CHAPTER FIVE: DISCUSSION Liyanage & Manage /Journal of Tropical Forestry and Environment Vol. 5. No 02 (2015) 13-25 13 Optimisation of Environmental Factors on Oil Degrading Bacteria Isolated from Coastal Water and Sediments in Sri Lanka G.Y. Liyanage1 and P.M. Manage1 * 1 Department of Zoology, University of Sri Jayewardenepura, Sri Lanka Date Received 25-11-2015: Date Accepted: 07-12-2015 Abstract Better understanding of the mechanisms of hydrocarbon degrading microorganisms and effect of some environmental factors is critical for the optimisation of the bioremediation processes. Temperature, pH, nitrate and phosphate are the major factors that influence there mediation process of bacterium. In the present study, optimisations some selected physico-chemical parameters (temperature, pH, nitrate and phosphate) were carried out on Bacillus cereus, Enterobacter sp. and Enterobacter ludwigii which were previously isolated as potential oil degraders. The bacteria showed maximum degradation of crude oil at 33 o C where the desirable pH was 8.6 for all the isolates except E. ludwigii (pH 5.4). A significant degradation (p < 0.05) of oil was detected by B. cereus (80% to 98%), Enterobacter sp. (73% to 90%) and E. ludwigii (70% to 83%) respectively with increasing of nitrate concentration from 0.1 to 2.5 ppm. Significant degradation of oil was not detected in the control and when bacteria were enriched with phosphate. Results of this study revealed that the bacterial remediation of oil is governed by nutritional status with special emphasis of nitrate enrichment in the environment. Thus, the results revealed that bacteria could be a useful tool to remove oil from the contaminated environment as eco-friendly, low cost application. Key words: bioremediation, crude oil, Bacillus cereus, Enterobacter sp., Enterobacter ludwigii 1. Introduction Oil spill pollution, a severe environmental problem across the world, is growing with increased levels of oil production and global transport. Its causes are either accidental or due to operation wherever oil is produced, transported, stored and used on sea or land (Mehrasbi et al., 2003). The hydrocarbons like PAHs (Poly Aromatic Hydrocarbons) have caused extensive damage to the local ecosystems since accumulation of pollutants in animals and plant tissues may cause progeny’s death or mutation (Alvarez et al., 1991). This is mainly due to an accumulation of PAHs through food chain. Ultimately the effect on human health in high because humans are frequently the last part of the food chain. Therefore, dealing with these problems and to adopt appropriate solutions, fundamental and advance researches with policies are needed. Hanson et al. (1997), Sartoros et al. (2005), Xiaojun et al. (2008) and Joel et al. (2011) documented that bioremediation is more affordable application to remove oil contamination when compare with available physical and chemical removal methods. Bioremediation is the treatment process that uses microorganisms such as yeast, fungi and bacteria to break down, or degrade hazardous substance into less toxic or nontoxic substances (Kim et al., 2005). * Correspondence: pathmalal@sjp.ac.lk Tel: +94 718538975 ISSN 2235-9370 Print/ISSN 2235-9362 Online © University of Sri Jayewardenepura 14 During the remediation process, specific microorganisms consume organic substances in crude oil as sole carbon source for their energy requirement (Bouwer and Zehnder, 1993) and the aerobic and anaerobic respiration mechanisms convert hazarders’ hydrocarbon in the environment to less harmful compounds such as carbon dioxide, methane and water (Farshid et al., 2011). Corynebacterium ulcerans, C. amycolatum, Bacillus badius and Micrococus varians were recorded as potential oil degradates (Omotayo et al., 2012) where Chin et al., (2008) documented that Yokenella spp., Alcaligenes spp., Roseomonas spp., Flavobacter spp., Sphinogo bacterium spp. and Moraxella spp. as petroleum hydrocarbon-degraders. Liyanage and Manage (2014) reported that B. cereus, Enterobacter sp. and E. ludwigii as crude oil degraders which were isolated from coastal environment in Sri Lanka. Bioremediation processes are governed by some environmental factors such as type and amount of crude oil present in the environment, environmental temperature, nutrients such as NO - 3, PO4 3- , pH, aeration, water acidity, type and population density of hydrocarbon degrading microorganisms and contaminant mobility (Dragun, 1998; Mace, 2012). Microbial growth rates in the environment are mainly