effects of humic acid on phytodegradation of petroleum hydrocarbons in soil simultaneously...

Journal of Environmental Sciences 2011, 23(12) 2034–2041

Effects of humic acid on phytodegradation of petroleum hydrocarbons in soilsimultaneously contaminated with heavy metals

Soyoung Park1, Ki Seob Kim2, Jeong-Tae Kim1, Daeseok Kang2, Kijune Sung2,∗

1. Department of Ocean Engineering, Pukyong National University, Busan 608-737, Korea. E-mail: [email protected]. Department of Ecological Engineering, Pukyong National University, Busan 608-737, Korea

Received 10 January 2011; revised 25 March 2011; accepted 19 May 2011

AbstractThe use of humic acid (HA) to enhance the efficiency of phytodegradation of petroleum hydrocarbons in soil contaminated with

diesel fuel was evaluated in this study. A sample of soil was artificially contaminated with commercially available diesel fuel to an

initial total petroleum hydrocarbons (TPH) concentration of 2300 mg/kg and four heavy metals with concentrations of 400 mg/kg for

Pb, 200 mg/kg for Cu, 12 mg/kg for Cd, and 160 mg/kg for Ni. Three plant species, Brassica campestris, Festuca arundinacea, and

Helianthus annuus, were selected for the phytodegradation experiment. Percentage degradation of TPH in the soil in a control pot

supplemented with HA increased to 45% from 30% without HA. The addition of HA resulted in an increases in the removal of TPH

from the soil in pots planted with B. campestris, F. arundinacea, and H. annuus, enhancing percentage degradation to 86%, 64%, and

85% from 45%, 54%, and 66%, respectively. The effect of HA was also observed in the degradation of n-alkanes within 30 days. The

rates of removal of n-alkanes in soil planted with B. campestris and H. annuus were high for n-alkanes in the range of C11–C28.

A dynamic increase in dehydrogenase activity was observed during the last 15 days of a 30-day experimental period in all the pots

amended with HA. The enhanced biodegradation performance for TPHs observed might be due to an increase in microbial activities

and bioavailable TPH in soils caused by combined effects of plants and HA. The results suggested that HA could act as an enhancing

agent for phytodegradation of petroleum hydrocarbons in soil contaminated with diesel fuel and heavy metals.

Key words: phytodegradation; TPHs; humic acid; n-alkanes; DHA; combined effects

DOI: 10.1016/S1001-0742(10)60670-5

Citation: Park S, Kim K S, Kim J T, Kang D, Sung K, 2011. Effects of humic acid on phytodegradation of petroleum hydrocarbons in

soil simultaneously contaminated with heavy metals. Journal of Environmental Sciences, 23(12): 2034–2041

Introduction

Soil contamination by petroleum hydrocarbons caused

by leaking storage tanks, spillage during their trans-

port, abandoned petrochemical manufacturing facilities,

and industrial facilities has attracted considerable public

attention over the past decades (Alkorta and Garbisu,

2001; Vasudevan and Rajaram, 2001; Peng et al., 2009).

Because petroleum hydrocarbons are mixtures of chemical

substances containing hazardous chemicals such as BTEX

(benzene, toluene, ethylbenzene, and xylenes) and PAHs

(polyaromatic hydrocarbons), they have brought up serious

issues regarding risks to human health and ecosystems

(Sarkar et al., 2005).

Phytodegradation, a biological treatment technique for

contaminated soils, involves microbial degradation of

petroleum hydrocarbons in the rhizosphere and uptake

of their byproducts by the plant root system. It is

cost-effective for remediation of soils contaminated by

petroleum hydrocarbons (Erickson et al., 1994; Glass,

* Corresponding author. E-mail: [email protected]

1997; Sung et al., 2001). Liste and Prutz (2006) reported

that hemp (Cannabis sativa), white mustard (Sinapis alba),

pea (Pisum sativum), pansy (Viola tricolor), and cress (Le-pidium sativum) are capable of enhancing the remediation

of soil contaminated with total petroleum hydrocarbons

(TPHs). According to Peng et al. (2009), Mirabilis jala-pa can be tolerant to petroleum contaminated soil and

effectively remove TPHs in the soil. The phytodegrada-

tion of TPH-contaminated soil by Astragalus adsurgensand Cyperus laxua was reported by Lin et al. (2008)

and Escalante-Espinosa et al. (2005), respectively. In all

the cases mentioned above, increased microbial activities

triggered by root exudates in the rhizosphere could be

associated with the degradation of organic contaminants

(Anderson et al., 1993; Sung et al., 2006).

