Metabolism of
13
C-labeled glucose in aggregates from soils with
manure application
M. Aoyama
a, D.A. Angers
b,*, A. N'Dayegamiye
c, N. Bissonnette
ba
Faculty of Agriculture and Life Science, Hirosaki University, Hirosaki, Aomori 036-8561, Japan
b
Centre de Recherche et de DeÂveloppement sur les Sols et les Grandes Cultures, Agriculture et Agroalimentaire Canada, 2560 boul. Hochelaga, Sainte-Foy, Que., Canada G1V 2J3
c
Institut de Recherche et de DeÂveloppement en Agro-environnement, 2700 rue Einstein, Sainte-Foy, Que., Canada G1P 3W8
Accepted 17 August 1999
Abstract
Soil microbial biomass and microbial products play an important role in the stabilization of soil structure and, in turn, as a feedback, structure is believed to be a signi®cant control of C dynamics in soils. We investigated the microbial mineralization and assimilation of added13C-[U]-glucose within macro- and microaggregates from surface soils (Humic Gleysol) obtained from long-term plots amended or not with cattle manure (20 Mg haÿ1yrÿ1 for 18 yr). Slaking-resistant macroaggregates (250±1000
mm) and microaggregates (53±250 mm) were separated by wet sieving and incubated with 13C-labeled glucose (1000 mg C gÿ1 soil) and (NH4)SO4(67mg N g
ÿ1
soil) for 14 d at 258C following a 7-d period of conditioning at 258C. The production of13 C-labeled CO2was measured periodically and the chloroform-labile C (microbial biomass) derived from glucose was determined at the end of the 14-d incubation. The added glucose was mineralized less but assimilated more in the microbial biomass of macroaggregates than in microaggregates, and this eect was generally greater in the manure-amended soil. Overall, the percentage of 13C-labeled glucose assimilated was inversely correlated r0:59 with that mineralized during the 14-d
incubation. The size of the native biomass 14 d after glucose addition followed the same trend as that of the glucose-derived biomass. Our results support the hypothesis that stable macroaggregates, especially those from manured-soil, support a greater microbial biomass than microaggregates and constitute `hot-spots' for the metabolism of readily-available substrates. # 2000 Elsevier Science Ltd. All rights reserved.
Keywords:Soil structure; Aggregates;13C-labeled glucose; Manure; Microbial biomass
1. Introduction
Evidence for the involvement of microorganisms in soil aggregate formation and stabilization comes from early work on the eects of adding organic materials to soil. Organic matter additions have little or no eect unless microorganisms are present (Lynch and Bragg, 1985). Close relationships between soil microbial bio-mass and aggregate stability have been observed in several studies (Drury et al., 1991; Sparling et al.,
1992; Edgerton et al., 1995). Organic matter of mi-crobial origin, especially fungal hyphal length or bio-mass (Tisdall and Oades, 1979; Chantigny et al., 1997; Beare et al., 1997), microbial polysaccharides (Rober-son et al., 1991; Haynes and Francis, 1993) and lipidic compounds (Dinel et al., 1992) is often related to changes in soil aggregate stability.
We have reported that the long-term application (18 yr) of cattle manure favored the formation of macro-aggregates resistant to slaking (Aoyama et al., 1999a). Although manure application increased the concen-tration of organic matter both in macro- and microag-gregates, manure-derived organic matter accumulated preferentially in macroaggregates, and both as
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* Corresponding author. Fax: +1-418-648-2402.
eral-associated and as particulate organic matter. We hypothesized that manure-derived organic matter ®rst enters the soil primarily as particulate material, and during decomposition is transformed within the macro-aggregate structure into mineral-associated material of microbial origin thereby contributing to the stabiliz-ation of macroaggregates (Aoyama et al., 1999a). If this hypothesis is true, for the manure-amended soil the microbial biomass within macroaggregates should be eectively maintained in greater amounts than in the control soil.