depending on the temperature and rates of degradation decrease with decreasing temperature. (Gibb et al., 2001). Toone et al., (2013) documented that degradation mechanisms are enzyme mediated and depend on intracellular and extracellular enzyme activity of the bacteria. Furthermore, the incomplete microbial degradation occurs when the ambient environment does not contain significant concentrations of nutrients such as nitrogen and phosphorous (Prince et al., 2002). Thus, inorganic nutrients are limiting factors for the natural biodegradation process (Trindade et al., 2002; Efsun et al., 2013). The majority of microorganisms thrive best in the pH range of 6 to 8 (Omatayo et al., 2012) and adjustment of pH range from 4.5 to 7.4 was resulted in a near doubling of biodegradation rates of hydrocarbon (Leahy and Colwell, 1990). Thus, the manipulation and optimisation of these factors are needed to enhance the remediation process of pollutants in the environment (Dragun, 1998; Boopathy, 2000). South Asian countries not only import much of oil for domestic consumption, but India, Maldives, Pakistan and Sri Lanka lie close to the main shipping route that connects the Middle East to the Far East (ITOPF, 2012). Several oil spills were recorded around the Sri Lankan Sea during past few years (MEPA, 2012). Spillage near to Panadura Sea area was the most recent event. Considering the danger and environment impact on biodiversity, numerous solutions have been proposed by national and international agencies to remove oil from contaminated areas. However, the main limitations to use available techniques are inefficient trace level adsorption, hazardousness of chemicals and non-accessibility due to high cost (Kishore. and Ashisk, 2007). Thus, to minimise the environmental effects of oil pollution, in situ bioremediation has been suggested (MEPA, 2012). Therefore, the present study discusses the in vitro optimisation of environmental factors for three crude oil degrading bacteria strains isolated from oil contamination sites in the coastal environment in Sri Lanka. Those bacteria strains were identified as Bacillus cereus (KM504128), Enterobacter sp. (KM4055978) and E. ludwigii (KM504129) using 16S RNA sequences. 2. Methodology 2.1. Materials and reagents Crude oil (100%) required for the present study was received from Sri Lanka Petroleum Corporation (Pvt) Ltd. Bacteriological grade chemicals were purchased from Sigma, Aldrich © while molecular grade chemicals were purchased from Promega © , USA. Liyanage & Manage /Journal of Tropical Forestry and Environment Vol. 5. No 02 (2015) 13-25 15 2.2 Bacteria for degradation studies Previously isolated oil degrading bacteria (Liyanage and Manage, 2014), identified as Bacillus cereus (KM504128), Enterobacter sp. (KM4055978) and E. ludwigii (KM504129) were used in the present study to optimise some selected physio-chemical environmental factors for bacterial degradation kinetics. 2.3 Preparation of bacteria cultures for optimization of environmental factors A loop of each bacterial strain was transferred into 5 ml of liquid Lurial Bertani (LB) medium and incubated at 25 o C. Exponentially growing cultures were centrifuged at 1000 rpm for 15 minutes. The resulted pellets were re-suspended in 0.01M Phosphate Buffer Solution (PBS) and kept overnight to let out residual carbon content. Then the suspensions were centrifuged at 1000 rpm for 15 minutes and the pellets were washed three times using PBS. Turbidity of all bacterial suspensions were equalized (A 590=0.35) using spectrophotometer (SPECTRO UV-VIS double beam PC) (Manage et al., 2009). Equalised bacterial suspensions were used to optimisation some of the physic-chemical factor such as temperature, pH, phosphate and nitrate. 2.4 Effect of temperature on crude oil degradation Filtered sterilised sea water was inoculated by 0.5 ml of overnight starved bacteria culture. Then 1% (v/v) crude oil was added to make final volume of 10 ml in universal bottles. Samples were incubated in three different temperatures at 23 o C, 28 o C and 33 o C respectively in the shaking incubator at 100 rpm. One milliliter of sub-samples was collected at two days intervals for a period of 14 days to determine the concentration of crude oil. 2.5 Effect of phosphate and nitrate concentration on crude oil degradation Phosphate concentration in sea water varies between 0.002-0.05 ppm while the nitrate levels were between 0.1-2.5 ppm (James, 2009; Liyanage and Manage, 2014). In order to determine the effect of phosphate on the crude