Rhizosphere effect of Galega orientalis in oil-contaminated soil

Soil Biology & Biochemistry 38 (2006) 817–827 www.elsevier.com/locate/soilbio

Rhizosphere effect of Galega orientalis in oil-contaminated soil A.H. Kaksonen1, M.M. Jussila*, K. Lindstro¨m, L. Suominen Department of Applied Chemistry and Microbiology, Viikki Biocenter, P.O. Box 56, FI-00014 University of Helsinki, Helsinki, Finland Received 20 December 2004; received in revised form 12 July 2005; accepted 22 July 2005 Available online 29 August 2005

Abstract Randomized lysimeters in an oil-contaminated field contained the following treatments: (1) Galega orientalis seeds inoculated with Rhizobium galegae HAMBI 540, (2) bioaugmentation with Pseudomonas putida PaW85, and (3) R. galegae -inoculated G. orientalis seeds plus bioaugmentation with P. putida PaW85. The bacterial abundance and diversity were analysed in composite samples after one growing season. A total of 208 m-toluate tolerating bacteria were isolated and screened with m-toluate tolerance and utilization tests, and the catechol test. Seventy-nine isolates were characterized with (GTG)5-PCR genomic fingerprinting and 16S rRNA gene PCR-RFLP ribotyping. Only 10% of the isolated strains were able to degrade m-toluate. Most of the m-toluate utilizing bacteria were catechol positive indicating the existence of a TOL plasmid. Rhizosphere effect of G. orientalis was manifested in oil-contaminated soil. G. orientalis and Pseudomonas bioaugmentation increased the amount of bacteria in oil-contaminated soil. G. orientalis especially together with Pseudomonas bioaugmentation increased the numbers of m-toluate utilizing and catechol positive bacteria in the soil samples indicating an increase in degradation potential. The rhizosphere of G. orientalis increased also the diversity of bacteria. More ribotypes were found in soils treated with G. orientalis and P. putida PaW85 compared to the untreated soil, but the diversity of the m-toluate utilizing bacteria did not significantly increase. q 2005 Elsevier Ltd. All rights reserved. Keywords: Rhizoshpere effect; Galega orientalis; Pseudomonas bioaugmentation; Culturable soil bacteria; Oil contamination; BTEX; Meta-toluate tolerance; Genetic diversity; (GTG)5 fingerprinting; 16S rRNA gene PCR-RFLP; in situ rhizoremediation; Ecology

1. Introduction Plants have been observed to enhance the bioremediation of oil-contaminated soils (Anderson et al., 1993; Radwan et al., 1995). The beneficial effect of plants on soil bioremediation may arise from various factors: Plants provide soil microbes with nutrients and easily degradable energy sources in form of root exudates and dead root cells. As many of the root exudates are acidic, including CO2 and amino acids, inorganic nutrients, otherwise unavailable for microbes, can be dissolved or weathered in the rhizosphere (Tate, 1995). The pH in the rhizosphere may differ by 1–2 units compared to bulk soil. Plants also affect the redox and * Corresponding author. Tel. C358 9 191 59279; fax: C358 9 191 59322. E-mail address: [email protected] (M.M. Jussila). 1 Present address: Tampere University of Technology, Institute of Environmental Engineering and Biotechnology, P.O. Box 541, FI-33101 Tampere, Finland.

0038-0717/$ - see front matter q 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.soilbio.2005.07.011

osmotic potential, and water content of the soil (Anderson et al., 1993). The rhizosphere effect consists of several aspects. Root exudates can stimulate the microbial communities in the rhizosphere. Often bacterial numbers have been observed to be above 109 cfu gK1 rhizosphere soil (Tate, 1995). The ratio of the bacteria in the rhizosphere and in bulk soil (R:S) is commonly 10–20, for some species even over 100 (Atlas and Bartha, 1993). On the other hand, root exudates can also create a selective pressure on the microbial community by stimulating those microbes that can grow efficiently with the provided energy sources. Thus, microbial diversity may also be smaller in the rhizosphere than in the bulk soil (Killham, 1994). Plants also provide microbes with surfaces to grow on and spread creating different ecological niches (Anderson et al., 1993). Biofilm formation in the rhizosphere may enhance microbial interactions and spread of plasmids by conjugation as different microbes grow in consortia on roots surfaces. In addition, transformation of plasmids has been observed to occur more often on solid surfaces compared to solutions (Sayler et al., 1990).

