evaluation of phyto-chemical remediation approaches …article.aascit.org/file/pdf/8920827.pdf ·...

American Journal of Agricultural Science

2016; 3(3): 48-58

http://www.aascit.org/journal/ajas

ISSN: 2381-1013 (Print); ISSN: 2381-1021 (Online)

Keywords Phytoremediation,

Phyto-chemical Remediation,

Petroleum Hydrocarbons,

RAPD,

ISSR,

Soil Microbial Communities

Received: April 21, 2016

Accepted: May 9, 2016

Published: June 15, 2016

Evaluation of Phyto-Chemical

Remediation Approaches to Remedy

Hydrocarbon from Oil Polluted Soils and Their Impact on Soil Microbial

Communities Using RAPD and ISSR

Markers

Shreen S. Ahmed1, Mohamed A. M. Atia

2, *, Gehan H. Abd El-Aziz

1,

Ashraf H. Fahmy3

1Soils, Water and Environment Research Institute, ARC, Giza, Egypt 2Genome Mapping Department, Agricultural Genetic Engineering Research Institute, ARC, Giza,

Egypt 3Plant Genetic Transformation Department, Agricultural Genetic Engineering Research Institute,

ARC, Giza, Egypt

Email address [email protected] (M. A. M. Atia) *Corresponding author

Citation Shreen S. Ahmed, Mohamed A. M. Atia, Gehan H. Abd El-Aziz, Ashraf H. Fahmy. Evaluation of

Phyto-Chemical Remediation Approaches to Remedy Hydrocarbon from Oil Polluted Soils and

Their Impact on Soil Microbial Communities Using RAPD and ISSR Markers. American Journal

of Agricultural Science. Vol. 3, No. 3, 2016, pp. 48-58.

Abstract

Soil contamination by petroleum hydrocarbons is one of the world’s most common

environmental problems. Remediation of the petroleum contaminated soil is essential to

maintain the sustainable development of soil ecosystem. In this study, we evaluate the

efficiency of different Phyto-chemical approaches for cleaning up hydrocarbon

contaminated soils and their effect on the soil properties, soil microbial communities

structure, grain yield, chemical composition of wheat plants (Triticum aestivum L). The

experiment included five treatments: phytoremediation (Phyto) and Phyto combination

with organic and inorganic compound. The degradation rate of total petroleum

hydrocarbons (TPHs) was in the following ascending order: Phyto + nitrogen (16.7%),

phytoremediation (40.0%), Phyto + potassium permanganate (61.5%), Phyto + bacteria

(63.7%), Phyto + humic acid (76.0%). Results revealed that yield, protein, fat,

macronutrients contents were decreased whereas; carbohydrate was increased as applied

of TPH in the soil compare to the control. Results also revealed that wheat grain that

grown in contaminated soil (Phyto) had higher concentrations of total petroleum

hydrocarbon compare to unpolluted soil (control) and Phyto combinations with organic

and inorganic compound treatments. It can be concluded that Phyto combination with

humic acid, bacteria and potassium permanganate was more effective for cleaning up

hydrocarbon contaminated soils than phytoremediation treatment separately. On the

other hand, Randomly Amplified Polymorphic DNA (RAPD) and Inter-simple sequence

repeats (ISSR) molecular marker systems were used to survey and explore the diversity

of soil microbial communities under different Phyto-chemical treatments. Cluster

analysis based on combined data of RAPD and ISSR fingerprinting was discussed. The

molecular phylogeny results exhibited the ability to differentiate and track genetic

American Journal of Agricultural Science 2016; 3(3): 48-58 49

variations in bacterial populations. Such approaches

represent a fundamental step for studying structure and

dynamics of microbial communities in contaminated

ecosystems.

1. Introduction

Soil contamination by petroleum hydrocarbons is one of

the world’s most common environmental problems [1]. Total

petroleum hydrocarbons (TPHs) are one of the most common

groups of persistent organic contaminants [2]. Generally, the

accumulation of contaminants in soils can have destructive

effects on both soil ecosystem and human health.

Contaminants present in soils can enter the food chain and

seriously affect animal and human health [3]. In today’s era

of heightened environmental awareness and good

stewardship of limited natural resources effort to clean up

contaminated sites involve series of remedial techniques or

approaches ranging from conventional physicochemical

techniques and natural attenuation to phytoremediation the

most emerging biotechnology approach [4].

