Identication of cellulolytic bacteria in soil by stable

isotope probing
Feth el Zahar Haichar,
1
Wafa Achouak,
1
Richard Christen,
2
Thierry Heulin,
1
Christine Marol,
1
Marie-France Marais,
3
Christophe Mougel,
4
Lionel Ranjard,
4
Jrme Balesdent
1
and
Odile Berge
1
*
1
CEA, DSV, DEVM, Laboratoire dEcologie Microbienne
de la Rhizosphre et des Environnements extrmes
(LEMiRE), UMR 6191 CNRS, CEA, Aix Marseille-Univ,
IFR-E 112, Saint-Paul-Lez-Durance F-13108, France.
2
Laboratoire de Biologie Virtuelle, Centre de Biochimie,
Parc Valrose, Universit de Nice, F- 06108 Nice,
France.
3
CERMAV-CNRS, 601 rue de la chimie BP 53, F- 38041
Grenoble Cedex 9, France.
4
INRA/Dijon, UMR INRA-Univ. Bourgogne, Laboratoire
de Microbiologie et Gochimie des Sols, France.
Summary
Plant residues, mainly made up of cellulose, are the
largest fraction of organic carbon material in terres-
trial ecosystems. Soil microorganisms are mainly
responsible for the transfer of this carbon to the atmo-
sphere, but their contribution is not accurately known.
The aim of the present study was to identify bacterial
populations that are actively involved in cellulose
degradation, using the DNA-stable isotope probing
(DNA-SIP) technique.
13
C-cellulose was produced by
Acetobacter xylinus and incubated in soil for 7, 14, 30
and 90 days. Total DNA was extracted from the soil,
the
13
C-labelled (heavy) and unlabelled (light) DNA
fractions were separated by ultracentrifugation, and
the structure of active bacterial communities was
analysed by bacterial-automated ribosomal intergenic
spacer analysis (B-ARISA) and characterized with
denaturing gradient gel electrophoresis (DGGE). Cel-
lulose degradation was associated with signicant
changes in bacterial community structure issued from
heavy DNA, leading to the appearance of new bands
and increase in relative intensities of other bands until
day 30. The majority of bands decreased in relative
intensity at day 90. Sequencing and phylogenetic
analysis of 10 of these bands in DGGE proles indi-
cated that most sequences were closely related to
sequences from organisms known for their ability to
degrade cellulose or to uncultured soil bacteria.
Introduction
Life on earth depends on photosynthesis, which results in
production of plant biomass having cellulose as the major
component (4060% of plant residues; Lynd et al., 2002).
Terrestrial plants contribute more than 70 10
9
Mg of
carbon annually to the global carbon budget (Paul and
Clark, 1989). Consequently, soil microorganisms that
catabolize the cellulosic material of plants inuence the
ow of energy from plant material to other trophic levels
and ultimately the release of CO
2
to the atmosphere.
Cellulose is a simple polymer, but it forms insoluble,
crystalline microbrils, which are highly resistant to enzy-
matic hydrolysis (Bguin and Aubert, 1994). The ability to
digest cellulose is restricted to microorganisms. Cellu-
lolytic organisms are widely distributed among many
genera in the domain Bacteria and in the fungal groups
within the domain Eucarya, although no cellulolytic
members of domain Archaea have yet been identied
(Lynd et al., 2002). Until now the investigation of cellu-
lolytic microorganisms in soil has been based on their
cultivation (Lednicka et al., 2000; Warnick et al., 2002;
Wirth and Ulrich, 2002) and most studies have involved
techniques based on dilution plating (Olson and Bakken,
1987) and detection of cellulolytic activity in vitro using a
range of different cellulose substrates, such as carboxym-
ethyl cellulose (CMC), substituted cellulose derivatives,
colloidal or amorphous cellulose, insoluble microcrystal-
line cellulose such as Avicel or lter paper (Wirth and
Ulrich, 2002).
Throughout the years, many features of cellulolytic bac-
teria have been described. Microbial degradation of
cellulose is characterized by the biosynthesis of multi-
component enzymes (Bguin and Aubert, 1994). These
cellulose hydrolysis enzymes can be divided into
three classes: endoglucanases (endo 1,4-b-D-glucan-4-
glucanohydrolase) that randomly attacks the cellulose
chain and splits b-1,4-glucosidic linkages, exoglucanases
(exo 1,4-b-D-glucan-4-cellobiohydrolase) that release
either cellobiose or glucose from the non-reducing end of
Received 2 October, 2006; accepted 5 October, 2006. *For
correspondence. E-mail odile.berge@cea.fr; Tel. (+33) 442257863;
Fax (+33) 442256648.