Petroleum hydrocarbons are highly hydrophobic with

low water solubility and thus have a strong attraction to

soil particles. Because microorganisms can degrade only

the bioavailable portion of contaminants (Al-Bashir et

al., 1990; Sung et al., 2001), low water solubility and

strong partition to solid phase may reduce bioavailable

petroleum hydrocarbons to degrading-microorganisms.

No. 12 Effects of humic acid on phytodegradation of petroleum hydrocarbons in soil simultaneously contaminated with heavy metals 2035

The poor bioavailability of petroleum hydrocarbons to

plants and microorganisms associated with their root

systems could be an important limiting factor for success-

ful phytodegradation. Phytodegradation of hydrophobic

organic compounds in polluted soils is a process that in-

volves interactions among soil particles, pollutants, water,

plants, and microorganisms in the rhizosphere (Sung et al.,

2004). Therefore, one of the feasible ways to increase the

bioavailability of hydrophobic organic compounds such

as petroleum hydrocarbon pollutants in soils is to use

surfactants (Fava et al., 1998).

Humic substances have surfactant-like micelle mi-

crostructures that can increase the solubility of organic

compounds and have potential for enhancing the degrada-

tion of hydrophobic organic compounds (Holman et al.,

2002). As a part of organic matter in soil, it comprises

relatively high molecular weight substances formed by

secondary synthetic reactions. Fava and Piccolo (2002)

showed that natural humic substances could enhance the

bioavailability of PCBs and thereby their aerobic biodegra-

dation in contaminated soils. Smith et al. (2009) found that

the presence of HA could lead to a direct increase in the

degradation of phenanthrene in soil. Therefore, humic acid

(HA) could act as a natural surfactant for enhancing the

bioavailability of TPHs in petroleum-contaminated soil for

successful phytodegradation.

Industrial processes could contaminate soils with both

TPHs and heavy metals (Adeniyi and Afolabi, 2002). If

soils are contaminated by both heavy metals and toxic

organic contaminants, it is very difficult to remedy using

bioremediation due to adverse effects of heavy metals on

organic matter degradation rate (Mitsch and Jørgensen,

2004; Zwolinski, 1994). Therefore, the application of

phytodegradation to degrade TPHs in soil simultaneous-

ly contaminated with heavy metals might be seriously

affected by the potential toxicity of heavy metals on mi-

croorganisms (Gadd and Griffiths, 1978). However, HA is

known to reduce the metal toxicity towards TPH-degrading

microorganisms because it forms stable complexes with

heavy metals in the water phase of soil (Chen, 1996;

Halim et al., 2003). This characteristic may contribute

to the improvement of phytodegradation efforts in se-

riously contaminated areas. There are only few studies

on HA-aided phytodegradation and even fewer ones on

phytodegradation of soils simultaneously contaminated

with heavy metals. This study investigated the effect of

HA as a natural surfactant on TPH phytodegradation and

a natural fertilizer, with a focus on the enhancement of

microbial activities in soils contaminated with both TPHs

and heavy metals.

