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Soil Biology & Biochemistry 50 (2012) 126e133

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Soil Biology & Biochemistry


journal homepage: www.elsevier.com/locate/soilbio

Linking microbial community to soil water-stable aggregation during crop residue


decomposition
C. Le Guillou a, b, c, D.A. Angers d, P.A. Maron e, P. Leterme a, b, c, S. Menasseri-Aubry a, b, c, *
a

INRA, UMR 1069 Sol Agro et hydrosystme Spatialisation, F-35000 Rennes, France
Agrocampus Ouest, UMR 1069 Sol Agro et hydrosystme Spatialisation, F-35000 Rennes, France
c
Universit europenne de Bretagne, France
d
Agriculture et Agroalimentaire Canada, Centre de Recherche sur les Sols et les Grandes Cultures, 2560 Boulevard Hochelaga, Qubec, Qubec, Canada G1V 2J3
e
INRA, UMR 1229 Microbiologie du Sol et de lEnvironnement, F-21065 Dijon, France
b

a r t i c l e i n f o

a b s t r a c t

Article history:
Received 21 December 2011
Received in revised form
7 March 2012
Accepted 9 March 2012
Available online 29 March 2012

The dynamics of soil water-stable aggregation (WSA) following organic matter (OM) addition are
controlled by microbial activity, which in turn is inuenced by carbon substrate quality and mineral N
availability. However, the role of microbial communities in determining WSA at different stages of OM
decomposition remains largely unknown. This study aimed at evaluating the role of microbial
communities in WSA during OM decomposition as affected by mineral N. In a 35-day incubation
experiment, we studied the decomposition of two high-C/N crop residues (miscanthus, C/N 311.3; and
wheat, C/N 125.6) applied at 4 g C kg1 dry soil with or without mineral N addition (120 mg N kg1 dry
soil). Microbial characteristics were measured at day 0, 7, and 35 of the experiment, and related to
previous results of WSA. Early increase in WSA (at 7 days) was related to an overall increase of the
microbial biomass (MBC) with wheat residues showing higher values in MBC and WSA than miscanthus.
In the intermediate stage of decomposition (from day 7 to 35), the dynamics of WSA were more associated with the dynamics of microbial polysaccharides and greatly inuenced by mineral N addition.
Mineral N addition resulted in a decrease or leveling off of WSA whereas it increased in its absence. We
suggest that opportunistic bacterial populations stimulated by N addition may have consumed binding
agents which decreased WSA or prevented its increase. To the contrary, microbial polysaccharide
production was high when no mineral N was added which led to the higher WSA in the late stage of
decomposition in this treatment. The late stage of decomposition was associated with a particular fungal
community also inuenced by the mineral N treatment. We suggest that WSA dynamics in the late stage
of decomposition can be considered as a narrow process3 where the structure of the microbial
community plays a greater role than during the initial stages.
2012 Elsevier Ltd. All rights reserved.

Keywords:
Bacteria
Fungi
Organic matter quality
N
Decomposition
Aggregate stability

1. Introduction
Water-stable aggregation (WSA) is an important characteristic
of soil functioning (Carter, 2002). It is a determinant factor of soil
susceptibility to erosion (Le Bissonnais, 1996), soils ability to
sustain plant germination and early growth (Angers and Caron,
1998) and is a mechanism of organic matter (OM) protection in
soils (Balesdent et al., 2000). Among factors affecting WSA, soil
organic matter is a major one (Tisdall and Oades, 1982) that can be
* Corresponding author. INRA, UMR Sol Agro et hydrosystme Spatialisation, 65
Route de Saint-Brieuc, CS 84215-35042 Rennes Cedex, France. Tel.: 33 2 23 48 54 73;
fax: 33 2 23 48 54 80.
E-mail address: Safya.Menasseri@agrocampus-ouest.fr (S. Menasseri-Aubry).
0038-0717/$ e see front matter 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.soilbio.2012.03.009

managed through agricultural practices such as returning crop


residue to soil (Lal, 2009). The effects of organic inputs on WSA are
generally related to their decomposability (Abiven et al., 2009) such
that easily decomposable residues have intense but transient
effects on WSA whilst less decomposable residues have smaller but
longer lasting effects on WSA.
The role of microorganisms in WSA following crop residue
addition has long been demonstrated (Martin and Waksman, 1940;
Angers and Chenu, 1998). Addition of crop residue rapidly stimulates microbial growth and activity which generate transient
binding agents mostly involving polysaccharides in the rst few
weeks (Tisdall and Oades, 1982). Fungi have often been shown to
have a dominant role in WSA (e.g. Metzger et al., 1987; Denef et al.,
2001) by producing polysaccharides (Chenu, 1989) but also

C. Le Guillou et al. / Soil Biology & Biochemistry 50 (2012) 126e133

physically enmeshing soil particles (Tisdall, 1991) and producing


hydrophobic materials which decrease aggregate wettability
(White et al., 2000), whereas the bacterial role has been mostly
associated with polysaccharide production (Lynch and Bragg, 1985).
However, some particular species within the fungal and bacterial
groups have been shown to be more effective than others in
determining WSA (Harris et al., 1966). For example, Waksman and
Martin (1939) showed that WSA of a soil inoculated with Aspergillus
niger was higher than with Rhizopus nigricans. Yet, the relationship
between WSA dynamics and temporal changes within the microbial communities following residue additions is still unclear
(Abiven et al., 2009).
On the other hand, plant residue incorporation has been shown
to inuence the soil microbial population dynamics (Bastian et al.,
2009). Population dynamics are complex as the initial biochemical
characteristics of the crop residue and soil mineral N availability
inuence the bacterial and fungal population succession during
crop residue decomposition (Baumann et al., 2009). For instance,
the amount of readily available carbon substrates has been positively related to the stimulation of Proteobacteria (Fierer et al.,
2007; Pascault et al., 2010). N supply has been shown to particularly affect the bacterial community (Ding et al., 2010; Ramirez
et al., 2010) but also to decrease soil fungal diversity (Allison
et al., 2007).
While the structure of soil microbial communities has been
related to crop residue decomposition, its relationship with WSA
dynamics has not yet been established. Recent methodological
advances in microbial structure analysis may improve our understanding of the role of microbial communities in determining WSA.
Better knowledge of these complex relationships will help
managing soil structure through manipulation of residue input and
their consequent effect on soil microorganisms.
In this study, we aimed at evaluating microbial community
characteristics related to WSA dynamics at different stages of crop
residue decomposition. Therefore, using the study of Le Guillou
et al. (2011), we examined over time (1) how OM quality interacting with soil mineral N inuenced bacterial and fungal communities and (2) how such changes in microbial community could be
related to the WSA dynamics.

