Biol Fertil Soils (2012) 48:325336 DOI 10.

1007/s00374-011-0629-2

ORIGINAL PAPER

Effects of long-term compost and fertilizer application on stability of aggregate-associated organic carbon in an intensively cultivated sandy loam soil
Hongyan Yu & Weixin Ding & Jiafa Luo & Ruilin Geng & Anwar Ghani & Zucong Cai

Received: 6 July 2011 / Revised: 20 September 2011 / Accepted: 30 September 2011 / Published online: 18 October 2011 # Springer-Verlag 2011

Abstract The study examined the influence of compost and mineral fertilizer application on the content and stability of soil organic carbon (SOC). Soil samples collected from a long-term field experiment were separated into macroaggregate, microaggregate, and silt + clay fractions by wet-sieving. The experiment involved seven treatments: compost, half-compost N plus half-fertilizer N, fertilizer NPK, fertilizer NP, fertilizer NK, fertilizer PK, and control. The 18-year application of compost increased SOC by 70.7121.7%, and mineral fertilizer increased by 5.4 25.5%, with no significant difference between control soil and initial soil. The C mineralization rate (rate per unit dry mass) in microaggregates was 1.522.87 mg C kg1 day1, significantly lower than in macroaggregate and silt + clay fractions (P<0.05). Specific C mineralization rate (rate per unit SOC) in silt + clay fraction amounted to 0.48 0.87 mg C g1 SOC day1 and was higher than in macroaggregates and microaggregates. Our data indicate that SOC in microaggregates is more stable than in macroaggregate and silt + clay fractions. Compost and mineral fertilizer application increased C mineralization rate in all aggregates compared with control. However, compost application significantly decreased specific C mineralization rate in microaggregate and silt + clay fractions by 2.6 28.2% and 21.925.0%, respectively (P<0.05). By contrast, fertilizer NPK application did not affect specific C
H. Yu : W. Ding (*) : R. Geng : Z. Cai State Key Laboratory of Soil and Sustainable Agriculture, Institute of Soil Science, Chinese Academy of Sciences, Nanjing 210008, China e-mail: wxding@issas.ac.cn J. Luo : A. Ghani Climate, Land and Environment, AgResearch, Hamilton 3240, New Zealand

mineralization rate in microaggregates but significantly increased that in silt + clay fractions. Carbon sequestration in compost-amended soil was therefore due to improving SOC stability in microaggregate and silt + clay fractions. In contrast, fertilizer NPK application enhanced SOC with low stability in macroaggregate and silt + clay fractions. Keywords Mineral fertilizer . Compost . Mineralization . Carbohydrate . Wet-sieving . Enzyme

Introduction The stability of soil organic C (SOC) is defined as its resistance to microbial decomposition and is correlated to soil C sequestration potential and soil fertility (Leinweber et al. 2008). The aggregate fractionation technique is used to distinguish SOC into different pools, including more easily decomposed and less easily decomposed. A wealth of studies have shown that SOC stability in aggregate generally increases with smaller aggregate size (Puget et al. 2000; Ashman et al. 2003) primarily due to physical protection in microaggregates and/or physico-chemical stabilization in the silt + clay fraction (Hassink 1997; Six et al. 2000; Jagadamma and Lal 2010). However, Razafimbelo et al. (2008) observed a significantly higher accumulation of the mineralized C in mesoaggregates (20200 m) than from macroaggregates (2002,000 m) during a 28-day incubation period in 11-year share-plowed soil with no residues and in a no-tilled soil with residue mulching. Liao et al. (2006) also reported a 1517% organic C loss in the silt + clay fraction 10130 years after grassland was converted into forestland. Gleixner et al. (2002), Dignac et al. (2005), and Bol et al. (2009) identified carbohydrate and protein, which are defined as labile organic C, in small aggregates,

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e.g., silt + clay fraction. Bayer et al. (2006) reported that within the silt + clay fraction, the strongest organo-mineral interactions were found in the clay fraction (<2 m), but the most recalcitrant organic C was in the fine silt fraction (202 m). Their finding suggests that in some cases the SOC in small size aggregate may not be as inert as expected. Carbon sequestration may also be variable due to physical protection in microaggregates or physico-chemical stabilization in the silt + clay fraction, depending on both soil type and management (Hassink 1997; Six et al. 2002). Organic waste and residue management are recommended to increase SOC by improving stabilization of organic C (Fonte et al. 2009; Liang et al. 2011). For example, Bidisha et al. (2010) and Majumder and Kuzyakov (2010) observed that applying manure for 20 years lowered the decomposability of SOC in soil, which facilitated C retention in cultivated soil. Six et al. (2000) and Chivenge et al. (2007) suggested that an observed increase in organic C in cultivated soils following manure or residue application could be attributed to physical protection of the newly added organic C by occlusion in microaggregates. In contrast, Razafimbelo et al. (2008) found that the increment of SOC in soil after an 11-year period of no-tillage, together with residue mulching, was due to increased organic C associated within the clay and silt fractions via physicochemical protection in aggregates. These inconsistent reports in the literature suggest that further work is needed to understand the organic C stability in aggregates and to accurately explain the accumulation process of organic C in soil. Potentially mineralizable C through incubation experiment is frequently reported as a measure of SOC stability. Since SOC labile pools are exhausted after 6 months to a year, depending on the soil material, some researchers suggested that short-term soil incubation studies are less reliable (Chen and Stark 2000). However, Chevallier et al. (2004) point out that if the protected SOC in aggregate was labile (refer to quality of SOC), long-term incubation would smoothen the importance of this SOC pool. In this study, therefore, we used short-term incubation to evaluate SOC stability in aggregates. In general, carbohydrate represents a main source of nutrients and energy for soil microorganisms and is easily mineralized (Jolivet et al. 2006). SOC decomposition has been determined by the activities of the C-cycle enzymes, including dehydrogenase, invertase, xylanases, cellulases, ligninases, and so on (Stemmer et al. 1999; Allison and Jastrow 2006). We also measured carbohydrate content and the activities of cellobiohydrolase (CBH), -glucosidase (BG), xylosidase, invertase, and polyphenol-oxidase (PPO) in aggregates. The objectives of this study were to (1) evaluate SOC stability in soil and aggregates and (2) understand the accumulation process of SOC in the compost-amended and fertilized soil.

