Highlights in Bioscience; Highlights in BioScience ISSN: 2682-4043 DOI:10.36462/H.BioSci.20218 Highlights in BioScience August 2020| Volume 3 http://bioscience.highlightsin.org/ Page 1 of 7 Research Article Open Access Department of Science Technology, Akwa Ibom State Polytechnic, Ikot Osurua, P.M.B 1200, Ikot Ekpene, Akwa Ibom State, Nigeria. Contacts of Authors * To whom correspondence should be addressed: Aniefon Alphonsus Ibuot Citation: James II, Ben MG, Jones AM, Akpan PS, Eka II, Oruk AE, Ibuot AA (2020). Characterization of hydrocarbon utilizing bacteria in waste engine oil-impacted sites. Highlights in BioScience Volume 3. Article ID 20218. dio:10.36462/H.BioSci.20218 Received: June 6, 2020 Accepted: August 18, 2020 Published: August 31, 2020 Copyright: © 2020 James et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and supplementary materials. Funding: The authors have no support or funding to report. Competing interests: The authors declare that they have no competing interests. Characterization of hydrocarbon utilizing bacteria in waste engine oil-impacted sites Iniobong Ime James, Mayen Godwin Ben, Agnes Monday Jones,Patience Saturday Akpan, Idorenyin Idorenyin Eka, Albert Ema Oruk, and Aniefon Alphonsus Ibuot* Abstract Changes in soil physicochemical properties and bacterial species present in soil contaminated with waste engine oil were evaluated at three auto-mechanical workshops in Uyo, Nigeria. This work was aimed at isolating and identifying hydrocarbon degrading bacteria from waste engine oil polluted soil, and assessing their hydrocarbon-utilizing ability. Waste engine oil pollution affected soils significantly with increases in soil physicochemical properties, and heterotrophic bacterial population counts. Eight bacterial species Corynebacterium kutscheri, Pseudomonas aeruginosa, Flavobacterium aquatile, Serratia odorifera, Micrococcus agilis, Staphylococcus aureus, Micrococcus luteus and Bacillus substilis were isolated by the selective enrichment technique and screened for hydrocarbon utilization capability in mineral salt media with 1% (v/v) waste engine oil as a sole carbon and energy source. The extent of bacterial growth observed was related to the ability of organisms to biodegrade hydrocarbons present in the medium bacterium species, which showed varying hydrocarbon utilization during the 15 days of incubation. Growth in hydrocarbon medium was the most efficient in cultures of Corynebacterium kutscheri. All isolates also showed variable emulsification ability, with Corynebacterium kutscheri, showing the highest ability. These results demonstrate the presence of indigenous bacteria in hydrocarbon-polluted soils and the potential toward the remediation of hydrocarbons. Keywords: Hydrocarbon-utilizing bacteria, selective enrichment technique, Corynebacterium kutscheri Introduction Petroleum utilization as fuel and petroleum products leads to severe environmental pollution [1]. Large-scale accidental spills pose a great threat to the ecosystem [2]. Soil pollution by petroleum hydrocarbons has been shown to produce pronounced changes in the physicochemical and microstructure of the oil-contaminated soil [3]. This affects parameters such as soil porosity, bulk density, and adsorption [4-5]. Fresh spills and/or high levels of pollutants may often result in the reduction of large sectors of soil microbial population, although soils with lower levels or old pollution may show an increase in numbers and diversity of microorganisms [6-7]. The diversity and the number of microorganisms at polluted soil sites may assist in the characterization of such a site, such as the toxicity of petroleum hydrocarbons to the microbiome, age of the spill and concentration of the pollutant [8]. https://creativecommons.org/licenses/by/4.0/ http://bioscience.highlightsin.org/ https://doi.org/10.36462/H.BioSci.20218 https://doi.org/10.36462/H.BioSci.20218 http://bioscience.highlightsin.org/ James et al., 2020 Characterization of hydrocarbon utilizing bacteria in waste engine oil-impacted sites Highlights in BioScience August 2020| Volume 3 http://bioscience.highlightsin.org/ Page 2 of 7 Additionally, microorganisms in soils exposed to hydrocarbon pollution usually exhibit a higher potential for biodegradation of such pollutant compounds than others with no history of such exposure. Percentages of hydrocarb- onoclastic microbes are quite low in soil when there is no oil spill, but may increase 1,000 fold after oil spill [9]. Conventional remediation methods do not seem to be able to address this problem, or tends to aggravate the problem [10]. Mechanical methods such as incineration, excavation and/or burial in secure land fill, as well as a host of other chemical decomprelocates osition methods are expensive, time consuming and only the pollution [11]. An efficient way of remediating the oil-contaminated sites could be employment of microorganisms, such as bacteria, microscopic algae, and fungi, isolated from polluted environments or enhanced from the organisms already present in the same environment [12-13]. Waste engine oil-polluted soils also serve as a source of indigenous bacteria capable of hydrocarbon degradation. The employment of