Directory UMM :Data Elmu:jurnal:S:Soil Biology And Chemistry:Vol32.Issue14.Dec2000:

Soil Biology & Biochemistry 32 (2000) 2011±2017
www.elsevier.com/locate/soilbio

Dissipation of 3±6-ring polycyclic aromatic hydrocarbons in the
rhizosphere of ryegrass
P. Binet, J.M. Portal, C. Leyval*
Centre de PeÂdologie Biologique, CNRS UPR 6831 associated with H. Poincare University, 17, rue Notre Dame des Pauvres,
B.P. 5 54501 Vandoeuvre-les-Nancy cedex, France
Accepted 22 April 2000

Abstract
Plants may contribute to the biodegradation of polycyclic aromatic hydrocarbons (PAHs) in contaminated soils. Different mechanisms
have been proposed, such as an increase in microbial numbers, but are not clearly elucidated. This study investigates the dissipation of a
mixture of eight PAHs, ranging from 3 to 6 rings, in the rhizosphere of ryegrass (Lolium perenne L.). Two pot experiments were conducted
with or without plants using soil spiked with 1 g kg 21 of PAHs in a growth chamber. The ®rst experiment was carried out shortly after spiking
and the second after 6 months of ageing. At the end of both experiments, the extractable concentrations of all PAHs were lower in
rhizospheric than non-rhizospheric soil. PAH dissipation was lower after soil ageing than before, but was still signi®cantly higher in the
rhizospheric soil, even for three of the high molecular weight PAHs. Total culturable micro¯ora were higher in the rhizospheric than nonrhizospheric soil, but was at the same level in spiked and non-spiked soil. The number of PAHs degraders, estimated by a modi®ed MPN
procedure, was not signi®cantly different in the freshly spiked rhizospheric and non-rhizospheric soils, but was signi®cantly higher in the
rhizosphere of the aged spiked soil. q 2000 Elsevier Science Ltd. All rights reserved.
Keywords: Dissipation; Micro¯ora; Polycyclic aromatic hydrocarbons; Quinones; Rhizosphere; Soil

1. Introduction
The soil environment in¯uenced by plant roots, or rhizosphere, represents a complex ecosystem with the potential to
accelerate biodegradation of organic contaminants, including polycyclic aromatic hydrocarbons (PAHs) (Aprill and
Sims, 1990; Anderson et al., 1993; Walton et al., 1994a).
Although a few studies indicated that the plant rhizosphere
is able to enhance degradation of 4-ring PAHs such as
pyrene (Schwab and Banks, 1994; Reilley et al., 1996),
they were performed with a limited number (2±4
compounds) of PAHs. To our knowledge, only Goodin
and Webber (1995) studied the degradation of a 5 ring
PAH (benzo(a)pyrene) in the rhizosphere. They reported
inconsistent degradation of benzo(a)pyrene in the rhizosphere. The studies of Schwab and Banks (1994) and Reilley et al. (1996) measured the disappearance with time of
PAHs in freshly spiked soils, where the availability of PAHs
may be higher than in industrial soils, where the contamination has a greater residence time.
Plants may contribute to the dissipation of PAHs by an
* Corresponding author. Tel.: 133-383-51-8463; fax: 133-383-57-6523.
E-mail address: leyval@cpb.cnrs-nancy.fr (C. Leyval).

increase in microbial numbers, improvement of physical

and chemical soil conditions, increased humi®cation and
adsorption of pollutants in the rhizosphere, but the impact
of each process has not been clearly elucidated. Several
studies, based on the hypothesis that root exudates increase
the rhizosphere microbial community, investigated the
signi®cance of plant microbial interactions for the degradation of PAHs. Walton et al. (1994b) speculated that when a
chemical stress is present in soil, a plant may respond by
increasing or changing exudation to the rhizosphere which
modi®es rhizospheric micro¯ora composition or activity. As
a result, the microbial community might increase the transformation rates of the toxicant. GuÈnther et al. (1996) noted
that in a soil polluted with PAHs and aliphatic hydrocarbons, microbial plate counts and soil respiration rates were
higher in the rhizosphere of ryegrass than in the bulk soil.
Reilley et al. (1996) showed that degradation of pyrene
increased in rhizosphere soil and that the highest pyrene
mineralisation rate was found when organic acids, typically
found in root exudates, were added to the soil. Nichols et al.
(1997) showed a selective enrichment of the bacterial populations that were organic compound degraders in the rhizosphere of alfalfa and bluegrass, in a soil amended with
organic compounds, including phenanthrene and pyrene.

