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Soil Biology & Biochemistry 32 (2000) 1405±1417
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Response of the bacterial community to root exudates in soil
polluted with heavy metals assessed by molecular and cultural
approaches
Jacek KozdroÂj a,*, Jan Dirk van Elsas b
a

b

Department of Microbiology, University of Silesia, Jagiellonska 28, 40-032 Katowice, Poland
Research Institute for Plant Protection (IPO-DLO), P.O. Box 9060, 6700 GW Wageningen, The Netherlands
Received 16 June 1999; received in revised form 28 October 1999; accepted 16 February 2000

Abstract
We have used PCR based on 16S rDNA sequences followed by denaturing gradient gel electrophoresis (PCR-DGGE) in
conjunction with cultivation-based methods to describe the e€ect of arti®cial root exudates (ARE), of which the composition
simulated maize root exudates, on the structural diversity of bacterial communities in various soils di€ering in the level of
contamination with heavy metals. The aim of this study was to evaluate the e€ects of organic compounds of a root exudates as
a potential mechanism for selectively enhancing speci®c bacterial populations in contaminated soils, leading to the development

of shifted communities di€ering in qualitative and quantitative composition. Soil microcosms were either just enriched with
ARE or enriched and, additionally, ¯ooded. To characterise the response of the soil micro¯ora to the enrichment, PCR-DGGE
was applied for assessment of the total bacterial community structure. Cultivation techniques were used to determine the
numbers of total heterotrophic bacteria as well as of pseudomonads (which are considered to be stimulated by components of
root exudates). The community structure of culturable bacteria was studied using the concept of r- and K-strategists, and
isolates from dominant colonies growing on King's B agar were identi®ed by MIDI-FAME pro®ling. The results obtained
showed a signi®cant e€ect of root exudates on the development of bacterial populations in soil contaminated with heavy metals.
Depending on their availability and conditions prevailing in the habitat (e.g. stronger enrichment by ¯ooding) di€erent bacterial
populations were stimulated, resulting in generation of di€erent community patterns by DGGE. The most signi®cant response to
root exudates occurred among the culturable fraction of the soil bacteria. Distribution of bacterial classes (i.e. majority of
colonies appeared after 24 h), values of EP (from 0.220 to 0.533) and CD (from 43 to 88) indices directly showed that the
culturable fraction of bacteria was highly a€ected by the organic mixture simulating root exudates. These exudates reduced the
bacterial diversity towards domination of r-strategists and the reduction of diversity was greater in soil with a higher
contamination level. Furthermore, ¯ooding of the soils enhanced the dominance of fast growing bacteria (over 70% formed
visible colonies after 24 h even on day 6) and reduced the community diversity (EP and CD indices were from about 0.291 to
0.425 and from 66 to 87, respectively). 7 2000 Elsevier Science Ltd. All rights reserved.
Keywords: Microbial community structure; Root exudates; Genetic ®ngerprint; Soil enrichment

1. Introduction
In heavily industrialized areas, numerous soil sites

are contaminated with prohibitively high concen-

* Corresponding author. Fax: +48-32-2555873.
E-mail address: kozdroj@us.edu.pl (J. KozdroÂj).

trations of heavy metals, which a€ect normal agricultural practices. A potential strategy to remediate these
soils is the use of plants to remove pollutants from the
habitat or to render them harmless (Salt et al., 1998).
Recently, di€erent metal tolerant plants such as
Thlaspi caerulescens, T. ochroleucum, Brassica juncea,
Hordeum vulgare, Avena sativa, and others have been
used for phytoextraction of Cd, Cu, Ni, Pb and Zn

0038-0717/00/$ - see front matter 7 2000 Elsevier Science Ltd. All rights reserved.
PII: S 0 0 3 8 - 0 7 1 7 ( 0 0 ) 0 0 0 5 8 - 4

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J. KozdroÂj, J.D. van Elsas / Soil Biology & Biochemistry 32 (2000) 1405±1417

from contaminated soil (Ebbs and Kochian, 1997,
1998; McGrath et al., 1998; Salt et al., 1998). Successful application of plants for remediation of polluted
soils depends on the phenotype and genotype of
plants, but interactions between the rhizosphere micro¯ora and plant roots are also of great signi®cance.
However, such microbiological aspects, to our understanding, have seldom been addressed (Burd et al.,
1998; Hasnain et al., 1993; Tichy et al., 1993).
In the rhizosphere, microbial biomass, activity and
community structure are highly in¯uenced by speci®c
physicochemical and biological characteristics prevailing in this habitat (Pearce et al., 1995; SoÈrensen, 1997).
Root exudates are known as one of the most important factors a€ecting these microbiological parameters.
These exudates comprise di€erent organic compounds;
their amounts and composition are dependent on plant
genotype, plant growth stage and environmental conditions such as CO2, light, pH, temperature, moisture
and nutrients (Grayston et al., 1996). Organic acids
occurring in root exudates enhance the mobilisation of
several metals through weathering and chelation
(Grayston et al., 1996; Vaugham et al., 1993). The bacterial communities in the rhizosphere, which can use
these substrates, di€er in composition and density
(SoÈrensen, 1997). However, the knowledge of microbial
diversity in the rhizosphere is far from complete, since

both traditional plating techniques and microscopical
techniques developed to date have important limitations (Liesack et al., 1997; Madsen, 1996; Wellington
et al., 1997). Therefore, the application of molecular
biological techniques to detect and identify microorganisms by molecular markers, such as 16S rRNA or
its corresponding gene (16S rDNA) is by far the most
widely used approach to explore microbial diversity
and to analyse the structure of microbial communities
(Bej and Mahbubani, 1996; Head et al., 1998; Hugenholtz et al., 1998; Liesack et al., 1997; van Elsas et al.,
1998).
A novel method, PCR followed by denaturing gradient gel electrophoresis (PCR-DGGE), was recently
proposed for studying complex microbial populations
(Muyzer et al., 1993). In this method, total microbial
DNA is extracted from soil and then the bacterial 16S
rDNA is ampli®ed by PCR with universal eubacterial
primers. The PCR-ampli®ed 16S rDNA fragments of
the same length but with di€erent sequences can be
separated in polyacrylamide gels containing a linearly
increasing gradient of denaturants. The patterns
obtained by DGGE or the related technique, temperature gradient gel electrophoresis (TGGE), provide information about the structural diversity of bacterial
groups, including the nonculturable ones (Muyzer and

