Directory UMM :Data Elmu:jurnal:S:Soil Biology And Chemistry:Vol32.Issue10.Sep2000:

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

Department of Microbiology, University of Silesia, Jagiellonska 28, 40-032 Katowice, Poland

b

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).72000 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-trations of heavy metals, which a€ect normal agricul-tural 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 matter72000 Elsevier Science Ltd. All rights reserved. PII: S 0 0 3 8 - 0 7 1 7 ( 0 0 ) 0 0 0 5 8 - 4

www.elsevier.com/locate/soilbio

* Corresponding author. Fax: +48-32-2555873.

from contaminated soil (Ebbs and Kochian, 1997, 1998; McGrath et al., 1998; Salt et al., 1998). Success-ful 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 under-standing, 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 prevail-ing in this habitat (Pearce et al., 1995; SoÈrensen, 1997). Root exudates are known as one of the most import-ant 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

con-ditions 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 bac-terial 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 limi-tations (Liesack et al., 1997; Madsen, 1996; Wellington et al., 1997). Therefore, the application of molecular biological techniques to detect and identify microor-ganisms 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; Hugen-holtz et al., 1998; Liesack et al., 1997; van Elsas et al., 1998).

A novel method, PCR followed by denaturing gradi-ent gel electrophoresis (PCR-DGGE), was recgradi-ently 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, tempera-ture gradient gel electrophoresis (TGGE), provide in-formation 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 continu-ously 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 respir-ation rates, but decreased the metabolic diversity of bacterial populations. Despite these observations, the knowledge about relationships between bacterial com-munity and root exudates is still limited. In addition, the possible roles of rhizosphere processes in phytore-mediation 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 com-pounds in root exudates as a potential mechanism for selective enhancement of bacterial populations in con-taminated 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

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 con-tained 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 pre-cipitation steps followed by Wizard (Promega, USA) spin column treatment (van Elsas et al., 1997).

Absor-bency measurements atA260and A280were determined

with a GeneQuant RNA/DNA calculator (Pharmacia, Sweden) and a small-volume quartz cuvette to

calcu-late the concentration (1 A260unitˆ50 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

mix-ture used contained 0.2mM of each primer, 200mM of

each dNTP, 5 ml of 10Sto€el bu€er (Perkin-Elmer,

USA), 5 U of AmpliTaq Sto€el fragment

(Perkin-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®ca-tion was performed for 40 thermal cycles in a touch-down scheme (Duarte et al., 1998) as follows: after initial denaturation of 4 min at 948C, each cycle

con-sisted 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, TAdecreased 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 electro-phoresis 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

pro-duct were loaded onto 6% (wt/vol) polyacrylamide

gels in 0.5TAE 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 transil-lumination 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

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 g}ÿ1_{) Amino acids (mg g}ÿ1₎

Glucose 7.080 Oxalic acid 1.960 Glutamic acid 0.068 Arabinose 2.760 Fumaric acid 3.660 Proline 0.061 Fructose 2.340 Malic acid 0.260 Alanine 0.057 Saccharose 1.530 Citric acid 0.460 Glycine 0.037 Succinic acid 0.310 Leucine 0.017 Benzoic acid 0.190 Serine 0.025 Tartaric acid 0.070 Arginine 0.030 Glutaric acid 0.030 Glutamine 0.012 Valine 0.010

a

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, K2HPO43H2O 1.8 g, MgSO47H2O 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-strate-gists in each sample. R-strateK-strate-gists 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 pro-duced 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:

H0ˆ ÿS…pi logpi†,

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

popu-lation). 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:

CDˆ …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 colo-nies 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

washing (Operating manual version 6.0, MIDI, USA). The organic phase containing cellular FAME was sep-arated by Hewlett Packard 6890 GC on a capillary col-umn 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 Sher-lock 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 one-way ANOVA (Statistica 7.0 PL). Since both the dupli-cates represented virtually identical pictures after PCR-DGGE, 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 character-istics, 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/

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

Assessed viaA260measurement. b

Values are means and standard deviations in the parenthesis.

c

A280values of 1.6±1.7. These DNA samples were su-ciently pure for subsequent PCR-DGGE analysis. PCR ampli®cation of 16S rDNA fragments success-fully generated 450 bp products visible as strong bands in the gel after electrophoresis (data not shown).

