American Journal of Agricultural Science 2016; 3(3): 48-58 http://www.aascit.org/journal/ajas ISSN: 2381-1013 (Print); ISSN: 2381-1021 (Online)

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. Ahmed 1, Mohamed A. M. Atia 2, *, Gehan H. Abd El-Aziz 1, 3 Ashraf H. Fahmy

1Soils, Water and Environment Research Institute, ARC, Giza, Egypt 2 Keywords Genome Mapping Department, Agricultural Genetic Engineering Research Institute, ARC, Giza, Egypt Phytoremediation, 3 Phyto-chemical Remediation, Plant Genetic Transformation Department, Agricultural Genetic Engineering Research Institute, Petroleum Hydrocarbons, ARC, Giza, Egypt RAPD, Email address ISSR, [email protected] (M. A. M. Atia) Soil Microbial Communities *Corresponding author

Citation Shreen S. Ahmed, Mohamed A. M. Atia, Gehan H. Abd El-Aziz, Ashraf H. Fahmy. Evaluation of Received: April 21, 2016 Phyto-Chemical Remediation Approaches to Remedy Hydrocarbon from Oil Polluted Soils and Accepted: May 9, 2016 Their Impact on Soil Microbial Communities Using RAPD and ISSR Markers. American Journal Published: June 15, 2016 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 of microbial communities of the soil across different represent a fundamental step for studying structure and treatments [11]. dynamics of microbial communities in contaminated Therefore, this study aims to: (1) evaluate and compare the ecosystems. efficiency of different Phyto-chemical approaches for cleaning up hydrocarbon contaminated soils (2) explore the 1. Introduction genetic diversity between microbial communities of different Phyto-chemically treated soils using RAPD and ISSR Soil contamination by petroleum hydrocarbons is one of markers. the world’s most common environmental problems [1]. Total petroleum hydrocarbons (TPHs) are one of the most common 2. Materials and Methods groups of persistent organic contaminants [2]. Generally, the accumulation of contaminants in soils can have destructive 2.1. Soil Used for Experiment effects on both soil ecosystem and human health. Contaminants present in soils can enter the food chain and Unpolluted surface soil (0-25 cm) was collected from an seriously affect animal and human health [3]. In today’s era Agricultural Research Station, Giza. The soil was air dried of heightened environmental awareness and good and ground to 20 meshes before used. Spent engine oil was stewardship of limited natural resources effort to clean up then added to a portion of the unpolluted soil with a dosage contaminated sites involve series of remedial techniques or of 2% of soil mass. approaches ranging from conventional physicochemical 2.2. Experimental Design techniques and natural attenuation to phytoremediation the most emerging biotechnology approach [4]. A greenhouse experiment was carried out to study the Phytoremediation is one of the best developed and effect of different Phyto-chemical remediation treatments on implemented approaches/technologies of bioremediation for the spent engine oil contaminated soil. Seven treatments were cleaning up the environmental pollution. Phytoremediation designed: has been proposed as a cost effective, non-intrusive, and 1). Control (unpolluted soil with wheat planting). environmental friendly technology for the restoration of soils 2). Phytoremediation (Phyto; polluted soil with wheat contaminated with TPH [5]. planting). Studying of the structure and dynamics of an ecosystem is 3). Phytoremediation + Potassium permanganate (PK) used as an indicator to measure the cumulative impact of addition to polluted soil (rate 0.9 M/Kg) (Phyto + PK). multiple stresses on population (s) and its adaptation to the 4). Phytoremediation + Nitrogen addition to polluted soil habitat. Microbial communities, capable of degrading (rate 0.2 g/kg) (Phyto + N). different pollutants in contaminated ecosystems, are relevant 5). Phytoremediation + Humic Acid (HA) addition to in microbial ecology for the development of bioremediation polluted soil (rate 15 g/kg) (Phyto + H). strategies. Analysis of biodiversity is particularly important 6). Phytoremediation + Pseudomonas aeruginosa bacteria when the soil ecosystems respond to changing environmental addition (Phyto + B; the soil was enriched with 30ml of conditions and such changes in the composition of the soil bacterial suspension of Ps. aeruginosa incubated for 48 micro-flora can be crucial for the functional integrity of the h at 28°C in 0.9% NaCl solution) [12]. soil as a main component in agriculture system. In recent Three replicates were sown for each treatment. Four years, several studies were performed to describe bacterial Kilograms crude oil contaminated soil was added. For wheat diversity and community changes in various pollutant- (Triticum aestivum L.) planting, 10 seeds were sown evenly degrading communities [6, 7, 8] and a number of molecular to the soil in each pot and covered with 2–3 cm of soil on the methods have been developed for describing and comparing top (4 Kg soil added in total for each pot). Pots were irrigated complex microbial communities [9]. Polymerase chain every day of field capacity. All of the experiment pots were reaction (PCR) has been successfully used for microbial placed in a greenhouse at 30°C. Seven days after seeds identification in the environmental context. germinated, 5 healthy seedlings were preserved in each pot PCR-Based molecular markers have been potentially used for further remediation. Soil samples were taken after 0, 15 to survey and explore the diversity of soil microbial 30, 45, 60, 75, 90, 105 and 120 days. Then the soil samples communities, bacterial taxonomy and phylogeny. Randomly were divided into two sub samples: one was used to study the amplified polymorphic DNA (RAPD) and Inter-simple variations of soil physicochemical properties and evaluation sequence repeats (ISSR) based detection of genetic of hydrocarbon