Smalla, 1998).
Recently, Griths et al. (1999) using community
DNA hybridisation, % G + C pro®ling and phospho-

lipid fatty acid analysis (PLFA), showed signi®cant
changes in microbial community structure in response
to synthetic root exudates, which were applied continuously to a soil held at constant water potential. The
microbial community structure changed consistently as
substrate loading increased, and fungi dominated over
bacteria at high substrate loading rates. Bossio and
Scow (1995) showed that carbon inputs into soil and
¯ooding increased counts of active bacteria and respiration rates, but decreased the metabolic diversity of
bacterial populations. Despite these observations, the
knowledge about relationships between bacterial community and root exudates is still limited. In addition,
the possible roles of rhizosphere processes in phytoremediation of polluted soil are poorly understood.
In this study, we used PCR-DGGE in conjunction
with cultivation-based methods to describe the e€ect of
arti®cial root exudates (ARE), which mimicked maize
root exudates, on the structural diversity of bacterial
communities in various soils di€ering in the level of

contamination with heavy metals. The aim of this
study was to evaluate the action of the organic compounds in root exudates as a potential mechanism for
selective enhancement of bacterial populations in contaminated soils, leading to the development of novel
communities. These root-dependent changes in the soil
micro¯ora composition may be an essential process in
successful phytoremediation of soils polluted with
heavy metals. The remediation activity that is thought
to be associated with plants is often due to the activity
of plant-associated microorganisms (O'Connell et al.,
1996).

2. Materials and methods
2.1. Soil
Composite soil samples (each prepared from eight
di€erent randomly-collected cores) were collected from
the surface (0±10 cm) at two sites (PS1 and PS19) of
Piekary Slaskie in Silesia, a highly industrialized region
of Poland. The soil with low contents of heavy metals
was the silt loam PS1 (37% sand, 54% silt, 11% clay,
3.4% organic C, pHKCl 6.2, 160 mg Pb kgÿ1, 4.4 mg

Cd kgÿ1, 330 mg Zn kgÿ1). The more contaminated
soil was silt loam PS19 (37% sand, 54% silt, 10%
clay, 4.6% organic C, pHKCl 6.3, 1830 mg Pb kgÿ1,
23.3 mg Cd kgÿ1, 2390 mg Zn kgÿ1). Concentrations
of heavy metals were determined by atomic absorption
spectrophotometry after soil extraction with aqua regia
(conc. HNO3/conc. HCl, 1:3, vol/vol). All soil samples
were sieved (2 mm-mesh) and stored moist at 48C
prior to use. For microcosm experiments, two air-dried
(about 10% moisture content) 100 g portions of PS1
and PS19 were placed into 300 ml Erlenmeyer ¯asks

J. KozdroÂj, J.D. van Elsas / Soil Biology & Biochemistry 32 (2000) 1405±1417

and wetted with either 20 ml of ARE or distilled water
(control), establishing a moisture content equal to
70% WHC (Alef and Nannipieri, 1995). One set of
soils was additionally ¯ooded with 35 ml of distilled
water, receiving a 50-mm layer of water over the soil.
The ¯asks were covered with para®lm and incubated

at room temperature, under gentle shaking (80 rpm) in
case of the ¯ooded microcosms. The ARE were based
on the sugar, organic acid and amino acid composition
of root exudates of maize (Kra€tczyk et al., 1984).
The concentrations of particular components of ARE
were recalculated from the data of Kra€tchyk et al.
(1984), considering their contents per 1 g of soil contained in 1 mm zone of the rhizosphere (Table 1).
2.2. DNA analysis
DNA was extracted from 1-g soil samples after 1, 3
and 6 days of PS1 and PS19 incubation by the method
of Saano and Lindstrom (1995). Puri®cation of the
crude extracts was by CsCl and potassium acetate precipitation steps followed by Wizard (Promega, USA)
spin column treatment (van Elsas et al., 1997). Absorbency measurements at A260 and A280 were determined
with a GeneQuant RNA/DNA calculator (Pharmacia,
Sweden) and a small-volume quartz cuvette to calculate the concentration (1 A260 unit ˆ 50 mg mlÿ1
double-stranded DNA) and the A260/A280 purity ratio
of DNA samples (Crecchio and Stotzky, 1998). DNA
quality (size) was checked by electrophoresis in 0.8%
(wt/vol) horizontal agarose gel run in 0.5% TBE bu€er
and stained with 0.9 mg mlÿ1 of ethidium bromide

(Sambrook et al., 1989).
A 1-ml volume (roughly 5±10 ng) of each extracted
DNA was ampli®ed by PCR with a Peltier thermal
cycler PTC 200 (MJ Research, USA). The PCR mixture used contained 0.2 mM of each primer, 200 mM of
each dNTP, 5 ml of 10  Sto€el bu€er (Perkin-Elmer,
USA), 5 U of AmpliTaq Sto€el fragment (Perkin-