Materials and methods Field experiment and soil sampling A long-term field experiment was established to evaluate the effect of compost and mineral fertilizer application on soil fertility and crop yield where two crops per year, winter wheat (Triticum aestivum L.) and summer maize (Zea mays L.), were cultivated. The experiment described in this paper started in September 1989 on a well-drained field at the Fengqiu State Key Agro-Ecological Experimental Station, Fengqiu County, Henan Province, China (3500 N, 11424 E). The 30-year mean annual temperature was 13.9C, with the lowest mean monthly value of 1.0C in January and highest mean monthly value of 27.2C in July. The mean precipitation was 615 mm. The soil, derived from alluvial sediments of the Yellow River, is classified as Aquic Inceptisol with 52% sand, 33% silt, and 15% clay (Ding et al. 2007). The experiment included seven treatments: compost (CM), half-compost N plus half-fertilizer N (HCM), fertilizer NPK (NPK), fertilizer NP (NP), fertilizer NK (NK), fertilizer PK (PK), and control (CK). The treatments were arranged in a randomized block design with four replicates. Each plot size was 9.55 m. The detailed experimental design and application amount of mineral fertilizers and compost were summarized in Table 1. The basal fertilizers (mineral fertilizers and compost) were evenly broadcast into the plowed soil (020 cm in depth) by tillage before sowing. The supplementary fertilizer urea was surface-applied by hand then brought into the plowed layer with irrigation water. For the experiments, compost was prepared by mixing wheat straw, oil cake, and cotton cake in a ratio of 100:40:45 and fermenting for 2 months at the experimental farm. This proportion was calculated on the basis of component C and N contents, with the goal of applying a total amount of organic C in composted manures (per hectare per season) equal to the amount in harvested wheat straw and organic N equivalent to 150 kg N ha1. These materials were machine-ground to ~5 mm in length before composting. The oil cakes and cotton cakes were the machine-dried residues of oil-harvested rapeseeds and cottonseeds, obtained from a commercial cooking oil business. The amounts of phosphorus and potassium were generally lower than the prescribed doses, so the compost was supplemented with calcium superphosphate and potassium sulfate prior to application. The compost had an average C/N ratio of approximately 8. After maize harvest, on 28 September 2007, eight soil samples were taken from the 020-cm layer at different positions in each plot using a 5-cm-diameter stainless steel soil sampler and were carefully mixed to form a composite. All samples were

Biol Fertil Soils (2012) 48:325336 Table 1 Experimental design and application amount of mineral fertilizers and compost Treatment Basal fertilizer N (kg Nha1) P (kg P2O5 ha1) K (kg K2O ha1)

327

Supplementary fertilizer urea (kg Nha1)

Total Compost Urea Total Compost Calcium superphosphate Total Compost Potassium sulfate Maize CK HCM CM NPK NP NK PK Wheat CK HCM CM NPK NP NK PK 0 75 150 60 60 60 0 0 90 150 90 90 90 0 0 75 150 0 0 0 0 0 75 150 0 0 0 0 0 0 0 60 60 60 0 0 15 0 0 0 0 0 0 75 75 75 75 0 75 0 75 75 75 75 0 75 0 25.5 51 0 0 0 0 0 22.5 45 0 0 0 0 0 49.5 24 75 75 0 75 0 52.5 30 75 75 0 75 0 150 150 160 0 150 150 0 150 150 160 0 150 150 0 32.5 65 0 0 0 0 0 31.5 63 0 0 0 0 0 117.5 85 150 0 150 150 0 118.5 87 150 0 150 150 0 75 0 90 90 90 0 0 60 0 60 60 60 0