0038-0717/00/$ - see front matter q 2000 Elsevier Science Ltd. All rights reserved.

PII: S 0038-071 7(00)00100-0

2012

P. Binet et al. / Soil Biology & Biochemistry 32 (2000) 2011±2017

However, bioremediation of the compounds was not estimated. Chaineau et al. (1995) showed a rapid adaptation of
the soil microbial community to degradation of hydrocarbons in an agricultural ®eld plot amended with drill cuttings,
and a speci®c diversity of the degraders, but did not
compare rhizospheric and non-rhizospheric soils. From
these data, it is not clear whether increased dissipation of
PAHs in the rhizosphere is due to a speci®c stimulation of
PAH degraders in the rhizosphere.
Field experiments should rather be used than pot experiments with spiked soils to study the feasibility of PAH
phytoremediation. However, soils contaminated with
PAHs rarely contain only PAHs, but contain also other
organic pollutants, possibly heavy metals, and are very
heterogeneous. Such complex systems cannot be used to
study the mechanisms involved in the biodegradation of
PAHs in the rhizosphere. Pot experiments with spiked

soils, especially using radio-labelled ( 14C or 13C) are useful
to follow the fate of known amounts of PAHs added to a
soil. With single compounds such as benzo-a-pyrene, it has
been performed (Goodin and Webber, 1995) and inconsistent degradation was reported. However, it is not realistic
for long-term experiments and for experiments with many
PAHs due to cost of the compounds (especially to have
uniform labelling). Further, the fate of a single (radiolabelled) PAH in the rhizosphere may not re¯ect the fate
of the same compound in a mixture of PAHs. A major
disadvantage of pot experiments with spiked soil is that
the availability of the PAHs may be different from PAH
contaminated ®eld sites. But the effect of ageing on the
dissipation of PAHs in the rhizosphere was never
investigated.
We investigated the dissipation of eight PAHs,
including 3±6-ring PAHs, in ryegrass rhizosphere in
pot experiments with soil freshly spiked and after
6 months of ageing. To estimate rhizosphere, spiking
and ageing effects on PAH degraders, total culturable
micro¯ora and PAH degraders were estimated, using a
modi®ed MPN procedure.

2. Materials and methods
2.1. Experimental design
Pregerminated ryegrass (Lolium perenne L., cv. Barclay)
seedlings were grown in pots containing 250 g of an agricultural soil, either spiked or un-spiked with PAHs. The soil
was a gleyic luvisol, with a pH of 6.6 and 15 g kg 21 C, and
has no previous history of exposure to PAHs or other
contaminants (Leyval and Binet, 1998). The agricultural
soil was spiked with a mixture of eight PAHs (1 g kg 21)
as described in Leyval and Binet (1998). The concentrations
of PAHs in the soil were, respectively, 200 mg kg 21 for
anthracene, phenanthrene, ¯uoranthene and chrysene, and
50 mg kg 21 for benzo(a)anthracene, benzo(k)¯uoranthene,

dibenzo(a,h)anthracene and benzo(g,h,i)perylene. Seeds of
ryegrass were surface sterilised with 30% H2O2 and pre
grown for 15 days in vermiculite. Two seedlings were
then transplanted to dark plastic pots containing 250 g of
soil. Seedlings were thinned to one after one week and the
soil was covered with a layer of coarse sand to minimize

