South African Journal of Botany 153 (2023) 1
10

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South African Journal of Botany

journal homepage: www.elsevier.com/locate/sajb

Remediation capacity of drought-tolerant plants and bacteria in

petroleum hydrocarbon-contaminated soil in Iran
Ronak ShirzadianGilana, Yahya Parvizib,*, Ebrahim Paziraa, Farhad Rejalic
a
Department of Soil Science, Science and Research Branch, Islamic Azad University, Tehran, Iran
b
Soil and Water Conservation Department, Soil and Water Conservation and Watershed Management Research Institute, AREEO, Tehran, Iran
c
Soil and Water Research Institute, Agriculture, Research, Education and Extension Organization, Karaj, Iran

A R T I C L E I N F O A B S T R A C T

Article History: Ex-Situ bioremediation techniques such as synthetic surfactants and bioreactors to detoxify petroleum con-
Received 16 April 2022 taminated soil are costly and have harmful environmental consequences. This study was conducted to evalu-
Revised 13 December 2022 ate the intrinsic bioremediation potential of native, compatible and drought-tolerant plants and bacteria for
Accepted 13 December 2022
removing total petroleum hydrocarbons (TPHs) from the soil. Furthermore, the potential of fertilizer for
Available online 16 December 2022
increasing the bioremediation capacity of these organisms was assessed. The study was performed as a
Edited by Dr. S.J. Siebert completely randomized factorial design experiment with four replications. The plant treatments were Opun-
tia ficus-indica (P1), Eucalyptus camaldulensis, (P2) and Nerium oleander (P3). The bacteria treatments were
Keywords:
Bacillus pumilus (B1), Pseudomonas putida (B2), and the combined use of these two bacteria and a fertilizer
Bioremediation
recommendation (B1B2F). The control treatment included only contaminated soil without plant, bacteria,
Soil pollution
and fertilizer. The results indicated that plant and bacteria treatments enhanced the decomposition of soil
Fertilizer
Plant-bacteria interaction TPHs significantly (a=0.01). P2B1B2F was found to be the most efficient treatment, removing 77.30% of soil
Eucalyptus camaldulensis TPHs. Eucalyptus camaldulensis cultivation along with P. putida (P2B2) reduced TPHs by 69.60%. P. putida
TPHs was more efficient than B. pumilus in reducing TPHs. Interactions between plants and bacteria increased root
and total dry weight of plants significantly (a=0.01). Application of bacteria and the fertilizer recommenda-
tion increased the decomposition rate of TPHs and improved phytoremediation efficiency. Rehabilitation by
Eucalyptus camaldulensis with bio-fertilization of soil using P. putida and chemical fertilizers is recom-
mended for the bioremediation of soil contaminated with TPHs.
© 2022 SAAB. Published by Elsevier B.V. All rights reserved.

1. Introduction Ex-situ soil remediation techniques involve expensive physico-

The leakage of pollutants from petrochemical industries, oil refin-
eries, oil and gas extraction wells, underground oil reservoirs and
refueling stations, as well as oil tanker accidents has caused soil pol-
lution in some areas of Iran (Hajabbasi, 2016). These pollutants pose
significant risks to human health and the environment (Euliss et al.,
2008). Crude oils are complex mixtures containing aliphatic and aro-
matic hydrocarbons predominantly and low concentrations of
asphaltenes, resins, and heavy metals. Many petroleum compounds
are considered to be toxic, mutagenic or carcinogenic (Abdel-Shafy
and Mansour, 2016). Some of these compounds degrade the chemi-
cal, physical, and biological qualities of soil (Dai et al., 2011; Hajab-
basi, 2016; Kosesakal et al., 2016). The findings of Anikwe et al.
(2017) showed the incubation of soils with petroleum for 90 days
increased soil bulk density (1.49 g/cm3) and lowered soil hydraulic
conductivity (8.22 mm/h) compared to the uncontaminated soils.
* Corresponding author.
E-mail address: yparvizi1360@gmail.com (Y. Parvizi).
chemical methods which may generate secondary pollution (Yoon
et al., 2006). In contrast, environment-friendly alternatives including
bioremediation by native plants and microorganisms have been pro-
posed to remediate petroleum-contaminated soils (Dadea et al.,
2017). Bioremediation is defined as the application of the metabolic
capacity of living organisms, including plants (phytoremediation)
and microorganisms, to detoxify harmful pollutants and convert
them to safe metabolites (Pawar et al., 2013). Phytoremediation can
be a promising method for the clean-up of petroleum hydrocarbons
from contaminated soil, with many advantages over other relevant
methods (Abdullah et al., 2020). The applications, advantages, limita-
tions, mechanisms, and new developments of phytoremediation
have been discussed thus far (Daryabeigi Zand and Hoveidi, 2016).
Xiao et al. (2015) found that plants such as Fire phoenix and Medicago
sativa reduced the polycyclic hydrocarbons in contaminated soil and
improved soil physico-chemical properties. Many researchers have
referred to the effectiveness of plants in the phytoremediation of oil-
polluted soils (Atagana, 2011; Zhang et al., 2010; Rajaei and Seyedi,
2018).