1 Materials and methods

1.1 Soil treatment

Soil was collected from a site at the campus of Pukyong

National University in Busan, Korea. In the laboratory,

the soil was air-dried and passed through a 2-mm sieve

prior to use. The pH was measured using an Orion 4

star pH electrometer (Thermo Electron Co., USA) in a

1:1 (W/V) soil:water paste (Thomas, 1996). The organic

matter content and cation exchange capacity (CEC) were

measured using the loss-on-ignition method (Nelson and

Sommers, 1996) and the 1 N-Acetic acid replacement

method (NAAS, 1988), respectively. The total organic car-

bon (TOC) was analyzed using a TOC analyzer (Shimadzu,

Japan). The contents of carbon, hydrogen, oxygen and

sulfur of the soil were analyzed using an element analyzer

(Vario macro/micro, Germany). The sand, silt, and clay

fractions were determined by the pipette method and

the soil texture was determined according to the textural

classification of the US Department of Agriculture. The

physicochemical properties of the soil used in this study

are summarized in Table 1.

The soil was artificially contaminated with commercial-

ly available diesel fuel with the initial TPH concentration

at 2300 mg/kg. It was also contaminated with PbCl2(99%, Kanto, Japan), CuCl2 (99%, Acros, Belgium),

CdCl2·2.5H2O (98%, Kanto, Japan), and NiSO4·6H2O

(99%, Kanto, Japan) to the levels of the Korean soil con-

tamination countermeasure standards for Pb (400 mg/kg),

Cu (200 mg/kg), Cd (12 mg/kg), and Ni (160 mg/kg).

Contaminated soil was kept for 7 days to stabilize and

then used as initial soil samples. Commercial HA (Daesin,

Korea) was added to the soil at an application dosage of

0.1% mass base. The composition of HA used was (73.1

± 2.5)% C, (6.2 ± 0.3)% H, (0.7 ± 0.2)% N, and (1.2 ±0.01)% S.

Three plant species of Brassica campestris, Festucaarundinacea, and Helianthus annuus were selected. They

were previously shown to have an ability to germinate and

grow in soils contaminated with TPH (5000 mg/kg), Pb

(400 mg/kg), Cu (200 mg/kg), Cd (12 mg/kg), and Ni (160

mg/kg) (Kim and Sung, 2011). Plants were germinated

and grown for 15 days in uncontaminated soil in a growth

chamber, and then 4 individuals of each species were trans-

planted into stainless pots containing the contaminated soil

(14.5-cm tall, 10.5-cm i.d.).

The transplanted plants were grown for 30 days under

greenhouse conditions with a daylight period of 14 hr,

a light intensity of (3500 ± 800) lux, a humidity of

45%–50% and a temperature of (28 ± 2)°C. During the

experimental period, the plants were watered every day

with distilled water (50 mL) to compensate for evapotran-

spiration loss.

To evaluate the effects of HA on TPH phytodegradation

Table 1 Physicochemical properties of the soil used in this study

Properties Value Properties Value

pH 7.71 H (%) 6.0 ± 0.7*

Organic matter content (%) 3.82 S (%) 12.3 ± 1.1*

CEC (meq/100 g) 19.14 Sand (%) 77.2

TOC (%) 0.22 ± 0.03* Silt (%) 8.2

Total N (mg/kg) 110 Clay (%) 14.6

P2O5 (mg/kg) 80 Soil texture Sandy loam

C (%) 17.7 ± 1.8*

* Data are expressed as mean ± SD. CEC: cation exchange capacity;

TOC: total organic carbon.

2036 Journal of Environmental Sciences 2011, 23(12) 2034–2041 / Soyoung Park et al. Vol. 23

in the contaminated soil with petroleum hydrocarbons and

heavy metals, a total of 10 pots were prepared for testing

with 6 different treatments: (1) a pot with uncontaminated

soil only; (2) a pot with uncontaminated soil amended with

HA; (3) a pot with contaminated soil only (control); (4)

a pot with contaminated soil amended with HA (control

+ HA); (5) 3 pots with contaminated soil and planted

with one of the three plant species (plant only); and (6)

3 pots with contaminated soil supplemented with HA, and

planted with one of the three plant species (plant + HA).

