Short communication

Soil microbial response during the phytoremediation

of a PAH contaminated soil

D.L. Johnsona, D.R. Andersonb, S.P. McGratha,*

aAgriculture and the Environment Division, Rothamsted Research, Harpenden, Herts. AL5 2JQ, UKbCorus RD and T, Environment Department, Swinden Technology Centre, Moorgate, Rotherham S60 3AR, UK

Received 26 June 2004; received in revised form 24 March 2005; accepted 5 April 2005

Abstract

The aim of this trial was to quantify and compare the responses of soil microbial communities during the phytoremediation of polycyclic

aromatic hydrocarbons (PAHs) in a laboratory trial. The experiment was conducted in 1-kg pots and planted treatments consisted of a mixed

ryegrass (Lolium perenne) and white clover (Trifolium repens) sward together with a rhizobial inoculum (Rhizobium leguminosarum bv.

trifolii). Throughout the 180-d experiment soil microbial biomass and communities of PAH degraders were monitored. PAH degradation was

enhanced in planted treatments that received a rhizobial inoculum. Microbial biomass was enhanced in planted treatments, but there were no

significant differences between treatments that had received a rhizobial inoculum and those that had not. However, numbers of PAH

degraders were greater in the treatment that had received a rhizobial inoculum.

q 2005 Elsevier Ltd. All rights reserved.

Keywords: Phytoremediation; Bioremediation; PAHs; Rhizosphere; Rhizoremediation; PAH degraders

There is mounting evidence that microbial activity in the

rhizosphere may increase the degradation of persistent

industrial chemicals such as polycyclic aromatic hydro-

carbons (PAHs) (Joner et al., 2001; Johnson et al., 2004).

A recent study by Johnson et al. (2004) demonstrated that

planting a soil with a mixed clover/ryegrass sward, together

with a rhizobial inoculum, enhanced the dissipation of

chrysene in a controlled experiment. It was suggested that

this may have been due to the growth response of microbial

communities within the soil. The aim of this study was to

test the hypothesis that the observed enhanced dissipation of

PAHs in the rhizosphere was due to the stimulation of the

microbial community within the soil rhizosphere. With this

in mind, several microbial techniques were employed to

monitor the soil microbial communities during a rhizo-

remediation laboratory trial.

The 16 US-EPA priority pollutants were examined in this

study. These compounds were defined as priority PAHs

0038-0717/$ - see front matter q 2005 Elsevier Ltd. All rights reserved.

doi:10.1016/j.soilbio.2005.04.001

* Corresponding author. Tel.: C44 1582 763 133; fax: C44 1582 760

981.

E-mail address: steve.mcgrath@bbsrc.ac.uk (S.P. McGrath).

based on their recalcitrance in the soil and their carcino-

genicity (IACR, 1983). Results presented here are a sum of

the total concentrations of extractable 16 US-EPA PAHs.

For PAH extraction wet soil samples were mixed with a

drying agent and extracted on a Dionex ASE200 accelerated

solvent extractor system. The sample was analysed by GC-

MS using internal standards to quantify soil PAH concen-

trations. Soil ammonium-N and nitrate-N were analysed by

continuous colourmetric flow analysis using a Skalar

SANPLUS system (Henriksen and Selmer-Olsen, 1970;

Krom, 1980). Standard error of means and the t-test were

calculated using the Genstat 5 package (1993).

An inoculum of an isolate of Rhizobium leguminosarum

bv. trifolii, selected for its tolerance to PAHs (Johnson et al.,

2004), was produced using a peat carrier. The inoculum and

seeds of the host legume (Trifolium repens) were planted into

soils together with ryegrass (Lolium perenne L.). The soil

used was collected in the vicinity of a coking plant and had a

total concentration of PAHs in excess of 1000 mg kgK1

when collected. A multifactorial greenhouse experiment

including unplanted treatments was set-up. There were four

treatments in this trial: planted with clover/ryegrass; planted

with clover/ryegrass and a rhizobial inoculum; no plants

with a rhizobial inoculum, no plants without an inoculum.

Soil Biology & Biochemistry 37 (2005) 2334–2336

www.elsevier.com/locate/soilbio

0

5000

10000

15000

20000

25000

30000

Planted +Rhizobia

Planted -Rhizobia

Unplanted +Rhizobia

Unplanted -Rhizobia

Treatment

Cel

ls g

-1

0 days180 days

Fig. 2. Most probable number of PAH degrading microbes in soil at the end

of the 180-d experiment.

