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a Soils and Crops Research and Development Centre, Agriculture and Agri-Food Canada, 2560 Hochelaga Blvd, Québec, QC, G1V 2J3 Canada
b Institut de Recherche et de Développement en Agroenvironnement (IRDA), Complexe Scientifique, 2700 rue Einstein, Québec, QC, G1P 3W8 Canada
c Département des Sols et de Génie Agro-alimentaire, Faculté des Sciences de l'Agriculture et de, l'Alimentation, UniversitéLaval, Québec, QC, G1K 7P4 Canada
* Corresponding author (angersd{at}agr.gc.ca).
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ABSTRACT |
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 TOPNOTES ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Abbreviations: CFA, organic carbon of fulvic acids • CHA, organic carbon of humic acids • CUHF, organic carbon of unhumified fraction • CPMS, composted paper mill sludge • MNF, mineral nitrogen fertilizer • PMS, fresh mixed paper mill sludge • UHF, unhumified fraction of organic matter
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INTRODUCTION |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Fungal hyphae and EPS may not persist for a long time in soil, and their effects on aggregate stability are often considered temporary (Tisdall and Oades, 1982). Consequently, it has been suggested that the improvement and maintenance of soil aggregate stability depends on the capacity of organic amendments to produce humic substances (Martens, 2000). Studies have shown significant correlations between aggregate stability and the soil content of various forms of humic substances (Chaney and Swift, 1984; Fortun et al., 1989). Piccolo and Mbagwu (1989) found that aggregate stability increased after the addition of humic substances to soil. In addition, Chaney and Swift (1984, 1986) showed that humic acids could stabilize soil aggregates under conditions where EPS were ineffective and that their binding effect was persistent.
The interactions between organic matter and soil structure are very complex, and the mechanisms that govern them are still being debated (Kay and Angers, 1999; Six et al., 2004). In a recent study, we showed the positive effects of applying mixed fresh paper mill sludge (PMS) and composted paper mill sludge (CPMS) on the formation and stabilization of soil macroaggregates (Bipfubusa et al., 2005). To gain information on the biochemical factors that control aggregate stabilization under these treatments, in this study we examined the organic C, total sugar, amino sugar, and humic substance contents fractions of different water-stable aggregate size classes.
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MATERIALS AND METHODS |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The experimental design was described by Bipfubusa et al. (2005). Briefly, a complete block design with six treatments and four replicates was used. The treatments included PMS and CPMS at a rate of 40 Mg ha–1 (wet basis) with or without a reduced rate of MNF(120 kg N ha–1), complete MNF (160 kg N ha–1), and an unamended, unfertilzed control. Detailed composition of the sludge is given in Bipfubusa et al. (2005). Briefly, the fresh sludge (PMS) contained 280 g dry matter kg–1, 334 g C kg–1 dry matter, and 11 g N kg–1 dry matter, while the CPMS contained 400 g dry matter kg–1, 281 g C kg–1 dry matter, and 8 g N kg–1 dry matter. Therefore the C application rates were 3.74 Mg C ha–1 for the PMS and 4.49 Mg C ha–1 for the CPMS. The PMS and CPMS were spread by hand in the spring and autumn of 2000 and in the autumn of 2001. All plots received 26 kg P ha–1 and 83 kg K ha–1. Inorganic mineral N (NH4NO3), superphosphate, and KCl were broadcast. Afterward, the organic amendments and mineral fertilizers were incorporated into the top 10 cm by disking. No organic amendment was added after 2002 so that the residual effect on subsequent crops could be investigated; however, all plots were treated with 120 kg N, 26 kg P, and 83 kg K ha–1. The plots were 3 by 10 m and separated by 1-m buffer zone. The plots included four rows of silage corn at 75-cm spacing.
Soil Sampling and Aggregate Preparation
Composite soil samples were obtained from five cores (5-cm diam.) collected from the topsoil layer (0–20 cm) of each experimental plot in October 2003, 2 yr after the last treatments. A portion was air dried and sieved to pass a 2-mm sieve, then ground to pass through a 0.15-mm sieve for organic C analysis. The other portion was prepared for aggregate analysis by separating 5- to 8-mm aggregates by dry sieving.