The supplement amount of mineral fertilizers added in the HCM and CM treatments was calculated according to the average N, P, and K contents in the compost for 18 years CK control, HCM half-compost N plus half-fertilizer N, CM compost, NPK fertilizer NPK, NP fertilizer NP, NK fertilizer NK, PK fertilizer PK

immediately stored in glass vials and kept on ice in coolers, directly transported to the laboratory, and stored at 4C before analysis. Soil fractionation Moist soil samples were gently broken apart along natural break points and passed through an 8-mm sieve. Plant and organic debris in the sieved soil were carefully removed with forceps. After thorough mixing, a subsample was dried at 105C to measure soil moisture. Another subsample was air-dried for analysis of the soil properties. The remaining moist soil was used for wet-sieving. The water-stable aggregates of moist soils were separated using the wet-sieving protocol (Elliott 1986). One hundred grams of oven-dried soil samples was submerged in deionized water on top of a 250-m sieve (aggregates with size >2,000 m were not found in the tested soil) for 5 min at room temperature. The sieve was manually moved up and down by 3 cm, and this process was repeated 50 times over a 2-min period. Floating organic material was discarded. The fraction remaining on the 250-m sieve was collected in a pre-weighed aluminum pan to obtain macroaggregates (>250 m). Water plus soil with particle size <250 m was poured through a 53-m sieve, and the sieving procedure was repeated. The fraction remaining on the 53-m sieve

was collected in another pre-weighed aluminum pan to obtain microaggregates (53250 m). All materials through the 53-m sieve were transferred into 250-ml centrifuge tubes and centrifuged at 3,750g for 30 min at 4C. The pellets were resuspended in deionized water and recentrifuged under the same conditions three times to exclude the possibly temporary adsorption of dissolved organic C by the mineral particles in silt + clay fraction. Finally, the pellets were collected to obtain the silt + clay fraction (<53 m), and centrifugal supernatants were decanted. An aliquot of each fraction was used to determine the moisture content. Another subsample of each fraction was dried at 50C and used to analyze the soil properties. The remaining moist sample was used for incubation and measurement of enzyme activities. Laboratory incubation Ten grams of bulk soil through 2-mm sieve or aggregates from each plot was placed in a 150-ml glass jar. All bulk soil samples were adjusted to 60% of water holding capacity, and the same amount of water was added to the aggregates. The water holding capacity of bulk soils was measured according to Cai et al. (2001). The jars were covered with a plastic wrap for air exchange and incubated in the dark at 25C. During incubation, soil moisture was

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adjusted every second day by adding deionized water (to maintain the initial weight). Carbon dioxide (CO2) emission rate was measured at 0.5, 1, 2, 3, 5, 7, 9, 11, 13, 15, 18, 21, 24, 28, and 32 days after the beginning of incubation. To measure CO2 emission, each bottle was sealed using an airtight butyl rubber stopper perforated by centered Perspex tubes for sampling. Immediately after and again 12 h after closure, 1 ml headspace gas of the jar was sampled using an airtight syringe for the measurement of CO2 concentration. The CO2 concentration was measured using a gas chromatograph equipped with a thermal conductivity detector operated at 60C (Agilent 7890, Santa Clara, CA, USA). Separation was done using a 177/149-m (80/100 mesh) Chromosorb 102 column (Advanced Minerals, Santa Barbara, CA, USA) at 40C. The temperature of the injecting port was 100C. The carrier gas (H2) flow rate was 80 ml min1. Carbon dioxide gas standards were supplied by the National Research Center for Certified Reference Materials, Beijing, China. The rates of CO2 emission were calculated by the linear model from the change of CO2 concentration in the jar over a 12-h period with an average chamber temperature (25C). Soil analysis Moist and air-dried soil was dried at 105C to determine the soil moisture content. Soil organic C contents in soil and aggregates were determined by the wet oxidationredox titration method (Carter 1993). The carbohydrate contents in soil and aggregates were determined according to Puget et al. (1999). Briefly, 1 g of finely ground soil or aggregate was hydrolyzed with a concentrated sulfuric acid solution. The supernatant was filtrated, and the filtrate was analyzed with the phenol-sulfuric acid method to determine the total sugar content (Doutre et al. 1978). Cellobiohydrolase (EC 3.2.1.91) activity was measured using the method of Desphaude et al. (1984). One gram (on an oven-dried basis) of soils or aggregates was incubated with p-nitrophenyl--D-cellobioside and acetate buffer (pH 5.0) in the dark at 50C for 30 min. The reaction was then stopped by adding 1.0 M Na2CO3. The color intensity of the filtered supernatants (obtained due to the production of p-nitrophenol) was measured at 410 nm on an ultravioletvisible light spectrophotometer (UV-1800, Mapada Instruments Co., Shanghai, China). The procedures of Tabatabai (1994) and Lama et al. (2004) were used to determine the activities of -glucosidase (EC 3.2.1.21) and xylosidase (EC 3.2.1.37), using p-nitrophenyl--D-glucoside and pnitrophenyl--D-xyloside as substrates, respectively. Briefly, 1 g (on an oven-dried basis) of soils or aggregates plus maleate buffer (pH 6.0) and substrate were incubated for 1 h in the dark at 37C. Then, 0.5 M CaCl2 and 0.2 M trishydroxymethylaminomethanesodium hydroxide (pH 12)

