Optimization of microbial assisted phytoremediation of soils contaminated with pesticides

Abstract

580 microbial strains were isolated from the rhizosphere of the plants Cucurbita pepo L. and Xanthium strumarium grown on soil contaminated with dichlorodiphenyltrichloroethane (DDT) and its metabolites. During the cultivation, two bacterial strains were selected because of their ability to grow on media containing 0.5-5.0 mg L-1 of dichlorodiphenyldichloroethylene (DDE) as the sole carbon source. They were identified as Bacillus vallismortis and Bacillus aryabhattai. Both of these species were shown to have a high capacity for the utilization of DDE - more than 90% of which was consumed after 21 days of cultivation. Laboratory experiments were carried out then to assess the possibility of using these strains for the decontamination of organochlorine pesticides (OCPs) contaminated soils. Inoculation of C. pepo and X. strumarium with our isolates B. vallismortis and B. aryabhattai resulted in a reduction of the pollutant stress to the plants as shown by an increase both in aboveground and in root biomass. The microorganisms enhanced the uptake and phytostabilization potential of C. pepo and X. strumarium and can be applied for the treatment of DDE contaminated soils.

Full text

Full Terms & Conditions of access and use can be found at
https://www.tandfonline.com/action/journalInformation?journalCode=bijp20
International Journal of Phytoremediation
ISSN: (Print) (Online) Journal homepage: https://www.tandfonline.com/loi/bijp20
Optimization of microbial assisted
phytoremediation of soils contaminated with
pesticides
Asil Nurzhanova , Togzhan Mukasheva , Ramza Berzhanova , Sergey Kalugin ,
Anel Omirbekova & Annett Mikolasch
To cite this article: Asil Nurzhanova , Togzhan Mukasheva , Ramza Berzhanova , Sergey
Kalugin , Anel Omirbekova & Annett Mikolasch (2020): Optimization of microbial assisted
phytoremediation of soils contaminated with pesticides, International Journal of Phytoremediation,
DOI: 10.1080/15226514.2020.1825330
To link to this article: https://doi.org/10.1080/15226514.2020.1825330
Published online: 01 Oct 2020.
Submit your article to this journal
View related articles
View Crossmark data

Optimization of microbial assisted phytoremediation of soils contaminated
with pesticides
Asil Nurzhanova
a
, Togzhan Mukasheva
b
, Ramza Berzhanova
b
, Sergey Kalugin
c
, Anel Omirbekova
b
, and
Annett Mikolasch
d
a
Institute of Plant Biology and Biotechnology, Almaty, Kazakhstan;
b
Faculty of Biology and Biotechnology, al-Farabi Kazakh National
University, Almaty, Kazakhstan;
c
Faculty of Chemistry and Chemical Technology, al-Farabi Kazakh National University, Almaty, Kazakhstan;
d
Institute of Microbiology, University Greifswald, Greifswald, Germany
ABSTRACT
580 microbial strains were isolated from the rhizosphere of the plants Cucurbita pepo L. and
Xanthium strumarium grown on soil contaminated with dichlorodiphenyltrichloroethane (DDT) and
its metabolites. During the cultivation, two bacterial strains were selected because of their ability
to grow on media containing 0.5–5.0 mg L
1
of dichlorodiphenyldichloroethylene (DDE) as the
sole carbon source. They were identified as Bacillus vallismortis and Bacillus aryabhattai.Bothof
these species were shown to have a high capacity for the utilization of DDE –more than 90% of
which was consumed after 21 days of cultivation. Laboratory experiments were carried out then
to assess the possibility of using these strains for the decontamination of organochlorine pesti-
cides (OCPs) contaminated soils. Inoculation of C. pepo and X. strumarium with our isolates B. val-
lismortis and B. aryabhattai resulted in a reduction of the pollutant stress to the plants as shown
by an increase both in aboveground and in root biomass. The microorganisms enhanced the
uptake and phytostabilization potential of C. pepo and X. strumarium and can be applied for the
treatment of DDE contaminated soils.
KEYWORDS
Inoculation; microorganism-
destructor; organochlorine
pesticides; phytoremedia-
tion; rhizosphere
Introduction
In Kazakhstan and in many other countries around the
world, surplus stocks of obsolete pesticides stored in now
deserted and crumbling warehouses, pollute the surrounding
soil and pose a serious threat to the environment (FAO
2001). A significant fraction of these pesticides is persistent
organic pollutants (POPs), which are of particular concern
due to their toxicity, persistence, and accumulation in the
fatty tissues of humans and animals (Letcher et al. 2010;
Laird et al. 2013; UNEP 2005). Prohibited pesticides are pre-
sent in the areas surrounding 65 former pesticide storage
facilities in the Almaty region of Kazakhstan. Soil in these
neighborhoods is contaminated with DDT and related com-
pounds like the 4,40- and 2,40-isomers, DDE and dichlorodi-
phenyldichloroethane (DDD) (Nurzhanova et al. 2013).
Such “hot spots”of pesticide pollution are a considerable
risk both to the environment and to human health.
One of the most effective methods for the elimination of
pollutants is phytoremediation. This is a cost-effective alter-
native to physical and chemical methods that require large
investments (Karthikeyan et al. 2004) and is used in many
developed countries to restore land contaminated with
POPs-pesticides. To date, a number of plant species have
been identified for the phytoremediation of soils contami-
nated with heavy metals, radionuclides, explosives and pesti-
cides (Kennen and Kirkwood 2015). As well, some members
of the genus Cucurbita (zucchini, pumpkin) have a unique
mechanism for the adsorption of 4.40-DDE metabolites in
their root system and their subsequent transfer into the
shoots (White et al. 2003); also possessed a high ability to
accumulate OCPs, namely: DDT and its metabolites, aldrin,
chlordane and endrin in the aboveground biomass (Zeeb
et al. 2006). The common cocklebur Xanthium strumarium
also tolerates DDT well, and is able to absorb it and its
metabolites from contaminated soils (Nwoko 2010; Arslan
et al. 2017; Nurzhanova et al. 2013). Many plant species that
have the ability to accumulate large concentrations of DDT
metabolites translocate them to the root system while others
transfer to the shoots (Mitra and Raghu 1989; White and
Kottler 2002; Nwoko 2010; Nurzhanova et al. 2013).
It has been reported that plants and their associated
microorganisms together play a crucial role in phytoreme-
diation of contaminated soils (Starkey 1958; Glick 1995;
Pilon-Smits 2005; Pascal-Lorber and Laurent 2011). A grow-
ing plant secretes a wide range of secondary metabolites,
such as sugars, polysaccharides, amino acids, tricarboxylic
acids, oxalic acid, fatty acids, sterols, phenols, and proteins,
into the rhizosphere, where they are available to soil micro-
organisms (Badri et al. 2009). On the other hand, com-
mensal microorganisms protect the plant from pathogenic
microbes and may produce phytohormones, such as auxins,
cytokinins, and gibberellins in the rhizosphere, which
CONTACT Anel Omirbekova anel.omirbekova@kaznu.kz al-Farabi Kazakh National University, Almaty, Kazakhstan.
ß2020 Taylor & Francis Group, LLC
INTERNATIONAL JOURNAL OF PHYTOREMEDIATION
https://doi.org/10.1080/15226514.2020.1825330