Present address: INRA, Unit Gochimie des
Sols et des Eaux, Europole mediterranneen de lArbois, BP 80, F-
13545 Aix-en-provence, France.
Environmental Microbiology (2007) 9(3), 625634 doi:10.1111/j.1462-2920.2006.01182.x
Journal compilation 2006 Society for Applied Microbiology and Blackwell Publishing Ltd
No claim to original French government works
cellulose, and cellobiase (b-glucosidase) that hydrolyses
cellobiose and other soluble cellodextrins to glucose
(Singh and Hayashi, 1995). These cellulases are either
free or cell-associated (Lynd et al., 2002). It has been
shown that maximal rates of cellulose degradation come
from mixtures of three or more cellulases with different
specicities acting synergistically, not from enzymes
acting alone (Reese et al., 1950; Wood et al., 1989).
Indeed, cellulase synergism has been demonstrated for
combinations of exoglucanases and endoglucanases
from different organisms (Barr et al., 1996) and for
enzymes from different bacteria (Kato et al., 2005). In soil,
it is likely that enzymes from many different organisms
function together during cellulose hydrolysis (Zhou and
Ingram, 2000). The ability to degrade cellulose aerobically
is widespread among several soil bacterial species in both
lamentous, e.g. Streptomyces and Micromonospora,
and non-lamentous, e.g. Cellulomonas, Bacillus,
Pseudomonas and Cytophaga, genera (Lynd et al., 2002;
Wirth and Ulrich, 2002). Although it is commonly believed
that the bulk of cellulose is degraded in aerobic environ-
ments, up to 510% is degraded under anaerobic condi-
tions exclusively by bacteria, e.g. Acetivibrio, Clostridium
and Ruminoccocus (Coughlan and Mayer, 1992). The
anaerobic bacteria use complexed cellulase systems, cel-
lulosomes, which allow concerted enzyme activity of dif-
ferent cellulases for highly efficient cellulose hydrolysis
(Lynd et al., 2002).
The aim of this study was to identify soil bacterial
species that are involved in the cellulose degradation,
using stable isotope probing (SIP; Radajewski et al.,
2000). We selected an agricultural soil from a long-term
eld experiment dedicated to carbon cycling studies. The
soil was amended with bacterial
13
C-cellulose and
molecular analysis was carried out on
13
C-labelled DNA
derived from cells that had incorporated
13
C-cellulose, or
breakdown products, after separation from
12
C-DNA by
ultracentrifugation.
Results
Biodegradation of cellulose
Degradation of the exogenous bacterial cellulose in our
soil was followed by CO
2
respiration in an experiment with
non-labelled bacterial cellulose. CO
2
respiration in soil
amended with cellulose was greater than that emitted by
bulk soil predicting that cellulose was biodegraded in
these conditions by soil organisms including bacteria and
eukaryotes (Fig. 1). On the basis of these data, we chose
four sampling dates (7, 14, 30 and 90 days) to study
the dynamics of cellulolytic bacterial communities in
13
C-
cellulose microcosm experiments by the DNA-SIP
approach.
DNA gradient
Centrifugation conditions optimized using
13
C-labelled
Escherichia coli DNA and unlabelled Rhizobium DNA
(data not shown), were applied for the separation of
nucleic acids extracted from soil microcosms. Figure 2
shows an example of the repartition of DNA and
13
C
labelling among gradient fractions of increasing density,
with DNA extracted from
13
C-cellulose microcosms after
7 days of incubation. DNA density prole presented a
unique peak ranging from 1.63 to 1.65 densities and,
13
C
labelling measured by isotope ratio mass spectrometry
(IRMS) increased slowly in the light fractions before a
dramatic rise showing the presence of labelled DNA
between 1.66 and 1.68 densities (Fig. 2). Similar proles
were obtained from DNA extracted from the other
microcosms. For all of them, one fraction from the ultra-
Fig. 1. Biodegradation of exogenous cellulose in a Versailles soil.
Temporal changes in CO2 efflux were determined from a soil
microcosm amended with unlabelled cellulose () and soil
microcosm without cellulose amendment (). Additional CO2 in
cellulose amended soil is mostly originated from cellulose C.
Fig. 2. Separation by CsCl density gradient centrifugation of light
and heavy DNA from a soil sample incubated with
13
C-cellulose
during 7 days. DNA distribution was quantied uorometrically ()
and d
13
C were measured by IRMS () within gradient fractions.