Aoyama and Itaya (1995) investigated the metab-olism of 14C-labeled glucose in soils that had been amended with dierent organic materials or 1000 mg Cu kgÿ1. The proportion of glucose-C incorporated into the microbial biomass increased in the presence of the previously added organic materials, irrespective of the presence of Cu. The proportion was lower in soils previously amended with stable organic matter (farm-yard manure) than in those with fresh organic matter (orchardgrass). Hence, the conversion eciency of sub-strates to the microbial biomass appeared to be closely associated with the quality of soil organic matter.
We measured the mineralization potential of organic matter in water-stable aggregates in the above men-tioned soils (Aoyama et al., 1999b). The proportions of mineralized C and N in the total aggregate C and N were markedly higher in macro- than in microaggre-gates, indicating that the organic matter in macroag-gregates was more available to soil microorganisms than that in microaggregates. The manure application did not markedly aect the proportion of the minera-lized C in the total aggregate C. However, the manure-derived particulate organic matter in macro-aggregates had a lower C-to-N ratio than the plant-derived par-ticulate organic matter in the non-amended plots (Aoyama et al., 1999a). Thus, the quality of organic matter in aggregates was dierent between the macro-and microaggregates macro-and was signi®cantly aected by the manure application.
Our aim was to see how the quality of organic mat-ter present in stable aggregates, as aected by long-term manure application, in¯uenced the metabolism of readily-available substrates. We therefore measured the microbial mineralization and assimilation of 13 C-labeled glucose within macro- and microaggregates using the soils amended or not with solid cattle man-ure for 18 yr.
2. Materials and methods
Soil samples (0±10 cm) used in this study were obtained from the control (no mineral or organic ferti-lizer) and manure-amended (20 Mg haÿ1 yrÿ1 for 18 yr) treatments of the long-term ®eld trials (started in
1978) at the St-Lambert-de-LeÂvis Soil Research Station of the QueÂbec Ministry of Agriculture, Fisheries and Food (MAPAQ) in the spring of 1996, as described by Aoyama et al. (1999a). The soil at this site is a Le Bras loam (Humic Gleysol). Details of the ®eld trial were reported by N'Dayegamiye (1996). The soil samples were taken from the three replicated plots per treatment and passed through a 6-mm screen in the ®eld. The ®eld-moist soils were used for the measure-ments of soil microbial biomass C and alkaline phos-phatase activity; the remainder was then air-dried in the laboratory and used for the separation of aggre-gate size fractions.
The microbial biomass C of the whole soil was measured using a modi®cation of the fumigation± extraction procedure proposed by Vance et al. (1987). Brie¯y, 20 g of the ®eld-moist soil was fumigated with alcohol-free chloroform for 24 h. Organic C was extracted from fumigated and unfumigated soils with 40 ml of 250 mM K2SO4and quanti®ed using a
Dohr-man DC-180 carbon analyzer (DohrDohr-man Co., Santa Clara, CA, USA). A kEC factor of 0.45 was used to
estimate microbial biomass C from extractable C (Wu et al., 1990). Alkaline phosphatase activity was measured using p-nitrophenyl phosphate as the sub-strate and soil samples were incubated at pH 11.0 for 1 h at 378C as described by Tabatabai (1994). The measurements of microbial biomass C and alkaline phosphatase activity were each replicated three times per plot.
For the incubation study using 13C-labeled glucose, the 250±1000 and 53±250 mm aggregates isolated from the control and manure-amended soils by Aoyama et al. (1999a) were used. The aggregates were separated by slaking of air-dried soil followed by wet sieving and dried in a forced air oven at 408C. The C and N con-tents of the aggregates were presented in Aoyama et al. (1999a).