promote the rapid growth of the plant (Anderson and Coats
1995; Tanimoto 2005; Abhilash et al. 2013). The C. pepo L.
root system secretes proteins, which have been shown to
play an important role in the process of biodegradation of
certain organochlorine compounds (Campanella and Paul
2000). The combination of plants with their associated com-
mensal microorganisms thus provide an encouraging model
for the destruction of toxic organic substances.
The main aim of this study was to isolate rhizobacteria
from the rhizosphere of X. strumarium and C. pepo growing
on soil artificially contaminated with metabolites of DDT
and to study the effect of microorganisms on the parameters
of biomass as well as phytoremediation of plants grown on
contaminated soil from the territory of the former ware-
house obsolete pesticides.
Materials and methods
Plants
Xanthium strumarium from the Kazakh flora and pumpkin
C. pepo L. as model plant, which are tolerant to DDT were
used in the study. Xanthium strumarium dominates in the
territory of the former warehouse obsolete pesticides in the
Almaty region, and is tolerant against the of pesticides.
Chemicals
The standard samples of 4.40-DDT (p,p0-dichlorodiphenyltri-
chloroethane), 4.40-DDE (p,p0-dichlorodiphenyldichloroethy-
lene) and 4.40-DDD (p,p0-dichlorodiphenyldichloroethane)
used during this work were 100.0 ± 1.0 mgml
1
solutions
in hexane.
Soil samples
The soils used for the experiment were of two types: one of
them were collected at the contaminated site, and the other
type was artificially contaminated soil in the laboratory. The
soil sampling at the contaminated and control sites was car-
ried out using the standard approach (ISO, 2017): from one
55 m testing square, five samples were taken at the depth
0-0.6 m using quartering.
The first soil was taken around the territory of the former
warehouse obsolete pesticides located in Kyzylkairat settle-
ment, Talgar district, Almaty region, Kazakhstan (GPS
4317058.700N77
11039.600E). The sampling was done at a
depth of 50–60 cm using the envelope method, and soil was
sifted through a sieve with a diameter of 3 mm. The control
soil was taken 1 km from Kyzylkairat settlement on the
regular agricultural land with the following coordinates: N
4451022.400,E78
42013.100.
The second soil was artificially contaminated by metabo-
lites DDT. The concentrations of various DDT metabolites
were selected on the basis of the average level of soil con-
tamination around of the former warehouse obsolete pesti-
cides (Table 1).
In order to prepare the artificially contaminated soil, the
clean soil samples were spiked with the solutions of pesti-
cides in 30% aqueous alcohol: the final concentration of
each pesticide was 0.6 mg of 4.40- DDE in 1 kg soil; 0.1 mg
of 4.40-DDD in1 kg soil; 0.7 mg of 4.40-DDT in 1 kg soil.
Control were added solutions in 30% aqueous alcohol.
After applying the solution, the soil was mixed and left for
1 month so that the solvents could completely evaporate.
Pot experiments
The pot experiment was carried out in the green house con-
ditions. Prior to the experiments, the artificially DDT-conta-
minated soil, contaminated soil from the territory of the
former warehouse of obsolete pesticides and control soils
were passed through a sieve with a pore diameter of 3 mm
followed by the thorough mixing. Then the bottom of the
pots was filled with a drain weighing 1.0 kg. Drainage was
covered with gauze and river sand weighing 1.0 kg, and
again covered with gauze. Then each pot was filled with the
soil weighing 3.0 kg. To prevent drying of the soil, it was
covered with one layer of sand. Each pot with the research
soil had a weight of 5 kg.
Seed of X. strumarium and C. pepo were planted in each
pot, 10 seeds per pot. Each experiment was set up in tripli-
cate. Growth was monitored for 7 months.
Isolation of microorganisms from the rhizosphere
of plants
To determine the population of microorganisms and to iso-
late pure cultures from the rhizosphere. The plants were dug
up with a shovel and, after extracting them from the soil,
they shook the soil that was loose on them from the roots
and left the soil firmly bound to the roots. 10 g of the soil
was put into flask with 100 ml of 0.1% sodium pyrophos-
phate. After settling of the suspension for 1 h prepare dilu-
tion and carry out sowing on the surface of dense nutrient
media (Somasegaran and Hoben 1994; Cavaglieri et al.
2009). Suspensions of 10
3
–10
5
dilutions were prepared and
inoculated on potato dextrose agar (PDA), tryptic soy agar
(TSA), starch casein agar (SCA) (Himedia Laboratories PvT
Ltd, India). For the selection of isolates, morphologically dis-
tinct bacterial colonies were selected from the TSA and
SCA medium.
Table 1. The average concentration of pesticides in soil.
Metabolite of DDT The residual amount of pesticides, lgkg
1
MAC, lgkg
1
Contaminated soil from the former warehouse pesticides
4.40-DDE 813 ± 17 100
4.40-DDD 284 ± 6 <1
4.40-DDT 907 ± 20 100
Sum 2004
Artificially contaminated soil
4.40-DDE 613 ± 17 100
4.40-DDD 105 ± 6 <1
4.40-DDT 707 ± 20 100
Sum 1425
MAC: Maximum allowable concentration values for the Republic of Kazakhstan
(Mynbayeva and Imanbekova 2013)
2 A. NURZHANOVA ET AL.

The structure of the bacterial community in the rhizo-
sphere of plants was characterized on the TSA medium
bythe frequency of individual genera. Primary identification
of bacteria was performed by studying the morphological
features of the colony and cells. The identification of the
genus of isolates was carried out according to morpho-
logical, physiological and biochemical properties
(Holt 1977).
Identification of species was carried out by determining
the nucleotide sequence of the 16S rRNA fragment of the
gene, followed by comparing this sequence with the sequen-
ces deposited in the international Gene Bank database
(Clayton et al. 1995).
16S rRNA sequences were deposited into the GenBank
database: the strain of Bacillus aryabhattai under the num-
ber MH478203, and the strain of Bacillus vallismortis under
the number MH478202.
Determination of microbial growth in the presence of
4.4’-DDE
The ability of isolates to grow in the presence of 4.40-DDE
was determined on solid and liquid mineral medium with
the following composition: Na
2
HPO
4
6.0 g, KHPO
4

3.0 g, NH
4
Cl 0.1 g, NaCl 0.5 g, H
2
O1 L with the
addition of 0.5 g of yeast extract.
4.40-DDE was added to solid and liquid medium to a final
concentration of 0.5, 1.0, 2.0, 3.0 and 5.0 mg per liter. Interest
in 4.40-DDE metabolite is associated with the fact that it is a
natural soil contaminant distributed in the upper soil hori-
zons. Among DDT metabolites, 4.40-DDE has a slightly low
value of hydrophobicity coefficient log Kow 6.51 in contrast
4.4-DDT (log Kow 6.91). Cultivation of bacteria was carried
out in 250-mL flasks containing 100 mL of medium. The ini-
tial number of bacteria for every approach was 10
8
CFU.
Quantitative assessment of the destruction of the
metabolite 4.40-DDE cultivating microorganisms in a
liquid medium
Microorganisms were cultivated in a liquid medium with
the following content: Na
2
HPO
4
6.0 g, KHPO
4
3.0 g,
NH
4
Cl 0.1 g, NaCl 0.5 g, H
2
O1 L. The initial content
of 4.40-DDE in a liquid medium was 480.4 ± 1.8 lgL
1
.
Cultivation of microorganisms (initial number of bacteria
10
8
CFU) was carried out in 500-mL flasks containing
100 mL of medium with intensive aeration on a circular
shaker at 220 rpm at a temperature of 30 C for 21 days.
Control cultures were grown on nutrient broth.
The residual amount of the metabolite 4.40-DDE during
the cultivation of microorganisms in liquid medium was
evaluated by gas chromatography.
Dry weight measurement
The biomass was determined by the dry weight method
described by Mikolasch et al. (2016). Biomass was expressed
in grams of dry matter.
Microbial inoculation of seeds
Seeds were first sterilized with 10% sodium hypochlorite
solution for 30 min, and then washed with sterile tap
water. Before inoculation to obtain biomass with a high
cell titer, bacteria were cultured in a liquid medium with
meat extract at a temperature of 30 C for 24 h. The cells
were harvested by centrifugation (12,000 g, 10 min),
washed twice with BD Phosphate Buffered Saline (pH 7.2).
Seeds were immersed in a suspension of 10
9
bacterial cells
per ml for 4–5 h. The following plant-microbe combina-
tions have been developed for seed bacterization:
C. pepo þB. vallismortis 114;C. pepo þB. aryabhattai 111;
X. strumarium þB. vallismortis 114;X. strumarium þB.
aryabhattai 111.
Chromatographic analyses of DDT metabolites
a. From nutrient medium: 20 ml of microbial nutrient
medium was extracted twice, each time with 10 ml of n-
hexane. The separated hexane layers were collected in
a flask.
b. From plants: For the extraction of pesticides (4.40–DDE,
4.40–DDD, 4.40–DDT) contained in plants, 1–2g of
plant sample was diced, placed in a 50 ml conical flask,
and shaken with 5–10 ml of n-hexane for 30 min. The
extract obtained was filtered through yellow ribbon fil-
ter paper.
c. From soil samples: For the extraction of pesticides
(4.40–DDE, 4.40–DDD, 4.40–DDT) from soil samples,
10 g of soil were placed in a stoppered bottle with a vol-
ume of 200 ml. 20 ml of n-hexane was added and the
mixture shaken for 60 min.
In all cases the extracts were concentrated to 1 ml.
Analysis was performed by gas chromatography with mass
spectrometric detection 7890 A/5973N (Agilent, USA). A
sample volume of 1 ll was injected using a Combi-PAL
autosampler (CTC Analytics AG, Switzerland). For the sep-
aration, a DB-35MS capillary column (Agilent, USA) with
a length 30 m. Helium carrier gas flow at 1.0 ml/min gave
an average linear flow velocity of 36 cm/s. The temperature
program ran from 40 C for 1 min, followed by a ramp at
20 C/min to 160 C which was then held for 3min, fol-
lowed by heating at 3 C/min to 250 C, and this tempera-
ture was then held for 5 min. Detection was carried out on
an electron capture detector, retention times corresponded
to OCPs (Agilent, USA).
Statistical analysis
Statistical analysis was conducted using the XLSTAT 2019
add-in to MS Excel 2019 software (Addinsoft, New York,
USA, 2020). Mathematically processed results were pre-
sented in the form M ± SE, where M was the arithmetic
mean, SE was the standard error.
INTERNATIONAL JOURNAL OF PHYTOREMEDIATION 3