https://doi.org/10.1016/j.sajb.2022.12.014
0254-6299/© 2022 SAAB. Published by Elsevier B.V. All rights reserved.
R. ShirzadianGilan, Y. Parvizi, E. Pazira et al.
Oil-contaminated environments in the west and southwest of Iran
near the Iraq border are characterized by extreme conditions, includ-
ing high temperatures, arid conditions, alkaline pH, and high salt con-
centrations. Hydrocarbon-degrading endemic microorganisms and
plants, adapted to grow in these extreme environments, play a key
role in the biological treatment of pollutants (Alvim and Pontes,
2018; Guzman et al., 2017). Different studies indicated the biodegra-
dation of a wide range of hydrocarbons, including aliphatic, aromatic,
and halogenated compounds, in various environments. Due to its
deep root system, compatibility with dry and warm conditions, and
fast growth, Eucalyptus camaldulensis tree is highly capable of reme-
diating petroleum-contaminated soils (Madejon et al., 2017). Ayuba
et al. (2020) studied the efficacy of Eucalyptus camaldulensis and
Chrysopogon zizanioides for their ability to decompose oil pollutants
from soils. Their analysis revealed that 40 mg of TPHs per 1 gram of
Eucalyptus camaldulensis root was accumulated after 12 weeks. Esco-
bar et al. (2017) showed the phytoremediation potential of Opuntia
ficus-indica, another drought-tolerant plant, which removed approxi-
mately 48% of soil TPHs in 20 weeks.
Petroleum hydrocarbons are biodegraded by rhizospheric micro-
organisms such as bacteria and fungi, as a sole source of carbon and
energy for their growth, and are converted into carbon dioxide and
biomass (McCutcheon and Schonnor, 2003). Viesser et al. (2020) eval-
uated the efficiency of three native bacteria, including Bacillus thurin-
giensis, Bacillus pumilus, and Rhodococcus hoagii isolated from
rhizosphere and incubated in petroleum-contaminated soil in labora-
tory assays. They found that the efficiency of these bacteria to
degrade TPHs after incubation was up to 90.77, 75.56, and 96.15%,
respectively. The efficiency of petroleum degradation by the genus
Bacillus is largely due to the ability of these microorganisms to pro-
duce biosurfactants (Bento et al., 2005)
The synergistic cooperation of plant roots and soil bacteria in rhi-
zosphere promotes the degradation of persistent TPHs in the phytor-
emediation process (Czarny et al., 2017). In the presence of carbon
sources provided by root secretions, bacteria produce enzymes that
are accountable for breaking hydrocarbon molecules (Saleem, 2016).
The efficiency of the phytoremediation process depends largely on
the presence and activity of the plant-dependent microbial commu-
nity. These organisms carry degradative genes required for the enzy-
matic decomposition of organic pollutants, and it has been reported
that the rhizosphere is the host of bacteria destroying TPHs (Andria
et al., 2009).
The possibility of obtaining synergistic effects between plants and
bacteria has increased the possibility of alternative techniques for
bioremediation (Guarino et al., 2020). Gouda et al. (2016) reported
that plant
bacteria interactions increased bioremediation efficiency
up to 99% in removing petroleum hydrocarbons. Based on the find-
ings reported by Afegbua and Batty (2019), plant growth-promoting
bacteria (PGPB), including Pseudomonas putida, significantly
enhanced the dissipation of polycyclic aromatic hydrocarbons (PAHs)
pollution in soil in M. sativa plant treatment after 65 days. Also, Sur-
yatmana et al. (2019) indicated that the Ramie plant and N-fixing
bacteria inoculants had removed up to 83.22% of petroleum hydro-
carbons from soil. Yang et al. (2020) emphasized that plant growth
and biomass directly affect the phytoremediation potential and that
bacteria can increase phytoremediation efficiency by promoting
plant growth. Therefore, native plants and bacteria adapted to
Table 1
physico-chemical properties and initial TPHs contamination of soil samples.
TPHs TNV EC £ 103 pH in P K
(%) (%) Ds/m saturation extract (ppm) (ppm)
0.53 24.00 1.39 7.60 3.80 200.00
South African Journal of Botany 153 (2023) 1
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environmental conditions have been used globally to remove petro-
leum hydrocarbons from soil.
A few studies have been conducted to investigate the capacities of
native and naturalized plants and bacteria, particularly in hot and dry
environments, to remediate petroleum pollutants (Gilan et al., 2022).
The present study aimed to investigate the synergistic remediation
potential of three drought-tolerant plants (Eucalyptus camaldulensis,
Nerium oleander, and Opuntia ficus-indica) with two environmen-
tally compatible and native bacterial species (Bacillus pumilus and
Pseudomonas putida) to remove TPHs from contaminated soil of one
of the oil fields in arid areas in the west of Iran. Moreover, to enhance
the phytoremediation efficiency and purification capacity of pollu-
tants, the appropriate fertilizer recommendation was considered. No
such significant research has been done in this region and similar
arid areas in Iran so far. Selected plant species that are compatible
with the experiment area show good vegetative growth in arid condi-
tions of the study region. Moreover, in previous studies, these species
showed high phytoremediation potential. Eucalyptus camaldulensis
is a single- or multi-stem tree or shrub. Nerium oleander is an ever-
green shrub of the Gentianales. Opuntia ficus-indica, a Mexican
native species, is one of the most commercially important cactus spe-
cies. These plants have adapted well to arid and semi-arid areas of
Iran and have been a domesticated plant grown in arid and semi-arid
parts of Iran for a long time.
The two bacterial species that had compatibility with hot and arid
conditions of the oil fields in the study area were selected. These bac-
teria were not pathogenic and showed high efficiency in the biore-
mediation of petroleum-contaminated soil) Chouychai et al., 2012;
Alves et al., 2018(. Furthermore, they had high potential for the pro-
duction of metabolites. Bacillus pumilus is a Gram-positive, aerobic,
and spore-forming bacterium commonly found in soil. B. pumilus
spores generally show high resistance to environmental stresses,
including exposure to UV radiation and desiccation. Pseudomonas
putida is a Gram-negative, rod-shaped, saprotrophic, and native soil
bacterium. Based on the 16S rRNA analysis, P. putida was taxonomi-
cally confirmed to be a Pseudomonas putida species (sensu stricto)
and was placed, along with several other species, in the P. putida
group to which it lends its name.
2. Materials and methods
2.1. Soil parameters
The pot experiment soil was collected from the petroleum exploi-
tation unit in an oil field in an arid region of the west of Iran (Naft-
shahr in Kermanshah Province) close to theIraq border. This study
was carried out in 2019. Based on the book Keys to Soil Taxonomy
(Twelfth Edition), the generic subgroup of the soil in this area is Gyp-
sic Haplustepts (Soil survey staff, 2014). The land use type is desert
rangeland. Before applying the treatments, the soil physical and
chemical properties were measured (Table 1). Soil texture and equiv-
alent calcium carbonate were measured using Bouyoucos hydrome-