Table 1
Composition of arti®cial root exudates (ARE) of maize based on
Kra€czyk et al. (1984)
Sugars (mg gÿ1)a

Organic acids (mg gÿ1)

Amino acids (mg gÿ1)

Glucose
Arabinose
Fructose
Saccharose

Oxalic acid
Fumaric acid
Malic acid
Citric acid
Succinic acid
Benzoic acid
Tartaric acid
Glutaric acid

Glutamic acid
Proline
Alanine
Glycine
Leucine
Serine
Arginine
Glutamine
Valine

7.080
2.760
2.340
1.530

1.960
3.660
0.260
0.460
0.310
0.190
0.070
0.030

0.068
0.061
0.057
0.037
0.017
0.025
0.030
0.012
0.010

a
All component concentrations were calculated per 1 g of soil contained in 1 mm zone of the rhizosphere.

1407

Elmer, USA), 3.75 mM MgCl2, 0.5 ml of 1% (vol/vol)
formamide, 0.25 mg T4 gene 32 protein (Boehringer,
Mannheim, Germany) and sterile Milli-Q water to a
®nal volume of 50 ml. The primers for PCR were
speci®c for conserved bacterial 16S rDNA sequences
(Heuer and Smalla 1997). PCR with primers R1401 (5 '
GCG TGT GTA CAA GAC CC-3 ') and F968GC (5 '
GC clamp-AAC GCG AAG AAC CTT AC-3 ') ampli®ed a bacterial 16S rDNA fragment from positions
968 to 1401 (Escherichia coli numbering). The GC-rich
sequence attached to the 5' end of primer F968GC
prevents complete melting of the PCR products during
subsequent separation on the denaturing gradient
during DGGE (Muyzer et al., 1993). PCR ampli®cation was performed for 40 thermal cycles in a touchdown scheme (Duarte et al., 1998) as follows: after
initial denaturation of 4 min at 948C, each cycle consisted of denaturation at 948C for 1 min, primer
annealing at TA for 1 min, and primer extension at
728C for 1 min. In the ®rst 10 cycles, TA decreased by
28C every second cycle from 658C in the ®rst cycle to
578C in the 10th. In the last 30 cycles, TA was 558C.
Cycling was followed by ®nal primer extension at 728C
for 10 min. PCR products were visualised by electrophoresis in 1.2% (wt/vol) agarose gels after ethidium
bromide (0.9 mg mlÿ1) staining (Sambrook et al.,
1989). Strong bands of the expected size (450 bp) were
subjected to DGGE analysis.
DGGE (Muyzer et al., 1993; Heuer and Smalla,
1997) was performed with an Ingeny phorU-2 system
(Leiden, Netherlands). Samples of 20 ml of PCR product were loaded onto 6% (wt/vol) polyacrylamide
gels in 0.5  TAE bu€er. The polyacrylamide gels were
made with a denaturing gradient ranging from 45% at
the top of the gel to 65% at the bottom (where 80%
denaturant contains 5.6 M urea and 32% formamide).
The electrophoresis was run for 16 h at 608C and 100
V. After the runs, gels were removed from the setup
and stained for 60 min with SYBR green I nucleic acid
gel stain (Molecular Probes, Netherlands). The stained
gels were immediately photographed on an UV transillumination table with a CCD camera and scanned
(Biozym, Netherlands). Digital images of the gels
showed banding patterns that were analysed by the 1/0
clustering method of the NT-SYS program (Exeter
Software, USA) by using the unweighted pair group
with mathematical averages (UPGMA). This method
allowed the construction of dendrograms that show
clustering trends among the soil samples analysed.
2.3. Culturable fraction analysis
Soil samples (5 g) were collected after 1, 3 and 6
days of incubation and placed in Erlenmeyer ¯asks
containing 45 ml of 0.1% sterile NaPP (pH 7.0) and 5
g of gravel. The ¯asks were shaken at 200 rpm for 30

1408

J. KozdroÂj, J.D. van Elsas / Soil Biology & Biochemistry 32 (2000) 1405±1417

min. Serial 10-fold dilutions of the soils in 0.85%
NaCl were plated in duplicate on 0.1-strength TSA for
total bacterial counts (De Leij et al., 1993; van Elsas et
al., 1994), and numbers of pseudomonads were
counted on King's B agar (Difco proteose peptone no.
3 20 g, K2HPO4
3H2O 1.8 g, MgSO4
7H2O 1.5 g, 87%
glycerol 15 ml, agar 15 g, demineralized water 1 `, pH
7.2). Plates were incubated at 278C for 10 days, and
cfu numbers of the bacteria studied were determined
after 6 days.
The bacterial community structure was characterised
following the method described by De Leij et al.
(1993). Colonies on 0.1-strength TSA were enumerated
on a daily basis for ®ve times and, in addition, on day
10. This way, several classes of culturable organism
were generated per plate. The number of bacteria in
each class was expressed as a proportion (%) of the
total count. The di€erent distributions of the classes
gave an insight into the distribution of r- and K-strategists in each sample. R-strategists are de®ned here as
fast growing bacteria forming visible colonies within
24 h in response to enrichment, while K-strategists are
characterised by slow growth and colonies are produced later.
To characterise the community composition in the
form of a single value two indices were calculated. The
eco-physiological (EP) index proposed by De Leij et
al. (1993) was calculated according to the following
equation:
H 0 ˆ ÿS…pi  log pi †,
where pi represents each of the six classes de®ned
above as a proportion of the total population in the
soil sample ( pi-population in class i per total population). The more even the distribution of the classes,
the higher the EP-index. The colony development
(CD) index proposed by Sarathchandra et al. (1997)
was calculated as follows:

