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a CRA-Instituto Sperimentale per la Nutrizione delle Piante, sezione di Gorizia, Via Trieste 23, I-34170 Gorizia, Italy
b CEBAS-CSIC, Departamento de Conservación de Suelos y Agua y Manejo de Residuos Orgánicos, Campus Universitario de Espinardo, E-30100 Espinardo, Murcia, Spain
* Corresponding author (claudio.mondini{at}entecra.it)
Received for publication February 8, 2006.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSREFERENCES |
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Abbreviations: BC, microbial biomass carbon • BNIN, microbial biomass ninhydrin-reactive nitrogen • CORG, total organic carbon • DH, degree of humification • HI, humification index • HLC, humic-like carbon • OM, organic matter • NHC, nonhumic carbon • NTOT, total nitrogen • TEC, total alkali-extracted organic carbon • WEOC, water-extracted organic carbon
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSREFERENCES |
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Despite the numerous researches performed on the subject there is, to date, no universally accepted method for the evaluation of compost stability (Itävaara et al., 2002). This is mainly due to the complexity of the process involved in the decomposition of OM and the wide range of physicochemical properties of the starting organic materials. Among the methods used for the evaluation of compost stability, respirometric techniques based on the measure of O2 consumption or CO2 production are widely accepted (Barberis and Nappi, 1996; Lasaridi and Stentiford, 1998; Wu and Ma, 2001; Francou et al., 2005). These methods have demonstrated to be effective to monitor stability, but they are time-consuming, too complex to be performed on-site, require sophisticated instrumentation, or provide only semiquantitative data.
Alternatively, the degree of decomposition of OM is determined by the analysis of different fractions of extracted OM, namely water-extracted and alkali-extracted OM. Water-extracted OM represents the most active fraction of compost OM since all biochemical reactions in compost occur in the aqueous phase (Chanyasak and Kubota, 1981; Bernal et al., 1998). The main constituents of water-extracted OM are sugars, carbohydrates, amino acids, polyphenols, enzymes, humic-like substances, and carboxylic acids, derived from OM decomposition and microbial synthesis (Leita and De Nobili, 1991; Ciavatta and Govi, 1993). Water-extracted OM has been shown to directly reflect the OM transformation process during composting (Chefetz et al., 1998). Therefore, a study of the changes occurring in water-extracted OM composition in terms of chemical fractions or specific compounds is thought to be useful for the evaluation of compost stability (Chefetz et al., 1998; Wu et al., 2000; Wu and Ma, 2002).
Alkali extraction is usually utilized to recover humic substances from soil and compost (Stevenson, 1994). During composting the most biodegradable material is decomposed and part of the remaining organic material is converted into humic-like substances. Humic-like substances are resistant to enzymatic decomposition due to their great complexity and lack of regular polymeric structure. Therefore, humification can be considered as the main process leading to the stabilization of compost and humification-related parameters have been extensively utilized for this aim (Sequi et al., 1986; Senesi et al., 1989; Ciavatta et al., 1990).
The evaluation of the degree of stabilization of OM could also be performed by means of parameters related to the microbial component of the compost (Wu et al., 2000). In the composting process, microorganisms are directly involved in the decomposition of organic residues and in the synthesis of humic-like substances. Microbial biomass size and activity have been shown to be related to the content of easily degradable substances (Belete et al., 2001). Furthermore, the introduction of the concept of microbial biomass as a unique functional entity (Powlson, 1994) had made possible the measurement of the content and activity of microorganisms in a substrate by means of easily determinable parameters.
Ligno-cellulosic wastes are usually used as bulking agents in the preparation of composting mixtures, but are less frequently composted alone due to the relative longer duration of the composting process caused by the high C/N ratio of these materials. As a consequence compost derived from ligno-cellulosic wastes are poorly studied and characterized. Nevertheless, compost prepared only with ligno-cellulosic wastes presents several advantages. Ligno-cellulosic wastes are more prone to source-separated collection than the municipal organic wastes. Composting of this kind of waste requires simpler composting technology than the separated organic fraction of municipal solid wastes. Composting can be safely performed by windrowing in open air plants without the risks usually associated to harmful emissions generated during the treatment of municipal solid wastes. Composts produced from ligno-cellulosic wastes have low heavy metal content and good physicochemical properties for their utilization as a peat substitute in nurseries.