oil degradation, filtered sterile sea water was prepared at final concentrations of 0.002 ppm and 0.05 ppm supplemented with KH2PO4. Filter sterilised sea water was inoculated by 0.5 ml of overnight starved bacteria culture and 1% (v/v) crude oil at final volume of 10 ml in universal bottles. The bottles were incubated at 33 o C in 100 rpm. The temperature was determined based on the results of the present study and literature (Efsun et al., 2013). One milliliter of sub-samples was removed at two days intervals for a period of 14 days and the concentration of crude oil was measured spectrophotometrically. Same set of experiment procedure was followed to study the effect of on remediation of crude oil by the bacterium. NH4NO3 enriched sea water with varying concentrations (0.1-2.5 ppm) were used as nitrate source. 2.6 Effect of pH on crude oil degradation Filtered sterile sea water in triplicate was supplemented with 1% (v/v) crude oil and 0.5 ml of exponentially growing culture of bacteria at final volume of 10 ml into the universal bottles. pH was adjusted to 5.4, 7.2 and 8.6 by adding 1.0M NaOH or 1.0M HCl where appropriated. The samples were incubated in the shaking incubator at 33 o C in 100 rpm. One millilitre of sub- samples was collected at two days intervals for a period of 14 days to determine the concentration of crude oil. 16 0 2 4 6 8 10 12 0 2 4 6 8 10 12 14 C r u d e o il c o n c e n tr a ti o n (g /m l) × 1 0 -3 Incubation time (days) 0 2 4 6 8 10 12 0 2 4 6 8 10 12 14 C r u d e o il c o n c e n tr a ti o n (g /m l) × 1 0 -3 Incubation time (days) 2.7 Estimation of crude oil concentration In order to estimate residual crude oil, 10 ml of analytical grade n-hexane was added to a flask containing 1 ml of sub-sample removed from the degradation experiment and then transferred in to a separating funnel to extract oil. The extracted aliquot was evaporated to dryness in the water bath at 69 o C and residue oil was dissolved in 10 ml of n-hexane and the concentration was measured according to Omatayo et al., (2012) using visible spectrophotometer at 400 nm. Concentration of oil in the sample was obtained using the pre-prepared standard curve and the dilution factor was considerate to calculate the actual concentration (Latha and Kalaivani, 2012). Recovery of analytical procedure was maintained to keep more than 95% before samples were subjected to final measurement (Liyanage and Manage, 2014). 2.8 Determination of degradation rates The degradation rates (h) of the bacteria were calculated using the equation bellow, where “Co” and “C” are the concentration of crude oil at the beginning and at the end of the time interval “t” respectively (Manage et al., 2000). 3. Results 3.1 Effect of water temperature on bacterial degradation of crude oil Figure 1: (a) (b) (c). Degradation of crude oil (0.01 gml -1 ) by bacteria in different temperatures. 23 o C (black square), 28º C (close circle) and 33º C (open circle). (a) B. cereus 0 2 4 6 8 10 12 0 2 4 6 8 10 12 14 C r u d e o il C o n c e n tr a ti o n (g /m l) × 1 0 -3 Incubation time (days) (b) Enterobacter sp. (c) E. ludwigii Liyanage & Manage /Journal of Tropical Forestry and Environment Vol. 5. No 02 (2015) 13-25 17 Figure 1 (a), (b) and (c) illustrate the degradation of crude oil by B. cereus, E. ludwigii and Enterobacter sp. at three different temperatures. When temperature increased to 28 o C and 33 o C the degradation of crude oil by the bacteria isolates were initiated without a lag phase was detected. On the other hand, degradation at 23 o C was slow in all bacteria strains and remained less than 60% except B. cereus following 14 days of incubation. Table 1: Degradation rates of crude oil by bacteria isolates in different temperature. Incubation time (days) Name of bacteria Degradation rate (day -1 ) 23 o C 28 o C 33 o C 0-2 B. cereus 0.540 ±0.008 0.740 ±0.008 1.510 ±0.002 Enterobacter sp. 0.470 ±0.007 0.652 ±0.008 0.812 ±0.011 E. ludwigii 0.350 ±0.005 0.581 ±0.008 0.678 ±0.003 2-4 B. cereus 0.480 ±0.008 0.642 ±0.008 1.213 ±0.002 Enterobacter sp. 0.450 ±0.007 0.583 ±0.008 0.585 ±0.011 E. ludwigii 0.420 ±0.005 0.564 ±0.008 0.572 ±0.003 4-6 B. cereus 0.475 ±0.008 0.689 ±0.008 1.012 ±0.002 Enterobacter sp. 0.472 ±0.007 0.595 ±0.008 0.523 ±0.011 E. ludwigii 0.465 ±0.005 0.578 ±0.008 0.575 ±0.003 6-8 B. cereus 0.515 ±0.008 0.712 ±0.008 1.112 ±0.002 Enterobacter sp. 0.532 ±0.007 0.612 ±0.008 0.586 ±0.011 E. ludwigii 0.486 ±0.005 0.593 ±0.008 0.590 ±0.003 8-10 B. cereus 0.315 ±0.008 0.432 ±0.008 0.765 ±0.002 Enterobacter sp. 