microorganisms in the biodegradation of hydrocarbons over chemical or conventional treatment is preferred for many reasons; end products are comparatively safer and cost-effectiveness [11]. Ogunbayo et al., [18] evaluated the effectiveness of bacteria indigenous to soil in remediating engine oil-polluted, soil and isolated Bacillus, Pseudomonas, Flavobacterium, Microc- occus and Rhodococcus species, with Pseudomonas and Rhodococcus species giving most favorable degradation effectiveness and efficiencies. This study therefore considered the isolation of indigenous bacterial communities in waste engine oil-polluted soil using selective enrichment technique, and the assessment of hydrocarbon-utilization capability in waste engine oil-augmented mineral salt medium. Materials and Methods Sample collection Waste engine oil –contaminated soil samples used in this study were collected from three auto-mechanic workshops within the mechanic village, Uyo, Akwa Ibom State, Nigeria. Composite soil samples were obtained at each sampling point using a soil auger from 0-10 cm below the soil surface. The soils were labeled “Unpolluted” for the unpolluted sample, “MA” for the mechanic workshop 1 sample, “MB” for the mechanic workshop 2 samples, and “MC” for the mechanic workshop 3 samples. This was followed by bulking and transportation to the laboratory in sterile polythene bags within six hours for isolation of organisms. Physico-chemical analysis of soil samples The soil pH was measured using HANNA Instruments Model 209 pH meter [14]. Moisture content was calculated on the basis of the air dry weight as described by AOAC [15]. Total organic carbon was calculated by weighing exactly 0.5 g of the soil sample into a flask, and 10 ml of 1.0 M K2Cr2O7 was added and swirled to mix. 20 ml conc. H2SO4 was added, gently swirled for a minute and allowed to stand for 20 minutes. The suspension was diluted to about 100 ml of distilled water. Five drops of o-phenanthroline indicator were added to each sample and was titrated with 0.5 M ferrous ammonium sulfate to a light blue end point. The reagent blank was also run and the titre values recorded, and used to calculate the organic carbon content [15]. The total hydrocarbon content (THC) was determined by first extracting hydrocarbons by acidifying 2 g of representative soil samples using H2SO4, and extracting upon addition of 20 ml of toluene in a separatory funnel. The contents of the funnel were shaken, and allowed to settle into two layers. The absorbance of the supernatant (extract) was read at 420 nm with UNICAM UV/VIS spectrophotometer (Spectronic 20D). Readings were recorded from the spectrophotometer and using the determined curve to obtain the figure [16]. Phosphorus was determined using the ascorbic acid method as described by AOAC, [15]. 50 ml of the soil dilution was pipetted into 250 ml Erlenmeyer flask, and 1 drop of phenolphthalein indicator was added. Exactly 5 N H2SO4 (148 ml conc. H2SO4 in 100ml H20) is added drop-wise to develop a red colour. Exactly 8 ml of combined reagents made up of 50 ml of 5 N H2SO4, 5 ml potassium antimonyl tartrate solution (1.372 g potassium antimonyl tartrate in 500 ml distilled water); 15 ml ammonium molybdate solution (20 g ammonium molybdate crystal in 500 ml distilled water); were added and thoroughly mixed, and allowed to stand for 20 min. The phosphorus content was determined by measuring the absorbance of the sample at 880 nm. The nitrogen concentration was determined according to the methods of Bremmer and Mulvaney, [17]. One milliliter of the soil sample was introduced into the standard kjeldahl flask containing 1.5 g CuSO4, and 1.5 g Na2SO4 as catalyst, alongside concentrated H2SO4. The flask was gently heated on a heating mantle, taking care to prevent frothing. The solution was transferred after heating to a 100 ml standard flask and made up to the mark with distilled water. A portion of this digest was pipetted into a semi micro-kjeldahl distillation apparatus and treated with 30 ml of 40% NaOH solution. The ammonia evolved was steam-distilled into a 100 ml conical flask containing 10 ml solution of saturated boric acid to which 4 drops of Tashirus indicator had been previously added. The tip of the condenser was immersed in the boric acid solution and the distillation continued until about two-thirds of the original volume was obtained. The tip of the condenser was finally rinsed with a few milliliters of distilled water. The distillate was then titrated with 0.1N HCl until a purple-pink end point was observed. A blank determination was also carried out in a similar manner without the sample, and the calculation done as follows: Nitrogen (%) = (Real titre – Blank titre) x 0.1 x 0.014 x 100 Weight of the sample Enumeration of total heterotrophic bacteria (THB) The THB population in the soil samples was enumerated by adopting the standard plate counts technique using the spread plate method as described by Ogunbayo et al., [18]. These involved spreading aliquots of a serially http://bioscience.highlightsin.org/ James et al., 2020 Characterization of hydrocarbon utilizing bacteria in waste engine oil-impacted sites Highlights in BioScience August 2020| Volume 3 http://bioscience.highlightsin.org/ Page 3 of 7 diluted 0.1 ml of 10-5 dilutions of the soil sample suspension on nutrient agar plates and the plates were incubated at 30 oC for 24 h. Similar aliquots were also incubated in minimal salt agar plates containing used engine oil as the sole source of carbon and energy. The plates were all incubated aerobically at 30 oC. The percentages of hydrocarbon-utilizing bacteria (HUB) relative to the total heterotrophic counts were noted. Enumeration of hydrocarbon utilizing bacteria (HUB) Oil-utilizing bacteria were isolated from polluted soil samples by enrichment in mineral salt medium (MSM) modified from Okpokwasili and Nwosu, [19] using waste engine oil as a carbon and energy source. The soil samples were sieved using a 2 mm mesh sieve. 