2. Materials and methods


The experimental design was previously described in details in
Le Guillou et al. (2011). Analyses of the present study were performed on samples collected in this previous experiment.

2.1. Soil and crop residues


The soil used in the experiment was sampled at the Champ Nol
experimental site of INRA (Institut National de la Recherche
Agronomique), near Rennes, France (48 070 N, 1430 W). The soil was
a Luvisol (FAO/ISRIC/ISSS, 1998) with 15.1% clay, 71.1% silt, and 13.8%
sand, a total C content of 9.7 g kg1, a C/N ratio of 8.8, and a pH in
water of 6.2. The sampled plot had been under maize/wheat rotation with mineral fertilization since 1995. In March 2009, the soil
was sampled in the 0e15 cm layer using a shovel. The soil was
gently sieved at 5 mm, air-dried, and stored at 4  C. Coarse visible
particles (roots and plant debris) were removed by hand.
The two crop residues were wheat straw (Triticum aestivum L.)
and miscanthus straw (Miscanthus  giganteus). These residues
were chosen for their high-C/N ratio and differences in biochemical
characteristics (Table 1). The residues were dried at 60  C and
ground to less than 1 mm.

127

Table 1
Biochemical characteristics of the crop residues.
Crop residue

C/N

Lignin/N

Van Soest (% dry mass)


Soluble

Hemicellulose

Cellulose

Lignin

Wheat
Miscanthus

125.6
311.3

21.2
88.1

13.2
6.3

34.0
28.1

45.6
52.4

7.2
13.2

2.2. Incubation and experimental treatments


The soil water content was adjusted to 200 g kg1 dry soil by
spraying ultraltered water and allowed to equilibrate for 3 d. The
remoistened soil was then preincubated at 25  C for 10 d to minimize variations in microbial activity due to changes in temperature
and water content conditions. Soil water content was readjusted to
217 g kg1 dry soil through the addition of ultraltered water for
the treatments without mineral N input and through the addition
of a KNO3 solution for the treatment with mineral N input. Initial
soil mineral N content was 24.2 mg N kg1 dry soil. Two mineral N
input rates were studied: 0 (N) and 120 (N) mg N kg1 dry soil.
Therefore, the initial average total soil mineral N content corresponded to: N 24.2 mg N kg1 dry soil, N 143.2 mg N kg1
dry soil.
Crop residues were mixed with the preincubated and Nadjusted soil at a rate of 4 g C kg1 dry soil. A control soil treatment
without crop residue input (unamended) and two crop residue
treatments were thus combined with two N treatments in a factorial design with three replicates. The incubation was performed in
hermetically closed 1-L jars. The contents of each jar were mixed
homogenously before incubation. The treatments were incubated
at constant temperature (25  C) for 35 d, and the soil water content
was maintained at 217 g kg1 dry soil with regular weighing of the
jars and addition of water when necessary.
2.3. Measurements
Destructive sampling was realized at 0, 7 and 35 d. Three jars for
each treatment were removed for analysis at each sampling date.
The content of each jar was homogenized and divided into
subsamples for analyses. The entire 0e5 mm soil fraction was used
for analyses. Subsamples for microbial genetic structure and
ergosterol analyses were stored at 20  C until analysis. Subsamples for hot-water extractable polysaccharides and WSA analyses
were oven-dried at 40  C for 24 h. Extraction for total microbial C
analysis was realized at the date of sampling and the extracts stored
at 20  C until analysis.
The WSA was obtained from Le Guillou et al. (2011) and the
method adapted from Angers et al. (2008). Briey, a 5-g subsample
(0e5 mm) of the dried soil was capillary-rewetted for 1 h and
transferred to the top of a nest of sieves (2, 1, and 0.5 mm in
diameter). The column was immersed in deionized water and
shaken vertically 20 times, with the column kept in the water. The
fraction remaining on each sieve was oven-dried at 40  C for 24 h
and weighed. The mean weight diameter (MWD) was calculated as
follows:

MWD

wi *xi

Where i corresponds to each fraction collected, wi is the dry weight


of the fraction collected relative to the total soil used, xi is the mean
diameter of the fraction collected.
Microbial biomass C (MBC) was estimated by the fumigationextraction method (Vance et al., 1987). No extraction coefcient
was used. A 0.025 mol L1 solution of K2SO4 (100 mL) was used to
extract labile organic C from the fumigated and non-fumigated

C. Le Guillou et al. / Soil Biology & Biochemistry 50 (2012) 126e133

2.4. Statistical analysis


Absolute values of variables measured on unamended soil
samples are presented in Table 2. As mineral N levels did not have
an effect on the measured properties of the unamended soils data
were expressed as the difference between the residue-treated and
unamended soils within each N treatment. The effects of residue
type and N treatment on microbial variables were tested by analysis
of variance (ANOVA) and differences between means were tested
with the LSD test. Prior to analysis, data were tested for homogeneity of variance using the Levenes test and log-transformed when
required.
ARISA data obtained from the 1D-Scan software were converted
into a table summarizing the band presence (i.e., peak) and intensity (i.e. height or area of peak) using the PrepRISA program
(Ranjard et al., 2003). This software enabled us to choose the
number of peaks (i.e. all detected populations vs. the most dominant populations), the prole resolution (between 1 bp and 10 bp),
and the method of evaluating peak intensity (area). We used 100
peaks, a 2 bp-resolution and the Gaussian peak area for a robust
analysis of bacterial and fungal communities (Ranjard et al., 2003).
Complex ARISA proles were analysed through a principal
component analysis (PCA) on a covariance matrix. PCA was performed using the ADE-4 software (Thioulouse et al., 1997).

