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Environmental Technology
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Lead-resistant strain KQBT-3 inoculants of Tricholoma

lobayensis Heim that enhance remediation of lead-
contaminated soil
a b a a a
Ying Li , Chui-xin Qin , Biyu Gao , Yuanjia Hu & Heng Xu
a
Key Laboratory of Bio-resources and Eco-environment (Ministry of Education), College of
Life Science, Sichuan University, Chengdu, Sichuan, People's Republic of China
b
Infinitus (China) Company Ltd, Guangzhou, Guangdong, People's Republic of China
Published online: 05 May 2015.

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To cite this article: Ying Li, Chui-xin Qin, Biyu Gao, Yuanjia Hu & Heng Xu (2015): Lead-resistant strain KQBT-3 inoculants
of Tricholoma lobayensis Heim that enhance remediation of lead-contaminated soil, Environmental Technology, DOI:
10.1080/09593330.2015.1034788

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 Environmental Technology, 2015
http://dx.doi.org/10.1080/09593330.2015.1034788

Lead-resistant strain KQBT-3 inoculants of Tricholoma lobayensis Heim that enhance

remediation of lead-contaminated soil
Ying Lia,† , Chui-xin Qinb,† , Biyu Gaoa , Yuanjia Hua and Heng Xua∗
a Key Laboratory of Bio-resources and Eco-environment (Ministry of Education), College of Life Science, Sichuan University, Chengdu,
Sichuan, People’s Republic of China; b Infinitus (China) Company Ltd, Guangzhou, Guangdong, People’s Republic of China
(Received 9 July 2014; accepted 14 December 2014 )

To enhance lead-detoxifying efficiency of Tricholoma lobayensis Heim, one lead-resistant strain KQBT-3 (Bacillus
thuringiensis) was applied owing to its excellent ability to tolerate Pb. KQBT-3 domesticated in liquid medium with increas-
ing lead concentrations could tolerate Pb(NO3 )2 up to a concentration of 800 mg L−1 . Pot experiments showed that the
KQBT-3 not only could promote the growth of T. lobayensis, but also could enhance its Pb accumulation ability under
heavy metal stress. Biomass and accumulation of Pb increased 47.3% and 33.2%, respectively. In addition, after inoculation
of KQBT-3, the significant decrease of malondialdehyde indicated KQBT-3 could alleviate lipid peroxidation in T. lobayen-
sis. What is interesting is that superoxide dismutase and peroxidase activities in T. lobayensis inoculated with KQBT-3
were increased, and the maximum increasing rate was 121.71% and 117.29%, respectively. However, the catalase activity
Downloaded by [FU Berlin] at 22:24 13 May 2015

increased slightly. This revealed that inoculating KQBT-3 further induced oxidative response in T. lobayensis due to Pb
accumulation. Therefore, the present work showed that KQBT-3 made a major contribution to promote growth and lead
uptake of T. lobayensis and alleviate the oxidative stress. This kind of auxiliary effect on macrofungi can be developed into
a novel bioremediation strategy.
Keywords: Tricholoma lobayensis Heim; lead-resistant bacteria; bioremediation; lead-contaminated soil; antioxidative
response

