EnvironmentAsia

The international journal published by the Thai Society of Higher Education Institutes on Environment
Available online at www.tshe.org/ea/index.html

EnvironmentAsia
Available
DOI
EnvironmentAsia
9(2) (2016) 128-133
online at www.tshe.org/EA
2 (2009) 50-54
10.14456/ea.2016.16

Genotoxicity Assessment of Mercuric Chloride in the Marine Fish Therapon jaruba

Effect of Indole-3-Acetic Acid-Producing Bacteria on Phytoremediation of Soil
Contaminated
Nagarajan with Phenanthrene
Nagarani, Arumugam Kuppusamy and Anthracene
Kumaraguru, by Mungbean
Velmurugan Janaki Devi
and Chandrasekaran Archana Devi
Waraporn Chouychai a, Thidarat Paemsom a, Chittra Pobsuwan a, Khanitta Somtrakoon b
c
Center for Marine and Coastal Studies,and
School
HungofLee
Energy, Environment and Natural Resources,
Madurai Kamaraj University, Madurai-625021, India
a
Biology Program, Faculty of Science and Technology, Nakhonsawan Rajabhat University, Nakhonsawan
60000, Thailand
Abstract b
Department of Biology, Faculty of Science, Mahasarakham University, Kantharawichai,
Mahasarakham 44150, Thailand
The aim cof the present
School study was Sciences,
of Environmental to standardize and to
University of assess
Guelph,the predictive
Guelph, value
Ontario, of 2W1,
N1G the cytogenetic
Canada analysis
by Micronucleus (MN) test in fish erythrocytes as a biomarker for marine environmental contamination. Micronucleus
frequency baseline in erythrocytes was evaluated in and genotoxic potential of a common chemical was determined
in fish experimentally exposed in aquarium under controlled conditions. Fish (Therapon jaruba) were exposed for 96
Abstract
hrs to a single heavy metal (mercuric chloride). Chromosomal damage was determined as micronuclei frequency in
fish erythrocytes. Significant increase in MN frequency was observed in erythrocytes of fish exposed to mercuric
The use of indole-3-acetic acid (IAA)-producing bacteria isolated from non-contaminated weed rhizosphere to enhance
chloride. Concentration of 0.25 ppm induced the highest MN frequency (2.95 micronucleated cells/1000 cells compared
plant growth and PAH phytoremediation capacity was investigated. IAA-producing bacterial isolates, designated NSRU1,
to 1 MNcell/1000 cells in control animals). The study revealed that micronucleus test, as an index of cumulative
NSRU2, and NSRU3, were isolated from the rhizosphere of Eleusine indica (Poaceae) and Chromolaena odorata
exposure, appears to be a sensitive model to evaluate genotoxic compounds in fish under controlled conditions.
(Asteraceae). The isolates were able to produce IAA in nutrient broth. However, when grown in the presence of 100 mg/l
of either phenanthrene or anthracene, the amount of IAA produced by each isolate was reduced significantly. Mungbean
Keywords: genotoxicity; mercuric chloride; micronucleus
seedlings were planted in 100 mg/kg phenanthrene - or anthracene-contaminated soil without or with inoculation
of 106 CFU/g dry soil with one of the bacterial isolates. Inoculation with either NSRU1 or NSRU2 was effective at
enhancing shoot length of mungbean in phenanthrene-contaminated soil on day 16. Also, inoculation with isolate
NSRU1 led to increased root dry weight of mungbean in phenanthrene-contaminated soil on day 30. Phenanthrene and
1. Introduction laboratory and field conditions. In 2006 Soumendra
anthracene degradation on day 16 and 30 in planted and inoculated soil ranged between 92 - 93.8% and 92.2 - 94.1%,
et al.,and
respectively, which were not significantly different from planted made an attemptsoil
uninoculated to (93.9
detectand
genetic biomarkers
94.9%). These data
In India,
showed about 200 tons
that IAA-producing of mercury
bacteria and its
could enhance in two fish
plant growth, but was unableLabeo
species, bata and
to increase PAHOreochromis
biodegradation
compounds are introduced
under the conditions tested. into the environment mossambica, by MN and binucleate (BN)
annually as effluents from industries (Saffi, 1981). erythrocytes in the gill and kidney erythrocytes
Mercuric
Keywords :chloride has been
IAA-producing used phytoremediation;
bacteria; in agriculture as polycyclic
a exposed to hydrocarbons
aromatic thermal power plant discharge at
fungicide, in medicine as a topical antiseptic and Titagarh Thermal Power Plant, Kolkata, India.
disinfectant, and in chemistry as an intermediate in The present study was conducted to determine
1. Introduction 45 min increased shoot and root lengths of cowpea 3 weeks
the acute genotoxicity of the heavy metal compound
the production of other mercury compounds. The
after seed germination. This strain could produce 23.8 - 104.8
contamination