Phytoremediation is one of the best developed and

implemented approaches/technologies of bioremediation for

cleaning up the environmental pollution. Phytoremediation

has been proposed as a cost effective, non-intrusive, and

environmental friendly technology for the restoration of soils

contaminated with TPH [5].

Studying of the structure and dynamics of an ecosystem is

used as an indicator to measure the cumulative impact of

multiple stresses on population (s) and its adaptation to the

habitat. Microbial communities, capable of degrading

different pollutants in contaminated ecosystems, are relevant

in microbial ecology for the development of bioremediation

strategies. Analysis of biodiversity is particularly important

when the soil ecosystems respond to changing environmental

conditions and such changes in the composition of the soil

micro-flora can be crucial for the functional integrity of the

soil as a main component in agriculture system. In recent

years, several studies were performed to describe bacterial

diversity and community changes in various pollutant-

degrading communities [6, 7, 8] and a number of molecular

methods have been developed for describing and comparing

complex microbial communities [9]. Polymerase chain

reaction (PCR) has been successfully used for microbial

identification in the environmental context.

PCR-Based molecular markers have been potentially used

to survey and explore the diversity of soil microbial

communities, bacterial taxonomy and phylogeny. Randomly

amplified polymorphic DNA (RAPD) and Inter-simple

sequence repeats (ISSR) based detection of genetic

polymorphism has been successfully utilized to identify

isolates, genetic diversity and population structure of

bacteria; to demonstrate genetic variation within the species;

and to elucidate the distribution of genes and population

structure of the species [10]. RAPD markers have also been

utilized for inter-specific and intra-specific genetic diversity

of microbial communities of the soil across different

treatments [11].

Therefore, this study aims to: (1) evaluate and compare the

efficiency of different Phyto-chemical approaches for

cleaning up hydrocarbon contaminated soils (2) explore the

genetic diversity between microbial communities of different

Phyto-chemically treated soils using RAPD and ISSR

markers.

2. Materials and Methods

2.1. Soil Used for Experiment

Unpolluted surface soil (0-25 cm) was collected from an

Agricultural Research Station, Giza. The soil was air dried

and ground to 20 meshes before used. Spent engine oil was

then added to a portion of the unpolluted soil with a dosage

of 2% of soil mass.

2.2. Experimental Design

A greenhouse experiment was carried out to study the

effect of different Phyto-chemical remediation treatments on

the spent engine oil contaminated soil. Seven treatments were

designed:

1). Control (unpolluted soil with wheat planting).

2). Phytoremediation (Phyto; polluted soil with wheat

planting).

3). Phytoremediation + Potassium permanganate (PK)

addition to polluted soil (rate 0.9 M/Kg) (Phyto + PK).

4). Phytoremediation + Nitrogen addition to polluted soil

(rate 0.2 g/kg) (Phyto + N).

5). Phytoremediation + Humic Acid (HA) addition to

polluted soil (rate 15 g/kg) (Phyto + H).

6). Phytoremediation + Pseudomonas aeruginosa bacteria

addition (Phyto + B; the soil was enriched with 30ml of

bacterial suspension of Ps. aeruginosa incubated for 48

h at 28°C in 0.9% NaCl solution) [12].

Three replicates were sown for each treatment. Four

Kilograms crude oil contaminated soil was added. For wheat

(Triticum aestivum L.) planting, 10 seeds were sown evenly

to the soil in each pot and covered with 2–3 cm of soil on the

top (4 Kg soil added in total for each pot). Pots were irrigated

every day of field capacity. All of the experiment pots were

placed in a greenhouse at 30°C. Seven days after seeds

germinated, 5 healthy seedlings were preserved in each pot

for further remediation. Soil samples were taken after 0, 15

30, 45, 60, 75, 90, 105 and 120 days. Then the soil samples

were divided into two sub samples: one was used to study the

variations of soil physicochemical properties and evaluation

of hydrocarbon degradation.