1.2 Determination of diesel in soil

Soil samples to determine TPH concentrations of soil

in the pots were collected from the rhizospheres after 1,

15, and 30 days. Total petroleum hydrocarbons in soil

were extracted using ultrasonic extraction according to US

EPA test method 3550B (US EPA, 1996a). In this test,

a 10-g soil sample was mixed with anhydrous sodium

sulfate to form a free-flowing powder. Next, 100 mL of

dichloromethane was added as an extraction solvent and

the sample was treated ultrasonically for 3 min using

an ultrasonic dismembrator (Fisher Scientific, USA) in

a pulse mode with a 50% duty cycle. The extract was

then decanted and filtered through filter paper (Advantec,

USA). The extraction was repeated twice, and the extract

was rinsed with the extraction solvent and concentrated

using a rotary evaporator N-1000S-W (Eyela, Japan).

A stock standard solution for TPH was prepared using

FTRPH calibration/window defining standard (Accustan-

dard, USA) and a stock standard solution for n-alkanes

were prepared using DRH-008S-R2 (Accustandard, USA).

The stock standard solutions were then used to prepare

calibration standards at five different concentrations for

both TPH and n-alkanes by the dilution method with a

minimal headspace.

The efficiency of extraction recovery of TPH was tested

using ortho-terphenyl (OTP) and nonatriacontane (C39)

(Supelco, USA). Two milliliters of OTP and nonatria-

contane were added in 10 g of uncontaminated soil and

extracted following the same method used in the sample

extraction. Extraction recovery efficiency of TPH was in

the range of 82%–97%.

Following the US EPA test method 8015B (US EPA,

1996b), the quantity of TPH in the extract was determined

using a gas chromatograph with a flame ionization detector

(Shimadzu GC 2010, Japan) equipped with a 30-m cap-

illary column (J&W DB-5, 0.32 mm i.d., 0.25 mm film

thickness).

Temperature conditions of GC-FID were 280°C for

injection port, 340°C for detector, and an oven temperature

program of 45°C (held for 2 min) to 310°C (held for 25

min) at a rate of 10°C/min. Helium was used as the carrier

gas at a flow rate of 1 mL/min.

The concentrations of TPHs and n-alkanes were deter-

mined separately. The concentration of TPHs, including

the unresolved portion of diesel, can provide an overall

phytodegradation efficiency supplemented with HA, while

the n-alkane concentration can indicate a specific degra-

dation pattern according to the molecular weight of diesel

components.

1.3 Soil microbial activity

Dehydrogenase activity in soil contaminated with or-

ganic contaminants can be used as an indirect indicator

of microbially-mediated remediation of soil because bio-

logical oxidation of organic compounds typically involves

a dehydrogenation catalyzed by dehydrogenase enzymes

(Page et al., 1982; Paul and Clark, 1989; Balba et al.,

1998). A colorimetric method was used to measure dehy-

drogenase activity in soil as it indicates the reduction of

TTC (2,3,5-triphenyltetrazolium chloride) to TPF (triph-

enylformazan).

A 3-g soil sample was placed in a 50-mL cap vial. After

adding 0.03 g CaCO3, 1 mL of 3% TTC solution and 2.5

mL of distilled water were added into the vial and mixed

thoroughly. After 24 hr of incubation at 37°C, red TPF

was extracted by adding 10 mL methanol and shaking

for 1 min. Extracted formazan was filtered through filter

paper (Whatman No. 42) in a funnel by adding methanol.

Methanol was added continuously until the red color

disappeared from the filter paper. The filtrate was then

diluted to 100 mL with methanol. The color intensity of

the filtrate was determined with a UV spectrophotometer

(Shimadzu, Japan) at 485 nm.

1.4 Statistical analysis

Because the experiments were conducted in a random-

ized block design without replicates, three factors that

possibly affect TPH degradation (addition of HA, presence

of plants, and time) were analyzed using ANOVA followed

by the least significant difference (SAS Version, 9.1).