of aquatic ecosystems by heavy HgCl in static systems. Mercuric chloride is toxic,
Indole-3-acetic acid (IAA) is a natural plant auxin which g/ml2 of IAA in broth supplemented with 1 g/l Trp for
metals and pesticides
exerts important effectshas gained
on plant increasing
growth attention
and development, solvable
48 h (Deepain water hence it can penetrate the aquatic
et al., 2010).
in
suchrecent decades.
as elongation, Chronic apical
differentiation, exposure to and
dominance and animals. Mutagenic studieshave
IAA-producing bacteria with native
been usedfish species
in phytoreme-
accumulation
flowering. IAAof these chemicals
biosynthesis in plantinoccurs
aquatic either represent
viabiota an important
diation to enhance effortin in
plant growth determining
polluted theas
soil as well
can result in tissue
Trp-dependent burdens thatpathway
or Trp-independent produce(Manoadverse and potential
accumulateeffects
heavy of toxic
metals in agents. This In
plant tissues. study
one was
study,
effects
Nemoto,not only
2012). in the directly
IAA-producing exposed
bacteria are a organisms, suspensions
group of plant carried out toofevaluate
the IAA-producing andmicronucleus
the use9 of the copper-tolerant
growth promoting bacteria
but also in human beings. (PGPB) generally detected in the Burkhoderia sp. strain LD-11 (10
test (MN) for the estimation of aquatic CFU/ml) werepollution
inoculated
rhizosphere and in plant tissues. Strains from
Fish provides a suitable model for monitoring several bacterial in surface soil near the plant root system
using marine edible fish under lab conditions. of 7 day-old
genera have been reported to produce IAA, such as Bacillus, flowering Chinese cabbage (Brassica campestris ssp.
aquatic genotoxicity and wastewater quality
Klebsiella, Pseudomonas, Rhizobium, Sphingomonas and chinensis) and sweet mustard (B. juncea) seedling. There
because of its ability to metabolize xenobiotics and 2. Materials and methods
Xanthomonas (Tsavkelova et al., 2006). IAA-producing were 2 plants of the same species/pot and each pot contained
accumulated pollutants. A micronucleus assay has
bacteria have been used to enhance crop growth. For example, soil contaiminated with 800 mg/kg copper and 1,000 mg/kg
been used of
immersion successfully
seeds of Tanzaniain several
grass in species (YM) 2.1.
(De Flora,
Yeast Manitol lead. Sample
After 42 Collection
days, bacterial inoculation was found to
et al.,containing
broth 1993, Al-Sabti8
10 CFU/ml and Metcalfe, Mesorhizibium
IAA-producing 1995). The increase copper accumulation in the shoots of sweet mustard
micronucleus
sp. or Bradyrhizobium (MN)sp.test and has been developed
then planted The fish
in soil led to and roots speciesChinese
of flowering selected for the
cabbage present
without study
any apparent
together
increased root with DNA-unwinding
dry weight of this plant onassays as
day 60 after enhancement
was collectedoffrom plantPudhumadam
growth. Bacterial inoculation
coast of Gulf also
of
perspective
germination. These methodsbacterialforstrains
masscould monitoring
produce aboutof increased lead
Mannar, accumulation
Southeast CoastinoftheIndia.
shoots Therapon
of flowering
0.2 - 4.0 g/ml and
clastogenicity of IAA in YM broth
genotoxicity supplemented
in fish and mussels Chinese belongs
with jarbua cabbage and to roots of sweetPerciformes
the order mustard (Huang of etthe
al.,
50 mg/l Trp for 48 h (Machado et al., 2013). In another 2013). In another study, Golubev et al. (2011) examined
(Dailianis et al., 2003). family Theraponidae. The fish species, Therapon
study, immersion ofhave
cowpea seeds in broth containing the effect of IAA-producing bacteria on PAH phytotoxicity.
The MN tests been successfully used as jarbua (6-6.3 cm in length and 4-4.25 g in weight)
108 CFU/ml of IAA-producing Enterobacter strain for These authors studied the effect of phenanthrene on the
a measure of genotoxic stress in fish, under both was selected for the detection of genotoxic effect
 W. Chouychai et al. / EnvironmentAsia 9(2) (2016) 128-133
IAA-producing and PAH-degrading Sinorrhizobium
meliloti and growth of sorghum in phenanthrene-contaminated
sand. The bacterial cells suspended in Ruakura nutrient
solution were applied to planted sand to final concentration
of 106 cell/g sterile sand. In the presence of 100 mg/kg
phenanthrene, the amount of IAA (measured by HPLC)
produced was 1.56 x 10-10 M in the uninoculated rhizosphere;
the amount increased to 2.18 x 10-10 M when S. meliloti
was inoculated. Furthermore, inoculation with S. meliloti
led to increased root biomass of sorghum grown in
100 mg/kg phenanthrene-contaminated sand but the effect
of bacterial inoculation on phenanthrene removal was not
reported (Golubev et al., 2011).
The toxicity of PAH on IAA production can adversely
affect the success of IAA-producing bacteria used in PAH
phytoremediation. This in turn can affect the positive effect
of these bacteria on plant growth and the plants capacity to
enhance PAH biodegradation. In this study, several bacteria
isolated from non-contaminated rhizosphere of Thai weeds
were shown to produce IAA when exposed to PAH.The
ability of these bacteria to enhance phenanthren and
anthracene phytoremediation in soil was assessed The
plant selected for phytoremediation was mungbean which
is a legume previously found to not tolerate PAH- and
engine oil-contaminated soil well (Chouychai et al., 2007).
We were interested in testing if the IAA-producing
bacterial isolates could enhance the growth of mungbean
in PAH-contaminated soil and if so whether improved
growth may translate into improved PAH degradation.
2. Materials and Methods
2.1 Isolation of IAA-producing bacteria phosphorus content (below 0.29 g/100 g soil). The soil
Bacteria used in this experiment were isolated from
the rhizospheric soil of Chromolaena odorata (Asteraceae)
and Eleusine indica (Poaceae), two common weeds
in Thailand. The weeds were collected from grassland
behind building 7 in Nakhonsawan Rajabhat University
(Nakhonsawan, Thailand). The 1-g soil sample from
the rhizosphere of each plant was placed in 9.5 ml of
sterile distilled water and shaken at 180 rpm for 2 h. The
suspension was serially diluted in sterile distilled water
and spread on Nutrient agar (NA) plates. After 48 h,
different colonies were picked and streaked onto fresh
NA plates to obtain single colonies. In total, 36 isolates
were obtained from the rhizospheric soil of both plants.
All isolated bacteria were tested for IAA production as
described by Ahmad et al. (2008). Samples of each
colony were grown individually in Nutrient Broth (NB)
supplemented with 0.05 g/l Trp for 72 h. The culture
was centrifuged at 1400 xg and two ml of supernantant
were transferred to a new tube and mixed with 2 ml of
Salkowskis reagent and 2 drops of orthophosphoric acid.
A pink color indicates the presence of IAA. Based on this
test, ten isolates were selected and subcultured individually
in fresh 50 ml NB media in a 125-ml flask for 3 days.
Then 106 cfu/ml (enumerated by plate count in NA) of each
culture were transferred to 50 ml NB supplemented with
129
0.05 g/l Trp in a 125-ml flask. The IAA concentration
was determined spectrophotometrically at 530 nm. IAA
concentrations in the samples were estimated from a
standard curve constructed with 1 - 100 g/ml IAA
(Fluka, purity 90%) dissolved in distilled water.
2.2 Effect of PAHs on IAA production
Each bacterial isolate was grown in 50 ml NB
supplemented with 0.05 g/l Trp in a 125-ml flask which
was shaken at 150 rpm at room temperature (25 0C) for
72 h. Then 106 cfu/ml of each culture were transferred to
NB supplemented with 0.05 g/l Trp with or without PAHs.
Phenanthrene (Sigma-Aldrich, purity 98%) or anthracene
(Fluka, purity 98%) dissolved in dimethyformamide was
added to separate flasks to final concentrations 100 mg/l
in NB. The experiment was done in triplicate. After 3 and
10 days, IAA production in the culture supernatant was
measured according to Ahmad et al. (2008) as described
above.
2.3 Soil preparation
Alkaline agricultural soil with no previous history of
hydrocarbon contamination was collected at the Khaorad
Agricultural Station, Faculty of Agricultural Technology and
Industrial Technology, Nakhonsawan Rajabhat University,
Nakhonsawan, Thailand as described in Chouychai and Lee
(2012). The soil was air-dried at room temperature (250C)
for at least 24 h to constant weight before use. The soil used
in this experiment was alkaline (pH 8.9) with low total
contained (per 100 g dry soil): 0.21 g total nitrogen (N),
0.13 g total potassium (K), and 1.78 g organic matter.
Soil was mixed with either phenanthrene or anthracene
individually dissolved in acetone to final concentration of
100 mg/kg.
2.4 Experimental design.
The following five experimental treatments were used
for each of the PAH-contaminated soil: (a) soil inoculated
with isolate NSRU1 and planted with mungbean, (b) soil
inoculated with isolate NSRU2 and planted with mungbean,
(c) soil inoculated with isolate NSRU3 and planted with
mungbean, (d) soil planted with mungbean, (e) soil which
received the NB only. The following four experimental
treatments were done in non-contaminated soil: (f) soil
inoculated with isolate NSRU1 and planted with mungbean,
(g) soil inoculated with isolate NSRU2 and planted with
mungbean, (h) soil inoculated with isolate NSRU3 and
planted with mungbean, (i) soil planted with mungbean.
Each uninoculated treatment received the same amount of
NB as that used in bacterial inoculation. Cells of each
isolate were inoculated into soil to final concentrations of
about 106 CFU/g dry soils (enumerated by plate count in
NA) and mixed thoroughly with a digger. Each treatment
was done in triplicate.
 W. Chouychai et al. / EnvironmentAsia 9(2) (2016) 128-133