626 F. Z. Haichar et al.
Journal compilation 2006 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 9, 625634
No claim to original French government works
centrifuge tubes was selected as representative of the
light DNA and another as representative of the heavy
DNA. These fractions corresponded to buoyant density of
1.64 g ml
-1
CsCl and 1.68 g ml
-1
CsCl respectively.
Structure of active cellulolytic community by
bacterial-automated ribosomal intergenic spacer
analysis (B-ARISA)
In order to analyse the structures of bacterial community,
bacterial-automated ribosomal intergenic spacer analy-
sis (B-ARISA) was performed on light and heavy DNA
fractions derived from
13
C-cellulose microcosms and
control microcosm after 7, 14, 30 and 90 days of incu-
bation (Fig. 3). B-ARISA ngerprints at each date
showed complex proles with intergenic spacer (IGS)
size ranging from 300 to 1000 bp (Fig. 3). These
B-ARISA proles were very similar for each fraction of
the triplicate
13
C-cellulose microcosms (M1, M2 and M3),
showing a good reproducibility of the experimental con-
ditions (Fig. 3).
Principal component analysis (PCA) was achieved for
each date and statistical ellipses were generated to
compare treatments (Fig. 4). At day 7, PCA analysis
showed that the slight differences between B-ARISA pro-
les obtained from light and heavy DNA in
13
C-cellulose
microcosms were not signicant (Fig. 4). In contrast, after
14 days of incubation, B-ARISA proles obtained from
light and heavy DNA were signicantly different indicating
changes in community structure (Figs 3 and 4). Principal
component analysis of SIP-B-ARISA proles clearly dis-
criminated between heavy and light fractions issued from
13
C-cellulose microcosms until day 90 (Fig. 4). In addition,
B-ARISA proles derived from heavy DNA were different
for control microcosm and
13
C-cellulose microcosms after
incubation for 14, 30 and 90 days (Fig. 3).
Identication of active cellulolytic bacteria by denaturing
gradient gel electrophoresis (DGGE)
To identify the bacteria involved in
13
C-cellulose degrada-
tion, light and heavy DNA derived from
13
C-cellulose
Heavy F.
Heavy F.
M
L H Light F.
M1 M2 M3 M1 M2 M3
Control
13 13
C-cellulose C-cellulose
M
Day 14 Day 30
L H
M1 M2 M3 M1 M2 M3
Light F. Heavy F.
Control
13 13
C-cellulose C-cellulose
M M
984 bp
867 bp
L H
M1 M2 M3 M1 M2 M3
Light F. Heavy F.
Control
13 13
C-cellulose C-cellulose
M
M
1051 bp
Day 90
540 bp
759 bp
654 bp
420 bp
300 bp
Day 7
984 bp
1051 bp
867 bp
540 bp
759 bp
654 bp
420 bp
300 bp
L H
Control 13 13
C-cellulose C-cellulose
Light F.
M1 M2 M3 M1 M2 M3 M M
Fig. 3. Bacterial-automated ribosomal intergenic spacer analysis (B-ARISA) banding proles of bacterial communities derived from light and
heavy DNA fractions from CsCl density gradients. Light F. and Heavy F., respectively, represent light DNA fractions (unlabelled DNA) and
heavy DNA fractions (labelled DNA) derived from triplicate
13
C-cellulose microcosms (M1, M2 and M3). L and H represent, respectively, light
and heavy DNA fractions obtained from control microcosm. Arrowheads indicate bands that appeared or increased in relative intensity in the
heavy DNA fraction in response to
13
C-cellulose amendment. M represents markers.
DNA-SIP of cellulolytic bacteria in soil 627
Journal compilation 2006 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 9, 625634
No claim to original French government works
microcosms and control microcosm at days 7, 14, 30 and
90 were analysed by denaturing gradient gel electro-
phoresis (DGGE) providing complex banding proles
(Fig. 5). After 7 days, the DGGE prole generated from
heavy DNA fraction differed from that obtained from light
DNA fraction with the appearance of new bands (5, 7, 6
and 9) and an increase in relative intensities of existing
band (4). These differences persisted at day 14 with a
general increase in relative intensity and the appearance
of novel bands (1, 2, 3, 8 and 10). The differences
detected at day 14 persisted throughout the incubation
period until day 30. In contrast, after 90 days of
13
C-
cellulose incubation we observed decrease in relative
intensities of bands 8 and 9, disappearance of bands 2
and 3 and increase in relative intensity of band 1. Dena-
turing gradient gel electrophoresis proles derived from
light DNA were similar for
13
C-cellulose microcosms and
the control microcosm, while DGGE proles derived from
heavy DNA were different for control microcosm and
13
C-
cellulose microcosms at each sampling date (Fig. 5). The
bands 110 were not detected in DGGE proles derived
from heavy DNA from the control microcosm (Fig. 5), and
therefore were selected for identication.