degradation. polymorphism has been successfully utilized to identify isolates, genetic diversity and population structure of 2.3. Analytical Methods bacteria; to demonstrate genetic variation within the species; Some Physical and chemical characteristics of the studied and to elucidate the distribution of genes and population soil was determined according to Page et al . [13]. Total structure of the species [10]. RAPD markers have also been petroleum hydrocarbon were extracted from soil and plant utilized for inter-specific and intra-specific genetic diversity 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 performed in a thermal cycler (Applied BioSystems, USA) analyzed using a Hewlett Packard (HP) 5890 Series II gas programmed for initial denaturation of 5 min at 94°C; 40 chromatograph (GC) with a 5971A mass selective detector cycles of 2 min denaturation at 94°C, 1 min annealing at (MSD), a HP 7673 autosampler, and HP Chemstation 36°C and 2 min extension at 72°C; and final elongation step software. The instrument was operated in the splitless mode at 72°C for 7min. The PCR products were electrophoresed on with 1 µL injections onto a 30 m x 0.25 mm x 0.25 µm RTX- 1.5% agarose gel containing ethidium bromide (0.5 µg/mL) 5 (5% phenylmethylsiloxane) capillary column. The run time in TBE buffer for 2 h at 100 V. After electrophoresis, the gels to elute all the target compounds was about 35 minutes, but were observed under an UV-transilluminator, documented in the full cycle time was about 60 minutes. In plant sample, Gel-Doc XR (Bio-Rad) and photographed. The size of the phosphorus content was determined by vanadomolybdate amplicons was determined using 100 bp DNA ladder plus. yellow method spectrophotometrically and potassium by flame photometer [15]. Heavy metal contents samples were 2.7. ISSR Marker Analysis extracted according to the method of Lindsay and Novell [16]. Total nitrogen was determined by micro-Kjeldahl Five ISSR primers were used in the present study. DNA method according to AOAC [17]. Crude protein was was amplified according to the following protocol. Each PCR calculated by multiplying the values of total nitrogen in 6.25. reaction mix of 25 µl contained the 30 ng template DNA, 2.5 Total lipid was determined according to AOAC [18]. Total µl of 10X PCR buffer, 1.5 µl of 25mM MgCl2, 2.5 µl of the carbohydrate was extracted according to Smith et al . [19] and dNTPs mix, 30 pmol of ISSR primer, 1.0 U Taq DNA determined using spectrophotometer according to Murphy polymerase (Promega, WI, USA). The amplification was [20]. All data were statistically analyzed using Costat performed in a thermal cycler (Applied BioSystems, USA) computer program according to procedures outlined by programmed for initial denaturation of 5 min at 94°C; 40 Snedecor and Cochran [21]. 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 2.4. Soil Sampling and Estimation of at 72°C for 7 min. The PCR products were electrophoresed Microbial Community 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 Soil samples were collected from both control and were observed under an UV-transilluminator, documented in treatments, and then stored at 4°C till DNA isolation and Gel-Doc XR (Bio-Rad) and photographed. The size of the molecular analysis. For each sample (control and treatments), amplicons was determined using 100 bp DNA ladder plus. 10 g was suspended into 90 mL of sterile doubled distilled water (ddH 2O) and stirred using shaker at 200 rpm for 1 hour. 2.8. Markers Data Analysis 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 The generated/ amplified bands were scored visually. The of LB broth medium and incubated at 37°C for 24 h for bands were scored as present (1) or absent (0) to create the microbial growth. The number of viable cells was determined binary data set. To estimate the genetic similarity, Jaccard’s by serial dilution technique and spectrophotometry as coefficient was used [24]. A dendrogram was generated by indirect approach to estimate the microbial community’s cluster analysis using the un-weighted pair group method of variations between control and treatments [22]. the arithmetic averages (UPGMA) using SPSS program V1.6. Support for clusters was evaluated by bootstrapping 2.5. Bacterial DNA Isolation analysis. One thousand permutation data sets were generated by re-sampling with replacement of characters within the The bacterial chromosomal DNA was extracted from combined 1/0 data matrix. whole bacterial community (Gram Positive and Gram Negative Bacteria) using Wizard Genomic DNA Purification Kit (Promega, WI, USA) according to manufacturer 3. Results instructions. The DNA was quantified with NanoDrop 3.1. Effect of Spent Engine Oil on Soil Spectrophotometer (Thermo Fisher Scientific Inc.). All Properties samples were adjusted to a concentration of 10 ng/µl for subsequent molecular analyses. The physicochemical properties of the tested soil shown in Table 1. Chemical properties of spent engine oil are 2.6. RAPD Marker Analysis presented in Table 2. Ten RAPD decamer primers (Operon Tech., Alameda, CA, Soil physicochemical analysis was done after the addition USA) were used in the present study. DNA was amplified of spent engine oil as source of hydrocarbon (PHCs). Results following the protocol of Adawy and Atia [23]. Each PCR of the physicochemical analysis of the soil samples were reaction mix of 25 µl contained the 30 ng template DNA, 2.5 reported in the table (3). Results of the pH revealed that the µl of 10X PCR buffer, 1.5 µl of 25mM MgCl2, 2.5 µl of the pH of the polluted soil sample was slight decrease compared dNTPs mix, 30 pmol of RAPD primer, 1.0 U Taq DNA to unpolluted soil as a control. The results of the organic polymerase (Promega, WI, USA). The amplification was carbon revealed that there was progressive increase in American Journal of Agricultural Science 2016; 3(3): 48-58 51