0.332 ±0.007 0.392 ±0.008 0.543 ±0.011 E. ludwigii 0.416 ±0.005 0.393 ±0.008 0.323 ±0.003 12-14 B. cereus 0.115 ±0.008 0.102±0.008 0.117±0.002 Enterobacter sp. 0.087±0.007 0.088 ±0.008 0.102 ±0.011 E. ludwigii 0.076 ±0.005 0.077 ±0.008 0.089 ±0.003 At 33 o C 92% removal of oil was detected by B. cerues, whereas Enterobacter sp. and E. ludwigii removed 79% and 88% respectively at 14 days of incubation. The highest degradation percentage of oil was recorded at 33 o C and it was significantly different from the removal percentages of crude oil obtained at 23° C and 28° C respectively. The rate of crude oil degradation by any bacterial isolate relies mainly on the incubation temperature. The highest degradation rate of oil was detected for all bacteria strains at 33 o C (Table 1) where B. cereus showed the highest degradation rate (1.510 ±0.002 d -1 ) after two days of incubation. Thus, 33 o C temperature was selected as optimum temperature to carry out the rest of experiments planned in the present study. Degradation rate of B. cereus was 0.540±0.008 d -1 and 0.740±0.008 d -1 at 23 o C and 28 o C respectively. Enterobacter sp. and E. ludwigii showed 0.812±0.011 d -1 and 0.678 ±0.003 d -1 degradation rates at 33 o C after 2 days of incubation respectively. In order to evaluate the possible influence of nutrients, especially nitrates and phosphates on bacterial degradation of crude oil, bacterial cultures were incubated separately in different concentrations of nitrate and phosphate with crude oil. 18 0 2 4 6 8 10 12 0 2 4 6 8 10 12 14 C r u d e o il c o n c e n tr a ti o n (g /m l) × 1 0 -3 Incubation time (days) (b) Enterobacter sp. 3.2 Effect of nutrients on degradation of crude oil The phosphate concentration in the sea water at the time of bacterial isolation was ranged between 0.002 to 0.05 ppm. Similar concentration range of phosphate was used to study degradation kinetics of oil in vitro in the present study. Significant degradation of oil with inoculation of phosphate was not recorded (Figure 2). The degradation rate of B. cereus varied between 0.735±0.002 d -1 - 0.755±0.008 d -1 at 0-2 days of incubation and maintained more or less constant degradation rate (0.642±0.008-0.692±0.005 d -1 ) at 2-8 days of incubation for each phosphate concentration. Then degradation rate increased from 0.642±0.008 d -1 to 0.756±0.003 d -1 at 8-10 days of incubation and then decreased to 0.102±0.002 d -1 at 10-14 days of incubation (Figure 2(a)). Degradation rate of oil by Enterobacter sp. was changed between 0.457±0.003 d -1 -0.682±0.021 d -1 at 0-8 days and was increased to 0.692±0.001 d -1 at 8-10 days. There after degradation rate was decreased from 0.692±0.021 d -1 to 0.069±0.003 d -1 at 10-14 days (Figure 2(b)). Significant difference of degradation rates was not detected between control and experiments setup for Enterobacter sp. as well. At each phosphate concentration E. ludwigii also showed less or more constant range of degradation (0.386 to 0.525 d -1 ) at 0-12 days and then gradually decrease (0.072 to 0.083 d -1 ) at 12-14 days of incubation (Figure 2(c)). Figure 2: Degradation of crude oil (0.01g/ml) by bacteria isolates with phosphate enrichment. (a) B. cereus (b) Enterobacter sp. (c) E. ludwigii; 0.002 ppm (close square), control (close circle) and 0.05 ppm (open square). 0 2 4 6 8 10 12 0 2 4 6 8 10 12 14 C r u d e o il c o n c e n tr a ti o n ( g /m l) × 1 0 -3 Incubation time (days) (a) B. cereus 0 2 4 6 8 10 12 0 2 4 6 8 10 12 14 C r u d e o il c o n c e n tr a ti o n (g /m l) × 1 0 -3 Incubation time (days) (c) E. Ludwigii Liyanage & Manage /Journal of Tropical Forestry and Environment Vol. 5. No 02 (2015) 13-25 19 0 2 4 6 8 10 12 0 2 4 6 8 10 12 14 C r u d e o il c o n c e n tr a ti o n (g /m l) × 1 0 3 Incubation time (days) 0 2 4 6 8 10 12 0 2 4 6 8 10 12 14 C r u d e o il c o n c e n tr a ti o n (g /m l) × 1 0 3 Incubation time (days) Table 2: Degradation rates of crude oil by bacteria with phosphate enrichment. Incubation time (days) Name of bacteria Degradation rate (day -1 ) Control 0.002 ppm 0.005 ppm 0-2 B. cereus 0.735 ±0.008 0.740 ±0.008 0.755 ±0.002 Enterobacter sp. 0.682 ±0.007 0.652 ±0.008 0.675±0.011 E. ludwigii 0.479 ±0.005 0.481 ±0.008 0.478 ±0.003 2-4 B. cereus 0.657 ±0.008 0.642 ±0.008 0.659 ±0.002 Enterobacter sp. 0.595 ±0.007 0.603 ±0.008 0.585 ±0.011 E. ludwigii 0.429 ±0.005 0.432 ±0.008 0.439 ±0.003 4-6 B. cereus 0.675 ±0.008 0.689 ±0.008 0.654 ±0.002 Enterobacter sp. 0.587 ±0.007 0.593 ±0.008 0.579 ±0.011 E. ludwigii 0.458 ±0.005 0.461 ±0.008 