Table 2
Absolute values of measured variables on unamended soil samples at day 0, 7 and 35
within N treatments (with (N) or without (N) mineral N supply). Values represent
the mean of three replicates. Values between brackets represent the standard deviation of the means.

Day 0
N
N
Day 7
N
N
Day 35
N
N

WSA (mm)

Microbial C
(mg kg1
dry soil)

Ergosterol
(mg kg1
dry soil)

Hot-water
extractable
polysaccharide
(mg kg1
dry soil)

0.5 (0.0)
0.5 (0.0)

60.8 (6.1)
56.6 (8.7)

0.7 (0.1)
0.7 (0.1)

0.9 (0.0)
0.9 (0.0)

0.4 (0.0)
0.4 (0.0)

70.2 (9.2)
58.9 (5.7)

1.0 (0.2)
0.8 (0.2)

0.7 (0.1)
0.6 (0.1)

0.3 (0.0)
0.4 (0.1)

60.5 (8.3)
55.8 (3.6)

0.5 (0.1)
0.6 (0.1)

0.5 (0.1)
0.9 (0.3)

Pearsons correlation analysis was used to explore statistical


relationships between microbial variables and WSA at day 7 and 35.
For the correlation analysis, complex ARISA proles were analysed
by calculating the Euclidian distance (Eudist) (Ranjard et al., 2000,
2008; Bressan et al., 2008) between treated and unamended soils
for each incubation time. This genetic distance, which can be
interpreted as the magnitude of genetic structure modications
induced by treatments, was evaluated by calculating the Euclidian
distance (Eudist) between treated and unamended soils divided by
the average Euclidian distances of intra-unamended soils and intratreated soils, as follows. Magnitude of modications, example of
calculation for each incubation date Average of inter-Eudist
(treated soils/unamended soils)/((Average of intra-Eudist treated
soils) (Average of intra-Eudist unamended soils))/2.
3. Results
3.1. Microbial biomass
The increase in the microbial biomass C occurred rapidly (Fig. 1)
whatever the N treatment considered and after 7 days was significantly higher in the wheat than the miscanthus treatment
(P < 0.01). Microbial biomass C remained high and stable between
days 7 and 35, except in the Wh N treatment where it decreased.
At day 35, microbial biomass decreased in the order:
Wh N > Wh N > Mc N Mc N (P < 0.01).

-1

fresh soil samples (24 g fresh soil). Microbial C was estimated as the
organic C extracted in the fumigated samples minus the organic C
extracted in the non-fumigated samples. Extractable C was
measured using a Shimadzu TOC 5050A total carbon analyzer .
Ergosterol content was determined according to the method of
Djajakirana et al. (1996) and Gong et al. (2001). Extraction from
3.5 g soil sample was realized with 120 mL ethanol and 4 g of glass
beads by shaking for 30 min on a rotative agitator. The extracts
were ltered through glass ber lters (Whatman GF/C, UK) and
evaporated under vacuum on a rotary evaporator at 40  C. The
residues were dissolved in 10 mL ethanol. Determination of
ergosterol content was performed by HPLC as described in Annabi
et al. (2007).
Hot-water extractable polysaccharide content was determined
according to the method of Puget et al. (1999). The samples (1-g
dried and ground soil sample) were extracted using 20 mL demineralized water at 80  C for 24 h. The supernatant was collected after
centrifugation (20 000 g) and the polysaccharide content measured
by a colorimetric method at 490 nm according to the phenol-H2SO4
method (Dubois et al., 1956).
Microbial DNA was extracted according to the method described
by Ranjard et al. (2003). The genetic structure of the microbial
communities was determined by using the DNA ngerprinting
technique, the automated ribosomal intergenic spacer analysis
(ARISA). The bacterial and fungal ribosomal IGS were, respectively,
amplied with two primers sets: S-D-Bact-1522-b-S-20/L-D-Bact132-a-A-18 and ITS1F/3126T, PCR conditions being described by
Ranjard et al. (2003). A uorescent-labelled primer was used for the
LiCor DNA sequencer (ScienceTec, Les Ulis, France) in B-ARISA and
F-ARISA, namely the IRD800 dye uorochrome (MWG SA Biotech,
Ebersberg, Germany). ARISA fragments were resolved on 3.7%
polyacrylamide gels and run under denaturing conditions for 15 h at
3000 V/60 W on a LiCor DNA sequencer (ScienceTec). The data were
analysed using the 1D-Scan software (ScienceTec). This software
converted uorescence data into electrophoregrams where peaks
represented PCR fragments. The height of the peaks was calculated
in conjunction with the median lter option and the Gaussian
integration in 1D-Scan, and represented the relative proportion of
the fragments in the total products.

Microbial C (mg.kg dry soil) (residue-treated


minus control within each N treatment)

128

120
100
80
60
40
Wh -N
Wh +N

20

Mc -N
Mc +N

0
0

10

15

20

25

30

35

40

Time (day)

Fig. 1. Microbial C during the incubation (residue-treated minus control within each N
treatment). Wheat (Wh) and Miscanthus (Mc) residues combined with 0 (N) and 120
(N) mg N kg1 dry soil addition rates. Error bars represent the standard error of the
means.