Uptake index is the mass of the pollutant present in the
plant’s tissues, it represents in micrograms (for pesticides)
and be calculated by the following formula:
Uptake index,lg
¼Concentration of pesticides in plant0s tissue ðstem=rootsÞ
Weight ðkgÞ
(1)
Bioaccumulation factors (BA) was expressed as the ratio
between the residual amount of pesticides in aboveground
of plant with respect to the residual amount of pesticides
soil calculated by following equation:
BAC ¼Caboveground=Csoil (2)
where, C
aboveground
and C
soil
are the residual amount of pesti-
cides in tissue of plant (lgkg
1
) and in soil (lgkg
1
),
respectively.
Translocation factor (TF) was calculated as the residual
amount of pesticides ratio in plant aboveground to the
residual amount of pesticides in the root using the equation
(Zu et al. 2005):
TF ¼Caboveground=Croot (3)
where, C
abovedround
and C
root
are the residual amount of pes-
ticides (lgkg
1
) in the aboveground of the plant and root,
respectively.
Results
The bacterial composition in the rhizosphere of plants
cultivated in the presence of pesticides
Determine the bacterial composition of rhizosphere in the
presence of pesticides, C. pepo L. and X. strumarium plants
were grown on soils containing 4.40-DDT and its metabo-
lites. Fresh roots of these plants were washed, sterilized and
ground. Suspensions of 10
3
–10
5
dilutions of the ground
roots were inoculate on PDA, TSA and SCA plates
(Table 2).
PDA is a general-purpose medium for micromycetes
(yeasts) that was supplemented with acid to inhibit bacterial
growth. TSA was used as a growth medium for the isolation
and cultivation of bacteria. TSA þPDA was used for the iso-
lation of Bacillus strains. SCA was used for the detection of
Actinomycetes. The number of microorganisms recovered
from the rhizosphere of X. strumarium grown in the pres-
ence of pesticides was the greatest on TSA and lowest on
SCA (Table 2). The number of microorganisms recovered
from C. pepo grown in the presence of pesticide also was
greatest on TSA, though differences among the other media
were not significant. In both cases, the numbers of microor-
ganisms recovered on TSA were roughly two orders of mag-
nitude greater than the numbers recovered on the other
media. For both plants grown in the absence of pesticide,
more microorganisms were recovered on TSA plates, and
roughly one order of magnitude less colonies were recovered
from the other media.
In both X. strumarium and C. pepo the numbers of
spore-forming bacteria recovered on TSA þPDA plates was
roughly the same as was the number of Actinobacteria
recovered on SCA plates. For plants grown in the presence
of pesticide, however, these values were roughly one order
of magnitude lower than for plants frown in the absence
of pesticides.
One of the most important characteristics of the rhizo-
sphere microbiota is its composition. For this analysis bac-
teria were assigned to three groups: dominant (recovered
from >60% of plants), medium abundance or sub-dominant
group (recovered from 30% to 60% of plants), and minor
(recovered from <30% of plants). Both for X. strumarium
and C. pepo bacteria of the genera Pseudomonas and
Bacillus predominated in the absence of pesticides. The gen-
era Mycobacterium,Arthrobacter,Rhodococcus and
Streptomyces were sub-dominant. Representatives of the gen-
era and Micromonospora were typical of the rhizosphere in
both species, though they were recovered from only 5% to
20% of the plants. The presence of pesticides in the soil
changed the ratios of the occurrence of different genera of
bacteria in the rhizosphere of the plants. Thus, the com-
munities of plant rhizosphere bacteria differ both in the
total number and in the frequency of occurrence of some of
the studied bacterial genera, depending on the growth con-
ditions. Bacteria of the genera Bacillus and Pseudomonas
gained an advantage, while representatives of the genus
Rhodococcus also now dominated in the rhizosphere of
plants in the presence of a pesticide.
Screening of rhizosphere microorganisms from plants
for their ability to grow in the presence of 4.4’-DDE
In order to develop a method of remediation using plants
and microorganisms, it is necessary to isolate pure cultures.
To this end 580 microbial strains were isolated from the
rhizosphere of X. strumarium and C. pepo plants, and were
characterized. Isolates were screened for their ability to grow
in the presence of various concentrations of 4.40-DDE jn
solid and in liquid mineral media a concentration of 0.5 mg
per l of medium was chosen for the initial selection of
Table 2. The number of microorganisms in the rhizosphere of plants grown in soil contaminated with 4.4’-DDT and its metabolites.
Plants Soil samples
Number of colonies forming microorganisms
Nutrient medium
TSA TSA þPDA SCA PDA
X. strumarium with pesticides 81.3 ± 4.5 10
3
42.1 ± 2.1 10
1
18.8 ± 1.5 10
1
23.8 ± 1.1 10
1
without pesticides 106.8 ± 12.4 10
3
98.7 ± 5.1 10
2
32.1 ± 2.1 10
2
61.3 ± 3.3 10
2
C. pepo L. with pesticides 39.9 ± 3.4 10
3
31.2 ± 1.7 10
1
16.5 ± 1.1 10
1
51.2 ± 3.2 10
1
without pesticides 112.9 ± 9.7 10
3
64.9 ± 6.5 10
2
48.1 ± 1.7 10
2
66.7 ± 8.1 10
2
4 A. NURZHANOVA ET AL.