The aim of the present work was the evaluation of parameters related to extracted OM and microbial biomass as reliable and simple indicators of the degree of stability of compost prepared with ligno-cellulosic wastes. The relationship of microbial biomass with chemical fractions of both water-soluble and alkali-extracted C was also investigated.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSREFERENCES |
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The composting process was performed in plastic boxes (106 x 90 x 42 cm deep; 0.4 m3) with holes to allow natural aeration and placed outdoors under a shed. During the process, manual turnings were done twice a week during the first month and weekly thereafter to allow aeration and homogenization of the mixture. The initial water content of the piles was adjusted to 65% and it was monitored weekly throughout the process. To keep the mixture at approximately the initial water content, watering was performed on Days 17, 29, 41, and 52 for mixture Y and after 19 and 41 d for mixture M. Water content of mixture CW remained in the recommended range during the process and therefore was never adjusted during the experiment. Temperature was monitored daily during the process by inserting a thermocouple into the piles. Total duration of the composting process, including maturation phase, was 21 wk for the three mixtures.
Experimental Design
One pile of adequate dimensions (0.4 m3) was prepared for each composting mixture as described in Mondini et al. (2004). The dimension of the pile ensures adequate homogenization of the initial mixture of raw wastes and self heating.
The sampling of the composting pile was performed by the combination of 5 subsamples (each subsample was approximately 0.2 kg) taken from the whole profile of the pile after 0, 7, 22, 49, 86, 112, and 149 d. By accurately mixing these subsamples, a sample (>1 kg) was obtained that is representative of the composting pile. This representative sample was used for the chemical and microbiological determinations that were performed in triplicate as described below. Chemical analyses were performed on air-dried samples and on the whole set of samples. Microbiological analyses were performed on moist samples collected on Days 22, 49, 86, 112, and 149. Results are expressed on an oven-dry sample basis (105°C, 24 h).
Chemical Analyses
Total organic C (CORG) and total N (NTOT) of the compost were determined by a dry combustion method with an NA 1500 elemental analyzer (Fisons, CE Instruments, Milan, Italy) on aliquots of dried samples ground to <0.5 mm.
Water-extracted organic C (WEOC) was extracted by shaking a sample ground to <2 mm with deionized and degassed water (water/solid ratio of 10:1) for 2 h at room temperature. Total alkali-extracted organic C (TEC) was extracted at 65°C for 24 h using a 0.1 M NaOH + 0.1 M Na4P2O7 solution (Grigatti et al., 2004). Both water and alkaline extracts were then centrifuged at 5000 x g and the supernatant filtered through a 0.45-µm Millipore filter (Millipore, Billerica, MA). In both of the extracts the humic-like acid C was separated from the fulvic-like acid and nonhumic carbon (NHC) by precipitation after acidification of the solution at pH < 2. NHC was separated from fulvic-like acid C by chromatography onto a column of polyvinylpyrrolidone (PVP, Aldrich, Germany) using the method suggested by Sequi et al. (1986). The fulvic-like acid C was then combined with the humic-like C to give the humic-like substances C (HLC). The organic C content was determined according to a wet dichromate oxidation procedure (Walkley and Black, 1934).
The following two humification parameters were calculated for alkaline extracts:
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Microbiological Analyses
Microbial biomass C (BC) and ninhydrin-reactive N (BNIN) were determined using the fumigation-extraction method (Vance et al., 1987). Moist samples (corresponding to approx. 25 g of compost dry weight) were fumigated with ethanol-free CHCl3 for 24 h at 25°C in a desiccator. After fumigant removal, the samples were extracted for 30 min with 400 mL of 0.5 M K2SO4 solution and then filtered through Whatman (Whatman International, Maidstone, UK) n° 42 filter paper. Nonfumigated samples were extracted as above at the time fumigation started. K2SO4–extracted organic C was determined by an automated UV persulphate oxidation procedure (Wu et al., 1990). BC was calculated as:
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K2SO4–extracted ninhydrin-reactive N (NNIN) was determined as described by Joergensen and Brookes (1990). BNIN was calculated as:
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Statistical Analysis
Data were checked for normal distribution and underwent univariate analysis of variance. Sample means were compared using the Student-Newman-Keuls test (Snedecor and Cochran, 1980). The relationships between variables were analyzed by the Pearson correlation coefficient. All statistical analyses were performed using the SPSS version 9.0 statistical package.
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSREFERENCES |
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The CORG/NTOT ratio declined in all 3 compost mixtures from initial values ranging from 22.4 to 33.5 to final values between 11.8 and 14.0. The larger changes of this ratio, indicating the intense transformations of the OM, were registered in the first week of composting in the case of compost mixture Y and in the initial 3 wk of composting for compost mixtures CW and M. After the sharp initial decline, the ratio decreased slowly in the 3 mixtures and remained nearly constant after the seventh week of composting. The CORG/NTOT ratios of the end products are consistent with a stabilized organic matrix not causing microbial immobilization of N when applied to the soil (De Bertoldi et al., 1983).