Aggregate Stability
The stability of macroaggregates was determined by wet sieving (Angers et al., 2008). Briefly, 50 g of 5- to 8-mm air-dried aggregates was put on the top of a series of sieves with decreasing openings (2 and 0.25 mm). The sieves were placed in a wet-sieving apparatus and sieved in deionized water for 10 min at 30 cycles min–1. Three water-stable aggregate size classes were obtained: large macroaggregates (2–8 mm), small macroaggregates (0.25–2 mm), and microaggregates (<0.25 mm). The microaggregates were recovered by centrifugation of the water used for sieving for 10 min at 3000 x g. The three fractions of aggregates were collected in a beaker and oven dried at 50°C to constant weight. The aggregate weight was corrected for the presence of sand. The stability of macroaggregates was estimated by the mean weight diameter of the water-stable aggregates, which was the sum of the mass of soil remaining in each sieve multiplied by the mean aperture of the adjacent meshes. The results were the means of two replicates. A part of the soil of each aggregate fraction was crushed to pass through a 0.15-mm sieve, and analyzed for organic C, total sugar, amino sugar, and humic substance contents.
Organic Carbon Contents
Total C contents of the whole soil and the three water-stable aggregate size classes were determined by dry combustion with a LECO CNS-1000 (LECO Corp., St. Joseph, MI.). Total C content was considered to represent organic C because this soil does not contain carbonates.
Humic Substance Analysis
Humic substances were extracted according to the method described by Schnitzer et al. (1981). A 10-g sample of aggregates crushed to pass through 0.15-mm sieve was placed in a 250-mL centrifuge tube containing 100 mL of 0.1 mol L–1 NaOH and 0.1 mol L–1 Na4P2O7 · 10H2O. The tubes were shaken for 24 h on a reciprocating shaker, and centrifuged for 20 min at 3000 x g. The supernatant was decanted and centrifuged for 15 min at 15,000 x g again. A 25-mL aliquot of the supernatant was acidified to pH 2 with 50% H2SO4, and the humic acids (HA) were allowed to precipitate for 24 h at 4°C. The precipitated HA were separated from fulvic acids (FA) through centrifugation for 15 min at 15000 x g. The precipitated HA were oven dried at 45°C and redissolved in 25 mL of 0.5 mol L–1 NaOH.
The FA were separated from unhumified organic matter fractions (UHF) by adsorption of FA onto polyvinylpyrolidone resin (Sequi et al., 1986). A 25-mL aliquot of FA extract was passed through a column containing 12 g of polyvinylpyrolidone resin (Sigma-Aldrich, Munich, Germany), previously purified and equilibrated by 0.5 mol L–1 NaOH and 0.005 mol L–1 H2SO4, respectively. The column was rinsed with 0.005 mol L–1 H2SO4 to remove all UHF, and sorbed FA were eluted with 0.5 mol L–1 NaOH.
The three fractions of humic substances (UHF, FA, and HA) were stored at 4°C until analysis. Organic C in the FA (CFA) and HA (CHA) was determined by the KMnO4 oxidation method (Nelson and Sommers, 1982), whereas UHF organic C (CUHF) was determined on a Technicon autoanalyzer (Technicon Instruments, Tarrytown, NY). The humification index (CUHF/(CHA+CFA) and polymerization index (CHA/CFA) were calculated as suggested by Sequi et al. (1986) and Orlov (1995), respectively.
Total Sugar Analysis
Total sugar content for each aggregate size class was determined following Angers et al. (1988). In short, a 2-g sample of aggregate crushed to pass through a 0.15-mm sieve was placed in a sealed 50-mL tube, and 8 mL of 12 mol L–1 H2SO4 was added. The suspension was slightly mixed and incubated for 2 h at room temperature. At the end of the incubation period, the solution was transferred to a 250-mL centrifuge tube with 192 mL of deionized water and incubated at 85°C for 24 h. After cooling, soil extracts were centrifuged for 20 min at 16,000 x g, filtered (Whatman no. 42), and stored at –20°C until analysis.
The concentration of total sugars was determined by the anthrone method according to Brink et al. (1960). A 5-mL aliquot of the soil hydrolysates was placed in a tube and 10 mL of 0.2% anthrone in 95% H2SO4 were added. After 15 min of color development, absorbance of the resulting solution was read at 625 nm using a Hitachi U-2010 spectrophotometer (Hitachi High Technologies, Schaumburg, IL). The standard curve was established with 50, 100, 150, and 200 mg L–1 of glucose.
Amino Sugar Analysis
Amino sugar analysis was performed according to the method of Chantigny et al. (1997) with minor modifications. A 1-g sample of aggregate crushed to pass through 0.15-mm sieve was placed in a 250-mL polypropylene copolymer centrifuge tube and hydrolyzed with 20 mL of 6 mol L–1 HCl in an oven at 105°C for 6 h. The hydrolysate was cooled on ice to room temperature and centrifuged for 10 min at 15,000 x g. The supernatant was decanted into a plastic vial and stored at –20°C until analysis.
