Crude oil is the most important source of energy worldwide; however, routine operations of extraction and drilling of this fossil energy resource cause serious environmental problems [1
]. Crude oil contains a wide range of compounds that pose a significant risk for the environment and human health and have cytotoxic, mutagenic, and carcinogenic effects [2
]. Reducing the petroleum hydrocarbon compounds in a polluted environment becomes a significant challenge for oil companies that are forced to conduct an adequate and effective treatment of these pollutant emissions. Thermal treatment, soil washing, soil vapor extraction, solidification, and stabilization are physical and chemical techniques used to treat petroleum hydrocarbon-polluted soil [3
]. However, they are often expensive, ineffective, and rarely neutral [4
Bioremediation of hydrocarbons in polluted soils is a promising treatment method [5
]. Based on the principle of complete mineralization or transformation of petroleum products into less toxic forms by different groups of microorganisms [7
], bioremediation is the most effective, non-invasive, the least expensive and eco-friendly technique [8
]. Conservation of soil texture and characteristics are among the advantages of bioremediation. Besides, physical and chemical properties of the soil, such as aeration, pH, water-holding capacity, and ion exchange capacity can be improved after bioremediation [11
This process occurring naturally can be accelerated by bio-stimulation. This strategy is based on stimulation of the catabolic activity of indigenous microorganisms by the addition of nutrient-rich organic and inorganic materials, supplying oxygen or other electron acceptors, and by maintaining suitable conditions of temperature, pH, and moisture [7
]. In arid areas, where soils are poor in organic and mineral nutrient matters, and are usually subjected to extreme environmental conditions (high temperatures and irradiance) [14
]; the rate of degradation of complex hydrocarbon compounds from crude oil polluted sites is usually limited by biodegrading microbiota [15
Bio-stimulants with promising results, like carob kibbles, sugarcane bagasse, sugarcane molasses, wheat straw, banana skin, yam peel, saw dust, spent brewing grain, rice husk, and coconut shell have been used earlier [16
Two important hypotheses have been proposed to elucidate the mechanisms involved in the enhanced removal of hydrocarbon petroleum products from soils using bio-stimulants [20
]. The first one suggested that, nutrient matters, especially nitrogen and phosphorus, usually considered as the limiting factors for the bioremediation of contaminated soils [21
], once provided by the bio-stimulants, enhance substantially the growth of hydrocarbon degrading bacteria [22
The second hypothesis is based on the ability of bio-stimulants to release biosurfactants that increase the bioavailability of poorly soluble hydrocarbon petroleum compounds [27
]. For instance, Yi and Crowley [27
] found that plants produced fatty acids acted as biosurfactants significantly enhancing pyrene and benzo[a
]pyrene degradation when added directly to polluted soil.
Furthermore, bioavailability is governed by the interactions between microorganisms and the environmental conditions (pH, temperature, etc.) as well as the physico-chemical interactions between polluting compounds and the soil matrix [30
]. Therefore, bioavailability of polluting hydrocarbons to degrading bacteria can be related to soil mineral composition, which is usually assessed using X-ray diffraction analysis [26
The aim of this work was to study the efficacy of two bio-stimulants, i.e., carrot peel waste and carob kibbles, to degrade crude oil polluted soil as a judicious alternative to expensive physical and chemical treatments.
2. Materials and Methods
2.1. Crude Oil Polluted Soil Origin
Crude oil polluted soil was collected at a disused oil-drilling quagmire in the Hassi Messaoud field (Algeria). Samples were collected at 0–50 cm depth using a stainless steel sampler, placed in appropriate containers thoroughly mixed therein.
2.2. Carrot Peel Waste Medium
Carrots, Daucus carota
, were purchased from a local vegetable market then peeled. The peels were mixed and macerated at a ratio of 1 kg in 2.5 L of distilled water at 85 °C for 45 min with continuous stirring [32
]. After filtration and decantation, the medium was autoclaved at 120 °C for 20 min and stored at 4 °C before its use as a bio-stimulating medium.
2.3. Carob Kibbles Medium
Dry pods of carob, Ceratonia siliqua
, were obtained from locality of Ighil-Ali (Bejaïa, Algeria). Pods were cut and manually de-seeded. Carob kibbles were then pitted and macerated at a ratio of 1 kg in 2.5 L of distilled water at 85 °C for 45 min with continuous stirring [32
]. The mash was filtered, decanted, autoclaved at 120 °C for 20 min, and stored at 4 °C before its use as a bio-stimulating medium.