PAH volatilisation and leaching. Three treatments were
carried out: vegetated pots with un-spiked and spiked soil
and un-vegetated pots with spiked soil. There were ®ve pots
per treatment randomly arranged in a growth chamber
(Conviron, 24/208C day/night, 16 h day, 80% RH, 200±
300 mmol s 21 m 22 PAR). Plants were harvested 40 days
after transplanting and dry weights estimated after drying
at 1058C. The ®rst experiment was performed 12 h after soil
spiking and the second one after ageing the same spiked soil
for 180 days (from June to December). During ageing, the
spiked soil was kept outdoors, in dark condition, at temperatures ranging from 5 to 258C and was maintained at 60% of
water holding capacity.
2.2. PAHs analysis
All the soil from the vegetated pots was considered as
rhizospheric soil. The soil from vegetated (rhizospheric soil)
and non-vegetated pots (non-rhizospheric soil) was carefully collected, homogenised and crushed. PAHs and a
few metabolites (anthraquinone, naphthoic acid and
benzo(a)anthraquinone) were extracted from soil using
Soxlhet method (50 g dry soil with 200 ml chloroform for
4 h). Soil extracts were ®ltered through a cellulose ®lter and

analysed using a 3400 CX Varian gas chromatograph
coupled to a mass spectrometer (ION TRAP Saturn III,
Varian GC±MS). Compounds were separated with a He
¯ow on a 30 m DB5 MS column, 0.25 mm internal diameter
and 0.25 mm ®lm thickness. The column oven temperature
was: 70±1508C at 108C min 21 and 150±3008C at
68C min 21. The Programmable Sample Injector (PSI)
temperature was set between 25 and 3008C at
1808C min 21. The mass spectrometer was operated at
70 eV in impact electronic mode. Detection and quanti®cation of the eight PAHs and of the metabolites were carried
out by Single Ion Monitoring (Table 1). The ion trap
temperature was set to 2208C. The concentrations are
expressed per unit soil dry weight. The initial extractable
concentration of PAHs (T0) was measured within 1 h after
spiking.
2.3. Enumeration of culturable PAHs degraders and total
micro¯ora
PAH degraders were enumerated using the most-probable-number (MPN) procedure (Wrenn and Venosa, 1995)
modi®ed for our study. A PAH mixture consisting of
phenanthrene (10 g l 21), anthracene (1 g l 21), ¯uorene

(1 g l 21) and ¯uoranthene (1 g l 21) was added to 96-well
microtiter plates (10 ml/well) as a solution in hexane before
the plates were ®lled with the growth medium. Hexane was

2013

P. Binet et al. / Soil Biology & Biochemistry 32 (2000) 2011±2017
Table 1
Some characteristics of the PAHs and m/z of ions used for quanti®cation, NA: not available
Compounds

Number of rings

Aqueous solubility (mg l 21)

Hydrophobicity log Kow

m/z of quanti®cation ion

PAHs added

Phenanthrene
Anthracene
Fluoranthene
Chrysene
Benzo(a)anthracene
Benzo(k)¯uoranthene
Dibenzo(a,h)anthracene
Benzo(g,h,i)perylene

3
3
3
4
4
4
5
6

1.6
0.075

0.265
0.006
0.01
NA
NA
NA

4.46
4.45
5.33
5.61
5.61
6.84
5.97
7.23

178
178
202
228

228
252
278
276

Oxidation intermediates
Naphthoic acid
Anthraquinone
Benzo(a)anthraquinone

2
3
4

NA
NA
NA

NA
NA
NA

198
180
258

evaporated for 1 h under a fumehood. Bushnell-Haas
medium (DIFCO) supplemented with 0.85% NaCl was
used as the growth medium (200 ml/well). At the end of
the experiments, a subsample (10 g) of soil from each pot
was diluted in 0.85% KCl (100 ml). Serial dilutions, ranging
from 10 23 to 10 26, were performed, and the plates were
inoculated by adding 25 ml of each dilution to one of the
12 rows of eight wells (40 wells per soil sample). Two rows
remained un-inoculated to serve as sterile controls. A
control was performed as above without PAHs. The plates
were incubated for 3 weeks at 288C in the dark. Positive
wells turned yellow or brown owing to the accumulation
of partial oxidation products of the aromatic substrates.
Colour change was measured spectrophotometrically as
(OD405±OD620). Total culturable micro¯ora was enumerated in separate 96-well microtiter plates ®lled with Nutrient
Broth (DIFCO) medium, using the same soil suspension and
dilution method. After one week, the growth of microbial
populations was determined spectrophotometrically by
measuring the absorbance at 620 nm. Wells were scored

positive when OD620 . 0.1 (OD620 of un-inoculated
wells ranged from 0 to 0.05). A computer program (using
standard Mac Crady tables) was used to calculate the MPN
for each sample, which is expressed as number per gram soil
dry weight.
All data means were compared by ANOVA or Student's
t-test for low replicate numbers …p , 0:05†:

