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Nature of carbohydrates associated with water-stable aggregates of two cultivated soils
PERGAMON
Soil Biology and Biochemistry 31 (1999) 55±63
Nature of carbohydrates associated with water-stable aggregates of two cultivated soils P. Puget a, *, D.A. Angers b, C. Chenu a a Unite de Science du Sol de Versailles, INRA, Route de Saint Cyr, 78026 Versailles, France Centre de Recherches et de DeÂveloppement sur les Sols et les Grandes Cultures, Agriculture et Agroalimentaire Canada, 2560 Bl. Hochelaga, Sainte Foy, GIV 2J3, Canada
b
Accepted 28 June 1998
Abstract The distribution and composition of carbohydrates were investigated in slaking-resistant aggregates and in particle size fractions of two silty soils from the Paris Basin, France. Three hydrolysis procedures, involving hot water, dilute acid and concentrated acid were used and hydrolysates were analysed on a HPLC system to further assess the origin of the carbohydrates. Results showed the predominantly plant origin of the particulate organic matter (>50 mm) and the microbial origin of the clay + silt fraction (<50 mm) carbohydrates. Total organic C content and carbohydrate content, both increased with aggregate size. The proportion of plant-derived carbohydrates also increased with aggregate size, which supported previous results on the distribution of particulate organic matter in aggregates. The clay + silt fraction (<50 mm), located within stable aggregates >50 mm, was enriched in carbohydrates produced by microorganisms. This is consistent with the hypothesis that aggregate stability is mediated partly by the extracellular polysaccharides of microorganisms developing on plant debris occluded within the aggregates. # 1998 Elsevier Science Ltd. All rights reserved. Keywords: Add keywords
1. Introduction Soil organic matter (SOM) has a well established role in the formation and stabilisation of soil aggregates. However, changes in aggregate stability following land use changes have been observed without changes in total SOM content which has led to the conclusion that only a small fraction of SOM was involved (Baldock and Kay, 1987; Haynes and Swift, 1990; Haynes et al., 1991). Extracellular polysaccharides from bacteria or fungi and roots mucilages are typically a very labile SOM fraction which is important as binding agents of soil aggregates (Cheshire, 1979; Oades, 1984). Isolated extracellular carbohydrates have been shown to be ecient in both the formation and stabilisation of soil aggregates (Robert and Chenu, 1992). Moreover, many investigators have found that the amount of carbohydrate in the soils is positively correlated with aggregate stability (Tisdall * Author for correspondence. E-mail: [email protected]. 0038-0717/98/$19.00 # 1998 Elsevier Science Ltd. All rights reserved. PII: S 0 0 3 8 - 0 7 1 7 ( 9 8 ) 0 0 1 0 3 - 5
and Oades, 1982; Angers and Mehuys, 1989; Haynes and Francis, 1993; Tisdall, 1994), though not all (e.g. Degens and Sparling, 1996). Soil carbohydrates may be extracted by numerous methods of which the most complete is, according to Cheshire (1979), the use of hot dilute sulphuric acid with a preliminary treatment with concentrated acid. This method is assumed to hydrolyse the whole polysaccharide fraction including cellulose and, therefore, to re¯ect the total carbohydrate content of most soils. Cheshire (1979) mentioned the potential usefulness of partial hydrolysis in studying the nature of carbohydrates. Hot-dilute acid, and hot-water are, considered not to solubilise or hydrolyse plant structural carbohydrates (Cheshire, 1979; Angers et al., 1988) and should therefore be relatively enriched in nonstructural plant carbohydrates and extracellular microbial carbohydrates. The correlation between hot water extractable carbohydrates and aggregate stability is usually better than that for total carbohydrates (Haynes et al., 1991; Haynes and Francis, 1993) or for
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carbohydrates hydrolysed with dilute acid (Angers et al., 1993). However, statistically signi®cant relationships do not always correspond to direct functional relationships. For instance, Kouakoua et al. (1997) found that hot water extractable C was positively correlated with aggregate stability, but the extraction of material from soil with hot water did not change the water stability of aggregates. Another approach to identify factors responsible for aggregate stability is to compare the composition of dierent size fractions from soil after immersion and sieving in water, i.e. comparing stable vs. unstable aggregates. Stable macroaggregates (>250 mm) contain more organic carbon (Elliott, 1986; Puget et al., 1995), more microbial biomass (Degens et al., 1994), more particulate organic matter (Cambardella and Elliott, 1993; Puget et al., 1996), more labile SOM (Elliott, 1986; Beare et al., 1994) and more young SOM (Puget et al., 1995; Angers and Giroux, 1996) than the microaggregates (<250 mm). However, the composition of aggregate fractions in carbohydrates did not always exhibit clear and consistent trends (Dormaar, 1984; Baldock et al., 1987; Hu et al., 1995). Our study was carried out ®rst to assess the eects of dierent extraction and hydrolysis procedures on the amounts and composition of soil carbohydrates, and second to determine whether water-stable macroaggregates have a carbohydrate signature dierent from the whole soil or from the microaggregate fractions. 2. Materials and methods 2.1. Soil Soil samples were collected from an experimental ®eld of the Institut Technique des CeÂreÂales et des Fourrages (ITCF) located at Boigneville, Essonne, France (48820 0 N, 2820 0 E). The soil was a loess (Typic Hapludalf) with 25% clay and 68% silt. One sample was taken from the 2±3 cm layer of a no-till plot (NT) and the other one was from the 0±30 cm layer of a conventionally tilled plot (T). The two samples were chosen to provide two dierent C contents: 9.5 mg g ÿ 1 for T and 25.1 mg g ÿ 1 for NT. Samples were collected in October 1992, before tillage for the T plot. 2.2. Particle size fractionation A signi®cant portion of the soil carbohydrate fraction is inherited from plants. To identify the carbohydrate signature of plant-derived carbohydrates, we separated plant debris, i.e., particulate organic matter (POM) from the soils using the approach of particle size fractionation developed by Balesdent et al. (1991).