D.L. Johnson et al. / Soil Biology & Biochemistry 37 (2005) 2334–2336 2335

The total soil microbial biomass was assessed using a

standard fumigation–extraction technique (Vance et al.,

1987). Soil microbial biomass was assessed in all treatments

at TZ0, 30, 60, 90, 120 and 180 days.

In addition to monitoring total soil microbial biomass, a

solvent-free microtitre plate method was applied to

determine the specific communities of PAH degraders in

the pots during remediation (Steiber et al., 1994). The

method allows a Most Probable Number estimation of PAH

degraders in the soil.

At the end of the 180-d experiment the planted pot

treatment had significantly (PZ!0.05) lower concen-

trations of total extractable PAHs (824 mg kgK1G17)

than the unplanted treatment (1017 mg kgK1G71). Data

are means and standard error of means (where nZ6). PAH

concentrations were only significantly lower than the

unplanted treatments in the planted treatments that had

received a rhizobial inoculum. Planted treatments with no

inoculum did not show a significant reduction in PAH

concentrations at the end of the 180-day trial relative to

similar unplanted treatments.

Soil pH did not change significantly over the 180-d

experiment. Similarly, soil nitrogen concentrations

(ammonium-N and nitrate-N) did not change significantly

during the experiment.

Soil microbial biomass did not differ between treatments

at the start of the experimental period (Fig. 1). However, by

the end of the experimental period there was a significantly

greater biomass in the inoculated planted treatments than in

the unplanted treatments (PZ!0.01). No significant

differences were observed between planted treatments and

planted inoculated treatments.

The most probable number of PAH degraders was

influenced by planting regime (Fig. 2). Initially there were

only small differences between the numbers of PAH

degraders. However, after 180-days, numbers of micro-

organisms capable of degrading PAHs were greater in the

planted treatments relative to the unplanted treatments.

Time (days)0 20 40 60 80 100 120 140 160 180 200

Soil

Mic

robi

al B

iom

ass

C (

mg

C k

g–1 s

oil)

0

100

200

300

400

500

600Planted with rhizobial inoculumUnplanted with rhizobial inoculum Unplanted without rhizobial inoculumPlanted without rhizobial inoculum

Fig. 1. Changes in soil microbial biomass throughout the 180-day pot trial.

Data are means and standard deviations.

However, unlike total soil biomass, the planted treatment

thathad receiveda rhizobial inoculum hadagreaternumberof

PAH degraders than the planted treatments with no inoculum.

Inoculated plots had an average of 1.5 clover nodules gK1

soil as opposed to 0.7 nodules gK1 soil in the planted

treatment that had received no inoculum. The presence of 0.7

nodules gK1 confirms that indigenous rhizobia were present

in the soil. However, both root and shoot growth were also

greater in the inoculated plots (Table 1), confirming that the

inoculum did have a positive impact upon plant vigour.

Previous laboratory tests designed to measure the in-vitro

degradation of PAHs had suggested that the strain of

R. leguminosarum used in this trial is unable to degrade the

PAHs as a sole carbon source. In the same trial, planted

sterile soils did not show significantly different concen-

trations of extractable chrysene with time (Johnson et al.,

2004). This suggests that the plants in the present trial did

not have a direct role in the dissipation of PAHs. Soil-borne

hydrophobic organic compounds such as PAHs, with a

relatively high log Kow, are rarely translocated from root to

shoot to a significant degree. Significantly lower extractable

concentrations of PAHs, observed in the planted treatment

that had received a rhizobial inoculum, are therefore,

unlikely to be a result of direct uptake by either the clover or

ryegrass or direct degradation by rhizobia.

The influence of the inoculated rhizobia is reflected in a

larger shoot and, most importantly, root biomass in the

inoculated pots (Table 1). Microbial measurements reveal

that the microbial biomass was significantly greater in planted

Table 1

Influence of rhizobial inoculum on root and shoot growth at end of the 180-

day pot trial

Root biomass mg gK1

soil

Shoot weight g per

pot

Planted/inoculated 8.21 (0.92) 2.23 (0.18)

Planted 4.13 (0.56) 1.59 (0.36)

Data are means on a dry weight basis with standard deviations in

parentheses (nZ10).