3.3. Hot-water extractable polysaccharides


Soil hot-water extractable polysaccharide content increased in
the rst 7 days of incubation in all treatments (Fig. 3). An early
signicant effect of N addition was only observed in the wheat
treatment, with signicantly more polysaccharide recovered in the
Wh N treatment compared to the Wh N (P < 0.05) at day 7.
Between day 7 and 35, soil polysaccharide content increased in
the N treatments whereas it decreased or leveled off in the N
treatments. At the end of the incubation (day 35), a strong effect of
N addition was observed, with signicantly lower polysaccharide
content recorded in the N treatments (P < 0.01). At day 35, hotwater extractable polysaccharide content decreased in the order:
Wh N Mc N > Mc N > Wh N.
3.4. Water-stable aggregation
As reported by Le Guillou et al. (2011), early water-stable
aggregation dynamics (day 7) were controlled by crop residue
quality with a signicantly higher level for wheat straw than miscanthus (P < 0.01) (Fig. 4). Late water-stable aggregation (day 35)
was controlled by the soil mineral N availability with a signicantly
higher aggregate MWD in the N treatments than the N treatments (P < 0.01).
3.5. Bacterial and fungal genetic structure

-1

ergosterol (mg.kg dry soil) (residue-treated


minus control within each N treatment)

Principal Component Analysis (PCA) of the bacterial genetic


structure proles showed that N addition induced a signicant
modication of bacterial communities in both wheat and miscanthus residue treatments since Wh N and Mc N treatments at
day 7 were discriminated from the other treatments and dates on
the rst axis of the factorial map (Fig. 5a and b). However, this

3.5
Wh -N
Wh +N

Mc -N
Mc +N

2.5
2
1.5
1
0.5
0
0

10

15

20

25

30

35

40

-1

The increase in ergosterol content was signicantly higher in the


Wh N treatment than the other treatments at day 7 (Fig. 2).
Ergosterol content greatly increased between day 7 and 35 (except
in the Wh N treatment where it decreased). As the microbial
biomass C remained stable between day 7 and 35, the contribution of
the fungal biomass to the total microbial biomass increased between
day 7 and 35 in every treatments (except in the Wh N treatment).
At day 35, ergosterol content decreased in the order:
Mc N z Mc N z Wh N > Wh N (P < 0.01).

129

1
Wh -N
Wh +N

0.8

Mc -N
Mc +N

0.6
0.4
0.2
0
-0.2
-0.4
0

10

15

25

20

30

35

40

Time (day)

Fig. 3. Hot-water extractable polysaccharide content during the incubation (residuetreated minus control within each N treatment). Wheat (Wh) and Miscanthus (Mc)
residues combined with 0 (N) and 120 (N) mg N kg1 dry soil addition rates. Error
bars represent the standard error of the means.

modication was resilient since all the treatments were grouped


together on the factorial map at day 35. Comparison of wheat and
miscanthus treatments at day 7 (Fig. 5c) and at day 35 (Fig. 5d)
showed evidence that N input was of critical importance in shaping
the structure of the bacterial communities after crop residue
incorporation since Mc N and Wh N treatments were signicantly discriminated from the other treatments on the rst axis of
the factorial map whatever the date considered.
Considering the fungal communities, a signicant modication
of the structure of the community following wheat residue incorporation was only observed at day 35 when mineral N was not
added (Fig. 6a). In the miscanthus treatment, a progressive modication of the structure of the fungal community was observed
with the different sampling dates being distributed on the rst axis
of the factorial map (Fig. 6b). An N effect on the fungal community
of the miscanthus treatment was only observed at day 35 and was
more evident on the PC1 and PC3 principal component plots (data
not shown), the total variance explained by PC2 and PC3 being
equivalent. Comparison of treatments at day 7 (Fig. 6c) and 35
(Fig. 6d) showed a consistent effect of residue incorporation on the
rst axis and of residue quality on the second axis. As observed for

MWD (mm) (residue-treated minus


control within each N treatment)

3.2. Ergosterol

hot-water polysaccharide (mg.kg dry soil)


(residue-treated minus control within each N treatment)

C. Le Guillou et al. / Soil Biology & Biochemistry 50 (2012) 126e133

1.6
Wh -N
Wh +N

1.4
1.2

Mc -N
Mc +N

1
0.8
0.6
0.4
0.2
0
0

10

15

20

25

30

35

40

Time (day)

Time (day)

Fig. 2. Ergosterol during the incubation (residue-treated minus control within each N
treatment). Wheat (Wh) and Miscanthus (Mc) residues combined with 0 (N) and 120
(N) mg N kg1 dry soil addition rates. Error bars represent the standard error of the
means.

Fig. 4. WSA expressed as the mean weight diameter (MWD) during the incubation
(residue-treated minus control within each N treatment). Wheat (Wh) and Miscanthus
(Mc) residues combined with 0 (N) and 120 (N) mg N kg1 dry soil addition rates.
Error bars represent the standard error of the means. Adapted from Le Guillou et al.
(2011).

130

C. Le Guillou et al. / Soil Biology & Biochemistry 50 (2012) 126e133

PC2 12%

Wh -N D7

PC2 14%

Mc -N D0

Mc +N D7
Wh +N D7

Wh +N D35
Wh -N D35

Mc -N D35

Mc +N D35

Mc -N D7
Wh -N D0

Wh +N D0

Mc +N D0

PC1 49%

PC2 14%

PC1 44%

PC2 16%

Un +N

Mc +N

Un -N

Wh -N
Mc +N

Mc -N

Wh +N

Wh +N
Un -N

Un +N

Mc -N
Wh -N

PC1 46%

PC1 26%

Fig. 5. Bacterial genetic structure analysis. Principal component analysis ordination (PC1 and PC2) of the bacterial genetic structure of the (a) wheat and (b) miscanthus treatments
from each sampling date and of the (c) day 7 and (d) day 35 considering every treatment. Statistical ellipses drawn over the plot replicates represent 90% condence. The percentage
of explained variations for the rst two axes is indicated within the gures. Unamended (Un), Wheat (Wh) and Miscanthus (Mc) residues treatments combined with 0 (N) (white
ellipses) and 120 (N) (grey ellipses) mg N kg1 dry soil addition rates at day 0 (D0), 7 (D7) and 35 (D35).