This method, which involves wet-sieving after soil dispersion in water by shaking with glass beads and density fractionation in water, enabled the separation of the POM, de®ned as a light fraction >50 mm, as well as a fraction <50 mm, consisting of particles of clay and silt-size (called hereafter clay + silt fraction). 2.3. Aggregate distribution: Water-stable aggregates Soils were also fractionated into water-stable aggregate size fractions using a wet-sieving method which allowed slaking to occur. Ten g of air dried soil were placed on the top of three stacked sieves (1 mm, 250 and 50 mm) submerged in water and gently wet-sieved using a mechanical agitator for 5 min (Puget et al., 1995). Fractions >0.25 mm are stable macroaggregates whereas <0.25 mm fractions are material deriving either from unstable macroaggregates or original ®ne fraction, which may be microaggregates or particles. All the fractions collected were dried at 608C and weighed. 2.4. Carbohydrate hydrolysis The analysis of carbohydrates requires their prior hydrolysis. This can be performed directly on soil samples or on solutions extracted from soil. We applied three procedures in order to recover and analyse three dierent carbohydrate fractions. A concentrated acid hydrolysis consisted in treating 1 g of ®nely ground soil material with 2 ml of 12 M H2SO4 at room temperature for 16 h with gentle shaking (Cheshire, 1979). This step was followed by dilution with 24 ml of distilled water to obtain a 1 M H2SO4 concentration that was placed in an oven at 1008C for 5 h. A dilute acid hydrolysis involved treating 1 g of ®nely ground soil material with 10 ml of 0.5 M H2SO4 at 808C for 24 h. A hot water extraction was carried out with 1 g of ®nely ground soil material suspended in 10 ml of distilled water at 808C for 24 h. Extracts were hydrolysed by adding H2SO4 to obtain a 0.5 M concentration as in the dilute acid hydrolysis procedure. For the POM fraction, 0.3 to 0.5 g was used. After hydrolysis, the suspensions were centrifuged at 24,500 g for 15 min. About 5 to 7 ml of clear solution was recovered and stored frozen. Before analysis, the solutions were neutralised to pH 7 with barium carbonate. These solutions were centrifuged to remove barium precipitates. 2.5. Liquid chromatography The preparation of samples and their separation by liquid chromatography have been described by Angers et al. (1988). HPLC analyses were performed on a Waters system ®tted with a Bio-Rad Aminex HPX-87P
P. Puget et al. / Soil Biology and Biochemistry 31 (1999) 55±63
column, guard cartridges as pre-column and a refractive index detector. With this HPLC system, we were able to separate ®ve of the eight most abundant monosaccharides commonly present in soils. Angers et al. (1988) have shown that two minor sugars, rhamnose and fucose, co-elute with galactose and arabinose, respectively. Ribose was not detectable. We used the ratio of mannose/xylose (M/X) to assess the relative origin of carbohydrates (SchlechtPietsch et al., 1994; Beare et al., 1997). The proportion of microbially-derived carbohydrates is assumed to increase with increasing M/X ratio. 2.6. Variability and statistical analysis Triplicate hydrolyses were performed on bulk soils, clay + silt fractions (<50 mm) and POM fractions; aggregate fractions were not replicated. The coecient of variation (CV) for total monosaccharides ranged between 2% and 8%. Widest CVs were found for the T sample with a CV of 15%. These results were in accordance with Angers et al. (1988), using the same system. One-way analysis of variance were performed in order to compare the methods of hydrolysis within each soil sample. Means were separated using a Tukey's honest signi®cant dierence mean separation test at p < 0.05. Student t-tests were used to compare the two soils within a same kind of procedure. 3. Results 3.1. Whole soils More carbohydrates were recovered from the bulk soils with an increase in strength of hydrolysis (Table 1). Hot water extracted about 2%, dilute acid 13% and concentrated acid, up to 19% of the total
57
soil C. Carbohydrate contents were higher in the NT than in the T sample for all procedures, but the contribution of carbohydrates to the total soil C content was not signi®cantly dierent between the samples. The distributions of monosaccharide in bulk soil samples varied with the kind of hydrolysis (Fig. 1). In the concentrated acid hydrolysates, glucose was the dominant sugar. Xylose and galactose were the second and third most abundant sugars. In the dilute acid hydrolysates, glucose was not the most abundant sugar and its content was lower than in the concentrated acid hydrolysates. The distributions and contents of the four other sugars were similar for the two types of acid hydrolysis. All individual sugars were more abundant in the NT than in the T sample. The M/X ratio was not signi®cantly dierent (Tukey's HSD at p > 0.05) among hydrolysis procedures for the T, whereas the NT sample exhibited a greater M/X ratio for the hot water extract than for the acid hydrolysates (Table 1). 3.2. Particle size fractions The NT sample had about 5 times more C as POM (73.8 g POM kg ÿ 1 soil) than the T sample (13.1 g POM kg ÿ 1 soil). The clay + silt fraction (<50 mm) of NT sample was also richer in C and N (14.4 mg C g ÿ 1 fraction and 1.48 mg N g ÿ 1 fraction) than the T sample (7.6 mg C g ÿ 1 fraction and 0.82 mg N g ÿ 1 fraction). As for the bulk samples, the amount of carbohydrate extracted from the fractions increased with the acidity of the hydrolysis procedure (Table 2). The POM as well as the clay + silt fractions from the NT were richer in polysaccharides than those from T sample. In the case of the acid hydrolysis, the sum of carbohydrates recovered from POM and clay + silt fractions represented between 90 and 108% of the
Table 1 Carbohydrate-C contents and monosaccharide ratios for the T and NT whole soils and the three hydrolysis procedures. Carbohydrate-C contents are the sum of the ®ve monosaccharides quanti®ed assuming 40% C in the monosaccharides. Results are expressed in mg carbohydrate-C g ÿ 1 soil and as a proportion of the total C content of the whole soil. Means of three replicates Hydrolysis procedure T 0±30 cm H2SO4 12 M/1 M H2SO4 0.5 M hot water NT 2±3 cm H2SO4 12 M/1 M H2SO4 0.5 M hot water
Carbohydrate-C content (mg C g ÿ 1 soil)
Carbohydrate-C/ total C (%)
M/X ratio
1.61 aa 1.32 a 0.19 b
18.36 a 13.41 a 1.81 b
0.65 a 0.65 a 0.50 a
4.69 a*b 3.21 b* 0.61 c*
19.28 a 12.78 b 2.41 c
0.42 a* 0.36 a* 0.75 b*
a Values of the same soil in the same column, following by the same letter are not signi®cantly dierent at p > 0.05 according to Tukey's honest signi®cant dierences mean separation test. b Values of NT soil, followed by * are signi®cantly dierent from corresponding values of T soil at p < 0.05 according to Student t mean comparison test.
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Fig. 1. The distribution of the ®ve monosaccharides in whole soils, clay + silt fraction (<50 mm) and POM fraction (>50 mm), expressed in mg carbohydrate C g ÿ 1 fraction, for T and NT samples and for the three hydrolysis procedures. Means of three replicates (except for the dilute acid hydrolysis of the clay + silt and POM fractions). Values within soil fraction and hydrolysis followed by the same letter are not signi®cantly dierent at p < 0.05 according to Tukey's HSD mean separation test. (T) Triangle indicates a signi®cant dierence between the two acid hydrolysis procedures for a same monosaccharide within the soils (two-tailed Student t-test at p < 0.05).