0.455 ±0.003 6-8 B. cereus 0.683 ±0.008 0.692 ±0.008 0.678 ±0.002 Enterobacter sp. 0.457 ±0.007 0.476 ±0.008 0.467 ±0.011 E. ludwigii 0.397 ±0.005 0.393 ±0.008 0.386 ±0.003 8-10 B. cereus 0.789 ±0.008 0.756 ±0.008 0.765 ±0.002 Enterobacter sp. 0.687±0.007 0.692 ±0.008 0.674 ±0.011 E. ludwigii 0.495 ±0.005 0.512 ±0.008 0.525 ±0.003 10-12 B. cereus 0.105 ±0.008 0.108±0.008 0.111±0.002 Enterobacter sp. 0.079±0.007 0.083 ±0.008 0.087 ±0.011 E. ludwigii 0.503 ±0.005 0.512 ±0.008 0.499 ±0.003 12-14 B. cereus 0.105 ±0.008 0.102±0.008 0.107±0.002 Enterobacter sp. 0.078±0.007 0.069 ±0.008 0.083 ±0.011 E. ludwigii 0.072 ±0.005 0.083 ±0.008 0.082 ±0.003 (b) Enterobacter sp. 0 2 4 6 8 10 12 0 2 4 6 8 10 12 14 C r u d e o il c o n c e n tr a ti o n (g /m l) × 1 0 3 Incubation time (days) (a) B. cereus (c) E. ludwigii Figure 3: Degradation of crude oil (0.01 g/ml) by bacteria in the presence of nitrate. (a) B. cereus (b) Enterobacter sp. (c) E. ludwigii; 1.0 ppm (black square), 0.1 ppm (open square) and 2.5 ppm (close circle). 20 Table 3: Degradation rates of crude oil by bacteria in the presence of nitrate. Incubation time (days) Bacteria Degradation rate (day -1 ) Control 0.1 ppm 2.5 ppm 0-2 B. cereus 0.742 ±0.008 0.738 ±0.008 0.749 ±0.002 Enterobacter sp. 0.654 ±0.007 0.664 ±0.008 0.672±0.011 E. ludwigii 0.572 ±0.005 0.578 ±0.008 0.583 ±0.003 2-4 B. cereus 0.603 ±0.008 0.598 ±0.008 0.612 ±0.002 Enterobacter sp. 0.575 ±0.007 0.583 ±0.008 0.585 ±0.011 E. ludwigii 0.323 ±0.005 0.357 ±0.008 0.378 ±0.003 4-6 B. cereus 0.597 ±0.008 0.621 ±0.008 0.632 ±0.002 Enterobacter sp. 0.632 ±0.007 0.648 ±0.008 0.663 ±0.011 E. ludwigii 0.356 ±0.005 0.392 ±0.008 0.401 ±0.003 6-8 B. cereus 0.595 ±0.008 0.626 ±0.008 0.628 ±0.002 Enterobacter sp. 0.432 ±0.007 0.412 ±0.008 0.441 ±0.011 E. ludwigii 0.345 ±0.005 0.357 ±0.008 0.489 ±0.003 8-10 B. cereus 0.621 ±0.008 0.791 ±0.008 0.845 ±0.002 Enterobacter sp. 0.689 ±0.007 0.698 ±0.008 0.732 ±0.011 E. ludwigii 0.534 ±0.005 0.567 ±0.008 0.592 ±0.003 10-12 B. cereus 0.374 ±0.008 0.432 ±0.008 0.692 ±0.002 Enterobacter sp. 0.112 ±0.007 0.231 ±0.008 0.541 ±0.011 E. ludwigii 0.521 ±0.005 0.572 ±0.008 0.579 ±0.003 12-14 B. cereus 0.015 ±0.008 0.132±0.008 0.147±0.002 Enterobacter sp. 0.079±0.007 0.081 ±0.008 0.083 ±0.011 E. ludwigii 0.071 ±0.005 0.076 ±0.008 0.082 ±0.003 A rapid degradation of crude oil was recorded by all three strains at 14 days of incubation with the increase of nitrate concentration in the medium from 0.1 to 2.5 ppm. Crude oil degradation percentage for B. cereus increased from 80% to 98% where Enterobacter sp. from 73% to 90% and in E. ludwigii from 70% to 83% respectively (Figure 3). Out of two nitrate concentrations, 2.5 ppm proved to be the most potent nitrate concentration which seems accelerates bacterial growth. The degradation rate of B. cereus gradually decreased from 0.749±0.05 to 0.628±0.03 d -1 during 0-8 days of incubation and thereafter increased rapidly to 0.845 d -1 during 8-10 days when medium contain 2.5 ppm nitrate. The lowest degradation rate (0.147 d -1 ) by the B. cereus was showed at 12-14 of incubation. The other two bacterial strains showed lower degradation rate throughout the incubation period compare to B. cereus. At 2.5 ppm nitrate concentration, oil degradation rate of Enterobacter sp. decreased from 0.672 to 0.441 d -1 during 0-8 days of incubation where more or less similar degradation rate was recorded by E. ludwigii (0.583 to 0.489 d -1 ).Thereafter, degradation rate was gradually increased by Enterobacter sp. (0.732d -1 ) and E. ludwigii (0.592 d -1 ) at 8-10 days of incubation and decreased again to 0.083 and 0.082 d -1 respectively for Enterobacter sp., E. ludwigii at 12-14 days of incubation. Liyanage & Manage /Journal of Tropical Forestry and Environment Vol. 5. No 02 (2015) 13-25 21 0 2 4 6 8 10 12 0 2 4 6 8 10 12 14 C r u d e o il c o n c e n tr a ti o n ( g /m l) × 1 0 -3 Incubation time (Days) 3.3 Effect of pH on degradation of crude oil Bacteria isolates employed in the present study showed different degradation rates in different pH. The highest degradation (84%) showed in acidic medium (pH 5.4) by E. ludwigii. In contrast B. cereus (94%) and Enterobacter sp. (88%) showed great degradation trends along with incubation in alkaline medium (pH 8.6). The highest degradation rate (0.767 d -1 ) was detected at 0-2 days of incubation and the degradation rate was decreased from 0.534±0.03 to 0.082±0.03 d -1 during 4-14 days of incubation, where degradation rate of Enterobacter sp. decreased gradually from 0.634±0.04 d -1 to 0.098±0.05 d -1 during 0-14 days when the medium was alkaline (pH= 8.6) at 33 o C (Table 4). Figure 4: Degradation of crude oil (0.01g/ml)by the bacteria in different pH. (a) B. cereus (b) Enterobacter sp.