organic matter of polluted soil; increase reached 4.6 fold than Table 3. Comparison of spent engine oil polluted and unpolluted soils before in unpolluted soil as a control. Similar trend was found in the planting. conductivity (EC) of the soils; the conductivity value of the Parameter Polluted Unpolluted soil after polluted was found maximum (2.98) and minimum Moisture content (%) 3.19 6.93 (1.78) in control. Data of moisture content of oil polluted Bulk density (gcm -3) 1.40 1.29 soils were lower than the control sample, decrease reached ECe dS m -1, soil paste extract 2.98 1.78 54%. The bulk density of spent engine oil treated soil pH 7.68 7.99 generally increased compared to unpolluted soil, increase NH4 (ppm) 99.4 49.4 reached 8.5%. The increase in bulk density of spent engine NO3 (ppm) 49.7 39.76 oil treated soil could be attributed to compaction resulting Organic carbon (%) 7.66 1.68 from oil contamination as well as reduced porosity. Also, Total N (%) 2.1 0.19 data of the some macro, micronutrient and heavy metals P % 1.28 1.02 revealed that there was increase in metals concentrations of K % 1.33 0.44 polluted soil relative to control (unpolluted soil). Na % 1.3 0.49 Ca (mgkg -1) 186 16 -1 Table 1. Some and chemical properties of the tested soil under different Mg (mgkg ) 8 6 experiments. Heavy metals Mn +2 9.88 9.18 Physical properties Value Fe +2 625.9 560 Coarse sand% 7.3 Co +2 0.615 0.382 Fine sand% 19.9 Ni +2 51.7 0.680 Silt% 38.3 Cu +2 7.51 3.9 +2 Clay% 34.5 Zn 29 10.6 Cd +2 2.5 0.2 Texture soil loamy clay Pb +2 108 2.86 Chemical properties TPH (mgkg -1) 17610 104 pH (1: 2.5, soil - water suspension) 7.99 Organic matter (%) 1.15 3.2. Effects of Remediation Treatments on Ece dS m -1, soil paste extract 1.78 Hydrocarbon Degradation Soluble cations (me/L) Five different treatments (Phyto, Phyto + N, Phyto + PK, Ca ++ 7.1 ++ Phyto + HA and Phyto + B) were individually used for Mg 3.9 degradation of soil polluted with TPH compared with + Na 5.1 control. The degradation rate of hydrocarbon (fig. 1) using + K 1.5 addition of different treatments was in the following Soluble anions (me/L) ascending order: Phyto + N (16.7%), Phytoremediation = CO3 - (40.0%), Phyto + PK (61.5%), Phyto + B (63.7%), Phyto + HCO3 - 4.1 Cl - 7.1 HA (76.0%). On the other hand, the effect of HA or B or PK SO4 = 6.4 without Phyto on TPH degradation was calculated and recorded as 36, 23.7 and 21.5%, respectively. HA and B Table 2. Chemical properties of spent engine oil. addition with Phyto significantly stimulated the degradation of hydrocarbon at the initial time. 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 Figure 1. Effect of different treatments on hydrocarbon degradation% in Pb (mg/kg) 105.3 soil. 52 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

