Biodegradation of premium motor spirit (PMS) by lipase from Bacillus thuringiensis and Lysinibacillus sphaericus

This study reported production of lipase by Bacillus thuringiensis and Lysinibacillus sphaericus. Bacteria isolates were screened on Bushnell-Hass Mineral Salt medium containing 1% PMS for oil degradation. Two potent isolates were identified using 16S rRNA as Bacillus thuringiensis and Lysinibacillus sphaericus. They were cultured for lipase production in a submerged medium. The crude lipases extracted were used for degradation of PMS. Optimum degradation of PMS 44.5% and 37.4% were obtained by lipase from Bacillus thuringiensis and Lysinibacillus sphaericus respectively at pH 7 and 35 in 20 days. This study therefore presented the use of bacterial lipase in degradation of PMS as a simple and effective approach.


Introduction
and lipolysis of cream and butter fat (Feng et al., tubes. The bacterial isolates were added and mixed by vortexing. Centrifugation was carried out at 10,000 rpm 2010).
for 5 min at 4 °C. The supernatant was discarded. 200 µl Other applications include paper, of sterile distilled water was added and vortex to pharmaceutical, cosmetics, detergent, leather, homogenize the pellets. The tubes containing the single cell protein production of fine chemicals, homogenized pellets were boiled at 100 °C for 10 waste water treatment, bakery products, and minutes. After boiling, the tubes were vortexed again biofuels industries (Bhavani et al., 2012). and centrifuged at 10,000 rpm for 5 minutes. The The direct use of microorganisms in supernatant were transferred into another pre-labeled bioremediation of oil polluted sites may be eppendorf tube by gentle aspiration using a technically difficult, hence the need to focus on micropipette. microbial enzymes is considered as alternative. This study therefore aimed at production of bacterial Polymerase chain reaction Fragments of the gene of interest, the 16S lipase and subsequent use for degradation of ribosomal gene, were amplified using standard PCR petrol.
protocol and the universal primer. The PCR reaction mixture (20 ìl) consisting of 4 ìl PCR master mix (Solis

Materials and Methods
Biodyne)), 0.5 ìl of each primer, 14.1 ìl nuclease free Sample Collection water and 1.5 ìl template DNA (Keramas et al., 2004). Samples were collected from oil contaminated Atlantic Seawater and Sediment Agarose gel electrophoresis sites. The collected samples were packed in sterile Agarose powder of (1.5 g) was added to 150 bottle to the laboratory. The entire sample was mls of 0.5x TAE buffer and dissolved by boiling using stored at refrigeration temperature before the microwave oven. The mixture was allowed to cool to experimental work. about 60ºC. Ethodium bromide (10 ml) was added and mixed by swirling gently it was then poured into Isolation of Bacteria electrophoresis tank with the comb in place to obtain a Soil and water sample were serially diluted gel thickness of about 4-5 mm. The tank was filled with and plated on Nutrient Agar medium, pH 7.0 by 1x TAE buffer. Thereafter the comb was removed. 10 µl spread plate method. Plates were incubated at of sample were mixed with 1µl of the 10x loading dye. 37°C for 48 hours. Pure cultures of the isolates were The samples were carefully loaded into the wells maintained on nutrient agar slants and were subcreated by the combs. The electrodes were connected cultured every 15 days.
to the power pack in such a way that the negative terminal was at the end where the sample was loaded.