for BG and 1.0 M Na2CO3 for xylosidase were added, respectively. The amount of p-nitrophenol produced was determined by spectrophotometry at 410 nm. Invertase (EC 3.2.1.26) activity was determined as suggested by Schinner and von Mersi (1990). One gram (on an oven-dried basis) of soils or aggregates was incubated with 1.2% (w/v) sucrose solution and acetate buffer (pH 5.5) in the dark at 50C for 3 h. After incubation, the contents were filtered, and 1 ml of the filtrate was used to estimate the amount of reducing sugars (i.e., as glucose) by using the 3,5dinitrosalicylic acid method (Miller 1972). Polyphenoloxidase (EC 1.14.18.1) activity was measured spectrophotometrically using pyrogallol as the substrate (Huang et al. 2009). One gram (on an oven-dried basis) of soils or aggregates and 1% pyrogallol was incubated for 2 h in the dark at 30C. After incubation, citrate buffer (pH 4.5) and ethyl ether were added to extract the reaction product, and the absorbance of the organic phase was measured at 430 nm. Enzyme activities were measured in soil samples of each treatment using controls made by mixing buffer with either soil fractions or substrate solution. The values were corrected by subtracting the combined absorption values for the sample and substrate controls from those for the analytical samples. The activity of CBH, BG, and xylosidase was expressed in mg p-nitrophenol (pNP) released kg1 dry soil 1. The activity of invertase and PPO was expressed in g glucose (GE) released kg1 dry soil h1 and g purple gallic (PG) released kg1 dry soil 1, respectively. Calculation and statistical analyses The SOC content in aggregates was not corrected for sand content as there was no obvious difference in particle size distribution between macroaggregates and microaggregates in all treatments. Soil organic C content in soil and aggregates was expressed as g Ckg1 soil and g Ckg1 aggregate, respectively. The sum of the mineralized C in macroaggregate, microaggregate, and silt + clay fraction was counted as follows: X Sum total mineralized C in aggregate
mass ratio of aggregate in soil

Specific C mineralization rate and specific activity of enzymes in soil and aggregates were calculated as follows: Specific C mineralization rate C mineralization rate = SOC content or Specific enzyme activity enzyme activity=SOC content

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Statistical analyses were performed using the SPSS 11.0 software package for Windows. The differences in SOC, carbohydrate, C mineralization rate, specific C mineralization rate, and enzyme activities in soil or aggregates among treatments and in different aggregates of the same treatment were examined using repeatedmeasures ANOVA. Regression analyses were used to test relationships between the C mineralization rate and SOC or carbohydrate content and between the specific C mineralization rate and specific enzyme activity in soil and aggregates.

The lowest carbohydrate content occurred in microaggregates (0.280.54 gC kg1 aggregate), and the highest was observed in macroaggregates (0.731.28 gC kg1 aggregate); the latter was significantly higher than the silt + clay fraction in the PK, NK, and CK treatments, but not in the CM, HCM, NPK, and NP treatments (Table 3). Fertilization increased carbohydrate content in bulk soil and microaggregates compared with the CK treatment, while compost application simultaneously substantially enhanced carbohydrate content in bulk soil and all aggregates. Mineralization of organic C in soil and aggregates

Results Soil organic C and carbohydrate content in soil and aggregates The recovery of the soil aggregate mass after wet-sieving ranged between 97.3% and 101.3%. The 18-year application of compost and mineral fertilizer increased SOC content by 70.7121.7% and 5.425.5%, respectively, and there was no significant difference between unfertilized soil (CK) and initial soil (Table 2). The SOC content in macroaggregates varied from 6.63 to 12.17 gC kg1 aggregate, which was significantly higher than those in microaggregate and silt + clay fractions. The SOC content in microaggregates was significantly lower than in bulk soil in all treatments and close to the value in the silt + clay fraction in the HCM and NP treatments. The SOC content in the silt + clay fraction was higher than those in microaggregates in treatments CM, NPK, NK, and PK. Fertilization significantly increased the SOC contents in bulk soil, macroaggregate, and silt + clay fractions, and compost application also elevated SOC content in microaggregates compared to the control treatment, CK.

The sum of the mineralized C in macroaggregate, microaggregate, and silt + clay fraction during the 32-day incubation period was equivalent to 100.8124.6% of the total mineralized C in bulk soil in all treatments. The C mineralization rates (per unit dry mass) in bulk soil in the HCM and CM treatment were 3.15 and 3.19 mg C kg1 soil day1, respectively. These were significantly higher than those in the other treatments and approximately 2.1 times higher than that in the CK treatment (Fig. 1). Fertilization also significantly elevated the C mineralization rate in all treatments except for the NK treatment. The mineralization rate of organic C over a 32-day experimental period in microaggregates was 1.522.87 mg C kg1 soil day1, significantly lower than the 3.536.26 mg C kg1 soil day1 in macroaggregates and the 2.025.24 mg C kg1 soil day1 in the silt + clay fraction. In comparison with the control treatment CK, the C mineralization rate in the HCM, CM, and NPK treatments was increased by 75%, 159%, and 139% in the silt + clay fraction, respectively, but only by 20%, 71%, and 36% in macroaggregates, respectively (Fig. 1). Compost application and fertilization had less effect on C mineralization rate in microaggregates, compared with macroaggregate and silt + clay fractions.