washing (Operating manual version 6.0, MIDI, USA).
The organic phase containing cellular FAME was separated by Hewlett Packard 6890 GC on a capillary column Ultra 2-HP (cross-linked 5% phenyl±methyl
silicone; 25 m, 0.22 mm ID; ®lm thickness, 0.33 mm)
with hydrogen as the carrier gas and analysed by Sherlock MIS software, using the aerobe method and
TSBA library version 3.9 (MIDI, USA).
2.4. Statistics
Prior to the DNA and cultural fraction analyses
each duplicate soil microcosm was sampled, and data
of bacterial counts (log cfu), EP and CD indices were
expressed as the means and treated statistically by oneway ANOVA (Statistica 7.0 PL). Since both the duplicates represented virtually identical pictures after PCRDGGE, the one showing better pattern was chosen for
presentation and a dendrogram drawing.

3. Results
Former work with di€erent methods of DNA
extraction from soil contaminated with heavy metals
showed that the direct method of bacterial cell lysis
with proteinase K at 378C (Saano and Lindstrom,
1995) and further extraction and puri®cation of the
crude extract yielded DNA pure enough for molecular
analysis with relatively high eciency. Therefore, this
method was chosen for DNA extraction from the PS
soils selected in this study. Two Silesian soils, i.e. PS1
and PS19, similar in structure and chemical characteristics, but di€erent in concentrations of heavy metals,
were used for the experiments. The addition of ARE
to the PS soils or additional enhancement of microbial
growth in the enriched soils by ¯ooding did not
increase the yields of extracted DNA (Table 2). After
puri®cation, DNA was colourless and showed A260/

CD ˆ …N1 =1 ‡ N2 =2 ‡ N3 =3 ‡ . . . ‡ N10 =10†  100,
where N1, N2, N3, . . .,N10 represent the proportion
(i.e. bacterial colonies appearing on each counting day
expressed as a proportion of the total number of colonies appearing over the 10 day period) of bacterial
colonies appearing on days 1, 2, 3, . . ., 10. The more
even the distribution of the classes is, the lower the
CD-index will be.
Dominant colonies growing on King's B agar were
selected and identi®ed based on whole-cell cellular
fatty acids, derivatized to methyl esters, i.e. FAME,
and analysed by gas chromatography (GC), using the
MIDI system (Microbial Identi®cation System, USA).
FAME were extracted from each isolate using the
standard and recommended procedure, consisting of
saponi®cation, derivatization, extraction and ®nal base

Table 2
Comparing yield of DNA (mg gÿ1 soil)a extracted from polluted soils
amended with arti®cial root exudates (ARE)b
Soil + treatment

Day 1

Day 3

Day 6

PS1 + ARE
PS1 + ARE + ¯ooding
PS1
PS19 + ARE
PS19 + ARE + ¯ooding
PS19

72.8
86.8
53.1
39.4
34.7
26.6

59.5 (36.7)
51.1 (28.3)
NDc
22.4 (11.7)
25.9 (3.1)
NDc

36.4 (11.4)
13.1 (9.2)
NDc
20.1 (19.9)
36.2 (3.0)
NDc

a

(28.4)
(29.3)
(15.4)
(23.8)
(0.4)
(1.3)

Assessed via A260 measurement.
Values are means and standard deviations in the parenthesis.
c
ND Ð DNA contents were not determined because we assumed
that DNA yields did not change in the nonenriched soils during several days.
b

J. KozdroÂj, J.D. van Elsas / Soil Biology & Biochemistry 32 (2000) 1405±1417

A280 values of 1.6±1.7. These DNA samples were suciently pure for subsequent PCR-DGGE analysis.
PCR ampli®cation of 16S rDNA fragments successfully generated 450 bp products visible as strong bands
in the gel after electrophoresis (data not shown).

1409

Samples collected from the soils enriched with ARE
or enriched and additionally ¯ooded showed variations
in banding patterns when analysed by PCR-DGGE
(Fig. 1). In all patterns, 24±31 bands of various intensities were detected per sample, with about 15 bands

Fig. 1. DGGE patterns of 16S rDNA fragments ampli®ed with DNA from soils polluted with heavy metals. The soils were either enriched with
ARE or enriched and additionally ¯ooded. (A) Untreated soils; lane 1, community pattern of PS1, lane 2, community pattern of PS19. (B)
Enriched soils; lanes 1±4, community patterns on day 6 of PS19 + ARE + ¯ooding, PS1 + ARE + ¯ooding, PS19 + ARE and PS1 + ARE,
respectively; lanes 5±8, community patterns on day 3 of PS19 + ARE + ¯ooding, PS1 + ARE + ¯ooding, PS19 + ARE and PS1 + ARE, respectively; lanes 9±12, community patterns on day 1 of PS19 + ARE + ¯ooding, PS1 + ARE + ¯ooding, PS19 + ARE and PS1 + ARE, respectively. Percent values indicate the percentage of denaturant at each position.