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

Data of residual hydrocarbon are presented in figure (2). respectively. On the other hand, wheat plants grown in spent Data showed that all treatments under investigation oil polluted soils with addition of HA recorded the highest decreased strongly hydrocarbon concentration. Percentages dry weight which was significantly different from the other of residual hydrocarbon at the end time of experiment (120 treatments. However, plants in the control experiments days) reached 59.9, 38.5, 23.9, and 36.3%, respectively. It recorded the highest dry weight. worth mention, Phytoremediation combined with HA, B Protein and fat content grain of wheat grown in the control and PK was more effective for cleaning up hydrocarbon experiment (unpolluted soil) recorded the highest values. The contaminated soils than phytoremediation individually. The values were significantly different from that of the other hydrocarbon degradation efficiency of Phyto + HA treatments. On the contrary, grain of wheat plants grown in treatment was more effective than others. At the end time the spent oil polluted soils produced (Phyto) the lowest (120 days), the hydrocarbon degradation rate increased at values of protein and fat (Table 4). HA treatment recorded different degrees under different treatments compared with the highest values in protein and fat followed with B and PK the control. treatments. Protein and fat contents were observed to be higher in the wheat plants grow in the control experiment 3.3. Effect of Spent Engine Oil on Growth (unpolluted soil). In contrast, percentage of protein and fat and Chemical Composition of Wheat was decreasing in the grain of the treated wheat, as the Plants addition of the spent oil (Phyto). Concerning the total Results of the study showed that there was significant carbohydrates (Table 4), data indicated that grain of wheat difference in the chemical composition of the wheat grain that grown in the spent oil polluted soils recorded the highest grown in the polluted soil, and those grown in the unpolluted value compared to control (unpolluted) and other treatments. soil. Wheat grown in spent engine oil treated soil (Table 4) Treatments of HA and B were recorded the same trended. recorded the lowest dry weight which was significantly Further, PK treatment recorded the higher value of different (P<0.05) from that of the control. Decline percent carbohydrate than HA and B treatments. reached 53.4 and 48.6% for plant and grain dry weight, Table 4. Effect of spent engine oil pollution on chemical composition % of wheat grains.

