Full Length Research Paper
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
Bioremediation of diesel-contaminated soil which involves the use of augmented bacteria and pig dung to reduce high levels of diesel to levels that can be harmless/safe and as a result, will minimize the subsequent damages caused to the environment. Bacteria either pure or mixed cultures used for the bioremediation of diesel-contaminated soils require nutrients, carbon and energy sources to grow and proliferate in harsh polluted environments (Lahel et al., 2016). Also, of importance is that, biodegradation/bioremediation rates of these microbes depend on the hydrocarbon composition and environmental conditions such as temperature, pH, moisture content, bioavailability of the pollutant, contamination levels and the presence of additional nutrients (such as pig dung in this research). Competition between indigenous and exogenous microorganisms for limited carbon sources, as well as antagonistic interactions and predation by protozoa and bacteriophage determines the ï¬nal outcome of the bioremediation process (Franco et al., 2014; Fernandez et al., 2016; Lahel et al., 2016). The soil sample (soil sample before pollution) in this study was nutrient deficient (Table 1). The moisture content was also low and this is an important factor that can have an adverse effect on the metabolic activities of microorganisms during biodegradation of hydrocarbon (Van hammed et al., 2003; Thompson et al., 2005; Abu et al., 2016).
The pH of the soil was 6.9, which was within the optimum range for microbial activities. It has been reported that such soil is ideal for hydrocarbon degrading microorganisms to be adapted in a bioaugmentation process (Lahel et al., 2016). The pig dung contains more nutrients (such as sodium, magnesium, calcium and nitrogen) as compared to that of the unpolluted soil sample. This is an additional nutrient source for the indigenous microorganisms and the inoculated bacteria. The presence of diesel in the soil samples had an adverse effect on the initial bacterial populations (Figure 1). However, some of the indigenous and the augmented bacteria that can be described as hydrocarbon utilizers (Table 2), soon adapted to the diesel-contaminated environment and utilized the diesel as a substrates for growth and this is the reason for increased bacterial population from the 9th day (Yakimov et al., 2007; Maduka and Okpokwasili, 2016). The bacterial activities in Tray “A” must have been boosted by additional nutrient from the pig dung and complimented by the inoculated crude oil degrading bacteria. These resulted in the rapid utilization of the diesel substrate (Figure 1). The bacterial population in tray “A” shows an initial decrease from day 0 to day 3, which is due to the toxicity of the diesel oil and a steady adapting lag phase from day 3 till day 9. The exponential phase is observed from day 9 to 15, having day 15 as peak growth. The observed reduction in the population density from days 15 to 18 is due to the exhaustion of nutrients (diesel oil depletion). The initial reduction in the population density from day 0 to day 3 in tray “B” can also be attributed to the toxic effect of diesel on the microorganisms present in the soil sample. The relative increase from days 3 to 9, then steady continuous increase in growth showing availability of more nutrient that can be utilized by the microorganisms’ presence. Due to the use of sterile soil in Tray “C”, the bacterial population was low at the early days and later increases steadily from day 6 till day 18 (still lower bacterial population as compared to those of Trays “A” and “B”). Table 3 shows the extent of diesel degradation/mineralization during the bioremediation of diesel-contaminated soil by bioaugmentation process supplemented with pig dung. Mineralization of diesel was found to be highest (73.82%) in tray “A” and lowest in tray “C” (35.76%). Bioaugmentation using the consortia of B. coagulans, C. koseri and S. ficaria supplemented with pig dung was effective in degradation of diesel-contaminated soil made up of 200 mL of diesel in 2 kg of soil approximately 10% (v/w) contamination. Nevertheless, this was not a complete removal of the diesel contaminant. Complete removal or higher degradation percentage will be preferred because the residue might be recalcitrant by-products from the initial diesel biodegradation which could lead to bioconcentration, bioaccumulation and biomagnification in a real life scenario. The by-products could also have toxic effect on soil organisms (Agnieszka and Zofia, 2010; Hou et al., 2013; Wu et al., 2016; Lahel et al., 2016). Microbial species capable of degrading all the constituents of crude oil/crude oil products are limited in number. Probably, because the inherent ability of each strain/species to degrade one or more hydrocarbon compound is individually confined to them. Hence, efficient degradation of hydrocarbon requires a consortium composed of various microbial strains (Yousseria et al., 2016). The consortium approach improves biodegradation ability. Microbial mixed cultures or consortia have a higher ability to adapt to stress conditions and therefore show increased microbial survival. In addition, they can increase the number of catabolic pathways available for diesel biodegradation and can easily prevent or reduce the accumulation of recalcitrant/toxic compounds from microbial degradation (Briceno et al., 2016). Therefore, increase in the concentration/dose of augmented bacteria could improve the amount of diesel mineralized. Higher microbial dose is believed to boost the adaptability and assimilation capacity of microbial population to the newly introduced soil, which in turn results in higher mineralization efï¬ciency (Trindade et al., 2002; Lahel et al., 2016). The soil sample in Tray “B”, which underwent natural attenuation, resulted in residual diesel of 59% meaning that 41% of the diesel was lost through the activities of indigenous organisms who had utilized the diesel (Bento et al., 2005; Silva et al., 2015). Pseudomonas spp., and Bacillus spp. which were some of the indigenous bacteria present in the soil sample, have actually been implicated many times in hydrocarbon degradation (Perfumo et al., 2007; Alfreda and Ekene, 2012; Collado et al., 2013). The indigenous organisms utilizing the diesel could take longer time. This will not be in the interest of maintaining healthy environment without harm to the organisms in that environment; hence, the need for effective method such as bioaugmentation and biostimulation. The impact of hydrocarbons on microorganisms may not be directly related to their toxicity. Several researchers have reported destruction of inorganic nutrient sources that are essential for microbial growth and catabolic activities due to the ability of hydrocarbons to react and form complexes with nitrates, sulphates and phosphates, thus making them unavailable to oil degrading organisms (Andrew and Jackson, 1996; Abu et al., 2016). The activities in tray “C”, was also probably influenced by the soil sorption property. Judging from the conditions in the tray and the bacterial load, it can be said that apart from the microbial activities, some other factors (soil sorption, adsorption, desorption and volatilization) could have influenced diesel concentration in soil sample in Tray “C”. The contaminated soil samples in the other two trays (tray “A” and ” B”) could also have been influenced by same factors. Soil potential to sorb a certain amount of diesel has been documented, especially the clay and humus compounds present in the soil (Lahel et al., 2016). Falciglia et al. (2011), also noted that adsorption and desorption efï¬ciency of diesel is usually affected by the type of soil texture.
CONCLUSION
The effect of augmented B. coagulans, C. koseri and S. ficaria in biodegradation of diesel-contaminated soil was more effective than natural attenuation in this present study. Their effectiveness can be improved by optimizing bioremediation parameters such as pH, temperature, substrate concentration and moisture content. Cell immobilization technique could as well enhance the degradation activities of these bacteria consortia. Also, the bacterial species isolated in this work had potential for hydrocarbon degradation, with further research, they can be used in the degradation of hydrocarbons. Since they do not occur as pure cultures in nature, the consortia of these organisms could show greater biodegrading abilities.
CONFLICT OF INTERESTS
The authors have not declared any conflict of interests.
ACKNOWLEDGMENT
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