Compost Science & Utilization, (2010), Vol. 18, No. 2, 105-110
Conformational Distribution of Dissolved Organic Matter Released from Compost by Repeated Water Extractions Edoardo Puglisi1, Riccardo Spaccini2, Alessandro Piccolo2, Marco Trevisan1* and Attilio A.M. Del Re1 1. Istituto di Chimica Agraria ed Ambientale, Università Cattolica del Sacro Cuore, Piacenza, Italy 2. Dipartimento di Scienze del Suolo della Pianta, e dell’Ambiente, Università di Napoli Federico II, Portici, Italy *E-mail contact:
[email protected] The dissolved organic matter (DOM) released from compost by repeated water extractions was quantified and DOM conformational changes by high performance size-exclusion chromatography (HPSEC) were followed. Three commercial composts were extracted consecutively 4 times with equal water: compost ratios. The total organic carbon content of each DOM sample was analyzed using the chemical oxygen demand method, and the conformational size-distribution assessed by means of HPSEC. Results showed that compost continues to release considerable amounts of DOM with repeated water extractions, and that subsequent DOM extractions resulted in organic matter with increasing molecular dimensions and presumably larger hydrophobicity. This may have important consequences on soil processes and functions such as organic matter dynamics and transport of pollutants.
Introduction Intensive soil tillage, use of mineral fertilizers and reduced applications of organic matter are amongst the most important causes of organic carbon decrease in agricultural soils. Organic matter levels in arable soils can be restored by amendment with compost (Spaccini et al. 2009). Compared to other organic fertilizers such as animal manure or sewage sludge, compost is a source of stabilized organic matter due to the humification and stabilization processes occurring during its formation (Hernández-Apaolaza et al. 2000). Soil amendment with compost can help to reduce erosion, alleviate soil compaction, control disease and pest infestation in plants, increase plant production, limit the use of chemical fertilizers, and contribute to decrease emission of CO 2 from soils (Barriuso et al. 1997; Piccolo et al. 2004). Compost application was also shown to reduce the xenobiotics hazard in soils (Crawford 1993; Puglisi et al. 2007; Puglisi et al. 2009). There are, however, some disadvantages associated with compost, including the possible presence of contaminants, the time necessary to produce quality compost, adverse effects on electrical conductivity, the possible release of pathogens, and a reduced nitrogen content compared to other organic fertilizers (Eghball and James 1999). Compost application to soil is bound to generate a net flux of dissolved organic matter (DOM) Compost Science & Utilization
through rainfall events, leaching and advancing microbial attack. DOM is commonly defined as the fraction of soil organic matter that passes through a 0.45 mm filter (Zsolnay 2003). It comprises a complex mixture of low-molecular weight compounds (free amino acids and sugars) and chemically heterogeneous biomolecules originated from dead cellular material (enzymes, aminosugars complexes, polyphenols, lignin residues, and alkanoic acids) (Tranvik and Jørgensen 1995; Puglisi et al. 2008). DOM is regarded as the most chemically and biologically active fraction of compost (Chefetz et al. 1998), and thus plays a major role in many important soil processes. Both organic and inorganic contaminants may adsorb to DOM with consequences for their mobility to ground waters (Haigh 1996; Kögel-Knabner and Totsche 1998), and their bioavailability and toxicity (Ohlenbusch et al. 2000). Association with DOM is strongly related to the characteristics of both pollutants and DOM. In particular, adsorption of contaminants onto DOM fractions depends on their molecular size and hydrophobicity of adsorbing molecules (Piccolo et al. 1996; Kögel-Knabner and Totsche 1998; Celano et al. 2008). Different methods can be used to characterize DOM. Total organic carbon content (TOC) is a quantitative measure of DOM content in water extracts of soils or compost. DOM samples have been studied by physical-chemical methods such as NMR specSpring 2010 105
Edoardo Puglisi, Riccardo Spaccini, Alessandro Piccolo, Marco Trevisan and Attilio A.M. Del Re