2.4. Experimental Design
In order to evaluate the effectiveness of hydrocarbon degradation, 4 mL of carrot peel waste or carob kibbles media were added to 200 g of crude oil polluted soil samples, resulting in slurry mixtures placed in circular cells and mixed thoroughly. Experiments were also conducted on unamended control soil samples for the sake of comparison. A total of 6 cells were used in the study and each treatment was carried out in duplicate. Cells containing treatment materials or the control were run in the open air and mixed every 3 days to ensure homogenous distribution during 45 days of remediation tests.
2.5. Characterization of Carrot Peel Waste and Carob Kibbles
Total soluble solids (TSS) were determined by desiccation in the oven at 105 °C until a constant weight while pH was determined using pH meter (Accumet AE150 instrument; Fisher Scientific, Illkirch, France). Calcium, magnesium, phosphorus, nitrate and nitrite were determined by digital titrator. All measurements were performed in triplicate, and the mean was used for analyses.
2.6. Physico-Chemical Characterization of Crude Oil
2.6.1. X-ray Diffraction (XRD)
The soil samples were prepared for XRD measurement by orienting them in a glass slide following standard procedure. The slides were air dried and placed in a desiccators containing silica gel to prevent rehydration. X-ray powder diffraction of the prepared materials was carried out using a Philips diffractometer with nickel-filtered Cu Kα radiation of wavelength 1.5406 Å. Powder diffraction patterns were obtained between 10° and 80° with a scan speed of 5 degree/min. Data were interpreted by reference to X’Pert accompanying software program High Score Plus in conjunction with data from the ICDD (International Centre for Diffraction Data) Powder Diffraction File [33
2.6.2. Gas Chromatographic (GC-FID) Analysis
The soil samples were extracted using methylene chloride solvent. Aliquot of extracts as well as pure fuel were analyzed using an Agilent 6890 Series II gas chromatograph equipped with a flame ionization detector and an on-column injector. Separation was achieved using a 25 m 0.32 mm internal diameter fused silica capillary column. The operating conditions were: Temperature program 40°–280 °C at 4 °C/min, injector temperature 280 °C, detector temperature 280 °C, and carrier gas: H2 (1 mL/min).
2.6.3. Physicochemical Parameters Measurements of Crude Oil Polluted Soil
The temperature and pH were determined using thermometer (MRC 201, France) and, pH meter (Accumet AE150 instrument; Fisher Scientific, Illkirch, France), respectively.
Total petroleum hydrocarbon (TPH) percentage of soil samples was measured by distillation using Fann distiller according to API recommendations [34
]. This method is used to determine the percentage of water/oil in the crude oil. 20 mL of each soil sample is placed in a distiller, then heated up to 800 °C. The vapors of water and oil are then condensed back into liquid form and collected (distillate). After about 30 to 60 min of decantation, the volumes of water and oil are read directly. After distillation, the remaining mass of mud is weighed. The percentages of water and oil are directly determined. Two replications were conducted for all measurements. The residual moisture of crude oil polluted soil samples was determined (in duplicate) by the difference in weight before and after drying in a vacuum oven at 105 °C for 3 h in the presence of P2
2.7. Microbiological Analysis
Two replicate samples from each crude oil polluted soil amended with carrot peel waste, carob kibbles, and unamended control soil were withdrawn at the end of second, fourth, and sixth week of the study for the enumeration of total aerobic heterotrophic bacteria. Aliquots of serially diluted samples (0.1 mL) were plated on nutrient agar medium (Oxoid). All inoculated plates were incubated aerobically at room temperature and counted after 48 h.
2.8. Statistical Analysis
Data were statistically analyzed by Data Analysis Tool pack of Microsoft Office Excel 2007 (Microsoft, New York, NY, USA). Excel was used for data management and exploratory data analyses. Excel was also used for drawing of graphs and bar charts regarding temperatures, pH, TPH, residual moisture, and microbiological analysis of crude oil polluted soil amended with carrot peel waste, carob kibbles, and unamended control soil during 45 days of treatments. p < 0.05 was used to judge statistical significance.