2 Results and discussion

2.1 Microbial dehydrogenase activity

Dehydrogenase activity (DHA), which is used as an

indicator for microbial degradation of organic matter,

increased in all of the pots subjected to different treatments

in the 30-day experiment (Fig. 1). DHA in the pots supple-

mented with HA was larger than that in the pots without

HA for contaminated soil until day 15. However, after 15

days of treatment, DHA in the pots with HA was greater

than that in the pots without HA for both contaminated

and uncontaminated soils. The effects of HA on DHA were

greater in the contaminated soil than the uncontaminated

one (Fig. 1a). This suggests that there is no inhibitory

effect of heavy metals on soil microbial activities (Balba

et al., 1998) and overall soil biogeochemical activities

increase because TPHs could be used as substrates for

microorganisms.

DHA in the pots with plants was greater than that in

the control pot and DHA increased with the addition of

HA (Fig. 1b–d). HA increased DHA greater than plants

did. Dynamic increases in DHA were observed for the

last 15 days of the 30-day experimental period in all pots

supplemented with HA, with an exception for the pots

that had F. arundinacea (Fig. 1c). The greatest increase


DH

A (

μg

TP

F/(

g d

ry s

oil

. day

))

2

4

6

8

10

12Uncontaminated soil

Uncontaminated soil + HA

Contaminated soil

Contaminated soil + HA

Contaminated soil only


Contaminated soil + B. campestris

Contaminated soil + B. campestris + HA

0 10 20 300

2

4

6

8

10

12Contaminated soil only


Contaminated soil + F. arundinacea

Contaminated soil + F. arundinacea + HA

Time (day)

0 10 20 30

Contaminated soil only


Contaminated soil + H. annuus

Contaminated soil + H. annuus + HA

Fig. 1 Changes in dehydrogenase activity (DHA) in the pots with different treatments for a 30-day experiment. HA: humic acid.

in DHA by HA amendment was observed in the pots

with B. campestris and H. annuus. In the pots containing

H. annuus, a continuous increase in DHA was measured

from the beginning of the experiment. Increase in DHA in

the pots supplemented with HA usually indicates that mi-

croorganisms are metabolically active and might contribute

to the biodegradation of TPHs.

The pot with H. annuus had 2.9-fold higher DHA than

the initial DHA of the control pot without HA, and it

increased to 5.8-fold with HA addition while the control

showed 3.8-fold higher DHA with HA addition at day 30

(Fig. 2).

2.2 Effect on TPH phytodegradation

Irrespective of treatments, the concentrations of TPH

recovered from soil in the pots decreased during the 30-

day incubation period (Fig. 3). However, the results shown

in Fig. 3 indicate that the degradation of TPH was greater

in the pots supplemented with HA than in the ones without

HA. Statistical analysis showed that both HA (p = 0.0201)

and time (p < 0.0001) affect the extent of TPH degradation.

The effect of plants on TPH degradation was the greatest

for H. annuus, but the combination of plants and HA had

the greatest for H. annuus and B. campestris. However,

the combined effect of HA and plants on TPH degradation

was not significant (p = 0.844). The effects of HA were

more conspicuous at day 30 than at day 15 and were

more pronounced when combined with plants than in the

controls (Fig. 3). These results suggest that enhanced phy-

todegradation induced by supplementing with HA requires

a certain period of time to exert an effect. The combined

effects of plants and HA appear to be greater than the

effects produced by plants or HA addition alone.

Only 30% of TPHs were degraded in the soil of the

control pot without HA. This could be attributed to natural

attenuation possibly by intrinsic microbial degradation,

volatilization and irreversible absorption by the contam-

inated soil. Comparison of TPH concentrations in the

control pot and the ones with plants illustrates the effect

of phytodegradation on the artificially contaminated soil

of this study (Fig. 4). TPHs were dissipated in phytodegra-

dation with B. campestris, F. arundinacea, and H. annuus,with TPH removal of 45%, 54%, and 66%, respectively

(Fig. 4). Peng et al. (2009) showed that a similar degree

of removal (19.75%–37.92% by natural attenuation and

41.61%–63.20% with Marabilis jalapa) occurred over a

127-day culture period.