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 inten-sities were detected per sample, with about 15 bands

shared among all samples. Generally, 37 band pos-itions 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). How-ever, when ¯ooded, PS1 and PS19 pro®les belonged to di€erent clusters. On day 6, they showed 91% simi-larity 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 simi-larity between the banding pro®les. This simisimi-larity 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), respect-ively. 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 num-bers decreased in PS19 on day 6. Hence, the numnum-bers 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 bac-teria growing on King's B agar (Fig. 4). However, sig-ni®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 bac-terial counts determined on 0.1-strength TSA.

Representatives of bacteria that formed the highest number of colonies on King's B agar were isolated

and identi®ed by MIDI-FAME pro®ling. Dominant

isolates from both PS soils were identi®ed as

Comamo-nas acidovorans. In PS1 enriched with ARE, Arthro-bacter oxydans and Stenotrophomonas maltophilia

dominated, whereasVariovorax paradoxusandC.

acid-ovoranswere isolated from ARE enriched PS19. When enriched PS1 and PS19 were additionally ¯ooded, the

dominant isolates were identi®ed as V. paradoxus,

Pseudomonas putidaandC. acidovorans, respectively.

Colonies of bacteria that developed on

0.1-strength TSA were generally represented by fast-growing 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 iso-lated from the unamended control soil. In the lightly contaminated PS1 soil, a shift towards slow-growing 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 incu-bation 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 incu-bation, while they constituted only 35% on day 1 (Fig. 5). Compared to the untreated PS1, higher

numbers of colonies were revealed after 48 h when PS19 was analysed.

Calculation of EP indices showed that the

enrich-ment 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 0.1-strength 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, es-pecially 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}

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; SoÈr-ensen, 1997). The excreted compounds are components of root exudates, which play an important role in car-bon ¯uxes into soil, a process also called rhizodeposi-tion (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

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 num-bers 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.

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 Day 3 Day 6

EP CD EP CD EP CD

PS1 ARE 0.492 (0.029) 69.2 (2.2) 0.533 (0.016) 55.0 (2.2) 0.636 (0.025) 43.4 (0.9) ARE + ¯ood 0.425 (0.033) 65.7 (0.4) 0.436 (0.021) 78.1 (1.8) 0.394 (0.051) 82.4 (4.3) Control 0.616 (0.016) 32.3 (1.8) 0.618 (0.024) 33.4 (1.5) 0.620 (0.020) 32.8 (2.0) PS19 ARE 0.362 (0.001) 65.9 (2.3) 0.220 (0.028) 87.8 (2.6) 0.377 (0.020) 78.2 (0.4) ARE + ¯ood 0.401 (0.008) 78.0 (0.2) 0.467 (0.039) 70.7 (0.4) 0.291 (0.006) 86.9 (1.9) Control 0.486 (0.013) 43.7 (3.1) 0.482 (0.011) 42.9 (3.0) 0.484 (0.015) 43.9 (2.6)

a

(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, how-ever, 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 mi-crobial 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 gen-ome sizes as well as di€erent lysis characteristics (Tre-vors, 1996). Hence, sometimes obscure and variable relationships between cell numbers and DNA yields have been found (Leung et al., 1995). The concen-trations 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 character-ised 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-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 abun-dance of major bacterial groups (populations), es-pecially 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 mol-ecular level, the observed e€ect of exudates may be small or undetectable. Duineveld et al. (1998) using PCR-DGGE found rather similar bacterial commu-nities between the chrysanthemum rhizosphere and bulk soil. Moreover, the DNA-based ®ngerprints indi-cated that the bacterial community structure,

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 bac-terial populations in soil was not strong enough to induce major shifts in community structure. The domi-nance of a reduced number of bacterial populations was also found in the rhizosphere of potato after ana-lysing 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 diver-sity 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 character-ised 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 bac-teria. 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 predomi-nance of fast growing bacteria over longer time, es-pecially in the lower polluted soil. This way, more bacterial populations responding to root derived com-pounds 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 sta-ted 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 struc-ture 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 contami-nants, temperature and other environmental factors (Anderson et al., 1995; Pearce et al., 1995).

The composition of root exudates a€ects the struc-tural diversity of bacterial communities not only

quali-tatively but also quantitatively. Di€erent species

(populations) of bacteria were positively a€ected by ARE under un¯ooded and ¯ooded conditions. This in-dicates 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 num-bers 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 rep-resented 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 het-erotrophic 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 inten-sively multiplying bacteria in response to added ARE, may have leached heavy metals into soil solution, increasing their availability (Banks et al., 1994; Ehr-lich, 1997; Jones and Darrah, 1994; Krishamurti et al., 1996).

