Isolation, screening and characterization of lipase from bacterial isolates and its application in detergents and oily waste water degradation

The present study was conducted to explore industrial potential of bacterial lipase. Seven isolates were isolated from oil contaminated soil samples. Isolated lipolytic bacteria were grown on lipolytic specific media comprising 1% (w/v) mustard oil. Egg-yolk test was carried out to determine primary lipase activity. The crude enzyme extracts of two isolates SI1 and S17 showing maximum lipolytic activity were prepared and applied for further practical applications. In oil displacement test


Introduction
Lipases also known as Triacylglycerol hydrolases are capable to perform hydrolysis of triaclyglycerols by converting them into free fatty acids, monoglyceroid, diglyceroid and partial acylglycerols. Lipases are the most flexible biocatalyst which brings about large variety of bioconversion reactions, such as hydrolysis, esterification, acidolysis and aminolysis [1]. These are insidious enzymes which are widely present in animals, plants, fungi and bacteria, having considerable physiological importance and industrial potential [2]. Among these, microbial lipases, particularly from bacteria, have considerable advantages over those obtained from plants and animals because of numerous vital characteristics. Many microbes belonging to Acinetobacter, Bacillus, Burkholderia, Idiomarina, Natronococcus, Pseudomonas and Staphylococcus genera have been described to have lipolytic activity [3]. Lipases obtained from microbes have particular aspects such as increased yield, cheap production, varied activities in term of reaction catalyzed, easy genetic manipulation or treatment, stable in presence of different organic solvents and wide substrate specificity due to which these can be applied in wide range of industrial application such as detergent industry and fatty waste water management [4]. It has been estimated that about 13 billion tons of detergents are using about 1000 tons of lipase enzymes annually. The practice of using lipases as detergent additives decrease the need of using synthetic detergents and increases the capability of household detergents to remove tough oily or greasy stains from clothes [5]. Lipases can be utilized efficiently as detergent additives due to their property of hydrolyzing lipids. Lipases are particularly applied in detergents because of their possession of following qualities: (1) Ability to catalyze fats in different combinations (2) capability to survive in comparatively harsh washing environment (pH 10-11, 30-60ºC); (3) being able to retain activity in presence of other destructive enzymes, surfactants and inhibitors, which are significant components of many detergent preparations [6]. Lipids (fats, oils and greases) are widely used organic matters in industrial processes and can form layer on the surface of water thereby diminishing oxygen supply rate so industrial and household wastewater containing oily contents can cause environmental issues, therefore their high concentration in wastewater can cause troubles in wastewater treatment processes [7]. Lipases are specifically engaged in wastewater treatment (decreasing lipid congested drains), pharmaceutical, dairy industry, leather (remove fats from skin), detergent (removal of oily stains) and medical (as diagnostic tool) industries [8]. Due to its ability to remove fatty residues and cleaning congested drains, lipases have huge applications in household and industrial laundry detergents [9]. Application of enzymes enhances washing efficacy, thus causing reduction in agitation period, frequently after a first round of soaking [10]. If lipase-producing bacteria are added into the treatment processes, the efficiency of oily wastewater treatment units can be increased. Penicillium, Yarrowia, Geotrichum, Bacillus, Acinetobacter and Serratia spp. are some of microbes, which are being widely used oily wastewaters treatment due to their lipolytic activity [11]. Different bacterial species are applied in bioremediation of crude oil especially Pseudomonas sp. which clean soil from this oil and thus play central role in biodegradation of crude oil [12]. In this context, present study was aimed to find out some of the wide industrial potential of bacterial lipase in order to further explore microbial world for human benefits.

Sample collection
Soil samples were collected from two locations. One sample was collected from oil and diesel contaminated soil of auto mechanic workshop and second sample was collected from kitchen waste, Mianwali, Pakistan. Each soil sample brought into the laboratory was labeled as S1 and S2 respectively and was further processed.

Isolation of lipolytic bacteria
Soil samples were diluted by using serial dilution method. Specific lipolytic media having following composition was prepared: Peptone Agar 2.9% (w/v), Yeast Extract 0.3% (w/v), Mustard Oil 1% (w/v), pH 7.0. The media was sterilized in autoclave for 15 minutes at 121ºC. After sterilization, media was poured in sterilized petri dishes. After solidification, 10ul of each dilution was spread on solidified media evenly by rotating spreader carefully, following spread plate method. These plates were incubated for 48 hours for isolation of lipolytic bacteria from different soil samples [13]. Pure culturing After 48 hours of incubation, lipolytic bacteria were identified by clear zone around colony. Seven isolates were identified (labeled as SI1, SI2, SI3, SI4, SI5, SI6, SI7). After that, isolates were repeatedly grown on specific media to get pure culture. Incubation was done at 37ºC for 24 to 48 hours.