1. Introduction efficiency.[5] According to previous investigations, macro-

Metals can be released to the environment through natu-
ral processes, but mainly through human activities, such as
agriculture, mining, and industry. Heavy metals could be
toxic to living beings when their concentration is above a
certain level. It brings about serious pollution, threatening
human health and the ecosystem.[1] Among heavy met-
als, lead (Pb) is a persistent environmental pollutant which
slowly accumulates, leading to biomagnifications at differ-
ent tropic levels in food chains, and is therefore referred
to as a cumulative poison.[2] Lead can cause a large
variety of toxic effects, including gastrointestinal, muscu-
lar, reproductive, neurological, behavioural, and genetic
malfunctions.[3] Thus, cleaning up lead-contaminated soil
or detoxifying lead with the fewest environmental side
effects is of great interest and practical methods are needed.
Recently, phytoremediation technologies have been
proved to be a quite efficient and cost-effective method
for remediating sites contaminated with toxic metals.[4]
However, phytoremediation is generally time-consuming
due to low biomass of hyperaccumulator, low bioavail-
ability of heavy metal in soils, and low phytoextraction
fungi possessed the capacity to accumulate large quantities
of heavy metals especially when they grew in polluted
areas such as those in close proximity to highways,[6]
smelting plants,[7] or sewage sludge landfills.[8] Besides,
mushroom, of which the growth cycle is short, could
be easily cultivated and shows multiple heavy metal
tolerances.[9,10] Furthermore, rational selection of effec-
tive inocula seems to be a promising avenue for biore-
mediation. Bacteria were always used in remediation of
contaminated soil because of their high surface area to
volume ratio and the ability to detoxify certain metals. Var-
ious resistance mechanisms of lead-resistant bacteria have
been documented, such as intracellular bioaccumulation,
extracellular sequestration, surface biosorption, and bio-
transformation of organo-lead.[11] However, only a few
studies have reported the application of assistant microor-
ganism can greatly increase the yield of macrofungi and
enhance its ability to uptake metals.[12,13]
Like other non-essential heavy metals, lead can cause
physiological stress response which lead to the generation
of free radicals when of a high concentration. Stress in turn

*Corresponding author. Email: xuheng64@sina.com

† The first two authors contributed equally to this paper.