carbohydrate-C hydrolysed from the bulk soil. However, in the hot water extraction, only 50 to 70% of carbohydrate-C was recovered as compared to the bulk soil sample. We assume that the missing carbohydrates had been solubilised during the POM and clay + silt fraction (<50 mm) separation procedure. The contribution of the POM to the carbohydrates extracted from bulk samples was similar in both acid hydrolyses: 20% and 22% for T sample and 50% and 55% for NT sample. Smaller values were observed for the hot-water extraction. Despite variations in the
recoveries, we can estimate that this contribution was between 9% and 20% for the T and 20±26% for the NT sample. Variations in these values depended upon the POM carbohydrates being expressed as a percentage of the carbohydrates analysed from the whole soils or as a percentage of the sum of carbohydrates in POM and clay + silt fraction. Concentrated acid hydrolysis yielded a large proportion of glucose (50±60% of the sum of the ®ve monosaccharides) and of xylose (12±25%) in the POM fraction (Fig. 1). Diluted acid yielded much less glu-
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Table 2 Carbohydrate-C contents and monosaccharide ratios for the clay + silt fractions and the particulate organic matter fractions, separated after complete dispersion of the T and NT whole soils, and for the three hydrolysis procedures. Results are expressed in mg carbohydrate-C g ÿ 1 of fraction and as a proportion of the total C content of the fraction. Means of three replicates (2 standard deviations) for the concentrated acid hydrolysis and the hot-water extraction Hydrolysis procedure T 0±30 cm H2SO4 12 M/1 M H2SO4 0.5 M Hot water
NT 2±3 cm H2SO4 12 M/1 M H2SO4 0.5 M Hot water
Soil fractions
Carbohydrate-C Content (mg C g ÿ 1 fraction)
Carbohydrate-C/ total C (%)
M/X ratio
Clay + silt <50 mm POM Clay + silt <50 mm POM Clay + silt <50 mm POM
1.37 (20.01) 24.93 (20.80) 1.00 20.90 0.07 (20.01) 1.22 (20.06)
18.17 16.06 13.22 13.46 0.90 0.78
1.16 0.56 1.18 0.14 0.40 0.34
(20.04) (20.04)
Clay + silt <50 mm POM Clay + silt <50 mm POM Clay + silt <50 mm POM
2.63 (20.04)*a 48.34 (20.80)* 2.01 30.66 0.35 (20.02)* 2.07 (20.02)*
18.53 25.41* 14.15 16.12 2.47* 1.09*
0.97 0.21 0.94 0.17 0.82 0.43
(20.053)* (20.00)*
(20.05) (20.03)
(20.06)* (20.04)
a Values of NT soil following by * are signi®cantly dierent from corresponding values of T soil at p < 0.05 according to Student t mean comparison test.
cose but slightly higher amounts of the other sugars especially xylose from the T sample. Glucose was the dominant sugar in the hot-water and concentrated acid extracts, whereas xylose was the most abundant one in the dilute acid extracts. Hydrolysates from POM fractions were in all cases characterised by a much smaller M/X ratio (Table 2) than the bulk samples (Table 1). More monosaccharides were extracted from POM of the NT sample as compared to the T sample, but for the three extractants their monosaccharide distribution was generally similar. In the hydrolysates of the clay + silt fractions (<50 mm), glucose was the dominant sugar followed by galactose (Fig. 1). The M/X ratio of the clay + silt
fraction (<50 mm) was larger than for whole soils or POM fractions (Tables 1 and 2), except for the hot water extracts. 3.3. Aggregate distribution: Water-stable aggregates As reported by Puget et al. (1995), the aggregate distributions showed that 80% of the soil was in aggregates >50 mm (Fig. 2). The NT sample exhibited a slightly greater proportion of aggregates >1 mm than the T sample. The C content of aggregate fractions increased with size as also reported by Puget et al. (1995). Total carbohydrate-C contents also increased with aggregate size for the three types of hydrolysis
Fig. 2. Water-stable aggregate size distributions obtained after slaking and wet sieving of T and NT soils. Mean (2standard deviations) of eight to ten replicates. (*) Asterisk indicates a signi®cant dierence between soils within aggregate fractions (two-tailed Student t-test at p < 0.05).
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Table 3 Carbohydrate-L contents and monosaccharide ratios mannose/xylose for the aggregate fractions obtained after wet sieving of T and NT soils and for the three hydrolysis procedures. Results are expressed in mg carbohydrate-L g ÿ 1 of aggregate fraction and as a proportion of the total L content of the aggregate fraction Hydrolysis procedure (%)
Aggregate size mm
H2SO4 12 M/1 M
T 0±30 cm
NT 2±3 cm
Carbohydrate-L content (mg L g ÿ 1 aggregate)
Carbohydrate-C/ L aggregate (%)
M/X ratio
Carbohydrate-C content (mg L g ÿ 1 aggregate)
Carbohydrate-C/ L aggregate (%)
M/X ratio
>1 0.250±1 0.05±0.250 < 0.05
4.94 1.68 1.28 1.05
21.34 16.85 16.55 19.06
0.29 0.68 0.76 0.91
9.01 5.87 3.73 1.31
24.29 20.44 19.48 16.86
0.31 0.37 0.55 0.63
H2SO4 0.5 M
>1 0.250±1 0.05±0.250 < 0.05
3.00 1.26 0.92 0.72
12.96 12.58 11.85 13.02
0.18 0.55 0.76 1.24
4.82 3.96 2.27 1.08
13.00 13.77 11.87 13.91
0.19 0.28 0.43 0.83
Hot water
>1 0.250±1 0.05±0.250 < 0.05
0.27 0.16 0.15 0.08
1.17 1.58 1.92 1.41
0.46 1.03 0.45 0.19
1.65 1.06 0.44 0.20
4.46 3.69 2.28 4.33
0.30 0.36 0.79 0.53
(Table 3). However, carbohydrates represented nearly the same percentage of total C in all aggregate fractions. The composition of the acid-hydrolysable carbohydrates varied with aggregate size (Fig. 3). In concentrated-acid hydrolysates, the proportion of glucose and xylose decreased and that of galactose increased as aggregate size decreased. In dilute acid hydrolysates, the most striking feature was the decrease in xylose and in hot-water extracts, the increase of galactose with decreasing aggregate size. These trends were con®rmed by the increasing M/X ratio with decreasing aggregate size, for the two acid hydrolyses (Table 3).
4. Discussion 4.1. Methods for carbohydrate extraction and hydrolysis and their signi®cance Three fractions of carbohydrates based on dierent hydrolysis procedures were compared in our study. As expected the amounts of carbohydrates recovered increased with the concentration of hydrolysis. Our results con®rmed that concentrated acid hydrolysed the cellulosic components, thereby releasing large amounts of glucose (Cheshire, 1979), whereas dilute acid did not hydrolyse cellulose but probably hydrolysed the hemicellulose leading to the greater release of xylose. The prior-treatment with 12 M H2SO4 may
cause degradation of some pentoses, such as xylose (Cheshire, 1979). The comparison of the dilute and concentrated acid hydrolysis on POM are consistent with this statement (Fig. 1). However, a complete account of pentoses with 12 M H2SO4 was not obtained for whole soil samples, or for clay + silt fractions (<50 mm) (Fig. 1). Hence, concentrated acid appears to provide a valuable estimate of total carbohydrates and dilute hydrolysis a good estimate for non-cellulosic carbohydrates. Hot water extracted about 2% of the C which represented about 10% of the total carbohydrates. Based on the observation that hot water extracts showed high sugar ratios ((G + M)/(X + A) > 2), Haynes and Francis (1993) assumed that these were mostly composed of extracellular microbial polysaccharides. Likewise, Feller et al. (1991) combined hot water extractions and transmission electron microscopy observations to show that the clay fractions from ferralitic soils were rich in amorphous polysaccharide material, with a morphology typical of that of microbial and plant exudates. This polysaccharide material was partly extracted with hot water and the extracts had high M/X ratios. In our study, the M/X ratio of hot water extracts was higher than that of other polysaccharide fractions only in the NT sample, presumably because of an abundant microbial biomass in this soil. The carbohydrate signatures showed that polysaccharides extractable by hot-water are of both
P. Puget et al. / Soil Biology and Biochemistry 31 (1999) 55±63
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Fig. 3. The distribution of the ®ve monosaccharides in the water-stable aggregate fractions of T and NT samples, expressed as a percentage of the sum of the ®ve monosaccharides.
microbial and plant origin. Root mucigels are soluble in hot water and may contribute to this fraction. The carbohydrate signature of the POM fractions compared with the clay + silt fractions showed, as expected, that the former contained a larger proportion of plant-derived carbohydrates (Angers and Mehuys, 1989; Guggenberger et al., 1990). POM accounted for 20% to 55% of the acid-hydrolysable carbohydrates of bulk soil and a lesser proportion (10% to 20%) of the hot water-soluble carbohydrates.
