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Paper in PDF(5920K) 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)
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