2.3. Analytical Methods

Some Physical and chemical characteristics of the studied

soil was determined according to Page et al. [13]. Total

petroleum hydrocarbon were extracted from soil and plant

samples then determined by UV-Spectrometer according to

50 Shreen S. Ahmed et al.: Evaluation of Phyto-Chemical Remediation Approaches to Remedy Hydrocarbon from Oil

Polluted Soils and Their Impact on Soil Microbial Communities Using RAPD and ISSR Markers

the procedure described by IOC [14]. The extracts were

analyzed using a Hewlett Packard (HP) 5890 Series II gas

chromatograph (GC) with a 5971A mass selective detector

(MSD), a HP 7673 autosampler, and HP Chemstation

software. The instrument was operated in the splitless mode

with 1 µL injections onto a 30 m x 0.25 mm x 0.25 µm RTX-

5 (5% phenylmethylsiloxane) capillary column. The run time

to elute all the target compounds was about 35 minutes, but

the full cycle time was about 60 minutes. In plant sample,

phosphorus content was determined by vanadomolybdate

yellow method spectrophotometrically and potassium by

flame photometer [15]. Heavy metal contents samples were

extracted according to the method of Lindsay and Novell

[16]. Total nitrogen was determined by micro-Kjeldahl

method according to AOAC [17]. Crude protein was

calculated by multiplying the values of total nitrogen in 6.25.

Total lipid was determined according to AOAC [18]. Total

carbohydrate was extracted according to Smith et al. [19] and

determined using spectrophotometer according to Murphy

[20]. All data were statistically analyzed using Costat

computer program according to procedures outlined by

Snedecor and Cochran [21].

2.4. Soil Sampling and Estimation of

Microbial Community

Soil samples were collected from both control and

treatments, and then stored at 4°C till DNA isolation and

molecular analysis. For each sample (control and treatments),

10 g was suspended into 90 mL of sterile doubled distilled

water (ddH2O) and stirred using shaker at 200 rpm for 1 hour.

The slurry was centrifuged at 5000 rpm for 5 min at 25°C.

Then, 1 ml of the supernatant was used to inoculate 100 mL

of LB broth medium and incubated at 37°C for 24 h for

microbial growth. The number of viable cells was determined

by serial dilution technique and spectrophotometry as

indirect approach to estimate the microbial community’s

variations between control and treatments [22].

2.5. Bacterial DNA Isolation

The bacterial chromosomal DNA was extracted from

whole bacterial community (Gram Positive and Gram

Negative Bacteria) using Wizard Genomic DNA Purification

Kit (Promega, WI, USA) according to manufacturer

instructions. The DNA was quantified with NanoDrop

Spectrophotometer (Thermo Fisher Scientific Inc.). All

samples were adjusted to a concentration of 10 ng/µl for

subsequent molecular analyses.

2.6. RAPD Marker Analysis

Ten RAPD decamer primers (Operon Tech., Alameda, CA,

USA) were used in the present study. DNA was amplified

following the protocol of Adawy and Atia [23]. Each PCR

reaction mix of 25 µl contained the 30 ng template DNA, 2.5

µl of 10X PCR buffer, 1.5 µl of 25mM MgCl2, 2.5 µl of the

dNTPs mix, 30 pmol of RAPD primer, 1.0 U Taq DNA

polymerase (Promega, WI, USA). The amplification was

performed in a thermal cycler (Applied BioSystems, USA)

programmed for initial denaturation of 5 min at 94°C; 40

cycles of 2 min denaturation at 94°C, 1 min annealing at

36°C and 2 min extension at 72°C; and final elongation step

at 72°C for 7min. The PCR products were electrophoresed on

1.5% agarose gel containing ethidium bromide (0.5 µg/mL)

in TBE buffer for 2 h at 100 V. After electrophoresis, the gels

were observed under an UV-transilluminator, documented in

Gel-Doc XR (Bio-Rad) and photographed. The size of the

amplicons was determined using 100 bp DNA ladder plus.

2.7. ISSR Marker Analysis

Five ISSR primers were used in the present study. DNA

was amplified according to the following protocol. Each PCR

reaction mix of 25 µl contained the 30 ng template DNA, 2.5

µl of 10X PCR buffer, 1.5 µl of 25mM MgCl2, 2.5 µl of the

dNTPs mix, 30 pmol of ISSR primer, 1.0 U Taq DNA

polymerase (Promega, WI, USA). The amplification was

performed in a thermal cycler (Applied BioSystems, USA)

programmed for initial denaturation of 5 min at 94°C; 40

cycles of 2 min denaturation at 94°C, 45 Sec. annealing at

50°C and 2 min extension at 72°C; and final elongation step

at 72°C for 7 min. The PCR products were electrophoresed

on 1.5% agarose gel containing ethidium bromide 0.5 µg/ml

in TBE buffer for 2 h at 100 V. After electrophoresis, the gels

were observed under an UV-transilluminator, documented in

Gel-Doc XR (Bio-Rad) and photographed. The size of the

amplicons was determined using 100 bp DNA ladder plus.