(c) E. ludwigii; pH= 5.4 (black square), pH= 8.6 (close circle) and pH=7.2 (open circle). Results of the present study revealed that favourable environmental factors often lead to increase the bioremediation rate of native crude oil degrading bacteria. Among the favourable factors; water temperature, nitrate and phosphate concentration and pH were considered as an essential component maintaining the balance of natural bacterial community. 0 2 4 6 8 10 12 0 2 4 6 8 10 12 14 C r u d e o il c o n c e n tr a ti o n (g /m l) × 1 0 -3 Incubation time (Days) (a) B. cereus (c) E. ludwigii 0 2 4 6 8 10 12 0 2 4 6 8 10 12 14 C r u d e o il c o n c e n tr a ti o n (g /m l) × 1 0 -3 Incubation time(Days) (c) E. ludwigii (b) Enterobacter sp. 22 Table 4: Degradation rates of bacterial strains in different pH values. Incubation time (days) Bacteria Degradation rate (day -1 ) 5.4 Control (7.2) 8.6 0-2 B. cereus 0.232 ±0.008 0.740 ±0.008 0.767 ±0.002 Enterobacter sp. 0.634 ±0.007 0.652 ±0.008 0.687 ±0.011 E. ludwigii 0.678 ±0.005 0.581 ±0.008 0.357 ±0.003 2-4 B. cereus 0.213 ±0.008 0.341 ±0.008 0.349 ±0.002 Enterobacter sp. 0.342 ±0.007 0.586 ±0.008 0.678 ±0.011 E. ludwigii 0.572 ±0.005 0.564 ±0.008 0.327 ±0.003 4-6 B. cereus 0.237 ±0.008 0.225 ±0.008 0.534 ±0.002 Enterobacter sp. 0.357 ±0.007 0.599 ±0.008 0.623 ±0.011 E. ludwigii 0.575 ±0.005 0.578 ±0.008 0.278 ±0.003 6-8 B. cereus 0.132 ±0.008 0.337 ±0.008 0.421 ±0.002 Enterobacter sp. 0.123 ±0.007 0.215 ±0.008 0.634 ±0.011 E. ludwigii 0.583 ±0.005 0.593 ±0.008 0.221 ±0.003 8-10 B. cereus 0.098 ±0.008 0.339 ±0.008 0.419 ±0.002 Enterobacter sp. 0.142 ±0.007 0.392 ±0.008 0.597 ±0.011 E. ludwigii 0.457 ±0.005 0.393 ±0.008 0.189 ±0.003 10-12 B. cereus 0.087 ±0.008 0.338 ±0.008 0.415 ±0.002 Enterobacter sp. 0.156 ±0.007 0.387 ±0.008 0.512 ±0.011 E. ludwigii 0.323 ±0.005 0.393 ±0.008 0.106 ±0.003 12-14 B. cereus 0.085 ±0.008 0.079±0.008 0.082 ±0.002 Enterobacter sp. 0.134 ±0.007 0.102 ±0.008 0.098 ±0.011 E. ludwigii 0.066 ±0.005 0.077 ±0.008 0.057 ±0.003 4. Discussion Sri Lanka is exposed as the worst polluter of the Indian ocean (NCEAS, 2012) and the irresponsible use of land practices, dumping oil waste to the sea, shipping activities are enhanced the contamination load of hydrocarbons, inorganic chemicals in the Indian ocean (MEPA, 2012). Therefore, the sites where the bacterial strains were isolated are more vulnerable to oil contamination with massive shipping activity and improper practices. Recently national and international agencies have received greater attention on oil pollutant status of sea and bioremediation application was suggested as an effective and eco-friendly solution (Mace, 2012). A limited number of research has been carried out up to date on microbial degradation of crude oil. Recent time, the contamination of crude oil in coastal area is becoming a problem and poses a potential threat to human health as well. Thus, application of microbes to remediate oil is important as natural method (Thavasi et al., 2011; Hassanshahian et al., 2012). Liyanage and Manage, (2014) reported isolation and characterization of oil degrading bacteria from coastal zone in Sri Lanka as a first report, and the present study was aimed to optimize the environmental factors that effect on the bacterial isolates. Bioremediation processes are governed by some environmental factors such as type and amount of crude oil present in the environment, environmental temperature, nutrients such as NO - 3, PO4 3- , pH, aeration, water acidity, type and population density of hydrocarbon degrading microorganisms and contaminant mobility (Dragun, 1998; Mace, 2012). Leahy and Colwell, (1990) reported the degradation Liyanage & Manage /Journal of Tropical Forestry and Environment Vol. 5. No 02 (2015) 13-25 23 of hydrocarbon reached 29.8% after 3 days of treatment at 30 o C, whereas at 20 o C, the degradation of TPH (Total Petroleum Hydrocarbon) reached only 7%. This indicates that bacteria prefer higher temperatures, which enhance bacteria growth, following accelerates bioremediation. In the present study, at 23°C, all strains showed low degradation of crude oil and pronounced degradation was detected with increase temperature at 33 0 C for all the bacterial strains. However, Alberty (2011) recorded that when temperature increases higher than 33 °C was resulted