4.2. Carbohydrate composition of aggregate fractions Wet sieving allows soil aggregates to be separated on the basis of stability in water. There was a clear relationship between the size of aggregates and their polysaccharide content for both soils and for total, non-cellulosic and hot-water soluble carbohydrates. However, total carbohydrates always represented the same proportion of the total organic C of the fractions. Similar results were reported by Baldock et al.
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Table 4 Carbohydrate-L contents and monosaccharide ratios mannose/xylose for the clay + silt fraction located within the water-stable aggregates >50 mm of T and NT samples, and for the three hydrolysis procedures. Results are expressed in mg carbohydrate-L g ÿ 1 of fraction and as a proportion of the total L content of the fraction. Values are obtained after calculation, by subtracting the amounts of carbohydrates associated with the aggregate fraction <50 mm recovered after aggregate wet-sieving, from the carbohydrate values of the clay + silt fraction obtained after complete dispersion of the whole soil Carbohydrate-L content (mg L g ÿ 1 fraction)
Carbohydrate-C/ total L (%)
M/X ratio
T 0±30 cm H2SO4 12 M/1 M H2SO4 0.5 M hot water
1.22 0.89 0.06
14.56 10.69 0.68
1.19 1.18 0.43
NT 2±3 cm H2SO4 12 M/1 M H2SO4 0.5 M Hot water
2.39 1.81 0.31
15.21 11.53 2.00
1.00 0.95 0.86
(1987), whereas Dormaar (1984) showed an enrichment in carbohydrates of the C with aggregate size at least for two soils. The nature of the carbohydrates, however, was not the same among the fractions. Monosaccharides indicating a POM signature decreased with decreasing aggregate size, consistent with the data of Baldock et al. (1987) and Hu et al. (1995). In a previous study, we have shown that stable aggregates were enriched in POM as compared with unstable ones (P. Puget, unpublished). Oades (1984) and Cambardella and Elliott (1993) suggested that POM was partly responsible for macroaggregate stability. Indeed POM may have an indirect role as an energy source for microorganisms, which can contribute to binding and stabilising aggregates, through physical enmeshment by hyphae and through binding by extracellular polysaccharides (e.g. Robert and Chenu, 1992; Dorioz et al., 1993; Tisdall, 1994). We can compare the carbohydrate contents and ratios of the clay + silt fraction obtained after complete dispersion of whole soil (Table 2) with those of the aggregate fraction <50 mm recovered after aggregate wet sieving (Table 3). The latter corresponds to clay and silt particles free in the soil, to free microaggregates or to clay and silt particles or microaggregates released from unstable aggregates upon slaking. This fraction was depleted in carbohydrates and its M/X ratio was also low compared to the clay + silt fraction separated from the whole soil (Table 3). This suggested that the clay + silt fraction (<50 mm) present in stable macroaggregates was enriched in polysaccharides of microbial origin compared to unstable ones. We esti-
mated the composition of clay + silt fractions located within stable aggregates >50 mm by dierence (Table 4). As might be inferred, these fractions are enriched in carbohydrates and in the case of NT soil, had a high M/X ratio suggesting a large proportion of microbially-derived carbohydrates. This fact is consistent with the studies of Roberson et al. (1995) showing a strong correlation of mineral-associated carbohydrates, which have a microbial signature, with aggregate stability. Cambardella and Elliott (1994) also found that the water-stable macroaggregates from cultivated grassland soils contain a silt-size SOM fraction, very enriched in labile compounds, and suggested that this enriched labile fraction (ELF) would be a byproduct of microbial activity and contribute to the macroaggregate stability.
Acknowledgements We thank Dr P. Nadeau and G. Devarennes for their contribution and assistance during HPLC determinations and C. Picot for preliminary assays of carbohydrate hydrolysis. C. Feller is acknowledged for his comments on the manuscript. This study is part of the Agriculture Agri-Food Canada-INRA Collaborative Research Program.
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P. Puget et al. / Soil Biology and Biochemistry 31 (1999) 55±63 Beare, M.H., Hu, S., Coleman, D.C., Hendrix, P.F., 1997. In¯uence of mycelial fungi on soil aggregation and organic matter storage in conventional and no-tillage soils. Applied Soil Ecology 5, 211±219. Cambardella, C.A., Elliott, E.T., 1993. Carbon and nitrogen distribution in aggregates from cultivated and native grassland soils. Soil Biology&Biochemistry 57, 1071±1076. Cambardella, C.A., Elliott, E.T., 1994. Carbon and nitrogen dynamics of soil organic matter fractions from cultivated grassland soils. Soil Science Society of America Journal 58, 123±130. Cheshire, M. V. (1979) Nature and Origin of Carbohydrates in Soils, Academic Press, London. Degens, B., Sparling, G., 1996. Changes in aggregation do not correspond with changes in labile organic C fractions in soil amended with C-14-glucose. Soil Biology & Biochemistry 28, 453±462. Degens, B.P., Sparling, G.P., Abbott, L.K., 1994. The contribution from hyphae, roots and organic carbon constituents to the aggregation of a sandy loam under long term clover-based and grass pastures. European Journal of Soil Science 45, 459±468. Dorioz, J.M., Robert, M., Chenu, C., 1993. The role of roots, fungi and bacteria, on clay particle organization. Geoderma 56, 179± 194. Dormaar, J.F., 1984. Monosaccharides in hydrolysates of waterstable aggregates after 67 years of cropping to spring wheat as determined by capillary gas chromatography. Canadian Journal of Soil Science 64, 647±656. Elliott, E.T., 1986. Aggregate structure and carbon, nitrogen and phosphorus in native and cultivated soils. Soil Science Society of America Journal 50, 627±633. Feller, C., Franc° ois, C., Villemin, G., Portal, J.M., Toutain, F., Morel, J.L., 1991. Nature des matieÁres organiques associeÂes aux fractions argileuses d'un sol ferrallitique. Comptes Rendus de l'AcadeÂmie des Sciences de Paris 312, 1491±1497. Guggenberger, G., Christensen, B.T., Zech, W., 1990. Land-use eects on the composition of organic matter in particle-size separates of soil: I. Lignin and carbohydrate signature. European Journal of Soil Science 45, 449±458. Haynes, R.J., Francis, G.S., 1993. Changes in microbial biomass C, soil carbohydrate composition and aggregate stability induced by growth of selected crop and forage species under ®eld conditions. Journal of Soil Science 44, 665±675.