2.8. Markers Data Analysis

The generated/ amplified bands were scored visually. The

bands were scored as present (1) or absent (0) to create the

binary data set. To estimate the genetic similarity, Jaccard’s

coefficient was used [24]. A dendrogram was generated by

cluster analysis using the un-weighted pair group method of

the arithmetic averages (UPGMA) using SPSS program

V1.6. Support for clusters was evaluated by bootstrapping

analysis. One thousand permutation data sets were generated

by re-sampling with replacement of characters within the

combined 1/0 data matrix.

3. Results 3.1. Effect of Spent Engine Oil on Soil

Properties

The physicochemical properties of the tested soil shown in

Table 1. Chemical properties of spent engine oil are

presented in Table 2.

Soil physicochemical analysis was done after the addition

of spent engine oil as source of hydrocarbon (PHCs). Results

of the physicochemical analysis of the soil samples were

reported in the table (3). Results of the pH revealed that the

pH of the polluted soil sample was slight decrease compared

to unpolluted soil as a control. The results of the organic

carbon revealed that there was progressive increase in


organic matter of polluted soil; increase reached 4.6 fold than

in unpolluted soil as a control. Similar trend was found in the

conductivity (EC) of the soils; the conductivity value of the

soil after polluted was found maximum (2.98) and minimum

(1.78) in control. Data of moisture content of oil polluted

soils were lower than the control sample, decrease reached

54%. The bulk density of spent engine oil treated soil

generally increased compared to unpolluted soil, increase

reached 8.5%. The increase in bulk density of spent engine

oil treated soil could be attributed to compaction resulting

from oil contamination as well as reduced porosity. Also,

data of the some macro, micronutrient and heavy metals

revealed that there was increase in metals concentrations of

polluted soil relative to control (unpolluted soil).

Table 1. Some and chemical properties of the tested soil under different

experiments.

Physical properties Value

Coarse sand% 7.3

Fine sand% 19.9

Silt% 38.3

Clay% 34.5

Texture soil loamy clay

Chemical properties

pH (1: 2.5, soil - water suspension) 7.99

Organic matter (%) 1.15

Ece dS m-1, soil paste extract 1.78

Soluble cations (me/L)

Ca++ 7.1

Mg++ 3.9

Na+ 5.1

K+ 1.5

Soluble anions (me/L)

CO3= -

HCO3- 4.1

Cl- 7.1

SO4= 6.4

Table 2. Chemical properties of spent engine oil.

Parameter Oil

Organic carbon% 5.98

Total nitrogen% 2.0

K% 0.98

Na% 0.81

P% 0.26

Mg (mg/kg) 1.7

Ca (mg/kg) 170

Mn (mg/kg) 0.7

Fe (mg/kg) 65.9

Co (mg/kg) 0.3

Ni (mg/kg) 50.8

Cu (mg/kg) 2.7

Zn (mg/kg) 19

Cd (mg/kg) 2.3

Pb (mg/kg) 105.3

Table 3. Comparison of spent engine oil polluted and unpolluted soils before

planting.

Parameter Polluted Unpolluted

Moisture content (%) 3.19 6.93

Bulk density (gcm-3) 1.40 1.29

ECe dS m-1, soil paste extract 2.98 1.78

pH 7.68 7.99

NH4 (ppm) 99.4 49.4

NO3 (ppm) 49.7 39.76

Organic carbon (%) 7.66 1.68

Total N (%) 2.1 0.19

P % 1.28 1.02

K % 1.33 0.44

Na % 1.3 0.49

Ca (mgkg-1) 186 16

Mg (mgkg-1) 8 6

Heavy metals

Mn+2 9.88 9.18

Fe+2 625.9 560

Co+2 0.615 0.382

Ni+2 51.7 0.680

Cu+2 7.51 3.9

Zn+2 29 10.6

Cd+2 2.5 0.2

Pb+2 108 2.86

TPH (mgkg-1) 17610 104

3.2. Effects of Remediation Treatments on

Hydrocarbon Degradation

Five different treatments (Phyto, Phyto + N, Phyto + PK,

Phyto + HA and Phyto + B) were individually used for

degradation of soil polluted with TPH compared with

control. The degradation rate of hydrocarbon (fig. 1) using

addition of different treatments was in the following

ascending order: Phyto + N (16.7%), Phytoremediation

(40.0%), Phyto + PK (61.5%), Phyto + B (63.7%), Phyto +

HA (76.0%). On the other hand, the effect of HA or B or PK

without Phyto on TPH degradation was calculated and

recorded as 36, 23.7 and 21.5%, respectively. HA and B

addition with Phyto significantly stimulated the degradation

of hydrocarbon at the initial time.