A 1-mL aliquot of the supernatant was dried completely by a rotary evaporator at 45 to 50°C under vacuum. The residue was redissolved in 980 µL of 99% o-phthaldialdehyde and 20 µL of mercaptoethanol. The precipitate was removed by centrifugation (3 min at 15,000 x g). The amino sugars (glucosamine, galactosamine, mannosamine, and muramic acid) were quantified by high performance liquid chromatography on a Waters HPLC (Waters Associates, Milford, MA) equipped with an autoinjector, a Waters analytical column no. 8NVC18 4µ, a column oven at 40°C, a Xenon lamp, and a fluorometric detector (Waters 474). The fluorometric detector was set at 338- and 425-nm wavelengths for excitation and emission, respectively. A 25-µL sample was injected manually and the flow rate was 1.0 mL min–1. The mobile phase (pH 5.3) consisted of 0.05 mol L–1 sodium citrate/0.05 mol L–1 NaOAc, 95% methanol (HPLC grade), and tetrahydrofuran at a ratio of 90:8.5:5. The column was cleaned with a 65% methanol solution and then reconditioned with the mobile phase.
Statistical Analysis
Data obtained were subjected to the GLM procedure of SAS (SAS Institute, 1985) using a randomized complete block design model. Single degree-of-freedom orthogonal contrasts were used to make preplanned comparisons among means for groups of treatments.
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RESULTS |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Humic Substances
Fulvic and Humic Acids
The soil aggregate CFA content varied from 0.90 to 3.10 g C kg–1 aggregate, while the CHA content varied from 2.03 to 4.08 g C kg–1 aggregate (Table 3
). The highest values of CHA content were observed for the >2-mm macroaggregates under the CPMS and the CPMS plus 120 kg N ha–1 treatments (average of 3.78 g C kg–1), which were significantly higher than those of the PMS and the PMS plus 120 kg N ha–1 treatments (average of 2.54 g C kg–1). Since the C application rate was slightly greater (20%) with the composted than with the fresh residue, we calculated a ratio of the CHA content to the amount of C added. The results still show a higher value for the composted sludge (0.84) than the fresh sludge (0.66), which suggests that the effect of the composts on CHA was not due only to the higher C loading rate.
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The applications of PMS and CPMS did not affect the CHA of the small macroaggregates (0.25–2 mm) but significantly increased their CFA content (Table 3). In this fraction, the highest CFA content was observed with the MNF treatment, which was significantly higher than those of the PMS and CPMS (Table 3). No relationships could be established, however, between these effects and aggregate stability.
Unhumified Fraction
The soil aggregate CUHF content varied from 1.56 to 2.13 g C kg–1 aggregate (Table 3). The application of PMS and CPMS had a positive effect on the CUHF content of the stable macroaggregate fractions compared with the control (Table 3). On the other hand, MNF treatment had no significant effect on the CUHF content. In comparison with CPMS, and despite a lower C loading rate, the application of PMS increased the UHF content of the macroaggregates >2 and 0.25 to 2 mm by 18 and 7%, respectively (Table 3). According to N'Dayegamiye and Watt (2000), this positive effect of PMS on CUHF would be related to their high content of easily degradable C, which would support microbial growth in the soil. Indeed, Stout et al. (1981) suggested that the UHF is partly composed of plant residues modified by the microorganisms, microbial bodies, or compounds newly synthesized by the microorganisms such as metabolites. The decrease in the content of CUHF of the >2-mm macroaggregates following the combined application of PMS and MNF (Table 3) confirms that this fraction consists of easily mineralizable organic compounds.
Humification Indices
The index of humification, CUHF/(CFA + CHA), varied between 0.3 and 0.5 (Table 4
), which is close to an average value of 0.5 proposed by Gigliotti et al. (2001). For both macroaggregate fractions, the results suggest that the organic matter of the soil amended with the CPMS was more humified. Indeed, contrary to the CPMS addition, the application of PMS resulted in an enrichment of the soil mostly in CFA and not in CHA (Table 3).
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Total Sugars
Total sugar content varied from 3.7 to 7.7 g C kg–1 aggregate (Table 5
), which is in the range of values found in Québec soils (Angers et al., 1988). Sugar contents generally increased with aggregate size (Table 5), which agrees with a previous study also performed on slaking-resistant aggregates (Puget et al., 1999). The application of PMS and CPMS significantly increased the total sugar content of the stable macroaggregates >2 mm by an average of 29% compared with the control, but there were no significant differences between the effect of composted and fresh sludge. Considering that carbohydrates determined by the anthrone reagent method are mainly hexoses and deoxyhexoses (Brink et al., 1960), which are predominantly of microbial origin (Cheshire, 1979), our results suggest that the application of PMS and CPMS has stimulated microbial activity in stable macroaggregates >2 mm. There were greater increases of total sugar contents in macroaggregates >2 mm, however, when PMS was applied in combination with MNF (Table 5), suggesting that the application of PMS along with inorganic N fertilizer led to increased microbial activity, and subsequently to an increase in microbial polysaccharide production. The small effect of adding mineral N fertilizer with CPMS on the sugar content of aggregates (Table 5) might be partly due to the low availability of labile C in these amendments.