© 2015 Taylor & Francis

 2 Y. Li et al.
induces the production of reactive oxygen species (ROS),
such as hydrogen peroxide, superoxide, and hydroxyl
radicals, causing lipid peroxidation (generation of
malondialdehyde (MDA)), which consequently increases
membrane permeability.[14,15] Under oxidative stress
conditions, living beings develop an antioxidant defence
system to protect them from metal-induced oxidative
injury. The ability of cells and tissues to withstand oxida-
tive stress mainly depends on the induction of antioxidant
enzymes such as superoxide dismutase (SOD), peroxidase
(POD), and catalase (CAT).[16] As a first line of defence
against ROS, SOD plays a pivotal role in decomposing
the superoxide radical to generate H2 O2 and O2 by dis-
proportionation, then POD and CAT can convert H2 O2 to
H2 O and O2 . Such biochemical stress responses have been
widely reported in different living beings.[15–17] Thus,
changes in the activities of antioxidant enzymes could be
an adaptive response to metal toxicity.
In the present work, KQBT-3 is a Pb-tolerant strain.
Downloaded by [FU Berlin] at 22:24 13 May 2015
The highest Pb-tolerant concentration of strain was mea-
sured after domestication. Tricholoma lobayensis Heim,
the selected macrofungi in this study, is a kind of commer-
cial edible mushroom, which could be easily cultivated.
Compared to other microthermal fungi (e.g. Agaricus bis-
porus), the growth temperature of T. lobayensis is higher
and soil microbes might be more active in this warm con-
dition. The purpose of this experiment was to elucidate
the role of KQBT-3 in Pb-bioremediation by T. lobayensis.
Hence, the effects of inoculating KQBT-3 on T. lobayen-
sis growth, Pb uptake, and antioxidant response when the
mushroom was exposed to artificial metal-stressed soil
with a series of concentrations of Pb were investigated in
pot experiments.
2. Materials and methods
2.1. Strain domestication and characterization
The medium for domesticating bacterial strain was the
sucrose-minimal salts low-phosphate (SLP) medium (1%
sucrose, 0.1% (NH4 )2 SO4 , 0.2% K2 HPO4 , 0.05% MgSO4 ,
0.01% NaCl, 0.05% yeast extract, and 0.05% CaCO3 ,
pH 7.2) supplemented with different concentrations of
Pb as Pb(NO3 )2 (lead nitrate). Luria-Bertani (LB) broth
(10 g tryptone, 5 g yeast extract, 10 g NaCl per litre
water, pH 7.0) was used to incubate the strain. A Pb-
resistant strain, named KQBT-3, used in this study was
kindly provided by the microbiology laboratory of Sichuan
University (Cheng Du, China). KQBT-3 was recovered in
liquid SLP medium at 28°C for 12 h with constant agita-
tion of 150 rev min−1 . One hundred microlitre of overnight
cultures was spread on the SLP plate with 50 mg L−1
Pb and incubated at 28°C. The colony growing vigor-
ously on SLP plates was restreaked on the same fresh
SLP plates with Pb of 50–1000 mg L−1 in 50 mg L−1
increment.
The characteristics of the bacteria were studied. Indole-
3-acetic acid (IAA) production was determined according
to the method of Jing et al.[13] Siderophores secreted to
the growth mediums by the isolated strains were detected
by the method of Schwyn and Neilands.[18] The phos-
phate solubilization ability was quantified according to the
method of Pikovskaya.[19]
2.2. Soil treatments and mushroom sown
Natural soil used in this study was collected from agri-
cultural fields in Chengdu. Soil was screened through a 2
mm diameter sieve after eliminating the visible weeds and
small stones. After the above steps, the soil was artificially
contaminated with Pb (200, 400, 800 mg kg−1 dried soil)
as Pb(NO3 )2 , and blank group(0 mg kg−1 Pb) was added
with the same volume of distilled water. Soil was spiked
for two months before the pot experiments to achieve an
equal distribution. The mycelia (mixed with compost) of
T. lobayensis were purchased from a mushroom production
site in Chengdu of China and then cultured at 25 ± 1°C in
the same room in order to maintain uniform growth.
For inoculation, the bacterial suspension of
KQBT-3 was prepared. After incubation in liquid LB
medium at 150 rev min−1 at 28°C, cells of KQBT-3 in
the exponential phase were collected by centrifugation at
6000g for 10 min, then washed with sterile distilled water,
and suspended in sterile distilled water. The density of
the bacterial suspension was adjusted to approximately
1 × 109 CFU mL−1 .
Every mycelia bag was put in a plastic pot contain-
ing 6 kg (dry weight) soil. All the pots were moistened
to maintain the water content of the soil. After 25 days,
mycelia began to grow on the surface of the soil. The sus-
pensions of the resistant microbe KQBT-3 (30 mL pot−1 )
then were sprayed into half the number of plastic pots
with a sprinkling can, and the other half were sprayed
with equal volume of distilled water as uninoculated con-
trols. For each heavy metal concentration (uninoculated
or inoculated treatments), three replicates were run. All
experiments were conducted in a greenhouse which was
used to keep the temperature constant and avoid direct
sunlight.
2.3. Chemical analysis
Before chemical analysis, soil was air dried at room tem-
perature for 72 h and then sieved to < 0.25 mm. Soil
pH was measured in 1:2.5 (w/v) suspensions of soil and
water. The cation exchange capacity (CEC) was deter-
mined by the methods described by Rhoades.[20] Water
content and organic matter (OM) contents were measured
following the standard methods described by Avery and
Bascomb.[21] To acquire the total Pb in the studied soil,