Table 2 Organic C content in bulk soil (g Ckg1 soil) and aggregates (g Ckg1 aggregate) as affected by the 18-year application of compost and mineral fertilizers Treatment Pre-soil CK HCM CM NPK NP NK PK Bulk soil 4.470.27 4.430.06 7.630.07 9.910.06 5.610.14 d d b a c Macroaggregate (>250 m) 6.630.06 eA 11.800.04 bA 12.170.13 aA 11.680.27 bA 10.970.07 cA 8.660.32 dA 8.920.11 dA Microaggregate (53250 m) 4.220.06 7.591.60 8.800.25 5.040.12 Silt + clay fraction (<53 m) 3.150.03 gC 7.320.11 bB 10.510.22 aB 5.770.07 cB 4.020.02 eB 3.430.03 fB 5.060.02 dB

bB aB aC bC

5.430.03 c 4.710.05 d 5.110.11 c

4.810.06 bB 3.660.05 bC 4.820.02 bC

Values are means (n=4) with standard error. Different lowercase letters within the column indicate significant differences between treatments at P<0.05 and different capital letters within the row indicate significant differences between aggregates for the same treatment at P<0.05 CK control, HCM half-compost N plus half-fertilizer N, CM compost, NPK fertilizer NPK, NP fertilizer NP, NK fertilizer NK, PK fertilizer PK

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Table 3 Carbohydrate content in bulk soil (g Ckg1 soil) and aggregates (g Ckg1 aggregate) as affected by the 18-year application of compost and mineral fertilizers Treatment CK HCM CM NPK NP NK PK Bulk soil 0.490.04 eC 0.780.06 bB 0.970.03 aB 0.600.02 cB 0.580.04 cdB 0.550.03 deB 0.510.07 eC Macroaggregate (>250 m) 0.730.02 eA 0.910.03 cA 1.280.10 aA 0.780.01 dA 0.870.06 cA 1.060.04 bA 1.110.07 bA Microaggregate (53250 m) 0.290.04 dD 0.540.05 aC 0.530.09 abC 0.460.01 bC 0.390.06 cC 0.320.03 dcD 0.280.18 dD Silt + clay fraction (<53 m) 0.590.03 dB 1.020.11 abA 1.210.02 aA 0.770.05 cA 0.910.03 bA 0.450.06 Ec 0.860.12 bcB

Values are means (n=4) with standard error. Different lowercase letters within the column indicate significant differences between treatments at P<0.05 and different capital letters within the row indicate significant differences between aggregates for the same treatment at P<0.05 CK control, HCM half-compost N plus half-fertilizer N, CM compost, NPK fertilizer NPK, NP fertilizer NP, NK fertilizer NK, PK fertilizer PK

The specific mineralization rate of organic C, which was expressed as the mineralization rate per unit SOC, ranged from 0.48 to 0.87 mg C g1 SOC day1 in the silt + clay fraction and was significantly higher than that in macroFig. 1 The C mineralization rate (a) and specific C mineralization rate (b) in bulk soil and aggregates during the 32-day incubation period. Treatments CK, HCM, CM, NPK, NP, NK, and PK represent control, compost, half-compost N plus half-fertilizer N, fertilizer NPK, fertilizer NP, fertilizer NK, and fertilizer PK, respectively. Vertical bars denote standard errors of the means (n=4). Different lowercase and capital letters indicate significant difference between aggregates for the same treatment and between treatments for the same aggregate at P<0.05, respectively. C mineralization rate=(cumulative mineralized C during the 32-day incubation period/32). Specific C mineralization rate=(C mineralization rate/SOC content)

aggregates (0.320.52 (0.280.57 application

(0.320.55 mg C g1 SOC day1), bulk soil mg C g1 SOC day1), and microaggregates mg C g1 SOC day1) (Fig. 1). Long-term of compost significantly reduced the specific C

a.
C mineralization rate (mg C kg-1 soil d-1)
C CD
8.0

A BC

D C

A B

A ABC A a b

AB B

CD A

BC D BCD B

C CD D

AB B

C a

AB B

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a a a b b a c

a
4.0

a b b b b

bb

b c b b

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CK Soil

HOM

OM

NPK

NP

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PK

Macroaggregate

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Specific C mineralization rate (mg C g-1 SOC d-1)
1.2

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AB B

B BC C

A C

AB AB BC

B A a

B B A a

B AB B

A C

A A B a b b

0.8

a a
0.4

a b b b b b

a b bb b b b b b bb

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HOM

OM

NPK

NP

NK Silt+clay fraction

PK

Macroaggregate

Microaggregate

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mineralization rate in microaggregate and silt + clay fractions by 2.628.2% and 25.021.9%, respectively, compared with treatment CK. Although amending fertilizer NPK slightly lowered the specific C mineralization rate in macroaggregates and microaggregates, it significantly increased the specific C mineralization rate in the silt + clay fraction (Fig. 1). Enzyme activities in soil and aggregates The mean recoveries of the activities of all enzymes except BG, in macroaggregate, microaggregate, and silt + clay fraction with the seven treatments, were 90.8102.2% compared with those in bulk soils. BG was the exception
Fig. 2 Activities of invertase, cellobiohydrolase (CBH), glucosidase (BG), xylosidase, and polyphenol-oxidase (PPO) in bulk soil and aggregates as affected by the 18-year application of compost and mineral fertilizes. Treatments CK, HCM, CM, NPK, NP, NK, and PK represent control, compost, halfcompost N plus half-fertilizer N, fertilizer NPK, fertilizer NP, fertilizer NK, and fertilizer PK, respectively. Vertical bars denote standard errors of the means (n=4). Different capital and lowercase letters indicate significant difference between aggregates for the same treatment and between treatments for the same aggregate at P<0.05, respectively
3.0