The aggregate samples equivalent to 20 g oven-dry soil were weighed into plastic vials and care-fully mixed with 20 g of silica sand ashed for 4 h at 5008C. Aggregate-sand mixtures were then inocu-lated with 1 ml of 1:10 aqueous suspension of fresh soil and wetted to a matric potential of ÿ50 kPa with deionized water. The water content of each aggregate±sand mixture at 50 kPa was determined gravimetrically by equilibration on a pressure plate. Each vial was covered with aluminum foil and maintained for 7 d at 258C to allow microbial ac-tivity to stabilize prior to the addition of 13 C-labeled glucose. Following the 7 d conditioning, 2 ml of an aqueous solution containing uniformly labeled
13
C-glucose (10.79 atom %, 10 mg C mlÿ1) and
(NH4)SO4 (670 mg N mlÿ1) was slowly pipetted onto
main-tain a moist atmosphere and a plastic vial conmain-taining 10 ml of 300 mM NaOH to trap CO2. The vials were
incubated for 14 d at 258C. The NaOH traps were changed on days 1, 3, 7 and 14. The total amount of CO2 in the NaOH trap was determined by back
ti-tration with standardized 60 mM HCl to pH 8.6 using an automatic titrator following precipitation of the carbonates by BaCl2added in excess. The BaCO3
pre-cipitate was immediately separated by vacuum ®l-tration on a glass ®ber ®lter (Whatman GF/A), carefully washed and dried at 808C, then ground in a mortar and pestle. For the determination of their atom % 13C, the BaCO3 crystals were converted to CO2 by
acidifying with phosphoric acid and the CO2analyzed
on a 20±20 isotope ratio mass spectrometer interfaced to an ANCA-GSL coupled with a DCP gas module (Europa Scienti®c, Crewe, UK) at the University of Saskatchewan Stable Isotope Facility.
At the end of the incubation, chloroform-labile C was estimated by the fumigation±extraction procedure as described above for whole-soil biomass C. A por-tion of each aggregate±sand mixture corresponding to 12.5 g oven-dry matter was fumigated. Both fumigated and non-fumigated samples were extracted with 25 ml of 250 mM K2SO4. The total amount of organic C in
the extract was determined by the Dohrman DC-180 carbon analyzer. A 10-ml aliquot of the K2SO4extract
was acidi®ed with 1 ml of 1 M H2SO4 and dried at
908C, then ground in a mortar and pestle. The deter-mination of the 13C atom % of the K2SO4 extracts
was adapted from Wanniarachchi (Ph.D. thesis, Uni-versity of Guelph, 1997). The K2SO4 crystals were
resolubilized and the extracts were dialyzed in distilled water ®rst for 16 h followed by an additional 4 h using 11.5 mm dia dialysis tubing with a molecular cut-o of 3500 Da (Spectra/Por). The dialyzed extracts were freeze-dried and analyzed for isotopic composition. In the aggregate incubation study, the dierence in extractable C between the fumigated and unfumigated soils for both the 13C-labeled and unlabeled C was not converted to biomass C, but is presented as chloro-form-labile C.
Residual soil samples were ground in a mortar and pestle after drying at 808C and also analyzed for isoto-pic composition. The eects of manure amendment
and aggregate size were tested using the two-way fac-torial analysis of variance.
3. Results
The total organic C and microbial biomass C con-tents, and alkaline phosphatase activity were signi®-cantly higher in the manure-amended than in the control soils (Table 1). However, the ratio of microbial biomass C-to-soil organic C was nearly constant (0.40±0.43%) irrespective of the amendment with man-ure. Besides, the alkaline phosphatase activity expressed per unit of total organic C was not signi®-cantly aected by the manure amendment.
Mineralization of native organic C proceeded stea-dily in all aggregate size fractions during the 14-d incu-bation (Fig. 1). Throughout the incuincu-bation, the mineralization rate was signi®cantly higher in the macroaggregates (250±1000 mm) than in the microag-gregates (53±250 mm) and was maintained at a higher rate in the manure-amended soil. The added 13 C-labeled glucose was very rapidly respired during the
Table 1
Total organic C (TOC), microbial biomass (MBC) and alkaline phosphatase activity (APA) of whole soils used Soil TOC
(%)
MBC (mg kgÿ1soil)
MBC/TOC (%)
APA
(mgp-nitrophenol kgÿ1soil hÿ1)
APA/TOC
(mgp-nitrophenol gÿ1TOC hÿ1)
Control 2.16 86 0.40 74 3.5
Manured 3.14 135 0.43 139 4.5
Probabilitya 0.03 0.02 0.61 < 0.01 0.15
aStudentt-test.