isolates. 289 of the isolates were able to grow in the presence
4.40-DDE in the concentration 0.5 mg L
1
). As the concen-
tration of pesticide in the medium was increased progres-
sively fewer isolates were able to grow, and at a
concentration of 5.0 mg L
1
only two of the isolates still
grew well (Table 3 and Figure 1.)
The two active isolates, strain 111 and strain 114, were
assigned to the genus Bacillus on morphological grounds
and species assignment was made based on 16S rRNA
sequence analysis. Strain 111 is B. vallismortis (identity
100%), and strain 114 B. aryabhattai (identity 100%).
The dry weight of cultures of B. vallismortis 111 and B.
aryabhattai 114 was determined after 21 days of cultivation
in liquid medium supplemented with 4.40-DDE as the sole
source of carbon and energy (Table 4).
The results showed an increase in biomass in the pres-
ence of 4.40-DDE. After 21 days, growth in the presence of
the pesticides was about 25–33% lower than in the
unamended controls.
To determine the extent of consumption of 4.40-DDE B.
vallismortis 111 and B. aryabhattai 114 were incubated in
liquid medium supplemented with 4.40-DDE (480.4 mgL
1
)
as the sole carbon source. After 14 and 21 days of incuba-
tion, the liquid media were extracted and the hexane
extracts were analyzed by chromatography.
This study of the decrease of 4.40-DDE in a liquid
medium showed that under these conditions the strain B.
aryabhattai 114 are capable of assimilating up to 89.3% of
the pesticide over an incubation period of 14 days and
93.4% over a period of 21 days.
The study the rhizobacteria influence on the efficiency
of phytoremediation of soil contaminated with OCPs
To increase the efficiency of phytoremediation using plant-
microbial assimilation soil from the territory of the former
warehouse of obsolete pesticides was used. OCPs concentra-
tions in the soil are summarized in Table 1. Extremely high
concentrations of OCPs sum equal to 2004 ± 43 lg
kg
1
exceeded the MAC up to 20 times were detected. In the
initial soil there were found 4.40-DDE (813 ± 17 lgkg
1
),
4.40-DDD (284 ± 6 lgkg
1
), and 4.40-DDT (907 ± 20 lg
kg
1
) metabolites. In Kazakhstan, the MAC for the pollu-
tants above in soil is 100 mgkg
1
.
Plant physiological parameters
The inoculation of plant seeds with these two bacterial
strains B. vallismortis 111 and B. aryabhattai 114 isolated
from the rhizosphere plants C. pepo and X. strumarium
stimulated the production of root biomass, and the produc-
tion of above ground biomass also increased, though to
lesser extent (Table 5).
The positive influence of strains on plant biomass grown
on the contaminated with pesticides relative to the control
Table 3. Growth of selected pure cultures on different concentrations of 4.4’-
DDE on solid medium.
Isolate ID
Concentration of the substrate 4.4’-DDE [mg L
1
]
1.0 2.0 3.0 5.0
115 5 5 5 2
12D 5 5 3 –
110 5 5 5 2
15D 5 5 2 –
114 5 5 5 4
11fflD5 5 ––
16D 5 5 2 –
20D 5 5 ––
111 5 5 5 3
21L 5 5 2 –
22L 5 5 2 –
23L 5 5 2 –
113 5 5 5 –
4A 5 5 ––
117 5 5 4 2
116 5 5 4 2
112 5 5 4 2
35 5 2 –
45 5 2 –
3TT 5 5 ––
3A5 532
Notes.5–severe turbidity of the medium; 4 –turbidity of the medium; 3 –
insignificant turbidity of the medium; 2 –slight turbidity of the medium; 1–
the medium was not turbid.
Control 2.0 mg L−1
3.0 mg L−1
5.0 mg L−1
11
4
111 113
111 113
111
111
113
114
114
114
113
Figure 1. Screening of the active isolates (ID numbers 111, 113 and 114) on increasing concentrations of 4.4’-DDE (only 111 and 114 isolates from 580 isolated
ones grew on solid medium with the addition of 5.0 mg L
1
of the 4.4’-DDE metabolite).
INTERNATIONAL JOURNAL OF PHYTOREMEDIATION 5

was revealed. These results suggest that, the studied in un-
inoculated and inoculated with microorganisms are able to
exhibit growth stimulating activity with respect to the plant.
However, a significant difference in biomass was found
between plants inoculated by B vallismortis 111 and B aryab-
hattai 114 strains. Noted that inoculation of seed C. pepo L.
by B aryabhattai 114 increased biomass compared with the
results obtained by inoculation by B. vallismortis 111 relative
to the control. On the contrary, increased the X. strumarium
biomass when the seeds were inoculated by strain B. vallis-
mortis 111 compared with the results obtained by inocula-
tion by B aryabhattai 114 relative to the control.
When determining the residual amount of DDT metabo-
lites in vegetative organs (aboveground and roots), it was
confirmed that the species X. strumarium and C. pepo have
a high accumulation capacity. The highest concentrations of
DDT metabolites are accumulated in the roots, which is typ-
ical for the phytoplants X. strumarium and C. pepo. The
species X. strumarium accumulated 1387 ± 100 lgkg
1
in
the roots without inoculation, 381 ± 39 lgkg
1
of DDT
metabolites in the aboveground biomass, and 1074 ± 92 lg
kg
1
and 412 ± 19 lgkg
1
in C. pepo, respectively (Table 6).
Inoculation of plant seeds with microorganisms increased
the ability of the resulting plants to assimilate DDT metabo-
lites from polluted soil. When X. strumarium was inoculated
by Bacillus vallismortis 111, the residual amount of DDT
metabolites in vegetative organs was 3568 lgkg
1
,by
Bacillus aryabhattai strain 114 2850 lgkg
1
. This residual
amount exceeded in comparison with the experiment with-
out inoculation (their residual amount in plant tissues grow-
ing on contaminated soil without inoculation) by almost 2
and 1.6 times, respectively. Similar results were obtained for
C. pepo. When calculating, the total absorption of DDT
metabolites without inoculation in the vegetative organs of
X. strumarium was 6.3 lg, 4.4 lg of which accumulated in
the aboveground biomass (leaves and stem). It has been
established that inoculation of seeds with microorganisms
enhances the process of absorption of pesticides from conta-
minated soil.
Upon inoculation of X. strumarium seeds by Bacillus val-
lismortis strain 111, the absorption of DDT metabolites
increased by 246% and was equal 15.5 lg; the amount of
DDT metabolites in the aboveground (7.9 lg) and roots
(7.5 lg) were practically at the same level, and during bacte-
rization with Bacillus aryabhattai 114–188% (absorption
index was 11.9 lg, including 6.8 lg in the aboveground and
5.1 lg in the roots). The Bacillus vallismortis strain 111
exhibited the greatest activity in this variant of the experi-
ment for enhancing the absorption of DDT metabolites
from contaminated soil. At the same time, this strain, upon
inoculation of the C. pepo model object, had an insignificant
effect on the absorption index of DDT metabolites from
contaminated soil: the absorption index increased by 135%
and amounted to 9.1 lg; in the aboveground 5.5 mg, and
in the roots 3.6 mg. At the same time, when this species
was inoculated with another strain of Bacillus aryabhattai
114, on the contrary, the absorption index increased to
170% and amounted to 11.4 lg (7.7 lg in the aboveground,
3.7 lg in the roots), which indicates the strains action speci-
ficity on plants.
We used the Bff to calculate the ability of C. pepo and
X. strumarium to absorb DDT metabolites. With these cal-
culations, the limited ability of accumulation of DDT metab-
olites in the tissue of C. pepo and X. strumarium was
confirmed. All BAvalues were lower than 1.
Introducing the B. vallismortis 111 bB aryabhattai 114
strains into contaminated soil-plant system enhances the
phytostabilization potential of plants that reflects in the
Table 4. Evaluation of the growth of B. vallismortis 111 and B. aryabhattai 114 strains in a liquid medium with 4.4’-DDE (the concentration is 480.4 mgL
1
).
Strains
Dry weight of biomass (g L
1
), p<0.05
Content of 4.40-DDE (mgL
1
), (p<0.05) Utilization of 4.40-DDE (%)
control (nutrient broth) with 4.40-DDE
days
0 21 21 0 14 21 14 21
B. vallismortis 111 0.9 ±0.03 6.5 ± 0.5 4.7 ± 0.4 480.4 ± 1.8 57.1 ± 0.3 23.5 ± 0.1 88.1 ± 0.9 95.1 ± 3.1
B. aryabhattai 114 0.6 ± 0.05 6.9 ± 0.5 4.6 ± 0.3 480.4 ± 1.8 51.5 ± 0.5 36.7 ± 0.2 89.3 ± 1.2 93.4 ± 2.9
Table 5. Effect of seed inoculation with effective bacteria on plant biomass (flowering period).
Mass of above ground Mass of root system
Experiment variants ± SE, g % relative to control ± SE, g % Relative to control
C. pepo
Control (non-contaminated soil) 12.2 ± 0.25 1.1 ± 0.06
Contaminated soil (control) 13.2 ± 0.23 108 1.2 ± 0.09 109
Contaminated soil þB. vallismortis 111 14.5 ± 0.2 118 1.8 ± 0.1 163
Contaminated soil þB aryabhattai 114 14.5 ± 0.18 118 1.9 ± 0.02 172
X. strumarium
Control (non-contaminated soil) 10.8 ± 0.59 1.2 ± 0.03
Contaminated soil (control) 11.5 ± 0,47 106 1.4 ± 0.02 116
Contaminated soil þB. vallismortis 111 13.8 ± 0.32 127 2.5 ± 0.05 250
Contaminated soil þB aryabhattai 114 13.1 ± 0.16 121 2.2 ± 0.03 220
Values are means ± SE. The probability of insignificant difference between control and experimental variants evaluated according to Student’s test:
0.001 <p<0.01; p<0.001; control and insignificantly different (p>0.05) experimental values are not marked. Every measurement was performed in
tree replicates (n¼3).
6 A. NURZHANOVA ET AL.