wheat treatment, an N effect occurred at day 35 of incubation with


Wh N treatment being discriminated on the rst axis (Fig. 6d).
4. Discussion
In a previous study (Le Guillou et al., 2011), we showed that
residue quality and N availability had differential and successive
effects on WSA dynamics during residue decomposition (Fig. 4).
The increase in WSA between 0 and 7 days of incubation was
related to residue quality with wheat straw showing greater values
than miscanthus residues, whereas between 7 and 35 days, the
availability of mineral N became the determining factor, with
greater values in the absence of added mineral N. It was also clear
that the formation of WSA was not simply related to the intensity of
residue mineralization (as expressed by soil respiration measurement) as the latter was stimulated by mineral N input but not WSA.
The role of the microbial biomass in determining WSA has long
been demonstrated under both eld and laboratory conditions
(Drury et al., 1991; Sparling et al., 1992; Edgerton et al., 1995). In
accordance with these ndings, the level of MBC was signicantly

correlated with WSA at day 7 (Table 3). Contrary to some studies on


the microbial contribution to WSA following high-C/N crop residue
addition (e.g. Bossuyt et al., 2001; Denef et al., 2001), early changes
in WSA were not related to the specic fungal biomass as measured
by ergosterol content. In these previous studies, the specic role of
fungi was established using biocide treatments. The main factor
limiting microbial growth is the availability of labile C (Reinertsen
et al., 1984; Bremer and Van Kessel, 1992). In our study, wheat
straw contained twice as much Van Soest-soluble C than the miscanthus residue which likely explains the greater MBC content in
the wheat residue amended soil. In the early stages of crop residue
decomposition, the easily decomposable compounds can be
metabolized by a large fraction of the total microbial community
(high functional redundancy when dealing with decomposition of
low-molecular weight carbon compounds) (McGuire and Treseder,
2010; Paterson et al., 2011) and thus contribute to the formation of
WSA. The implication of the global microora in increasing WSA
could not be related specically to microbial polysaccharides, and
therefore probably involved a combination of binding agents such
as a direct effect of lamentous microorganisms, adhesion of

C. Le Guillou et al. / Soil Biology & Biochemistry 50 (2012) 126e133

PC2 16%

131

PC2 13.1%

Mc -N D0

Wh +N D35
Mc -N D35

Mc +N D0
Mc +N D35

Wh +N D7
Mc -N D7
Wh -N D35

Wh +N D0
Wh -N D7
Mc +N D7
Wh -N D0

PC1 38%

PC2 18%

c
Mc +N

PC1 29%

Mc -N

PC2 21%

Un +N
Un -N

Un -N

Wh -N

Wh +N

Wh -N

Mc +N

Un +N
Mc -N
Wh +N
PC1 25%

PC1 37%

Fig. 6. Fungal genetic structure analysis. Principal component analysis ordination (PC1 and PC2) of the fungal genetic structure of the (a) wheat and (b) miscanthus treatments from
each sampling date and of the (c) day 7 and (d) day 35 considering every treatment. Statistical ellipses drawn over the plot replicates represent 90% condence. The percentage of
explained variations for the rst two axes is indicated within the gures. Unamended (Un), Wheat (Wh) and Miscanthus (Mc) residues treatments combined with 0 (N) (white
ellipses) and 120 (N) (grey ellipses) mg N kg1 dry soil addition rates at day 0 (D0), 7 (D7) and 35 (D35).

microbial cells to soil particles or excretion of lipidic compounds


(Lynch and Bragg, 1985). The early increase in WSA is therefore
linked to an increase of the overall MBC and, as proposed by Abiven
et al. (2008), could be related to the easily degradable soluble
fraction of the residue.
Between 7 and 35 days, the dynamics of WSA were associated
with changes in hot-water extractable polysaccharide such that at
35 days the two parameters were signicantly correlated (Table 3).
This is in agreement with the common understanding that polysaccharides play an important role in determining WSA (Haynes
and Swift, 1990). However, polysaccharides are easily

decomposable organic compounds (Martin, 1971) and have been


categorized as transient aggregate binding agents (Tisdall and
Oades, 1982). From day 7 to 35 in the treatments with N, WSA
under wheat reached a similar low level as that of miscanthus
which also corresponded to low levels of hot-water extratable
polysaccarides. In parallel, the addition of mineral N resulted in the
stimulation of specic bacterial populations (particularly at day 7).
Bacteria are generally considered as the main decomposers of labile
C compounds (de Boer et al., 2005; Poll et al., 2008) and are also
stimulated by N addition (Meidute et al., 2008). This suggests that
the early stimulation of microbial respiration by mineral N addition

Table 3
Correlation coefcients (r) between the mean weight diameter (MWD) and measured microbial variables at day 7 and 35 (data are expressed as the difference between the
residue-treated and control soils within each N treatment). Bacterial and fungal genetic structure are expressed as the Euclidian distance between treated and control soils
within each N treatment for each incubation time. CeCO2 rate data are from Le Guillou et al. (2011). (ns non signicant, yP < 0.1, *P < 0.05, **P < 0.01).

Day 7
Day 35

CeCO2 rate

Microbial C

Ergosterol

Hot-water
extractable
polysaccharide

Bacterial
genetic
structure

Fungal
genetic
structure

ns
ns

0.76**
ns

ns
ns

ns
0.67*

ns
0.79**

ns
0.53y

132

C. Le Guillou et al. / Soil Biology & Biochemistry 50 (2012) 126e133

(particularly in the wheat treatment) observed in Le Guillou et al.


