. Aranda). http://dx.doi.org/10.1016/j.jenvman.2014.08.024 0301-4797/© 2014 Elsevier Ltd. All rights reserved.

originally poor soils such as in the Mediterranean. The role of organic matter and its application to the soil has changed in modern agriculture, where chemical fertilizers are now the major source of nutrients for crops. Deterioration of soil quality in these areas would therefore be related primarily to inappropriate farming techniques (Moreno et al., 2009). In several studies, the use of environmentally-friendly agricultural practices has proven to be effective in restoring or improving soil quality in olive grove areas in southern Spain by reducing the mechanical disturbance of soil, protecting the soil surface with mulch cover, maintaining vegetative cover, adding organic matter to the soil, etc. (e.g., Castro et al., 2008; Aranda et al., 2011; Calero et al., 2013). The agronomic value of biochar and other emerging technologies, such as hydrothermal carbonization (Poerschmann, 2013), should also be considered in the near future.

V. Aranda et al. / Journal of Environmental Management 147 (2015) 278e285

In Andalusia, the extensive olive crop leads to a huge annual production of olive-mill pomace (around four million tons), the main by-product of the two-phase olive oil extraction system (García-Ruiz et al., 2012). Olive-mill pomace is a semi-solid or pasty material with very little porosity, very moist, acidic in reaction, rich in organic matter, potassium and hydrosoluble carbohydrates, which may contain lipid compounds and organic phytotoxic and anti-microbial compounds. The phosphorous content is low as are micronutrients and nitrogen, so the C/N ratio is usually high, with a mean of 48, which is far from the optimum established for composting (25e35) (Alburquerque et al., 2004; Mechri et al., 2011). Its direct application as an organic amendment in agriculture could cause serious environmental problems in soils or in surface waters, which limits its general use in soil improvement. The stability of organic amendments has been considered of paramount
s importance in the incorporation of organic matter into soil (Nicola et al., 2012). Under controlled conditions, composting produces a stabilized organic matter enriched in “humic-like substances” and free of phytotoxic compounds and pathogens. In addition, composting allows longer persistence of the organic carbon from amendments in the soil (Bernal et al., 1998). The composted olivemill pomace thus acquires acceptable maturity, stability, and detoxification (Alburquerque et al., 2006). Agricultural practices based on periodic inputs of organic amendments are strongly recommended for Mediterranean agroecosystems. According to García-Ruiz et al. (2012), composted olive-mill pomace contains a large amount of organic matter, and thus might be useful as an amendment to agricultural soils, potentially lowering the need for nitrogen, phosphorus and potassium fertilizers, improving a range of soil properties, and reducing loss of agricultural production. Apart from its positive effect of storing carbon in soil, it would also help prevent problems associated with erosion. Hence, olive-mill pomace compost application could be considered an attractive strategy for soil C
nchez-Monedero et al., 2008). sequestration (Sa In spite of the enormous amounts of waste from olive-oil mills every year, their potential as an organic amendment for environmental remediation of agricultural soils has scarcely been evalu
ntara, 2013). Some studies ated (Lozano-García and Parras-Alca have provided information on the beneficial effects of olive-mill pomace compost on the physical, chemical and biological proper
ntara, 2011; Garcíaties of the soil (Lozano-García and Parras-Alca
ntara, 2013; Ruiz et al., 2012; Lozano-García and Parras-Alca
mez-Mun ~ oz et al., 2013), and have demonstrated a general inGo crease in fertility and protection from erosion. However, in-depth studies on the structural evolution of the soil organic matter after application of this compost, and its relationship with soil biochemical activity linked to nutrient cycles continue to be scarce and incomplete. Therefore, the main goals of this study were to analyze samples of olive grove soil organically amended with olive-mill pomace cocompost by Pyrolysis-GC to find out essential molecular information about the chemical-structural changes in the organic matter, extent of humification to evaluate organic matter quality, and some soil enzyme activities very sensitive to soil management. This study on the application of this organic amendment was carried out on two different soil typologies (carbonated and silicic) in southern Spain, after 17 years of application in the field. 2. Material and methods 2.1. Site description and soil sampling The studied olive farms were productive private farms located in
n Province (southern Spain). The average annual Andújar, Jae