3. Results
Shoot and root biomass were signi®cantly lower in the
spiked soil than in the control un-spiked soil, and lower in
the experiment without soil ageing than in the experiment
after soil ageing (Fig. 1). There was a signi®cant effect of
PAHs on plant dry weights, although plants grown in spiked
soils showed no outward signs of phytotoxicity. Ryegrass
formed a dense ®brous root system, ranging from 0.45 to
1.5 mg g 21 soil dry weight after 40 days, in all soils irrespective of treatment.

Fig. 1. Shoot and root dry weight of ryegrass after 40 days in spiked soil with or without ageing and in un-spiked soil. Different letters above column indicate
signi®cant difference between treatments.

2014

P. Binet et al. / Soil Biology & Biochemistry 32 (2000) 2011±2017

Table 2
Extractable concentrations (mg kg 21) of the eight PAHs and anthraquinone at different times in the spiked soil. T0: just after spiking, T180 days: 180 days after
spiking, mean of ®ve replicates ^ SE, * indicates signi®cant difference …p , 0:05† between rhizospheric and non-rhizospheric soil. PHE: phenanthrene; ANT:
anthracene; FLT: ¯uoranthene; BaANT: benzo(a)anthracene; CHY: chrysene; BkFLT: benzo(k)¯uoranthene; dBahANT: dibenzo(a,h)anthracene; BghiPL:
benzo(g,h,i)perylene; ANQ: anthraquinone
T0

T0 1 40 days

T180 days

Non-rhizospheric soil

Rhizospheric soil

187
175
204
200
52
48
51
48
965

100 ^ 26
19 ^ 2
150 ^ 16
163 ^ 11
35 ^ 2
40 ^ 2
42 ^ 3
32 ^ 3
582

57 ^ 7 *
10 ^ 1 *
85 ^ 9 *
100 ^ 11 *
20 ^ 2 *
25 ^ 2 *
25 ^ 3 *
17 ^ 1 *
339

Oxidation intermediate
ANQ
0

107 ^ 15

60 ^ 8 *

PAHs added
PHE
ANT
FLT
CHY
BaANT
BkFLT
dBahANT
BghiPL
Total

At the end of the ®rst experiment (T0 1 40 days), the
extractable concentrations of PAHs in the non-rhizospheric
soil were lower than the extractable T0 values (Table 2). The
extractable concentrations of phenanthrene and anthracene
were low (50 and 10% of initial extractable concentrations
in non-rhizospheric soil, and 28 and 5% in rhizospheric soil)
but for ¯uoranthene, benzo(a)anthracene, chrysene,
benzo(k)¯uoranthene, dibenzo(a,h)anthracene and benzo
(g,h,i)perylene they were higher (ranging from 65 to 85%
in rhizospheric and non-rhizospheric soil). The total extractable concentrations of the PAHs after the ®rst experiment
decreased to 60% in non-rhizospheric soil. After 180 days
of ageing it had decreased to 43%. During the second
experiment, after the soil ageing period, the extractable
concentrations in non-rhizospheric soil decreased to 60%