(2011) may be associated to the stimulation of an opportunistic
bacterial community developing rapidly on labile compounds. Very
early, Harris et al. (1963) observed that sucrose-amended treatments maintained WSA longer than sucrose-amended treatments
with mineral N supply and attributed it to the metabolism of
aggregating agents induced by N supply. We suggest that the
leveling off of WSA between 7 and 35 days in the treatments with
added N may be related to the stimulation of an opportunistic
bacterial population (Table 3) and its catabolic activity on labile
binding agents.
To the contrary, in the treatments without added mineral N, the
clear increase in hot-water extractable polysaccharide between
days 7 and 35 is consistent with the prediction that microorganisms produce polysaccharides when their respiratory activity is
limited by lack of N (Knapp et al., 1983; Hadas et al., 1998). The
increase in time of the contribution of the fungal biomass to the
overall microbial biomass and the succession of fungal populations
observed over time in our incubation suggest the development of
particular fungal communities specialized in the decomposition of
more complex organic compounds such as found in the latter
stages of residue decomposition (McMahon et al., 2005; McGuire
and Treseder, 2010). A smaller number of more specialized microorganisms are able to decompose more complex compounds, and
fungi are generally considered to have the enzymatic material
capable of decomposing such compounds (Swift et al., 1979). The
inuence of mineral N on fungal populations at the late stage of
decomposition suggests that specic fungi populations may be
involved in the synthesis of polysaccharides. This phase in the WSA
dynamics could be considered as a narrow process where the
structure of the microbial community may play a greater role than
during the initial stages of decomposition (Table 3), likely because
of a decrease in the functional redundancy when dealing with
specialized metabolic pathways involved in the decomposition of
complex C-substrates (Maron et al., 1998). Martin and Anderson
(1943) suggested that fungal species appearing in the latter stage
of organic residue decomposition could be the most effective at soil
aggregation. Caesar-TonThat and Cochran (2000) and CaesarTonThat (2002) have illustrated ability of a saprophytic lignindecomposing basidiomycete fungus in forming stable aggregates
through the production of extracellular binding agents. In accordance, considering the small effect of miscanthus residues on the
overall microbial biomass - which can be explained by its recalcitrant biochemical composition - the fungal populations found at
day 35 in the absence of N are likely very efcient at forming WSA.
In conclusion, our study covered diverse situations of OM
decomposition inuenced by a cross combination of C quality
(residue type and stage of decomposition) and N availability. We
propose a conceptual model (Fig. 7) to summarize our understanding of the microbial contribution to the transient soil waterstable aggregation following OM inputs. Early changes in WSA
induced by OM addition may be more related to carbon substrate
quality than to mineral N addition, and involve the total microbial
biomass more than a specialized one (linked to microbial biomass
rather than community diversity). In the intermediate stage of
decomposition, mineral N addition resulted in a leveling off of WSA
whereas it increased in the absence of mineral N. We suggest that
an opportunistic bacterial community stimulated by N addition
metabolized binding agents which decreased WSA or prevented its
increase. To the contrary, microbial polysaccharide production was
high when no mineral N was added which led to the higher WSA in
the late stage of decomposition. These observations suggest that
microorganisms may play a dual role in controlling WSA dynamics
by being both producers as well as degraders of aggregate binding
agents, depending largely on the availability of mineral N and the

Water-stable aggregate

II

Time
week
C quality

OM input

Total microbial

month

Water-stable aggregate

r = 0.76 **

N availability

biomass
Microbial
community

II

r = 0.67 *

Polysaccharide

structure

Fig. 7. This conceptual diagram depicts our understanding of the microbial contribution to the transient soil water-stable aggregation dynamics following organic matter
(OM) inputs. ( ) denotes the controls on the level of water-stable aggregates at
different stages of OM decomposition. In phaseI (week(s)) (continued line), the early
increase in water-stable aggregation is controlled by OM inputs biochemical characteristics (C-substrate quality) and related to the increase in the total microbial biomass.
In phaseII (dotted line) (month(s)), the level of water-stable aggregates is controlled by
N availability and related to the level of microbial polysaccharides. The amount of
polysaccharides would be determined by the balance between production and
consumption which are inuenced by the fungal and bacterial populations stimulated
(microbial community structure).

degradability of the C-substrates entering the soil. The late stage of


decomposition was associated with a particular fungal community
which was also inuenced by the mineral N treatment. As the
microbial biomass remained constant, we suggest that WSA
dynamics in the late stage of decomposition could be considered as
a narrow process where the structure of the microbial community rather than the total biomass may play a greater role than
during the initial stages.
Acknowledgements
We are grateful to Mlanie Lelivre, Samuel Dequiedt, Armelle
Racap, Laurence Carteaux, Sylvain Busnot and Yannick Fauvel for
their help during the experiment. We are grateful to the College
Doctoral International de lUniversit Europenne de Bretagne
(College%20Doctoral%20International%20de%20lUniversit%
20Europenne%20de%20Bretagne) for their nancial support
during the project.
References
Abiven, S., Menasseri, S., Angers, D.A., Leterme, P., 2008. A model to predict soil
aggregate stability dynamics following organic residue incorporation under
eld conditions. Soil Science Society of America Journal 72, 119e125.
Abiven, S., Menasseri, S., Chenu, C., 2009. The effects of organic inputs over time on
soil aggregate stability e a literature analysis. Soil Biology and Biochemistry 41,
1e12.
Allison, S.D., Hanson, C.A., Treseder, K.K., 2007. Nitrogen fertilization reduces
diversity and alters community structure of active fungi in boreal ecosystems.
Soil Biology and Biochemistry 39, 1878e1887.
Angers, D.A., Caron, J., 1998. Plant-induced changes in soil structure: processes and
feedbacks. Biogeochemistry 42, 55e72.
Angers, D.A., Chenu, C., 1998. Dynamics of soil aggregation and C sequestration. In:
Lal, R., Kimble, J.M., Follett, R.F., Stewart, B.A. (Eds.), Soil Processes and the
Carbon Cycle. CRC Press, Boca Raton, FL, pp. 199e206.
Angers, D.A., Bullock, M.S., Mehuys, G.R., 2008. Aggregate stability to water. In:
Carter, M.R., Gregorich, E.G. (Eds.), Soil Sampling and Methods of Analysis,
second ed. CRC Press, Boca Raton, FL, pp. 811e820.
Annabi, M., Houot, S., Francou, C., Poitrenaud, M., Le Bissonnais, Y., 2007. Soil
aggregate stability improvement with urban composts of different maturities.
Soil Science Society of America Journal 71, 413e423.