279

rainfall in the area is 480 mm, falling mainly in autumn and spring. The climate is Mediterranean with a mean annual temperature of 17.9
C, cool winters, and hot, very dry summers. Olive tree density on the study farms varied between 90 and 100 trees per hectare, trees were 35e45 years old, and distributed in a regular arrangement with a typical canopy cover of about 30% of the farm area. The management practices applied on these farms were those most commonly used in the area. Two of the farms were fertilized with the organic amendment and two with mineral fertilizer. On the farms with organic management, 6e10 Mg ha1 compost had been applied once every autumn for the last 17 years. The cocompost was about 50% olive-mill pomace, air-dried to less than 20% of its moisture, and 50% olive leaves and manure mixed and heaped in 3-m-high  6-m-diameter piles, turned regularly every 15 days to prevent anaerobic processes, and matured for seven months. The moisture content was checked periodically and maintained at 40e60% by adding the necessary amount of water and the excess water leached was recirculated. The co-compost was always evenly spread over the soil in the intercanopy and mowed (very superficial chisel passes to control plant cover). The application period and rate varied depending on co-compost availability, climate conditions, and olive waste production. On the farms where the co-compost was applied, management was organic with no mineral fertilization or pesticides, and was characterized by no-till soils and plant cover maintenance. Fertilization of the farms which did not receive co-compost consisted of the application of 50e70 kg N ha1 as urea or ammonium sulfate under the tree canopy in the early spring. Conventional management was also characterized by no-till soils, and herbicides were used for weed control. Four farms near each other were sampled, two with calcareous soils (with and without co-compost application) and two with silicic soils (with and without co-compost application). Calcareous and silicic soils were from calcarenitic and quartzitic parent material, respectively. The dominant soils in the calcareous area were Eutric Regosols (FAO, 2006) and in the silicic area Dystric Leptosols (FAO, 2006). Olive farms which received co-compost were comparable to those which received no compost (control soils) in terms of climate, slope, orientation, soil type, and tree density and age. Sampling at each farm consisted of random selection of three intercanopy locations, and taking a random soil sample composed of four subsamples (from the 0e10 cm surface layer) within a 5 m radius in each location. The original co-compost was also kept for further analysis. 2.2. Soil analyses All analytical soil sample data refer to the fine-earth fraction (<2 mm). The chemical analyses used followed the standard procedures, and were as outlined by the American Society of Agronomy and Soil Science Society of America (Page et al., 1982; Klute, 1986). Organic carbon (OC) content was determined by the Walkley-Black's method with dichromate oxidation (1 N K2Cr2O7 and 0.5 N Fe(NH4)2(SO4)2 as titrant for excess Cr2O2 7 ). Total N was measured with the Kjeldhal's method, employing a potassium sulfate-catalyst mixture (K2SO4eCuSO4eSe), concentrated H2SO4 and 10 N NaOH; finally, titration with 0.01 N H2SO4. The pH was measured by potentiometry in distilled water (1:2.5, w/v). Electrical conductivity (EC) at 25
C was determined in water extracts (1:5, w/v). Calcium carbonate equivalent was determined with a Bernard calcimeter, reacting carbonates with hydrochloric acid (10% HCl, w/w). Soil available P was extracted with 0.5 N NaHCO3 (pH 8.5), using a mixed reagent of (NH4)6Mo7O24 and K(SbO) C4H4O6, and ascorbic acid as color-developing reagent; finally,

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phosphorus was determined by colorimetry. The CEC was determined by saturating the soils with Na, its extraction and analysis by flame photometry. Exchangeable basic cations were extracted by 1 N NH4OAc (pH 7), and the concentrations determined by atomic absorption spectrometry. Clay and sand contents were determined using the Robinson's pipette method after eliminating carbonates, soluble salts, organic matter and iron oxides, and finally, dispersion with Na-hexametaphosphate. 2.3. Soil enzyme activities Dehydrogenase (DH-ase) activity was measured using 0.4% 2-piodophenyl-3-pnitrophenyl-5-tetrazolium chloride (INT) as substrate; iodonitrotetrazolium formazan (INTF) produced in the reduction of INT was measured by means of a spectrophotometer at 490 nm (Masciandaro et al., 2000). For hydrolytic enzymes, total enzyme activity was measured in soil samples and extracellular enzyme activity in dialyzed pyrophosphate extracts at pH 7 prepared as reported by Ceccanti et al. (2008). b-glucosidase and phosphatase activities were determined using p-nitrophenyl phosphate disodium and p-nitrophenyl glucopyranoside as substrates, respectively, by extraction and determination of p-nitrophenol (PNP) in a spectrophotometer at 398 nm (Tabatabai, 1994). 2.4. Soil organic matter fractionation and pyrolysis-gas chromatography Fractionation of soil organic matter followed the IHSS procedure (Swift, 1996). Total extractable carbon (TEC) for humic and nonhumic substances was extracted from soil by mechanically shaking the samples with a solution of 0.1 M NaOH and Na4P2O7 (pH 14) for 24 h at 60
C (1:10, w/v). The extracts were centrifuged and filtered (Millipore 0.45 mm). The TEC fraction was separated into humic acids (HA) and fulvic acids (FA) by precipitation with H2SO4 (for HA determination) and purification with polyvinylpolypyrrolidone to eliminate non-humic carbon, respectively. Water-soluble carbon (WSC) was extracted in distilled water (1:5, w/v) for 1 h at 60
C. Pyrophosphate extractable carbon (PEC) was obtained following the Ceccanti et al. (2008) method, with sodium pyrophosphate as the extracting solution (0.1 M; pH 7.1; 1:10, w/v) at 37
C for 24 h, and then filtering the extract using a 0.22 mm Millipore membrane. PEC in the extract was retained by an ultrafiltration AMICON PM10 cut-off membrane (10 KDa). The C content in TEC, FA, HA, WSC, and PEC was determined by dry combustion with RC-412 multiphase carbon (LECO Corp.). Free lipids were extracted with petroleum ether in 250-mL Soxhlet extractors filled with 50 g of soil sample, and changing the extraction liquid every 4 h. The total extract was dehydrated with anhydrous Na2SO4, evaporated under reduced pressure and finally weighed. Pyrolysis-gas chromatography (Py-GC) was used for separation and quantification of pyrolytic fragments from bulk soil samples. Fifty micrograms of a representative soil sample, air-dried and ground (<100 mm mesh) were inserted in pyrolysis quartz microtubes in a CDS Pyroprobe 190. The Py-GC system consisted of a platinum coil probe and a quartz sample holder. Pyrolysis was carried out at 800
C for 10 s, and heated at a rate of 10
C ms1 (nominal conditions). The probe was coupled directly to a Carlo Erba 6000 gas chromatograph with a flame ionization detector (FID). Chromatographic conditions were as follows: a 3 m  6 mm, 80/100 mm mesh, SA 1422 (Supelco, Inc.) POROPAK Q packed column, with a temperature program of 60
C, increasing to 240
C by 8
C min1. The pyrograms were quantified using seven peaks corresponding to volatile fragments (Ceccanti et al., 1986): acetic acid (K),