Figure 1. Effect of different treatments on hydrocarbon degradation% in

soil.



Figure 2. Interaction effect of different treatment and time on residual hydrocarbon in soil.

Data of residual hydrocarbon are presented in figure (2).

Data showed that all treatments under investigation

decreased strongly hydrocarbon concentration. Percentages

of residual hydrocarbon at the end time of experiment (120

days) reached 59.9, 38.5, 23.9, and 36.3%, respectively. It

worth mention, Phytoremediation combined with HA, B

and PK was more effective for cleaning up hydrocarbon

contaminated soils than phytoremediation individually. The

hydrocarbon degradation efficiency of Phyto + HA

treatment was more effective than others. At the end time

(120 days), the hydrocarbon degradation rate increased at

different degrees under different treatments compared with

the control.

3.3. Effect of Spent Engine Oil on Growth

and Chemical Composition of Wheat

Plants

Results of the study showed that there was significant

difference in the chemical composition of the wheat grain

grown in the polluted soil, and those grown in the unpolluted

soil. Wheat grown in spent engine oil treated soil (Table 4)

recorded the lowest dry weight which was significantly

different (P<0.05) from that of the control. Decline percent

reached 53.4 and 48.6% for plant and grain dry weight,

respectively. On the other hand, wheat plants grown in spent

oil polluted soils with addition of HA recorded the highest

dry weight which was significantly different from the other

treatments. However, plants in the control experiments

recorded the highest dry weight.

Protein and fat content grain of wheat grown in the control

experiment (unpolluted soil) recorded the highest values. The

values were significantly different from that of the other

treatments. On the contrary, grain of wheat plants grown in

the spent oil polluted soils produced (Phyto) the lowest

values of protein and fat (Table 4). HA treatment recorded

the highest values in protein and fat followed with B and PK

treatments. Protein and fat contents were observed to be

higher in the wheat plants grow in the control experiment

(unpolluted soil). In contrast, percentage of protein and fat

was decreasing in the grain of the treated wheat, as the

addition of the spent oil (Phyto). Concerning the total

carbohydrates (Table 4), data indicated that grain of wheat

that grown in the spent oil polluted soils recorded the highest

value compared to control (unpolluted) and other treatments.

Treatments of HA and B were recorded the same trended.

Further, PK treatment recorded the higher value of

carbohydrate than HA and B treatments.

Table 4. Effect of spent engine oil pollution on chemical composition % of wheat grains.

Treatment Dry weight (g/pot)

Carbohydrates% Fat% Protein% Plant grain

Control (unpolluted soil) 13.10A 6.17A 67.47D 1.54A 11.33B

Phytoremediation 6.33E 3.17E 72.50A 1.02E 10.63C

Potassium permanganate 8.00D 3.60D 69.43B 1.22D 11.62B

Humic acid 9.40B 5.23B 68.17C 1.40B 12.86A

Bacteria 8.43C 4.07C 68.60C 1.32C 11.61B

LSD at 0.050 0.1191 0.1031 0.6440 0.0595 0.6101


Table 5. Effect of spent engine oil pollution on macronutrients % of wheat plant and soil after harvesting.

Treatment Plant Soil

N P K N P K

Control (unpolluted soil) 1.76B 0.23A 0.40B 0.10C 0.41D 0.32C

Phytoremediation 1.66C 0.16B 0.36B 0.41B 1.12A 0.97B

Potassium permanganate 1.81B 0.20AB 0.51A 0.39B 0.97C 1.18A

Humic acid 2.00A 0.21AB 0.45AB 1.60A 1.12A 0.95B

Bacteria 1.81B 0.21AB 0.43AB 0.36B 0.98BC 0.93B

LSD at 0.050 0.10 0.06 0.10 0.15 0.06 0.06

3.4. Effect of Spent Engine Oil Pollution on

Macronutrients of Wheat Plant and Soil

After Harvesting

Data of the effect of spent engine oil pollution on

macronutrients of wheat grain and soil are presented in

Table (5). Macronutrient contents (Nitrogen, phosphorus,

and potassium) of wheat grown in the unpolluted soil

recorded the higher values than macronutrient contents of

wheat plants grown in the spent oil polluted soils. Decline

percent reached 5.68, 30.4, and 10.0%, respectively. On the

other hand, significant effect was observed when wheat

plants grown in spent oil polluted soils with addition of

different treatments.