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Haynes, R.J., Swift, R.S., 1990. Stability of soil aggregates in relation to organic constituents and soil water content. Journal of Soil Science 41, 73±83. Haynes, R.J., Swift, R.S., Stephen, R.C., 1991. In¯uence of mixed cropping rotations (pasture-arable) on organic matter content, stable aggregation and clod porosity in a group of soils. Soil and Tillage Research 19, 77±87. Hu, S., Coleman, D.C., Beare, M.H., Hendrix, P.F., 1995. Soil carbohydrates in aggrading and degrading agroecosystems: in¯uences of fungi and aggregates. Agriculture Ecosystems and Environment 54, 77±88. Kouakoua, E., Sala, G.H., BartheÁs, B., Larre-Larrouy, M.C., Albrecht, A., Feller, C., 1997. La matieÁre organique soluble aÁ l'eau chaude et la stabilite de l'agreÂgation. Aspects meÂthodologiques et application aÁ des sols ferrallitiques du Congo. European Journal of Soil Science 48, 239±247. Oades, J.M., 1984. Soil organic matter and structural stability mechanisms and implications for management. Plant and Soil 76, 319± 337. Puget, P., Chenu, C., Balesdent, J., 1995. Total and young organic matter distributions in aggregates of silty cultivated soils. European Journal of Soil Science 46, 449±459. Puget, P., Besnard, E., Chenu, C., 1996. Une meÂthode de fractionnement des matieÁres organiques particulaires des sols en fonction de leur localisation dans les agreÂgats. Comptes Rendus de l'AcadeÂmie des Sciences de Paris 322, 965±972. Roberson, E.B., Sarig, S., Shennan, C., Firestone, M.K., 1995. Nutritional management of microbial polysaccharide production and aggregation in an agricultural soil. Soil Science Society of America Journal 59, 1587±1594. Robert, M., Chenu, C., 1992. Interactions between soil minerals and microorganisms. In Soil Biochemistry, 7, Eds. Bollag, J. M., Stotzky, G., pp. 307±393. Marcel Dekker, New York. Schlecht-Pietsch, S., Wagner, U., Anderson, T.H., 1994. Changes in composition of soil polysaccharides and aggregate stability after carbon amendments to dierent textured soils. Applied Soil Ecology 1, 145±154. Tisdall, J. M., 1994. Possible role of soil microorganisms in aggregation in soils. 159, 115±121. Tisdall, J.M., Oades, J.M., 1982. Organic matter and water-stable aggregates. Journal of Soil Science 33, 141±163.
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Nature of carbohydrates associated with water-stable aggregates of two cultivated soils P. Puget a, *, D.A. Angers b, C. Chenu a a Unite de Science du Sol de Versailles, INRA, Route de Saint Cyr, 78026 Versailles, France Centre de Recherches et de DeÂveloppement sur les Sols et les Grandes Cultures, Agriculture et Agroalimentaire Canada, 2560 Bl. Hochelaga, Sainte Foy, GIV 2J3, Canada
b
Accepted 28 June 1998
Abstract The distribution and composition of carbohydrates were investigated in slaking-resistant aggregates and in particle size fractions of two silty soils from the Paris Basin, France. Three hydrolysis procedures, involving hot water, dilute acid and concentrated acid were used and hydrolysates were analysed on a HPLC system to further assess the origin of the carbohydrates. Results showed the predominantly plant origin of the particulate organic matter (>50 mm) and the microbial origin of the clay + silt fraction (<50 mm) carbohydrates. Total organic C content and carbohydrate content, both increased with aggregate size. The proportion of plant-derived carbohydrates also increased with aggregate size, which supported previous results on the distribution of particulate organic matter in aggregates. The clay + silt fraction (<50 mm), located within stable aggregates >50 mm, was enriched in carbohydrates produced by microorganisms. This is consistent with the hypothesis that aggregate stability is mediated partly by the extracellular polysaccharides of microorganisms developing on plant debris occluded within the aggregates. # 1998 Elsevier Science Ltd. All rights reserved. Keywords: Add keywords
1. Introduction Soil organic matter (SOM) has a well established role in the formation and stabilisation of soil aggregates. However, changes in aggregate stability following land use changes have been observed without changes in total SOM content which has led to the conclusion that only a small fraction of SOM was involved (Baldock and Kay, 1987; Haynes and Swift, 1990; Haynes et al., 1991). Extracellular polysaccharides from bacteria or fungi and roots mucilages are typically a very labile SOM fraction which is important as binding agents of soil aggregates (Cheshire, 1979; Oades, 1984). Isolated extracellular carbohydrates have been shown to be ecient in both the formation and stabilisation of soil aggregates (Robert and Chenu, 1992). Moreover, many investigators have found that the amount of carbohydrate in the soils is positively correlated with aggregate stability (Tisdall * Author for correspondence. E-mail: [email protected]. 0038-0717/98/$19.00 # 1998 Elsevier Science Ltd. All rights reserved. PII: S 0 0 3 8 - 0 7 1 7 ( 9 8 ) 0 0 1 0 3 - 5
and Oades, 1982; Angers and Mehuys, 1989; Haynes and Francis, 1993; Tisdall, 1994), though not all (e.g. Degens and Sparling, 1996). Soil carbohydrates may be extracted by numerous methods of which the most complete is, according to Cheshire (1979), the use of hot dilute sulphuric acid with a preliminary treatment with concentrated acid. This method is assumed to hydrolyse the whole polysaccharide fraction including cellulose and, therefore, to re¯ect the total carbohydrate content of most soils. Cheshire (1979) mentioned the potential usefulness of partial hydrolysis in studying the nature of carbohydrates. Hot-dilute acid, and hot-water are, considered not to solubilise or hydrolyse plant structural carbohydrates (Cheshire, 1979; Angers et al., 1988) and should therefore be relatively enriched in nonstructural plant carbohydrates and extracellular microbial carbohydrates. The correlation between hot water extractable carbohydrates and aggregate stability is usually better than that for total carbohydrates (Haynes et al., 1991; Haynes and Francis, 1993) or for
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P. Puget et al. / Soil Biology and Biochemistry 31 (1999) 55±63
carbohydrates hydrolysed with dilute acid (Angers et al., 1993). However, statistically signi®cant relationships do not always correspond to direct functional relationships. For instance, Kouakoua et al. (1997) found that hot water extractable C was positively correlated with aggregate stability, but the extraction of material from soil with hot water did not change the water stability of aggregates. Another approach to identify factors responsible for aggregate stability is to compare the composition of dierent size fractions from soil after immersion and sieving in water, i.e. comparing stable vs. unstable aggregates. Stable macroaggregates (>250 mm) contain more organic carbon (Elliott, 1986; Puget et al., 1995), more microbial biomass (Degens et al., 1994), more particulate organic matter (Cambardella and Elliott, 1993; Puget et al., 1996), more labile SOM (Elliott, 1986; Beare et al., 1994) and more young SOM (Puget et al., 1995; Angers and Giroux, 1996) than the microaggregates (<250 mm). However, the composition of aggregate fractions in carbohydrates did not always exhibit clear and consistent trends (Dormaar, 1984; Baldock et al., 1987; Hu et al., 1995). Our study was carried out ®rst to assess the eects of dierent extraction and hydrolysis procedures on the amounts and composition of soil carbohydrates, and second to determine whether water-stable macroaggregates have a carbohydrate signature dierent from the whole soil or from the microaggregate fractions. 2. Materials and methods 2.1. Soil Soil samples were collected from an experimental ®eld of the Institut Technique des CeÂreÂales et des Fourrages (ITCF) located at Boigneville, Essonne, France (48820 0 N, 2820 0 E). The soil was a loess (Typic Hapludalf) with 25% clay and 68% silt. One sample was taken from the 2±3 cm layer of a no-till plot (NT) and the other one was from the 0±30 cm layer of a conventionally tilled plot (T). The two samples were chosen to provide two dierent C contents: 9.5 mg g ÿ 1 for T and 25.1 mg g ÿ 1 for NT. Samples were collected in October 1992, before tillage for the T plot. 2.2. Particle size fractionation A signi®cant portion of the soil carbohydrate fraction is inherited from plants. To identify the carbohydrate signature of plant-derived carbohydrates, we separated plant debris, i.e., particulate organic matter (POM) from the soils using the approach of particle size fractionation developed by Balesdent et al. (1991).