acetonitrile (E1), benzene (B), toluene (E3), pyrrole (O), furfural (N), and phenol (Y) with retention times (minutes) of: E1, 17.7; K, 20.4; B, 26.3; O, 27.7; E3, 31.4; N, 33.9; Y, 41.8. Peak areas were normalized and expressed as relative abundances, so the area under each peak referred to the percentage of the total of the seven peaks. Peak purity of the selected major volatile fragments was checked by coupling the same chromatographic system to an HP 8000A mass detector under the same operating conditions. A numeric index of similarity (Sij) between the relative abundances (I) of the homologous peaks (k) in two pyro-chromatograms (i and j) was calculated using the following expression: Sij ¼ (S(Ii/Ij) k)/n where Ii < Ij, and n is the number of peaks (Ceccanti et al., 1986). The index varied from 0 to 1: the higher the index, the greater the similarity. Four conventional levels have been suggested (Ceccanti et al., 2007): very high (>0.85), high (0.75e0.85), medium (0.70e0.75), and low (0.60e0.70). Finally, the compounds diversity was calculated with the Shannon-Weaver’s diversity index, as reported by García et al. (1993). 2.5. Statistical analysis The results were expressed as the mean of three replicates, grouped by type of management (organic or conventional) and previously considering the soil type (carbonated or silicic). A Student's t-test was used to verify whether two means were significantly different, and the KolmogoroveSmirnov and Levene's tests were performed to assess normality and equality of variances. Correlations between variables were analyzed with the Pearson's correlation coefficient. These analyses were done with Statgraphics Centurion XVI software (StatPoint Technologies, Inc.). 3. Results and discussion 3.1. Effects of composted olive-mill pomace amendments on physicochemical and biochemical soil properties Table 1 shows the results of the physicochemical analysis of the surface soil samples. The coarse fragments content was significantly higher in silicic soil samples, characteristic that was inherited from parent soil material. Differences found in other textural parameters, like clay and sand, in each soil type were unremarkable. After 17 years of olive-mill pomace co-compost application, total organic carbon (TOC), total N, and the C/N ratio in the carbonated soil, were markedly higher under organic management,
pez-Pin ~ eiro et al., 2008; as already found in previous studies (Lo García-Ruiz et al., 2012). As expected, soil pH was significantly higher in carbonated soils. However, the effect on this parameter after application of cocompost was strongly influenced by soil type. In carbonated soils, the organic amendment lowered the pH, and on the contrary, in silicic soil pH increased. Therefore, the application of co-compost seems to have a buffering effect, and the pH becomes balanced at better levels for the availability of nutrients assimilable by olive trees. Concerning EC, even though the salinity of the co-compost was relatively high (1.22 dS m1), the amendment had little or no effect on soil EC. Soil available phosphorus in the compost-amended soils was higher, probably due to the high P content in composted material. The highly significant increase in P availability in the carbonated soils is probably related to more favorable physicochemical conditions as the pH goes down (below 8) with the organic amendment. A moderately acidic pH may explain the increased availability of phosphorus in silicic soils under both organic and conventional management.