at 79.4%. Except PPO, enzyme activities in microaggregates were lower than that in macroaggregates, silt + clay fraction, and bulk soil in all treatments (Fig. 2). The highest PPO activity was observed in microaggregates in all treatments, except NPK and PK. Long-term compost amendment significantly increased the activities of invertase in the bulk soil, microaggregates, and silt + clay fraction, and CBH, BG, and xylosidase in bulk soil and all aggregates. The increase of enzyme activities from compost application in macroaggregates or in the silt + clay fraction was more pronounced than that in microaggregates. Adding mineral fertilizers NPK increased the activities of invertase, CBH, BG, and xylosidase in bulk soil and

Invertase activity -1 -1 (g GE kg h )

2.5 2.0 1.5 1.0 0.5 0.0 80

BC B C C C B BC a c cd cd d

b c

A B B A AA B a b c d d e f

CC D C CBC

B A A B BB A a a b c bc d

bc

a a

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CBH activitiy -1 -1 (mg pNP kg h )

C CABB B B A
60 40 20 0 200 150 100 50

A A AAAAA a b c a c d e

C DBB BCA

B B A B BB A a

a ab a bc bc c c B C C B BBB a b e c d e e

b a a b ab ab b b BDDC CBA d c d d d

BG activity -1 -1 (mg pNP kg h )

A A A AA A A a b c d e f ef

B B B C BC B B a

a e bc

b b cd e de f c cd ef de

Xylosidase activity -1 -1 (g pNP kg h )

0 60 50 40 30 20 10 0 1.0

B B B B AB BAB a a ab c bcbc bc

A A A A A A AB a a b c c c c

BCCABBB

B A A A ABB A a a bc b c b

a b

ab ab b b ab

PPO activity -1 -1 (g PG kg h )

0.8 0.6 0.4 0.2 0.0

AABA A B ABB a c b b

D B B AB BA a

B A A AA A A a

C C A ABC A a

d d d

c c

b d d

b b b b

c c

b b

c d

Bulk soil
CK HCM

Macroaggregate
CM

Microaggregate
NPK NP

Silt+clay fraction
NK PK

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all aggregates. The application of unbalanced fertilizers had no obvious effect on the activities of CBH, BG, xylosidase in bulk soil and all aggregates, and invertase in bulk soil, microaggregates, and the silt + clay fraction. In macroaggregates, compost or unbalanced fertilizers application significantly decreased invertase activity compared with CK. In contrast, PPO activities in all aggregates were reduced by fertilization, regardless of the fertilizer type. Relationship between C mineralization rate and SOC, carbohydrate, or enzyme activity Correlation analysis showed that the C mineralization rate was significantly logarithmically correlated to SOC content in bulk soil (R2 =0.760, P=0.010) and the silt + clay fraction (R2 =0.735, P=0.014) and marginally significantly correlated with SOC content in microaggregates (R2 =0.546, P=0.058). The C mineralization rate was also significantly correlated with the carbohydrate content in the bulk soil (R2 =0.633, P= 0.32) and silt + clay fraction (R2 =0.604, P=0.040). The specific C mineralization rate was significantly correlated with the specific activities of -glucosidase in macroaggregates and with -glucosidase, polyphenol-oxidase, and invertase activities in the silt + clay fraction (Table 4). No significant relationship was observed between specific C mineralization rate and the specific activities of any of the tested enzymes in microaggregates.

Discussion Stability of aggregate-associated organic C The specific C mineralization rate in microaggregates was lower than the rates in the macroaggregate and silt + clay fractions (Fig. 1), which suggested that organic C in microaggregates was more stable than it was in macroaggregates and also in the silt + clay fraction. Lisboa et al. (2009) postulated that for a forest-to-pasture chronosequence, the turnover time of organic C in the slow-cycling C pool of microaggregates (53250 m) amounted to 498 years, which is longer than the predicted time of 210 years for the silt fraction (253 m). It has been proposed that excluding microbes and enzymes from pores is the key protection mechanism for occluded organic C in microaggregates (Sollins et al. 1996). Zimmerman et al. (2004) and McCarthy et al. (2008) reported that mesopores of less than 0.1 m in diameter, which could account for up to 21% of the total pore volume in microaggregates, were inaccessible to exoenzymes. The organic C in mesopores is therefore protected from decomposition. McCarthy et al. (2008) further pointed out that even if some pores in microaggregates were suitable habitats for small bacteria and accessible to exoenzymes, the enzymes produced might not be physiologically capable of decomposing organic C due to the tortuosity of the pore network. In the present study, we found lower invertase, CBH, BG, and xylosidase

Table 4 Correlation between specific C mineralization rate and specific activities of invertase, cellobiohydrolase, glucosidase, xylosidase, and polyphenol-oxidase in soil and aggregates affected by long-term application of compost and mineral fertilizers