C. Le Guillou et al. / Soil Biology & Biochemistry 50 (2012) 126e133


Balesdent, J., Chenu, C., Balabane, M., 2000. Relationship of soil organic matter
dynamics to physical protection and tillage. Soil and Tillage Research 53,
215e230.
Bastian, F., Bouziri, L., Nicolardot, B., Ranjard, L., 2009. Impact of wheat straw
decomposition on successional patterns of soil microbial community structure.
Soil Biology and Biochemistry 41, 262e275.
Baumann, K., Marschner, P., Smernik, R.J., Baldock, J.A., 2009. Residue chemistry and
microbial community structure during decomposition of eucalypt, wheat and
vetch residues. Soil Biology and Biochemistry 41, 1966e1975.
Boer, W.d., Folman, L.B., Summerbell, R.C., Boddy, L., 2005. Living in a fungal world:
impact of fungi on soil bacterial niche development. FEMS Microbiology
Reviews 29, 795e811.
Bossuyt, H., Denef, K., Six, J., Frey, S.D., Merckx, R., Paustian, K., 2001. Inuence of
microbial populations and residue quality on aggregate stability. Applied Soil
Ecology 16, 195e208.
Bremer, E., Van Kessel, C., 1992. Seasonal microbial biomass dynamics after addition
of lentil and wheat residues. Soil Science Society of America Journal 56,
1141e1146.
Bressan, M., Mougel, C., Dequiedt, S., Maron, P.-A., Lemanceau, P., Ranjard, L., 2008.
Response of soil bacterial community structure to successive perturbations of
different types and intensities. Environmental Microbiology 10, 2184e2187.
Caesar-TonThat, T.C., Cochran, V.L., 2000. Soil aggregate stabilization by a saprophytic lignin-decomposing basidiomycete fungus e I. microbiological aspects.
Biology and Fertility of Soils 32, 374e380.
Caesar-TonThat, T.C., 2002. Soil binding properties of mucilage produced by
a basidiomycete fungus in a model system. Mycological Research 106, 930e937.
Carter, M.R., 2002. Soil quality for sustainable land management. Agronomy Journal
94, 38e47.
Chenu, C., 1989. Inuence of a fungal polysaccharide, scleroglucan, on clay microstructures. Soil Biology and Biochemistry 21, 299e305.
Denef, K., Six, J., Bossuyt, H., Frey, S.D., Elliott, E.T., Merckx, R., Paustian, K., 2001.
Inuence of dry-wet cycles on the interrelationship between aggregate,
particulate organic matter, and microbial community dynamics. Soil Biology
and Biochemistry 33, 1599e1611.
Ding, X.L., Zhang, X.D., He, H.B., Xie, H.T., 2010. Dynamics of soil amino sugar pools
during decomposition processes of corn residues as affected by inorganic N
addition. Journal of Soils and Sediments 10, 758e766.
Djajakirana, G., Joergensen, R.G., Meyer, B., 1996. Ergosterol and microbial biomass
relationship in soil. Biology and Fertility of Soils 22, 299e304.
Drury, C.F., Stone, J.A., Findlay, W.I., 1991. Microbial biomass and soil structure
associated with corn, grasses, and legumes. Soil Science Society of America
Journal 55, 805e811.
Dubois, M., Gilles, K.A., Hamilton, J.K., Rebers, P.A., Smith, F., 1956. Colorimetric
method for determination of sugars and related substances. Analytical Chemistry 28, 350e356.
Edgerton, D.L., Harris, J.A., Birch, P., Bullock, P., 1995. Linear relationship between
aggregate stability and microbial biomass in 3 restored soils. Soil Biology and
Biochemistry 27, 1499e1501.
FAO/ISRIC/ISSS, 1998. World Reference Base for Soil Resources. Food and Agriculture
Organization of the United Nations, Rome, Italy.
Fierer, N., Bradford, M.A., Jackson, R.B., 2007. Toward an ecological classication of
soil bacteria. Ecology 88, 1354e1364.
Gong, P., Guan, X., Witter, E., 2001. A rapid method to extract ergosterol from soil by
physical disruption. Applied Soil Ecology 17, 285e289.
Hadas, A., Parkin, T.B., Stahl, P.D., 1998. Reduced CO2 release from decomposing
wheat straw under N-limiting conditions: simulation of carbon turnover.
European Journal of Soil Science 49, 487e494.
Harris, R.F., Allen, O.N., Chesters, G., Attoe, O.J., 1963. Evaluation of microbial activity
in soil aggregate stabilization and degradation by the use of articial aggregates. Soil Science Society of America Journal 27, 542e545.
Harris, R.F., Chesters, G., Allen, O.N., 1966. Dynamics of soil aggregation. In:
Norman, A.G. (Ed.), Advances in Agronomy. Academic Press, pp. 107e169.
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, 73e83.
Knapp, E.B., Elliott, L.F., Campbell, G.S., 1983. Carbon, nitrogen and microbial
biomass interrelationships during the decomposition of wheat straw e
a mechanistic simulation-model. Soil Biology and Biochemistry 15, 455e461.
Lal, R., 2009. Soil quality impacts of residue removal for bioethanol production. Soil
and Tillage Research 102, 233e241.
Le Bissonnais, Y., 1996. Aggregate stability and assessment of soil crustability and
erodibility .1. theory and methodology. European Journal of Soil Science 47,
425e437.
Le Guillou, C., Angers, D.A., Leterme, P., Menasseri-Aubry, S., 2011. Differential and
successive effects of residue quality and soil mineral N on water-stable