V. Aranda et al. / Journal of Environmental Management 147 (2015) 278e285

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Table 1 Physicochemical parameters of the surface soil samples and co-compost. Variable

Carbonated soils organica

CF (%) Clay (%) Sand (%) TOC (%) N (%) C/N EC (dS m1) pH (H2O) CaCO3 (%) P (mg kg1) Ca2þ ex. (cmolc kg1) Mg2þ ex. (cmolc kg1) Naþ ex. (cmolc kg1) Kþ ex. (cmolc kg1) Base saturation (%) CEC (cmolc kg1) WSC (mg C/kg soil) PEC (mg C/kg soil) TEC (mg C/kg soil) HA (mg C/kg soil) FA (mg C/kg soil) HA/FA ratio Free Lipids (g kg1)

9 7 71 2.83 0.21 14 0.32 7.7 8.7 37.3 15.27 0.74 1.08 0.44 85 20.67 642 9076 26,828 13,308 6510 2.1 0.371

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

Silicic soils conventionalb

1.15* 2.08* 1.44 0.30* 0.01* 0.87 0.06 0.13* 1.15* 8.39* 0.41* 0.13 0.08 0.0* 5.51 1.53* 142.21* 1141* 2303* 3339* 394* 0.51* 0.01*

14 16 69 0.44 0.04 11 0.35 8.3 23.7 3.3 7.80 0.61 1.29 0.16 84 11.67 404 1711 4836 1698 1738 1.0 0.332

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

2.08* 3.21* 2.03 0.03* 0.01* 2.11 0.05 0.02* 2.52* 2.31* 0.81* 0.05 0.33 0.02* 4.04 1.15* 37.34* 236* 1148* 173* 333* 0.11* 0.01*

organica 64 7 65 4.97 0.41 12 0.30 6.5 1.7 32.7 13.34 2.99 0.71 0.57 82 21.0 786 5500 35,595 15,931 3957 4.0 0.512

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

Co-compost conventionalb

9.54 5.20 3.13 1.10* 0.08* 0.89 0.02* 0.06* 0.58 6.43 3.30* 0.56* 0.44 0.12* 11.85* 2.0* 123.89* 1624 4047* 4035* 1022* 0.03* 0.03*

52 9 69 0.56 0.05 12 0.21 5.1 1.7 23.0 1.38 0.49 0.85 0.19 45 6.33 328 1721 4642 526 575 0.9 0.409

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

9.81 2.52 0.68 0.20* 0.02* 2.07 0.04* 0.15* 0.58 5.0 0.60* 0.17* 0.41 0.04* 7.64* 1.53* 184.37* 55 1461* 138* 107* 0.10* 0.01*

27.9 1.69 17 1.22 8.8 5000 278.44# 58.39# 38.87# 18,648 9249 181,040 114,846.7 2114.4 54.3 0.379

Data are mean values of three soil samples composed of four subsamples (mean ± standard deviation); -: data not available. CF: coarse fragments; TOC: total organic carbon; N: total nitrogen; EC: electrical conductivity at 25
C; P: available phosphorus; ex.: exchangeable bases; CEC: cation exchange capacity; #: bases in meq/100 g; WSC: water soluble carbon; PEC: pyrophosphate-extractable carbon (pH 7.1); TEC: total extractable carbon; HA: humic acids; FA: fulvic acids. * Values differed significantly at 0.05 probability level (t-test). a Organic management and 17 years of olive-mill pomace co-compost application. b Conventional management without olive-mill pomace co-compost application.

Also all exchangeable base cations (Ca2þ, Mg2þ and Kþ) content increased in soils amended with olive-mill pomace co-compost, with the exception of exchangeable-Naþ concentration (which tended to decrease), suggesting that the organic amendment seems to reduce its content. Coinciding with a previous study by Nasini et al. (2013), of special interest is the significant increase in exchangeable-Kþ in the compost-treated soils. Thus application of this type of compost could be used as an alternative source of potassium in soils where this element is deficient. The organic amendment had a very favorable influence on base saturation, which was especially significant in silicic soils (over 50%), and cation exchange capacity (over 20 cmolc kg1). The more resistant organic matter pool (humic substances) was evaluated by assaying total extractable carbon (TEC) and its components: fulvic acids, which represent the least stable part of humic matter, and humic acids, which represent the most stable fraction of humic matter. As expected, organic fertilization affected TEC, HA and FA contents in both carbonated and silicic soils more positively than mineral fertilization. A similar trend may also be observed for the PEC fraction (C extracted with pyrophosphate at neutral pH), which represents the most active form of humic matter, able to link enzyme complexes (Ceccanti et al., 2008). The HA/FA ratio was significantly higher in organic management (particularly in silicic soils), showing that carbon sequestration potential is higher in stable organic forms. These results again confirm the positive effect of co-compost application on chemical fertility of the soils studied. All soil enzyme activities and metabolic potential (DH-ase/WSC ratio) were significantly higher in the samples from organic management (Table 2). DH-ase activity is considered one of the most important enzyme activities used as an indicator of overall microbial activity, because it is intracellular in all living microbial cells and is linked to the microbial respiratory process (Masciandaro et al., 2000). DH-ase activity clearly decreased under conventional management, probably due to herbicides and/or tillage, as