ter method (Gee and Bauder, 1990) and Richards method (Richards,
1954), respectively. Soil organic carbon was determined using
Walkley and Black method (Nelson et al., 1996). Soil reaction (pH) in
saturated soil dough (soil to water ratio is 1:2.5) (McLean, 1982) was
measured. Electrical conductivity (EC) was determined using an
OC Mn Fe Zn Cu Sand Silt Clay
(%) (ppm) (ppm) (ppm) (ppm) (%) (%) (%)
0.51 4.80 3.76 1.16 1.10 48.00 33.40 18.60
2
R. ShirzadianGilan, Y. Parvizi, E. Pazira et al.
electrical conductivity meter in a 1:2.5 (soil to water ratio) soil-water
extract (Page et al., 1992). Also, available phosphorus, potassium, and
soil microelement content (Fe, Zn, Mn and Cu) were measured using
the Olsen P solution method, the method applied by Dewis and Frai-
tas (1984), and an atomic absorption apparatus in DTPA-extract sam-
ples, respectively.
2.2. Soil properties
Surface soil (0
25 cm) was collected to conduct the pot experi-
ment. Some physico-chemical properties of soil used in this study are
shown in Table 1. Low organic carbon, pore mineral content, and
high sand content are diagnostic properties of this soil. Crude oil con-
tamination of 3.5% was considered as the most common rate in the
study area based on initial soil test (Table 1). This level of contami-
nant was calculated by the amount of crude oil and was uniformly
mixed with soil. To do this, about 12 liters of petroleum (the density
value: 0.81 g cm
3) was mixed with the required 272 kg of soil to cre-
ate an oil pollution concentration of 3.5%.
12  0:81
oilpollutionconcentration ¼ ¼ 3:5%ð1Þ
272
OC: Organic carbon, EC: Electrical conductivity, TNV: Total neu-
tralizing value, TPHs: total petroleum hydrocarbons
Table 2 shows some physical and chemical properties of crude oil
used in this experiment. This mixture was exposed to the air for three
days until the oil covered soil surface and the mixture became
completely uniform. The oil contents in soil samples of different
treatments were quantified gravimetrically (Fatima et al., 2018).
2.3. Treatments
This experiment was designed as a factorial experiment with a
completely randomized design (CRD). It was carried out under green-
house conditions by combining 4 plant treatments, 4 bacteria-nutri-
tion treatments, and 1 control treatment with four replications, and a
total of 68 pots were considered. The dimensions of the pots were
20 cm £ 18 cm (diameter £ height). Each pot contained 4 kg of soil.
To adapt to the conditions of the target area, the temperature of the
greenhouse was set between 24 and 33 °C during fall and winter and
about 27 and 38 °C during spring and summer. The composition of
the treatments is described below.
Plant treatments
Plant treatments were considered in the following 4 levels:
P0: No plant
P1: Opuntia)Opuntia ficus-indica(
P2: Eucalyptus (Eucalyptus camaldulensis)
P3: Oleander (Nerium oleander)
Bacteria and nutrition treatments
Bacteria and nutrition treatments were considered in the follow-
ing 4 levels:
B0: No bacteria inoculation-control
B1: Inoculation with Bacillus pumilus
B2: Inoculation with Pseudomonas putida
Table 2
Properties of used crude oil.
Oil fraction Molecular Stock
weight (mol%)
Residual crude oil (C8-C20) 188 60.0
C21fraction 500 14.2
Separator Gas (N2, CO2 and c1-c7)
25.8
Total 100.0
South African Journal of Botany 153 (2023) 1
10
B1B2F: The combined use of the inoculation of the two above-
mentioned bacterial species and a fertilizer recommendation based
on soil test.
Bacterial populations tend to decrease in soil under various stress
conditions (Abdul Rahman, 2021). Bacterial inoculation is used to
compensate for this reduction. For good bioremediation, the amount
of soil bacteria should be increased to the required level for improv-
ing the result of bioremediation. In this research, the powder culture
medium of bacteria was used. The culture media contained all ele-
ments which were not selective and most bacteria needed for
growth; therefore, they were used for the general cultivation and
maintenance of bacteria kept in laboratory culture collections. The
bacteria were kept in the culture medium until the end of the growth
stage, being 24 and 72 h for Pseudomonas putida and Bacillus pumilus,
respectively. The bacteria did not leave their reproductive environ-
ment and were used in the culture medium.
The fertilizer recommendation included soil enrichment based on
the soil test and the appropriate fertilizer recommendation of micro
and macro elements. Urea, triple superphosphate, and potassium sul-
fate fertilizers were applied as the sources of nitrogen, phosphorus,
and potassium, respectively. The Flourish fertilizer was used as a
source of microelements. The compounds of the Flourish fertilizer
were Ethylenediamine tetraacetic acid (EDTA) chelated of iron, man-
ganese, zinc, and copper by 3, 2, 3, and 0.35%, respectively, and 0.2%
boron solution. Fertilizers were mixed with soil. Subsequently, they
were distributed in pots for cultivation. Moreover, the control treat-
ment (no plant cultivation, no bacterial inoculation, and no enrich-
ment) was considered to investigate and compare the effect of
different treatments on the reduction of soil oil pollutants.
2.4. Experimental operations
The operations in this research were carried out in two phases:
field study and laboratory operations. In the field study, random soil
sampling around the oil exploitation units was done. Then, the infor-
mation about native or adapted plants to the conditions of the region
was collected. For this purpose, through field investigation, plants
with desirable growing conditions were identified and evaluated in
the oil-contaminated areas. In the field study, the selected plants
showed good vegetative growth in the area under study. The physical
and chemical characteristics of the soil samples were examined in the
laboratory. Furthermore, the amounts of oil pollutants in the soil
were determined. For this experiment, one-year-old seedlings were
prepared and cultivated. Fig. 1 shows some of the seedlings 4 months
after the start of the experiment.
The amount of bacterial inoculum to supply the bacterial popula-
tion was 1 g of inoculant per 1000 g of soil. This amount of inoculant
was added to each pot for providing the population of bacteria (about
108 bacteria per 1 gram of inoculant) (Santos et al., 2019). At planting
time, 4 gs of powder inoculums of each two selected bacteria were
added to the experimental pots at a depth of 2 cm. In the B1B2F treat-
ment, fertilizer need was calculated based on the soil test and after
considering nutritional requirements. Two-thirds of the required fer-
tilizers were given to each pot at the time of cultivation. Moreover,
during the growing season, one-third of calculated fertilizers was
uniformly given to the treatments twice (once every 30 days) at a
density of 2 gs/liter during the irrigation. Operations during the
growing season included irrigation to reach field capacity (FC) and
control of pests and diseases, which were uniformly applied to all
treatments until the vegetative period was completed (about 12
Specific gravity in months). FC moisture content (27%) was determined in laboratory
15.6 °C(gram/cm3)
using pressure plates.