Dry weight (g/pot) Treatment Carbohydrates% Fat% Protein% Plant grain Control (unpolluted soil) 13.10 A 6.17 A 67.47 D 1.54 A 11.33 B Phytoremediation 6.33 E 3.17 E 72.50 A 1.02 E 10.63 C Potassium permanganate 8.00 D 3.60 D 69.43 B 1.22 D 11.62 B Humic acid 9.40 B 5.23 B 68.17 C 1.40 B 12.86 A Bacteria 8.43 C 4.07 C 68.60 C 1.32 C 11.61 B LSD at 0.050 0.1191 0.1031 0.6440 0.0595 0.6101 American Journal of Agricultural Science 2016; 3(3): 48-58 53

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

Plant Soil Treatment N P K N P K Control (unpolluted soil) 1.76 B 0.23 A 0.40 B 0.10 C 0.41 D 0.32 C Phytoremediation 1.66 C 0.16 B 0.36 B 0.41 B 1.12 A 0.97 B Potassium permanganate 1.81 B 0.20 AB 0.51 A 0.39 B 0.97 C 1.18 A Humic acid 2.00 A 0.21 AB 0.45 AB 1.60 A 1.12 A 0.95 B Bacteria 1.81 B 0.21 AB 0.43 AB 0.36 B 0.98 BC 0.93 B LSD at 0.050 0.10 0.06 0.10 0.15 0.06 0.06

3.6. Estimation of Soil Microbial Community 3.4. Effect of Spent Engine Oil Pollution on in Different Treatments Macronutrients of Wheat Plant and Soil After Harvesting The variation in soil microbial community content between control (un- polluted soil) and different treatments of polluted Data of the effect of spent engine oil pollution on soil were indirect estimated through determine the number of macronutrients of wheat grain and soil are presented in viable cells via spectrophotometry analysis (Table 6). Table (5). Macronutrient contents (Nitrogen, phosphorus, and potassium) of wheat grown in the unpolluted soil Table 6. The variation in soil microbial community content between control recorded the higher values than macronutrient contents of and treatments of polluted soil as determined via spectrophotometry wheat plants grown in the spent oil polluted soils. Decline analysis. percent reached 5.68, 30.4, and 10.0%, respectively. On the Treatment Optical Density other hand, significant effect was observed when wheat Control 1.2 plants grown in spent oil polluted soils with addition of Phytoremediation 0.8 different treatments. Phyto + PK 1.5 Phyto + Humic 1.7 3.5. Content of Hydrocarbons by Wheat Phyto + Bacteria 2.3 Grown Under Different Treatments Phyto + Nitrogen 1.1

Content of TPH by wheat grown in different field- As shown in table (6), the most enriched soil with microbial contaminated soils was investigated. TPH concentrations in community/content comparing with control was as following: grain correlated positively with the corresponding Phyto + Bacteria treatment, Phyto + Humic treatment, Phyto + concentrations in soils (Figure 3). Result of the experiment PK treatment, and Phyto + Nitrogen treatment. While, the indicated that wheat grain that grown contaminated soils Phytoremediation treatment exhibited the lowest enriched soil (phyto) had higher concentrations of total petroleum with microbial community/content comparing with control. hydrocarbon compare to unplanted soil (control). Increase 3.7. Analysis of Variations in Microbial percent reached 80.6% related to unpolluted soil. On the Community Using RAPD and ISSR other hand, progressive effect was observed when wheat Markers plants grown in spent oil polluted soils (phyto) with addition of different treatments (PK, HA, and B). All Molecular markers analysis of six DNA samples represent treatments recorded the lower values in hydrocarbon control and five phyto-chemical treatments were performed contents than plants grown in polluted soil. Decline percent by using 10 RAPD decamer primers and 5 ISSR primer in in hydrocarbon at these treatment reached 75.3, 85.7, and order to explore the effect of the different treatments on 75.6%, respectively. 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 Figure 3. Hydrocarbon content in wheat grains under different treatments of the Phyto. + Nitrogen treatment. Meanwhile, the second cluster polluted soil and their control. involved only the Phyto. + Bacteria treatment. 54 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

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.