T180 days 1 40 days
Non-rhizospheric soil

Rhizospheric soil

80
35
90
120
22
30
30
20
427

20 ^ 2
7.5 ^ 1
28.5 ^ 3
113 ^ 6
15.5 ^ 1
28 ^ 2
29 ^ 2
18 ^ 2
259.5

13 ^ 2
8.5 ^ 2
18.5 ^ 3 *
86 ^ 11 *
12 ^ 2
23 ^ 3
23 ^ 3 *
15 ^ 3
199

46

20 ^ 2

13 ^ 1

of the concentration at T180 days and to 26% of the T0
concentration. However, the PAH decrease concerned
mainly phenanthrene, anthracene and ¯uoranthene.
Total extractable PAHs in the freshly spiked soil were
signi®cantly lower in rhizospheric than in non-rhizospheric
soil (Fig. 2). The decrease was signi®cant for all eight
PAHs. After soil ageing, the rhizosphere effect was less
pronounced but concentrations of extractable ¯uoranthene,
chrysene and dibenzo(a,h)anthracene were signi®cantly
lower in rhizospheric than in non-rhizospheric soil (Fig.
3). After the second experiment, the total extractable PAH
concentration was also lower in rhizospheric than nonrhizospheric soil (Table 2).
At the end of the experiment with aged spiked soil, dissipation of anthracene, chrysene, benzo(k)¯uoranthene,

Fig. 2. Extractable concentrations of PAHs (mg kg 21) after 40 days in spiked soil without ageing as a percentage of the initial extractable concentration (T0
numbers above columns). * indicates signi®cant difference between rhizospheric soil and non-rhizospheric soil. PHE: phenanthrene; ANT: anthracene; FLT:
¯uoranthene; BaANT: benzo(a)anthracene; CHY: chrysene; BkFLT: benzo(k)¯uoranthene; dBahANT: dibenzo(a,h)anthracene; BghiPL: benzo(g,h,i)perylene.

P. Binet et al. / Soil Biology & Biochemistry 32 (2000) 2011±2017

2015

Fig. 3. Extractable concentrations of PAHs (mg kg 21) after 40 days in spiked soil after ageing as a percentage of the extractable concentration after the ageing
period (number above column). * indicates signi®cant difference between rhizospheric and non-rhizospheric soil. PHE: phenanthrene; ANT: anthracene;
FLT: ¯uoranthene; BaANT: benzo(a)anthracene; CHY: chrysene; BkFLT: benzo(k)¯uoranthene; dBahANT: dibenzo(a,h)anthracene; BghiPL: benzo(g,h,i)perylene.

rhizospheric soil (Table 3). No PAH degraders, within the
dilution range tested (from 10 23 to 10 26), were scored for
the un-spiked soil.

dibenzo(a,h)anthracene, benzo(g,h,i)perylene was lower
than in the ®rst experiment with non-aged spiked soil, but
dissipation of ¯uoranthene was higher.
Anthraquinone was clearly identi®ed and quanti®ed in
the spiked soil at the end of the ®rst and second experiments.
The concentration of anthraquinone was higher in the nonaged than in the aged spiked soil. In the freshly spiked soil,
it was signi®cantly higher in non-rhizospheric than rhizospheric soil (Table 2). Metabolites of phenanthrene, and
benzo(a)anthracene (e.g. naphthoic acid and benzo(a)anthraquinone) were also clearly identi®ed with GC±MS in
the spiked soil, but were not quanti®ed. However, these
metabolites were not identi®ed in the un-spiked soil.
In the freshly spiked soil, the numbers of culturable PAH
degraders in the rhizospheric and non-rhizospheric soils
were not signi®cantly different (Table 3). In the aged spiked
soil, the number of culturable PAH degraders was higher
in rhizospheric than non-rhizospheric soil. However, the
percentage of the total culturable micro¯ora able to degrade
a mixture of PAHs was similar in rhizospheric and non-

4. Discussion
Previous experiments with a mixture of aliphatic hydrocarbons or with PAHs such as anthracene and pyrene
(Schwab and Banks, 1994; GuÈnther et al., 1996; Reilley et
al., 1996) showed a very rapid dissipation of these
compounds in the rhizosphere of several plants in the
early stages (40 days) followed by slower rates. The authors
also reported that degradation of pyrene was much faster in
a spiked soil than in an industrial soil. However, these
experiments were performed with a mixture of only 2±4
PAHs. We studied the fate of eight PAHs in the rhizosphere,
and showed that ryegrass was able to accelerate the dissipation of a range of PAHs, including 5 and 6 ring PAHs such
as dibenzo(a,h)anthracene and benzo(g,h,i)perylene which