Screening of Hydrocarbon Degrading Bacteria
The electrophoresis was allowed to run at 60-100 V The isolated bacteria were inoculated on an until the loading dye migrates about three-quarter of enrichment medium that contains mineral salt the electrodes. Electrodes were turned off and medium (MSM) supplemented with single disconnected. The gel was observed on a UV-transhydrocarbon compound as sole carbon source (1% illuminator (Keramas et al., 2004). petrol and diesel). The MSM composition was made up of basal salt medium and trace element solution.
Screening of the Isolates for Lipase Activity The basal medium contain (g/L): K HPO , 1.8; incubation, the culture was centrifuged at 10,000 rpm for carried out by extracting DNA (1 ml) of bacterial 20 min at 4ºC and the cell free culture supernatant fluid isolate using the method of (Keramas et al., 2004).
was used as the sources of extracellular enzyme. The Sterile distilled water was added into the eppendorf lipase activity in the supernatant was determined by carbon source. This corresponds to the findings of Latha the colorimetric method.
and Kalaivani (2012) where the isolates were able to utilized hydrocarbons-as their carbon source. A total of Determination of Lipase biodegradation of PMS 5 bacterial colonies were selected and isolated. Only The degrading activities of each enzyme two out of five 5 isolates showed good clear zone in the were obtained using Mineral salt broth (MSB) in olive agar medium with phenol red as indicator. Plate 1 which 40 ml of each hydrocarbon (PMS) was added shows gel electrophoresis of DNA extraction of bacteria and incubated at room temperature for 20 days.
isolates using 100bp marker. Their nucleotide The enzyme activity was measured by taking the sequences were 97% and 99% identical to optical density (O.D) readings at 600 nm after 20 Lysinibacillus sphaericus strain 2362 and Bacillus days against mineral salt medium as blank.
thuringiensis strain VITSJ-01 respectively. In the screening medium, Bacillus thuringensis Optimization studies on degradation shows maximum lipase production which produced 2.6 Optimization studies of the enzymes on u/ml followed by Lysinibacillus sphaericus which degradation of hydrocarbons was done, effect of produced 1.96 u/ml was selected for further research temperature (20 ºC, 25ºC, 30ºC, 35ºC, 40ºC, 45ºC, while others showed less than 1.96 u/ml (Table 2). It 50ºC), effect of pH (5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0) was reported that maximum lipase production was at 72 and degradation time (5 days, 10 days, 15 days, 20 hours for Bacillus coagulans (PrasanthKumar et al., days). Degradation was carried out using the 2007). Bacterial strain isolated in this study was method described previously in section 2.7.
i d e n t i f i e d a m o n g h y d r o c a r b o n d e g r a d i n g microorganisms for crude oil. The results obtained Gravimetric Analysis clearly showed that lipase produced from this The amount of oil in culture was estimated microorganism had biodegradable abilities and values using the Gravimetric method. Diethyl ether and of degraded PMS varied after incubation at 20 days. acetone were taken in 1:1 ratio and was mixed with Table 3 presented degradation of PMS with free culture. The mixture was allowed to vaporize at lipase of Bacillus thuringiensis and Lysinibacillus room temperature. The oil residue obtained was sphaericus. Lipase from Bacillus thuringiensis degraded weighed and taken as the gravimetric value for 44.5% PMS while free lipase from Lysinibacillus further calculation. (Marquez-Rocha et al., 2001) sphaericus degraded 37.4% PMS. The result of pH on degradation of PMS showed that degradation increased progressively with increase in pH from 5-8.5 reaching a maximum at 7 for PMS degradation from lipase of Weight of PMS oil present originally Bacillus thuringiensis and Lysinibacillus sphaericus Where, the weight of PMS oil degraded ( = (figure 1). A comprehensive review of all bacterial lipase original weight of PMS oil -weight of residual PMS done by Gupta et. al. (2004), states that maximum oil obtained after evaporating the extract).
activity of lipases at pH values higher than 7 has been observed in many cases. Effect of temperature on lipase Extraction and analysis of residual oil activity of the crude enzyme on degradation of PMS Biodegradation of petroleum hydrocarbon showed that degradation of PMS increased in liquid culture was collected for analysis by gas progressively with increase in temperature from 20oC chromatography (GC HP 680 series GC system, reaching a maximum at 35oC for lipase of Bacillus US90704303) (Marquez-Rocha et al., 2001).

Results and Discussion
An optimum temperature of 40ºC for Many bacterial species are present in the phenantheren degradation was reported by soil normally and adapting to the soil conditions and Stringfellow and Aitken (1994). Effect of degradation almost the bacteria that degrade the oil present in time on lipase activity of the crude enzyme on the soil contaminated with oil, which help in degradation of PMS was presented in Figure 3, an cleaning the soil from oil products (Cesarini et al., increase in degradation was observed along with the 2014). Prominent among the bacteria found in the increase in time from 5 to 20 days. Siddiqui et. al. (2001) oil contaminated soil is Bacillus spp. This agrees reported the percentage degradation for individual nwith earlier reports by Kumar et al. (2012)