2.5 Pot experiment

3.2 Effect of PAHs on IAA production
Ten-day old mungbean seedlings were planted in soil
containing 100 mg of either phenanthrene or anthracene Different amounts of IAA were produced by each of
per kg dry soil at one plant per pot. This phenanthrene the 3 bacterial isolates when cultured in NB supplemented
concentration has been reported that there were toxic to with 0.05 g/l Trp for 3 days. Isolate NSRU1 produced
mungbean root (Chouychai et al., 2007). The diameter of the highest amount of IAA (37.9 9.1 g/ml), while isolates
the pot was 7 inches, with each pot holding 1 kg dry weight NSRU2 and NSRU3 produced lower amounts of 17.4 and
of soil. The pots were kept in a nursery in which the 15.9 g/ml, respectively. When 100 mg/l phenanthrene
temperature was around 24oC during the day and 21oC at were added to the culture, the amounts of IAA produced
night and exposed to natural sunlight. Each pot was watered by isolates NSRU1, NSRU2 and NSRU3 were reduced to
every day with about 20 ml water for 30 days. The experiment 10.6, 4.8, and 7.7 g/ml, respectively (Table 1). Addition
was conducted in triplicate. On days 16 and 30, the mungbean of anthracene reduced the amount of IAA produced by
plants were removed and the pots terminated. The length, each strain to 12.5 - 6.3 g/ml. The amounts of IAA
fresh weight, and dried weight of shoot and root of mungbeans remaining in the medium on day 10 were generally lower
were measured. than those on day 3. In particular, the amounts of IAA
One g of soil sample was collected from each treatment remaining were below the detection limit in the supernatant
on days 16 and 30 for analysis of remaining PAHs. The of isolate NSRU3 grown in the presence of the PAHs
amount of phenanthrene or anthracene remaining in soil was (Table 1).
determined by gas chromatography using a Shimadzu model
AOC-5000 GC equipped with a mass spectroscopic 3.3 Effect of IAA-producing bacteria on mungbean
detector (Shimadzu MS-QP2010) according to Somtrakoon growth
et al. (2014). One g of soil was mixed with an equal amount
of anhydrous Na2SO4, and subjected to Soxhlet extraction In non-contaminated soil, growth of mungbean
prior to GC analysis. Phenanthrene and anthracene were appeared normal and was not affected by inoculation
separated on a Rtx-5MS capillary column (30 m x 25 mm, with any of the 3 IAA-producing bacterial isolates
I.D. = 25 m) as described in Somtrakoon et al. (2014). The (Tables 2-4). The presence of phenanthrene in soil
detection limit of this analysis was 0.4 mg/kg. led to decreased root length of mungbean on day 16
to 3.9 3.0 cm from that in non-contaminated soil
3. Results (8.4 3.0 cm). Inoculation with isolates NSRU1 or
NSRU2 led to increased shoot length of mungbean
3.1 Isolation of IAA-producing bacteria in phenanthrene-contaminated soil on day 16 to 24.1
3.2. Effect of PAHs on IAA production and 26.0 cm, respectively, from that in non-inoculated
Three bacterial isolates which showed the highest treatment (21.0 cm). On day 30, only the treatment
IAA concentrations were
Different selectedoffor
amounts IAAusewere produced bywhich
in subsequent each ofwas
theinoculated
3 bacterial with isolate
isolates whenNSRU1
culturedshowed
in
NB supplemented with 0.05 g/l Trp for 3 days. Isolate NSRU1 produced the highest amount15.2
experiment. All were Gram-positive bacteria, and they increased root dry weight of mungbean to 3.7 mg
of IAA
were(37.9
designated
9.1 NSRU1,
g/ml), NSRU2, and NSRU3.
while isolates NSRU2 NSRU1
and NSRU3 in phenanthrene-contaminated
produced lower amounts of soil;
17.4this
andvalue
15.9 was
was isolated from the rhizosphere of E. Indica and its significantly higher than the root dry weight of mungbean
g/ml, respectively. When 100 mg/l phenanthrene were added to the culture, the amounts of IAA
colonies were white, circular, transparent, raised and smooth. in other treatments (Table 4). In anthracene-contaminated
produced by isolates NSRU1, NSRU2 and NSRU3 were reduced to 10.6, 4.8, and 7.7 g/ml,
NSRU2 and NSRU3 were isolated from the rhizosphere soil, innoculation of any of the isolates did not result in
of C.respectively (Tableof1).NSRU2
odorata. Colonies Addition
were ofyellow,
anthracene reduced
circular, the amount of IAA produced by each strain to
altered growth parameters of mungbean (Tables 2-4).
12.5 - 6.3 g/ml. The amounts of IAA remaining
undulated, opaque and umbonate while colonies of NSRU3 in the medium on day 10 were generally lower than
those on day 3. In particular,
were yellow, circular, and umbonate. the amounts of IAA remaining were below the detection limit in the
supernatant of isolate NSRU3 grown in the presence of the PAHs (Table 1).