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ern
et al. (2003) indicated the negative effect of mineral N fertilization on the soil microbial biomass under silage corn production.
Amino Sugars
The amino sugar contents of the aggregate fractions ranged from 396 to 563 mg C kg–1 aggregate (Table 6
) and represented 1.8 to 3.2% of their total organic C. The sum of glucosamine and galactosamine accounted for 90 to 93% of total amino sugar, and mannosamine and muramic acid accounted for 7 to 10%. Generally, the amino sugar content was greater in the macro- than in the microaggregates (Table 6).
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DISCUSSION |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The >2-mm size fraction was the most influenced by the application of the organic residues. The millimeter-size aggregate fraction has been found to be highly responsive to organic residue addition in this soil (N'Dayegamiye et al., 1997; Aoyama et al., 1999) but also in other soils and under other management practices in Québec (Angers, 1998). The large macroaggregates (>2 mm) were also generally enriched in the various organic matter fractions. Isotopic studies have shown that newly added organic matter is preferentially incorporated in the stable macroaggregates (Puget et al., 1995; Angers and Giroux, 1996). This enrichment supports the hypothesis that organic matter from PMS and CPMS favored binding of microaggregates into macroaggregates, as proposed by the hierarchical soil aggregation model (Tisdall and Oades, 1982).
The HA contents of the aggregates >2 mm was greater in the soil amended with composted residue than with the fresh material and, conversely, their glucosamine content was greater with the application of fresh residues. We hypothesize that the effects of the fresh sludge are attributable to their greater lability and, consequently, to their capacity to stimulate the soil microflora, in particular fungi, as quantified in this study by the glucosamine content. Fungi have been shown to play a significant role in macroaggregate stabilization (Tisdall and Oades, 1982). Several mechanisms have been invoked, such as a direct effect on cohesion by physical enmeshment by the hyphae or by the production of extracellular products that can increase cohesion or reduce wettability.
Contrary to fresh PMS, the application of the CPMS resulted in HA enrichment of the >2-mm macroaggregates. The addition of humic substances contributes to binding soil particles and increases soil aggregate stability (Piccolo and Mbagwu, 1989). In an incubation study, Annabi et al. (2007) showed a rapid effect of composted organic matter on aggregates, which they attributed to the diffusion of humic material within the aggregate, and a consequent increase in aggregate cohesion.
Our results illustrate the beneficial effects of both fresh and composted organic residues on aggregate stability under field conditions. The effects of three consecutive applications lasted for at least 2 yr after the last application. The effects were quantitatively similar for both residues but the mechanisms involved appear to be different. Microorganisms, in particular fungi, were a more important factor of stable macroaggregation in the soil amended with fresh sludge, while humic substances played a greater role in the compost-amended soil.
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ACKNOWLEDGMENTS |
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NOTES |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Received for publication February 8, 2007.
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REFERENCES |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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ern, J., J. Balík, D. Pavlíková, M. Zitková, and K. Skora. 2003. The influence of organic and mineral nitrogen fertilizers on microbial biomass nitrogen and extractable organic nitrogen in long-term experiments with maize. Plant Soil Environ. 49:560–564.This article has been cited by other articles:
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  A. N'Dayegamiye Soil Properties and Crop Yields in Response to Mixed Paper Mill Sludges, Dairy Cattle Manure, and Inorganic Fertilizer Application Agron. J., July 7, 2009; 101(4): 826 - 835. [Abstract] [Full Text] [PDF]
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 B. M. Wallace, M. Krzic, T. A. Forge, K. Broersma, and R. F. Newman Biosolids Increase Soil Aggregation and Protection of Soil Carbon Five Years after Application on a Crested Wheatgrass Pasture J. Environ. Qual., January 13, 2009; 38(1): 291 - 298. [Abstract] [Full Text] [PDF]
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J. Nyiraneza, M. H. Chantigny, A. N'Dayegamiye, and M. R. Laverdiere Dairy Cattle Manure Improves Soil Productivity in Low Residue Rotation Systems Agron. J., January 8, 2009; 101(1): 207 - 214. [Abstract] [Full Text] [PDF]
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