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 Figure 5. Phylogenetic analysis based on combined data obtained from ISSR soil pH together with increases in soil organic carbon and and RAPD markers. organic matter on crude oil polluted soils have been recorded American Journal of Agricultural Science 2016; 3(3): 48-58 55

[26]. Increases in total nitrogen, NH 4, and NO 3 have also contamination affects on the growth parameters of wheat been observed on these soils that polluted with PHCs these plants. Results showed that there was significant difference data agreement to Marinescu et al . [27]. The increase in in the chemical composition of the wheat grain grown in the percent organic carbon and Nitrogen of spent engine oil polluted soil, and those grown in the unpolluted soil. This treated soil relative to control could be attributed to structural could be as a result of a hydrophobic layer over the roots of spent engine oil that applied to soil. Okonokhua et al . [28] forward by the spent engine oil, which may have limited reported increase in carbon and nitrogen of spent oil treated water and nutrients absorption necessary for synthesis of soil relative to control. The highest values of P, K, Na, Ca protein and fat in plant. This observation is in line with the and Mg were recorded in polluted soil compared to findings of Ogbuehi et al. [40] and Agbogidi et al. [41] who unpolluted soil. Also there were increases of heavy metals reported that reduction in protein, crude fiber and at contents content in polluted soil sample than in unpolluted soil. of cassava and maize respectively was due to impairment of Reduced soil pH caused by the presence of PHC in soils also photosynthetic activities through cell injury and disruption of favours the availability of heavy metals which may be cell membrane caused by properties of crude oil. Also, absorbed by crops growing on this soil and this can be toxic carbohydrates increased as results of hydrocarbon treatments. to them [29]. These data may be attributed to the nature of These findings may be due to the effect of hydrocarbon the polluting substance as well as the initial soil properties pollutants on metabolism, mobilization and translocation of [30]. Generally, soil that is polluted with spent engine oil as carbohydrates. source of hydrocarbon (PHCs) is different from unpolluted soils. These change due to changes in their biological as well 4.4. Effect of Spent Engine Oil Pollution on as physicochemical properties [31]. Oil pollution could lead Macronutrients of Wheat Plant and Soil to significant changes in soil chemical properties, such as After Harvesting TPH, TOC, C/N and C/P ratios [32]. Macronutrient contents of wheat grown in the unpolluted 4.2. Effects of Remediation Treatments on soil recorded the higher values than macronutrient contents Hydrocarbon Degradation of wheat plants grown in the spent oil polluted soils. These data agree with Agbogidi et al ., [41] who reported that In the present study, the treatments of Phyto-Chemical petroleum products are known to reduce nitrogen availability remediation enhanced the degradation of TPH significantly in the soil. This could be the cause of adverse effect on the and obviously prolonged the validity of Phyto-Chemical plant growth parameters in diesel oil polluted soil. The effect compared with the Phyto separately. Hydrocarbon has been of addition of nutrient amendment on diesel polluted soil was reported to bind to humic substances strongly depending on found to ameliorate the soil condition and enhanced the the aromaticity of the humic material [33]. Humic substances growth performance of plant. The adverse effects could be possess many functional groups and have good sorption due to disruption of the absorption and uptake of nutrients by characteristics. From the bioremediation point of view this petroleum products of the polluted soil [42]. These nutrients usually leads to immobilization and consequent decrease in (nitrogen, phosphorus, and potassium) are essential to plant pollutant toxicity [34]. On the other hand, humic substances growth and development hence reduction in their can increase bioavailability of pollutants for degrading bioavailability will lead to reduction in plant growth. microorganisms among other, by acting as surfactants [35]. In Similarly, reduction in some essential plant nutrients such as the presence of permanganate ions, chemical oxidation can nitrogen and phosphorus in PHC-polluted soil [43] may occur [36]. In potassium permanganate oxidation, hydrocarbon affect proper crop development on these soils. PHCs alter the which are in contact with the soil matrix components are fertility status of soils and hence reduce their ability to oxidized