®rst 3 d of incubation. By d 3, the 13C respired accounted for about 40% of the added 13C, and there-after the mineralization proceeded relatively slowly. A large dierence in the amount of respired 13C among the aggregate size fractions was observed only on d 1. The amount of respired13C by d 1 was signi®cantly (P = 0.002) greater in the manure-amended soil than in the control, while the dierence between the macro-and microaggregates was signi®cant only at the 0.07 level. On d 3 and 7, there were no signi®cant eects of manure amendment and aggregate size on the cumu-lative amount of respired 13C. By d 14, more of the added 13C was respired in the control soil than in the manure-amended soil with no signi®cant dierences between the macro- and microaggregates.
At the end of the 14 d, on average, the macroaggre-gates had more native chloroform-labile C, but the dierence between the aggregate sizes was not statisti-cally signi®cant (Fig. 2). In contrast, the amount of chloroform-labile C derived from glucose was signi®-cantly larger in the macro- than in the microaggre-gates. Glucose addition to the manure-amended soil signi®cantly increased both the size of the native and glucose-derived fractions of chloroform-labile C. The signi®cant manure±aggregate size interaction observed for the chloroform-labile C derived from glucose indi-cated a greater eect of manure in the macroaggre-gates. The chloroform-labile 13C accounted for 20± 30% of the total chloroform-labile C. The proportion of the total chloroform-labile C derived from glucose was higher in the manure-amended than in the control soils.
Between 53 and 56% of the added 13C had been respired after 14 d (Table 2). The proportion of the glucose C left in soil was signi®cantly higher in macro-than in microaggregates. The recovery of the added
13
C ranged from 109 to 119%. The chloroform-labile
13
C accounted for up to 3.2% of the added 13C. The percentage of glucose-13C respired was less and the proportion incorporated into the chloroform-labile fraction was greater in the manure-amended than in control soils. However, the dierence between the macro- and microaggregates was signi®cant at the 0.05 level only for the 13C incorporated into the chloro-form-labile fraction. Furthermore, there was a signi®-cant (P< 0.05) negative linear correlation between the respired and chloroform-labile fraction of added 13C (Fig. 3).
4. Discussion
The ratio of microbial biomass C-to-soil organic C in arable soils has found to be constant under steady-state conditions (Anderson and Domsch, 1989). For arable agricultural soils the size of the biomass is gen-erally between 1 and 3% of total soil organic C (Jen-kinson and Ladd, 1981; Anderson and Domsch, 1989). However, the ratio of microbial biomass C-to-soil or-ganic C found in our study was lower (0.40±0.43%) and no signi®cant dierence was detected between the control and manure-amended soils. These low values are attributable to the recalcitrant nature of organic matter in the soils as inferred from their high C-to-N ratios (Aoyama et al., 1999a). Alkaline phosphatase activity, which has been proposed as a satisfactory index of microbial activity (Frankenberger and Dick, 1983), was also higher in the manure-treated soil. However, the activity expressed per unit of soil organic C was not signi®cantly dierent between the soils. Thus, although the application of manure increased both the size and activity of microbial biomass in the ®eld soils, the quality of whole-soil organic matter appeared not to be greatly dierent between the con-trol and manure-amended soils.
The mineralization rate of native C was signi®cantly higher in the macro- than in the microaggregates and
Fig. 3. Relationship between the percentage of13C mineralized and
that incorporated in the chloroform-labile C. Fig. 2. Chloroform-labile C derived from native C and from glucose
this eect was greater in manure-treated soil. The over-all pattern with regards to treatment eects was similar to the results obtained for the aggregates without sub-strate addition by Aoyama et al. (1999b). However, the amount of mineralized C was 2±3 times higher in our study than in Aoyama et al. (1999b). The greater decomposition in the current study is likely due to the priming eect of glucose addition on the decompo-sition of native C (Wu et al., 1993). Similarly in the whole soil (Table 1), the size of the native microbial biomass (chloroform-labile C in Fig. 2) in both aggre-gate fractions was greater in the manure-treated soil than in the non-amended soil. Although not statisti-cally signi®cant, the size of the biomass was also larger in the macro- than in the microaggregates. Gupta and Germida (1988) and Franzluebbers and Arshad (1997) also reported that macroaggregates had more minera-lizable C and biomass C than microaggregates.