also found in previous studies (Reinecke et al., 2002; García-Ruiz et al., 2008). Similarly, hydrolytic enzyme activities related to the C (bglucosidase) and P (phosphatase) cycles, respectively, were lower in the conventional treatment. Of the hydrolytic enzymes, bglucosidase increased the most after the organic treatments, suggesting greater stimulation of the C cycle, as confirmed by the positive and significant correlation between TOC and b-glucosidase (Table 3). On the other hand, phosphatase (total and extracellular forms) was particularly high in silicic soils, where available P content was observed to be higher. In addition, the positive correlation of b-glucosidase and phosphatase with DH-ase activity suggests that these enzymes may be considered strongly dependent on microbial metabolism (Macci et al., 2013). In fact, extracellular activities followed the same trend as the total, and were higher under organic management where there were more available organic substrates (WSC), thus suggesting interaction between the available energy-rich compounds and the biochemical energy accumulated and preserved in humuseenzyme complexes (Masciandaro and Ceccanti, 1999). Additional supply of OM and mainly labile C (e.g., WSC) to soil can stimulate biochemical activity.
nez, 2002; These results coincide with those of other authors (Jime Bastida et al., 2006; Moreno et al., 2009). Correlations between TOC content and enzyme activities (Table 3) suggest improved conditions for microbial activity with the increase in soil organic carbon from compost application, particularly in silicic soil. Other correlations, often very significant, were also found between enzyme activities and the CEC and HA/FA ratio (Table 3), and therefore, with soil chemical fertility and more advanced humification, thereby emphasizing that the organic management system similarly improved the chemical and biochemical properties in both type of soils. The net effect of an increase in biological activity in silicic soils, which was higher than in carbonated soils, led to better availability

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Table 2 Biological properties and metabolic potential of the surface soil samples and co-compost. Variable

Carbonated soils organicb

b-glucosidase (mg PNF/g∙h) Dehydrogenase (mg INTF/g∙h) Phosphatase (mg PNF/g∙h) a Extracellular b-glucosidase (mg PNF/g∙h) a Extracellular phosphatase (mg PNF/g∙h) DH-ase/WSC ratio

Silicic soils conventionalc

756 ± 104.37* 5.64 ± 0.69* 305.33 60.12 18.98 0.0092

± ± ± ±

39.27* 8.27* 4.33* 0.002*

64 ± 12.77* 1.05 ± 0.44* 25.00 2.16 6.20 0.0025

± ± ± ±

12* 0.41* 4.15* 0.001*

organicb 1034 ± 176.95* 3.36 ± 0.78* 1893 56.28 69.61 0.0042

± ± ± ±

Co-compost conventionalc

198.64* 8.85* 12.11* 0.001*

41 ± 43.09* 0.24 ± 0.21* 259.67 1.87 22.77 0.0011

± ± ± ±

108.9* 1.08* 6.78* 0.001*

339 12.36 1263 69.70 106.31 0.0007

Data are mean values of three soil samples composed of four subsamples (mean ± standard deviation). Metabolic potential [dehydrogenase (DH-ase)/water soluble carbon (WSC) ratio]. *Values differed significantly at 0.05 probability level (t-test). a Assayed in PEC: pyrophosphate-extractable carbon (pH 7.1). b Organic management and 17 years of olive-mill pomace co-compost application. c Conventional management without olive-mill pomace co-compost application.

of nutrients and higher soil pH after application of the organic amendment. On the contrary, as mentioned by García-Ruiz et al. (2012) for carbonated soils, the decrease in pH in soils receiving this type of compost may be due to the phenolic and carboxylic groups resulting from organic matter decomposition, which act as weak acids. 3.2. Chemical-structural properties of bulk soil organic matter after co-compost amendments The Py-GC technique is simple and rapid, and has been used successfully to study changes in SOM quality after the application of different amendments (Leinweber and Schulten, 1998; Ceccanti et al., 2007; Andreetta et al., 2013). The relative abundance of major pyrolitic peaks and indices of bulk soil surface samples are shown in Table 4. The percentage of acetonitrile (E1) was significantly higher in conventional management, in both carbonated and silicic soils, and higher in organically amended carbonated than in silicic soils. Increased acetonitrile, mostly derived from condensed nitrogenous humic structures (Marinari et al., 2007), suggests that OM was enriched in resistant and more stable N-compounds as a consequence of degradation of the labile substrate (mineralizable fraction) over time. Larger amounts of N-bearing compounds in the conventional management samples could be due to their greater persistence and stability after intensive agricultural management, although the nitrogen fertilization used in these soils could increase heterocyclic N forms content, as suggested by Dieckow et al. (2006). Therefore, the amounts of mineralizable compounds, such as furfural (N) and acetic acid (K), which derive mainly from cellulose, lipids and easily degradable C compounds, decrease with increasing acetonitrile (E1). The percentage of furfural (N), which is related to the carbohydrate content, reflects some changes in the amended soils, and is lower in silicic soils. As these compounds are more susceptible to microbial attack, there would be stronger biological activity in this medium and more stable organic matter. The percentage of acetic acid (K) was higher in the amended soils than in conventionally managed soils. Acetic acid may be derived from carbohydrates or lipids, hence the enrichment of SOM with this kind of organic compounds. In agreement with previous studies employing sewage sludge (Nicol
as et al., 2012), the higher relative abundance of acetic acid in the soils amended with olivemill pomace co-compost in silicic soils may be the result of the formation of lipids linked to humic substances, coinciding with the higher presence of free lipids in these amended soils (Table 1). There was somewhat more abundance of pyrrole (O), a pyrolysis product from proteins, in organically amended soil samples, characterized by more microbiological activity than in the