and their concentration will decrease [37]. support proper crop growth and development [44]. From the Permanganate ions quickly oxidize hydrocarbon alkene results, it can be concluded that HA, B and PK addition to carbon-carbon double bonds [36]. Ferrarese et al . [38] showed Phyto are effective remediation materials for diesel oil that the oxidation reactions were frequently rapid and appear to polluted soil and at the same time restored the fertility of the be completed within few hours. However, in order to assess soil, thus enhancing plant growth and timber productivity. the total removal efficiency of different reactants including potassium permanganate, the reactions were not quenched and 4.5. Content of Hydrocarbons by Wheat were allowed to continue until the complete consumption of all Grown Under Different Treatments chemicals before being analysed. The resulting products of This study was investigated the content of TPH by wheat chemical oxidation may or may not be more biologically toxic grown in different field-contaminated soils. Naturally, uptake than the original compound [39]. of hydrocarbon plant increase as the concentration of 4.3. Effect of Spent Engine Oil on Growth hydrocarbon soil increase and translocation of hydrocarbon and Chemical Composition of Wheat that depended on their chemical properties [45]. Results Plants indicate that wheat plant was effective and promising for the removal of TPH from highly contaminated soil. Additives of The results clearly showed that Spent Engine Oil organic and inorganic compound may promote plant growth 56 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 even in oil contaminated soils and thereby positively affect cultured under these stress conditions as compared to the phytoremediation efficiency. Moreover, the improvement of normal agricultural field soils, which is certainly affecting soil nutrient conditions through this addition can further soil fertility and productivity. enhance hydrocarbon biodegradation. Since the main Finally, the availability of simple molecular techniques mechanism of phytochemical in oil-polluted soils is based on such as (RAPD, ISSR, ….ect.) for fast and reliable genotypic the stimulation of soil micro-organisms, it can be assumed characterization should increase our knowledge of ecology, that the higher root biomass obtained with plants provides a structure and dynamics of microbial communities in larger rhizosphere for the microbial population and, contaminated ecosystems. Documentation of microbial therefore, an enhanced degradation of petroleum diversity at petroleum-impacted sites will help to formulate hydrocarbons in soils [46]. Tejada et al . [47] also observed novel strategies for efficient and effective reclamation of that oil degradation could possibly be further enhanced by contaminated sites. improving plant growth through fertilizer optimization. 4.6. Analysis of Variations in Microbial 5. Conclusion Community Using RAPD and ISSR This study recommend avoiding the uses of Markers phytoremediation approach separately for cleaning up Petroleum hydrocarbons in nature are degraded by diverse hydrocarbon contaminated soils due to the high accumulation groups of soil microorganisms, which have capability to ratio of TPH in plant grains, which consequently can represent utilize hydrocarbons as a sole source of carbon and energy. a toxic ratio for human and animal consumption. Therefore, Exploration and documentation of microbial diversity in a approaches combining the phytoremediation with other TPH-contaminated soil is crucial because it helps to identify organic and inorganic compound (such as humic acid, novel bacterial strains capable of degrading a wide range of potassium permanganate and bacteria) were recommended due pollutants. Moreover, they give a background about bacterial to their ability to degrade the TPH in contaminated soil diversity and community changes in various pollutant- without accumulate a higher ratio inside the wheat plant grain. degrading communities. Moreover, the exploration of the Moreover, additives of organic and inorganic compounds to effect of different Phyto-chemical treatments on the soil phytoremediation treatment represent a significant positive microbial communities represent a key and initial step for effect on the microbial communicates in contaminated soil. developing any bioremediation strategy. The obtained results from RAPD and ISSR marker References systems successfully revealed a discriminative pattern [1] US EPA. (2000). Introduction to phytoremediation. between the DNA isolated from soil microbial communities Environmental Protection Agency, USA. Page 5. of control and treatments. 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