Effect of Petroleum Hydrocarbons Contamination on Soil Microorganisms and Biodegradation

The experiment under lab condition was conducted to investigate the effect of petroleum hydrocarbon contamination on soil microorganisms and biodegradation. The experiment was conducted in 12 treatment and three replication. The treatments include soil contaminated with Kerosene, Diesel, lubricate oil and used lubricate oil in three concentration 0%, 5%, and 10%. contamination with hydrocarbons significantly decreased the count of heterotrophic bacteria and there was reversal relationship between the contaminant concentration and the count of heterotrophic bacteria. The results indicated soil contamination with hydrocarbons significantly increased the count of Hydrocarbon Utilizing Bacteria (HUB). The effect of petroleum hydrocarbons on fungi count differed according to the type of hydrocarbons, concentration of contaminant and the periods of incubation. Our results show that soil contamination by hydrocarbons caused significant increase in actinomycetes count at all hydrocarbon types and concentrations except 5% used and non-used lubrication oil. The higher percentage of biodegradation in hydrocarbons contaminated soil 49.6% was recorded in soil contaminated with 5% kerosene while the lower percentag10.8% was recorded with soil contaminated 10% lubricate oil.


INTRODUCTION
Considering the rapid growth in the world's population, crude oil continues to be the main source of energy and raw material for industries worldwide (Abbasian et al., 2016). The increasing demand on petroleum products drives a surge in production, transportation and refinery -resulting in an inevitable toll on the environment (Gallego et al., 2001). Indeed, increased oil production and distribution is associated with higher incidents of pollution due to oil spillage (Hussain and Gondal, 2008). Furthermore, natural causes such as superficial waters and floods can spread this contamination to other areas (Zakaria, 2002). Crude oils typically consist of various types of hydrocarbons (aliphatic and aromatic). While these hydrocarbons contain plenty of carbon and hydrogen atoms, they are very nutritionally poor and insufficient to sustain many microorganism species (Abbasian et al., 2016;Sathishkumar et al., 2008;Viñas et al., 2005). The toxicity of these hydrocarbons to microorganisms means that contaminated soil may experience drastic changes to the population and abundances of various microbial species (Abbasian et al., 2015;Megharaj et al., 2011). The extent of these changes to the microbial populations is dependent on several factors including: the composition of the microbial community prior to the contamination, the chemical composition of the contaminant (crude oil), and the physiochemical factors of the contamination site (Abbasian et al., 2016;Rahman et al., 2002a). Certain microbial species face a challenge when grown in the presence of hydrocarbons due to their hydrophobicity and insolubility -leading to restriction of absorption by these cells, ultimately leading toxicity (Abbasian et al., 2015). Moreover, these hydrocarbons affect the microorganisms membrane fluidity; thus, microorganism exposed to petroleum products must adapt their membrane structure to survive (Jin et al., 2014). The extent of these changes are depending on the type of hydrocarbon contaminant and their carbon chain length (Abbasian et al., 2015). Some hydrocarbon-degrading microorganisms learn to adapt by producing and secreting a surfactant compounds to emulsify the hydrocarbon and form micelles -to be taken up later in various ways (Abbasian et al., 2015;Bustamante et al., 2012). Microorganisms are a key player in maintaining sustainable environment and ecosystem (Varjani, 2017). Microorganisms are known to be the most effective tools to naturally degrade crude oil after a spillage episode. Bioremediation is a process in which microorganism are utilized to convert dangerous organic pollutants from crude oil into environmentally friendly compounds such as CO 2 and H 2 O (Ron and Rosenberg, 2014;Varjani, 2017). In the process of biodegradation, oleophilic microbes are utilized and administrated to degrade the hydrocarbons and remediate the site of spillage (Varjani and Upasani, 2013). since microorganisms are naturally effective to degrade petroleum products, it is important to understand the changes in microbial diversity post soil contamination with various petroleum products (Abbasian et al., 2016). In this study, we aimed to understand the changes in indigenous soil microbial diversity and abundances upon the addition of kerosene, diesel, and non-used and used lubricate oil. Our study will also evaluate the ability of the various soil indigenous microorganism species to biodegrade of these petroleum products.

MATERIALS AND METHODS Soil Sampling and Analysis
The soil was collected from Basheka area 12 km Northeast of Mosul. The soil collected from surface area (0-20)cm. The soil air died sieved with 2 mm sieve to remove debris. The physical and chemical properties of the soil were determined as following. Soil texture by hydrometer method, Soil Reaction (pH) by glass electrode method (1:2.5 soil water suspension) (CON 720), soluble salts by Electrical Conductivity (conductivity bridge method) (Inolab pH 720), Organic Content by rapid titration method (Black et al., 1965), available phosphorus by Olsen's method (Olsen et al., 1954) Ca and Mg by titration with EDTA (Graham et al., 1962), Potassium and sodium by flame photometer (PFP 7) (Jackson, 1973). Some of soil physical and chemical properties were recorded in (Table 1).

Experimental Design
200 gm of sieved soil was placed in 250 ml volumetric flask. The petroleum hydrocarbon treatments comprised of four different petroleum fractions Kerosene(K), Diesel (D), lubricate oil (O) and used lubricate oil(UO) ). Treatment were applied at 0%, 5% and 10% levels. The petroleum hydrocarbon contamination involved twelve treatments in a completely randomized design replicated three times. All components were thoroughly mixed with the soil. The moisture content of soil was brought to 65% water holding capacity. Soil was turned weekly and distilled water was added to maintain them 65% of WHC. The beakers were incubated at a temperature of 25C. Soil sampling was carried out at one month and subsequently biweekly intervals for 12 weeks (Alrumman et al., 2015).