troscopy, pyrolysis-GC-MS, and electrospray ionisation (ESI) LC-MS. These have been used to obtain both quantitative and qualitative information on the carbon compounds and their mass distribution in natural organic matter (Piccolo and Spiteller 2003; Kim et al. 2003; Spaccini and Piccolo 2009). High Pressure Size Exclusion Chromatography (HPSEC) is often applied to rapidly assess the molecular size distribution of DOM and compare the conformational properties of differently-treated samples (Kitis et al. 2002; Smejkalova and Piccolo 2005). Despite the large agricultural utilization of compost, very few studies have focused on changes in the release of DOM over time, especially following compost application and subsequent leaching events. Given the importance of DOM in many ecological processes outlined above, it is important to assess how much DOM is released by compost in time, and how its molecular characteristics change with time. Borken et al. (2004) studied the effects of a single application of mature compost on DOM output in seepage water from nutrient-depleted forest soils. They found that compost application increased the cumulative DOM output at 10 cm, and that most DOM was adsorbed at a depth of 0- to 20- cm, especially in mineral soils with low organic matter content. Agassi et al. (1998) studied the effects of consecutive rainstorms on the percolation rate of soils amended with sludge compost and found that sludge compost reduced the percolation rate compared to control. Ertunç et al. (2002) analyzed by HPSEC the molecular size distribution of DOM samples leached from a biowaste compost contaminated with simazine after 29 and 200 days of ageing. Chromatograms showed a shift of the molecular size distribution towards lower molecular weight compounds as compost ageing proceeded. Changes in DOM composition from a soil amended with municipal waste compost for 6 years were analysed by Gigliotti et al. (1997). They found that compost DOM hydrophobic molecules were reduced since they were adsorbed onto the soil surface, whereas hydrophilic (e.g.: polysaccharide) molecules were less adsorbed and increased in DOM. The assessment of DOM released from commercial composts by repeated water extractions simulating rainfall events may provide useful information on the long-term behaviour of compost following application in the field, particularly regarding changes in the quantity and quality of DOM releases over time. In this work, we thus evaluated the quantity of DOM released from compost by repeated water extractions and followed DOM changes by HPSEC. 106 Compost Science & Utilization
Materials and Methods Compost Three commercial compost samples were used for this study. Two were green compost, originating from bog-moss peat (COM 1 and COM 3). The third one was a mixed compost containing cattle and horse manure and humus (COM 2, manure:humus ratio 3:1). The three samples showed similar chemical properties (Table 1). Organic carbon content ranged from 20 to 28%. Total nitrogen levels were also similar, averaging 1.2 %. The C/N ratio varied and was greater for COM 3 than for the rest of compost samples. TABLE 1. Chemical properties of the composts used for this study COM 1
COM 2
COM 3
Water content (%)
48
28
35
pH (H O) 2
7
7.4
7.8 28.5
Organic carbon (%)
27
20
Total nitrogen (%)
1.4
1.2
1.6
C/N
12
16.5
23.7
Extraction of Dissolved Organic Matter (DOM) All experiments were conducted in triplicate. DOM was extracted from the composts with a slight modification of the method of Gigliotti et al. (2002), using a soil:water ratio of 1:2. All experiments were conducted in triplicate. Compost (80 g) and deionised water (160 ml) were placed in a vessel and mixed on a mechanical shaker at 190 rpm for 20 hours. Samples were then centrifuged at 8000 rpm for 1 hour. The supernatant was separated from the solid sediment and filtered to 0.45 mm using Albet® cellulose acetate membrane filters. Deionised water was then added to the solid sediments in the vessel to give the original volume and extraction was repeated three additional times, in order to obtain DOM successively released from compost. An interval of 24 hours was allowed between each extraction. Sodium azide (NaN3, 0.002 M) was added to each DOM sample to minimize microbial activity. Samples were refrigerated at 4°C until analysis. The total organic carbon (TOC) content of each DOM sample was assessed using the chemical oxygen demand (COD) method (Standard methods 1998). Three replicates per sample were analyzed for TOC content. High Pressure Size Exclusion Chromatography (HPSEC) A Biosep S2000 Phenomenex column (300 x 7.8 mm) with a nominal molecular weight range between Spring 2010