3.5. Content of Hydrocarbons by Wheat

Grown Under Different Treatments

Content of TPH by wheat grown in different field-

contaminated soils was investigated. TPH concentrations in

grain correlated positively with the corresponding

concentrations in soils (Figure 3). Result of the experiment

indicated that wheat grain that grown contaminated soils

(phyto) had higher concentrations of total petroleum

hydrocarbon compare to unplanted soil (control). Increase

percent reached 80.6% related to unpolluted soil. On the

other hand, progressive effect was observed when wheat

plants grown in spent oil polluted soils (phyto) with

addition of different treatments (PK, HA, and B). All

treatments recorded the lower values in hydrocarbon

contents than plants grown in polluted soil. Decline percent

in hydrocarbon at these treatment reached 75.3, 85.7, and

75.6%, respectively.

Figure 3. Hydrocarbon content in wheat grains under different treatments of

polluted soil and their control.

3.6. Estimation of Soil Microbial Community

in Different Treatments

The variation in soil microbial community content between

control (unpolluted soil) and different treatments of polluted

soil were indirect estimated through determine the number of

viable cells via spectrophotometry analysis (Table 6).

Table 6. The variation in soil microbial community content between control

and treatments of polluted soil as determined via spectrophotometry

analysis.

Treatment Optical Density

Control 1.2

Phytoremediation 0.8

Phyto + PK 1.5

Phyto + Humic 1.7

Phyto + Bacteria 2.3

Phyto + Nitrogen 1.1

As shown in table (6), the most enriched soil with microbial

community/content comparing with control was as following:

Phyto + Bacteria treatment, Phyto + Humic treatment, Phyto +

PK treatment, and Phyto + Nitrogen treatment. While, the

Phytoremediation treatment exhibited the lowest enriched soil

with microbial community/content comparing with control.

3.7. Analysis of Variations in Microbial

Community Using RAPD and ISSR

Markers

Molecular markers analysis of six DNA samples represent

control and five phyto-chemical treatments were performed

by using 10 RAPD decamer primers and 5 ISSR primer in

order to explore the effect of the different treatments on

structure of soil microbial community comparing with

control (Figure 4). The RAPD reactions produced 138

scorable total bands, out of which 113 found to be

polymorphic. For ISSR, used primer yielded 56 total bands,

out of which 51 bands were polymorphic.

A dendrogram based on UPGMA analysis of the

fingerprints/amplicons obtained from both RAPD and ISSR

markers was constructed (Figure 5). The dendrogram comprise

two main clusters, the first cluster (The major) was subsequently

divided into two subclusters; the first subcluster comprised two

sub-subclusters. The first sub-subcluster including the

Phytoremediation treatment and Phyto. + Humic acid treatment.

Meanwhile, the second sub-subcluster including the control and

Phyto. + PK treatment. While, the second subcluster comprised

the Phyto. + Nitrogen treatment. Meanwhile, the second cluster

involved only the Phyto. + Bacteria treatment.



Figure 4. Agarose gel illustrate the RAPD and ISSR pattern variations of soil microbial communities content between control and treatments as determined

via spectrophotometry analysis.

Figure 5. Phylogenetic analysis based on combined data obtained from ISSR

and RAPD markers.

4. Discussion

4.1. Effect of Spent Engine Oil on Soil

Properties

Oil pollution could lead to significant changes in soil

physiochemical properties, such as bulk density, soil organic

carbon and organic matter, holding capacity, moisture content

and hydraulic conductivity, NH4, and NO3. These data are

agreed with that of Kayode et al. [25] reported increased bulk

density in soil contaminated with spent lubricant oil. The

hydrophobic nature of PHCs influences the water holding

capacity and moisture content of soils. Studies have shown

that soils polluted with PHCs are characterized by lower

water holding capacity, moisture content and hydraulic

conductivity compared with unpolluted soils, also reduced

soil pH together with increases in soil organic carbon and

organic matter on crude oil polluted soils have been recorded


[26]. Increases in total nitrogen, NH4, and NO3 have also

been observed on these soils that polluted with PHCs these

data agreement to Marinescu et al. [27]. The increase in

percent organic carbon and Nitrogen of spent engine oil

treated soil relative to control could be attributed to structural

of spent engine oil that applied to soil. Okonokhua et al. [28]

reported increase in carbon and nitrogen of spent oil treated

soil relative to control. The highest values of P, K, Na, Ca

and Mg were recorded in polluted soil compared to

unpolluted soil. Also there were increases of heavy metals

content in polluted soil sample than in unpolluted soil.