Gram-negative copiotrophic organisms such as pseu-domonads may be strongly stimulated in the rhizo-sphere by root exudates. Results of this study clearly showed that this group dominated among culturable heterotrophic bacteria in both enriched soils. The ad-dition 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

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-dichlorophenoxyace-tic acid-degrading Variovorax paradoxus from a

con-taminated soil.

De Leij et al. (1993) proposed a simple method based on the growth response when exposed to sub-strate for determination of the community structure of the culturable fraction of soil micro¯ora. The method applied in this study showed that both pol-luted soils enriched with ARE under un¯ooded and ¯ooded conditions were dominated by fast growing r-strategists that are characteristic for environments rich in easily available nutrients. In contrast, the soils una-mended with ARE were dominated by K-strategists characteristic for nutrient-poor and uncrowded habi-tats. Fast growing bacteria (r-strategists) are often predominant on young immature roots and in the rhi-zosphere, 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; Sar-athchandra et al., 1997). The possible faster disap-pearance 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 indi-ces 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, Sar-athchandra et al. (1997) found that the CD-index was of greater relevance, and related better to the r±K con-cept 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 diver-sity towards domination of r-strategists and the

re-duction 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 sig-ni®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) various bacterial populations can be stimu-lated resulting in the generation of di€erent commu-nity patterns. The stimulation of speci®c populations in contaminated soil by organic substrates exuded by plant roots may be an important result of phytoreme-diation. 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 diver-sity in the rhizosphere and soil. Nevertheless, selected species or a consortium of species can stimulate phy-toremediation 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 sig-ni®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). Com-parison 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).

Acknowledgements

This work, except identi®cation of bacterial isolates, was performed with ®nancial support for Jacek Koz-droÂj 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 Keij-zer-Wolters for valuable technical assistance.

References

Anderson, T.A., White, D.C., Walton, B.T., 1995. Degradation of hazardous organic compounds by rhizosphere microbial commu-nities. In: Singh, V.P. (Ed.), Biotransformations: Microbial Degradation of Health Rrisk Compounds. Elsevier, New York, pp. 205±225.

Banks, M.K., Walters, C.Y., Schwab, A.P., 1994. In¯uence of or-ganic acids on leaching of heavy metals from contaminated mine tailings. Journal of Environmental Science and Health A 29, 1045±1056.

Bej, A.K., Mahbubani, M.H., 1996. Current development and appli-cations of nucleic acid technology in the environmental sciences. In: Dangler, Ch.A. (Ed.), Nucleic Acid Analysis. Principles and Bioapplications. Willey-Liss, New York, pp. 231±273.

Bossio, D.A., Scow, K.M., 1995. Impact of carbon and ¯ooding on the metabolic diversity of microbial communities in soils. Applied and Environmental Microbiology 61, 4043±4050.

Burd, G.I., Dixon, D.G., Glick, B.R., 1998. A plant growth-promot-ing bacterium that decreases nickel toxicity in seedlgrowth-promot-ings. Applied and Environmental Microbiology 64, 3663±3668.

Chiarini, L., Bevivino, A., Dalmastri, C., Nacamulli, C., Tabacchioni, S., 1998. In¯uence of plant development, cultivar and soil type on microbial colonization of maize roots. Applied Soil Ecology 8, 11±18.

Crecchio, C., Stotzky, G., 1998. Binding of DNA on humic acids: e€ect on transformation of Bacillus subtilis and resistance to DNase. Soil Biology and Biochemistry 30, 1061±1067.

De Leij, F.A.A.M., Whipps, J.M., Lynch, J.M., 1993. The use of col-ony development for the characterization of bacterial commu-nities in soil and on roots. Microbial Ecology 27, 81±97. Di Giovanni, G.D., Neilson, J.W., Pepper, I.L., Sinclair, N.A., 1996.

Plasmid diversity within a 2,4-dichlorophenoxyacetic acid-degrad-ing Variovorax paradoxus population isolated from a contami-nated soil. Journal of Environmental Science and Health A 31, 963±976.

Duarte, G.F., Rosado, A.S., Seldin, L., Keijzer-Wolters, A.C., van Elsas, J.D., 1998. Extraction of ribosomal RNA and genomic DNA from soil for studying the diversity of the indigenous bac-terial community. Journal of Microbiological Methods 32, 21±29. Duineveld, B.M., Rosado, A.S., van Elsas, J.D., van Veen, J.A.,

1998. Analysis of the dynamics of bacterial communities in the rhizosphere of the chrysanthemum via denaturing gel electrophor-esis and substrate utilization patterns. Applied and Environmental Microbiology 64, 4950±4957.