The mineralization of 13C-labeled glucose proceeded rapidly during the ®rst 3 d of incubation. In general, initial rapid mineralization of glucose in soil ceases within 3±5 d (e.g. Shen and Bartha, 1996; Webster et al., 1997). On d 1, the amount of 13C respired was greater in the manure-amended than in the control soil aggregates, and greater in the macro- than in the microaggregates. The increase in CO2 production
im-mediately after the addition of easily decomposable substrates, such as glucose, is referred to as ``substrate-induced respiration'' and is proportional to the size of the microbial biomass (Anderson and Domsch, 1978). Since the respired 13C on d 1 followed the same trend as the amount of native microbial biomass C at the end of the incubation, the dierence in the mineraliz-ation rate of13C during the initial period of incubation re¯ects the dierence in the size of the microbial bio-mass, and indicates greater biomass in macroaggre-gates, especially those from the manure-treated soil.
Overall, the added 13C-labeled glucose was minera-lized less but assimilated more in the biomass of macroaggregates than in microaggregates, and this eect was generally greater in the manure-amended
soil. Therefore, the percentage of 13C assimilated was inversely correlated r0:59
with that mineralized
during the 14-d incubation. These results suggest that macroaggregates had a higher conversion of glucose to the microbial biomass than microaggregates and this conversion was even greater in the manure-treated than in the control soil. As mentioned before, the con-version eciency of glucose to the microbial biomass is aected by the quality of soil organic matter with more labile organic matter leading to greater conver-sion eciency (Aoyama and Itaya, 1995). Therefore, the higher eciency observed for the macroaggregates from the manured soil is probably attributable to more available C to soil microorganisms than in the microaggregates from either the control or manure-treated soils. Some of our previous ®ndings from this experiment support this conclusion. In particular, the carbon within macroaggregates was more labile than that in the microaggregates, and there was a tendency for this eect to be greater in the manured soil (Aoyama et al., 1999b). Also, the POM located in macroaggregates of the manured soil had a lower C-to-N ratio than that from the control soil which would indicate greater lability (Aoyama et al., 1999a).
Guggenberger et al. (1999) reported that an incu-bation of microaggregates with granular starch resulted in the formation of macroaggregates and in an increase in the microbial biomass, especially in the fungal biomass, in macroaggreagates. They thus suggested that microhabitats enriched in substrates acted as `hot spots' for fungal growth and gation. Our results suggest that stable macroaggre-gates, especially those from the manure-amended soil, presented a greater potential for microbial assimilation of readily-available substrate than microaggregates and as such constitute microbial `hot-spots'. We hypoth-esized that when manure enters the soil, its particulate fraction is decomposed and acts as a nucleus for aggre-gate formation and stabilization (Aoyama et al., 1999a). The present study con®rms the enhanced mi-crobial biomass in macroaggregates from manured soil
Table 2
Distribution of13C-labeled glucose between the respired and chloroform-labile (CL) fraction determined 14 days after glucose addition
Fraction Respired (%) Left in soil (%) Recovery (%) CL (%)
Macroaggregates
Control 55.6 57.7 113.3 1.9
Manured 52.5 66.0 118.5 3.2
Microaggregates
Control 55.7 54.0 109.7 1.6
Manured 54.5 54.8 109.3 1.9
F-probability
Manure 0.02 0.13 < 0.01
Aggregate size 0.17 0.03 < 0.01
and suggest that this provides a feedback mechanism by which available substrates are metabolized and assimilated in the microbial biomass of macroaggre-gates.
Acknowledgements
We thank P. Jolicoeur for his technical assistance. M. Aoyama thanks the Ministry of Education, Science, Sports and Culture of Japan for providing a visiting fellowship during the course of this study.
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