one gram of treated or untreated soil samples was digested
with a mixture of 6:2 HNO3 /H2 O2 (v/v) using a microwave
 Environmental Technology
oven (SINEO, MDS-6) following the procedure of Tüzen
et al. [22] and then diluted with demineralized water. The
bioavailable (EDTA-extractable) levels of Pb in treated and
untreated soil were determined by the method of Tarvainen
and Kallio.[23] The concentrations of lead in the digest and
extract were determined using a graphite furnace for flame
atomic absorption spectrophotometer (Varian, SpectrAA-
220Z) with argon as inert gas. The limit of quantification
for lead was 1.0 mg L−1 (graphite furnace atomic absorp-
tion spectrometry). The blanks were run in triplicate to
check the precision of the method with each set of samples.
Ten days after inoculating with KQBT-3, the fresh
fruiting bodies of T. lobayensis were harvested, and the
harvest time lasted for 20 days. After being washed thor-
oughly with distilled water, each fruiting body was cut
into halves. One half was dried at 60°C for heavy metal
analysis, and the other was used to detect MDA and antiox-
idant enzymes. The dried mushrooms were powdered and
sieved to < 0.25 mm, then digested with a solution of con-
Downloaded by [FU Berlin] at 22:24 13 May 2015
centrated HNO3 and 30% H2 O2 (3:1, v/v) as described
before.
2.4. Activities of antioxidant enzymes
Fresh mushroom (1.0 g) was quick-frozen in liquid nitro-
gen and then ground using pre-chilled mortar and pestle.
The resultant paste was extracted in 50 mM phosphate-
buffered saline (PBS) (pH 7.8). The homogenate was
centrifuged at 10,000g for 15 min at 4°C, and then the
supernatant was diluted with PBS buffer to 5 mL. The
resulting diluent was named as the cell-free extract.
SOD activity was assayed by measuring its ability to
inhibit the photoreduction of nitroblue tetrazolium (NBT)
to blue formazan (absorption peak: 560 nm) under light
following the method of Beauchamp and Fridovich.[24]
The 3 mL reaction mixture contained 0.05 mL 50 mM
PBS (pH 7.8), 2.5 mL 13 μM methionine, 0.25 mL 63 μM
NBT, 0.15 mL 13 μM riboflavin, and 0.05 mL of cell-free
extract. Enzymatic activities were expressed as U g−1 fresh
weight (fw). POD activity was determined by the method
of Omran.[25] The reaction mixture in a total volume of 3
mL contained 50 mM PBS (pH 6.0), 0.2% H2 O2 , 2% gua-
iacol (prepared before use), and 0.05 mL cell-free extract.
Enzyme activity was defined as μmol of H2 O2 reduced
min−1 g−1 fw based on the decline in absorbance at 470
nm. CAT activity was determined according to Beers and
Sizer.[26] The reaction mixture (2 mL) consisted of 0.2%
H2 O2 and 1.9 mL H2 O as well as 0.05 mL cell-free extract.
Decrease in the absorbance was taken at 240 nm. Enzyme
activity was expressed as μmol of H2 O2 reduced min−1 g−1
fw.
2.5. Lipid peroxidation
According to the method of Heath and Packer, MDA
level (as an index of lipid peroxidation) was measured
3
spectrophotometrically by reaction with thiobarbituric acid
(TBA).[27] The cell-free extract (0.6 mL) was mixed with
1 mL of 20% trichloroacetic acid containing 0.5% TBA
and incubated at 95°C for 0.5 h. After quickly cooling, the
mixture was centrifuged at 10,000g for 10 min and then
the absorbance of the supernatant was recorded at 532 nm
and 600 nm. The non-specific absorbance at 600 nm was
subtracted from the 532 nm absorbance. The concentration
of MDA was calculated from the extinction coefficient of
155 mM−1 cm−1 and expressed as mmol/g fw.
2.6. Data analysis
Data analysis was performed with SPSS 18.0. Duncan’s
multiple range test (DMRT) was performed to determine
the significant differences (p < .05) between treated and
control groups. One-way analysis of variance was per-
formed followed by Tukey’s test.
3. Result
3.1. Domestication of Pb-resistant strain KQBT-3
KQBT-3 isolated from the heavy meta-contaminated soil
was characterized as Bacillus thuringiensis in previous
experiments. Colonies of strain KQBT-3 on LB agar
were milk-white, circular with irregular margins, and
2.0–3.0 mm in diameter after 24 h of incubation at 28°C.
After domestication and screening, KQBT-3 could toler-
ate Pb(NO3 )2 up to a concentration of 800 mg L−1 . It was
noted that such tolerance was a character as the result
of adaptation, because the highest tolerant Pb concentra-
tion was measured based on the results of the original
strain inoculated at a series of medium, in which the
concentrations of Pb raised gradually. However, the orig-
inal strain only could grow in the SLP plate containing
Pb(NO3 )2 at a concentration of 0–400 mg L−1 without
the domesticating process. In addition, the KQBT-3 strain
had the capacity to produce IAA (9.38 ± 0.28 mg/mL),
siderophore (15.11 ± 0.071 mg/mL), and solubilize inor-
ganic phosphate (36.91 ± 0.78 mg/mL).
3.2. Soil properties and effects of KQBT-3 on the
growth and Pb accumulation of T. lobayensis
The basic physicochemical properties of the untreated and
treated soil are listed in Table 1.
Dry mass of T. lobayensis under different treatments
is shown in Figure 1. It can be seen that Pb content in
soil affected the growth of T. lobayensis. With the increase
of Pb concentration, biomass of the fruiting body pre-
sented a downward trend. But an obvious increasing trend
was observed at each metal concentration when KQBT-
3 was inoculated. Under the treatment of 800 mg kg−1
Pb, dry mass increased by 47.4% compared to uninocu-
lated mushroom. As shown in Figure 2, Pb content in the
fruiting body of T. lobayensis was increased as the heavy
 4 Y. Li et al.