Irrigation operation in spring and summer, autumn, and winter
0.913 was done once every 2
3 days, once a week, and once every ten


days, respectively, in such a manner that water would not come out
0.812
of the bottom of the pots. At harvest time, plant samples were
3
R. ShirzadianGilan, Y. Parvizi, E. Pazira et al. South African Journal of Botany 153 (2023) 1
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Fig. 1. Experimental pots of cultivated plants: a) Nerium oleander b) Opuntia ficus-indica c) Eucalyptus camaldulensis.

harvested by cutting them from the soil surface. Subsequently, after the end of the experiment was calculated according to Eq. (2). Conse-
separating soil from the roots and washing them, harvested plants quently, the percentage of TPHs degradation was calculated from
(including shoot and root) were transferred to laboratory for analy- Eq. (3).
ses. Plant samples were dried in an oven for 48 h at 50 °C. The dry
100  0:44
weight of the plant was measured. remainedTPHs ¼ ¼ 22:68% ð2Þ
1:94

2.5. Determination of TPHs 100%
22:68% ¼ 77:32% ð3Þ

The soil samples were collected from the soil surrounding root
growth zone. They were sieved using a 0.5 mm sieve and were trans-
ferred to the laboratory for measuring the remaining TPHs. The
amount of TPHs in each soil sample was compared with their pre-
cultivation amount, and the reduction was calculated as the amount
of TPHs decomposed due to the related treatment. Soil TPHs were
measured using gravimetric method and ultrasonic bath. To do this,
the soil was passed through a 35-mesh sieve, 5 gs of the soil was
weighed, and 2 gs of sodium sulfate was added to it.
The mixture was transferred to a 40 ml glass centrifuge tube.
40 ml of the dichloromethane solvent was added and was placed for
1 hour in an ultrasonic bath (3550 EPA) at 40 °C (Schwartz et al.,
2012). Afterwards, the mixture was centrifuged at 1500 rpm for
5 min. The extract was separated and was air-dried at room tempera-
ture for 24 h. After the evaporation of the solvent, the materials
remaining at the bottom of the container were weighed and recorded
as the amount of oil in the soil. The amount of oil extracted from each
treatment sample was compared with the amount of oil in the soil in
the control treatment, and its reduction was determined in percent-
age terms. The amount of TPHs remaining in the soil of each pot at
2.6. Statistical analyses of data
The variables measured in this research included concentrations
of soil TPHs and root, shoot, and total dry weight of plants. These var-
iables were subjected to the normality test analysis using the Kolmo-
gorov-Smirnov normality test. Statistical analyses of measured
variables, including descriptive statistics, correlation between the
variables, analysis of variance, and mean comparison using Duncan’s
test were done. SAS, SPSS, and Excel software were employed for the
statistical analyses.
3. Results
3.1. Variables statistical description
The minimum and maximum concentrations of TPHs in different
treatments were 0.34 and 1.94%, respectively (Table 3). The average
concentration was 0.84%, highlighting the effects of the treatments
and the significant reduction of petroleum hydrocarbons in all

Table 3
Some descriptive statistics indices of the measured variables.