decreasing of TF values. It was shown that TF are lower
than 1, which indicates a limited ability to accumulate pesti-
cides in the aboveground organs of C. pepo and X. struma-
rium. Furthermore, it was noted that the seed inoculation of
plants with these bacterial strains decreased migration in the
system “soil –roots –aboveground.”For example, transloca-
tion factor of C. pepo decreased from 0.38 to 0.27, and that
of X. strumarium –from 0.27 to 0.19.
Discussion
Diversity of bacteria in the rhizosphere of plants grown
in the presence of DDT and its metabolites
The taxonomic composition of microorganisms capable of
transforming DDT is diverse, and includes representatives of
the genera Alcaligenes, Arthrobacter, Bacillus,
Corynebacterium, Enterobacter, Pseudomonas,
Stenotrophomonas, Streptomyces and mycelium fungi of the
genera Fusarium and Phlebia (Mitra et al. 2001; Xiao et al.
2011; Xie et al. 2011; Pan et al. 2016; Cyco
net al. 2017;
Wang et al. 2017,2018; Nasution and Bakti 2018). However,
there are fewer microorganisms are capable of using DDE
(Bumpus et al. 1993; Aislabie et al. 1997; Hay and
Focht 1998).
In our studies, the genera Pseudomonas and Bacillus rep-
resented the dominant group, Mycobacterium,Arthrobacter
and Streptomyces the medium abundance group, and
Micromonospora the minor group of the rhizosphere micro-
biota, regardless of whether DDT polluted or pristine soil
was used. In DDT polluted soils the genus Rhodococcus also
dominated in the rhizosphere whereas this genus is of lower
abundance in the rhizosphere of plants grown in pristine
soils. All analyzed genera differ in the total number of
occurrences in pristine and polluted soils. A plant is a com-
plex ecosystem, which accumulates distinct microbe popula-
tions into the rhizosphere. In our studies, the genera
Rhodococcus is enriched in the rhizosphere of polluted soils.
Bacterial strains from the genus Rhodococcus have been
shown previously to be able to degrade organochlorinated
and organophosphorus pesticides (Cyco
net al. 2017).
Rhodococci strains carry out hydroxylations, oxidations,
dehydrogenations, epoxidations, hydrolyzes, dehalogenations,
ring attacks, and cleavage of aromatic ring systems both by
ortho- and meta-path way (De Carvalho and Da
Fonseca 2005).
Despite the enrichment of Rhodococci in pesticide conta-
minated soil, Pseudomonas and Bacillus strains still domi-
nated the rhizosphere of plants grown both in polluted and
in pristine soils. Bacillus species, in particular, B. subtilis
have been used in mixed cultures for the degradation of pes-
ticides like DDT (Betancur-Corredor et al. 2015; Sariwati
et al. 2017). The actual degradation processes in these co-
cultures are probably due to the fungi present in the mixed
cultures, because various brown-rot fungi are known to
transform DDT to DDD, DBP and other products
(Purnomo et al. 2011). In these cases, the initial reaction is
a reductive dechlorination of DDT to DDD followed by oxi-
dative dechlorination.
Our two new isolates B. vallismortis 111 and B. aryabhat-
tai 114 were able to utilize more than 90% of 4.40-DDE in
liquid medium after 21 days. To the best of our knowledge
there is nothing described for the degradation or transform-
ation of DDT or 4.40-DDE by these two species of Bacillus.
However, B. subtilis is in a list of methoxychlor (1,1,1-tri-
chloro-2,2-bis(4-methoxyphenyl) ethane an organochloride
insecticide) transformer (Satsuma and Masuda 2012). The
methoxychlor is dehalogenated by all of the listed strains. B.
aryabhattai on the other hand is known to produce the
manganese peroxidase enzyme and is able to degrade color
and lignin from pulp and paper mill waste water (Zainith
et al. 2019). B. vallismortis has been reported to be able to
produce a temperature and pH stable spore laccase enzyme,
and is able to degrade dyes and PAHs like pyrene, phenan-
threne and fluorine (Tony et al. 2009; Ling et al. 2011;
Zhang et al. 2012,2013). The manganese peroxidase and the
laccase are enzymes, can both perform the one-electron
transfer reactions that are necessary for dechlorination reac-
tions. These enzymes may also be responsible for the trans-
formation of 4.40-DDE by our B. aryabhattai 114 and B.
Table 6. Pesticide concentrations, uptake index, Bff and TF without and with inoculation of isolated bacterial strains (flowering period).
Experiment variants Samples
Concentration of
metabolites DDT,
lgkg
1
Sum of metabolites
DDT in tissue of
plant, lgkg
1
Uptake index, lgBff TF
Polluted soil Soil 2004 ± 43
C.pepo Pumpkin
Polluted soil
without strains
Aboveground 412 ± 19 1486 5.4 0.20 ± 0.01 0.38± 0.02
Root 1074 ± 92 1.3
Polluted soil þB.
vallismortis 111
Aboveground 380 ± 16 2380 5.5 0.18 ± 0.02 0.19± 0.01
Root 2000 ± 57 3.6
Polluted soil þB
aryabhattai 114
Aboveground 533 ± 16 2485 7.7 0.26 ± 0.04 0.27± 0.03
Root 1952 ± 77 3.7
X.strumarium
Polluted soil
without strains
Aboveground 381 ± 39 1768 4.4 0.19 ± 0.02 0.27± 0.02
Root 1387 ± 100 1.9
Polluted soil þB.
vallismortis 111
Aboveground 573 ± 29 3568 7.9 0.28 ± 0.05 0.19± 0.02
Root 2995 ± 91 7.5
Polluted soil þB
aryabhattai 114
Aboveground 520 ± 44 2850 6.8 0.26 ± 0.04 0.22± 0.01
Root 2330 ± 123 5.1
Values are means ± SE. The probability of insignificant difference between control and experimental variants evaluated according to Student’s test:
0.01 <p<0.05; 0.001 <p<0.01; insignificantly different (p>0.05) experimental values are not marked. Every measurement was performed in tree repli-
cates (n¼3).
INTERNATIONAL JOURNAL OF PHYTOREMEDIATION 7