Table 3
Enumeration of total and PAH degrading culturable micro¯ora at the end of the experiments with the PAH spiked soil. Different letters indicate signi®cant
differences between treatments in column at 5% level (mean of ®ve replicates ^ SE), ND: not determined; d.l.: detection limit
PAHS degraders
Nb g 21 soil dry
weight

Total micro¯ora
Nb g 21 soil dry
weight

Exp. with freshly spiked soil
Non-rhizospheric spiked soil
Rhizospheric spiked soil

6 (^5) £ 10 6
4 (^3) £ 10 5

ND
ND

Exp. with soil ageing
Non-rhizospheric spiked soil
Rhizospheric spiked soil

5 (^2) £ 10 6a
2.3 (^0.7) £ 10 7b

2.5 (^0.7) £ 10 7b
1.5 (^0.3) £ 10 8a

20
15

Rhizospheric non-spiked soil

d.l.

1.5 (^0.2) £ 10 8a

±

PAH degraders/total
micro¯ora (%)

2016

P. Binet et al. / Soil Biology & Biochemistry 32 (2000) 2011±2017

have a low solubility and bioavailability (Table 1). Our
results showed that the decrease of the total extractable
PAHs in the rhizosphere was higher in the experiment without soil ageing (66%) than the one with soil ageing (53%).
This difference could be due to the decreased bioavailability
of the PAHs with the ageing process (Hatzinger and Alexander, 1995). The ryegrass rhizosphere was able to improve
dissipation of 3±6-ring PAHs, but the dissipation was higher
for 3-ring PAHs (phenanthrene and anthracene) than for the
other high molecular weight compounds con®rming that
they were more recalcitrant, particularly after the ageing
process. However, the 5±6-ring PAHs still decreased in
the rhizospheric soil after ageing while they did not in the
non-rhizospheric soil.
The increased dissipation of the eight PAHs in the rhizosphere may also be due to a decreased extractability of the
PAHs with formation of bound residues. Walton et al.
(1994b) suggested that rhizosphere could stabilise pollutants by polymerisation reactions such as humi®cation.
They cited an experiment with 14C PAHs which showed
that 14C in fulvic/humic acids was higher in rhizosphere
than nonrhizosphere soil. A recent study reported the formation of bound residues during microbial degradation of
anthracene in soil (KaÈstner et al., 1999).
These experiments were carried out within a relatively short
time scale (40 days), which may not be long enough for drawing conclusions about the end point of the enhanced dissipation. However, a longer time scale would lead to unrealistic
root density in the pots compared to a ®eld situation. Under
these experimental conditions, a signi®cant dissipation of
PAHs was observed in pots without plants, which could
be attributed to a ªgrowth chamber effectº or rapid dissipation of PAHs after placing the pots in the growth chamber.
Only a small percentage of the soil micro¯ora is culturable (Bakken, 1985), and the microbial numbers obtained
with the microplate techniques are underestimated.
However, total culturable micro¯ora and PAH degraders
were estimated using the same method and these numbers
can be compared. The number of culturable PAH degraders
was higher in rhizospheric than non-rhizospheric soil only
in the second experiment where the spiked soil had been
ageing for 6 months. The results of the ®rst experiment
suggest that the increased transformation of PAHs in the
rhizosphere may not be attributed only to an increased
number of these degraders. An adaptation period may be
necessary for the indigenous micro¯ora in the rhizosphere
of the polluted soil to degrade PAHs. Chaineau et al. (1995,
1996) showed a rapid adaptation period of 16 days in
laboratory conditions for the soil micro¯ora to degrade
hydrocarbons from drill cuttings, but a longer period
(6 months) in a ®eld experiment. The percentage of PAH
degraders was similar in rhizospheric and non-rhizospheric
soils, suggesting that they are not speci®cally stimulated
in the rhizosphere, but mainly stimulated by the presence
of PAHs. After 180 days of ageing, the total culturable
micro¯ora in the rhizosphere of the spiked soil was at the