Aggregate Bulk soil

Enzyme type Invertase Cellobiohydrolase -Glucosidase Xylosidase Polyphenol-oxidase Invertase Cellobiohydrolase -Glucosidase Xylosidase Polyphenol-oxidase Invertase Cellobiohydrolase -Glucosidase Xylosidase Polyphenol-oxidase Invertase Cellobiohydrolase -Glucosidase Xylosidase Polyphenol-oxidase

Equation

R2 0.694 0.509 0.705 0.879 0.098 0.448 0.300 0.741 0.365 0.007 0.166 0.261 0.125 0.128 0.049 0.525 0.288 0.618 0.170 0.862

P 0.020 0.072 0.018 0.002 0.495 0.100 0.203 0.013 0.151 0.861 0.364 0.241 0.436 0.432 0.633 0.065 0.214 0.036 0.358 0.003

y 1:451lnx 3:233 y 0:104x 1:548 y 0:796lnx 0:282 y 0:122x 0:827

y 0:059x 3:316 y 2:002lnx 4:044 y 1:303lnx 0:834 y 1:883e2:632x

Macroaggregate (>250 m)

Microaggregate (53250 m)

Silt + clay fraction (<53 m)

Biol Fertil Soils (2012) 48:325336

333

activities in microaggregate than that in macroaggregates and silt + clay fractions. However, the activities of PPOdegrading macromolecular compounds like lignin were higher in microaggregate (Fig. 2). Therefore, we argue that the micro-environment in microaggregates may not be favorable for microbial life and enzyme production, which results in slow rates of organic C decomposition. In macroaggregates, the specific C mineralization rate was higher than that in microaggregates (Fig. 1), indicating that organic C associated with macroaggregates was more easily decomposed than that in microaggregates. Correlation analysis showed that the specific C mineralization rate in macroaggregates was significantly correlated with the specific activity of -glucosidase, an enzyme driving the hydrolysis of cellobiose (Turner et al. 2002), and marginally significantly with that of invertase (Table 4). In general, enzymes have high specificity for their substrate, and they can even distinguish isomers of the same substrate molecule. The mineralization rate of organic C in the macroaggregate fraction might therefore be primarily controlled by the decomposition of cellulose and sucrose. Lignin and hemicellulose might be rich in macroaggregate, resulting in a temporary increase in SOC (Klbl and Kgel-Knabner 2004). We found that the specific C mineralization rate in the silt + clay fraction was higher than that in microaggregates (Fig. 1). This disagrees with the results of Six et al. (2000) and Allison and Jastrow (2006), who reported that the stability of organic C in aggregates increased with a reduction in aggregate size. It has also been reported that the organic C associated with silt and clay particles is very recalcitrant, with turnover times ranging from 400 to 1,000 years (Buyanovsky et al. 1994). However, Baldock et al. (1987) and Puget et al. (1999) reported a number of carbohydrates and high activities for carbohydrate hydrolytic enzymes in the silt + clay fraction. Zimmerman et al. (2004) and Allison and Jastrow (2006) suggested that the low degradation capacities of these enzymes were probably due to their adsorption onto mineral surfaces, resulting in a loss of functional activity. In contrast, Mikha and Rice (2004) observed that the percentage of total organic C mineralized to C-CO2 in the 2053-m fraction over a 28day incubation period was 3.33%, higher than the 2.77% in microaggregates (53250 m) and just slightly lower than the 3.704.35% in small (2502,000 m) and large (>2,000 m) macroaggregates. This was in spite of the fact that the labile organic C content in the 2053-m fraction was significantly lower than those in other fractions. Mutuo et al. (2006) also monitored higher C mineralization rate in the 2053-m fraction than in the 53 212-m fraction in an arenosol of western Kenya. John (2003) reported that parts of organic C in the clay fraction had a high turnover rate, which was believed to be caused

by a relatively high enrichment of organic C from fresh residues and/or microbial biomass. In the present study, we found that the carbohydrate content in the silt + clay fraction was significantly higher than that in the bulk soil and in microaggregates (Table 3). The C mineralization rate was also significantly correlated with carbohydrate content, and the specific C mineralization rate was significantly correlated with the specific activities of invertase and -glucosidase (Table 4). We suggested that carbohydrates in the silt + clay fraction are not as stable as previously reported (Zimmerman et al. 2004; Allison and Jastrow 2006) and are more easily decomposed than in microaggregates. This may be due to the enzymes responsible for degradation being incompletely adsorbed onto mineral surfaces in our studied soil and therefore capable of retaining some level of activity in the silt + clay fraction. Another possibility is that the increase of carbohydrates may stimulate the growth and activity of microorganisms, which in turn increased the synthesis of enzymes in the silt + clay fraction. Effect of fertilizer on stability of aggregate-associated organic C A large number of studies have shown that C mineralization rates are significantly linearly correlated with SOC content in bulk soil, regardless of soil management practices (Cochran et al. 2007; Zhao et al. 2008), and in aggregates, independent of aggregate size (Chevallier et al. 2004; Barreto et al. 2009). However, in our study, we found that there was a significantly logarithmic, rather than linear, correlation between C mineralization rate and SOC content in bulk soil. We also found that the specific C mineralization rate in compost alone-amended soil was lower than in CK (control) and mineral fertilizer-amended soils (Fig. 1). Our findings suggest that the long-term application of compost more efficiently improved the stability of SOC than did the mineral fertilizer. This finding agrees with the results of Bidisha et al. (2010), who observed that a 20-year addition of farmyard manure at 60 t ha1 year1 decreased the specific C mineralization rate in soil compared to soil with and without the addition of mineral fertilizer. As discussed above, the organic C associated with microaggregates was the most stable that was found in any of the aggregate fractions. The rapid increase of SOC in compost-amended soil was primarily attributed to an accumulation of organic C in microaggregates (Table 2). Said-Pullicino et al. (2007) noted that the increased organic C derived from added compost in microaggregates included lignin-derived phenols, microbial-derived carbohydrates, and so on. In contrast, the water-extractable organic C in compost, especially microbial-derived carbohydrates, was generally stabilized in the silt + clay fraction (Zimmerman