Conformational Distribution of Dissolved Organic Matter Released from Compost by Repeated Water Extractions
1 and 300 kDa was used to measure the relative molecular size distribution of DOM samples. Two replicates from each sample were analysed. The HPSEC system comprised a Shimadzu LC-10AD Pump and a UV/VIS Perkin-Elmer LC295 detector set to 280 nm. Chromatograms were recorded using a PE TotalChrom 6.2 software for peak integration. The computer hardware, pump system and detectors were interfaced by a Perkin-Elmer Nelson 900 Series Interface. The mobile phase was a phosphate buffer (pH 7), consisting of solutions of 0.1 M NaH2PO4 and 0.1 M NaOH eluted at a flow rate of 0.6 mL.min-1. A sodium azide aqueous solution (0.5 g L-1) was added as bacteriostatic agent. DOM solutions (100 µL) were injected through a Rheodyne 7125 rotary injector and eluted at a constant flow rate of 0.6 ml.min-1. Before the HPSEC analyses, each DOM sample was diluted to a final con-1 centration of 100 mg L of organic carbon. To test the reproducibility of column behaviour, each DOM sample was run twice. Polystyrene sulphonate standards of known molecular weights (3800, 6780, 16900, 30900, 123000 Da) were used to calibrate the HPSEC column by dissolving the standards in the same solvent used to analyze DOM samples and. Number-average molecular weights (Mw) were calculated for each DOM sample using the following equation:
where Mw and ni are the molecular weight and the height of each fraction eluted at the i-th volume in the chromatogram, respectively. Void (Vo) volume was 12.59 mL and total volume (Vt) was 17.90 mL.
Statistical Analyses TOC data were analysed using analysis of variance (ANOVA, PROC GLM, SAS Institute 1985). All significant effects were confirmed by Tukey’s test for comparison of means.
Results and Discussion COD results for DOM samples are reported in Figure 1. The coefficient of variation amongst replicates was, in most cases, below 25 %, indicating good reproducibility of the COD method. The three composts released different quantities of DOM. Carbon released per g of -1 compost for the first extraction ranged -1 from 1.02 mg g for COM 1 to 2.93 mg g for COM 3. In all cases, the quantity of released DOM decreased with each subsequent extraction, but all compost samth ples still released DOM after the 4 extraction. ConCompost Science & Utilization
FIGURE 1. Releases of DOM from commercial composts with repeated water extractions. Data are expressed as mg of TOC released per g of compost.
centration recovered in the 4th extraction as a percentage of the first extraction varied among compost materials (63.5%, 39.0% and 40.7% for COM 1, COM 2 and COM 3, respectively). No clear correlation was found between the original carbon content in each compost (Table 1), and the amount of DOM extracted in the four steps (Figure 1). Although COM 3 was the material with more organic carbon content (28.5) % and the one that released more DOM in four extractions (5.0 mg g-1), in the other two cases COM 1 had a higher canon cntent than COM 2 (27 against 20 %), but the amounts of DOM released were similar (2.5 and 2.4 mg g-1 respectively). The observations were confirmed using ANOVA (by SAS software, SAS Institute 1985). Three effects on the release of DOM were studied: the effect of compost type (classification variable COMPOST), repeated extraction (EXTRACT), and their interaction (COMPOST * EXTRACT). All three effects were significant (Table 2). Tukey’s test for comparison of means for the COMPOST effect indicated that COM 3 released a significantly larger quantity of DOM than the other compost samples (average values of 0.85 mg g-1 for COM 1, 1.00 mg g-1 for COM2 and 1.96 mg g-1 for COM 3). Regarding the EXTRACTION effect, a significant decrease was observed between the first two (1.96 and TABLE 2. ANOVA performed on DOM concentrations in water extracts of compost. The effects of compost type, repeated extraction and their interaction were assessed. Degrees of Freedom
F Value
Pr > F
Compost
2
101.20