1410

J. KozdroÂj, J.D. van Elsas / Soil Biology & Biochemistry 32 (2000) 1405±1417

shared among all samples. Generally, 37 band positions were recognised in the DGGE gel. The highest
number of bands (31) was detected in the ¯ooded
PS19 on day 3. The other pro®les were not signi®cantly di€erent as to the number of bands detected. In
the control soils, 23 and 27 bands were found in PS1
and PS19, respectively. The di€erence in band intensity
was presumed to indicate numerical di€erences
between the target molecules.
Clustering of the pro®les revealed that all pro®les
were about 78% similar, with some trends with respect
to the clustering above this level (Fig. 2). One day
after soil enrichment with ARE, the pro®les of PS1
and PS19 were almost identical (99% similarity). However, when ¯ooded, PS1 and PS19 pro®les belonged to
di€erent clusters. On day 6, they showed 91% similarity and formed one cluster (Fig. 2). The pro®les
were the most di€erent on day 3 when they did not
show any trend with respect to the clustering. At the
end of soil microcosms' incubation, PS1 and PS19 pro®les formed two distinct clusters comprised of either
the ¯ooded soils or those only enriched with ARE.
Clustering of the banding patterns was not associated
with concentrations of heavy metals in the soils studied
(Fig. 2). The enrichment of the contaminated soils
with ARE or additional ¯ooding generated banding
pro®les that maintained 71% similarity to the pro®les
representing the bacterial communities of the untreated
soils.
The dendrogram represents only the qualitative similarity between the banding pro®les. This similarity
does not consider the intensities of bands. Each pro®le
showed several di€erent bands of high intensity. On

day 6, ¯ooding of the enriched PS1 and PS19 soils led
to the appearance of ®ve and two dominant bands in
the pro®les (at around 53±56% denaturant), respectively. In addition, two distinct bands (at around 50±
51% denaturant) were found in the ¯ooded PS19 after
6 days of incubation (Fig. 1). A single intensive band
around 62% denaturant was detected in the pro®le of
ARE enriched PS19 on day 3. This band, albeit less
intensive, was also present in the pro®les of enriched
PS1 and PS19 after 6 days of incubation (Fig. 1).
Determination of counts of culturable bacteria
showed a signi®cant positive e€ect of soil enrichment
with ARE under unsaturated and ¯ooded conditions.
The numbers of total heterotrophic bacteria did not
di€er signi®cantly between PS1 and PS19 when the
soils were enriched with ARE or left untreated (Fig. 3).
On day 6, the cfu counts decreased in ARE treated
PS1 and PS19. When these soils were ¯ooded, cfu
counts increased signi®cantly compared to the
un¯ooded conditions. In the PS1 soil, cfu counts of
total heterotrophs increased during incubation with
ARE after ¯ooding (Fig. 3). In contrast, the cfu numbers decreased in PS19 on day 6. Hence, the numbers
of total heterotrophic bacteria were highest in ¯ooded
PS19 on day 1 but in ¯ooded soil PS1 on day 6.
Similar trends in cfu counts were found for the bacteria growing on King's B agar (Fig. 4). However, signi®cant di€erences between cfu counts in un¯ooded
PS1 and PS19 soils enriched with ARE were found on
day 6. This was in contrast with the results of the bacterial counts determined on 0.1-strength TSA.
Representatives of bacteria that formed the highest
number of colonies on King's B agar were isolated

Fig. 2. Genetic similarity of microbial-community pro®les obtained with PCR-DGGE from soils contaminated with heavy metals. The soils PS1
and PS19 were either enriched with ARE or enriched and additionally ¯ooded. 1±4, the communities on day 6 in PS19 + ARE + ¯ooding, PS1
+ ARE + ¯ooding, PS19 + ARE and PS1 + ARE, respectively; 5±8, the communities on day 3 in PS19 + ARE + ¯ooding, PS1 + ARE +
¯ooding, PS19 + ARE and PS1 + ARE, respectively; 9±12, the communities on day 1 in PS19 + ARE + ¯ooding, PS1 + ARE + ¯ooding,
PS19 + ARE and PS1 + ARE, respectively; 13, the community in PS1 untreated; 14, the community in PS19 untreated.

J. KozdroÂj, J.D. van Elsas / Soil Biology & Biochemistry 32 (2000) 1405±1417

and identi®ed by MIDI-FAME pro®ling. Dominant
isolates from both PS soils were identi®ed as Comamonas acidovorans. In PS1 enriched with ARE, Arthrobacter oxydans and Stenotrophomonas maltophilia
dominated, whereas Variovorax paradoxus and C. acidovorans were isolated from ARE enriched PS19. When
enriched PS1 and PS19 were additionally ¯ooded, the
dominant isolates were identi®ed as V. paradoxus,
Pseudomonas putida and C. acidovorans, respectively.
Colonies of bacteria that developed on 0.1strength TSA were generally represented by fastgrowing organisms when they were isolated from
both ARE enriched or additionally ¯ooded PS soils
(Fig. 5). By contrast, higher numbers of visible
colonies were formed from 48 h onwards, when isolated from the unamended control soil. In the
lightly contaminated PS1 soil, a shift towards slowgrowing bacteria was found over time when the soil
was treated with ARE (Fig. 5). When the soil was
additionally ¯ooded, populations that formed visible
colonies after 24 h were dominating during incubation of the soil. In contrast, bacteria forming
colonies after 24 h dominated in PS19 enriched
with ARE after 3 and 6 days of the soil incubation, while they constituted only 35% on day 1
(Fig. 5). Compared to the untreated PS1, higher

1411

numbers of colonies were revealed after 48 h when
PS19 was analysed.
Calculation of EP indices showed that the enrichment of soil with ARE or additional ¯ooding
decreased the EP values as compared with those of the
unamended controls (Table 3). For the soils enriched
with ARE under un¯ooded conditions, EP values were
signi®cantly lower for total heterotrophic bacteria in
PS19 than in PS1. With the exception of the data
obtained on day 3, similar trends were observed for
the soils that were ¯ooded. However, the di€erences
between PS1 and PS19 were either small or not signi®cant. Generally, EP indices of the bacterial populations
from ¯ooded soils were lower than those from the soils
only enriched with ARE (Table 3).
CD values of the bacterial community on 0.1strength TSA were signi®cantly higher for both the
ARE enriched and the treated and subsequently
¯ooded soils compared with the untreated soils. Total
heterotrophic bacteria in treated and untreated soil
PS19 showed higher CD values than those of PS1, especially on day 3 and 6 (Table 3). Compared to the
EP indices, signi®cant di€erences between CD values
of the bacterial community originating from ¯ooded
PS1 enriched with ARE were found during incubation.
The same observation was found for bacteria isolated
from ARE enriched PS19, when CD values obtained