133

aggregation during crop residue decomposition. Soil Biology and Biochemistry


43, 1955e1960.
Lynch, J.M., Bragg, E., 1985. Microorganisms and soil aggregate stability. Advances in
Soil Science 2, 134e170.
Maron, P.A., Schimel, J.P., Gulledge, J, 1998. Microbial community structure and
global trace gases. Global Change Biololgy 4, 745e758.
Martin, J.P., Waksman, S.A., 1940. Inuence of microorganisms on soil aggregation
and erosion. Soil Science 50, 29e48.
Martin, J.P., 1971. Decomposition and binding action of polysaccharides in soil. Soil
Biology and Biochemistry 3, 33e41.
Martin, T.L., Anderson, D.A., 1943. Organic matter decomposition, mold ora, and soil
aggregation relationships. Soil Science Society of America Journal 7, 215e217.
McGuire, K.L., Treseder, K.K., 2010. Microbial communities and their relevance for
ecosystem models: decomposition as a case study. Soil Biology and Biochemistry 42, 529e535.
McMahon, S.K., Williams, M.A., Bottomley, P.J., Myrold, D.D., 2005. Dynamics of
microbial communities during decomposition of carbon-13 labeled ryegrass
fractions in soil. Soil Science Society of America Journal 69, 1238e1247.
Meidute, S., Demoling, F., Bth, E., 2008. Antagonistic and synergistic effects of
fungal and bacterial growth in soil after adding different carbon and nitrogen
sources. Soil Biology and Biochemistry 40, 2334e2343.
Metzger, L., Levanon, D., Mingelgrin, U., 1987. The effect of sewage-sludge on soil
structural stability e microbiological aspects. Soil Science Society of America
Journal 51, 346e351.
Pascault, N., Ccillon, L., Mathieu, O., Hnault, C., Sarr, A., Lvque, J., Farcy, P.,
Ranjard, L., Maron, P.-A., 2010. In situ dynamics of microbial communities
during decomposition of wheat, rape, and alfalfa residues. Microbial Ecology 60,
816e828.
Paterson, E., Sim, A., Osborne, S.M., Murray, P.J., 2011. Long-term exclusion of plantinputs to soil reduces the functional capacity of microbial communities to
mineralise recalcitrant root-derived carbon sources. Soil Biology and
Biochemistry 43, 1873e1880.
Poll, C., Marhan, S., Ingwersen, J., Kandeler, E., 2008. Dynamics of litter carbon
turnover and microbial abundance in a rye detritusphere. Soil Biology and
Biochemistry 40, 1306e1321.
Puget, P., Angers, D.A., Chenu, C., 1999. Nature of carbohydrates associated with
water-stable aggregates of two cultivated soils. Soil Biology and Biochemistry
31, 55e63.
Ramirez, K.S., Lauber, C.L., Knight, R., Bradford, M.A., Fierer, N., 2010. Consistent
effects of nitrogen fertilization on soil bacterial communities in contrasting
systems. Ecology 91, 3463e3470.
Ranjard, L., Poly, F., Combrisson, J., Richaume, A., Gourbire, F., Thioulouse, J., Nazaret, S.,
2000. Heterogeneous cell density and genetic structure of bacterial pools associated
with various soil microenvironments as determined by enumeration and DNA
ngerprinting approach (RISA). Microbial Ecology 39, 263e272.
Ranjard, L., Lejon, D.P.H., Mougel, C., Schehrer, L., Merdinoglu, D., Chaussod, R., 2003.
Sampling strategy in molecular microbial ecology: inuence of soil sample size
on DNA ngerprinting analysis of fungal and bacterial communities. Environmental Microbiology 5, 1111e1120.
Ranjard, L., Nowak, V., Echairi, A., Faloya, V., Chaussod, R, 2008. The dynamics of soil
bacterial community structure in response to yearly repeated agricultural
copper treatments. Research in Microbiology 159, 251e254.
Reinertsen, S.A., Elliott, L.F., Cochran, V.L., Campbell, G.S., 1984. Role of available
carbon and nitrogen in determining the rate of wheat straw decomposition. Soil
Biology and Biochemistry 16, 459e464.
Sparling, G.P., Shepherd, T.G., Kettles, H.A., 1992. Changes in soil organic-C, microbial-C and aggregate stability under continuous maize and cereal cropping, and
after restoration to pasture in soils from the Manawatu region, New-Zealand.
Soil and Tillage Research 24, 225e241.
Swift, M.J., Heal, O.W., Anderson, J.M., 1979. Decomposition in Terrestrial Ecosystems. Blackwell Scientic Publications, Oxford, 372 pp.
Thioulouse, J., Chessel, D., Doldec, S., Olivier, J.-M., 1997. ADE-4: a multivariate
analysis and graphical display software. Statistics and Computing 7, 75e83.
Tisdall, J.M., Oades, J.M., 1982. Organic matter and water-stable aggregates in soils.
Journal of Soil Science 33, 141e163.
Tisdall, J.M., 1991. Fungal hyphae and structural stability of soil. Australian Journal of
Soil Research 29, 729e743.
Vance, E.D., Brookes, P.C., Jenkinson, D.S., 1987. An extraction method for measuring
soil microbial biomass C. Soil Biology and Biochemistry 19, 703e707.
Waksman, S.A., Martin, J.P., 1939. The role of microorganisms in the conservation of
the soil. Science 90, 304e305.
White, N.A., Hallett, P.D., Feeney, D., Palfreyman, J.W., Ritz, K., 2000. Changes to
water repellence of soil caused by the growth of white-rot fungi: studies using
a novel microcosm system. FEMS Microbiology Letters 184, 73e77.