Sequencing and phylogenetic analysis of the 10 bands
showed a broad phylogenetic distribution and indicated
that most correspond to organisms known to be able to
degrade cellulose (Fig. 6).
Discussion
In this study we identied, by DNA-SIP, bacteria involved
in cellulose degradation in a complex soil environment. To
our knowledge, this is the rst application of DNA-SIP to
investigate assimilation of an insoluble, high-molecular-
weight organic compound. Highly
13
C-labelled pure cellu-
lose, structurally similar to that produced by plants (Wong
et al., 1990), was readily obtained by the cellulose-
producing bacteria Acetobacter xylinus. Isotope ratio
mass spectrometry analyses demonstrated that some
microbial populations incorporated the
13
C isotope into
their DNA, conrming in situ assimilation of this bacterial
13
C-cellulose in soil.
Statistical analysis of B-ARISA proles obtained inde-
pendently from the triplicate microcosms showed that sig-
nicant differences obtained between heavy and light
DNA were not due to random error but to the effect of the
treatments. These analyses allowed us to identify repre-
sentative labelled populations by sequencing specic
DGGE bands.
Density gradient centrifugation was employed to sepa-
rate unlabelled and labelled DNA (Lueders et al., 2004;
Freitag et al., 2005), but this can also lead to fractionation
through differences in nucleic acid GC-content (Holben
et al., 2004). Comparison of heavy DNA DGGE proles
derived from control and
13
C-cellulose microcosms at
Fig. 4. Principal component (PC1 PC2)
plots generated from B-ARISA proles of
bacterial communities obtained from DNA
extracted from
13
C-cellulose microcosms at 7,
14, 30 and 90 days after isopycnic CsCl
density centrifugation and fractionation. ,
independent triplicate of heavy DNA fraction;
, independent triplicate of light DNA fraction.
Ellipses represent 90% condence limits.
PC1 37%
PC1 41%
PC2 19%
7 days
PC2 16%
PC1 39%
14 days
PC2 19%
30 days PC2 14%
PC1 47%
90 days
628 F. Z. Haichar et al.
Journal compilation 2006 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 9, 625634
No claim to original French government works
each date provided condence that changes observed in
13
C-cellulose microcosms were not due to GC effects
(Fig. 5). Therefore, these changes could be related to
13
C
incorporation into DNA of bacteria involved in cellulose
degradation.
When using techniques involving substrate amend-
ment, it is difficult to determine whether increases in
bacterial growth result from assimilation of the substrate
by primary utilizers, or utilization of degradation products
by secondary utilizers. Stable isotope probing-based
time-course experiments allow to analyse the ow of
13
C
through microorganisms in the environment and to
examine microbial food chains and succession of popu-
lations (Dumont and Murrell, 2005). Mahmood and
colleagues (2005) used a SIP-based time-course experi-
ment in an elegant study to determine the primary
consumers of pentachlorophenol (PCP). In our study,
DNA-SIP detected labelled bacteria as soon as day 7
corresponding to early stages of cellulose degradation
(Fig. 1). At this sampling time, similarities between the
majority of sequences of excised DGGE bands (4, 5, 6,
7 and 9) and known cellulose degraders (Fig. 6) suggest
that these organisms represent the primary utilizers of
cellulose. Indeed, sequences determined from these
bands are most closely related, respectively, to Dyella,
Mesorhizobium sp., Sphingomonas sp., and uncultured
Deltaproteobacterium affiliated to Myxobacteria (Fig. 6).
Dyella, particularly Dyella koreensis sp. nov. recently
isolated by An and colleagues (2005) from soil around
the roots of bamboo plants in Korea, has b-glucosidase
activity and can utilize cellulose as sole source of carbon
and energy. Mesorhizobium belongs to the rhizobia
group, which is known to produce cellulolytic and pecti-
nolytic enzymes that can break glycosidic bonds present
in plant cell wall (Hubbell et al., 1978; Mateos et al.,
1992). Cellulases produced by rhizobia appear to be
involved in legume infection process (Higashi and Abe,
1980) but little attention has been paid to their potential
ability to degrade organic compounds found in soil
during growth as free-living saprophyte (Germida, 1988).