vallismortis 111. Furthermore, strains of B. aryabhattai are
also able to degrade organophosphate pesticides like chlor-
pyrifos (Pailan et al. 2015).
Improving phytoremediation of soil contaminated with
OCPs by using plant-microbe symbiotic systems
The inoculation of plants with active microbial destructors
has been previously described as an effective way of increas-
ing tolerance of plants to contaminants. At the same time, it
enhances the effectiveness and speeds up the process of
cleaning contaminated soils (Aislabie et al. 1997; Chaudhry
et al. 2005).
In our studies, in a greenhouse, two species of plants, C.
pepo and X. strumarium, were germinated with/without
inoculation with rhizobacteria B. vallismortis strain 111 and
B. aryabhattai strain 114 on pesticide-contaminated soil,
delivered from the territory of former pesticide storages
(Almaty region, Talgar district, the village of Kyzyl-Kairat).
We studied the effect of seed inoculation with rhizobacteria
on plant biomass, accumulation and absorption of DDT
metabolites in vegetative plant organs (aboveground biomass
and root) from contaminated soil in relation to plants grow-
ing on contaminated soil without inoculation. It was found
that inoculation of seeds with rhizobacteria increases the
plant biomass, mainly the root system, thereby increasing
the adsorbent surface of the root system. It was noted that
the effect of rhizobacteria on plant biomass is species-spe-
cific. The best indices of morphological parameters in the
species C. pepo were revealed upon inoculation of plant
seeds with strain B aryabhattai 114, and for X. strumarium
with strain B. vallismortis 111. During bacterization of seeds
of X. strumarium with B. vallismortis strain 111, the mass of
the root system increased practically by 1.8 times (from
1.4 ± 0.02 to 2.5 ± 0.05 g), and in the combination of C. pepo
with the B aryabhattai strain 114 1.6 times (from
1.2 ± 0.09 to 1.9 ± 0.02 g), respectively. In particular, certain
plant growth promoting bacteria associated with plant roots
may have some beneficial effects on plant growth and nutri-
tion. Growth-stimulating rhizosphere microorganisms are
used in agriculture as “biofertilizers,”and the effect of
increased biodegradation of pollutants in the root zone has
led to the use of plants and related microorganisms to clean
up the environment (Glick 1995; Hutchinson et al. 2001;
Karthikeyan et al. 2004;Wuet al. 2006).
In this work, we show that bacteria B. aryabhattai 114
and B. vallismortis 111 isolated from the rhizosphere of X.
strumarium and C. pepo can make a significant contribution
to the degradation of DDT metabolites. Pesticides from con-
taminated soil accumulated in the tissues of C. pepo and X.
strumarium even without B. vallismortis 111 and B aryab-
hattai 114, but inoculation of plant seeds with these strains
significantly increased the absorption of DDT metabolites
from contaminated soil: the residual amount of pesticides in
the vegetative organs of C. pepo when inoculated with rhizo-
bacteria relatively without inoculation from 1486 to 2485 lg
kg
1
,X. strumarium –from 1768 to 3568 lgkg
1
,
respectively.
Bioaccumulation coefficient (BAC) and translocation
coefficient (TF) are widely used parameters to characterize
the phytoremediation process (Zaier et al. 2010). The trans-
location factor describes the ability of plants to translocate
pollutants from roots to aboveground tissues (Zu et al.
2005), and a high translocation factor is always favorable for
phytoremediation. For a better understanding of the process,
we determined the metabolites of DDT for the aboveground
biomass and the roots separately. The calculated BAC and
TF data are presented in Table 6. When the plant was
grown in combination with rhizobacteria, all BAC and TF
values were below 1, indicating a limited ability to accumu-
late pesticides in aboveground biomass. The obtained BAC
and TF values are always <1, which indicates that the plants
accumulated DDT metabolites mainly in the root system.
These results confirm that X. strumarium and C. pepo,
together with B. vallismortis 111 and B aryabhattai 114,
enhance the ability to phytostabilize DDT metabolites in
soil. However, pollutants have also been found, albeit at
lower concentrations, in the aboveground part of the plants.
In our study, seed inoculation by B. vallismortis 111 and B.
aryabhattai 114 reduced migration from soil –root –above-
ground, which emphasized the main accumulation of pesti-
cides in the root system: in the combination of X.
strumarium with the B. vallismortis strain 111 TF decreased
from 0.27 ± 0.02 to 0.19 ± 0.02 (0.001 <p<0.01); B aryab-
hattai strain 114 –up to 0.22 ± 0.01 (0.01 <p<0.05).
A similar pattern was characteristic for the inoculation of
C. pepo seeds with rhizobacteria in relation to the experi-
ment without inoculation. We assume that the use of rhizo-
bacteria in combination with plants can provide a high
efficiency of the technology of phytostabilization of DDT
metabolites in soil. The plants may provide optimal condi-
tions for the development of rhizobacteria, offering nutrients
and residence, enabling them to feed on contaminants in
the rhizosphere (Kuiper et al. 2004). In this scenario, the
joint use of plants and appropriate bacteria strengthens the
role of each partner. The bacteria may help the host to over-
come the stresses caused by the presence of the pollutant,
while the plant may provide nutrients that increase the
microbial population and hence increase degradation of the
organic pollutants (Weyens et al. 2009).
Plants and microorganisms have significant potential for
catabolic activity toward DDT and its metabolites. The com-
bined metabolic potential of microorganisms and plants may
be exploited as a safe and economic means to restore the
fertility of soil contaminated with DDT and its metabolites.
Phytoremediation technologies may be particularly effective
in the rehabilitation of soils over large areas, in which the
pollutants can be gradually removed from the soil.
Conclusion
Phytoremediation of soils contaminated with DDT metabo-
lites using plants and microorganisms may provide a solu-
tion to an urgent environmental problem. The isolation and
identification of microorganisms associated with the rhizo-
sphere of plants growing in polluted soil, their introduction
8 A. NURZHANOVA ET AL.