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In a previous study, Suominen et al. (2000) evaluated the bioremediation potential of a nitrogen-fixing leguminous plant goat’s rue, Galega orientalis, and its microsymbiont, Rhizobium galegae HAMBI 540, in BTEX (benzene, toluene, ethyl benzene and xylenes) contaminated soils in microcosm and mesocosm scale. The G. orientalis-R. galegae symbiosis showed good potential for revegetation of oil-polluted soils. In some cases bioremediation of oil-contaminated soil may be enhanced by bioaugmentation of soil with oildegrading microbes, but so far there is little proof of its usefulness (Pritchard, 1992). The bioaugmentation microbes used may have been poor competitors or unable to move efficiently through the soil pores to reach the target chemicals. Alternatively, substrate concentrations in the environment may have been too low to support the growth of the introduced strain or the strain may ignore the target pollutant if other substrates are present (Pritchard, 1992). Bioaugmentation with Pseudomonas putida PaW85 (pWW0) did not improve plant growth in the previous study by Suominen et al. (2000), but the possible rhizosphere effect was not studied. Oil-degrading bacteria have been previously studied with phenotypic methods and numerical taxonomy (Atlas, 1981). Genotypic methods have enabled a rapid analysis of numerous isolates (Jussila et al., 2006). For example 16S rRNA gene PCR-RFLP ribotyping and rep-PCR fingerprinting have been found to be rapid screening methods for bacteria (Versalovic et al., 1991; Vaneechoutte et al., 1992; Jussila et al., 2006). Jussila et al. (2006), compared the accuracy and reproducibly of repPCR using different primers: REP, ERIC, (GTG)5, and BOX. (GTG)5 fingerprints were found to be both distinctive and reproducible for both Gram-negative and Gram-positive bacteria. In this work, oil-contaminated soil was used in a fieldlysimeter study where the effect of legume plant treatment, bioaugmentation or both of them together were assayed to further elucidate the rhizoremediation (bioremediation using rhizosphere) process. The objectives of the present study were (1) to enumerate bacteria and isolate m-toluate tolerating bacterial strains, (2) to phenotypically characterize the isolated strains by determining their capability to grow on m-toluate and to express a key enzyme, catechol 2,3-dioxygenase, in the TOL pathway needed for BTEX degradation by Gram-negative bacteria, and (3) to study the genetic diversity of culturable m-toluate tolerating bacteria during in situ rhizoremediation by using (GTG)5 fingerprinting, 16S rRNA gene PCR-RFLP and partial 16S rRNA gene sequencing. Our hypotheses were: (1) the legume plant increases bacterial numbers and diversity in oil-contaminated rhizosphere and (2) bioaugmentation with conjugative Pseudomonas increases bacterial diversity of mtoluate utilizing bacteria in Galega rhizosphere.

2. Materials and methods 2.1. Soil properties Coarse sieved oil-contaminated soil originating from Pikku-Huopalahti, an industrial estate in Helsinki, Finland, was used. The soil type was fine sand according to the Finnish classification (Elonen, 1971). The dry weight of the soil was 880 mg gK1 material and organic content 8.2 mg gK1 dry material. The pH of the soil was 7.2 or 8.2 as determined using 10 mM CaCl2-solution or distilled water, respectively, and the water holding capacity (WHC) was 33%. The initial concentrations of mineral oils and petrolether extractable compounds in the soil, as determined according to the Finnish standard SFS 3009 (1980), were 140–260 mg kgK1 and 440–570 mg kgK1, respectively. 2.2. Soil preparation for the lysimeter experiment, seeds and inoculations, and sampling For the lysimeter experiment, 24 plastic cylinders (400 mm wide, 600 mm high) were filled with gravel (bottom 100 mm) and oil-contaminated soil (top 400 mm) (Fig. 1). A wooden frame filled with the same gravel and oilcontaminated soil surrounded the lysimeters. The soils were covered with 100 mm of non-nitrogenous peat (pH 6.5) to enhance the germination of the seeds. Finally, fertilizer (NPK 0-5-3CCa 17, Mg 6, Kemira Agro Oy, Finland) was added to the soils (0.5 kg mK2). Seeds of Galega orientalis, inoculated with Rhizobium galegae HAMBI 540 (Lindstro¨m, 1989), were sown to 10 lysimeters, 30 seeds to each (treatment G) (Fig. 1). Four lysimeters were bioaugmented with Pseudomonas putida PaW85 (HAMBI 1828) harbouring the conjugative TOL plasmid pWW0 (Williams and Murray, 1974; Meulien and Broda, 1982) (treatment P). In addition, 10 lysimeters were treated with both R. galegae -inoculated G. orientalis seeds and P. putida (treatment GP). The soil surrounding the lysimeters was left as an untreated control (treatment O). The soil was covered with thin gauze and watered during the first days of the experiment. After a 5-month growing season (June–October) the soil was sampled with an earth drill (: 1 cm) to 30 cm depth. The composite samples of each of the four treatments consisted of 12 subsamples pooled together. These composite samples were stored at C4 8C to be used directly for analysis. 2.3. Growth media and isolation of bacteria Viable bacterial numbers in the composite samples were determined by cultivation on tryptone yeast extract agar (TY) (Beringer, 1974). In addition, m-toluate tolerating bacteria were counted using three different m-toluate (4.6 g lK1) containing media: TY medium (TYm), and two DEF8-based media (Lindstro¨m and Lehtoma¨ki, 1988)

A.H. Kaksonen et al. / Soil Biology & Biochemistry 38 (2006) 817–827

A Top view

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B Side view

4500 mm

O

G

P

GP

600 mm 4500 mm

Treatments =O =G =P = GP

Wooden frame Ø 400 mm plastic tube

Galega orientalis seeds (30) inoculated with Rhizobium galegae Non-nitrogenous peat (100 mm) and organic fertilizer (0.5 kg m-2) Pseudomonas putida inoculum (4 g) Oil contaminated soil (400 mm) Gravel (100 mm) Asphalt