This method, which involves wet-sieving after soil dispersion in water by shaking with glass beads and density fractionation in water, enabled the separation of the POM, de®ned as a light fraction >50 mm, as well as a fraction <50 mm, consisting of particles of clay and silt-size (called hereafter clay + silt fraction). 2.3. Aggregate distribution: Water-stable aggregates Soils were also fractionated into water-stable aggregate size fractions using a wet-sieving method which allowed slaking to occur. Ten g of air dried soil were placed on the top of three stacked sieves (1 mm, 250 and 50 mm) submerged in water and gently wet-sieved using a mechanical agitator for 5 min (Puget et al., 1995). Fractions >0.25 mm are stable macroaggregates whereas <0.25 mm fractions are material deriving either from unstable macroaggregates or original ®ne fraction, which may be microaggregates or particles. All the fractions collected were dried at 608C and weighed. 2.4. Carbohydrate hydrolysis The analysis of carbohydrates requires their prior hydrolysis. This can be performed directly on soil samples or on solutions extracted from soil. We applied three procedures in order to recover and analyse three dierent carbohydrate fractions. A concentrated acid hydrolysis consisted in treating 1 g of ®nely ground soil material with 2 ml of 12 M H2SO4 at room temperature for 16 h with gentle shaking (Cheshire, 1979). This step was followed by dilution with 24 ml of distilled water to obtain a 1 M H2SO4 concentration that was placed in an oven at 1008C for 5 h. A dilute acid hydrolysis involved treating 1 g of ®nely ground soil material with 10 ml of 0.5 M H2SO4 at 808C for 24 h. A hot water extraction was carried out with 1 g of ®nely ground soil material suspended in 10 ml of distilled water at 808C for 24 h. Extracts were hydrolysed by adding H2SO4 to obtain a 0.5 M concentration as in the dilute acid hydrolysis procedure. For the POM fraction, 0.3 to 0.5 g was used. After hydrolysis, the suspensions were centrifuged at 24,500 g for 15 min. About 5 to 7 ml of clear solution was recovered and stored frozen. Before analysis, the solutions were neutralised to pH 7 with barium carbonate. These solutions were centrifuged to remove barium precipitates. 2.5. Liquid chromatography The preparation of samples and their separation by liquid chromatography have been described by Angers et al. (1988). HPLC analyses were performed on a Waters system ®tted with a Bio-Rad Aminex HPX-87P
P. Puget et al. / Soil Biology and Biochemistry 31 (1999) 55±63
column, guard cartridges as pre-column and a refractive index detector. With this HPLC system, we were able to separate ®ve of the eight most abundant monosaccharides commonly present in soils. Angers et al. (1988) have shown that two minor sugars, rhamnose and fucose, co-elute with galactose and arabinose, respectively. Ribose was not detectable. We used the ratio of mannose/xylose (M/X) to assess the relative origin of carbohydrates (SchlechtPietsch et al., 1994; Beare et al., 1997). The proportion of microbially-derived carbohydrates is assumed to increase with increasing M/X ratio. 2.6. Variability and statistical analysis Triplicate hydrolyses were performed on bulk soils, clay + silt fractions (<50 mm) and POM fractions; aggregate fractions were not replicated. The coecient of variation (CV) for total monosaccharides ranged between 2% and 8%. Widest CVs were found for the T sample with a CV of 15%. These results were in accordance with Angers et al. (1988), using the same system. One-way analysis of variance were performed in order to compare the methods of hydrolysis within each soil sample. Means were separated using a Tukey's honest signi®cant dierence mean separation test at p < 0.05. Student t-tests were used to compare the two soils within a same kind of procedure. 3. Results 3.1. Whole soils More carbohydrates were recovered from the bulk soils with an increase in strength of hydrolysis (Table 1). Hot water extracted about 2%, dilute acid 13% and concentrated acid, up to 19% of the total
57
soil C. Carbohydrate contents were higher in the NT than in the T sample for all procedures, but the contribution of carbohydrates to the total soil C content was not signi®cantly dierent between the samples. The distributions of monosaccharide in bulk soil samples varied with the kind of hydrolysis (Fig. 1). In the concentrated acid hydrolysates, glucose was the dominant sugar. Xylose and galactose were the second and third most abundant sugars. In the dilute acid hydrolysates, glucose was not the most abundant sugar and its content was lower than in the concentrated acid hydrolysates. The distributions and contents of the four other sugars were similar for the two types of acid hydrolysis. All individual sugars were more abundant in the NT than in the T sample. The M/X ratio was not signi®cantly dierent (Tukey's HSD at p > 0.05) among hydrolysis procedures for the T, whereas the NT sample exhibited a greater M/X ratio for the hot water extract than for the acid hydrolysates (Table 1). 3.2. Particle size fractions The NT sample had about 5 times more C as POM (73.8 g POM kg ÿ 1 soil) than the T sample (13.1 g POM kg ÿ 1 soil). The clay + silt fraction (<50 mm) of NT sample was also richer in C and N (14.4 mg C g ÿ 1 fraction and 1.48 mg N g ÿ 1 fraction) than the T sample (7.6 mg C g ÿ 1 fraction and 0.82 mg N g ÿ 1 fraction). As for the bulk samples, the amount of carbohydrate extracted from the fractions increased with the acidity of the hydrolysis procedure (Table 2). The POM as well as the clay + silt fractions from the NT were richer in polysaccharides than those from T sample. In the case of the acid hydrolysis, the sum of carbohydrates recovered from POM and clay + silt fractions represented between 90 and 108% of the
Table 1 Carbohydrate-C contents and monosaccharide ratios for the T and NT whole soils and the three hydrolysis procedures. Carbohydrate-C contents are the sum of the ®ve monosaccharides quanti®ed assuming 40% C in the monosaccharides. Results are expressed in mg carbohydrate-C g ÿ 1 soil and as a proportion of the total C content of the whole soil. Means of three replicates Hydrolysis procedure T 0±30 cm H2SO4 12 M/1 M H2SO4 0.5 M hot water NT 2±3 cm H2SO4 12 M/1 M H2SO4 0.5 M hot water
Carbohydrate-C content (mg C g ÿ 1 soil)
Carbohydrate-C/ total C (%)
M/X ratio
1.61 aa 1.32 a 0.19 b
18.36 a 13.41 a 1.81 b
0.65 a 0.65 a 0.50 a
4.69 a*b 3.21 b* 0.61 c*
19.28 a 12.78 b 2.41 c
0.42 a* 0.36 a* 0.75 b*
a Values of the same soil in the same column, following by the same letter are not signi®cantly dierent at p > 0.05 according to Tukey's honest signi®cant dierences mean separation test. b Values of NT soil, followed by * are signi®cantly dierent from corresponding values of T soil at p < 0.05 according to Student t mean comparison test.