Table 1. The physicochemical properties of the untreated and treated soil.

Parameter Blank Pb200 Pb400 Pb800

pH 7.46 ± 0.03 7.36 ± 0.02 7.28 ± 0.03 7.44 ± 0.02

Water-holding capacity (%) 12.76 ± 0.61 11.97 ± 1.57 11.88 ± 0.89 11.67 ± 2.03
CEC (cmol kg−1 ) 21.45 ± 0.26 21.36 ± 1.18 22.03 ± 0.93 22.19 ± 1.34
OM (g kg−1 ) 18.64 ± 0.19 18.77 ± 0.98 18.03 ± 1.47 18.93 ± 1.22
Total Pb (mg kg−1 ) 44.05 ± 2.54 221.19 ± 11.09 416.13 ± 14.29 806.01 ± 9.41
Extracted Pb (mg kg−1 ) 9.31 ± 0.43 67.32 ± 5.89 117.98 ± 9.57 219.30 ± 10.35
Total Cd (mg kg−1 ) 0.12 ± 0.03 < 0.05 < 0.05 < 0.05
Extracted Cd (mg kg−1 ) 0.05 ± 0.04 < 0.05 < 0.05 < 0.05

Note: Data shown represent the mean ± standard deviation from three replicates. The contents of heavy metal
at undetectable levels are marked as ‘ < 0.05’.
Downloaded by [FU Berlin] at 22:24 13 May 2015

Figure 1. Effects of inoculation with KQBT-3 on T. lobayensis
biomass in Pb-containing soil with 0 (blank), 200, 400, and 800
mg kg−1 .
Note: All the values are mean of triplicates ± SD. Different let-
ters indicate significant differences assessed by DMRT (P < .05)
(a–d denote significance among different Pb concentrations; A,
B denote significance between the uninoculated control and
inoculated treatment, respectively).
Figure 2. Effects of inoculation with KQBT-3 on Pb accumu-
lation in T. lobayensis in Pb-containing soil with 0 (blank), 200,
400, and 800 mg kg−1 .
Note: All the values are mean of triplicates ± SD. Different let-
ters indicate significant differences assessed by DMRT (P < .05)
(a–d denote significance among different Pb concentrations; A,
B denote significance between the uninoculated controls and
inoculated treatments, respectively).