334

Biol Fertil Soils (2012) 48:325336

et al. 2004; Allison and Jastrow 2006), or it was combined with insoluble organic matter and organic components in compost (which were well matured and more resistant as a result of the fermenting process) to form microaggregates (Tisdall and Oades 1982; Helfrich et al. 2008). One possibility is that the reduced organic C biodegradability in microaggregates could be attributed to the accumulation of recalcitrant organic C derived from compost. We also found that long-term application of compost somewhat increased the population of bacteria but significantly reduced the abundance of actinomycetes in microaggregates compared to treatment with mineral fertilizer (data not shown). By measuring phospholipids fatty acid, Poll et al. (2003) also measured a shift towards a more bacteria-dominated community in the coarse sand fraction of a manured Luvic Phaeozem. Vikman et al. (2002) demonstrated that the ability of bacteria to degrade recalcitrant organic C is weaker than that of fungi and actinomycetes. Therefore, another possible explanation for the observed increase in SOC stability in our CM treatment was the microbial community shift to bacterial domination in microaggregates. The application of compost also improved the stability of organic C, even labile organic C such as carbohydrate, in the silt + clay fraction compared to mineral fertilizer (Fig. 1). Among carbohydrates, polysaccharides produced by microorganisms are more enriched in the silt + clay fraction in cultivated soils compared with plant-sourced polysaccharides (Puget et al. 1999; Kiem et al. 2003). Martin et al. (1966) and Greenland and Oades (1975) found that a microorganism-derived polysaccharide, like Azotobacter polysaccharide, was relatively resistant to decomposition. As a consequence, the polysaccharides formed by microorganisms became the main source of the recalcitrant organic compounds in soil (Bottner et al. 1998; Spaccini et al. 2000). In the present study, the compost had been fermented for 2 months before use. During composting, the proportion of the labile, hydrophilic, plant-derived organic compounds gradually reduced. In contrast, that of more stable, hydrophobic moieties, including lignin-derived phenols and microbial-derived carbohydrates, increased in water-extractable organic matter (Said-Pullicino et al. 2007). When these microbial-derived carbohydrates are incorporated into the soil, they are not usually utilized by soil microorganisms and can become stabilized by mineral particles. In contrast, as previously described, applying mineral fertilizer NPK accelerated the decomposition of native organic C in our soil (Ding et al. 2010). This was especially obvious in the silt + clay fraction (Fig. 1), of which the newly increased organic C was mainly derived from root residues and exudates, as no other exogenous organic materials were amended in mineral fertilizer Nadded soils. Manna et al. (2007) demonstrated that fertilizer NPK application significantly promoted the growth and

activities of microorganisms, which in turn accelerated the mineralization of organic C in the silt + clay fraction. Therefore, we argue that the application of fertilizer NPK temporarily increased organic C in the silt + clay fraction, such as carbohydrates. In this fraction, organic C, which is mainly sourced from root residues and exudates, may not be as stable as it is in compost-amended soil where increased organic C also stemmed from recalcitrant organic C in compost. This had resulted in the low level of accumulation of organic C that we observed in the silt + clay fraction in the NPK treatment.

Conclusion The lowest C mineralization rate and specific C mineralization rate was in microaggregates, indicating that the most stable organic C in aggregate fractions was associated with microaggregates. The specific C mineralization rate in the silt + clay fraction was significantly higher than in other two aggregates in all treatments except CM and CK. Therefore, we conclude that organic C in the silt + clay fraction in our soil was not stable as expected. Compared to mineral fertilizer, long-term compost application significantly increased the stability of organic C in the microaggregate and silt + clay fractions, but not in the macroaggregate fraction. Carbon sequestration in compostamended soil therefore may mainly correlate the accumulation of organic C in the microaggregate and silt + clay fractions. In contrast, fertilizer NPK application temporarily increased organic C (with low stability) in the macroaggregate and silt + clay fractions compared to that in the control treatment, CK. Further study is required to measure the structure of soil organic matter and microbial community in macroaggregate, microaggregate, and silt + clay fraction in soil amended with compost and mineral fertilizer to understand C sequestration mechanism.
Acknowledgements We would like to thank Dr. Andrea Donnison for reviewing this paper. This study was financially supported by the Chinese Academy of Sciences (KZCX2-YW-439), the Natural Science Foundation of China (40725003, 41001173), and the Natural Science Foundation of Jiangsu province (SBK200922477, BK2008057, BK2009338).

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