Table 1. IAA production of selected bacterial isolates cultured in NB containing 0.05 g/l tryptophan and 100
mg/l PAH for 3-10 days. Starting cell concentration of each bacterial isolate was 106 CFU/ml.

IAA concentration (g/ml)

Bacterial isolate
Without PAHs 100 mg/l phenanthrene 100 mg/l anthracene
Day 3
NSRU1 37.9 9.1a 10.6 0.8b 12.5 0.9b
a b
NSRU2 17.4 2.2 4.8 0.3 6.3 0.6b
a b
NSRU3 15.9 4.0 7.7 0.7 8.1 0.3b
Day 10
NSRU1 21.9 3.4a 12.1 1.8b 4.5 0.2b
a b
NSRU2 14.3 1.4 4.6 1.5 B.D.
NSRU3 5.0 2.6 B.D. B.D.
Different lower case letters showed significant difference (P<0.05) between treatments in same strain.
B.D. = below detection limit

3.3. Effect of IAA-producing bacteria on mungbean growth

130
In non-contaminated soil, growth of mungbean appeared normal and was not affected by
inoculation with any of the 3 IAA-producing bacterial isolates (Tables 2-4). The presence of
phenanthrene in soil led to decreased root length of mungbean on day 16 to 3.9 3.0 cm from that in
 Table 2. Shoot and root length of mungbean grown in non-contaminated or PAH-contaminated soil with
different
Table bacterial
2. Shoot strain
and root inoculation
length of mungbean grown in non-contaminated or PAH-contaminated soil with
different bacterial strain inoculation
Table 2. Shoot and root length of mungbean
Shoot length grown
(cm)in non-contaminated or PAH-contaminated
Root length (cm)soil with
different bacterial strain inoculation
Treatment
NCS Shoot length PHE (cm) ANT NCS Root length PHE(cm) ANT
Treatment
Day 16 NCS PHE
Shoot length (cm) ANT NCS PHE
Root length (cm) ANT
Treatment
DayNon-inoculated
16 22.3 2.1a 21.0 2.1b 21.6 1.4b 8.4 3.0a 3.9 0.5a* 7.3 1.1a
NCS a a PHE b a ANT b b NCSa a PHEa* a ANTa a
NSRU1
Non-inoculated 24.2
22.3 2.3 21.0
2.1 24.1 1.1 21.6
2.1 21.3 1.3 8.48.7
1.4 2.5 3.95.8
3.0 1.5 7.39.4
0.5 2.2
1.1
Day 16
NSRU2 22.4 1.9
a a
26.0 3.2
a a
25.1 0.2
b a
7.0 1.4
a a
4.3 1.4
a a
9.6 a a
2.4
NSRU1 24.2 2.3 aa 24.1 1.1 bb 21.3 1.3 a,b b 8.7 2.5 aa 5.8 1.5 a*a 9.4 2.2 a
Non-inoculated
NSRU3 22.3
24.4 2.1
1.5a 26.0
a 21.0
20.0 2.1
2.4a 25.1
a 21.6
23.60.21.4 8.4
1.2 b 7.08.0
a 3.0 3.9
1.8a 4.36.1
a 0.5 7.3
2.2a 9.69.0
a 1.1
a a
3.2
NSRU2 22.4 1.9 3.2 1.4 1.4 2.4
NSRU1
Day 30 24.2 2.3 24.1 1.1 21.3 1.3 8.7 2.5 5.8 1.5 9.4 2.2a
NSRU3 24.4 1.5a aa 20.0 2.4b aa 23.6 1.2a, b aa 8.0 1.8a aa 6.1 2.2a aa 9.0 3.2a aa
NSRU2
Non-inoculated 22.4
23.4 1.9
2.7a 26.0
22.2 3.2
2.0b 25.1
25.0 0.2
1.3a, b 7.0
7.1 1.4
1.9a 4.3
6.0 1.4
1.0a 9.6
6.8 2.4
2.4a
DayNSRU3
30 24.4 1.5 20.0 2.4 23.6 1.2 8.0 1.8 6.1 2.2 9.0 3.2
NSRU1 a a a a a a a a a a a a
Non-inoculated 23.4 2.7 a 22.2 2.0 a 25.0 1.3 a 7.1 1.9 a 6.0 1.0 a 6.89.3
24.2 2.8 21.8 0.4 26.3 1.3 8.3 1.9 5.8 0.6 2.44.2
Day 30
NSRU2 23.5 1.6 23.6 1.7 22.6 1.8 a a
NSRU1 24.2 2.8 a
a 21.8 0.4 a
a 26.3 1.3 a
a 8.38.0 1.2
1.9 a
a 5.86.5 0.7
0.6 a
a 9.34.5 1.6
4.2 a
Non-inoculated
NSRU3 23.4
25.1 2.7
1.8 a 22.2
19.5 1.7 2.0
3.8 a* 25.0
25.6 1.3
2.9 a 7.1 1.9 a 6.0 1.0 a 6.8 2.4
a a
NSRU2 23.5 1.6 a
a 23.6 a
a 22.6 1.8 a
a 8.08.1 1.8
1.2 a
a 6.57.8 2.8
0.7 a
a 4.56.2 0.8
1.6 a
Different 24.2 showed
NSRU1lower case letters 2.8
a 21.8 0.4
significant 26.3 (P<0.05)
a* difference 1.3
a 8.3 1.9
between 5.8 soil
a the same 0.6
aon the 9.3
same 4.2
day;
a
NSRU3 25.1 1.8 a 19.5 3.8 a 25.6 2.9 a 8.1 1.8 a 7.8 2.8 a 6.2 0.8
* NSRU2
showedlower
significant 23.5 1.6(P<0.05)
difference 23.6from 1.7non-contaminated
22.6 1.8 soil8.0 1.2 6.5 0.7 4.5 1.6a
Different case letters showed
NSRU3NCS = non-contaminated a significant
25.1 1.8 soil, 19.5PHE 3.8difference
a* (P<0.05)
25.6 2.9 a between thea same soil on the