Our results suggest that rhizobia could play an important
role in degrading cellulose originating from plant in soil
during their saprophytic life. Sphingomonas sp. are ubiq-
uitous in the environment and are frequently isolated
from soil (Kelley et al., 2004). Mnnist and Hggblom
(2006) isolated Sphingomonas sp. from soil, stream
Day 7 Day 90 Day 14
L H
Control
Day 30
4
5
6
7
8
9
10
L H
13
C-cellulose
L H
Control
L H
Control
2
3
1
L H
13
C-cellulose
L H
13
C-cellulose Control
L H L H
13
C-cellulose
Fig. 5. Stable isotope probing (SIP)-based DGGE banding proles of bacterial 16S rRNA genes PCR-amplied from heavy (labelled) and light
(unlabelled) DNA fractions obtained by ultracentrifugation of CsCl density gradient of DNA extracted from control microcosm and
13
C-cellulose
microcosms at 7, 14, 30 and 90 days of incubation. Arrowheads indicate bands 110 that appeared and increased in relative intensity in the
heavy DNA fraction in response to
13
C-cellulose amendment (for phylogenetic analysis, see Fig. 6).
DNA-SIP of cellulolytic bacteria in soil 629
Journal compilation 2006 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 9, 625634
No claim to original French government works
water and lake sediments and some Sphingomonas iso-
lates exhibited cellulase activity. Uncultured Deltaproteo-
bacterium clone BIfdi19 was isolated from biolters used
for waste gas treatment in an animal-rendering plant.
Biolter packing material consisting of tree roots and
tree bark may be a signicant source of cellulose for the
microbial community (Friedrich et al., 2002). Moreover,
this uncultured Deltaproteobacterium is affiliated to the
cultivated group of Myxobacteria known to be special-
ized in degradation of cellulose (Yan et al., 2003). These
bacteria which are expected to be the primary consum-
ers of cellulose were detected until day 90. They
increased in abundance until day 30 before to decrease
at the end of the experiment.
At day 14, in addition to these bacteria, ve labelled
populations appeared in DGGE proles (bands 1, 2, 3, 8
and 10). Their sequences were related to uncultured
Xiphinematobacteriaceae, unidentied bacterium affili-
ated Bacteroidetes, uncultured Gammaproteobacterium
isolated from soil and to Streptomyces sp. Some of these
taxa are known for their ability to degrade cellulose and
could be also primary consumers of cellulose. Streptomy-
ces spp. are widely distributed in soil and are known to
play an important role in cellulose degradation (Wach-
inger et al., 1989; Semdo et al., 2000; Wirth and Ulrich,
2002). Most streptomycetes isolated from soil are unable
to produce the cellulase complex, producing only endo-
glucanase, and exoglucanase activity is provided by other
cellulolytic bacteria for complete hydrolysis of cellulose
(Semdo et al., 2000). In the Bacteroidetes group, most of
the bacteria are widely distributed in soil and exhibit cel-
lulase activity (Lednicka et al., 2000; Navarrete-Bolanos
et al., 2003). These populations detected at day 14 fol-
lowed the same dynamics as those detected at day 7.
They persisted until day 30 before to decline after 90 days
of incubation. These ndings could result from dilution of
DGGE gel band 10 {DQ923854}
uncultured Bacteroidetes bacterium E1C12 {AY632522}
uncultured Flavobacterium sp OF70 {AY177767}
DGGE gel band3 {DQ675012}
uncultured bacterium {AF010082}
uncultured Bacteroidetes bacterium LiUU-9-73 {AY509370}
DGGE gel band 1 {DQ675010}
uncultured soil bacterium HSBNT21_G01 {DQ128660}
uncultured Xiphinemato bacteriaceae bacterium EB1006{AY395325}
DGGE gel band 5 {DQ675014}
Mesorhizobium sp. GN25 {DQ862066}
Mesorhizobium sp. casi-1 {AY490133}
DGGE gel band 9 {DQ675016}
Myxobacterium sp. AT3-01 {AB246772}
uncultured bacterium BIfdi19 {AJ318128}
DGGE gel band 8 {DQ675015}
Streptomyces sp. SO6 {AY566563}
Streptomyces sp. SM4 SM4 {DQ887331}
DGGE gel band 2 {DQ675011}
uncultured bacterium S206D {AM158345}
gamma proteobacterium YU21-B {AB174846}
DGGE gel band 4 {DQ675013}
Dyella japonica XD22 {AB110497}
Dyella japonica 200D13 {AM086243}
uncultured Crater Lake bacterium CL0-92 {AF316779}
DGGE gel band 6 {DQ923856}
Sphingomonadaceae bacterium Ellin7157 {AY673323}
uncultured bacterium ABW-169 {AY456773}
DGGE gel band 7 {DQ923855}
0.05
+* 62%
+* 43%
+* 99%
+* 58%
+*99%
+ 61%
+* 99%
+* 100%
+* 100%
+* 100%
+* 98%
+* 100%
+* 100%
+* 100%
+* 72%
Fig. 6. Phylogenetic tree obtained using the neighbour-joining algorithm, reecting the relationships of 16S rRNA gene sequences obtained
from the bands 110 of DGGE ngerprints from heavy DNA fractions (see Fig. 5). Branches also supported by parsimony and
maximum-likelihood are indicated, respectively, by plus symbols (+) and asterisks (*). Percentages above branches correspond to 1000
bootstrap replicates using the neighbour-joining algorithm. GenBank accession numbers of all sequences are indicated.