metal stress increased with or without adding KQBT-3.
Additionally, it could be seen that the ability of accumu-
lating Pb of T. lobayensis was obviously promoted after
inoculating KQBT-3. Especially at the concentration of
800 mg kg−1 Pb, accumulation of Pb increased by 33.2%
compared to the uninoculated controls. Furthermore, in
control soil, the decrease of biomass was not very evi-
dent at the low concentration of Pb (200, 400 mg kg−1 ),
and the bioaccumulation increased along with increasing of
the concentration of Pb, but the increasing extent reduced.
However this phenomenon has improved in inoculating
treatments.
3.3. Effects of KQBT-3 on MDA content in T.
lobayensis
In order to assess the potential influence of inoculation
KQBT-3 on preventing membrane damage, MDA content
was analysed as a measure of lipid peroxidation. As Figure
3 shows, MDA content in T. lobayensis gradually increased
with increasing Pb concentration in contaminated soil,
indicating a concentration-dependent free radical genera-
tion relation. The maximum rate of MDA production was
found at the highest concentration of Pb. However, MDA
production of inoculated treatments was lower than that
of uninoculated treatments at the same Pb concentration.
Under heavy metal pressure, MDA content decreased from
17.01% to 21.94%.
3.4. Effects of KQBT-3 on the activity of antioxidant
enzymes
In the present study, the activity of SOD showed an
increasing trend with gradual increasing of stress of Pb,
and the highest activity was obtained under the treatment
of 800 mg kg−1 Pb (Figure 4). Besides, it was found that
 Environmental Technology
Figure 3. Effects of inoculation with KQBT-3 on the MDA
content of T. lobayensis under different treatments at various con-
centrations.
Note: All the values are mean of triplicates ± SD. Values
Downloaded by [FU Berlin] at 22:24 13 May 2015
with different lower-case letters indicating significant difference
between treatments while values with different upper-case let-
ters indicate significant difference within treatment, according to
DMRT (P < .05).
Figure 4. Effects of inoculation with KQBT-3 on the SOD
activity of T. lobayensis under different treatments at various con-
centrations.
Note: All the values are mean of triplicates ± SD. Values with
different lower-case letters indicate significant difference between
treatments while values with different upper-case letters indi-
cate significant difference within treatment, according to DMRT
(P < .05).
inoculating KQBT-3 promoted SOD activity in T. lobayen-
sis after long-term exposure to Pb. The more serious the
Pb-contamination was, the greater positive influence of
inoculation KQBT-3 made. At the concentration of 800 mg
kg−1 Pb, the inoculated treatment was found to be about
1.37 times higher than non-inoculated controls. POD and
CAT exhibited a similar type of response in general, show-
ing a progressively increasing trend initially, and then a
5
Figure 5. Effects of inoculation with KQBT-3 on the POD
activity of T. lobayensis under different treatments at various con-
centrations.
Note: All the values are mean of triplicates ± SD. Values with
different lower-case letters indicate significant difference between
treatments while values with different upper-case letters indi-
cate significant difference within treatment, according to DMRT
(P < .05).
Figure 6. Effects of inoculation with KQBT-3 on the CAT
activity of T. lobayensis under different treatments at various con-
centrations.
Note: All the values are mean of triplicates ± SD. Values with
different lower-case letters indicate significant difference between
treatments while values with different upper-case letters indi-
cate significant difference within treatment, according to DMRT
(P < .05).
decreased trend at high-level Pb concentration (Figures 5
and 6). Hence, a maximum increase was obtained under the
treatment of 400 mg kg−1 Pb after adding KQBT-3. Like-
wise, inoculation of KQBT-3 also enhanced the POD and
the CAT activities by 126.26% and 104.33%, respectively.
Besides, POD activity displayed a substantial increase
compared to uninoculated controls, while CAT activity did
not present a significant change.
 6 Y. Li et al.
4. Discussion
Accumulation of heavy metals by the wild growing
mushroom has been reported in Coprinus comatus, Tri-
choloma terreum, Agaricus campestris, etc.[28,29] Simi-
larly, Demirbaş reported that three mushrooms showed a