conventionally managed soil samples. Coinciding with Nierop et al. (2001), this refractory N-compound probably originates largely from microbes. Benzene (B) and phenol (Y) are usually associated with condensed aromatic structures and less mineralized organic substances. They can also derive from proteins, lignin, tannins, polysaccharides and phenolic compounds. Toluene (E3), associated with partially humified materials, derived from pyrolyzates of proteins, lignin and polysaccharides (Nierop, 2001). The abundance of benzene, phenol and toluene did not appear to be associated with management or with type of soil, although a stronger tendency to accumulate phenol in the silicic soil medium could be related to slightly modified lignin and lower degradation rates. Using pyrolytic indices from the relative abundances of some of the Py-GC peaks (Table 4), structural changes in SOM composition due to the influence of management and soil type could easily be
s identified (e.g., Ceccanti et al., 2007; Marinari et al., 2007; Nicola et al., 2012; etc.). N/O (furfural/pyrrole) and O/Y (pyrrole/phenol) are considered mineralization indices of the easily mineralizable and less mineralizable fractions of the organic matter, respectively. The N/O index expresses the ratio between furfural, which is the pyrolysis product of polysaccharides, and pyrrole, which derives from nitrogenous compounds, humified organic matter and microbial cells. The higher the ratio, the lower the mineralization of organic matter is. In the conventionally managed soil samples, especially in the silicic medium, a significantly low OM mineralization rate (i.e., lowest rate of decomposition) is observed that could be related to less biological activity, as shown by the negative correlations between the N/O index with b-glucosidase and extracellular b-glucosidase activity and DH-ase activity (Table 3). This lower biological activity could in turn be conditioned by a less favorable geochemical environment. The conditions that affect OM, causing less mineralization, which are therefore negatively correlated to the N/O index, are decreases in the soil parameters: pH, exchangeable-Ca2þ and exchangeable-Kþ, CEC and base saturation (Table 3). On the other hand, the higher the O/Y ratio, the higher the mineralization of organic matter is. The O/Y index expresses the ratio between nitrogenous compounds and ligno-cellulosic materials. The samples studied contribute similar information, although opposite the N/O index, corroborated by the significant negative correlation between the two indices (Table 3). According to this index, the samples from organic management with co-compost showed higher mineralization in both carbonated and silicic soil types. This increased mineralization of OM leads to the improvement of soil fertility from the accumulation of nitrogen, phosphorous and other essential plant nutrients in the soil, as shown in Table 1. Furthermore, the negative correlation between the O/Y

V. Aranda et al. / Journal of Environmental Management 147 (2015) 278e285

283

Table 3 Coefficients of correlation matrix between physicochemical, biochemical and chemical-structural variables.

b-glucosidase Dehydrogenase Phosphatase Ext. b-glucosidase Ext. phosphatase N/O TOC 0.9028*** pH 0.0933 CaCO3 0.4417 Ca2þ ex. 0.7687** þ 0.9210*** K ex. base sat. 0.4626 CEC 0.8864*** HA/FA ratio 0.9225*** b-glucosidase e dehydrogenase phosphatase ext. b-glucosidase ext. phosphatase N/O B/E3 O/Y AL/AR comp. diversity

0.5289 0.3415 0.1358 0.7305** 0.6005* 0.5486 0.6965* 0.4856 0.6735* e

0.8543*** 0.2628 0.5665 0.3821 0.7648** 0.1909 0.5692 0.9346*** 0.7854** 0.2528 e

0.8567*** 0.1871 0.3934 0.8529*** 0.8620*** 0.5154 0.9188*** 0.7903** 0.9379*** 0.7505** 0.6047* e

0.8349*** 0.3706 0.6591* 0.3183 0.7183** 0.1257 0.5024 0.8806*** 0.7193** 0.2317 0.9737*** 0.5726 e

