Bioremediation of Crude Oil Contaminated Soils Using Surfactants and Hydrocarbonoclastic Bacteria

A study of the rate of crude oil remediation in soils with the application of surfactants and hydrocarbonoclastic bacterial population was


INTRODUCTION
Soil, or any other environmental component, contaminated with pollutants can cause extensive damage to local systems since the accumulation of pollutants in animals and plant tissues may cause mutations or even death [1]. April, [2] suggested that bioremediation is cost effective amongst other methods for remediating soil contamination while working with filamentous fungi. Being an evolving remedial method, it involves the use of biological agents, like microorganisms, to detoxify or remove pollutants from the environment including the products of petroleum industry [3,4]. Although, microorganisms are found everywhere in the environment, oil degrading organisms are most abundant in areas where there have been petroleum seeps or spillages metabolizing petroleum hydrocarbons as food and energy source [5]. The rate at which microbial cells can convert crude oil contaminants during bioremediation depends on the rate of its uptake and metabolism and the rate of transfer to the cell (mass transfer). Increased microbial conversion capacities do not lead to higher biotransformation rates when mass transfer is a limiting factor [6]. These bioavailability problems can be overcome by the use of surfactants [7], which increase the availability of contaminants for microbial degradation. Biosurfactants act by partitioning preferentially at interphases and exhibiting high surface and emulsifying activities [8].
Varied works on locally sourced biosurfactants have been reported with high effectiveness some examples are Essien [9] using wood ash and sawdust, Agbor [10] experimenting with plantain peels and cocoa pod husk etc. The aim of this study was to check the remedy of crude oil soil polluted with microbial and surfactant enhancements.

Sample Collection and Processing
The soil samples were collected following the protocol of Okop, [11] with modifications. Five to ten (5-10) centimeters deep topsoil was collected with shovel into clean bucket from a site at the Pharmacy farm and transported to the Microbiology Postgraduate laboratory, University of Uyo Town campus. Bonny light crude oil was sourced from Elf Petroleum, Nigeria. The wood ash was obtained from charcoal residue. The wood lump was chopped and ashed in the oven at 100°C. The char residue was grounded, sieved and stored in airtight containers at room temperature. The commercial surfactant Tween® 80 (Ariaria market in Aba, Abia State, Nigeria) was also considered.
The soil was divided into six portions and all received 5% crude oil contamination. The six portions (each weighing 1000 g) were then separated into two, out which half was used for biostimulation with surfactants only while the other half was used for bioaugmentation with surfactants inclusive. The surfactants were then added at 2.5% concentration. Soils with and without crude oil contamination served as controls set ups. They were contained in perforated wood boxes and kept outside the laboratory.
Throughout the monitoring period there was constant tilling and moistening of the soil samples for aeration, optimum microbial growth and even distribution of contaminant for increased microbial-contaminant contact using a scapular and sterile water. For each soil sample 30ml of sterile water was used for moistening every three days.

Physicochemical properties of samples
The physicochemical properties of the soil was measured using standard methods to check if the soil was fit for microbial growth and capable of supporting bioremediation in the soil. The Walkely and Black methods for organic carbon as reported by Osuji et al. [12]; the Bowman [13] method for available phosphorus; the Kjeldhal method for soil total nitrogen as reported by [14]. Also the surfactants were also analysed for their chemical component, starting with their emulsification index using the Batista [15] method, nitrogen, organic matter, pH and phosphorus.

Preparation of isolates used for bioaugmentation
Organisms were isolated from previously contaminated soil at 5% contamination (this was to simulate a minor spill and ensure microbial survival and adaptation) using Mineral salt agar (MSA) and re-incubated into fresh nutrient agar plates. Their morphology and pigmentation were done visually, while cell shape and Gram stain were determined. Other biochemical tests were carried out as described in the Bergey's manual of determinative bacteriology. These cultured organisms were later introduced to a set of sterilized soil. The isolates identified were of the Pseudomonas, Corynebacterium, Bacillus and Alicaligene genera.