Fig. 1. The field experiment with oil-contaminated soil. (A) Top view of the lysimeter field. (B) Side view of the lysimeters.

amended with root and rhizosphere extract. Root and rhizosphere extract concentrations (lK1) were 100 ml and 200 ml for the rich DEF8 (DEF8mRich) and for the minimal DEF8 (DEF8mMini) media, respectively. In contrast to the DEF8mMini, DEF8mRich contained also glucose (1 g lK1), mannitol (1 g lK1) and DEF-vitamin solution (1 ml lK1). All four media contained 25 mg lK1 cycloheximide to inhibit fungal growth. The root extract was prepared according to Zaat et al. (1988) with the modifications made by Suominen et al. (2003). The rhizosphere extract was prepared as described by Jussila et al. (2006). Both the root and the rhizosphere extract were short-time stored at C4 8C until used. Four parallel dilution series for each composite sample were made using Ringer buffer, and from each dilution two replicate plates were cultured on TY, TYm, DEF8mRich and DEF8mMini. Half of the replicates was incubated at C15 8C and the other half at C28 8C for 7 d. A total of 208 bacterial isolates was randomly picked from the m-toluate containing TYm plates incubated at C15 8C. The bacterial strains were subcultured at least three times on m-toluate containing media and Gram-stains were prepared to ensure the purity of the strains before characterization. For longtime storage the strains were freeze-dried and deposited in HAMBI (H), the Microbial Culture Collection at the Division of Microbiology, Department of Applied Chemistry and Microbiology, University of Helsinki, Finland. 2.4. Metabolic characterization of m-toluate tolerating bacterial strains The isolated bacterial strains were tested for tolerance of m-toluate by culturing them on TY media containing different m-toluate concentrations (5, 7 and 9 g lK1) at C15 8C for 7 d. The ability of the isolates to use m-toluate as

the sole carbon source was tested by culturing the bacteria on basic DEF8 medium, containing 4 g lK1 m-toluate, at room temperature (C22 8C) for 5 d. The presence of a catabolic key enzyme, catechol 2,3-dioxygenase (C23O), encoded by a gene on TOL plasmids (Williams et al., 1988), was tested as a colour reaction with 250 mM catechol (Jussila et al., 2006), which turns yellow when 2-hydroxymuconic semialdehyde is formed by the action of the enzyme. 2.5. DNA extraction Total genomic DNA was extracted from 1.5 ml bacterial cultures, grown to saturation in TY broth with 2–5 g lK1 mtoluate, using the following combination of two methods (Wilson, 1994; Chomczynski and Sachhi, 1996) with slight modifications essentially made by Nick et al. (1999). Cells were collected by centrifugation and the pellet was resuspended in 100 ml Tris–EDTA buffer (10 mM Tris, 5 mM EDTA; pH 8) containing 1 mg lysozyme (Sigma) and incubated at room temperature for 15 min. After incubation, 600 ml of guanidium thiocyanate solution (4 M guanidium thiocyanate, 5 mM EDTA, 40 mM Tris–HCl, 2% 2-mercaptoethanol; pH 7.5) was slowly pipetted into the mixture simultaneously mixing with the tip of the pipette. The tube was inverted until a clear lysate was formed. 700 ml of cooled phenol–chloroform–isoamylalcohol (25:24:1) was added to the lysate, and the mixture was mixed vigorously for 30 s to generate an emulsion. The different phases were separated by centrifugation at 14,000 rev minK1 for 2 min. The aqueous phase was collected and nucleic acids were precipitated with 700 ml isopropanol. The DNA precipitate was pelleted by centrifugation at 14,000 rev minK1 for 5 min. The pellet was washed with 200 ml 70% ethanol, dried briefly and resuspended in 200 ml Tris–EDTA buffer containing 2 ml DNase-free RNase (0.5 mg ml K1;

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2.7. 16S rRNA gene PCR-RFLP ribotyping 16S rRNA gene PCR-RFLP was ribotyped as described by Jussila et al. (2006). For PCR amplifications Taq DNA polymerase (2.5 U; Promega) was used. 2.8. Partial sequencing of the 16S rRNA gene The 16S rRNA gene of selected isolates was partially (1000 bp) sequenced. DNA sequences between the primers pA and pE’-B (2 mM each) were amplified as described by Edwards et al. (1989). PCR products were sequenced using the solid-phase method as described by Hultman et al. (1991) with an automated ALF DNA sequencer (Pharmacia) at the Institute of Biotechnology, University of Helsinki. The determined sequences were analyzed with the Wisconsin Package Version 9.1 of the Genetics Computer Group, Inc. (GCG-package) using the FastA program. The two 16S rRNA gene nucleotide sequences obtained for the bacterial strains were deposited in the DDBJ/EMBL/ GenBank sequence databases under the accession numbers AF501369 (strain P49ZHAMBI 2377) and AF501370 (strain G38ZHAMBI 2376). 2.9. Statistical analysis The statistical significance of the differences in lnconverted bacterial counts between different composite samples was tested with one- or two-way analysis of variances (ANOVA) using Excel 7.0. 3. Results 3.1. Plant germination The germination rate of the Galega seeds was 38% 2.5 months after sowing. Bioaugmentation with P. putida