However, TPH degradation in the control pot amended

with HA increased to 45%, as compared to 30% in the

control without HA added. Moreover, the addition of HA

increased TPH removal from soil in the pots planted with

B. campestris, F. arundinacea, and H. annuus to 86%,

64%, and 85%, respectively (Fig. 4). The proportional

decrease in the TPH concentration of soil after HA was

added was the greatest (2.8-fold higher) in the pots that

had B. campestris. The effect of HA on TPH degradation

in soil was the lowest for the control pot. This indicates

that HA had a greater effect on phytodegradation of soil

contaminated with TPH than on natural attenuation.

When no HA was added, the increasing order of TPH


0.0

1.0

2.0

3.0

4.0

5.0

6.0

7.0

Control

Treatment

DH

A ra

tio (

trea

tmen

t/co

nta

min

ated

only

)

Day 15

Day 30

B. campestris H. annuus F. arundinacea H. annuus Control B. campestris F. arundinacea

Contaminated soil Contaminated soil + HA

Fig. 2 Ratio of dehydrogenase activity in different treatments at day 15 and day 30 to that in the control without both plants and HA at day 1.

0

500

1000

1500

2000

2500

Control

Treatment

TP

H (

mg/k

g)

Without HA

With HA

B. campestris F. arundinacea H. annuus

Day 1 Day 15 Day 30 Day 1 Day 15 Day 30 Day 1 Day 15 Day 30 Day 1 Day 15 Day 30

Fig. 3 TPH concentrations recovered from artificially contaminated soil in the pots with different treatments after 1, 15, and 30 days.

removal efficiency with respect to plant species was H. an-nuus > F. arundinacea > B. campestris. When HA

was added, more TPH were removed from soil in the

pots containing B. campestris and H. annuus than that

in the pots with F. arundinacea. Except for the case of

B. campestris, TPH removal from the pots with plants

but no HA added was greater than that in the control

pot with the HA addition. This result suggests that the

efficiency of phytodegradation of soil contaminated with

diesel oil could be higher than that of natural attenuation

supplemented with HA.

Even if HA added to the artificially contaminated

soil at the beginning of the experiment might have

provided adsorption surface for hydrocarbons, enhanced

phytodegradation seems to have contribution to a decrease

in TPH concentrations as reflected in increased dehydro-

genase activity by root exudates together with HA added.

Dissolved humic substances can facilitate the desorption

of hydrophobic pollutants from the soil solid phase and

increase bioavailability of contaminants in phytodegrada-

tion (Iglesias-Jimenez et al., 1997; Kastner and Devliegher,

1996; Kastner and Mahro, 1996; Fava and Piccolo, 2002).

2.3 Effect on n-alkanes (C9–C28) degradation

The effects of plants and HA on the removal of n-alkanes

in the range of C9–C28 (n-nonane ∼ n-octacosane) in soil

were evaluated by comparing n-alkane concentrations in

the control pots and the ones with plants +HA (Fig. 5).

Compared to the control pot, the effect of plants on the

removal of n-alkanes could not be established within 30

experimental days in the pots planted with B. campestris(Fig. 5a). However, the addition of HA to the pots planted

with B. campestris resulted in a more conspicuous decrease

in the concentration of n-alkanes than that in the control


0

10

20

30

40

50

60

70

80

90

100

F. arundinacea

Treatment

TP

H r

emoval

(%

)

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

TP

H r

emev

al r

atio

(tr

eatm

ent/

contr

ol)

Removal

Removal ratio

Control B. campestris H. annuus F. arundinacea Control B. campestris H. annuus

Contaminated soil + HAContaminated soil

Fig. 4 TPH removal from artificially contaminated soil in the pots with different treatments, and ratio of TPH removal in the planted pots to that in the

control after 30 days of experiment.