Variables N Range Min. Max. Mean Standard deviation C.V Variance Skewness Kurtosis

TPHs (%) 68 0.760 0.340 1.960 0.855 0.193 0.220 0.037
0.685
0.526
SDW (g) 68 75.290 8.550 75.290 20.820 10.492 0.005 110.090 1.570 6.415
RDW (g) 68 21.050 1.300 21.050 9.760 5.994 0.610 35.932 0.315
1.407
TDW (g) 68 92.020 13.100 92.020 30.590 12.033 0.004 144.805 0.650 2.239

4
R. ShirzadianGilan, Y. Parvizi, E. Pazira et al. South African Journal of Botany 153 (2023) 1
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Table 4
Pearson correlation coefficient (r) between plant dry weight variables (root, shoot and
total) and soil TPH values.
Variables TPHsb SDW RDW
TPHs 1
0.176
0.219
SDW 1
0.010
RDW 1 0.392*
TDW
*Correlation is significant at the 0.01 level.
b
TPHs: total petroleum hydrocarbons, SDW: shoot dry weight, RDW: root dry
weight, and TDW: total dry weight.
treatments. Statistical indices as well as oblique indices of the means
such as kurtosis and skewness showed that the data were distributed
normally. Also, the Kolmogorov-Smirnov normality test results con-
firmed the normal distribution of the data. Variability indices (includ-
ing variance, standard deviation, and range of the data) in shoot dry
weight (SDW), root dry weight (RDW) as well as total dry weight
(TDW) variables were relatively high due to the intrinsic differences
between plant species. Opuntia ficus-indica had the lowest root dry
weight, while Eucalyptus camaldulensis had the highest root and total
dry weight.
The results of the Pearson correlation coefficient between the var-
iables revealed that there is a negative correlation between the
amounts of residual TPHs and root, shoot, and total dry weight of
plant indices (Table 4). In other words, dry weight indices are
decreased with increasing residual TPHs. Although the amount of the
correlation coefficient was not significant, the negative values indi-
cated the negative effects of petroleum contaminants on the growth
of plants in the treatments containing plants. It could be noted that
plants with more potential of biomass production caused higher
decomposition of TPHs and reduced residual soil TPHs.
3.2. Soil TPHs
Table 5 shows the analysis of variance of TPHs variables and
explain the effects of plant and bacteria treatments and their interac-
tions on soil TPHs. In this regard, the effects of plants and bacteria as
well as their interactions on soil TPHs were statistically significant at
99% probability level. In other words, plant types and the use of bac-
teria, separately and in combination with each other, resulted in a
reduction in petroleum hydrocarbon amounts in soil.
Table 6 statistically compares and addresses the main effects of
the treatments and their interactions on the residual TPHs means
using Duncan’s test. The results indicated that in the absence of bac-
teria and fertilizer recommendation, the cultivation of Eucalyptus
camaldulensis was more successful than other plant species in
decreasing petroleum pollutants. The combined use of two bacteria,
particularly with fertilizer recommendation, significantly increased
the decomposition of pollutants and phytoremediation capacity in all
plant treatments. The B1B2F treatment decreased soil TPHs by
approximately 70% in two plant (Opuntia ficus-indica and Nerium
Table 5
ANOVA table of main effects of treatments and their interactions on soil TPHs.
Sources of variation (treatment) df Mean square F Sig.
TDW
Plants 3 0.084 14.260 0.00