Numbers of cultivable bacteria in the soil were counted using four different growth media and two incubation temperatures (Fig. 2). The total bacterial counts on TY were 3-15 times higher than the counts on the selective m-toluate containing media. The composite sample GP obtained from the soil treated with both Galega orientalis H540 and Pseudomonas putida PaW85, had the highest bacterial count determined on all the different media. However, the difference between G and GP treatments was not statistically significant on TY medium. Bacterial counts of composite samples obtained from treatments with G. orientalis (G) or P. putida (P) were higher than those of the composite samples from the untreated soil (O) in both temperatures. Both G. orientalis and P. putida significantly (ANOVA: **P! 0.01) increased the bacterial counts of the composite samples as determined on all the media. The Galega plants stimualted growth of bacteria including different types of m-toluate tolerants (Fig. 2). Especially the GP treatment increased the number of mtoluate tolerating bacteria. The proportion of m-toluate tolerating bacteria from the total culturable bacteria was A

C

B 80 70 60 50 40 30 20 10 0

TY

O

G P Treatment

GP

D 20

DEF8mRich

15 10 5 0 O

G P Treatment

GP

Bacterial counts (x 106 cfu g-1)

(GTG)5-PCR was fingerprinted as described by Jussila et al. (2006).

3.2. Total and m-toluate tolerating/degrading bacterial plate counts

Bacterial counts (x 106 cfu g-1)

2.6. (GTG)5-PCR fingerprinting

PaW85 did not have a significant effect on the germination (data not shown).

Bacterial counts (x 106 cfu g-1)

Boehringer Mannheim). RNA was digested at C37 8C for 30 min. The RNA-free DNA-solution was further treated with 50 ml of 5 M NaCl and 50 ml of preheated (C60 8C) 10% CTAB solution containing 0.7 M NaCl, and incubated at C60 8C for 30 min. The mixture was cooled on ice for 10 min and purified by extracting twice with 300 ml cooled chloroform–octanol (24:1). The DNA was precipitated with 800 ml 99% ethanol and centrifuged at 14,000 rev minK1 for 5 min. The pellet was washed with 70% ethanol, dried and resuspended in 50 ml 5 mM Tris, pH 8.0. Standard techniques were used for estimating the DNA concentrations in agarose gel (Sambrook et al., 1989).

Bacterial counts (x 106 cfu g-1)

820

20 TYm 15 10 5 0 O

G P Treatment

GP

20 DEF8mMini 15 10 5 0 O

G P Treatment

GP

Fig. 2. Bacterial counts of composite samples determined on different growth media and at incubation temperatures of C15 8C (dark columns) and C28 8C (light columns). (A) total bacterial plate counts; B–D, culturable m-toluate tolerating bacterial counts. Treatments: G, soil treated with R. galegae H540 inoculated Galega orientalis; P, soil treated with Pseudomonas putida PaW85; GP, soil treated with both R. galegae H540 inoculated G. orientalis and P. putida PaW85; and O, untreated soil. Error bars represent standard deviations. m, medium containing 4.6 g lK1 mtoluate.

A.H. Kaksonen et al. / Soil Biology & Biochemistry 38 (2006) 817–827

0.8 Counts on TYm / counts on TY

15˚C 0.6

28˚C

0.4 0.2 0 O

G P Treatment

GP

Fig. 3. The proportion of m-toluate tolerating bacteria from the total culturable bacteria as obtained from colony counts on TY media with and without m-toluate. Treatments: G, soil treated with R. galegae H540 inoculated Galega orientalis; P, soil treated with Pseudomonas putida PaW85; GP, soil treated with both R. galegae H540 inoculated G. orientalis and P. putida PaW85; and O, untreated soil. Error bars represent standard deviations. m, medium containing 4.6 g lK1 m-toluate.

higher in O and GP treatments compared to G and P treatments (Fig. 3). 3.3. Isolation, morphology and m-toluate degradation capacity of the bacterial strains A total of 208 bacterial isolates was randomly picked from the terminal dilutions of m-toluate containing TYm plates incubated at C15 8C for 7 d. These plates were selected, because no visible differences in the colony morphologies on the various m-toluate containing plates incubated at different temperatures was observed. The incubation temperature of C15 8C was considered to be closer to real field conditions as compared to C28 8C. Bacterial strains were screened for their morphology, ability to tolerate and degrade m-toluate, and the expression of the enzyme catechol 2,3-dioxygenase (C23O). Most of the 208 isolated m-toluate tolerating strains were Gramnegative rods probably due to favourable growth medium. Almost all the m-toluate tolerating isolates grew on TY agar containing 5 g lK1 m-toluate (Table 1). Over 95% of the isolates tolerated 7 g lK1 m-toluate and over 50% tolerated it at concentration as high as 9 g lK1. Depending