According to both monitoring parameters, there were two clear phases during the composting of ligno-cellulosic wastes: an initial active phase of approximately 7 or 8 wk, characterized by high temperatures and significant OM transformations (particularly intense during the first 3 wk), and a second phase characterized by lower temperatures corresponding to the typical maturation phase of composting, where OM reached an appropriate stabilization degree (Bernal et al., 1998).
Water-Extracted Organic Carbon
Dynamics of water-extracted OM in the three composting mixtures were monitored by means of the content of WEOC and its separation in HLC and NHC fractions (Fig. 2). All three starting mixtures presented similar concentrations of WEOC, ranging between 28.0 and 28.6 g kg–1. More than 50% of the initial WEOC was degraded during the first week of composting in piles CW and M. The decline was less pronounced in compost Y where WEOC steadily decreased during the whole thermophilic phase. After the initial decrease of the WEOC, this fraction showed little transformations in all the cases and remained practically constant.
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The concentrations of WEOC in the end products were influenced by the origin of the composting materials, reaching final values of 10.6, 19.2, and 12 g kg–1 for compost CW, Y, and M, respectively (Fig. 2). This behavior of rapid initial decrease followed by stabilization with curing time was slightly different from previous works performed on different types of wastes, where changes in WEOC occurred steadily during the whole composting process (Leita and De Nobili, 1991; Garcia et al., 1992a; Garcia et al., 1992b; Wu et al., 2000; Sánchez-Monedero et al., 2001).
On the evidence that stable or mature composts contain less WEOC than the corresponding immature compost, several authors have proposed WEOC as an indicator of degree of OM decomposition and suggested threshold values for composts with an adequate level of stability (Inbar et al., 1990; Garcia et al., 1992a; Hue and Liu, 1995; Chefetz et al., 1998; Wu et al., 2000; Wu and Ma, 2002). However, WEOC of composting mixtures studied in the present experiment did not reflect changes in the OM during the process, since it remained almost unaltered. Consequently, WEOC is not considered a suitable stability index to monitor the performance of ligno-cellulosic waste composting.
Several authors (Garcia et al., 1992a; Hue and Liu, 1995) have proposed the ratio WEOC/NTOT as a parameter for the evaluation of compost stability, regardless of the nature of the starting materials. The normalization of WEOC data implied in the calculation of the ratio allows for the comparison of compost produced from different starting materials.
In this work, WEOC/NTOT decreased markedly during the first week of compost from initial values ranging from 1.53 to 2.28 to values ranging from 0.70 to 0.86. Afterward, the ratio slowly decreased to final values in the range between 0.35 and 0.56 (Fig. 2). Therefore, in the case of ligno-cellulosic wastes studied in the present experiment, this ratio only reflected changes in water-extracted OM during the first weeks of composting, as already described in the case of WEOC. The threshold value of this ratio to be used as a compost stability index has been established in the range between 0.55 and 0.70 (Hue and Liu, 1995; Sánchez-Monedero et al., 2001). In the composting mixtures studied in the present experiment, this value was reached after only a few weeks of composting limiting, again, the suitability of this ratio based on WEOC as a stability index for these materials.
Alkali-Extracted Organic Carbon
The alkali-extracted fraction of OM has been traditionally used to follow humification processes by monitoring changes occurring in the content of humic-like substances during composting. Several humification parameters, obtained from fractionation of the TEC in HLC and NHC, are commonly used as conventional stability parameters for a wide range of organic materials.
In the present work, TEC and HLC increased in all three mixtures during the active phase of composting (7 to 8 wk) (Fig. 3). After the initial increase, TEC and HLC remained almost unchanged until the end of the experiment. NHC of the alkali extracts followed the same trend observed for WEOC, remaining almost unchanged during the whole composting process.
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Adani et al. (1995, 1999) questioned the suitability of current methodologies to study the humification process during composting because of the way in which experimental results are expressed and the analytical limitations of the alkaline extraction procedure.
First, HLC are reported in relative terms (i.e., as units of C per units of dry matter of compost) giving rise, in most of the cases, to an increase of HLC during composting. However, Adani et al. (1999), calculating the absolute amount of HLC from data of several previously published works, found a net decrease in the amount of HLC in all the cases. They concluded that humic-like substances were apparently formed because of a concentration effect due to OM degradation during composting. Contrary to these findings, the calculation of the absolute amount of HLC in the present experiment (Fig. 4) resulted in an increase in HLC during the whole composting process in the 3 ligno-cellulosic waste mixtures, confirming the synthesis of new humic-like substances.