a decreasing degradation ability of bacterial strains. It is likely that at high temperatures, bacterial cells are unable to produce crude oil degrading enzymes which may result slow degradation rates (Amer et al., 2014). The studies carried out by Zaccone et al., (2002) have clearly described the effect of seasonal variance of nutrients in water which enhance the metabolic activities of heterotrophic bacteria. Odu, (1978) documented that, after 12 weeks of incubation oil degradation by bacteria was not significantly increased in the media which was treated with phosphate. Similar results were obtained from the present study as the degradation of oil by the bacterium was not significant when the media was supplemented with phosphate (Figure 2). Zahra and Alireza, (2005) documented that NH4NO3 is the best nitrogen source which maximize the degradation of oil by bacteria. Results of the present study also showed that increasing of nitrate concentration in the medium from 0.1 to 2.5 ppm, enhance the oil degradation by all bacterial species (Figure 3). Ronald, (1996) documented that phosphate concentration on oil degradation is less effective compare with nitrogen. The analysis of the effect of phosphate and nitrate concentrations on crude oil degradation percentage showed that phosphate concentration does not have a potent on removal of crude oil where nitrate performs a major role on degradation process; through the degradation rate was subjected to fluctuation (Figure 3 and Figure 4). Biochemical reactions of microorganisms are catalysed by enzymes (Zahra and Alireza, 2012) and it is well known fact that enzymatic reactions occur within a suitable pH range for microorganisms which are sensitive to alterations of pH (Zahra and Alireza, 2012; Ron and Rosernberg, 2014). Results of the present study also revealed that all the bacteria isolates except E. ludwigii showed the highest degradation when the medium was alkaline (pH=8.6) (Figure 4) where similar condition was recorded by Diaz-Ramirez et al., (2013) for degradation of oil by the Bacillus sp. in alkaline media as well. Thus, the results of the present study showed the effect of pH is one of the limiting factors for bio stimulation which enhance the bacterial bio remediation process in the natural environment. In addition to the results revealed that favourable environmental factors often lead to increase the bioremediation rate of native crude oil degrading bacteria. Further researches in this field can result in the development of most efficient and less time consuming microbial technologies which are important for developing country like Sri Lanka as accelerated development has lounged international shipping harbours and fishing harbours along the coast of the country. Put in harnessing microbes to degradation of petroleum became world interest and scientists are being established soon remediation methods efficient, economic, versatile and environmentally sound treatment to remove hydrocarbons form the contaminated environment. The results of the present study have showed that the bacterium B. cereus, Enterobacter sp. and E. ludwigii can be used as microbial agents to remove crude oil from contamination sites of the environment and temperature, pH and nitrate concentrations are critically effect on natural oil degradation process in the environment. Acknowledgement Authors acknowledge the financial assistance of University of Sri Jayewardenepura. 24 References Amer, R., El-Gendy, N. S., Taha, T., Farag, S. and Adel Fattah, Y. 2014. Biodegradation of crude oil and its kinetics using indigenous bacterial consortium isolated from contaminated area in Egyptian Mediterranean ecosystem. Jokull Journal, 64(4): 42-52. Alvarez. P.J.J., Anid. P.J. and Vogel, T.M. 1991. Kinetics of aerobic biodegradation of benzene and toluene in sandy aquifer material. Biodegradation, 2: 43-51. Boopathy. R. (2000) Factors limiting bioremediation technology. Bioresource Technology. 74: 63-67. Bouwer. E.J. and Zehner. A.J.B. 1993. Bioremediation of organic compounds; putting microbial metabolism to work. Trends Biotechnology, 11: 360-367. Chin. K.J., Sharma. M.L., Russel. L.A. and Lovely, D.R. 2008. Quantifying expression of a dissimilatory (bi) sulphite reductase gene in petroleum-contaminated marine harbour sediments. Microbial Ecology, 55: 489-499. Dragun. J. 1998. The soil chemistry of hazardous materials. 