630 F. Z. Haichar et al.
Journal compilation 2006 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 9, 625634
No claim to original French government works
label through respiration of cellulose and its degradation
products.
The detection of Xiphinematobacter species, an
obligate cytoplasmic symbiont of nematodes widely
distributed in soil (Vandekerckhove et al., 2000), was
unexpected and evidenced the spreading of label through
the microbial food chain. It is most likely that this symbiont
became labelled by cross-feeding on
13
C-metabolites
derived from cellulose degradation by the nematode
(Bera-Maillet et al., 2000; Goellner et al., 2001) or through
predation of primarily cellulose degraders by the
nematode. This last hypothesis could be supported by the
increase of the Xiphinematobacter population at day 90.
Previous studies have advocated DNA-SIP as an ideal
tool to identify microorganisms involved in organic com-
pound degradation (Padmanabhan et al., 2003; Singleton
et al., 2005). Here we report the rst application of SIP-
based time-course technique to analyse the dynamics of
bacterial communities involved in biodegradation of non-
soluble polymeric substrate in soil system.
Our experiment, over time, had apparently evidenced a
stimulation and enrichment of bacteria most closely
related to known cellulose degraders. These ndings
underscore the importance of conducting time-course
analyses in DNA-SIP.
Identication of known soil cellulolytic bacteria by DNA-
SIP as well as uncultured bacteria and bacteria not pre-
viously known to degrade cellulose in soil contributes to
the validation of this approach.
For DNA-SIP, minimum of two cell divisions in combi-
nation with the assimilation of carbon depending mostly
on the
13
C-substrate are prerequisites for a highly
13
C-
labelled genomic DNA. RNA-SIP, in contrast, has the
advantage of high sensitivity, as
13
C incorporation into
RNA is greater and occurs earlier than into DNA (Man-
eeld et al., 2002). The application of RNA-SIP will allow
detection of cells which become activated but have not yet
divided, or that might be metabolically active but not
growing. In future SIP studies it may be advisable to
improve our label application strategy in order to probe
active community rather than enrichment (for review, see
Friedrich, 2006).
This study focused on degradation of cellulose by bac-
teria and, to our knowledge, no prior study has identied
in situ active cellulose degrading bacteria. It would also be
of great interest to identify cellulose-degrading soil fungi,
which are likely to contribute to cellulose metabolism
(Lynd et al., 2002).
Stable isotope probing provides an important new tool
for investigating members of microbial communities that
are directly involved in biodegradation of polymeric sub-
strates, in contrast to established molecular analysis
which does not distinguish between active and dormant
organisms.
Experimental procedures
Production of bacterial
13
C-cellulose
Acetobacter xylinus was used to produce pure
13
C-cellulose
from glucose. Acetobacter xylinus strain ATCC 12733
T
was
grown on minimal medium supplemented with 2%
13
C-
glucose (
13
C = 99 atom%, Martek Biosciences Corporation)
as sole carbon source for 28 days at 30C under static
conditions. Bacteria were pre-cultured on
13
C to minimize the
proportion of inoculum carbon (
12
C) in the nal product. Bac-
terial cellulose was synthesized as opaque pellicles on the
surface of the growth medium and was puried according to
Gromet-Elhanan and Hestrin (1962) and then freeze-dried.
The
13
C labelling of the bacterial cellulose was determined by
IRMS as 99% atom
13
C.