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The dynamics observed for both water-soluble and alkali-extracted fractions of the OM should be related to the humification mechanisms occurring during composting. The most accepted humification pathways in composting mixtures involve either the partial transformation of the original lignin of the raw wastes (lignin theory) or the condensation of polyphenols, derived from lignin or synthesized by microorganisms, and amino compounds (polyphenol theory) (Veeken et al., 2000). In the present work where composting mixtures were prepared with ligno-cellulosic materials, lignin transformation was likely to be the predominant humification pathway. This pathway mainly includes demethylation and oxidation of side chains of the insoluble macromolecules (Stevenson, 1994) and consequently it would not generate significant amounts of water-soluble intermediates. This mechanism is consistent with the pattern observed for the WEOC that remained almost unchanged after the first weeks of composting when the readily degradable substances were mostly depleted. According to the lignin theory, the newly formed humic-like acids present similar molecular weight as the original lignin, but enriched in oxygen-containing functional groups (COOH, CO, OH) that increase solubility in alkali (Stevenson, 1994). This is consistent with the observed increase in HLC during the composting of ligno-cellulosic waste materials.
Adani et al. (1999) suggested that the main process leading to HLC formation in compost is the transformation of the original lignin contained in the raw wastes. As a consequence, a high content of lignin in the starting materials is favorable for the production of high quality compost with a higher content of HLC.
The condensation mechanism, involving the initial breakdown of the insoluble biopolymers (including lignin) into their monomeric structural units and further polymerization to produce humic molecules (Stevenson, 1994), leads to the formation of water-soluble intermediate compounds that are likely to affect WEOC dynamics, or at least some of its fractions (HLC and NHC). Since WEOC and its components remained almost unchanged throughout the composting process (Fig. 2), this mechanism was likely to exert only a minor role on the formation of HLC in ligno-cellulosic materials.
Microbial Biomass
Microbial biomass C was determined in the 3 piles as an indicator of the degree of biochemical transformations of the OM during composting. The BC in compost Y and CW decreased at different rates during the process: the decline was slight in compost CW, whereas it was remarkable and constant in compost Y (Fig. 5). The BC of compost M showed a very different behavior, with an initial increase up to a maximum reached after 86 d, followed by a marked decrease of BC to a value significantly lower to that recorded after 22 d.
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The limitation of WEOC to monitor the amount of available C in ligno-cellolosic materials could be explained on the basis of the lignin pathway as the main humification mechanism operating in the composting process of this kind of materials (see above). In this context the products of lignin degradation would represent the predominant source of energy and C for the microorganisms. Lignin is a macromolecule characterized by a low rate of degradation, due to the complexity of the structure, large size, and insolubility in water. As a consequence, WEOC is not likely to be significantly affected by products of lignin degradation. Therefore, the amount of C available for microorganisms in compost from ligno-cellulosic materials would be determined by the rate of lignin degradation rather than WEOC.
Compost stability is a reflection of the progress of the composting process and depends on the extent to which microbial degradation has taken place (Epstein, 1997). Therefore, compost stability is a function of the microbial availability of easily degradable substances. The BNIN/NTOT ratio has been proposed as an index of compost stability on the assumption that the amount of microbial N with respect to NTOT is a function of the quantity of substances easily degradable available for microorganisms (De Nobili et al., 1996). In soil, higher values of this ratio were found when the content of available substrate was increased by soil amendment with OM (Powlson et al., 1987). Furthermore, the normalization of BNIN data implied in the calculation of the ratio allows for comparison of composting materials resulting from different starting materials, characterized by a different content of OM and NTOT.
The BNIN/NTOT ratio decreased in the three compost heaps with significant variations in samples of different ages (Fig. 5). According to this ratio, compost stability was reached after 86 to 112 d of composting. Furthermore the correlation of BNIN/NTOT with the time of composting was linear (r = –0.91, r = –0.96, r = –0.85; P < 0.01 for CW, Y, and M, respectively). Values of BNIN/NTOT in the end products ranged between 0.6 and 1.3%, in agreement with values found in compost with a good degree of stabilization (De Nobili et al., 1996; Mondini et al., 1997, 2002).
The reliability of the BNIN/NTOT ratio as a compost stability parameter was also supported by its significant and inverse relationship with the HLC fraction in alkaline extracts (r = –0.69; P < 0.01). The ratio was inversely correlated with the increase in HLC, the main process leading to the stabilization of OM. The observed correlation is consistent with a decrease of available C with increasing amount of HLC.
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CONCLUSIONS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSREFERENCES |
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Stability parameters obtained by the alkali-extracted fraction of the OM (DH and HI) and from microbial biomass determination (BNIN/NTOT) have shown to be reliable for the characterization of the ligno-cellulosic waste composting process and for the definition of the stability level of end products. In particular, humification parameters HI and DH were effective in detecting an increase in the absolute and relative content of the HLC during composting, whereas BNIN/NTOT was a more reliable indicator of available C for microorganisms than WEOC.
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSREFERENCES |
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