Ebbs, S.D., Kochian, L.V., 1997. Toxicity of zinc and copper to

Brassica species: implications for phytoremediation. Journal of Environmental Quality 26, 776±781.

Ebbs, S.D., Kochian, L.V., 1998. Phytoextraction of zinc by oat (Avena sativa), barley (Hordeum vulgare), and indian mustard (Brassica juncea). Environment Science and Technology 32, 802± 806.

Ehrlich, H.L., 1997. Microbes and metals. Applied Microbiology and Biotechnology 48, 687±692.

Grayston, S.J., Vaughan, D., Jones, D., 1996. Rhizosphere carbon ¯ow in trees, in comparison with annual plants: the importance of root exudation and its impact on microbial activity and nutri-ent availability. Applied Soil Ecology 5, 29±56.

Griths, B.S., Ritz, K., Ebblewhite, N., Dobson, G., 1999. Soil mi-crobial community structure: e€ects of substrate loading rates. Soil Biology and Biochemistry 31, 145±153.

Hasnain, S., Yasmin, S., Yasmin, A., 1993. The e€ects of lead-resist-ant pseudomonads on the growth of Triticum aestivumseedlings under lead stress. Environmental Pollution 81, 179±184.

Head, I.M., Saunders, J.R., Pickup, R.W., 1998. Microbial evol-ution, diversity, and ecology: a decade of ribosomal RNA analy-sis of uncultivated microorganisms. Microbial Ecology 35, 1±21. Heuer, H., Smalla, K., 1997. Application of denaturing gradient gel

electrophoresis and temperature gradient gel electrophoresis for studying soil microbial communities. In: van Elsas, J.D., Trevors, J.T., Wellington, E.M.H. (Eds.), Modern Soil Microbiology. Marcel Dekker, New York, pp. 353±373.

Hugenholtz, P., Goebel, B.M., Pace, N.R., 1998. Minireview Ð Impact of culture-independent studies on the emerging phyloge-netic view of bacterial diversity. Journal of Bacteriology 180, 4765±4774.

Jones, D.L., Darrah, P.R., 1994. Role of root derived organic acids in the mobilization of nutrients from the rhizosphere. Plant and Soil 166, 247±257.

Kra€tczyk, I., Trolldenier, G., Beringer, H., 1984. Soluble root exu-dates of maize: in¯uence of potassium supply and rhizosphere microorganisms. Soil Biology and Biochemistry 16, 315±322. Krishamurti, G.S.R., Cieslinski, G., Huang, P.M., van Rees, K.C.J.,

1996. Studies on soil rhizosphere: speciation and availability of Cd. Chemical Speciation and Bioavailability 8, 23±28.

Leung, K., Trevors, J.T., van Elsas, J.D., 1995. Extraction and ampli®cation of DNA from the rhizosphere and rhizoplane of plants. In: Trevors, J.T., van Elsas, J.D. (Eds.), Nucleic Acids in the Environment: Methods and Applications. Springer-Verlag, Heidelberg, pp. 69±87.

Liesack, W., Janssen, P.H., Rainey, F.A., Ward-Rainey, N.L., Stackebrand, E., 1997. Microbial diversity in soil: the need for a combined approach using molecular and cultivation techniques. In: van Elsas, J.D., Trevors, J.T., Wellington, E.M.H. (Eds.), Modern Soil Microbiology. Marcel Dekker, New York, pp. 375± 433.

Madsen, E.L., 1996. A critical analysis of methods for determining the composition and biogeochemical activities of soil microbial communities in situ. In: Stotzky, G., Bollag, J.M. (Eds.), Soil Biochemistry, Vol. 9. Marcel Dekker, Inc, New York, pp. 287± 370.

Maloney, P.E., van Bruggen, A.H.C., Hu, S., 1997. Bacterial com-munity structure in relation to the carbon environments in lettuce and tomato rhizospheres and in bulk soil. Microbial Ecology 34, 109±117.

McGrath, S.P., Shen, Z.G., Zhao, F.J., 1998. Heavy metal uptake and chemical changes in the rhizosphere ofThlaspi caerulescens

andThlaspi ochroleucum grown in contaminated soils. Plant and Soil 188, 153±159.