Incubation of soil supplemented with bacterial
13
C-cellulose
The soil used is a eutric Cambisol from Versailles, France
(I.N.R.A. Unit de Science du sol), continuously cropped with
winter wheat for 8 years, representative of large areas in
northern France, and studied for soil carbohydrate dynamics
(Derrien et al., 2006). Sieved (1 mm), air-dried soil was
moistened (120 g deionized water kg
-1
) and pre-incubated at
24C for 14 days in the dark to reach a biological steady
state. Soil moisture was then adjusted to 170 g kg
-1
, prior to
13
C-cellulose input (2 g kg
-1
soil dry weight, conditioned in
small pieces). The added
13
C-cellulose represented approxi-
mately 6% of the native soil C. Soil amended with
13
C-
cellulose was incubated in triplicate microcosms (M1, M2 and
M3, 100 g soil in each) at 24C in the dark. Control micro-
cosm was set up in the same manner, but was not amended
with cellulose. After incubation for 7, 14, 30 and 90 days, 24 g
of soil was sampled from each microcosm and preserved at
-20C. A parallel incubation of non-labelled bacterial cellu-
lose was performed to follow cellulose degradation over time
by measuring CO
2
efflux.
DNA extraction
For all incubation dates, nucleic acids were extracted from
3 g of soil from each microcosm according to Ranjard and
colleagues (2003). DNA samples were visualized and quan-
tied by electrophoresis in a 1% agarose gel. Yields of total
DNA extracted from microcosms were between 1.5 and
2.5 mg g
-1
soil. DNA was preserved frozen (-20C).
Isopycnic centrifugation and gradient fractionation
Heavy and light DNA were separated by density gradient
ultracentrifugation using CsCl as described by Lueders and
colleagues (2004). The gradient mixture consisted of mixing
3.95 ml of CsCl solution containing exactly 1.9 g of solid CsCl
(Sigma) for every millilitre of TE (0.1 M Tris-HCl, pH 8; 1 mM
EDTA) with 0.95 ml of DNA solution in TE. Density gradient
centrifugation was performed in 4.9 ml tubes in a NVT 90
rotor (Beckman) at 20C, 140 000 g, for 16 h. Centrifuged
gradients were fractionated from top to bottom by using 1 ml
DNA-SIP of cellulolytic bacteria in soil 631
Journal compilation 2006 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 9, 625634
No claim to original French government works
syringes tted with 21-gauge needles into fractions of
approximately 300 ml. Buoyant density was determined by
weighing and DNA was quantied in CsCl density fractions
with the PicoGreen assays (Molecular Probes). Nucleic acids
were puried from CsCl salts using geneClean Kit (Qbiogene,
Montreal, Qubec).
Isotope ratio determination
The
13
C labelling in CsCl density fractions was measured by
IRMS (Delta +, Thermonnigan) coupled with an elemental
analyser (Delta + and Cono, Thermonnigan, Thermo-
electron corp, Bremen). One to two micrograms of sample of
CsCl density fraction was placed into 5 9 mm tin capsules
(Microanalysis) and dried for 2 h at 60C. Tin capsules were
then submitted to mass spectrometry.
d
13
C () was determined using the equation:

13
sample standard
C ( ) = ( ) [ ] R R 1 1000
where R =
13
C/
12
C. The R
standard
was Pee Dee Belemnite
(Wang and Hsieh, 2002).
B-ARISA ngerprinting and PCA analysis
The bacterial ribosomal IGS were amplied with forward
primer 72 F (TGC GGc TGGATCACC TCC TT) uorescently
labelled at its 5 end with the IRD 800 uorochrome (MWG SA
Biotech, Ebersberg, Deutschland) and reverse primer 38 R
(CCG GGT TTC CCC ATT cGG). Polymerase chain reaction
(PCR) conditions were as described by Ranjard and col-
leagues (2001). The concentration of labelled PCR product
was estimated by agarose gel electrophoresis, and between
1 ml and 3 ml of the product was added to deionized forma-
mide (2 ml) and denatured at 90C for 2 min. B-ARISA frag-
ments were resolved under denaturing conditions for 14 h at
3000 V on a LiCor DNA sequencer (ScienceTec, Les Ulis,
France), on 3.7% polyacrylamide gels. Data were converted
into B-ARISA covariance matrix using the 1 D-Scan (Science
Tec) and PrepRISAprogram (Ranjard et al., 2001) and analy-
sed using PCA ADE-4 software (Thioulouse et al., 1997).
Statistical ellipses representing 90% condence on PCAplots
were used to statistically compare the B-ARISA proles.