Reduced soil pH caused by the presence of PHC in soils also

favours the availability of heavy metals which may be

absorbed by crops growing on this soil and this can be toxic

to them [29]. These data may be attributed to the nature of

the polluting substance as well as the initial soil properties

[30]. Generally, soil that is polluted with spent engine oil as

source of hydrocarbon (PHCs) is different from unpolluted

soils. These change due to changes in their biological as well

as physicochemical properties [31]. Oil pollution could lead

to significant changes in soil chemical properties, such as

TPH, TOC, C/N and C/P ratios [32].

4.2. Effects of Remediation Treatments on

Hydrocarbon Degradation

In the present study, the treatments of Phyto-Chemical

remediation enhanced the degradation of TPH significantly

and obviously prolonged the validity of Phyto-Chemical

compared with the Phyto separately. Hydrocarbon has been

reported to bind to humic substances strongly depending on

the aromaticity of the humic material [33]. Humic substances

possess many functional groups and have good sorption

characteristics. From the bioremediation point of view this

usually leads to immobilization and consequent decrease in

pollutant toxicity [34]. On the other hand, humic substances

can increase bioavailability of pollutants for degrading

microorganisms among other, by acting as surfactants [35]. In

the presence of permanganate ions, chemical oxidation can

occur [36]. In potassium permanganate oxidation, hydrocarbon

which are in contact with the soil matrix components are

oxidized and their concentration will decrease [37].

Permanganate ions quickly oxidize hydrocarbon alkene

carbon-carbon double bonds [36]. Ferrarese et al. [38] showed

that the oxidation reactions were frequently rapid and appear to

be completed within few hours. However, in order to assess

the total removal efficiency of different reactants including

potassium permanganate, the reactions were not quenched and

were allowed to continue until the complete consumption of all

chemicals before being analysed. The resulting products of

chemical oxidation may or may not be more biologically toxic

than the original compound [39].

4.3. Effect of Spent Engine Oil on Growth

and Chemical Composition of Wheat

Plants

The results clearly showed that Spent Engine Oil

contamination affects on the growth parameters of wheat

plants. Results showed that there was significant difference

in the chemical composition of the wheat grain grown in the

polluted soil, and those grown in the unpolluted soil. This

could be as a result of a hydrophobic layer over the roots

forward by the spent engine oil, which may have limited

water and nutrients absorption necessary for synthesis of

protein and fat in plant. This observation is in line with the

findings of Ogbuehi et al. [40] and Agbogidi et al. [41] who

reported that reduction in protein, crude fiber and at contents

of cassava and maize respectively was due to impairment of

photosynthetic activities through cell injury and disruption of

cell membrane caused by properties of crude oil. Also,

carbohydrates increased as results of hydrocarbon treatments.

These findings may be due to the effect of hydrocarbon

pollutants on metabolism, mobilization and translocation of

carbohydrates.

4.4. Effect of Spent Engine Oil Pollution on

Macronutrients of Wheat Plant and Soil

After Harvesting

Macronutrient contents of wheat grown in the unpolluted

soil recorded the higher values than macronutrient contents

of wheat plants grown in the spent oil polluted soils. These

data agree with Agbogidi et al., [41] who reported that

petroleum products are known to reduce nitrogen availability

in the soil. This could be the cause of adverse effect on the

plant growth parameters in diesel oil polluted soil. The effect

of addition of nutrient amendment on diesel polluted soil was

found to ameliorate the soil condition and enhanced the

growth performance of plant. The adverse effects could be

due to disruption of the absorption and uptake of nutrients by

petroleum products of the polluted soil [42]. These nutrients

(nitrogen, phosphorus, and potassium) are essential to plant

growth and development hence reduction in their

bioavailability will lead to reduction in plant growth.

Similarly, reduction in some essential plant nutrients such as

nitrogen and phosphorus in PHC-polluted soil [43] may

affect proper crop development on these soils. PHCs alter the

fertility status of soils and hence reduce their ability to

support proper crop growth and development [44]. From the

results, it can be concluded that HA, B and PK addition to

Phyto are effective remediation materials for diesel oil

polluted soil and at the same time restored the fertility of the

soil, thus enhancing plant growth and timber productivity.