Egg-yolk test for primary lipolytic activity
The nutrient agar was melted, cooled to 55°C and yolk suspension was added to prepare egg yolk testing media. The saturated copper sulphate (CuSO4) was added after incubating plates for two days, and kept them for 20 mins. After that, he excess CuSO4 solution was removed and the plates were kept in incubator for drying. The lipolysis was confirmed by appearance of greenish-blue color of copper soaps of fats [14]. Lipase production The basal salt media was prepared for inoculation of lipolytic bacteria having following components: 10% Na2HPO4.7H2O (w/v), 2.5 % KH2PO4 (w/v), 0.6 % NaCl, 3 Mm MgSO4.7H2O, 1 % NH4Cl (w/v), 2.5% Glucose) (w/v) with 2.5 % additional Olive oil substrate (v/v).The pH of media was kept 7 and then incubation was done at 37°C24-48 hrs [15]. Preparation of crude lipase 24 hrs old culture was used to prepare inoculum by transferring loopful of cells into production media and the flasks were foe 24hr at 30⁰C in shaker incubator at a rate of 100 r.p.m for lipase synthesis. The crude enzyme were extracted from media by centrifugation at 12000 r.p.m and supernatant was obtained. Further studies of crude enzyme were carried out. Growth on low cost media All seven isolates were grown on two low cost media M1 and M2 (Table 1). Media were prepared, sterilized and all isolates were streaked to check their growth on low cost media. . For extraction of crude lipase enzyme extract, 100 ml of broth culture was prepared for each isolate (peptone 0.3g, yeast extract 0.1g, Sodium Chloride 0.05g, tween 80 0.5g per 100ml) , and then sterilization was carried out for 15 minutes at 121ºC. After cooling, media was Inoculated with isolates and incubated at 30ºC for 72 hours. The culture was then subjected to centrifugation at 7000 rpm for 30 minutes and the crude enzyme extract (supernatant) was collected. Petri dishes (150mm diameter) were filled with 40ml of distilled water and then 10ul of five different oils (weathered hydrocarbons) was added such as engine oil, vegetable oil, mustard oil, mobile oil and olive oil. Then 10ul of cell free culture supernatant of each isolate was placed on the center of each type of oil films. Diameter of clear halo zone was measured, after 30 sec of incubation and readings were noted in triplicates.

Characterization studies of lipase pH effect on lipase activity
The pH range of 4 to 11 was selected to detect optimum pH where enzymes exhibit maximum activities. Citrate phosphate buffer (4.0 7.0), Glycine-NaOH buffer (7.0 9.0) and Tris-HCl buffers (9.0 11.0) were used for pH adjustments.

Temperature effect on enzymatic activities
To study temperature effect on lipase activity, the activity assay was conducted at temperature range of 20 to 70°C to find out temperature for maximum enzyme activity.

Effects of commercial detergents on lipase activity
The compatibility of lipase in presence of detergents (Ariel, Bright, surf excel, Bonus, Tide, Sufi, Express power) was assayed by adding these at a concentration of 7mg/mL in reaction mixture to find out relative activity (%). The diluted detergents were heated for 40 minutes 60∘C to inactivate lipases already present detergents before adding enzyme preparation. The enzyme sample (40U/mL) aliquot was subjected to incubation with detergent solution in Tris-HCl buffer for 1 hour at 37⁰C to determine enzyme stability and activity. The relative activity (%) was recorded and comparison was done with enzyme sample without detergent (control). The activity of enzyme studied without detergent, incubated by having similar conditions was defined as relative activity of control and it was taken as100%.

Determination of Lipase activity in presence of metal ions, surfactants and inhibitors
The lipase activity in presence of metal ions such Ca2+, Mg2+, Zn2+, Na+, Ba+, Li+, K+ (5Mm Final concentration), surfactants such as Sodium dodecyl sulphate (SDS), Tween 20, Tween 80, (0.1% and 0.5% final concentration) and inhibitors such as Ethylenediaminetetraacetic acid (EDTA) and β-mercaptoethanol (1mM and 5 mM of final concentration) were studied by preincubation of lipase with each of these for 15 minutes. Residual activities were noted down as mentioned above and compared with control.