0.0

0.2

0.4

0.6

0.8

1.0

1.2Day 1 Day 30

Conce

ntr

atio

n r

atio

(C

/Cco

ntr

ol)

0.0

0.2

0.4

0.6

0.8

1.0

0.0

0.2

0.4

0.6

0.8

1.0

a b

c d

e f

n-Alkane

C9 C10 C11 C12 C13C14 C15 C16 C17 C18 C19 C20 C21 C22 C23 C24 C25 C26 C27 C28 C9 C10 C11 C12 C13 C14 C15 C16 C17 C18 C19 C20 C21 C22 C23 C24 C25 C26 C27 C28

Fig. 5 Ratios of n-alkanes (C9–C28) concentrations in the planted pots with or without HA added to that in the control. B. campestris (a), B. campestris+ HA (b), F. arundinacea (c), F. arundinacea + HA (d), H. annuus (e), and H. annuus + HA (f).


pot. The effect of HA on the removal of n-alkanes was

found in the range of C14–C27 one day after HA was

added. This trend was obvious for n-alkanes in the range

of C11–C28 at day 30. HA had the largest effect on the

concentration of n-heptadecane (C17) (Fig. 5b).

For n-alkanes of C12–C23, phytodegradation seems to

have contribution to their removal as demonstrated in the

pots planted with F. arundinacea and H. annuus at day

30 even though it was not established at the beginning

of the experiment (Fig. 5c, e). However, compared to the

control pot, the addition of HA to both pots resulted in

a decrease in the concentration of n-alkanes for a more

broad range of C11–C28 at day 30. In both pots, HA had

the largest effect on the concentration of n-heptadecane

(C17) as in the pots planted with B. campestris (Fig. 5d, f).

The concentrations of n-alkanes after the addition of HA

decreased more in soils with B. campestris and H. annuusthan that with F. arundinacea. This implies that the effect

of HA on phytodegradation may depend on plant species.

Decrease in n-alkane concentrations in the experimental

soil with a wider range of n-alkanes dissipated at day

30 might be due to increased microbial activities and

bioavailable TPH in soils produced by combined effects

of plants and HA. This was attributed to the potential

increase of specific microorganisms such as dioxygenase-

expressing rhizosphere bacteria (Liste and Prutz, 2006).

3 Conclusions

Low TPH bioavailability and degradability are key lim-

iting factors that control the phytodegradation efficiency

of petroleum contaminated soil. Heavy metal toxicity may

also constrain the use of phytodegradation in simultane-

ously contaminated soil with heavy metals and TPHs. This

study evaluated the use of HA-aided phytodegradation in

enhancing the removal of TPH in an experimental soil

contaminated with TPH and heavy metals. TPH removal in

the soil of the control pot amended with HA was 45%, as

compared to 30% observed in the control pot with no HA

added. Moreover, the addition of HA resulted in decreased

TPH concentrations in the soil in the pots planted with

B. campestris, F. arundinacea, and H. annuus.In these pots, the percentage degradation of TPH in-

creased to 86%, 64%, and 85% from 45%, 54%, and

66%, respectively. Combined effects of plants and HA

were also observed for the degradation of n-alkanes of

C11–C28. During the experimental period of 30 days, the

removal of n-alkanes from the soil in the pots planted with

B. campestris, F. arundinacea, and H. annuus increased

after HA was added. Increased levels of DHA suggest that

heavy metal toxicity has negligible effects on microbial

TPH degradation. As suggested by Chen (1996) and Halim

et al. (2003), the addition of HA might have partially

contributed to reducing heavy metal toxicity on TPH-

degrading microbes by forming stable complexes with

heavy metals in the soil water phase.

The enhanced remedial performance for TPHs in this

study might be due to the combined effect of plants and HA

that caused increase in microbial activities and bioavailable

TPH in soil. Furthermore, the combined effect of plants

and HA seems to enhance chemical extractability and

degradability by increasing specific microorganisms. The

results of this study suggest that HA could function as an

enhancing agent in phytodegradation of soil contaminated

with TPH and heavy metals.

Acknowledgments

This work was supported by the Korea Research Foun-

dation (KRF) grant funded by the Korean Government

(MOEHRD) (No. KRF-2007-521-F00006) and MEST

(No. 2009-0075072).

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