4.5. Content of Hydrocarbons by Wheat

Grown Under Different Treatments

This study was investigated the content of TPH by wheat

grown in different field-contaminated soils. Naturally, uptake

of hydrocarbon plant increase as the concentration of

hydrocarbon soil increase and translocation of hydrocarbon

that depended on their chemical properties [45]. Results

indicate that wheat plant was effective and promising for the

removal of TPH from highly contaminated soil. Additives of

organic and inorganic compound may promote plant growth



even in oil contaminated soils and thereby positively affect

phytoremediation efficiency. Moreover, the improvement of

soil nutrient conditions through this addition can further

enhance hydrocarbon biodegradation. Since the main

mechanism of phytochemical in oil-polluted soils is based on

the stimulation of soil micro-organisms, it can be assumed

that the higher root biomass obtained with plants provides a

larger rhizosphere for the microbial population and,

therefore, an enhanced degradation of petroleum

hydrocarbons in soils [46]. Tejada et al. [47] also observed

that oil degradation could possibly be further enhanced by

improving plant growth through fertilizer optimization.

4.6. Analysis of Variations in Microbial

Community Using RAPD and ISSR

Markers

Petroleum hydrocarbons in nature are degraded by diverse

groups of soil microorganisms, which have capability to

utilize hydrocarbons as a sole source of carbon and energy.

Exploration and documentation of microbial diversity in a

TPH-contaminated soil is crucial because it helps to identify

novel bacterial strains capable of degrading a wide range of

pollutants. Moreover, they give a background about bacterial

diversity and community changes in various pollutant-

degrading communities. Moreover, the exploration of the

effect of different Phyto-chemical treatments on the soil

microbial communities represent a key and initial step for

developing any bioremediation strategy.

The obtained results from RAPD and ISSR marker

systems successfully revealed a discriminative pattern

between the DNA isolated from soil microbial communities

of control and treatments. The cluster analysis results

exhibited that the Phyto. + Bacteria treatment was clustered

individually, this may be due to the directional enrichment of

soil microflora with particular type of bacteria (Pseudomonas

aeruginosa bacteria addition). In this context, La Rosa et al.,

[22] studied microbial diversity in a polycyclic aromatic

hydrocarbon-impacted soil by 16S rRNA gene sequencing

and amplified fragment length polymorphism (AFLP)

analysis. They results showed that AFLP marker had the

ability to differentiate and track related closely microbes is

fundamental for studying structure and dynamics of

microbial communities in contaminated ecosystems.

While, Patel and Behera [10] assessed the genetic diversity

between 18 metagenomes of Coal mine spoil and their

impact on the microbial ecosystem using twenty RAPD

decamer primers. They results indicated that different coal

mine spoils, through microbiologically distinct, are

interlinked in a sequence as per the age series which reflect

the enrichment of genetic diversity due to the reclamation

progress with the age of coal mine spoil. Also, Tilwari et al.,

[11] investigated the microbial diversity of industrially

contaminated and uncontaminated agriculture field soil using

random amplified polymorphic DNA (RAPD) analysis. They

results confirmed the effects of pollution on the distribution

and biodiversity of soil microorganisms where most of the

native beneficial microorganisms were disappeared or not

cultured under these stress conditions as compared to the

normal agricultural field soils, which is certainly affecting

soil fertility and productivity.

Finally, the availability of simple molecular techniques

such as (RAPD, ISSR, ….ect.) for fast and reliable genotypic

characterization should increase our knowledge of ecology,

structure and dynamics of microbial communities in

contaminated ecosystems. Documentation of microbial

diversity at petroleum-impacted sites will help to formulate

novel strategies for efficient and effective reclamation of

contaminated sites.

5. Conclusion

This study recommend avoiding the uses of

phytoremediation approach separately for cleaning up

hydrocarbon contaminated soils due to the high accumulation

ratio of TPH in plant grains, which consequently can represent

a toxic ratio for human and animal consumption. Therefore,

approaches combining the phytoremediation with other

organic and inorganic compound (such as humic acid,

potassium permanganate and bacteria) were recommended due

to their ability to degrade the TPH in contaminated soil

without accumulate a higher ratio inside the wheat plant grain.

Moreover, additives of organic and inorganic compounds to

phytoremediation treatment represent a significant positive

effect on the microbial communicates in contaminated soil.

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