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P. Puget et al. / Soil Biology and Biochemistry 31 (1999) 55±63
Fig. 1. The distribution of the ®ve monosaccharides in whole soils, clay + silt fraction (<50 mm) and POM fraction (>50 mm), expressed in mg carbohydrate C g ÿ 1 fraction, for T and NT samples and for the three hydrolysis procedures. Means of three replicates (except for the dilute acid hydrolysis of the clay + silt and POM fractions). Values within soil fraction and hydrolysis followed by the same letter are not signi®cantly dierent at p < 0.05 according to Tukey's HSD mean separation test. (T) Triangle indicates a signi®cant dierence between the two acid hydrolysis procedures for a same monosaccharide within the soils (two-tailed Student t-test at p < 0.05).
carbohydrate-C hydrolysed from the bulk soil. However, in the hot water extraction, only 50 to 70% of carbohydrate-C was recovered as compared to the bulk soil sample. We assume that the missing carbohydrates had been solubilised during the POM and clay + silt fraction (<50 mm) separation procedure. The contribution of the POM to the carbohydrates extracted from bulk samples was similar in both acid hydrolyses: 20% and 22% for T sample and 50% and 55% for NT sample. Smaller values were observed for the hot-water extraction. Despite variations in the
recoveries, we can estimate that this contribution was between 9% and 20% for the T and 20±26% for the NT sample. Variations in these values depended upon the POM carbohydrates being expressed as a percentage of the carbohydrates analysed from the whole soils or as a percentage of the sum of carbohydrates in POM and clay + silt fraction. Concentrated acid hydrolysis yielded a large proportion of glucose (50±60% of the sum of the ®ve monosaccharides) and of xylose (12±25%) in the POM fraction (Fig. 1). Diluted acid yielded much less glu-
P. Puget et al. / Soil Biology and Biochemistry 31 (1999) 55±63
59
Table 2 Carbohydrate-C contents and monosaccharide ratios for the clay + silt fractions and the particulate organic matter fractions, separated after complete dispersion of the T and NT whole soils, and for the three hydrolysis procedures. Results are expressed in mg carbohydrate-C g ÿ 1 of fraction and as a proportion of the total C content of the fraction. Means of three replicates (2 standard deviations) for the concentrated acid hydrolysis and the hot-water extraction Hydrolysis procedure T 0±30 cm H2SO4 12 M/1 M H2SO4 0.5 M Hot water
NT 2±3 cm H2SO4 12 M/1 M H2SO4 0.5 M Hot water
Soil fractions
Carbohydrate-C Content (mg C g ÿ 1 fraction)
Carbohydrate-C/ total C (%)
M/X ratio
Clay + silt <50 mm POM Clay + silt <50 mm POM Clay + silt <50 mm POM
1.37 (20.01) 24.93 (20.80) 1.00 20.90 0.07 (20.01) 1.22 (20.06)
18.17 16.06 13.22 13.46 0.90 0.78
1.16 0.56 1.18 0.14 0.40 0.34
(20.04) (20.04)
Clay + silt <50 mm POM Clay + silt <50 mm POM Clay + silt <50 mm POM
2.63 (20.04)*a 48.34 (20.80)* 2.01 30.66 0.35 (20.02)* 2.07 (20.02)*
18.53 25.41* 14.15 16.12 2.47* 1.09*
0.97 0.21 0.94 0.17 0.82 0.43
(20.053)* (20.00)*
(20.05) (20.03)
(20.06)* (20.04)
a Values of NT soil following by * are signi®cantly dierent from corresponding values of T soil at p < 0.05 according to Student t mean comparison test.
cose but slightly higher amounts of the other sugars especially xylose from the T sample. Glucose was the dominant sugar in the hot-water and concentrated acid extracts, whereas xylose was the most abundant one in the dilute acid extracts. Hydrolysates from POM fractions were in all cases characterised by a much smaller M/X ratio (Table 2) than the bulk samples (Table 1). More monosaccharides were extracted from POM of the NT sample as compared to the T sample, but for the three extractants their monosaccharide distribution was generally similar. In the hydrolysates of the clay + silt fractions (<50 mm), glucose was the dominant sugar followed by galactose (Fig. 1). The M/X ratio of the clay + silt
fraction (<50 mm) was larger than for whole soils or POM fractions (Tables 1 and 2), except for the hot water extracts. 3.3. Aggregate distribution: Water-stable aggregates As reported by Puget et al. (1995), the aggregate distributions showed that 80% of the soil was in aggregates >50 mm (Fig. 2). The NT sample exhibited a slightly greater proportion of aggregates >1 mm than the T sample. The C content of aggregate fractions increased with size as also reported by Puget et al. (1995). Total carbohydrate-C contents also increased with aggregate size for the three types of hydrolysis
Fig. 2. Water-stable aggregate size distributions obtained after slaking and wet sieving of T and NT soils. Mean (2standard deviations) of eight to ten replicates. (*) Asterisk indicates a signi®cant dierence between soils within aggregate fractions (two-tailed Student t-test at p < 0.05).
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P. Puget et al. / Soil Biology and Biochemistry 31 (1999) 55±63
Table 3 Carbohydrate-L contents and monosaccharide ratios mannose/xylose for the aggregate fractions obtained after wet sieving of T and NT soils and for the three hydrolysis procedures. Results are expressed in mg carbohydrate-L g ÿ 1 of aggregate fraction and as a proportion of the total L content of the aggregate fraction Hydrolysis procedure (%)
Aggregate size mm
H2SO4 12 M/1 M
T 0±30 cm
NT 2±3 cm
Carbohydrate-L content (mg L g ÿ 1 aggregate)
Carbohydrate-C/ L aggregate (%)
M/X ratio
Carbohydrate-C content (mg L g ÿ 1 aggregate)
Carbohydrate-C/ L aggregate (%)
M/X ratio
>1 0.250±1 0.05±0.250 < 0.05
4.94 1.68 1.28 1.05
21.34 16.85 16.55 19.06
0.29 0.68 0.76 0.91
9.01 5.87 3.73 1.31
24.29 20.44 19.48 16.86
0.31 0.37 0.55 0.63
H2SO4 0.5 M
>1 0.250±1 0.05±0.250 < 0.05
3.00 1.26 0.92 0.72
12.96 12.58 11.85 13.02
0.18 0.55 0.76 1.24
4.82 3.96 2.27 1.08
13.00 13.77 11.87 13.91
0.19 0.28 0.43 0.83
Hot water
>1 0.250±1 0.05±0.250 < 0.05
0.27 0.16 0.15 0.08
1.17 1.58 1.92 1.41
0.46 1.03 0.45 0.19
1.65 1.06 0.44 0.20
4.46 3.69 2.28 4.33
0.30 0.36 0.79 0.53
(Table 3). However, carbohydrates represented nearly the same percentage of total C in all aggregate fractions. The composition of the acid-hydrolysable carbohydrates varied with aggregate size (Fig. 3). In concentrated-acid hydrolysates, the proportion of glucose and xylose decreased and that of galactose increased as aggregate size decreased. In dilute acid hydrolysates, the most striking feature was the decrease in xylose and in hot-water extracts, the increase of galactose with decreasing aggregate size. These trends were con®rmed by the increasing M/X ratio with decreasing aggregate size, for the two acid hydrolyses (Table 3).