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on the treatment, 10–50% of the cultivable bacteria tolerated m-toluate (Fig. 3). Nevertheless, there was no statistically significant difference in the m-toluate tolerance between isolates obtained from different treatments. Still, in the treatments O and GP, with 9 g lK1 m-toluate in the medium, the amount of tolerants was higher than in G or P (Table 1). Interestingly, in these O and GP treatments the proportion of m-toluate tolerating bacteria from the total culturable bacteria was high (Fig. 3). Most importantly, the amount of m-toluate degrading and catechol positive bacteria was highest in soils treated with G. orientalis (both G and GP treatment) (Fig. 4). However, the difference between G and GP treatments was not statistically significant. Only about 10% of the m-toluate tolerating isolates grew with m-toluate as a sole carbon source (Table 1). The catechol test revealed the presence of C23O of TOL like plasmids in about 10% of the m-toluate tolerating isolates. In the further study with the selected strains (Fig. 6), 77% of the m-toluate utilizing strains showed a positive reaction in the catechol test. Only 35% of the catechol positive strains did utilize m-toluate as a sole carbon source. Three percent of the isolates had yellow pigmentation on TY media, and thus, the existence of C23O could not be determined by the catechol test. 3.4. The genetic diversity of the bacterial strains with (GTG)5-PCR fingerprinting and 16S rRNA gene PCR-RFLP ribotyping A total of 79 bacterial strains was selected to be further characterized with (GTG)5-PCR fingerprinting and 16S rRNA gene PCR-RFLP ribotyping. The selected strains included all the strains from one random dilution series from each composite sample, and all the strains that used m-toluate as a sole carbon source and/or were catechol positive. A strain-specific genomic fingerprint was obtained with the (GTG)5-primer for all the characterized 79 bacterial strains. (GTG)5-PCR fingerprint patterns of some strains were as shown in Fig. 5. In the UPGMA (unweighted pair group method with averages) dendrogram, the isolates from different

Table 1 The percentage valueGstandard deviation of m-toluate tolerating, as the sole carbon source utilizing and catechol positive strains among the isolated bacterial strains (nZ52 for each treatment) obtained from different composite samples Treatmenta

O G P GP

Tolerance

Degradation

Expression of C23Ob

5 g lK1 m-toluate on TY agar

7 g lK1 m-toluate on TY agar

9 g lK1 m-toluate on TY agar

4 g lK1 m-toluate as the sole C source on DEF8 agar

Catechol positive

100G0 98G4 100G0 100G0

96G8 96G8 98G4 98G4

79G12 60G13 54G29 75G12

6G12 17G7 8G9 12G8

12G4 (0)c 17G13 (1)c 8G15 (2)c 15G14 (4)c

a Treatments: G, soil treated with R. galegae H540 inoculated Galega orientalis; P, soil treated with Pseudomonas putida PaW85; GP, soil treated with both R. galegae H540 inoculated G. orientalis and P. putida PaW85; and O, untreated soil. b Catechol 2,3-dioxygenase. c The number of strains that grew as yellow colonies on TY agar and thus could not be tested for catechol reaction.

A.H. Kaksonen et al. / Soil Biology & Biochemistry 38 (2006) 817–827 30 25

meta -toluate degrading

20 15 10 5 0 O

G P Treatment

GP

B

Bacterial counts (x 105cfu g-1)

A

Bacterial counts (x 105cfu g-1)

822

in the same main cluster as P. putida PaW85 and R. galegae H540. All catechol positive isolates and most of the mtoluate utilizing isolates also appeared in this cluster, while all the Gram-positive isolates appeared in the other main cluster. None of the isolates had the same genotype as P. putida PaW85 or R. galegae H540.

30 25

catechol positive

20 15 10 5 0 O

G P Treatment

GP

Fig. 4. The bacterial counts of (A) m-toluate degrading and (B) catechol positive bacteria in different composite samples. Treatments: G, soil treated with R. galegae H540 inoculated Galega orientalis; P, soil treated with Pseudomonas putida PaW85; GP, soil treated with both R. galegae H540 inoculated G. orientalis and P. putida PaW85; and O, untreated soil. Error bars represent standard deviations.

treatments were dispersed (Fig. 6). The inoculant P. putida PaW85 was not detected among the characterized isolates. The 16S rRNA gene PCR-RFLP ribotyping (Fig. 7) of the 79 bacterial strains resulted in 5–10 different ribotypes per enzyme depending on the enzyme used. The most powerful enzymes in differentiating the isolates were AluI, HinfI and MspI, whereas the least powerful was TaqI. When the data obtained with the 10 restriction enzymes was combined, the 79 bacterial strains were divided into 24 ribotypes (Fig. 8). The number of strains in each ribotype varied between 1 and 15. Strains isolated from the O, G, P, and GP treatments appeared in 8, 10, 13 and 10 ribotypes, respectively. M-toluate utilizing bacteria were found in nine ribotypes and catechol positive isolates in seven ribotypes (only Gram-negatives), six of which also included m-toluate utilizing strains. The number of different ribotypes was highest in the bioaugmented soil (P) and lowest in the untreated soil (O) (Fig. 8). In the UPGMA dendrogram of the 16S rRNA gene PCRRFLP analysis, the 79 bacterial strains were divided into two main clusters at the similarity level of 70% (Fig. 8). Seventy-one percent of the characterized isolates appeared kb 8.13 6.56 6.44 3.68 2.61 2.56 2.13 2.00 1.95 1.61 1.42 1.28 0.99 0.97 0.89 0.60 0.51 0.43

