Journal of Environmental Protection, 2012, 3, 748-759 doi:10.4236/jep.2012.38089 Published Online August 2012 (http://www.SciRP.org/journal/jep) Factorial Designs Application to Study Enhanced Bioremediation of Soil Artificially Contaminated with Weathered Bonny Light Crude Oil through Biostimulation and Bioaugmentation Strategy Samuel E. Agarry * , Oladipupo O. Ogunleye Biochemical Engineering and Biotechnology Research Laboratory, Department of Chemical Engineering, Ladoke Akintola Univer- sity of Technology, Ogbomosho, Nigeria. Email: { * sam_agarry, ooogunleye}@yahoo.com Received May 12 th , 2012; revised June 9 th , 2012; accepted July 11 th , 2012 ABSTRACT The objective of this study was designed to evaluate the effects of biostimulation and bioaugmentation amendment agents (NPK fertilizer, Tween 80 and mixed culture) on the bioremediation of tropical soil samples artificially con- taminated with Weathered Bonny Light Crude Oil (WBLCO). Response Surface Methodology (RSM) with Box Behnken Design (BBD) was used with three levels and three factors of NPK fertilizer (2 - 6 g), Tween 80 (5 - 15 mg/l) and mixed culture (0.5 - 1.5 g/l) as independent variables and WBLCO removal as dependent variable (response) in a six weeks remediation period. The results showed that the rate of WBLCO removal generally increased with increase in the amount of NPK fertilizer, Tween 80 and mixed culture (biomass), respectively. A statistically significant (P < 0.0001) second-order quadratic regression model for WBLCO removal (using design-expert statistical program (v. 6.0.8)) with a coefficient of determination, R (=0.9996) was obtained. Numerical optimization technique based on de- sirability function was carried out to optimize the bioremediation process. The optimum values for biostimulation and bioaugmentation amendment agents to achieve a predicted maximum WBLCO removal of 84.88 percent were found to be: NPK fertilizer, 4.25 g; Tween 80, 10.22 mg/l and mixed culture, 0.46 g/l. At this optimum point, the observed WBLCO removal was found to be 83.79 percent. The statistical analyses and the closeness of the experimental results and model predictions show the reliability of the regression model and thus, biostimulation and bioaugmentation of in- digenous microbial density and activity can reduce remediation period of petroleum hydrocarbon contaminated envi- ronment and subsequently the cost of remediation. Keywords: Bioremediation; Box-Behnken Designs; Crude Oil; Second-Order Quadratic Regression Model 1. Introduction Crude oil is an extremely complex mixture of aliphatic and aromatic hydrocarbons, including volatile compo- nents of gasoline, petrol, kerosene, lubricating oil and solid asphaltene residues. In developed and developing countries, contamination of soil and marine environment by crude oil and petroleum products has become a seri- ous problem. The main sources of this contamination can originate from natural oil seepage and human activities including extraction, transportation, utilization of petro- leum (crude oil and natural gas), oil field installations, petroleum plants (refining), liquid fuel distribution and storage devices, transportation equipment for petroleum products, airports and illegal drillings in pipe lines [1-3]. The scale of the hazards imposed on the natural envi- ronment depends on the surface of the area contaminated by petroleum products, their chemical composition, and the depth at which pollutants occur. Crude oil causes a variety of risks when released into the environment. It is physically, chemically and bio- logically harmful to soil because of the presence of many toxic compounds, such as polycyclic aromatic hydrocar- bons, benzene and its substituted and cycloalkane rings, in relatively high concentrations. The fate and effects of spilled crude oil and its products in soils have already been the subject of several studies [4-6]. Biodegradation of hydrocarbon compounds is one of the most important processes involved in the weathering and eventual re- moval of oil from the environment, particularly for its non-volatile components. Thus, potentially biodegrada- * Corresponding author. Copyright © 2012 SciRes. JEP