Microbial Count
In estimating microbial population, standard plate count methods were used to prepare nutrient agar (NA) for estimation of the heterotrophic bacteria, potato dextrose agar (PDA) for estimation of fungi, Starch casein nitrate (SCN) agar for estimation of actinomycetes, and Luria bertani agar (LBA) ( in LBA yeast extract was replaced with kerosene, diesel or lubricate oil as carbon source) for estimation of hydrocarbon utilizing bacteria (HUB). One gram each of the soil samples were measured into the test tube containing 9 ml sterile distilled water and serially diluted to dilution factor 10 5 and 1 ml of the last dilutions was pipette into sterile plate with appropriate medium which were incubated at 28˚C -30˚C. All plated were incubated inverted wise. Microbial counted were done at 48 hours for NA and 72 hours for PDA and 72 hours for LBA and 6 days for(SCN) (Stanley et al., 2015;Desai, 2015;Adesina and Adelasoye, 2014).

Hydrocarbons Determination
To extract the hydrocarbons from soil.10 g of soil was placed in a dry baker and add 10 ml of organic solvent (hexane). The solvent filtered and placed in a weighted baker and dried in the 80 C. The precipitate was weighted. the percentage of remaining hydrocarbons in soil were calculated (Mishra et al., 2001)

Statistical Analysis
ANOVA and correlation analyses were carried out. The means were compared using Duncan's test at p <0.05 after, ANOVA. The relationships between HUB and percentage of hydrocarbon biodegradation were determined using Pearson's correlation analysis.

RESULTS AND DISCUSSION Soil Properties
Table (1) shows some physical and chemical properties for soil under study.  Fig. (1) shows the total aerobic heterotrophic bacterial counts for the control (uncontaminated) soil ranged from 0.2.18 x 10 5 colony forming unit (CFUg -1 ) soil to 2.83 x 10 5 CFUg -1 soil while counts for 5%, 10% kerosene, 5%, 10% diesel oil, 5%, 10% non-used lubrication oil and 5%,10% used lubrication oil contaminated soils ranged from 1.49 x 10 5 to 1.87x10 5 CFU -1 soil, 0.96 x 10 5 CFUg -1 soil to 1.63 x 10 5 CFUg -1 soil and 1.29 x 10 5 CFUg -1 to 1.79 x 10 5 CFUg -1 soil 1.03 x 105 to 1.32 x 10 5 CFUg -1 , 1.05 x 10 5 to 1.32 x 10 5 CFUg -1 soil and 0.9 x 10 5 to1.13 x 10 5 CFUg -1 soil, 1.19 x 105 to 1.51 x 10 5 CFUg 1soil and 0.88 x 105 to 1.16 x 105 CFUg -1 soil respectively. The results indicated soil contamination with hydrocarbons significantly decreased the count of heterotrophic bacteria and there was reversal relationship between the contaminant concentration and the count of heterotrophic bacteria. After twelve weeks incubation. Soil contamination with 5% and 10% of each hydrocarbon kerosene, 5 and10% diesel, 5 and 10% non-used lubricate oil and 5 and 10% used lubricate oil decreased heterotrophic bacteria 23%, 39%, 24%, 44%, 44%,52%, 42% and 50% respectively and these results are consistent with (Kucharski et al., 2004;Wyszkowska and Kucharski, 2001;Adesina and Adelasoye, 2014) who concluded the hydrocarbon contamination soil cause decrease of heterotrophic bacteria count and increase heavy metals concentration and they suggested decreases in both count and diversity of bacteria communities in hydrocarbon contaminated soils are caused by presence of toxin effect for heavy metals. Our results show that there were no significant differences in count of heterotrophic bacteria between soil contaminated with non-used and used lubricate oil

Hydrocarbons Biodegradation
The percentage of hydrocarbons biodegradation in the soil contaminated with 5% and 10% of kerosene, diesel, lubricate oil and used lubricating oil is shown in (Table 2). At the end of twelve weeks the result indicate the higher percentage of biodegradation 49.6% was recorded in soil contaminated with 5% kerosene while the lower percentage of biodegradation 10.8% was recorded with soil contaminated 10% non-used lubricate. The percentage of hydrocarbons biodegradation in the soil contaminated with 5% diesel, 5% non-used lubricate oil and 5% used lubricate oil were 32%, 28% and 24% respectively. However the biodegradation of Kerosene 10%, diesel 10%, and used lubricate oil were 30%, 25.6% and 12.4% respectively. Positive correlation was found between HUB count and percentage of hydrocarbons biodegradation. Our results show there was reversal relationship between hydrocarbons biodegradation percentage and the contaminant concentration and this consistence with (Boldu-Prenafeta et al., 2004;Ambrazaitiene et al., 2013;Abioye et al., 2012;Rahman et al., 2002b). High concentration of contaminant causing decreasing in biodegradation percentage due to high concentration can be inhibitory of microorganisms by toxic effects (Abioye et al., 2012;Rahman et al., 2002b;Ijah and Antai, 2003) for this reason reported that bioremediation is a useful method of soil remediation if contaminant concentrations are moderate. Also the results show there are differences between biodegradation percent of hydrocarbons and these results agreed with (Ambrazaitiene et al., 2013;Boldu-Prenafeta et al., 2004) who concluded the rate of biodegradation depends on the microbial population, the type, structure and level of contamination. The biodegradation percentage in hydrocarbons contaminated soil was in the order of kerosene > Diesel> lubricate oil > used lubricate oil. The kerosene, diesel, and lubricate oil consisted mainly of n-alkanes with chain length of C10-C16, C10-C22 and C15 -C50 respectively. These result consistence with (Wang et al., 1998) who concluded the biodegradation rate of n-alkane inverse proportional with chain length and branched alkanes less ability to biodegradation. The results show there was no huge differences in biodegradation percentage between non-used and used lubricate oil.