3.5. Phylogenetic affiliation of the selected bacterial strains Based on the 16S rRNA gene PCR-RFLP dendrogram, two bacterial strains (G38 and P49) representing the two main clusters were selected for the partial sequencing of the 16S rRNA gene. Both of the strains utilized m-toluate as a sole carbon source, and in addition, Gram-negative strain G38 was catechol positive. The colonies of the Grampositive strain P49 were yellow on TY agar, so its ability to degrade catechol could not be tested. Thirteen other strains had a similar 16S rRNA gene PCR-RFLP genotype as G38 strain, 6 of which utilized m-toluate as a sole carbon source and were also catechol-positive. On the basis of the partial sequencing of 16S rRNA gene, strain G38 was Pseudomonas migulae with 99.2% similarity. Strain P49 was Arthrobacter aurescens with 99.1% similarity.

4. Discussion 4.1. The effect of Galega rhizosphere and Pseudomonas bioaugmentation on the bacterial abundance Galega orientalis but also Pseudomonas putida increased the overall number of bacteria in the field experiment with oil-contaminated soil. G. orientalis especially together with Pseudomonas bioaugmentation, increased the amount of m-toluate tolerants, which may indicate an increased efficiency in oil degradation under these favourable rhizosphere conditions.

M 1 2 3 4 5 6 7 8 M 9 10 11 12 13 14 15 16 17 M

M = λsinI marker 1 = isolate O12 2 = isolate O46 3 = isolate O53 4 = isolate O55 5 = isolate G8 6 = isolate G16 7 = isolate G19 8 = isolate G22 9 = isolate G25 10 = isolate G28 11 = isolate P1 12 = isolate P4 13 = isolate P6 14 = isolate P8 15 = isolate P22 16 = isolate GP1 17 = isolate GP3

Fig. 5. (GTG)5-PCR fingerprinting patterns of some m-toluate tolerating bacterial strains from the field scale lysimeter experiment.

A.H. Kaksonen et al. / Soil Biology & Biochemistry 38 (2006) 817–827 Similarity % 20

30

40

50

60

70

80

90

823

100 Pseudomonas putida PaW85 GP6 GP8 G20 P1 G18 P11 GP13 G22 X O55 Bacillus macroides (99.2 %) P10 G25 G28 O18 GP35 GP9 O43 O49 P16 O31 G48 P2 P26 GP24 P13 GP23 O57 P8 O52 O56 O50 O27 O47 G7 P7 GP5 P3 P22 P6 G47 G52 G5 G19 P12 GP16 G26 P4 GP1 GP12 GP10 G35 G38 Pseudomonas migulae (99.2 %) GP33 G13 GP7 GP26 G24 P5 GP2 Arthrobacter aurescens (99.4 %) P23 O12 G8 P49 Arthrobacter P9 aurescens (99.1 %) O51 O54 O28 G21 O46 G23 GP3 Rhizobium galegae H540 O58 G17 GP4 GP11 Arthrobacter histidinolovorans (99.4 %) O48 P47 O53 G16 G27

Fig. 6. UPGMA dendrogram of the m-toluate tolerating bacterial strains and reference strains generated from the (GTG)5-PCR analysis after similarity matrix calculations based on the Pearson product-moment correlation coefficient. C, grows on m-toluate as a sole carbon source; X, catechol-positive; &, Grampositive.

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A.H. Kaksonen et al. / Soil Biology & Biochemistry 38 (2006) 817–827 HinfI

kb 2.65 1.61 1.20 0.68 0.52 0.46 0.40 0.35 0.22 0.18 0.13

M 1

2

3

4

5 M 1

2

RsaI 3 4

5 M M= pGEM marker 1 = isolate P10 2 = isolate P4 3 = isolate P49 4 = isolate G5 5 = isolate GP3

Fig. 7. 16S rRNA gene PCR-RFLP patterns of some m-toluate tolerating bacterial strains obtained by digestion with the restriction endonucleases HinfI and RsaI.