into natural ecosystems may be an important step in the
development of the phytoremediation technology of soils
contaminated with persistent organic pesticides.
Using this approach, we isolated B. aryabhattai and B.
vallismortis from the rhizosphere of X. strumarium and C.
pepo and showed that they are able to utilize 4.40-DDE.
When investigating the potential of isolated strains of B.
aryabhattai and B. vallismortis in partnership with X. stru-
marium and C. pepo, it was evaluated for the recovery of
soils contaminated with DDT metabolites. The seeds of ino-
culated and non-inoculated plants were grown on pesticide-
contaminated soil from the former obsolete pesticide storage
sites. The results showed that bacterial inoculation improves
plant growth and the ability to phytoremediation: this is
manifested in an increase in the biomass of the plant root
system by 63–150% and in the aboveground biomass by
18–37% when growing on soil contaminated with DDT
metabolites, compared with plants without bacterial inocula-
tion. The species X. strumarium and C. pepo demonstrated
the ability to accumulate DDT metabolites mainly in the
root system and in a small amount in the aboveground part,
nevertheless after the bacterization, the residual amount of
metabolites in the root system increases significantly: after
bacterization of seeds of X. strumarium by B. vallismortis
strain 111, residual the amount of DDT metabolites in the
roots increased almost 2 times, and after bacterization of
seeds of C. pepo by B aryabhattai strain 114 –by 1.7 times
compared with the experiment without inoculation. These
results show that combinations of X. strumarium and C.
pepo and rhizobacteria can be effectively used to phytostabi-
lize soils contaminated with DDT metabolites and confirm
that plant-microbial interactions in the rhizosphere can play
an important role in the removal of resistant chemicals in
the environment.
Further research should be directed to the study of the
mechanism of interaction of the system “soil-plant-
rhizobacteria.”Knowledge of the metabolic activity of rhizo-
bacteria and their diversity in the rhizosphere of tolerant
plants may lead to new strategies for the phytoremediation
of soil contaminated with OCPs.
Funding
The work was carried out as part of a scientific and technical program
of the Ministry of Education and Science of RK “Develop methods of
phytoremediation of soils contaminated with pesticides based on the
design of microbial and plant associations”. We thank R. Jack
(Institute of Immunology, University of Greifswald) for help in prepar-
ing the manuscript.
References
Abhilash PC, Dubey RK, Tripathi V, Srivastava P, Verma JP, Singh
HB. 2013. Remediation and management of POPs-contaminated
soils in a warming climate: challenges and perspectives. Environ Sci
Pollut Res Int. 20(8):5879–5885. doi:10.1007/s11356-013-1808-5.
Aislabie JM, Richards NK, Boul HL. 1997. Microbial degradation of
DDT and its residues—a review. N Z J Agric Res. 40(2):269–282.
doi:10.1080/00288233.1997.9513247.
Anderson TA, Coats JR. 1995. Screening rh1zosphere soil samples for
the ability to mineralize elevated concentrations of atraz1ne and
metolachlor. J Environ Sci Health B. 30(4):473–484. doi:10.1080/
03601239509372948.
Arslan M, Imran A, Khan QM, Afzal M. 2017. Plant-bacteria partner-
ships for the remediation of persistent organic pollutants. Environ
Sci Pollut Res Int. 24(5):4322–4336. doi:10.1007/s11356-015-4935-3.
Badri DV, Weir TL, van der Lelie D, Vivanco JM. 2009. Rhizosphere
chemical dialogues: plant-microbe interactions. Curr Opin
Biotechnol. 20(6):642–650. doi:10.1016/j.copbio.2009.09.014.
Betancur-Corredor B, Pino NJ, Cardona S, Pe~
nuela GA. 2015.
Evaluation of biostimulation and Tween 80 addition for the bio-
remediation of long-term DDT-contaminated soil. J Environ Sci
(China). 28:101–109. doi:10.1016/j.jes.2014.06.044.
Bumpus JA, Powers RH, Sun T. 1993. Biodegradation of DDE (1,1-
dichloro-2,2-bis(4-chlorophenyl)ethene) by Phanerochaete chrysospo-
rium. Mycol Res. 97(1):95–98. doi:10.1016/S0953-7562(09)81144-1.
Campanella B, Paul R. 2000. Presence, in the rhizosphere and leaf
extracts of zucchini (Cucurbita pepo L.) and Melon (Cucumis melo
L.), of Molecules, capable of increasing the apparent aqueous solu-
bility of hydrophobic pollutants. Int J Phytorem. 2(2):145–158. doi:
10.1080/15226510008500036.
Cavaglieri L, Orlando J, Etcheverry M. 2009. Rhizosphere microbial
community structure at different maize plant growth stages and root
locations. Microbiol Res. 164(4):391–399. doi:10.1016/j.micres.2007.
03.006.
Chaudhry Q, Blom-Zandstra M, Gupta S, Joner EJ. 2005. Utilising the
synergy between plants and rhizosphere microorganisms to enhance
breakdown of organic pollutants in the environment. Environ Sci
Pollut Res Int. 12(1):34–48. doi:10.1065/espr2004.08.213.
Clayton RA, Sutton G, Hinkle PS, Jr, Bult C, Fields C. 1995.
Intraspecific variation in small-subunit rRNA sequences in
GenBank: why single sequences may not adequately represent pro-
karyotic taxa. Int J Syst Bacteriol. 45(3):595–599. doi:10.1099/
00207713-45-3-595.
Cyco
n M, Mrozik A, Piotrowska-Seget Z. 2017. Bioaugmentation as a
strategy for the remediation of pesticide-polluted soil: a review.
Chemosphere. 172:52–71. doi:10.1016/j.chemosphere.2016.12.129.
De Carvalho CCCR, Da Fonseca MMR. 2005. The remarkable
Rhodococcus erythropolis. Appl Microbiol Biotechnol. 67(6):715–726.
doi:10.1007/s00253-005-1932-3.
FAO 2001. Baseline study on the problem of obsolete pesticides stocks. No.
9. Rome: Viale delle Terme di Caracalla; [accessed 2016 Nov 21].
http://www.fao.org/docrep/003/x8639e/x8639e00.htm#TopOfPage.
Glick BR. 1995. The enhancement of plant growth by free-living bac-
teria. Can J Microbiol. 41(2):109–117. doi:10.1139/m95-015.
Hay AG, Focht DD. 1998. Cometabolism of 1,1-dichloro-2,2-bis(4-
chlorophenyl)ethylene by Pseudomonas acidovorans M3GY grown
on biphenyl. Appl Environ Microbiol. 64(6):2141–2146. doi:10.1128/
AEM.64.6.2141-2146.1998.
Holt JG. 1977. The shorter Bergey’s manual of determinative bacteri-
ology. 8th ed. Baltimore (MD): The Williams and Wilkins Co.
Hutchinson SL, Schwab AP, Banks MK. 2001. Phytoremediation of
aged petroleum sludge: effect of irrigation techniques and schedul-
ing. J Environ Qual. 30(5):1516–1522. doi:10.2134/jeq2001.3051516x.
Karthikeyan R, Davis LC, Erickson LE, Al-Khatib K, Kulakow PA,
Barnes PL, Hutchinson SL, Nurzhanova AA. 2004. Potential for
plant-based remediation of pesticide-contaminated soil and water
using nontarget plants such as trees, shrubs, and grasses. Crit Rev
Plant Sci. 23(1):91–101. doi:10.1080/07352680490273518.
Kennen K, Kirkwood N. 2015. Phyto: principles and resources for site
remediation and landscape design. London: Routledge.
Kuiper I, Lagendijk EL, Bloemberg GV, Lugtenberg BJ. 2004.
Rhizoremediation: a beneficial plant-microbe interaction. Mol Plant
Microbe Interact. 17(1):6–15. doi:10.1094/MPMI.2004.17.1.6.
Laird BD, Goncharov AB, Chan HM. 2013. Body burden of metals and
persistent organic pollutants among Inuit in the Canadian Arctic.
Environ Int. 59:33–40. doi:10.1016/j.envint.2013.05.010.
Letcher RJ, Bustnes JO, Dietz R, Jenssen BM, Jørgensen EH, Sonne C,
Verreault J, Vijayan MM, Gabrielsen GW. 2010. Exposure and
INTERNATIONAL JOURNAL OF PHYTOREMEDIATION 9