Muyzer, G., Smalla, K., 1998. Application of denaturing gradient gel electrophoresis (DGGE) and temperature gradient gel electro-phoresis (TGGE) in microbial ecology. Antonie van Leeuwenhoek 73, 127±141.

Muyzer, G., De Waal, E.C., Uiterlinden, A.G., 1993. Pro®ling of complex microbial populations by denaturing gradient gel electro-phoresis analysis of polymerase chain reaction-ampli®ed genes coding for 16S rRNA. Applied and Environmental Microbiology 59, 695±700.

following seed bacterization with Burkholderia cepacia MCI 7. FEMS Microbiology Ecology 23, 183±193.

O'Connell, K.P., Goodman, R.M., Handelsman, J., 1996. Engineering the rhizosphere: expressing a bias. Trends in Biotechnology 14, 83±88.

Operating manual version 6.0. Sherlock Microbial Identi®cation System. MIDI, New York, USA.

Pearce, D., Bzin, M.J., Lynch, J.M., 1995. The rhizosphere as a bio-®lm. In: Lappin-Scott, H.M., Costerton, J.W. (Eds.), Microbial Bio®lms. Cambridge University Press, Cambridge, pp. 207±220. Saano, A., Lindstrom, K. 1995. Small scale extraction of DNA from

soil with spun column cleanup. In: Akkermans, A.D.L., de Bruijn, F.J., van Elsas, J.D. (Eds.), Molecular Microbial Ecology Manual. Kluwer Academic Publishers, Dordrecht, pp. 1.3.4: 1-6. Salt, D.E., Smith, R.D., Raskin, I., 1998. Phytoremediation. Annual

Review of Plant Physiology and Plant Molecular Biology 49, 643±668.

Sambrook, J., Fritsch, E.F., Maniatis, T., 1989. Molecular Cloning Ð A Laboratory Manual. Cold Spring Harbor, New York. Sarathchandra, S.U., Burch, G., Cox, N.R., 1997. Growth patterns

of bacterial communities in the rhizoplane and rhizosphere of white clover (Trifolium repensL.) and perennial ryegrass (Lolium perenne L.) in long-term pasture. Applied Soil Ecology 6, 293± 299.

Smalla, K., Wachtendorf, U., Heuer, H., Liu, W-E., Forney, L., 1998. Analysis of Biolog GN substrate utilization patterns by mi-crobial communities. Applied and Environmental Microbiology 64, 1220±1225.

SoÈrensen, J., 1997. The rhizosphere as a habitat for soil microorgan-isms. In: van Elsas, J.D., Trevors, J.T., Wellington, E.M.H. (Eds.), Modern Soil Microbiology. Marcel Dekker, New York, pp. 21±45.

Tate III, R.L., 1995. Soil Microbiology. Wiley, New York.

Tichy, R., Kuzel, S., Riha, V., Kolar, L., 1993. A possible use of soil microorganisms for bioindication of cadmium toxicity of plants. Environmental Technology 14, 379±384.

Trevors, J.T., 1996. Genome size in bacteria. Antonie van Leeuwenhoek 69, 293±303.

Trojanovska, S., Brotz, M.L., Bhave, M., 1997. Detection of heavy metal ion resistance genes in Gram-positive and Gram-negative bacteria isolated from a lead-contaminated site. Biodegradation 8, 113±124.

van Elsas, J.D., Wolters, A.C., Clegg, C.D., Lappin-Scott, H.M., Anderson, J.M., 1994. itness of genetically modi®ed Pseudomonas ¯uorescens in competition for soil and root coloni-zation. FEMS Microbiology Ecology 13, 259±272.

van Elsas, J.D., Duarte, G.F., Rosado, A.S., Smalla, K., 1998. Microbiological and molecular methods for monitoring microbial inoculants and their e€ects in the environment. Journal of Microbiological Methods 32, 133±154.

van Elsas, J.D., MaÈaÈntynen, V., Wolters, A., 1997. Soil DNA extrac-tion and assessment of the fate of Mycobacterium chlorophenoli-cum strain PCP-1 in di€erent soils by 16S ribosomal RNA gene sequence based most-probable-number PCR and immuno¯uores-cence. Biology and Fertility of Soils 24, 188±195.

Vaugham, D., Lumsden, D.G., Linehan, D.J., 1993. In¯uence of dis-solved organic matter on the bio-availability and toxicity of metals in soils and aquatic systems. Chemical Ecology 8, 185± 201.