same level as in the non-spiked soil, but was higher than in
non-rhizospheric soil. Schwab and Banks (1994) also
showed a signi®cant increase of total micro¯ora in the
rhizosphere compared to non-rhizospheric soil in PAHspiked and non-spiked soil. GuÈnther et al. (1996) found
the same result in an experiment with aliphatic hydrocarbons and PAHs but did not observe an enhancement of PAH
degradation in planted soil. Also, Lee and Banks (1993)
noted that the enhancement of microbial counts in planted
soil was higher in soil contaminated with aliphatic hydrocarbons than in soil contaminated with PAHs.
The presence of anthraquinone indicated an oxidation of
anthracene in all treatments (Field et al., 1992; Kotterman et
al., 1994; Field et al., 1995). This oxidation of PAHs to
corresponding quinone could be associated with peroxydase
or laccase activity (Cerniglia, 1997; Majcherczyk et al.,
1998). Anthraquinone concentrations were lower in soil
after ageing and in rhizospheric soil than in freshly spiked
or non-rhizospheric soil. This result could be explained by
decreased extractability of the compound or increased
degradation during the ageing process and also in the rhizosphere soil. The decrease of anthraquinone concentration
with time may also be explained by the development of a
micro¯ora able to degrade quinones. Several studies
observed the biotransformation of PAHs to corresponding
quinones, which are more available and could be easily
degraded by bacteria (Brodkorb and Legge, 1992; Field et
al., 1992; Kotterman et al., 1998).
5. Conclusions
Our study showed that ryegrass rhizosphere potentially
enhances dissipation or biotransformation of a large range
of PAHs including 5 and 6-ring PAHs. The ryegrass rhizosphere enhanced this process in an aged spiked soil, where
the remaining compounds were more recalcitrant to biodegradation. Whether part of the dissipation of PAHs in the
rhizosphere is due to the formation of bound residues
remains to be investigated. Experiments using 14C or 13C
labelled PAHs could be used for that purpose. The increased
PAH dissipation in rhizospheric soil was associated with an
enhancement of PAH degraders. Although ryegrass appears
to facilitate a general rhizospheric effect, it did not appear to
stimulate PAH degraders. Microbial adaptation to PAHs in
soil and in the rhizosphere, and their role in the increased
biodegradation or biotransformation of PAHs in the rhizosphere need to be sorted out to make recommendations for
phytoremediation.
Acknowledgements
The authors thank Bernadette GeÂrard, Genevieve Jeandat,
Thidar Myint and TheÂreÁse Orel for technical assistance, the
GIS-CNRS Sol Urbain, the French Ministry of Environment
and the European Commission for ®nancial support.

P. Binet et al. / Soil Biology & Biochemistry 32 (2000) 2011±2017

References
Anderson, T.A., Guthrie, E.A., Walton, B.T., 1993. Bioremediation.
Environ. Sci. Technol. 27, 2631±2636.
Aprill, W., Sims, R.C., 1990. Evaluation of the use of prairie grasses for
stimulating polycyclic aromatic hydrocarbon treatment in soil. Chemosphere 20, 253±265.
Bakken, L.R., 1985. Separation and puri®cation of bacteria from soil. Appl.
Environ. Microbiol. 49, 1482±1487.
Brodkorb, T.S., Legge, R.L., 1992. Enhanced biodegradation of phenanthrene in oil tar-contaminated soils supplemented with Phanerochaete
chrysosporium. Appl. Environ. Microbiol. 58, 3117±3121.
Cerniglia, C.E., 1997. Fungal metabolism of polycyclic aromatic hydrocarbons: past, present and future applications in bioremediation. J. Ind.
Microbiol. Biotechnol. 19, 324±333.
Chaineau, C.H., Morel, J.L., Oudot, J., 1995. Microbial degradation in soil
microcosms of fuel oil hydrocarbons from drilling cuttings. Env. Sci.
Technol. 29, 1615±1621.
Chaineau, C.H., Morel, J.L., Oudot, J., 1996. Land treatment of oil-based
drill cuttings in an agricultural soil. J. Environ. Qual. 25, 858±867.
Field, J.M., de Jong, E., Costa, G.F., de Bont, J.A.M., 1992. Biodegradation
of polycyclic aromatic hydrocarbons by new isolates of white rot fungi.
Appl. Environ. Microbiol. 58, 2219±2236.
Field, J.A., Boelsma, F., Baten, H., Rulkens, W.H., 1995. Oxidation of
anthracene in water/solvent mixtures by the white rot fungus Bjerkandera sp. strain BOS55. Appl. Microbiol. Biotechnol. 44, 234±240.
Goodin, J.D., Webber, M.D., 1995. Persistence and fate of anthracene and
benzo(a)pyrene in municipal sludge treated soil. J. Environ. Quality 24,
271±278.
GuÈnther, Th., Dornberger, U., Fritsche, W., 1996. Effect of ryegrass on
biodegradation of hydrocarbons in soil. Chemosphere 33, 203±215.
Hatzinger, P.B., Alexander, M., 1995. Effect of aging of chemicals in soil
on their biodegradability and extractability. Environ. Sci. Technol. 29,
537±545.
KaÈstner, M., Streibich, S., Beyrer, M., Richnow, H.H., Fritsche, W., 1999.
Formation of bound residues during microbial degradation of [ 14C]
anthracene in soil. Appl. Environ. Microbiol. 65, 1834±1842.