0.247 Bacteria 3 0.427 72.310 0.00
0.915* Plants and bacteria 9 0.060 10.177 0.00
Replications 3 0.001 0.232 0.87
1
oleander) treatments (Table 6). The best treatment was the cultiva-
tion of Eucalyptus camaldulensis by applying Pseudomonas putida,
Bacillus pumilus, and the full fertilizer recommendation. This treat-
ment reduced soil TPHs by almost 77.3%.
According to Table 6, in the B1B2F treatment of Eucalyptus camal-
dulensis plant, the percentage of TPHs in the soil is 0.44%. The con-
tamination percentage of the control treatment was initially 3.5%.
Due to the native crude oil degradation capacity of soil, during the
experiment the average of contamination percentage reached 1.94%
in 4 repetitions. Therefore, the amount of TPHs remaining in soil in
the B1B2F treatment in Eucalyptus camaldulensis pots was 22.68%.
The percentage of TPHs degradation is calculated using Eq. (3) and is
equal to 77.3%.
Pseudomonas putida was more successful than Bacillus pumilus in
reducing soil TPHs. However, the simultaneous use of these two bac-
teria had better results in this experiment (Table 6).
Evaluating the interactions between the treatments proved that
the cultivation of Eucalyptus camaldulensis in the soil treated with the
combined use of two bacteria and the complete fertilizer recommen-
dation had the highest TPHs decomposition capacity (Fig. 2). The
results of the effects of interactions between treatments showed that
the separate use of Eucalyptus camaldulensis and B2 reduced petro-
leum pollutants by 54.60 and 50%, respectively. However, the use of
B2 along with the cultivation of Eucalyptus camaldulensis resulted in
a 70% reduction in the level of petroleum contaminants in the soil.
*The difference of averages with common letters in each column
is not statistically significant.
The treatment including bacteria, fertilizer recommendation, and
the cultivation of Eucalyptus camaldulensis resulted in the highest
removal of petroleum hydrocarbons by 77.3% in this one-year experi-
ment (Fig. 3). Moreover, the decomposition percentage of petroleum
hydrocarbons in the complete fertilizer protocol and bacterial treat-
ments for Nerium oleander (P1B1B2F) and Oputia ficus-indica
(P3B1B2F) plants was the same (Fig. 3). The Pseudomonas putida
treatment in the presence of Eucalyptus camaldulensis (P2B2) had the
highest degradation of TPHs (69.60%), marking a significant differ-
ence between this treatment and P1B2 and P3B2 treatments.
This finding indicated a positive and significant interaction between
Pseudomonas putida and the cultivation of Eucalyptus camaldulensis.
On the one hand, the positive interaction between the B1 treatment
and the Nerium oleander plant (the degradation of TPHs by 54.10% in
the experiment) was more evident than that of the other two plants.

Table 6
Effects of different treatments and their interactions on soil TPHs (%) using Duncan’s grouping test.

Bacteria Plants Control SD

P1Ó P2 P3 P0
e f e
B1B2F 0.62§0.06 * 0.44§0.04 0.63§0.05 0.64§0.07e
0.58D 0.06
B2 1.01§0.11b 0.59§0.07e 0.87§0.09d 0.97§0.1d
0.86C 0.12
B1 0.997§0.06bc 1.00§0.1b 0.89§0.08bcd 0.99§0.1d
0.90C 0.08
B0 1.01§0.12b 0.88§0.08cd 0.92§0.09bcd 1.94§0.13a
1.18B 0.10
Control




1.94A
0.91C 0.73E 0.83D 1.14B 1.94A
SD 0.11 0.08 0.08 0.35
*The difference of averages with common uppercase or lowercase letters in each row and column is not statistically significant.
B1: Bacillus pumilus, B2:Pseudomonas putida, F: fertilizer, P0: no plant, P1: Opuntia ficus-indica, P2: Eucalyptus camaldulensis, P3: Nerium oleander, and SD: standard deviation.

5
R. ShirzadianGilan, Y. Parvizi, E. Pazira et al.
Fig. 2. Effects of interactions between Pseudomonas putida (B2), Bacillus pumilus (B1), and plant treatments on soil TPHs (%).
Fig. 3. Comparison of decomposition percentage of TPHs means using Duncan’s grouping test.
On the other hand, the other two plants were able to decompose
approximately 48.50% of petroleum hydrocarbons in the presence of
B1. In the absence of bacteria and fertilizers (P2), Eucalyptus camaldu-
lensis degraded soil TPHs by 54.60%, as compared to the other two
plants in the experiment (Fig. 3)
(Control: no bacteria, B1: Bacillus pumilus, B2: Pseudomonas
putida, F: fertilizer, P1: Opuntia ficus-indica, P2: Eucalyptus camaldu-
lensis, and P3: Nerium ole ander)
*The difference of averages with common letters in each column
is not statistically significant.
A comparison of the two bacteria indicated that the TPH degrada-
tion efficiency of Pseudomonas putida (B2) was greater than that of
Bacillus pumilus (B1) (Figs. 3 and 4). The TPH degradation efficiency
was significantly improved by applying soil enrichment along with
the fertilizer recommendation (Fig. 4). In other words, the enrich-
ment protocol increased the efficiency of bacteria and plants, indicat-
ing that it is extremely effective in improving the degradation of
petroleum contaminants in soil.
3.3. Dry weight
In this experiment, different plant species with different develop-
mental and physiological characteristics have been used. Hence, the
6
South African Journal of Botany 153 (2023) 1
10
investigated plant species differed considerably in terms of plant dry
weight indices. However, the important point in this analysis is that
the effects of bacteria and plant-bacteria interactions on shoot dry
weight were statistically significant at 95 and 99% probability levels,
respectively. Furthermore, plant-bacteria interactions had a signifi-
cant effect on root dry weight and total dry weight at a 99% probabil-
ity level (Table 7).
Fig. 5 compares shoot dry weight means and shows the effects of
bacteria on shoot dry weight means using Duncan’s test. The B1B2F
treatment, i.e. the combined use of two bacteria and the proposed
fertilizer recommendation, resulted in an average 37% increase in
root dry weight in all cultivated plants.
*The difference of averages with common letters in each column
is not statistically significant.
Another important result of this study is the effects of interactions
between bacteria and plant treatments on the total dry weight of the
cultivated plants. The lowest dry weight was observed in Opuntia
ficus-indica plant without bacteria treatment. However, this plant
responded positively to the use of bacteria and had an average 70%
increase in dry weight, provided that both bacteria were used in com-
bination with the fertilizer recommendation. The highest dry weight
belonged to the Eucalyptus camaldulensis plant with Bacillus pumilus
(B1) (Table 8).
R. ShirzadianGilan, Y. Parvizi, E. Pazira et al. South African Journal of Botany 153 (2023) 1
10

Fig. 4. TPHs decomposition percentage of contaminated soils without plant cultivation and containing Pseudomonas putida (B2), Bacillus pumilus (B1) and fertilizer (F) enrichment.