DGGE analysis
16S rRNA genes were amplied using a nested PCR
approach. The rst PCR amplication step was performed
using universal bacterial primers fD1 (5-AGAGTTTGA
TCCTGGCTCAG-3, position 827 on the E. coli rrs gene),
and S17 (5-GTTACCTTGTTACGACTT-3, position 1492
1509 on the E. coli rrs gene). For the second PCR step, PCR
products were amplied using primers 375f-GC and 518r
(Muyzer et al., 1993) to generate 233 bp products for DGGE
analysis. Polymerase chain reaction amplication conditions
were performed according to Rangel-Castro and colleagues
(2005).
Denaturing gradient gel electrophoresis analysis of the
nal PCR products was carried out using the DCode
universal mutation detection system (BIO-Rad Laboratories,
France) using polyacrylamide gels with 3260% denaturing
gradients. Gels were run for 16 h at 75 V in 1 TAE buffer at
a constant temperature of 60C. Following electrophoresis,
the gels were silver-stained and scanned as described by
McCaig and colleagues (2001).
Recovery and purication of DNA template from DGGE
bands
Representative bands appearing and increasing in relative
intensity in the heavy DNA proles were excised and
sequenced. The bands that migrated to a similar position in
the gel were presumed to have similar sequences and there-
fore only representative bands (with identical migratory posi-
tion) were excised for subsequent DNA sequence analysis.
Denaturing gradient gel electrophoresis gels were run as
described above, except that they were stained with a
1:10 000 dilution of SYBR Green I nucleic acid stain (Sigma-
Aldrich, USA) in 1 TAE for 20 min. The bands of interest
were excised with a sterile razor while the gels were illumi-
nated under ultraviolet. DNA was eluted from excised bands
by incubation of the band in 25 ml of sterile H
2
O at 65C for
30 min followed by centrifugation at 5000 g for 1 min
(Mahmood et al., 2005). The eluted DNA (1 ml) was used as
template for PCR amplication under the conditions
described by Rangel-Castro and colleagues (2005), except
with 25 amplication cycles, rather than 35. In most cases, as
mentioned by Mahmood and colleagues (2005), the PCR
product analysed by DGGE yielded a number of bands in
addition to the band of interest, necessitating further rounds
of band excision, PCR amplication and DGGE analysis to
check purity. The correct migration positions of PCR products
of puried bands were conrmed by DGGE analysis of these
products and environmental PCR products on the same gel.
Sequencing and phylogenetic analysis
Polymerase chain reaction products were sequenced with
518r primer (Muyzer et al., 1993) at Genome express
(Meylan, France). Each band sequence was blasted at NCBI
against the Bacteria division in order to retrieve the 10 most
similar sequences (no Filter). These sequences were auto-
matically aligned, the alignments were then carefully adjusted
by hand (using SeaView, Galtier et al., 1996). A well-aligned
domain of 95 positions common to every sequence was used
for phylogeny, using a neighbour-joining method (Gascuel,
1997). Each band sequence was also similarly blasted
against a local database of sequences corresponding to cul-
tured strains. The ve most similar sequences were retained
and analysed as above.
From these two analyses the two closest sequences to
band sequences are retained for the nal analysis. These
sequences were analysed using the BIONJ algorithm
(Gascuel, 1997), DNADIST (Kimura-2 parameters correc-
tion), DNAPARS, DNAML (with G option) and consens from
the PHYLIP package. A bootstrap analysis of 1000 replica-
tions was also performed using BIONJ algorithm. In the
maximum-likelihood tree, every branch indicated with an
asterisk (*) in Fig. 6 was signicantly positive at P < 0.01. The
tree was nally drawn using TreeDyn (http://www.treedyn.
org/).
632 F. Z. Haichar et al.
Journal compilation 2006 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 9, 625634
No claim to original French government works
Accession numbers
The sequences from this study were deposited in the
GenBank nucleotide sequence database under the Acces-
sion No. DQ675010DQ675016 and DQ923854DQ923856.
Acknowledgements
We thank J.I. Prosser for the critical review of this article and
D. Rumeau for advices concerning ultracentrifugation. We
are grateful to J.I. Prosser, J.I. Rangel-Castro and G.W. Nicol
for their help in DGGE method set-up and for discussions. We
thank also A. De Groot for critical reading of this article. This
work was supported by a CNRS pHD grant and by the
MICROGER program. MICROGER is funded jointly by the
INRA cologie pour la Gestion des cosystmes et de leurs
Ressources (ECOGER) project of the ANR ECosphre COn-
tinentale (ECCO) National Program, and by the ADEME
Gestion Biologique et Sols Department.
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