Fig. 3. Numbers (log cfu gÿ1 dry soil) of culturable heterotrophic bacteria in soils polluted with heavy metals. The numbers were determined
after 6 days of incubation at 278C. The soils PS1 and PS19 were either enriched with ARE or enriched and additionally ¯ooded. (A) Soil +
ARE; (B) soil + ARE + ¯ooding; (C) control.

J. KozdroÂj, J.D. van Elsas / Soil Biology & Biochemistry 32 (2000) 1405±1417

1412

Fig. 4. Numbers (log cfu gÿ1 dry soil) of culturable bacteria growing on King's B agar isolated from soils polluted with heavy metals. The numbers were determined after 6 days of incubation at 278C. The soils PS1 and PS19 were either enriched with ARE or enriched and additionally
¯ooded. (A) Soil + ARE; (B) soil + ARE + ¯ooding; (C) control.

on day 1 and 6 were compared (Table 3). Generally,
CD values of the bacterial community were higher for
¯ooded soils compared to the soils that were only
enriched with ARE.

4. Discussion
The excretion of di€erent organic compounds and
the sloughing o€ of root hairs and epidermal cells are
major factors responsible for the stimulation of micro-

organisms in the rhizosphere (Pearce et al., 1995; SoÈrensen, 1997). The excreted compounds are components
of root exudates, which play an important role in carbon ¯uxes into soil, a process also called rhizodeposition (Grayston et al., 1996; Pearce et al., 1995). The
rhizodeposition e€ect was studied in this work, in
which a mixture of sugars, organic acids and amino
acids was added to polluted soils. To enhance the
possible e€ect of these ARE on the indigenous soil
micro¯ora, and to make soil conditions more similar
to those of a rhizosphere in water-logged conditions

Table 3
Values of eco-physiological (EP) and colony development (CD) indices of culturable heterotrophic bacteria isolated from soils contaminated with
heavy metals and enriched with arti®cial root exudates (ARE)a
Soil

Treatment

Day 1
EP

PS1

PS19

a

ARE
ARE + ¯ood
Control
ARE
ARE + ¯ood
Control

0.492
0.425
0.616
0.362
0.401
0.486

Day 3
CD

(0.029)
(0.033)
(0.016)
(0.001)
(0.008)
(0.013)

69.2
65.7
32.3
65.9
78.0
43.7

EP
(2.2)
(0.4)
(1.8)
(2.3)
(0.2)
(3.1)

Values are the means and standard deviations in the parenthesis.

0.533
0.436
0.618
0.220
0.467
0.482

Day 6
CD

(0.016)
(0.021)
(0.024)
(0.028)
(0.039)
(0.011)

55.0
78.1
33.4
87.8
70.7
42.9

EP
(2.2)
(1.8)
(1.5)
(2.6)
(0.4)
(3.0)

0.636
0.394
0.620
0.377
0.291
0.484

CD
(0.025)
(0.051)
(0.020)
(0.020)
(0.006)
(0.015)

43.4
82.4
32.8
78.2
86.9
43.9

(0.9)
(4.3)
(2.0)
(0.4)
(1.9)
(2.6)

J. KozdroÂj, J.D. van Elsas / Soil Biology & Biochemistry 32 (2000) 1405±1417

(e.g. reduced oxygen potential, permanent easy access
to nutrients), the soils were ¯ooded and incubated as
slurries.
The total yields of DNA extracted from these soils
did not change in response to soil enrichment, however, the numbers of culturable heterotrophic bacteria
increased signi®cantly. This is contradictory to the
statement that concentrations of microbial DNA in
rhizosphere and bulk soil is related to microbial cell
densities, especially when the DNA content of microbial cells can be described using an average number
(Leung et al., 1995). However, for estimates of DNA
yields obtained from the rhizosphere it is important to
note that di€erent bacterial species have di€erent genome sizes as well as di€erent lysis characteristics (Trevors, 1996). Hence, sometimes obscure and variable
relationships between cell numbers and DNA yields
have been found (Leung et al., 1995). The concentrations of heavy metals in polluted soils also did not
have an e€ect on bacterial counts and, consequently,
DNA yields and purity. This could result from the fact
that both soils (i.e. PS1 and PS19), containing di€erent
concentrations of total heavy metals, likely characterised similar contents of bioavailable forms. In contrast,
Griths et al. (1997) reported that soils contaminated
with heavy metals, especially Pb, Zn and Cd, charac-

1413

terised by a lower microbial biomass and DNA yields,
compared to the uncontaminated control. However,
DNA extracted per unit of microbial biomass C was
increased in Cu and Ni contaminated soil, and
decreased by Pb, Zn and Cd. The authors did not ®nd
any correlation between DNA yields and microbial
biomass. Extractability of DNA may depend on the
type and physiological status of the microorganisms
present, and these factors may depend on the history
of soil contamination. Hence, a signi®cant e€ect of the
contamination on soil micro¯ora should be expected
even if no di€erences in culturable bacterial counts are
found.
The results obtained with PCR-DGGE showed that
soil enrichment with ARE slightly shifted the abundance of major bacterial groups (populations), especially when the soil was additionally ¯ooded. Root
exudates are known to a€ect microbial density and
species richness (SoÈrensen, 1997; Tate, 1995). However,
when analysing the whole bacterial community at molecular level, the observed e€ect of exudates may be
small or undetectable. Duineveld et al. (1998) using
PCR-DGGE found rather similar bacterial communities between the chrysanthemum rhizosphere and
bulk soil. Moreover, the DNA-based ®ngerprints indicated that the bacterial community structure, compris-