8.1 1.8soil and7.8 2.8 a same
6.2 day;
0.8a
* Symbol: = phenanthrene-contaminated ANT = anthracene-
showed significant difference (P<0.05) from non-contaminated soil
Different lower case letters showed significant difference (P<0.05) between the same soil on the same day;
contaminated
Symbol:
* NCS = soil.
non-contaminated soil, PHE = phenanthrene-contaminated soil and ANT = anthracene-
showed significant difference (P<0.05) from non-contaminated soil
contaminated soil.
Symbol: NCS =and
Table 3. Shoot non-contaminated
root fresh weight soil, PHE = phenanthrene-contaminated
of mungbean grown in non-contaminated soilor
and ANT = anthracene-
PAH-contaminated soil with
contaminated
different soil. strain inoculation
bacterial
Table 3. Shoot and root fresh weight of mungbean grown in non-contaminated or PAH-contaminated soil with
different bacterial strain inoculation
Table 3. Shoot and root fresh weight Shootoffresh
mungbean grown in non-contaminated or PAH-contaminated
weight (mg) Root fresh weight (mg)soil with
different bacterial strain inoculation
Treatment
NCS Shoot freshPHE weight (mg) ANT NCS Root fresh weight
PHE (mg) ANT
Treatment
Day 16 NCS PHE ANT NCS PHE ANT
a Shoot fresh weighta (mg) a aRoot fresh weight b (mg)
Non-inoculated
Day 16Treatment 473.8 64.9 382.3 156.7 372.5 28.6 113.9 20.4 66.7 15.3 118.0 52.9a
NSRU1 NCS
504.9 78.3
a
a PHE
443.3 51.3 a
a ANT
548.3 28.6
227.6
a
a NCS
132.6 31.1
a
a PHE
133.3 28.9
b
a ANT
159.5 75.2a
a
Non-inoculated 473.8 64.9 a 382.3 156.7 a 372.5 a 113.9 20.4 a 66.7 15.3 ab 118.0 52.9 a
NSRU2
Day
NSRU1 16 428.3 52.5
a 480.0 99.0
a 381.6 7.1a 103.4
504.9 78.3 aa 443.3 51.3 aa 548.3 227.6 aa 132.6 31.1 aa 133.3 28.9 ab 31.2
a 120.0 14.1a 123.2 25.1
159.5 75.2a aa
b
NSRU3
Non-inoculated
NSRU2 481.8
473.8
428.3 52.559.7
64.9
a 373.3
382.3
480.0 156.7
85.0
99.0 a 522.9
372.5
381.6 28.6
7.19.4
a 119.0
113.9
103.4 23.0
20.4
31.2 a 116.7
66.7 15.3
5.8
ab 148.1
118.0 15.3
52.9
a
a a a a 120.0 14.1 a 123.2 25.1
NSRU1
Day 30
NSRU3 504.9 78.3
a 443.3
481.8 59.7 a 373.3 85.0 a 51.3
a 548.3 227.6
522.9 9.4 a a 132.6 31.1 133.3 28.9 159.5
119.0 23.0 a 116.7 5.8 ab 148.1 15.3a a
a ab 75.2a
NSRU2
Non-inoculated 428.3
492.3 52.5
79.5 a 480.0 244.6
550.2 99.0 a 381.6 85.7
461.3 7.1 a 103.4
113.4 31.2
10.5aa 120.0
110.514.1
9.1aba 123.2
116.7 25.1
28.5aa
DayNSRU3
30 481.8 59.7 a
a 373.3 85.0 a
a 522.9 9.4 a
a 119.0 23.0 a 116.7 5.8 a 148.1 15.3 a
NSRU1
Non-inoculated 652.0 79.5
492.3 224.2
a 495.1 125.5 a 614.2 82.2a 166.3 79.8
a 159.4 9.1
47.0
a 167.7 28.5
105.7
a
a 550.2 244.6 a 461.3 85.7 a 113.4 10.5 a 110.5 a 116.7 a
NSRU2
Day 30
NSRU1 557.1 72.2 627.8 115.5 519.1 16.8 180.0 51.9 167.2 73.0
652.0 224.2a a a 495.1 125.5a aa 614.2 82.2a a a 166.3 79.8a aa 159.4 47.0a a a 167.7 105.7a aa 89.0 20.7
NSRU3
Non-inoculated
NSRU2 616.1
557.1 72.2
492.3 107.6
79.5
a 451.3 244.6
550.2 203.8
a 597.3
461.3 16.8
112.6
85.7
a 208.5
113.4 49.7
10.5
a 136.6
110.5 67.5
9.1a 175.4
116.7 28.0
28.5
a
a 627.8 115.5 a 519.1 a 180.0 51.9 a 167.2 73.0 a 89.0 20.7 a
NSRU1
Different
NSRU3 lower case 652.0
letters 224.2
showed 495.1
a significant 125.5
difference 614.2
(P<0.05) 82.2
between 166.3
the same 79.8
a on the159.4
soil
616.1 107.6 a 451.3 203.8 a 597.3 112.6 a 208.5 49.7 a 136.6 67.5 a 175.4 28.0a a
a a same
day47.0a 167.7 105.7
NSRU2
Symbol:lower 557.1
NCS = non-contaminated 72.2 soil, 627.8 115.5
PHEdifference 519.1
= phenanthrene-contaminated 16.8 180.0
soil andsoil
ANT 51.9 167.2 73.0 89.0 20.7
Different
NSRU3 case letters showed
616.1 107.6 significant
a
451.3 203.8a (P<0.05)