0.5993* 0.7626** 0.3360 0.8850*** 0.5946* 0.9097*** 0.8642*** 0.5751 0.6576* 0.5787* 0.3646 0.6718* 0.2516 e

B/E3

O/Y

0.4961 0.3742 0.6749* 0.7952** 0.3203 0.3554 0.7030* 0.8332*** 0.4320 0.4200 0.8170** 0.8266*** 0.7185** 0.7427** 0.4898 0.3137 0.5420 0.5277 0.4676 0.8200** 0.4041 0.0464 0.5421 0.6508* 0.3492 0.0252 0.8863*** 0.8494*** e 0.7463** e

AL/AR

Comp. Diversity

0.8405*** 0.2049 0.3448 0.8809*** 0.8614*** 0.5473 0.9137*** 0.7258** 0.8627*** 0.7559** 0.5139 0.9485*** 0.4902 0.6469* 0.4608 0.6374* e

0.4796 0.7557** 0.9457*** 0.0097 0.5346 0.4517 0.1662 0.4595 0.4640 0.2026 0.5028 0.4350 0.5688 0.2745 0.3342 0.2690 0.3987 e

Parameter abbreviations from Tables 1, 2 and 4. Statistical significance: *P < 0.05; **P < 0.01; ***P < 0.001; n ¼ 12.

Table 4 Relative abundance (%) of main pyrolytic peaks and indices of bulk soil surface samples and co-compost. Variable

Carbonated soils organica

E1 (%) K (%) B (%) O (%) E3 (%) N (%) Y (%) AL (%) AR (%) N/O O/Y B/E3 AL/AR Compounds Diversity

16.51 7.61 5.93 24.25 22.70 12.70 10.29 36.82 63.18 0.52 2.38 0.26 0.58 2.65

± ± ± ± ± ± ± ± ± ± ± ± ± ±

Silicic soils conventionalb

1.21* 1.53* 0.44 0.31* 1.07 2.20 1.13 0.53* 0.53* 0.09 0.27* 0.03 0.01* 0.02*

33.77 2.90 5.95 14.97 23.05 10.57 8.80 47.23 52.77 0.71 1.70 0.26 0.90 2.47

± ± ± ± ± ± ± ± ± ± ± ± ± ±

1.75* 0.26* 0.27 1.29* 1.34 1.89 0.44 1.83* 1.83* 0.13 0.06* 0.01 0.07* 0.04*

organica 12.28 14.54 4.65 22.93 19.30 11.84 14.46 38.66 61.34 0.52 1.60 0.24 0.63 2.69

± ± ± ± ± ± ± ± ± ± ± ± ± ±

Co-compost conventionalb

0.95* 2.31* 0.50* 0.86* 1.21 0.88* 1.74 2.29* 2.29* 0.05* 0.21* 0.04* 0.06* 0.02

21.97 8.31 7.21 9.87 19.88 17.04 15.72 47.32 52.68 1.77 0.64 0.36 0.90 2.69

± ± ± ± ± ± ± ± ± ± ± ± ± ±

1.30* 1.21* 1.20* 2.31* 0.63 1.52* 1.90 1.38* 1.38* 0.29* 0.20* 0.05* 0.05* 0.03

12.80 12.82 3.77 25.65 18.77 12.43 13.79 38.02 61.98 0.48 1.86 0.20 0.61 2.66

Data are mean values of three soil samples composed of four subsamples (mean ± standard deviation). E1 ¼ acetonitrile; K ¼ acetic acid; B ¼ benzene; O ¼ pyrrole; E3 ¼ toluene; N ¼ furfural; Y ¼ phenol. Aliphatic (AL ¼ E1 þ K þ N) and Aromatic (AR ¼ B þ O þ E3 þ Y) compounds. Pyrolytic indices of mineralization (furfural/pyrrole N/O; pyrrole/phenol O/Y), humification (benzene/toluene B/E3), and aliphatic/aromatic (AL/AR) ratios. *Values differed significantly at 0.05 probability level (t-test). a Organic management and 17 years of olive-mill pomace co-compost application. b Conventional management without olive-mill pomace co-compost application.

index and the AL/AR index (Table 3) could confirm that the O/Y index represents the mineralization of stable OM derived from the organic amendment, which is in agreement with Marinari et al. (2007). Thus under more favorable geochemical conditions, in the silicic soils, the more stable OM would remain in a lower state of biodegradation. The benzene/toluene index (B/E3) is related to the intensification of humification, because benzene derives mainly from pyrolyitic degradation of condensed aromatic structures, while toluene comes from non-condensed aromatic rings with aliphatic chains. This index was higher in conventionally managed silicic soil samples. A positive correlation found between the B/E3 index and the N/O index (Table 3) is due to a decrease in mineralization in more humified and stable OM. The fact that the B/E3 indices were similar in all treatments except for conventionally managed silicic soil samples could be related to greater recalcitrance of OM, given the significantly higher N/O index in this same environment. The almost unvarying B/E3 index in the samples analyzed could also suggest that the soil management practices did not influence the

