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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
No. 12 Effects of humic acid on phytodegradation of petroleum hydrocarbons in soil simultaneously contaminated with heavy metals 2037
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 + HA
Contaminated soil + B. campestris
Contaminated soil + B. campestris + HA
0 10 20 300
2
4
6
8
10
12Contaminated soil only
Contaminated soil + HA
Contaminated soil + F. arundinacea
Contaminated soil + F. arundinacea + HA
Time (day)
0 10 20 30
Contaminated soil only
Contaminated soil + HA
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
2038 Journal of Environmental Sciences 2011, 23(12) 2034–2041 / Soyoung Park et al. Vol. 23
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
No. 12 Effects of humic acid on phytodegradation of petroleum hydrocarbons in soil simultaneously contaminated with heavy metals 2039
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).
2040 Journal of Environmental Sciences 2011, 23(12) 2034–2041 / Soyoung Park et al. Vol. 23
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).
References
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