D.L. Johnson et al. / Soil Biology & Biochemistry 37 (2005) 2334–23362336

treatments than in unplanted treatments (Fig. 1). However,

there is no significant difference between the biomass of

planted treatments that had received a rhizobial inoculum and

those that had not. In contrast, the numbers of PAH degraders

increased in the presence ofa rhizobial inoculum(Fig.2).This

suggests that selective enhancement of PAH degraders within

the rhizosphere leads to enhanced PAH loss.

Earlier studies (e.g. Siciliano et al., 1998) have shown

enhanced losses of PAHs in planted soils and suggested that

the mechanism by which this occurs is via plant root

exudates stimulating the microbial community involved in

the dissipation of aromatic compounds. Leigh et al. (2002)

went one step further and demonstrated that seasonal fine

root death releases several flavones which act as substrates

for polychlorinated biphenyl (PCB) degrading bacteria.

Thus, upon death, fine roots may serve not only as injectors

of bacterial substrates but also may facilitate soil aeration

through the formation of air channels left after root

senescence. Corgie et al. (2004) found that phenanthrene

degradation declined with distance from plant roots.

Although the authors found the influence of planting regime

on numbers of PAH degraders was less important than the

presence or absence of phenanthrene. It therefore follows

that any improvement of root growth through the use of a

growth stimulating rhizobial inoculum will lead to a

stimulation of the growth and activity of bacteria capable

of degrading PAHs.

In conclusion, our results support the hypothesis that the

enhanced dissipation of PAHs in the rhizosphere was due to

the stimulation of the microbial community within the soil

rhizosphere. However, this loss was only greater in soils that

received a rhizobial inoculum. It is therefore likely that

rhizobia play an important role in the rhizoremediation of

high molecular weight PAHs. It would appear that microbes

responsible for PAH degradation are selectively enhanced

within the rhizosphere of soil that has received a rhizobial

inoculum. The exact mechanisms involved in this process

are not revealed and further work is required to further

elucidate the processes involved.

Acknowledgements

This work was funded by a BBSRC grant. Rothamsted

Research receives grant-aided support from the UK

Biotechnology and Biological Sciences Research Council.

References

Corgie, S.C., Beguiristain, T., Leyval, C., 2004. Spatial distribution of

bacterial communities and phenanthrene degradation in the rhizosphere

of Lolium perenne L. Applied and Environmental Microbiology 70 (6),

3552–3557.

Henriksen, A., Selmer-Olsen, A.R., 1970. Automatic methods for

determining nitrate and nitrite in water and soil extracts. Analyst 95,

514–518.

IACR, 1983. Monographs on the Evaluation of the Carcinogenic Risk of

Chemicals to Humans: Polynuclear Aromatic Hydrocarbons, vol. 32.

WHO, Lyons, France.

Johnson, D.L., Maguire, K.L., Anderson, D.R., McGrath, S.P., 2004.

Enhanced dissipation of chrysene in planted soil: the impact of a

rhizobial inoculum. Soil Biology & Biochemistry 36, 33–38.

Joner, E.J., Johansen, A., Loibner, A.P., Dela Cruz, M.A., Szolar, O.H.J.,

Portal, J.M., Leyval, C., 2001. Rhizosphere effects on microbial

community structure and dissipation and toxicity of polycyclic aromatic

hydrocarbons (PAHs) in spiked soil. Environmental Science and

Technology 35, 2773–2777.

Krom, M., 1980. Spectrophotometric determination of ammonia; a study of

modified Berthelot reaction using salicyclate and chloroisocyanurate.

The Analyst 105, 305–316.

Leigh, M.B., Fletcher, J.S., Fu, X., Schmitz, F.J., 2002. Root turnover: an

important source of microbial substrates in rhizosphere remediation of

recalcitrant contaminants. Environmental Science and Technology 36,

1579–1583.

Siciliano, S.D., Germida, J.J., Siciliano, S.D., 1998. Biolog analysis and

fatty acid methyl ester profiles indicate that pseudomonad inoculants

that promote phytoremediation alter the root-associated microbial

community of Bromus biebersteinii. Soil Biology & Biochemistry 30,

1717–1723.

Steiber, M., Haeseler, F., Werner, P., Hartmann, F., 1994. A rapid screening

method for microorganisms degrading polycyclic aromatic hydrocar-

bons in microplates. Applied Microbiology and Biotechnology 40,

753–755.

Vance, E.D., Brookes, P.C., Jenkinson, D.S., 1987. An extraction method

for measuring microbial biomass C. Soil Biology & Biochemistry 19,

703–707.