effects assessment of persistent organohalogen contaminants in arc-
tic wildlife and fish. Sci Total Environ. 408(15):2995–3043. doi:10.
1016/j.scitotenv.2009.10.038.
Ling J, Zhang G, Sun H, Fan Y, Ju J, Zhang C. 2011. Isolation and
characterization of a novel pyrene-degrading Bacillus vallismortis
strain JY3A. Sci Total Environ. 409(10):1994–2000. doi:10.1016/j.sci-
totenv.2011.02.020.
Mikolasch A, Reinhard A, Alimbetova A, Omirbekova A, Pasler L,
Schumann P, Kabisch J, Mukasheva T, Schauer F. 2016. From oil
spills to barley growth - oil-degrading soil bacteria and their pro-
moting effects. J Basic Microbiol. 56(11):1252–1273. doi:10.1002/
jobm.201600300.
Mitra J, Mukherjee PK, Kale SP, Murthy NBK. 2001. Bioremediation of
DDT in soil by genetically improved strains of soil fungus Fusarium
solani. Biodegradation. 12(4):235–245. 10.1023/a:1013117406216.
Mitra J, Raghu K. 1989. Effects of DDT on the growth of crop plants.
Environ Pollut. 61(2):157–170. doi:10.1016/0269-7491(89)90033-X.
Mynbayeva BN, Imanbekova TG. 2013. Assessment of standards of
soil’s contamination by heavy metals (analytical review). Bull Natl
Acad Sci Republic Kazakhstan. 4:29–39.
Nasution L, Bakti D. 2018. Identification of fungi originated from soil
polluted by Dichloro Diphenyl Trichloroethane (DDT). E&ES.
205(1):12021.
Nurzhanova A, Kalugin S, Zhambakin K. 2013. Obsolete pesticides and
application of colonizing plant species for remediation of contami-
nated soil in Kazakhstan. Environ Sci Pollut Res Int. 20(4):
2054–2063. doi:10.1007/s11356-012-1111-x.
Nwoko CO. 2010. Trends in phytoremediation of toxic elemental and
organic pollutants. Afr J Biotechnol. 9(37):6010–6016.
Pailan S, Gupta D, Apte S, Krishnamurthi S, Saha P. 2015. Degradation
of organophosphate insecticide by a novel Bacillus aryabhattai strain
SanPS1, isolated from soil of agricultural field in Burdwan, West
Bengal, India. Int Biodeterior Biodegradation. 103:191–195. doi:10.
1016/j.ibiod.2015.05.006.
Pan X, Lin D, Zheng Y, Zhang Q, Yin Y, Cai L, Fang H, Yu Y. 2016.
Biodegradation of DDT by Stenotrophomonas sp. DDT-1: character-
ization and genome functional analysis. Sci Rep. 6(1):21332. doi:10.
1038/srep21332.
Pascal-Lorber S, Laurent F. 2011. Phytoremediation techniques for
pesticide contaminations. In: Alternative farming systems, biotech-
nology, drought stress and ecological fertilisation. Netherlands:
Springer. p. 77–105. doi:10.1007/978-94-007-0186-1_4.
Pilon-Smits E. 2005. Phytoremediation. Annu Rev Plant Biol. 56(1):
15–39. doi:10.1146/annurev.arplant.56.032604.144214.
Purnomo AS, Mori T, Takagi K, Kondo R. 2011. Bioremediation of
DDT contaminated soil using brown-rot fungi. Int Biodeterior
Biodegradation. 65(5):691–695. doi:10.1016/j.ibiod.2011.04.004.
Sariwati A, Purnomo AS, Kamei I. 2017. Abilities of co-cultures of
brown-rot fungus Fomitopsis pinicola and Bacillus subtilis on biodeg-
radation of DDT. Curr Microbiol. 74(9):1068–1075. doi:10.1007/
s00284-017-1286-y.
Satsuma K, Masuda M. 2012. Reductive dechlorination of methoxy-
chlor by bacterial species of environmental origin: evidence for pri-
mary biodegradation of methoxychlor in submerged environments.
J Agric Food Chem. 60(8):2018–2023. doi:10.1021/jf2048614.
Somasegaran P, Hoben HJ. 1994. Handbook for rhizobia: methods in
legume-rhizobium technology. New York: Springer Science &
Business Media.
Starkey RL. 1958. Interrelations between microorganisms and plant
roots in the rhizosphere. Bacteriol Rev. 22(3):154–172. doi:10.1128/
MMBR.22.3.154-172.1958.
Tanimoto E. 2005. Regulation of root growth by plant hormones -
roles for auxin and gibberellin. Crit Rev Plant Sci. 24(4):249–265.
doi:10.1080/07352680500196108.
Tony BD, Goyal D, Khanna S. 2009. Decolorization of textile azo dyes
by aerobic bacterial consortium. Int Biodeterior Biodegradation.
63(4):462–469. doi:10.1016/j.ibiod.2009.01.003.
UNEP 2005. Riding the World of POPs: A guide on the Stockholm
Convention on Persistent Organic Pollutants. [accessed 2016 Sep
20]:[17 p.]. Available at: http://www.pops.int/documents/guidance/
beg_guide.pdf.
Wang BB, Liu WX, Liu XY, Franks AE, Teng Y, Luo YM. 2017.
Comparative analysis of microbial communities during enrichment
and isolation of DDT-degrading bacteria by culture-dependent and
-independent methods. Sci Total Environ. 590-591:297–303. doi:10.
1016/j.scitotenv.2017.03.004.
Wang X, Sun L, Wang H, Wu H, Chen S, Zheng X. 2018. Surfactant-
enhanced bioremediation of DDTs and PAHs in contaminated
farmland soil. Environ Technol. 39(13):1733–1744. doi:10.1080/
09593330.2017.1337235.
Weyens N, van der Lelie D, Taghavi S, Newman L, Vangronsveld J.
2009. Exploiting plant-microbe partnerships to improve biomass
production and remediation. Trends Biotechnol. 27(10):591–598.
doi:10.1016/j.tibtech.2009.07.006.
White JC, Kottler BD. 2002. Citrate-mediated increase in the uptake of
weathered 2,2-bis (p-chlorophenyl)1,1-dichloroethylene residues by
plants. Environ Toxicol Chem. 21(3):550–556. doi:10.1002/etc.
5620210312.
White JC, Mattina MJI, Lee WY, Eitzer BD, Iannucci-Berger W. 2003.
Role of organic acids in enhancing the desorption and uptake of
weathered p,p0-DDE by Cucurbita pepo. Environ Pollut. 124(1):
71–80. doi:10.1016/S0269-7491(02)00409-8.
Wu CH, Wood TK, Mulchandani A, Chen W. 2006. Engineering
plant-microbe symbiosis for rhizoremediation of heavy metals. Appl
Environ Microbiol. 72(2):1129–1134. doi:10.1128/AEM.72.2.1129-
1134.2006.
Xiao P, Mori T, Kamei I, Kondo R. 2011. A novel metabolic pathway
for biodegradation of DDT by the white rot fungi, Phlebia lindtneri
and Phlebia brevispora. Biodegradation. 22(5):859–867. doi:10.1007/
s10532-010-9443-z.
Xie H, Zhu L, Xu Q, Wang J, Liu W, Jiang J, Meng Y. 2011. Isolation
and degradation ability of the DDT-degrading bacterial strain KK.
Environ Earth Sci. 62(1):93–99. doi:10.1007/s12665-010-0500-z.
Zaier H, Ghnaya T, Lakhdar A, Baioui R, Ghabriche R, Mnasri M,
Sghair S, Lutts S, Abdelly C. 2010. Comparative study of Pb-phy-
toextraction potential in Sesuvium portulacastrum and Brassica jun-
cea: tolerance and accumulation. J Hazard Mater. 183(1–3):609–615.
doi:10.1016/j.jhazmat.2010.07.068.
Zainith S, Purchase D, Saratale GD, Ferreira LFR, Bilal M, Bharagava
RN. 2019. Isolation and characterization of lignin-degrading bacter-
ium Bacillus aryabhattai from pulp and paper mill wastewater and
evaluation of its lignin-degrading potential. 3 Biotech. 9(3):1–11.
doi:10.1007/s13205-019-1631-x.
Zeeb BA, Amphlett JS, Rutter A, Reimer K. 2006. Potential for phytor-
emediation of polychlorinated biphenyl-(PCB)- contaminated soil.
Int J Phytorem. 8(3):199–221. doi:10.1080/15226510600846749.
Zhang C, Diao H, Lu F, Bie X, Wang Y, Lu Z. 2012. Degradation of
triphenylmethane dyes using a temperature and pH stable spore lac-
case from a novel strain of Bacillus vallismortis. Bioresour Technol.
126:80–86. doi:10.1016/j.biortech.2012.09.055.
Zhang C, Zhang S, Diao H, Zhao H, Zhu X, Lu F, Lu Z. 2013.
Purification and characterization of a temperature- and pH-stable
laccase from the spores of Bacillus vallismortis fmb-103 and its
application in the degradation of malachite green. J Agric Food
Chem. 61(23):5468–5473. doi:10.1021/jf4010498.
Zu YQ, Li Y, Chen JJ, Chen HY, Qin L, Schvartz C. 2005.
Hyperaccumulation of Pb, Zn and Cd in herbaceous grown on lead-
zinc mining area in Yunnan, China. Environ Int. 31(5):755–762. doi:
10.1016/j.envint.2005.02.004.
10 A. NURZHANOVA ET AL.