In previous studies plant rhizosphere has been found to increase the bacterial concentrations (Atlas and Bartha, 1993; Lee and Banks, 1993), which is consistent with our results. However, the observed effects of bioaugmentation have been contradictory (Vogel, 1996). In this study, P. putida PaW85 may have increased the bacterial numbers by providing a conjugative plasmid, which helps other bacteria to adapt to the oil-contaminated environment. The assessment of the benefits of P. putida bioaugmentation is complicated by many factors that also affect the bioremediation efficiency. Significant factors may be physical and chemical properties of the soil, the properties of the soil

contaminants (bioavailability, concentration and toxicity), microbial ecology (predation and competition), and the way in which the bioaugmentation is done (Vogel, 1996). According to Atlas (1981), population levels of hydrocarbon utilizers and their proportions within a microbial community appear to be a sensitive indicator of environmental exposure to hydrocarbons. In unpolluted ecosystems, hydrocarbon utilizers generally constitute less than 0.1% of the microbial community; in oil-polluted ecosystems, they can constitute up to 100% of the viable microorganisms. In the present study, the exposure of soil to oil compounds was the same in all four treatments. Thus the observed differences in bacterial counts are due to the different treatments, not the contamination history. 4.2. M-toluate tolerance and degradation capacity of the isolated bacterial strains Despite tolerating m-toluate excellently, 90% of the isolated strains only tolerated but did not degrade m-toluate. Similarly, in a previous greenhouse experiment the 20% minority of the tolerants were also able to degrade m-toluate (Jussila et al., 2006). Most of the m-toluate utilizing strains had the degradation pathway where C23O was involved indicating the presence of a TOL plasmid. However,

Similarity % 70

80

90

100 16S rDNA (0/G/P/GP) m+ c+ g+ Genotype 1 (1/2/0/2) 0 0 0 2 (4/2/4/5) 4 9 0 3 (6/3/0/0) 2 3 0 4 (0/3/0/0) 3 3 0 5 (0/0/1/1) 0 0 0 6 (1/0/1/0) 1 2 0 7 (1/0/1/1) 0 1 0 8 (2/5/2/5) 8 7 0 9 (1/0/0/0) 0 0 0 10 (0/0/1/0) 0 0 0 11 (0/0/1/0) 1 1 0 Pseudomonas putida PaW85 Rhizobium galegae HAMBI 540 12 (0/0/1/0) 0 0 0 13 (0/0/0/1) 1 0 1 14 (0/0/1/1) 0 0 2 15 (0/0/1/0) 0 0 0 16 (0/0/0/1) 0 0 0 17 (0/0/0/1) 0 0 0 18 (0/1/0/0) 0 0 0 19 (0/2/3/1) 0 0 0 20 (3/2/0/0) 1 0 0 21 (0/1/0/0) 0 0 0 22 (0/0/1/0) 1 0 1 23 (0/1/0/0) 0 0 0 24 (0/0/1/0) 0 0 0

G38

P49

Fig. 8. UPGMA dendrogram showing the relationships between the m-toluate tolerating bacterial strains characterized by RFLP analysis (Dice band-matching correlation coefficient) of the 16S rRNA gene with ten restriction enzymes. The genotypes are numbered in order of occurrence. In parenthesis are the numbers of strains representing each genotype obtained from different composite soil samples (O/G/P/GP). Numbers of bacterial strains utilizing m-toluate as a sole carbon source (mC), being catechol-positive (cC), and being Gram-positive (gC) are denoted. Treatments: G, soil treated with R. galegae H540 inoculated Galega orientalis; P, soil treated with Pseudomonas putida PaW85; GP, soil treated with both R. galegae H540 inoculated G. orientalis and P. putida PaW85; and O, untreated soil.

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the breakdown of m-toluate by Gram-negative bacteria was not restricted to the TOL pathway. The others and also the Gram-positive degraders may have another degradation pathway encoded in a plasmid or on the chromosome. In addition, only part of the catechol positive degraders could use m-toluate as a sole carbon source indicating the presence of a different kind of substrate specificity. The amount of heavy tolerants (9 g lK1) might have favoured the higher proportion of m-toluate tolerating bacteria in O and GP soils by transferring m-toluate degradation plasmids. When isolates were grown on TY agar and thereafter transferred again on TYm media, most of the isolates did not tolerate as high concentrations of mtoluate as before. This may be due to the loss of a plasmid or parts of it containing the tolerance and/or degradation genes. One of the criteria, by which the degradation of m- and p-toluate by P. putida (arvilla) mt-2 was originally judged to be plasmid-specified, was the loss of function at efficiencies higher than those normally found for mutations (Bayley et al., 1977). Most m-toluate degrading and catechol positive bacteria were found in soils where Galega plant was growing, which may indicate an increased efficiency in oil biodegradation. For example Sayler et al. (1985) have showed a correlation between the enhanced rates of PAH (polyaromatic hydrocarbons) mineralization in oil-contaminated sediments and an increase in the number of colonies containing DNA sequences which hybridize to TOL (toluate oxidation) and NAH (naphthalene oxidation) plasmid probes using the colony hybridization technique. However, in the present study, the variation between the bacterial counts obtained from different dilution series was high. Still, Palmroth et al. (2005) showed recently that the utilization of diesel fuel by soil bacteria was higher in contaminated soil, especially when vegetated, than in uncontaminated soil. In addition, diesel fuel disappeared more rapidly in the legume mixture treatment (white clover, Trifolium repens and pea, Pisum sativum) than in other plant treatments (Palmroth et al., 2002). 4.3. The effect of Galega rhizosphere and Pseudomonas bioaugmentation on the bacterial composition According to Killham (1994) the microbial diversity in the rhizosphere can be lower than in the bulk soil. In our study, the rhizosphere of G. orientalis did not reduce the diversity of bacteria, since more different ribotypes were found in the G. orientalis containing soils compared to the untreated soil. The rhizosphere of G. orientalis seemed to increase the diversity of bacteria. However, the diversity of the m-toluate degrading bacteria did not significantly increase. The number of different ribotypes was highest in the bioaugmented soil and lowest in the untreated soil. In bioaugmented soils, also Gram-positive ribotypes were observed. This might indicate a positive effect of Pseudomonas bioaugmentation to also activate Gram-