Fig. 5. Community structure of culturable fraction of bacteria in soil contaminated with heavy metals. The soils PS1 and PS19 were either
enriched with ARE or enriched and additionally ¯ooded. (A) Soil + ARE; (B) soil + ARE + ¯ooding; (C) control. Fast growing bacteria (rstrategists) form visible colonies within 24 h, and slow growing bacteria (K-strategists) form colonies later.

1414

J. KozdroÂj, J.D. van Elsas / Soil Biology & Biochemistry 32 (2000) 1405±1417

ing several dominant groups, was stable in soil and the
rhizosphere both in time and space. The authors
suggested that the potential impact of the root on bacterial populations in soil was not strong enough to
induce major shifts in community structure. The dominance of a reduced number of bacterial populations
was also found in the rhizosphere of potato after analysing of TGGE pro®les (Heuer and Smalla, 1997). In
contrast, the presence of speci®c organic compounds in
root exudates may even decrease the microbial diversity in the rhizosphere leading to the dominance of a
few species. This was shown by Smalla et al. (1998)
when 16S rDNA TGGE pro®les of potato rhizosphere
bacterial communities that had responded to di€erent
organic substrates of BIOLOG reaction wells were
compared with those of the untreated control.
In this study, soil enrichment with ARE not only
a€ected the number of dominant bacterial types but
also caused changes in the structural diversity. The
soils contaminated with di€erent concentrations of
heavy metals (i.e. PS1 versus PS19) could be characterised by bacterial communities that showed a more
similar composition when the soils were treated in the
same way than when the same soil was treated with
ARE under di€erent conditions. This indicates that the
structural diversities of bacterial communities were
relatively similar in these soils. However, the reaction
of soil bacteria to the mixture of organic compounds,
simulating the rhizodeposition e€ect of root exudates,
was dependent on the ``strength'' of the enrichment.
When the soils were ¯ooded, organic compounds of
ARE were probably more available for the soil bacteria. In contrast, ARE might have been sorbed on
soil particles after addition to un¯ooded soil, resulting
in a di€erent response of the indigenous bacteria.
Under un¯ooded conditions, a fast reaction of soil
bacteria to the enrichment with ARE could be
observed, whereas ¯ooding resulted in the predominance of fast growing bacteria over longer time, especially in the lower polluted soil. This way, more
bacterial populations responding to root derived compounds could be detected. Duineveld et al. (1998)
reported that the e€ect of roots on dominating soil
bacterial groups was marginal because these bacteria
were probably oligotrophic and responded slowly to
the changes brought about by root exudates. They stated that each soil type may have its typical set of
dominant bacterial groups, and this mainly determines
the DNA-based pro®les of bacterial communities
obtained in the rhizosphere. In contrast, Maloney et
al. (1997) applying a physiological approach, found
that the qualitative and quantitative composition of
root exudates strongly a€ected the community structure of lettuce and tomato rhizospheres. Their results
agree with the DNA-based ®ngerprints of the present
study. However, the development of the microbial

community in the rhizosphere depends on soil type,
plant species, plant growth stage, presence of contaminants, temperature and other environmental factors
(Anderson et al., 1995; Pearce et al., 1995).
The composition of root exudates a€ects the structural diversity of bacterial communities not only qualitatively but also quantitatively. Di€erent species
(populations) of bacteria were positively a€ected by
ARE under un¯ooded and ¯ooded conditions. This indicates that a longer access to the organic compounds
of ARE could stimulate the development of speci®c
bacterial populations living in micropores, where these
substrates become available after ¯ooding. However,
the stronger intensity of some bands in the DGGE
pro®les could be associated not only with higher numbers of speci®c bacterial species. Since one band may
represent more than one species, the increase of some
bands' intensity could be connected with the detection
of higher number of di€erent species with similar
rDNA sequences, which were stimulated by ARE
(Heuer and Smalla, 1997). These populations represented by dominant bands may be of special interest,
when bacterial species that speci®cally respond to root
exudates in a heavy metal containing background are
to be obtained.
A common reaction of the rhizosphere micro¯ora to
organic compounds exuded by plant roots is an
increase in the numbers of culturable cells (Grayston
et al., 1996). This result was also found in this study.
Two soils contaminated with di€erent concentrations
of heavy metals showed similar numbers of total heterotrophic bacteria. However, di€erences were revealed
when ARE enriched soils were additionally ¯ooded,
which increased substrate availability for indigenous
bacteria. In comparison with PS1 on day 6, the lower
numbers of bacteria in PS19 could result from the
impact of higher concentrations of heavy metals
released from soil colloids during incubation. Organic
acids contained in ARE and those produced by intensively multiplying bacteria in response to added ARE,
may have leached heavy metals into soil solution,
increasing their availability (Banks et al., 1994; Ehrlich, 1997; Jones and Darrah, 1994; Krishamurti et al.,
1996).
Gram-negative copiotrophic organisms such as pseudomonads may be strongly stimulated in the rhizosphere by root exudates. Results of this study clearly
showed that this group dominated among culturable
heterotrophic bacteria in both enriched soils. The addition of ARE to the PS soils induced the development
of other pseudomonads than Comamonas acidovorans
that dominated in untreated soils. In addition, a
Gram-positive isolate, Arthrobacter oxydans, appeared
in PS1 amended with ARE. The isolates identi®ed in
this study were also detected by other authors in various contaminated soils. Arthrobacter-like strains