2017

Kotterman, M.J.J., Heessels, E., De Jong, E., 1994. The physiology of
anthracene biodegradation by the white-rot fungus Bjerkandera sp.
strain BOS55. Appl. Microbiol. Biotechnol. 42, 179±186.
Kotterman, M.J.J., Vis, E.H., Field, J.A., 1998. Succesive mineralization
and detoxi®cation of benzo(a)pyrene by the white rot fungus Bjerkandera sp. strain BOS55 and indigenous micro¯ora. Appl. Environ.
Microbiol. 64, 2853±2858.
Lee, E., Banks, M.K., 1993. Bioremediation of petroleum contaminated soil
using vegetation: a microbial study. J. Environ. Sci. Health 28, 2187±
2198.
Leyval, C., Binet, P., 1998. Effect of polycyclic aromatic hydrocarbons in
soil on arbuscular mycorrhizal plants. J. Environ. Qual. 27, 402±407.
Majcherczyk, A., Johannes, C., HuÈtterman, A., 1998. Oxidation of polycyclic aromatic hydrocarbons (PAHS) by laccase of Trametes versicolor. Enzyme Microb. Technol. 22, 335±341.
Nichols, T.D., Wolf, D.C., Rogers, H.B., Beyrouty, C.A., Reynolds, C.M.,
1997. Rhizosphere microbial population in contaminated soils. Water
Air Soil Pollut. 95, 165±178.
Reilley, K.A., Banks, M.K., Schwab, A.P., 1996. Dissipation of polycyclic
aromatic hydrocarbons in the rhizosphere. J. Environ. Qual. 25, 212±
219.
Schwab, A.P., Banks, M.K., 1994. Biologically mediated dissipation of
polycyclic aromatic hydrocarbons in the root zone. In: Anderson,
T.A., Coats, J.R. (Eds.). Bioremediation through rhizosphere technology, American Chemical Society, Washington, DC, pp. 132±141.
Walton, B.A., Guthrie, E.A., Hoylman, A.M., 1994. Toxicant degradation
in the rhizosphere. In: Anderson, T.A., Coats, J.R. (Eds.). Bioremediation through rhizosphere technology, American Chemical Society,
Washington, DC, pp. 11±26.
Walton, B.A., Hoylman, A.M., Perez, M.M., Anderson, T.A., Johnson,
T.R., Guthrie, E.A., Christman, R.F., 1994. Rhizosphere microbial
communities as a plant defense against toxic substances in soils. In:
Anderson, T.A., Coats, J.R. (Eds.). Bioremediation through rhizosphere
technology, American Chemical Society, Washington, DC, pp. 82±92.
Wrenn, B.A., Venosa, A.D., 1995. Selective enumeration of aromatic and
aliphatic hydrocarbon degrading bacteria by a most-probable-number
procedure. Can. J. Microbiol. 42, 252±258.