597.3 between
112.6a the same on=the
208.5 49.7 a
anthracene-contaminated
same day
136.6 67.5a
soil
175.4 28.0a
Symbol: NCS = non-contaminated soil, PHE = phenanthrene-contaminated soil and ANT = anthracene-contaminated soil
Different
Table 4. lower
Shootcase
andletters showed
root fresh significant
weight differencegrown
of mungbean (P<0.05) between the same soil
in non-contaminated oron
100themg/kg
same dayeach PAH-
Symbol: NCS =soil
contaminated non-contaminated
with differentsoil, PHE =strain
bacterial phenanthrene-contaminated
inoculation soil and ANT = anthracene-contaminated soil
Table 4. Shoot and root fresh weight of mungbean grown in non-contaminated or 100 mg/kg each PAH-
contaminated soil with different bacterial strain inoculation
Table 4. Shoot and root fresh weightShootof mungbean
dried weight grown
(mg) in non-contaminated Rootor 100
driedmg/kg
weight each PAH-
(mg)
Treatmentsoil with different bacterial strain inoculation
contaminated
NCSShoot driedPHE weight (mg) ANT NCSRoot dried weight PHE (mg) ANT
Treatment
Day 16 NCS Shoot dried PHE weight (mg)ANT NCS Root dried PHEweight ANT
a a a ab b (mg)
Non-inoculated
Treatment
Day 16 45.9 6.7 39.5 12.1 37.9 7.0 5.5 0.6 4.5 1.2 7.0 3.5a
NSRU1 NCS
51.0 a
7.1 39.5
a
PHE
46.8 8.6a a ANT
57.2 7.0 a
25.0 5.57.1
a
NCS a
1.4 4.58.9
ab
PHE a
3.5 7.07.0
b
ANT a a
1.3
Non-inoculated 45.9 6.7 12.1 37.9 0.6 1.2 3.5
Day 16
NSRU2 50.5 a a
8.1a 46.8 42.6 8.6 a
11.8a 57.2
a 51.0 a
12.9a 7.14.6
a b
0.6ab 8.95.6
a b
2.3b 7.07.5
a a a
0.4
NSRU1 51.0 7.1 25.0 1.4 3.5 1.3 a
Non-inoculated
NSRU3 45.9
47.9 8.16.7 a
11.1a 42.6
a 39.5
40.1 12.1
a a
7.9a 51.0 37.9
54.5 7.0 a
3.4 a 4.6
a 5.5 0.6
5.5 0.6 ab
1.0 a 5.67.5
b 4.5 1.2 a
1.9a 7.57.9
b 7.0 3.5
a a
1.6
NSRU2 50.5 11.8 12.9 2.3 0.4
NSRU1
Day 30 51.0 7.1 46.8 8.6 57.2 25.0 7.1 1.4 8.9 3.5 7.0 1.3a
NSRU3 47.9 11.1a a a 40.1 7.9a aa 54.5 3.4a aa 5.5 1.0ab ba 7.5 1.9a bb 7.9 1.6a aa
NSRU2
Non-inoculated 50.5 14.2
62.2 8.1 42.6
70.5 11.8
23.0a 51.0
79.9 12.9
28.8a 4.6 0.6
10.9 2.7ab 7.1 5.6
0.9
2.3 14.1 7.5 0.4
2.1a
DayNSRU3
30 47.9 11.1 a
a 40.1 7.9 a 54.5 3.4 a 5.5 1.0 a 7.5 1.9 a
a 7.9 1.6
NSRU1 85.9 34.7
a 66.0 19.3a 102.4 29.0
a 19.5 9.2a 15.2 3.7
b 16.2 a a
2.7
Non-inoculated 62.2 14.2 70.5 23.0 79.9 28.8 10.9 2.7 7.1 0.9 14.1 2.1
Day 30
NSRU2 74.5 19.0
a a
85.0 24.5 a a
64.8 23.9 a a
17.7 5.2 a a
8.3 2.1 a b
12.8 1.1a a
NSRU1 85.9 34.7 a 66.0 19.3 a 102.4 29.0 a 19.5 9.2 a 15.2 3.7 b 16.2 2.7 a
Non-inoculated
NSRU3 62.2
92.5 14.2
27.7
a a 70.5 23.0
58.6 26.3 a a 79.9 28.8
101.3 27.8 a a 10.9
17.5 4.02.7
a a 7.1 0.9
5.9 0.9b b 14.1 2.1
19.4 1.6a a
NSRU2 74.5 19.0 a 85.0 24.5 a 64.8 23.9 a 17.7 5.2 a 8.3 2.1 a 12.8 1.1 a
NSRU1
Different 85.9 34.7 66.0 19.3 102.4 29.0 19.5 9.2 15.2 3.7 16.2 2.7
NSRU3 lower case letter
92.5 showed
27.7 a significant
58.6 26.3difference
a
a 101.3 (P<0.05)
27.8a a between
17.5 4.0 same
a
a soil
5.9 in0.9
same
b
b day.
19.4 1.6a a
Symbol: 74.5 19.0a soil,
NSRU2NCS = non-contaminated 85.0PHE
24.5
= 64.8 23.9
phenanthrene 17.7 5.2
-contaminated soil and8.3
ANT 2.1
= 12.8 1.1
anthracene-
Different
NSRU3 lower case letter
92.5showed
27.7 asignificant difference
58.6 26.3 a (P<0.05)
101.3 27.8 between
a
17.5 same
4.0 soil
a in same
5.9 0.9 day.
b
19.4 1.6a
contaminated
Symbol: NCS = soil
non-contaminated soil, PHE = phenanthrene -contaminated soil and ANT = anthracene-
Different lower case letter showed significant difference (P<0.05) between same soil in same day.
contaminated soil
Symbol: NCS = non-contaminated soil, PHE = phenanthrene -contaminated soil and ANT = anthracene-
contaminated soil