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positive degraders. Notable is that P. putida PaW85 did not form, however, any superior population in the soils, which is consistent with the results of the previous greenhouse experiment (Jussila et al., 2006). Both Gram-negative and Gram-positive bacteria play important roles in oil degradation. In this study, the phylogenetically identified strains P. migulae and A. aurescens were able to degrade m-toluate. Pseudomonas and Arthrobacter are genera commonly found in oilcontaminated soils (Atlas, 1981). Representatives of both genera are also capable of producing plant stimulating organic compounds (Atlas and Bartha, 1993). The communication, i.e. signal exchange, between tolerant soil bacteria and plant might explain partly the favourable conditions prevailing in oil-contaminated Galega rhizosphere. An interesting finding was that only half of the strains representing P. migulae ribotype were catechol positive and able to degrade m-toluate as a sole carbon source and thus, most likely contained the TOL plasmid. The observation that various strains had differences in their degradation abilities was found earlier for P. oryzihabitans strains (Jussila et al., 2006). Yet in this study, it was not verified whether the TOL plasmid had conjugated from P. putida PaW85 to the other Pseudomonas species or whether these species harboured an indigenous TOL plasmid. The horizontal transfer of degradative plasmids could play an important role for rhizosphere bacteria to survive well in oilcontaminated soils (Sarand et al., 2000). 16S rRNA gene PCR-RFLP analysis clustered catechol positive bacteria and most of the m-toluate utilizing bacteria in one of the two main clusters, whereas the strains were more dispersed in the (GTG)5-PCR dendrogram. Although it is possible to assort isolates by (GTG)5-PCR analysis even strain specifically, the usefulness of the method in ecological studies may be restricted to contemplating only relationships of strains that are close relatives (Judd et al., 1993; Louws et al., 1994). On the other hand, Jussila et al. (2006) found it useful to analyze Gram-negative and Grampositive bacteria separately in rep-PCR to be able to group rhizosphere bacteria at genus level. However, the grouping of genetically heterogeneous genera (Rhodococcus/Nocardia, Bacillus) was not fully successful probably due to obscurity in taxonomic situation of a few members of the genus (Gu¨rtler et al., 2004). In the future, molecular methods will be used to describe both the diversity of bacteria and their degradation genes in order to estimate the intrinsic remediation potential of contaminated soil and thus, if necessary, the choice of methods for enhanced bioremediation.

5. Conclusions Our hypotheses were that (1) the legume plant increases bacterial numbers and diversity in oil-contaminated

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rhizosphere and (2) bioaugmentation with conjugative Pseudomonas increases bacterial diversity of m-toluate utilizing bacteria in Galega rhizosphere. Rhizosphere effect of Galega orientalis was manifested in oil-contaminated soil. Indeed, our first hypothesis was supported, since G. orientalis increased not only the total bacterial numbers but also the numbers of m-toluate utilizing bacteria. Also the bacterial diversity, when measured as the amount of ribotypes, was increased in Galega rhizosphere. However, the diversity of m-toluate utilizing bacteria did not significantly increase. On the contrary, our second hypothesis was not supported: the bioaugmentation with P. putida PaW85 did not significantly increase the diversity of m-toluate utilizing bacteria. However, Pseudomonas increased the overall bacterial diversity, especially the amount of Gram-positive ribotypes. Pseudomonas bioaugmentation could also increase bacterial numbers and especially together with Galega plant the amount of m-toluate utilizing bacteria, which might have been triggered by conjugation. In addition to bacterial numbers and diversity, a third evidence of the rhizosphere effect was the fact that only a part of P. migulae strains were able to degrade m-toluate as the sole carbon source. This gives rise to a new hypothesis that a conjugative plasmid exists among the rhizosphere bacteria. This will be tested further in future work. Taken together, rhizoremediation seems to work in such a way that the legume stimulates bacterial proliferation and m-toluate degradation, which may facilitate roots to grow deeper into the contaminated soil.

Acknowledgements Katri Ma¨kela¨inen, Sirpa Tiikkainen, Eeva-Liisa Ko¨ssi, Seppo Kaijalainen and Gao Junlian are thanked for their help in the laboratory. We also thank Zewdu Terefework and Giselle Nick for sharing their knowledge of the GelCompar software. This work was supported by the Academy of Finland, Maj and Tor Nessling Foundation and the University of Helsinki.

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