10 g of the sieved soil samples was inoculated into 100 ml sterile MSM. 1 ml of the waste engine oil was added to the medium as a sole source of carbon and energy, and the culture was incubated on a rotary shaker at 170 rpm for 1 week. The enrichment procedure was repeated for three cycles. At the end of each enrichment cycle, 1 ml of the culture was diluted serially 10-fold down the gradient to 10-5 and plated. Pure cultures of the isolates were obtained by plating 1 ml of the 10-5 dilution of the third enrichment cycle onto MSM agar plates, and incubating at 30 oC (± 2) for 48 h. Pure cultures obtained by this procedure were stored in slants at 4 oC until further identification. Characterization and identification of bacteria Isolates were identified on the basis of colonial characteristics, Gram’s reaction and cell biochemical reactions as described by Cheesbrough [20]. Identification used the taxonomic schemes of Holt et al. [21]. Hydrocarbon utilization screening of bacteria To determine the ability of the isolates to utilize engine oil as the sole carbon and energy source, the growth patterns of isolates in mineral salt medium in the presence of 1% (v/v) of the waste engine oil (5.0 mL in 100 mL MSM) were determined according to Onuoha et al. [22]. Waste engine oil-augmented MSM was dispensed into 250 ml Erlenmeyer, and inoculated with 0.1 ml of 24 h cultures of the bacterial isolates. Incubation was done at 30 oC for 15 days. Growth patterns were determined monitoring changes in pH, optical density and total viable count at 5-day intervals during the incubation. The pH of the medium was measured using the pH meter (HANNA Instruments). Growth was also monitored by measuring the optical density (OD) at 600 nm using the spectrophotometer (Spectrumlab). Total viable counts of the cultures were obtained by incubation of 0.1 ml of the cultures using the spread plate technique on nutrient agar plates at 30 oC for 24 h. Emulsification activity of bacteria The emulsification index (E24) of the isolates was determined according to the methods of Ganesh and Lin, [23], by adding 1ml of waste engine oil to the same amount of culture media as used for degradation assay, mixing the vortex for 2 min and leaving to stand for 24 h. The percentage of emulsification index was obtained as follows: E24 = Height of the emulsified layer x 100 Total height of the liquid column Results Physicochemical properties of soil samples The results of the physicochemical analysis of the different soil sample are shown in Table 1. The high amounts of organic carbon (5.32 ± 2.65% in MA, 9.79 ± 0.51% in MB and 7.29 ± 3.09% in MC), and THC (2933.76 ± 404.27 mg/kg in MA, 3122.72 ± 131.00 mg/kg in MB and 3202.61± 675.07 mg/kg in MC), compared to the unpolluted soil sample (3.7 ± 2.43% organic carbon content and 39.97± 13.49 mg/kg THC) is indicative of heavy pollution of the mechanical workshop samples with petroleum hydrocarbons. Soil samples from mechanical workshop 1 contained higher amounts of nitrates (0.25 ± 0.03 mg/g), while samples from mechanical workshop 2 contained the highest amounts of phosphates (10.74 ± 0.88 mg/g) and THC (3202.61± 675.07 mg/kg). The pH values of the soil samples indicate all soil samples as moderately acidic to acidic (from pH 5.78 to pH 6.79). Bacterial count of soil samples The total heterotrophic bacterial count and hydrocarbon utilizing bacterial count of the original soil samples is shown in Table 2. A higher THB count was recorded in polluted soil samples (4.4 ± 1.90 x 107 CFU/gfrom MA sample, 6.0 ± 0.23 x 107 CFU/g from MB sample and 4.5 ± 0.03 x 107 CFU/g from MC sample) than in the unpolluted soil sample (1.9 x 107 CFU/g). Higher THB (6.0 ± 0.23 x 107 CFU/g) and HUB (5.2 ± 0.25 x 107 CFU/g) counts were observed in the MB sample than in other similar polluted samples indicative of its extent of pollution. Hydrocarbon utilising bacterial counts were slightly lower in all samples than the corresponding heterotrophic bacterial counts. Characterization and identification of bacteria The identified bacterial isolates were Corynebacterium kutscheri, Pseudomonas aeruginosa, Micrococcus agilis, Flavobacterium aquatile, Staphylococcus aureus, Micrococcus luteus, Serratia odorifera and Bacillus substilis, as shown in Table 3 and 4. Hydrocarbon utilization potential of bacteria Table 5 shows the changes in pH of MSM during growth of bacteria isolates in hydrocarbon. Decreases in pH (to