Table 7 3.4. Discussion

Variance analysis of main effects of treatments and their interactions on root, shoot,
and total dry weight (g) variables.
Based on the results of the present study, the separate use of each
Sources Df Mean square F Sig. of plants or bacteria in this study decomposed about half of the TPHs
contaminants from the soil after one year. Using bacteria and plants
Shoot dry weight (SDW)
Plants 2 200.137 2.740 0.080 together led to the decomposition of more than two-thirds of the ini-
Bacteria 3 196.799 2.694 0.050 tial contaminants from the soil over a one-year period. Comparing
Interactions between plants and bacteria 6 259.015 3.546 0.010 the three plants revealed that Eucalyptus camaldulensis (without the
Root dry weight (RDW)
NPK fertilizer and bacteria) was the most efficient plant in removing
Plants 2 623.363 82.320 0.000
Bacteria 3 2.808 0.371 0.770
soil TPHs. In a greenhouse study, Barcellos et al. (2016) showed that
Interactions between plants and bacteria 6 26.802 3.539 0.010 Eucalyptus urograndis and Pinus taeda are particularly suitable for
Total dry weight (TDW) removing petroleum hydrocarbons from contaminated soils with a
Replications 3 55.931 1.115 0.298 sandy texture. They observed that these plants assisted in reducing
Plants 2 252.289 5.031 0.010
approximately 91
98% of petroleum hydrocarbons in the soil. More-
Bacteria 3 51.038 1.018 0.396
Interactions between plants and bacteria 6 157.753 3.146 0.010 over, Gotelli et al. (2020) found that the forestation of Eucalyptus dun-
nii effectively decontaminated 98.10% of initial TPHs from the soil in

Fig. 5. Effects of bacteria on shoot dry weight means (g) using Duncan’s grouping test.