different ability to accumulate Pb, Cd, Cu, and Hg from
artificially enriched soils.[30] Furthermore, Bioaugmen-
tation as a soil bioremediation approach is a promising
method to improve biomass production and the rate of
extraction of heavy metals. It had been reviewed that plant
microbe partnerships could enhance the total biomass of
plant and phytoextraction of heavy metals such as Cd,
Pb, Zn, Cr, Cu, Ni, and so on.[31,32] Similar to previ-
ous studies in plant, in our work, a significant increase of
the dry mass of T. lobayensis and accumulation of Pb was
observed after inoculating KQBT-3, especially in the seri-
ous heavy metal pollution. When the heavy metal pollution
was light, the advantage of adding strains was not obvious.
However, heavy metal stress for indigenous microorgan-
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isms strengthened with increasing of Pb content, so the
advantage of Pb-resistant strain was more obvious.
T. lobayensis inoculated with KQBT-3 exhibited high
abilities to remedy heavy metal-contaminated soil as plant
and plant-growth-promoting microbes do. The positive
influence of inoculating KQBT-3 on mushroom growth
and accumulation of heavy metals may be due to the
production of IAA, siderophores, and solubilization of
phosphate. Strain KQBT-3 producing IAA may indirectly
promote metal accumulation by increasing the biomass of
T. lobayensis. Siderophores may be important for the mobi-
lization of the heavy metal in the rhizosphere. They not
only showed high affinity for ferric iron but also formed
complexes with bivalent heavy metal ions that could be
assimilated by the T. lobayensis.[33] IAA and phosphate
solubilization could reduce soil pH and solubilize inor-
ganic phosphorus of soil, but also played an important role
in improving the bioavailability of soil metals.[32] Pre-
sumably, the increased accumulation of Pb in T. lobayensis
in the presence of KQBT-3 could be due to more Pb uptake
under acidic soil conditions, which developed as a result
of activity of phosphate solubilization in soil. In addition,
the environment of contaminated soil has been improved
because of Pb removal. This was beneficial for the growth
of KQBT-3. These results concurred with the earlier obser-
vations. For C. comatus grown in soil inoculated with
heavy metal-mobilizing bacteria, Pb content in the fruit-
ing body was increased depending on Pb solubilization and
IAA production.[13] Ling-yun Ji also found that inocula-
tion of heavy metal-solubilizing microorganisms M6 and
K1, respectively, could promote the growth of T. lobayen-
sis and total Cd and Zn accumulations due to the pro-
duction of IAA and siderophores, and solubilize inorganic
phosphate by heavy metal-solubilizing strains.[34]
Heavy metals affect plant growth through the gen-
eration of free radicals and ROS, which pose constant
oxidative damage to cellular structures by degrading
important cellular components.[35] The induced oxida-
tive stress (e.g. Lipid peroxidation) that could damage the
membranes was investigated by monitoring MDA content.
Lipid peroxidation causes the breakdown of functional and
structural integrity of biological membranes, then increases
the permeability of plasma membrane, leakage of K+
ions, oxidation of amino acid, and eventually causes cell
death.[36] MDA is a low molecular weight decomposition
product of peroxidized polyunsaturated fatty acids.[37]
Thus, MDA has been recognized as a reliable indicator
of lipid peroxidation.[15] When coping with heavy metal
stress, the increase in MDA content in the present study
was in agreement with earlier studies in which differ-
ent plant species were exposed to different metals.[38,39]
The obvious decrease of MDA in T. lobayensis inoculated
with KQBT-3 indicated lipid peroxidation in T. lobayen-
sis was alleviated by microbes. Alleviation of Pb-induced
oxidative stress may be attributed to KQBT-3 scavenging
the free radicals. A similar result was observed in ear-
lier studies. Likewise, Oudemansiella radicata had higher
MDA content in control treatments (exposure to heavy
metals) compared to siderophore-containing filtrates treat-
ments.[39] MDA content in the Solanum nigrum L. was
significantly higher than that in application of citric acid
and cadmium-resistant strain treatments under cadmium
stress.[40]
Macrofungi possess a defence system to mitigate and
