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 perenneL.). Two pot experiments were conducted with or without plants using soil spiked with 1 g kg21of 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 non-rhizospheric 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.q2000 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 rhizo-sphere, represents a complex ecosystem with the potential to accelerate biodegradation of organic contaminants, includ-ing 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 rhizo-sphere. The studies of Schwab and Banks (1994) and Reil-ley 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 contamina-tion has a greater residence time.
Plants may contribute to the dissipation of PAHs by an
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 degrada-tion 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 trans-formation rates of the toxicant. GuÈnther et al. (1996) noted that in a soil polluted with PAHs and aliphatic hydrocar-bons, 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 popu-lations that were organic compound degraders in the rhizo-sphere of alfalfa and bluegrass, in a soil amended with organic compounds, including phenanthrene and pyrene.
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However, bioremediation of the compounds was not esti-mated. Chaineau et al. (1995) showed a rapid adaptation of the soil microbial community to degradation of hydrocar-bons 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 experi-ments 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 or13C) 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 inconsis-tent 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 (radio-labelled) 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 perenneL., cv. Barclay) seedlings were grown in pots containing 250 g of an agri-cultural soil, either spiked or un-spiked with PAHs. The soil was a gleyic luvisol, with a pH of 6.6 and 15 g kg21C, 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 kg21) as described in Leyval and Binet (1998). The concentrations of PAHs in the soil were, respectively, 200 mg kg21 for anthracene, phenanthrene, ¯uoranthene and chrysene, and 50 mg kg21for 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 s21m22 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 tempera-tures 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 care-fully 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 0.25mm ®lm thickness. The column oven temperature
was: 70±1508C at 108C min21 and 150±3008C at 68C min21. The Programmable Sample Injector (PSI) temperature was set between 25 and 3008C at 1808C min21. The mass spectrometer was operated at 70 eV in impact electronic mode. Detection and quanti®ca-tion 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-prob-able-number (MPN) procedure (Wrenn and Venosa, 1995) modi®ed for our study. A PAH mixture consisting of phenanthrene (10 g l21), anthracene (1 g l21), ¯uorene (1 g l21) and ¯uoranthene (1 g l21) was added to 96-well microtiter plates (10ml/well) as a solution in hexane before
the plates were ®lled with the growth medium. Hexane was
evaporated for 1 h under a fumehood. Bushnell-Haas medium (DIFCO) supplemented with 0.85% NaCl was used as the growth medium (200ml/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 1023 to 1026, were performed, and the plates were inoculated by adding 25ml 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 enumer-ated 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 g21soil dry weight after 40 days, in all soils irre-spective of treatment.
P. Binet et al. / Soil Biology & Biochemistry 32 (2000) 2011±2017
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.
Table 1
Some characteristics of the PAHs andm/zof ions used for quanti®cation, NA: not available
Compounds Number of rings Aqueous solubility (mg l21) Hydrophobicity logKow m/zof quanti®cation ion
PAHs added
Phenanthrene 3 1.6 4.46 178
Anthracene 3 0.075 4.45 178
Fluoranthene 3 0.265 5.33 202
Chrysene 4 0.006 5.61 228
Benzo(a)anthracene 4 0.01 5.61 228
Benzo(k)¯uoranthene 4 NA 6.84 252
Dibenzo(a,h)anthracene 5 NA 5.97 278
Benzo(g,h,i)perylene 6 NA 7.23 276
Oxidation intermediates
Naphthoic acid 2 NA NA 198
Anthraquinone 3 NA NA 180
At the end of the ®rst experiment (T0140 days), the
extractable concentrations of PAHs in the non-rhizospheric soil were lower than the extractableT0values (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 extrac-table 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%
of the concentration at T180days 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 non-rhizospheric soil (Table 2).
At the end of the experiment with aged spiked soil, dissi-pation of anthracene, chrysene, benzo(k)¯uoranthene,
P. Binet et al. / Soil Biology & Biochemistry 32 (2000) 2011±2017
Table 2
Extractable concentrations (mg kg21) of the eight PAHs and anthraquinone at different times in the spiked soil.T
0: just after spiking,T180days: 180 days after spiking, mean of ®ve replicates^SE, * indicates signi®cant difference p,0:05between 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 T0140 days T180days T180days140 days
Non-rhizospheric soil Rhizospheric soil Non-rhizospheric soil Rhizospheric soil
PAHs added
PHE 187 100^26 57^7* 80 20^2 13^2
ANT 175 19^2 10^1* 35 7.5^1 8.5^2
FLT 204 150^16 85^9* 90 28.5^3 18.5^3*
CHY 200 163^11 100^11* 120 113^6 86^11*
BaANT 52 35^2 20^2* 22 15.5^1 12^2
BkFLT 48 40^2 25^2* 30 28
^2 23^3
dBahANT 51 42^3 25^3* 30 29
^2 23^3*
BghiPL 48 32^3 17^1* 20 18
^2 15^3
Total 965 582 339 427 259.5 199
Oxidation intermediate
ANQ 0 107^15 60^8* 46 20
^2 13^1
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 non-aged than in the non-aged spiked soil. In the freshly spiked soil, it was signi®cantly higher in non-rhizospheric than rhizo-spheric soil (Table 2). Metabolites of phenanthrene, and benzo(a)anthracene (e.g. naphthoic acid and benzo(a)an-thraquinone) 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-rhizospheric soil (Table 3). No PAH degraders, within the dilution range tested (from 1023to 1026), were scored for the un-spiked soil.
4. Discussion
Previous experiments with a mixture of aliphatic hydro-carbons 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 dissipa-tion of a range of PAHs, including 5 and 6 ring PAHs such as dibenzo(a,h)anthracene and benzo(g,h,i)perylene which
P. Binet et al. / Soil Biology & Biochemistry 32 (2000) 2011±2017
Fig. 3. Extractable concentrations of PAHs (mg kg21) 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.
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 g21soil dry weight
Total micro¯ora Nb g21soil dry weight
PAH degraders/total micro¯ora (%)
Exp. with freshly spiked soil
Non-rhizospheric spiked soil 6 (^5)£106 ND
Rhizospheric spiked soil 4 (^3)£105 ND
Exp. with soil ageing
Non-rhizospheric spiked soil 5 (^2)£106a 2.5 (^0.7)£107b 20
Rhizospheric spiked soil 2.3 (^0.7)£107b 1.5 (^0.3)£108a 15
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 with-out 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 Alex-ander, 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 rhizo-sphere 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 pollu-tants 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 forma-tion of bound residues during microbial degradaforma-tion 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 draw-ing conclusions about the end point of the enhanced dissipa-tion. 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 dissipa-tion of PAHs after placing the pots in the growth chamber. Only a small percentage of the soil micro¯ora is cultur-able (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 PAH-spiked and non-PAH-spiked soil. GuÈnther et al. (1996) found the same result in an experiment with aliphatic hydrocar-bons 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 hydro-carbons 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 rhizo-sphere 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 rhizo-sphere enhanced this process in an aged spiked soil, where the remaining compounds were more recalcitrant to biode-gradation. 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 rhizo-sphere 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.
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