J. KozdroÂj, J.D. van Elsas / Soil Biology & Biochemistry 32 (2000) 1405±1417

dominated among culturable bacteria isolated from
soil contaminated with Zn (Heuer and Smalla, 1997).
Heavy metal resistant representatives of Arthrobacter
and Alcaligenes (Variovorax ) were found in highly
lead-contaminated sites (Trojanovska et al., 1997). Di
Giovanni et al. (1996) isolated 2,4-dichlorophenoxyacetic acid-degrading Variovorax paradoxus from a contaminated soil.
De Leij et al. (1993) proposed a simple method
based on the growth response when exposed to substrate for determination of the community structure
of the culturable fraction of soil micro¯ora. The
method applied in this study showed that both polluted soils enriched with ARE under un¯ooded and
¯ooded conditions were dominated by fast growing rstrategists that are characteristic for environments rich
in easily available nutrients. In contrast, the soils unamended with ARE were dominated by K-strategists
characteristic for nutrient-poor and uncrowded habitats. Fast growing bacteria (r-strategists) are often
predominant on young immature roots and in the rhizosphere, where a high release of readily available
growth and energy substrates occurs (Chiarini et al.,
1998; De Leij et al., 1993; Nacamulli et al., 1997; Sarathchandra et al., 1997). The possible faster disappearance of ARE in PS1 due to microbial activity
could explain the shift in community structure
towards K-strategists observed on day 6, compared
with microbial community in PS19. The level of soil
contamination with heavy metals could be a major
factor in this process.
The e€ect of the metal concentrations on culturable
bacterial populations in soil was evident when EP indices were calculated. A lower diversity was found in
PS19 than in PS1, especially when the soil was not
¯ooded. The uneven distribution of classes within the
culturable bacterial community in contaminated soils
amended with ARE was also con®rmed by CD indices.
In addition, this index indicated further reduction of
diversity among r-strategists, while the EP indices
showed similar values, suggesting no changes were
occurring in bacterial community. Furthermore, Sarathchandra et al. (1997) found that the CD-index was
of greater relevance, and related better to the r±K concept than the EP-index. Generally, both indices
directly showed that the culturable fraction of bacteria
was highly a€ected by the organic mixture simulating
root exudates. These exudates reduced bacterial diversity towards domination of r-strategists and the reduction of diversity was greater in the higher
contaminated soil. Also, ¯ooding of the soils enhanced
the dominance of fast growing bacteria and reduced
the community diversity.
The results obtained in this study clearly showed signi®cant e€ect of root exudates on the development of
bacterial populations in soil contaminated with heavy

1415

metals. Depending on their availability and conditions
prevailing in the habitat (e.g. stronger enrichment by
¯ooding) various bacterial populations can be stimulated resulting in the generation of di€erent community patterns. The stimulation of speci®c populations
in contaminated soil by organic substrates exuded by
plant roots may be an important result of phytoremediation. Identifying of these species by reampli®cation,
sequencing and/or hybridisation with speci®c probes
would be an interesting endeavour in this strategy.
These isolates, if metal-resistant or metal-transforming
could be studied for a plant growth promoting activity
and might be useful for reintroduction into polluted
soil and use in remediation processes. The other
approach based on the application of a consortium of
microorganisms actively responding to root exudates
and indicating a remediative capacity is also worth
considering. It is dicult to predict whether such
approaches result in the reduction of microbial diversity in the rhizosphere and soil. Nevertheless, selected
species or a consortium of species can stimulate phytoremediation directly by immobilization of heavy
metals, reducing amount of bioavailable metals that
could be toxic for plants. In addition, an indirect
stimulation of phytoremediation by the bacteria used
likely occur due to the bacterial-mediated stimulation
of plant growth. Thus, the plant can accumulate
higher amounts of heavy metals inside tissues and/or
enhance immobilization of the metals outside due to
increased exudation of di€erent organic compounds
by roots.
The results of this study indicate that the most signi®cant response to root exudates occurred among the
culturable fraction of soil bacteria. However, this does
not imply that these bacteria will be the dominant
ones in the rhizosphere (Duineveld et al., 1998). Comparison between total community pro®les and those of
the culturable fractions obtained by PCR-DGGE
showed only a few bands in common (Heuer and
Smalla, 1997). Culturable bacteria are considered to
represent only a small fraction of the rhizosphere
micro¯ora, which, however, can very quickly respond
to root exudates (Duineveld et al., 1998).
This study indicates that root-derived organic compounds will signi®cantly a€ect the development of the
rhizosphere micro¯ora. This impact may be very important in order to restore biological components of
contaminated soil to the form characteristic for an
agriculturally productive soil. Further studies that are
more precise are needed to assess the potential role for
bacteria in phytoremediation of the heavy metal polluted soils. The interactions between the root exudates
and soil micro¯ora occurring in the rhizosphere seem
to be key for successful application of plants in bioremediation of polluted sites.

1416

J. KozdroÂj, J.D. van Elsas / Soil Biology & Biochemistry 32 (2000) 1405±1417

Acknowledgements
This work, except identi®cation of bacterial isolates,
was performed with ®nancial support for Jacek KozdroÂj by a ``MOE'' grant to work in the laboratory of
Jan Dirk van Elsas at IPO-DLO, Wageningen. The
authors thank Ludwina Lankwarden and Anneke Keijzer-Wolters for valuable technical assistance.

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