repair the oxidative damage initiated by ROS, which is
similar to plants. Mushroom was able to respond to ele-
vated levels of ROS to activate their antioxidative defence
systems.[15] Enzymes such as SOD, CAT, and POD can
be activated against ROS in several organisms following
heavy metal stress.[41] SOD is an efficient scavenger of
ROS. It could convert the superoxide into peroxide and
oxygen by the dismutation, and then the H2 O2 could be
degraded to H2 O and O2 by POD and CAT working at
different locations in the cell.[15] POD functions in the
apoplast of lignifying tissues, whereas CAT is present in
peroxisomes and mitochondria.[41]
As indicated above, inoculation of KQBT-3 could sig-
nificantly enhance Pb accumulation in T. lobayensis, thus
improving SOD activity. These phenomena were also
reported in previous studies carried out with other plant
species and metals. Yang Gao found that SOD activity sig-
nificantly correlated with Cd accumulation in S. nigrum
L.[40] The Cu-tolerant bacteria could promote SOD in
Triticumaestivum L. to reduce superoxide radicals.[42]
Likewise, AM fungi had been successfully used to relieve
oxidative stress of Pb in seedlings of Zea mays L., as shown
by a higher SOD activity in the mycorrhizal seedlings.[43]
In our experiments, because the accumulation of H2 O2
in T. lobayensis was increased when the KQBT-3 was inoc-
ulated, the generation of POD and CAT were increased
in heavy metal-stressed soil. Literature on POD and
 Environmental Technology
CAT activity increase by inoculating microbe in plants
under metal stress conditions is scanty. In contrast, some
authors have reported antioxidant enzyme activities dis-
played an obvious decrease in the plants which were
inoculated.[16,42] But at the concentration of 800 mg kg−1
Pb, both POD and CAT experienced a reduction. This
decrease in antioxidant enzymes in T. lobayensis could
be explained partially by the fact that T. lobayensis may
have been submitted to severe oxidative stress under seri-
ous heavy metal contamination. Additionally, the change
of POD was more obvious than CAT after inoculation
with KQBT-3. The reason may be that inoculating KQBT-
3 substantially promoted the POD activity to respond to
Pb toxicity. The generation of CAT was linked to a self-
detoxification mechanism in T. lobayensis, but little influ-
enced by application of KQBT-3. It revealed that POD
played an important role in elimination of H2 O2 when T.
lobayensis inoculated with KQBT-3 responded to heavy
metal stress.
Downloaded by [FU Berlin] at 22:24 13 May 2015
5. Conclusion
In the present study, KQBT-3 (B. thuringiensis) was able to
tolerate a metal-stressed environment with a concentration
of 800 mg kg−1 Pb, which was the result of domestica-
tion. In addition, the effects of KQBT-3 on T. lobayensis
growth, Pb uptake, and antioxidative response in lead-
contaminated soil in greenhouse conditions were mainly
investigated in this study. First, KQBT-3 not only could
promote the growth of T. lobayensis, but also could help it
to accumulate Pb; second, lipid peroxidation in T. lobayen-
sis under Pb stress was alleviated by KQBT-3. Finally,
the strain increased the activities of antioxidant enzymes
to reduce the oxidative damage induced by the excessive
Pb accumulation in T. lobayensis. The activities of SOD
and POD displayed an obvious increase, while CAT activ-
ity had no significant change compared to non-inoculated
control treatments. Based on the above-mentioned results,
it can be concluded that KQBT-3 played an important
role in accumulating Pb and relieving the toxicity of
Pb in T. lobayensis. The present work demonstrated that
this macrofungi-microbe method can effectively detox-
ify heavy metal-contaminated soil at lesser cost without
environmental side effects.
Acknowledgements
The authors wish to thank Professor Guanglei Cheng and Dong
Yu from Sichuan University for their technical assistance.
Disclosure statement
No potential conflict of interest was reported by the authors.
Funding
This study was financially supported by the National Natural
Science Foundation of China [Nos. 41171253, J1103518), the
7
National High Technology Research and Development Program
of China [Nos. 2013AA06A210, 2006AA06Z361], Science and
Technology Development Project of Infinitus (China) Company
Ltd and Chengdu Longquanyi District Science and Technology
Bureau.
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