4. Discussion 4.1. Methods for carbohydrate extraction and hydrolysis and their signi®cance Three fractions of carbohydrates based on dierent hydrolysis procedures were compared in our study. As expected the amounts of carbohydrates recovered increased with the concentration of hydrolysis. Our results con®rmed that concentrated acid hydrolysed the cellulosic components, thereby releasing large amounts of glucose (Cheshire, 1979), whereas dilute acid did not hydrolyse cellulose but probably hydrolysed the hemicellulose leading to the greater release of xylose. The prior-treatment with 12 M H2SO4 may
cause degradation of some pentoses, such as xylose (Cheshire, 1979). The comparison of the dilute and concentrated acid hydrolysis on POM are consistent with this statement (Fig. 1). However, a complete account of pentoses with 12 M H2SO4 was not obtained for whole soil samples, or for clay + silt fractions (<50 mm) (Fig. 1). Hence, concentrated acid appears to provide a valuable estimate of total carbohydrates and dilute hydrolysis a good estimate for non-cellulosic carbohydrates. Hot water extracted about 2% of the C which represented about 10% of the total carbohydrates. Based on the observation that hot water extracts showed high sugar ratios ((G + M)/(X + A) > 2), Haynes and Francis (1993) assumed that these were mostly composed of extracellular microbial polysaccharides. Likewise, Feller et al. (1991) combined hot water extractions and transmission electron microscopy observations to show that the clay fractions from ferralitic soils were rich in amorphous polysaccharide material, with a morphology typical of that of microbial and plant exudates. This polysaccharide material was partly extracted with hot water and the extracts had high M/X ratios. In our study, the M/X ratio of hot water extracts was higher than that of other polysaccharide fractions only in the NT sample, presumably because of an abundant microbial biomass in this soil. The carbohydrate signatures showed that polysaccharides extractable by hot-water are of both
P. Puget et al. / Soil Biology and Biochemistry 31 (1999) 55±63
61
Fig. 3. The distribution of the ®ve monosaccharides in the water-stable aggregate fractions of T and NT samples, expressed as a percentage of the sum of the ®ve monosaccharides.
microbial and plant origin. Root mucigels are soluble in hot water and may contribute to this fraction. The carbohydrate signature of the POM fractions compared with the clay + silt fractions showed, as expected, that the former contained a larger proportion of plant-derived carbohydrates (Angers and Mehuys, 1989; Guggenberger et al., 1990). POM accounted for 20% to 55% of the acid-hydrolysable carbohydrates of bulk soil and a lesser proportion (10% to 20%) of the hot water-soluble carbohydrates.
4.2. Carbohydrate composition of aggregate fractions Wet sieving allows soil aggregates to be separated on the basis of stability in water. There was a clear relationship between the size of aggregates and their polysaccharide content for both soils and for total, non-cellulosic and hot-water soluble carbohydrates. However, total carbohydrates always represented the same proportion of the total organic C of the fractions. Similar results were reported by Baldock et al.
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P. Puget et al. / Soil Biology and Biochemistry 31 (1999) 55±63
Table 4 Carbohydrate-L contents and monosaccharide ratios mannose/xylose for the clay + silt fraction located within the water-stable aggregates >50 mm of T and NT samples, and for the three hydrolysis procedures. Results are expressed in mg carbohydrate-L g ÿ 1 of fraction and as a proportion of the total L content of the fraction. Values are obtained after calculation, by subtracting the amounts of carbohydrates associated with the aggregate fraction <50 mm recovered after aggregate wet-sieving, from the carbohydrate values of the clay + silt fraction obtained after complete dispersion of the whole soil Carbohydrate-L content (mg L g ÿ 1 fraction)
Carbohydrate-C/ total L (%)
M/X ratio
T 0±30 cm H2SO4 12 M/1 M H2SO4 0.5 M hot water
1.22 0.89 0.06
14.56 10.69 0.68
1.19 1.18 0.43
NT 2±3 cm H2SO4 12 M/1 M H2SO4 0.5 M Hot water
2.39 1.81 0.31
15.21 11.53 2.00
1.00 0.95 0.86
(1987), whereas Dormaar (1984) showed an enrichment in carbohydrates of the C with aggregate size at least for two soils. The nature of the carbohydrates, however, was not the same among the fractions. Monosaccharides indicating a POM signature decreased with decreasing aggregate size, consistent with the data of Baldock et al. (1987) and Hu et al. (1995). In a previous study, we have shown that stable aggregates were enriched in POM as compared with unstable ones (P. Puget, unpublished). Oades (1984) and Cambardella and Elliott (1993) suggested that POM was partly responsible for macroaggregate stability. Indeed POM may have an indirect role as an energy source for microorganisms, which can contribute to binding and stabilising aggregates, through physical enmeshment by hyphae and through binding by extracellular polysaccharides (e.g. Robert and Chenu, 1992; Dorioz et al., 1993; Tisdall, 1994). We can compare the carbohydrate contents and ratios of the clay + silt fraction obtained after complete dispersion of whole soil (Table 2) with those of the aggregate fraction <50 mm recovered after aggregate wet sieving (Table 3). The latter corresponds to clay and silt particles free in the soil, to free microaggregates or to clay and silt particles or microaggregates released from unstable aggregates upon slaking. This fraction was depleted in carbohydrates and its M/X ratio was also low compared to the clay + silt fraction separated from the whole soil (Table 3). This suggested that the clay + silt fraction (<50 mm) present in stable macroaggregates was enriched in polysaccharides of microbial origin compared to unstable ones. We esti-
mated the composition of clay + silt fractions located within stable aggregates >50 mm by dierence (Table 4). As might be inferred, these fractions are enriched in carbohydrates and in the case of NT soil, had a high M/X ratio suggesting a large proportion of microbially-derived carbohydrates. This fact is consistent with the studies of Roberson et al. (1995) showing a strong correlation of mineral-associated carbohydrates, which have a microbial signature, with aggregate stability. Cambardella and Elliott (1994) also found that the water-stable macroaggregates from cultivated grassland soils contain a silt-size SOM fraction, very enriched in labile compounds, and suggested that this enriched labile fraction (ELF) would be a byproduct of microbial activity and contribute to the macroaggregate stability.
Acknowledgements We thank Dr P. Nadeau and G. Devarennes for their contribution and assistance during HPLC determinations and C. Picot for preliminary assays of carbohydrate hydrolysis. C. Feller is acknowledged for his comments on the manuscript. This study is part of the Agriculture Agri-Food Canada-INRA Collaborative Research Program.
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