more stable part of soil organic matter. More research about soil organic matter evolution in relation to soil type in this agricultural soil environment is necessary. The AL/AR index (Aliphatic/Aromatic compounds), expresses the ratio between the sum of aliphatic products (acetic acid, furfural, and acetonitrile) related to the presence of easily metabolizable materials, and the sum of aromatic compounds (benzene, toluene, and phenol), more resistant and stable to biodegradation. A decrease in this ratio would coincide with highly stable humic substances and advanced humification. With further humification, humic substances may become highly aromatic, with development of polycondensed rings (Ding et al., 2002). This argument is confirmed by the negative correlation between the AL/AR index and the HA/FA ratio (Table 3), which is related to aromaticity and the degree of polymerization of humic substances, and therefore, has been used as an index for describing the intensity of humification (Stevenson, 1994). Organic management of soil with application of olive-mill pomace co-compost to both carbonated and silicic soils, showed a

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lower AL/AR index, and therefore, higher aromaticity in humic substances. Brunetti et al. (2004) and Plaza et al. (2007) found that in the final steps of olive-mill pomace composting, condensed aromatic compounds, COOH, phenolic-OH content and aromaticity increased. According to García-Ruiz et al. (2012), these recalcitrant compounds might contribute to the low rate of carbon decomposition of this compost when applied to olive grove soils under field conditions, improving the physical and chemical fertility. Hence, this type of ligno-cellulosic co-compost is relatively resistant to degradation, enriching the soil with compounds that are not easily degradable, and could also be an appropriate strategy for sequestering organic carbon into the soil. The correlations between CEC and the AL/AR index (Table 3), and between CEC and the HA/FA ratio (data not shown), showed the positive relationship between aromaticity of humic substances incorporated in the soil by the organic amendment and chemical fertility of the soil. The samples from conventionally managed carbonated soils showed significantly less diversity of compounds generated during pyrolysis (Table 4). This result could be due to selective degradation of organic compounds on this type of soil and with soil management making it relatively easy degradation of added organic matter. Another possibility, given the high negative correlation between diversity and carbonates content (Table 3), is that this soil component favors a type of humification with the insolubilization of low-molecular-weight humic precursors and physical retention of particulate fractions. This may be due to a calcium-saturated medium typical of calcareous substrates in an environment with clear climate limitations. The result would be a less transformed SOM, with reduced variety and fewer organic compounds detected by Py-GC. This “carbonate protection” has been mentioned by other authors in natural calcareous soils (Miralles et al., 2007) and in olive
rez-Lomas et al., 2010) in the Mediterranean area. grove soils (Pe Both aspects should be studied further. Finally, the index of similarity (Sij) can be used to quantify the differences between samples related to management and soil type (carbonated or silicic). Table 5 shows low similarity (0.647) and high similarity (0.784) between the conventional soils and organic soils, respectively. This result suggests that either the chemicalstructural characteristics of organic matter in the two soils were modified differently by mineral fertilization, or that these soils probably maintain their native characteristics. On the other hand, organic management affected the organic matter characteristics similarly in both carbonated and silicic soils. When types of management are compared, the greater similarity of carbonated soils (0.736) and less similarity of silicic soils (0.684) suggests stronger influence of management type in the chemical-structural characteristics of silicic soils. 4. Conclusions In this study, the use of olive-mill pomace co-compost in organic management was effective in improving the soil quality in both types of soils studied (carbonated and silicic). Moreover, Table 5 Pyrolytic index of similarity (Sij) of bulk soil surface samples. Soil samples group

conventionalb carbonated

organica silicic

conventionalb silicic

organica carbonated conventionalb carbonated organica silicic

0.736 e e

0.784 0.619 e

0.739 0.647 0.684

a Organic management and 17 years of olive-mill pomace co-compost application. b Conventional management without olive-mill pomace co-compost application.

the ability of a soil to recover its physicochemical and biochemical properties, as well as increase the quality and stabilization of OM, after the change to organic management with application of olive-mill pomace co-compost was found to be generally more favorable in soils developed over silicic material. This should make it possible to define long-term soil conservation strategies, as long as the influence of the soil type in each zone is clearly established. Application of olive-mill pomace co-compost could be an important strategy for maintenance of the olive grove agroecosystem, as it would increase the amount of organic matter, producing a positive effect on soil properties and carbon storage. This strategy could also help to prevent environmental problems related to erosion in large agricultural zones in the Mediterranean region. Recycling of organic waste in agriculture could be crucial to maintaining the productivity of soil in this region, where organic matter content is very deficient. Therefore, the use of this type of transformed by-product (olive-mill pomace co-compost) from the extraction of olive oil could be an attractive solution for avoiding degradation of agricultural soil and sustainable elimination of olive-mill pomace, increasing the agro-environmental viability of the olive grove.

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