Microbial counts
Soil sample (1 gram) was gotten every two weeks to check for Total Heterotrophic Count in the soils. The soil samples were diluted in sterile water using ten-fold dilution and pour plated with nutrient agar in petri dishes. The set up was incubated for 24 hours, the bacterial density was reported as mean of triplicate determination and recorded as cfu/g of soil.

Total residual hydrocarbon content
The remediation is usually calculated by subtracting the residual amount from the initial amount. One gramme of the contaminated soil sample was mixed in 10 ml of hexane and shaken for ten minutes using a mechanical shaker. The solution was filtered using Whatman No 1 filter paper and the filtrate diluted by taking 1ml of the extract into 50 ml hexane. The absorbance of the solution was read at 460 nm with Mamotte 701 Spectrophotometer using nhexane as blank [16].

RESULTS AND DISCUSSION
The experimental soil was slightly acidic at pH of 5.3, while the surfactants had pH of 10.8, 7.8 and 4.9 for palm fruit bunch ash, wood ash and Tween 80 respectively. Kamalu and Isirimah [17] suggested that soils samples in the Niger Delta region are usually slightly acidic. Udoetok [18] and Udosen [19] reported similar results for the palm fruit bunch ash and wood ash respectively. The biosurfactants' addition to the soil aided to increase the pH of the soil toward alkalinity, thereby, enabling optimal microbial growth and crude oil degradation [19]. The acidity of Tween 80 reflected in the extension of the microbial adaptation period with initial lowered counts in the soil mixtures [20]. From Table 1, Tween 80 surfactant also had the lowest chemical content while palm fruit bunch ash had good nutrients levels followed by wood ash. The high chemical content of the biosurfactants (wood and palm fruit bunch ashes) showed they could be used to fortify less fertile soils [18,19] and contained nutrients for the growth of crude oil degrading organisms [4].
Presented in Table 2 is the microbial counts for the control, biostimulated and bioaugmented soil samples for the 90 days. There was observable difference in the microbial counts from the two processes of biostimulation and bioaugmention. While microbial numbers in bioaugmented soils reduced drastically from their initial counts [21], the ones in the biostimulated portions increased steadily [22,23]. The bioaugmented isolates reduced in numbers due to adaptation to the new environment and contaminant stress, while the biostimulated indigenous organisms only took time to produce the enzymes needed for degradation of crude oil. The counts from the soil portions over the 90 day period followed the trend observed by Bahrampour and Moghanlo [24], Essien and Udosen [25]; Atlas [22] with low initial counts before acclimatization, then increased microbial number during heightened degradation. These numbers rescinded with lowering contaminant levels, increased waste metabolites and probable toxic degradation byproducts [26,27].
The residual hydrocarbon content of each contaminated soil sample revealed that biodegradation took place to different extents. Table 3 revealed that the bioaugmented samples had higher crude oil content reductions compared to the reductions observed in biostimulated soils. The highest reduction was observed in bioaugmented soil treated with palm bunch ash (94.54%), while the lowest reduction was seen in contaminated soil without surfactant treatment. The reduction results obtained in treated soils with 'trained' consortia population were higher than those obtained from soils with indigenous population. This result is in contrast to the observations of Demque [28] who used acclimatized indigenous bacterial populations to treat diesel contaminated soils.

CONCLUSION
Microbial growth and hydrocarbon reduction results from this study showed a lot can be achieved with the application of cheap and readily sourced agricultural/industrial by-products like the wood and palm fruit bunch ashes in bioremediation of crude oil contaminated soils.
With the 2015 projection of 15.63 x10 6 m 3 day -1 world petroleum consumption by the United States Energy Information Administration [29] high rate of oil spills are inevitable. Speedy and efficient removal of these contaminants from polluted environment would curtail the negative impact spilled crude oil or its products could bring to fore, on human, plant and environmental health. Taking into account the environmental friendliness of these locally sourced surfactants, on the one hand and the re-use of substances considered 'waste' to ensure a cleaner environment, locally sourced biosurfactants have high prospects in the oil and gas sector. The addition of surfactant and use of 'trained' bacterial cells showed high effectiveness in contaminated soil remediation.