7
R. ShirzadianGilan, Y. Parvizi, E. Pazira et al.
Table 8
Effects of interactions between plants and bacteria on total dry weight (g) using Dun-
can’s grouping test.
Bacteria N Subset*
1 2 3 4
Ó
P1Bo 4 18.85
P1B2 4 20.02 20.02
P1B1 4 21.68 21.68
P2B2 4 28.83 28.83
P3B1 4 29.07 29.07
P2B1B2F 4 30.82
P3B1B2F 4 31.39
P1B1B2F 4 31.91
P3Bo 4 33.37
P2B0 4 35.93
P3B2 4 36.52
P2B1 4 36.72
Sig. 0.57 0.08 0.06
* Means are displayed for groups in homogeneous subsets.
Ó
B0: no bacteria, B1: Bacillus pumilus, B2: Pseudomonas Putida, F: fertilizer, P1:
Opuntia ficus-indica, P2: Eucalyptus camaldulensis, and P3: Nerium oleander.
10 years. Lou et al. (2016) reported the high phytoremediation effi-
ciency of Eucalyptus globulus trees grown in different environments.
They also observed that in one year, Eucalyptus globulus produced the
highest biomass. Madejon et al. (2017) stated that the Eucalyptus
camaldulensis plant is perfectly capable of clearing heavy metals from
contaminated soils due to its strong root system and rapid growth.
The cultivation of Eucalyptus camaldulensis using the combination
of two bacteria and the fertilizer recommendation was found to be
the most efficient TPHs decomposer, removing 77.3% of TPHs from
the soil in this experiment. The bacteria and the fertilizer recommen-
dation directly increased the decomposition rate of TPHs and
enhanced root system development, ultimately improving plant bio-
mass and plant phytoremediation efficiency. Agnello et al. (2016)
reported that the combined use of plants and bacteria was the most
advantageous option for the treatment of TPH-contaminated soils, as
compared to the separate application of natural attenuation, bioaug-
mentation, or phytoremediation. The highest degree of TPH removal
was obtained by bioaugmentation-assisted phytoremediation treat-
ment (68%) followed by bioaugmentation (59%), phytoremediation
(47%), and natural attenuation (37%).
The cultivation of Eucalyptus camaldulensis using P. putida inocula-
tion decomposed about two-thirds (69.60%) of petroleum contami-
nants and removed them from the soil ecosystem. Pseudomonas
putida acts as a bacterium promoting plant growth and phytoreme-
diation efficiency for the remediation of polycyclic aromatic hydro-
carbons (Afegbua and Batty, 2019). On the other hand, carbon
sources supplied by root secretions enhance the production of
responsible enzymes by microorganisms for the decomposition of
petroleum hydrocarbons (Saleem, 2016). The possibility of obtaining
synergistic effects between plants and microorganisms has increased
the possibility of alternative techniques for bioremediation (Guarino
et al., 2020). Moreover, Czarny et al. (2017) reported the synergistic
cooperation of plant roots and soil bacteria, promoting the degrada-
tion of TPHs.
P. putida was more efficient than B. pumilus, but B. pumilus had
higher compatibility with Nerium oleander than Eucalyptus camaldu-
lensis and Opuntia ficus-indica plants and could decontaminate more
TPHs from the soil. Bento et al. (2005) emphasized that B. pumilus
was described as a producer of lipopeptide surfactin. This compound
is responsible for reducing surface and interfacial tensions that facili-
tate TPHs degradation.
The interactions between plant and bacteria treatments had sig-
nificant effects on root and total dry weight at a level=0.01. Ho et al.
(2007) reported the positive effects of the Pseudomonas fluorescens
bacterium on the degradation of polycyclic aromatic hydrocarbons in
South African Journal of Botany 153 (2023) 1
10
the rhizosphere. Jamil et al. (2014) indicated that Bacillus lichenifor-
mis improves the biochemical attributes of plants, can protect them
from the toxic effects of contaminated soil, and can be used to
improve phytoremediation efficiency. Moreover, planting signifi-
cantly enhanced the survival of bacteria cells in both bulk and rhizo-
spheric soil, and this, in turn, significantly improved the degradation
of soil TPHs (Chouychai et al., 2012).
Applying fertilizers rehabilitated plants and increased their slow
28.83 growth induced by the toxicity of petroleum hydrocarbons in the
29.07 soil. Petroleum degradation in the rhizosphere was enhanced by
30.83 applying the medium amount of fertilizers, particularly when plants
31.39
were exposed to intermediate and high petroleum contamination
31.91
(Andrew et al., 2014). The combined use of bacteria and the fertilizer
recommendation significantly increased phytoremediation efficiency
in all treatments. This finding was in agreement with those reported
by Asim et al. (2016) stating that TPHs degradation was 59% higher in
0.16
the combined application of bacterial consortium and fertilizer than
the untreated plant. Also, Daryabeigi Zand and Hoveidi (2016)
reported a positive effect of N + P fertilizer on the degradation of
four-ring PAHs in a creosote polluted soil.
The positive effects of bacteria to promote the phytoremediation
efficiency of cultivated plants were validated. This improvement may
be due to the enhancement effects of bacteria in plant metabolic
activities (Motesharezadeh et al., 2017), development of the root sys-
tem, and ultimately the increase in plant biomass and dry weight
(Saha et al., 2021).
The bacterial consortium can enhance the tolerance of plants to
oily conditions by promoting plant metabolites and TPHs degrada-
tion. In this study, soil TPHs were degraded by 47.90% using Opuntia
ficus-indica. Escobar et al. (2017) reported a similar finding (48% soil
TPHs degradation in the phytormediation process using this plant).
The remediation potential of the combinations of plants and bacteria
can be influenced by fertilizer, irrigation and suitable temperature
conditions (Sharma et al., 2022). Applying the biological and chemical
fertilizer (B1B2F) treatment enhanced the phytoremediation poten-
tial of Opuntia ficus-indica by approximately 68.10%. This finding is
similar to the report by Tang et al. (2020) showing that bacterial
treatments generally had comparable or better efficiencies than
chemical fertilization treatments.
Bacterial treatments did not have a significant effect on root and
total dry weight. However, employing bacteria and the fertilizer rec-
ommendation significantly increased shoot dry weight. This increase
in biomass production can help increase plant phytoremediation
capacity. This finding is similar to those reported by Alikhani et al.
(2011). Moreover, Weyens et al. (2015) found that Pseudomonas
putida had significant positive effects on the production of poplar bio-
mass and on poplar performance in phytoremediation.
4. Conclusion
This paper investigated and compared the remediation potential
of native and environmentally friendly plants and bacteria, including
indigenous or exotic species for removing petroleum pollutants from
petroleum-contaminated soils in the west of Iran. The cultivation of
Eucalyptus camaldulensis using the chemical fertilizer and both bacte-
ria is suggested in this research as the best treatment to decompose
the largest amount of petroleum pollutants from the soil. Although
the application of Pseudomonas putida was more effective than Bacil-
lus pumilus, Bacillus pumilus had better symbiosis with Nerium olean-
der plant to decompose TPHs from soil. The direct effects of bacteria
on the decomposition and removal of petroleum pollutants from soil
were found in this study.
Cultivated Eucalyptus camaldulensis, a plant with the best adapta-
tion to the climatic conditions of the study region, is capable of
decomposing about two-thirds of petroleum contaminants from the
soil. This ability can be promoted with inoculation by P. putida
8
R. ShirzadianGilan, Y. Parvizi, E. Pazira et al.
bacterium. Moreover, using the fertilizer recommendation, particu-
larly for macroelements such as phosphorus and potassium fertilizers
and microelements such as zinc, can enhance phytoremediation effi-
ciency because fertilization could increase plant biomass and
improve the resistance of plants to drought and environmental
stresses. It is suggested that results of this research be validated in a
field condition experiment. Furthermore, the efficiency of best treat-
ments found in this study was validated and compared under differ-
ent drought stress conditions. This research finding showed that
plants adapted to extreme conditions are highly effective in soil
bioremediation. Therefore, further studies are suggested to identify
better performing plant species in order to achieve greater phytore-
mediation of oil contaminated soil.
Author contributions
Throughout the present study, Yahya Parvizi roles: conceptuali-
zation, formal analysis, data curation, project administration, supervi-
sion and writing. Ronak Shirzadian Gilan roles: Investigation,
resources, original draft, software, literature review. Ebrahim Pazira
roles: supervision and editing the data and the manuscript. Farhad
Rejali roles: resources, supervision, and review and editing.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
Acknowledment
The authors would like to appreciate the Western Oil and Gas
Exploitation Company in Iran for their support throughout this study.
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