2 nd edition. Amherst Scientific Publisher, Amherst. MA. Efsun. D., Fatma. O.T. and Huseyin. S.B. 2013. Bioremediation of petroleum contaminated soil. Biological Environmental Science, 7: 39-47. Farshid. K., Sara. R. and Yaghoob. T. 2011. Evaluation of bioremediation of naphthalene using native bacteria isolated from oil contaminated soils in Iran. Biological Research, 6: 610-616. Gibb. A., Chu. R., Wong. K. and Goodman. R.H. 2001. Bioremediation kinetics of crude oil at 5 0 C. Environmental Engineering, 127(a): 818-824. Hanson. K.G., Nigam. A. and Kapadia. M. 1997. Bioremediation of crude oil contamination with Acinetobactersp. Microbiology, 35. 191-193. Hassanshahian, M., Mehdi, G., and Cappello, S. 2012. "Isolation and characterization of crude-oil- degrading bacteria from the Persian Gulf and the Caspian Sea. Marine pollution bulletin, 64.1: 7-12. ITOPF. (2012).www.itopf.com/information-services/date-and-statistics/ statistics/#top. [Accessed 05 August 2015]. Joel. E.K., Prakash. O., Will. A. and Stefan. J. 2011. Hydrocarbon degrading bacteria and the bacterial community response in Gulf of Mexico beach sands impacted by the deep water horizon oil spill. Applied and Environmental Microbiology, 77: 7962-7974. Kishore. D. and Ashisk. M. 2007. Crude petroleum oil biodegradation efficiency of Bacillus subtilis and Pseudomonas aeruginosa strains isolated from a petroleum contaminated soil. Bioresource Technology, 98(7): 1339-1345. Kim. S.J., Choi. D.H. and Sim. D.S. 2005. Evaluation of bioremediation effectiveness on crude oil contaminated sand. Chemosphere, 59: 845-852. Leahy. J.G. and Colwell. R.R. 1990. Microbial degradation of hydrocarbons in the environment. Microbiology and Molecular Biology Reviews, 54: 305-315. Liyanage. Y. and Manage. P.M. 2014. Isolation and characterisation of oil degrading bacteria from coastal water and sediments, 19 th International Forestry and Environment Symposium Proceeding, University of Sri Jayewardenepura, Sri Lanka, pp 129. Liyanage & Manage /Journal of Tropical Forestry and Environment Vol. 5. No 02 (2015) 13-25 25 Mace. G.B. 2012. Ecological impacts of the deep water horizon oil spill; Implications for immunotoxicity. Toxicologic Pathology, 40: 315-320. Manage. P.M., Christine E., Linda. and Lawton, A. 2009. Biodegradation of Microcystin –LR by natural bacterial population. Applied Environmental Microbiology, 75(21): 5928-5932. Manage. P.M., Zen’ichiro. K. and Shin-ichi. 2000. Algicidal effect of the bacterium Alcaligenes denitrification on Microcystis spp. Aquatic Microbial Ecology, 22: 111-117. NCEAS. 2012 Available at:www.nceas.ucsb.edu/product. [Accessed 05 August 2015]. MEPA. 2012. Oil spill events. www.mepa.gov.lk/web/index.php? option=com_content&view =article&id=56&itemid. [Accessed 12 August 2015]. Widdel. F. and Rabus. R. 2001. Anaerobic biodegradation of saturated and aromatic hydrocarbons. Current Opinion in Biotechnology, 12: 259-276. Omatayo. A.E., Ojo. O.Y. and Amund. O.O. 2012. Crude oil degradation by microorganisms in soil composts. Microbiology, 7: 209-218. Prince. R.C., Clark. J.R. and Lee. K. 2002. Bioremediation effectiveness; Removing hydrocarbons while minimizing environmental impacts. 9 th International Petroleum Environmental Conference. IPEC. Albuquerque. NM. Ronald. M. Atlas. 1996. Petroleum biodegradation and oil spill bioremediation. Oceanographic Literature Review, 43(6): 351-357. Ron, E.Z. and Rosenberg, E. 2014. Enhanced bioremediation of oil spills in the sea. Current Opinion in Botechnology, 27: 191-194. Santos. H.F., Carmo. F.L., Paes. J.E., Rosado. A.S. and Pexioto, R.S. 2011. Bioremediation of mangroves impacted by petroleum. Water, Air and Soil Pollution, 216. 329-350. Thavasi, R., Singaram J. and Ibrahim M. B. 2011. Application of biosurfactant produced from peanut oil cake by Lactobacillus delbrueckii in biodegradation of crude oil. Bioresource Technology, 1023: 3366-3372. Trindade. P., Sobral. L.G., Rizzo. A.C., Leite. S.G.F. and Lemos. L.S. 2002. Evaluation of the biostimulation and bioaugmentation techniques in the bioremediation process of petroleum hydrocarbon contaminated soils. 9 th International Petroleum Environmental Conference. IPEC. Albuquerque. NM. Toone. E., Winde. H., Steffan. R.J. and Ramos, J.L. 2013. Environmental biotechnology. Current Opinion in Biotechnology, 24: 367-549. Xiaojun. Li., Peijun. Li., Xin. Lin., Chungui. Z. and Qi, L. 2008. Biodegradation of aged polycyclic aromatic hydrocarbons by microbial consortia in soil and slurry phases. Hazardous Materials, 150. 21-26. Zahra. Y. and Alireza. F. 2012. Survey effect of pH on isolated petroleum degrading bacteria. Agriculture; Research and Review, 2(5): 652-65.