131
 W. Chouychai et al. / EnvironmentAsia 9(2) (2016) 128-133

3.4 Effect of IAA-producing bacteria on PAH removal
In non-planted soil, phenanthrene and anthracene
were degraded rapidly with the extent of degradation
reaching 81 and 42%, respectively on day 16. Planting
with mungbean increased the removal of phenanthrene
and anthracene from soil to 93.9 and 94.9%, respectively,
on day 16 (Table 5). Further inoculation of planted soil
with bacterial strains did not result in faster rate or extent
of phenanthrene and anthracene degradation relative
to that seen in planted soil. This trend was seen also on day
30 (Table 5). Likely, the PAHs were degraded so rapidly
that any differences were masked and the removal of both
PAHs seems to depend on mungbean and soil indigenous
bacteria more than the inoculated IAA-producing bacteria.
4. Discussion
In this study, 100 mg/l phenanthrene and anthracene
were found to adversely affect IAA production by the 3
bacterial isolates. IAA production by NSRU 1 was inhibited
by 72.0 and 67.0%, while IAA production by NSRU2 and
NSRU3 was inhibited by 72.4% and 63.8%, and 51.6%
and 49.0% when phenanthrene and anthacene, respectively,
were added to the cultures. There were previous reports that
IAA production by IAA-producing bacteria was sensitive
to pesticides. For example, addition of 0.04 - 0.12 mg/l
hexaconazole reduced IAA production by Mesorhizobium
sp. MRC4 culture by 36.4 - 52.3% from that produced by the
culture in a pesticide-free medium (44.0 g/ml) (Ahemad
and Khan, 2012). In another study, IAA production by
Gluconacetobactor diazotrophicus strain PAL5 was reduced
by 77.6 - 93.2% to 0.63 - 0.19 g/ml in the presence of
either 6.0 mg/l Monocrotophos or 2.8 mg/l Dichorvos from
that produced in a pesticide-free medium (2.81 g/ml).
Interestingly, addition of 2.5 mg/l lindane completely
inhibited IAA production by G. diazotrophicus PAL5
(Madhaiyan et al., 2006).
Although pesticides could reduce IAA production, some
authors reported the use of IAA-producing bacteria to
improve plant growth in pesticide-contaminated soil. For
example, inoculation of Mesorhizobium sp. MRC4
significantly increased the dry weight of chickpea grown in
0.6 mg/kg Fibronil or 3.9 mg/kg pyrifroxyfen-contaminated
soil to 2.67 and 2.61 g/plant from those of chickpea in
un-inoculated soil of 1.42 and 1.08 g/plant, respectively.
In our study, bacterial inoculation led to increased shoot
length and dry root weight of mungbean grown in
phenanthrene-contaminated soil on day 16. NSRU1 which
produced the highest amount of IAA seemed to be the most
beneficial to mungbean in phenanthrene-contaminated soil.
The removal of PAHs in planted soil was reported in
many literatures. The main mechanism was the stimulating
microbial growth by plant root. Exudation of organic
acid, nutrient and detergent from root could enhance
biodegradation rate by microorganism in soil (Siciliano
and Germida, 1998). The inoculation of IAA-producing
bacteria did not seem to enhance phenanthrene or anthracene
biodegradation when compared with planted and
uninoculated soil. This may be because both PAHs tested
in this experiment were degraded rapidly in mungbean
planted soil. The ability of synthetic IAA to increase PAH
removal from soil has been reported. Application of 2.4 mg/kg
IAA to ryegrass planted in 200 mg/kg fluoranthrene increased
fluoranthene removal from soil. The amount of fluoranthene
remaining in soil planted with ryegrass and treated with
IAA was 100 mg/kg while the amount remaining in soil
planted with ryegrass without IAA treatment was 130 mg/kg
soil. IAA could increase fluoranthene removal by enhance
fluoranthene uptake to shoot and increase number of soil
microorganism (Li et al., 2015). However, application with

Table 5. Percentages of phenanthrene or anthracene remaining in soil grown with mungbean and non-inoculated
or inoculated with IAA producing bacteria in phenanthrene or anthracene -spiked soil for 30 days

% remaining
Plant
Day 16 Day 30
Phenanthrene
Non-planted soil 19.0 1.0a 15.2 3.8a
b
Planted soil 6.1 2.1 2.8 1.8b
b
Planted soil + NSRU1 6.2 1.6 3.6 2.7b
b
Planted soil + NSRU2 7.3 4.9 2.0 0.8b
b
Planted soil + NSRU3 8.0 1.8 1.4 1.1b*
Anthracene
Non-planted soil 58.4 6.0a 8.4 6.0a*
b
Planted soil 5.1 0.6 6.6 2.4a
b
Planted soil + NSRU1 5.9 4.0 3.0 1.1a
b
Planted soil + NSRU2 7.8 7.6 5.0 3.6a
b
Planted soil + NSRU3 8.8 3.3 5.2 0.8a
Different lower case letter showed significant difference (P<0.05)
between same PAH in same day and *showed significant difference
(P<0.05) between PAH-remaining on day 16 and 30.

4. Discussion

In this study, 100 mg/l phenanthrene and anthracene were found to adversely affect IAA
production by the 3 bacterial isolates. IAA production
132 by NSRU 1 was inhibited by 72.0 and 67.0%,
while IAA production by NSRU2 and NSRU3 was inhibited by 72.4% and 63.8%, and 51.6% and
49.0% when phenanthrene and anthacene, respectively, were added to the cultures. There were
 W. Chouychai et al. / EnvironmentAsia 9(2) (2016) 128-133
indolebutyric acid, which is auxin also, did not enhance
corn growth but could increase hexachlorocyclohexane
removal in soil within 30 days (Chouychai et al., 2015).
The role of IAA on phytoremediation process in PAH-
contaminated soil should be studied in more detail.
5. Conclusions
In summary, IAA-producing bacteria isolated
from non-contaminated weed rhizosphere exhibited
some tolerance to PAHs and produced IAA in PAH-
containing media. In soil experiment, inoculation
with these IAA-producing bacterial strains led to
enhanced root growth of mungbean in phenanthrene-
contaminated soil but not PAH biodegradation. Given
the potential benefit of IAA-producing bacteria to
phytoremediation, further studies are warranted.
These may include inoculum size and selecting
conditions in which PAHs were poorly degraded
to further assess the potential beneficial effect of
IAA-producing bacteria.
Acknowledgements
We thank Nakhonsawan Rajabhat University for
infrastructure and equipment support for this research
References
Ahemad M, Khan MS. Effects of pesticides on plant growth
promoting traits of Mesorhizobium strain MRC4. Journal
of the Saudi Society of Agricultural Sciences 2012; 11(1):
63-71.
Ahmad F, Ahmad I, Khan MS. Screening of free-living
rhizospheric bacteria for their multiple plant growth
promoting activities. Microbiological Research 2008;
163(2): 173-81.
Chouychai W, Lee H. Phytotoxicity assay of crop plants to
lindane and alpha-endosulfan contaminants in alkaline
Thai soil. International Journal of Agriculture and
Biology 2012; 14(5): 734-38.
Chouychai W, Thongkukiatkul A, Upatham S, Lee H,
Pokethitiyook P, Kruatrachue M. Phytotoxicity assay
of crop plants to phenanthrene and pyrene contaminants
in acidic soil. Environmental Toxicology 2007; 22(6):
597-604.
Chouychai W, Kruatrachue M, Lee H. Effects of plant
growth regulators on phytoremediation of hexachlo-
rocyclohexane-contaminated soil. International
Journal of Phytoremediation. 2015; 17(11): 1053-59.
Deepa CK, Dastager SG, Pandey A. Isolation and
characterization of plant growth promoting bacteria
from non-rhizospheric soil and their effect on cowpea
(Vigna unguiculata (L.) Walp.) seedling growth. World
Journal of Microbiology and Biotechnology 2010;
26(7): 1233-40.
133
Golubev SN, Muratova AY, Wittenmayer L, Bondarenkova
AD, Hirche F, Matora LY, Merbach W, Turkovskaya
OV. Rhizosphere indole-3-acetic acid as a mediator in
the Sorghum bicolor-phenanthrene-Sinorhizobium
meliloti interactions. Plant Physiology and Biochemistry
2011; 49(6): 600-08.
Huang G, Tian HH, Liu HY, Fan XW, Liang Y, Li YZ.
Characterization of plant-growth-promoting effects and
concurrent promotion of heavy metal accumulation in
the tissues of the plants grown in the polluted soil by
Burkholderia strain LD-11. International Journal of
Phytoremediation 2013; 15(10): 991-1009.
Li W, Xu L, Wu J, Ma L, Liu M, Jiao J, Li H, Hu F. Effects
of indole-3-acetic acid (IAA), a plant hormone, on the
ryegrass yield and the removal of fluoranthene from
soil. International Journal of Phytoremediation. 2015;
17(1-6): 422-28.
Machado RG, De S ELS, Bruxel M, Giongo A, da Silva
Santos N, Nunes AS. Indoleacetic acid producing
rhizobia promote growth of Tanzania grass (Panicum
maximum) and Pensacola grass (Paspalum saurae).
International Journal of Agriculture and Biology
2013; 15(5): 827-34.
Madhaiyan M, Poonguzhali S, Hari K, Saravanan VS, Sa
T. Influence of pesticide on the growth rate and
plant-growth promoting traits of Gluconacetobacter
diazotrophicus. Pesticide Biochemistry and Physiology.
2006; 84(2): 143-54.
Mano Y, Nemoto K. The pathway of auxin biosynthesis in
plants. Journal of Experimental Botany 2012; 63: 2853-72.
Siciliano SD, Germida JJ. Mechanisms of phytoremediation:
biochemical and ecological interactions between plants
and bacteria. Environmental Reviews 1998; 6(1): 65-79.
Somtrakoon K, Chouychai W, Lee H. Comparing anthracene
and fluorene degradation in anthracene and fluorene-
contaminated soil by single and mixed plant cultivation.
International Journal of Phytoremediation 2014; 16
(4): 415-28.
Tsavkelova EA, Klimova SY, Cherdyntseva TA, Netrusov
AI. Microbial producers of plant growth stimulators
and their practical use: a review. Applied Biochemistry
and Microbiology. 2006; 42(2): 117-26.
Received 10 May 2016
Accepted 1 June 2016
Correspondence to
Dr.Waraporn Chouychai
Biology Program, Department of Science,
Faculty of Science and Technology,
Nakhonsawan Rajabhat University,
Nakhonsawan, 60000
Thailand
Tel: +66-5621-9100, ext. 2528
E-mail: waraporn.c@nsru.ac.th; chouychai@yahoo.com
Copyright of EnvironmentAsia is the property of Thai Society of Higher Education Institutes
on Environment and its content may not be copied or emailed to multiple sites or posted to a
listserv without the copyright holder's express written permission. However, users may print,
download, or email articles for individual use.