Applications Application of lipase enzyme extract in removal of oil from synthetic and real waste water
Pure culture of two highly efficient lipolytic isolates (SI1 and SI7) selected from seven isolates on the basis of oil displacement test were applied in the removal of oil from synthetic and real waste water by using method stated by [11]. Media for Inoculum preparation of these two isolates were prepared by adding Yeast extract 0.3g, NaCl 0.05g, Tween 80 0.5ml, in 100ml of distilled water. After sterilization, media was inoculated with SI1 and SI7 and incubated at 37ºC for 24 hours. Synthetic waste water was prepared (Ammonium nitrate 0.4g, Sodium chloride 0.5g, KH2PO4 0.2g per 100ml )with various concentration of olive oil (0.1, 1.5 and 2.0ml).This synthetic waste water and 100ml of real waste water was sterilized at 121ºC. 5ml of inoculum was inoculated in real and synthetic waste water and four readings were taken by titration method at 0, 24, 48, and 72 hours of incubation. After this, 10 ml from inoculated real and synthetic wastewater was taken at each intervals of incubation and added 20ml of acetone: alcohol (1:1) mixture to stop reaction. Than mixture was titrated ( Fig. 1) with 0.1M NaOH using phenolphthalein indicator, until it got pink color. Noted that point and readings were determined. Application of lipase enzyme extract in oily stains removal Application of lipase enzyme extract (for SI1 and SI7)) as a detergent additive was carried out on cotton cloth pieces (10×10 cm) blemished with weathered hydrocarbons (olive oil and mustard oil) following method described by [10]. The experimental groups were set to study oily stain removal efficacy of lipase enzyme as follow: 1. Flask with distilled water (100 ml) + stained cloth (stained with olive and mustard oil). 2. Flask with distilled water (100 ml) + stained cloth (stained with olive and mustard oil) + 1 ml of detergent surf excel (10mg/ml). 3. Flask with distilled water (100 ml) + stained cloth (stained with olive and mustard oil) + 1 ml surf excel detergent (10mg/ml) + 2 ml of lipase enzyme extract. All flasks were kept in incubator at 60°C for 15 minutes to check oily stain removal efficiency. Cloth pieces were visually examined after treatment.

Statistical analysis
All experiments were performed in triplicates.

Results and Discussion
Isolation and morphological examination A total of 7 morphologically distinct colonies were isolated (Fig. 2) by using lipolytic selective agar medium from the soil samples and labeled as SI1, SI2, SI3, SI4, SI5, SI6, and SI7. Analysis of pure colonies of lipolytic bacteria based on morphological characteristics was carried out ( Table 2). Growth of each isolated strains on semi solid agar media showed that all strains were motile.

Egg-yolk screening test for primary lipolytic activity
The bacterial lipase activity can be detected by using egg-yolk suspension. Lipolysis can be observed by appearance of, shimmering 'pearly layer' covering the colonies and a 'confined' opalescence in the in solid media containing egg yolk suspension. Greenishblue unsolvable copper soaps is formed by reaction of copper sulphate with the fatty acids, visible in both shining layer and opalescence in the media. Lipase destroys the fats within the egg yolk, which results in Greenish blue color of colony appears due to activity of lipase destroying fats of egg yolk of the after addition of copper II sulphate solution.

Growth on low cost media
On the media M1 and M2, all seven isolates (SI1, SI2, SI3, SI4, SI5, SI6 and SI7) were grown. After incubating for 48h, the results showed that on M1 media each isolates showed positive growth, but on M2 media SI5 and SI7 showed negative growth, while other SI1, SI2, SI3, SI4, and SI6 gave positive results.

Oil displacement test
Oil displacement test showed that all seven isolated strains were lipolytic but SI1 and SI7 (Fig. 3, 4, 5) showed increased lipolytic activity and hence these were used for further experiments to check their potential for practical applications [7] Zone of Hydrolysis were measured for all isolates to quantify their lipolytic potential (Table 3).

Figure 6. (a) Relative activity (%) of SD1 at optimum pH; (b) Relative activity of SI7 at optimum pH
The SI1 lipases was found to have optimum activity at 60⁰C while SI7 crude lipase was had showed maximum activity at 40⁰C (Fig.  7).

Application of Lipase Enzymes in the removal of oil from synthetic and real waste water
The ability of two isolated strains (SI1 & SI7) that offered the highest lipolytic activity to degrade oily waste water was assessed. Flasks with synthetic and real waste water were prepared and incubated at 30°C and oil removal rate was observed after 24 to 48 hrs of incubation. Titration values recorded at equal interval of time for isolate SI1 and SI7 in synthetic and real waste water are shown (Fig. 8 , 9, 10, 11). Ammonium sulfate (NH4SO4) can also be used as a nitrogen source [30]. But Sulfuric acid is produced due to ammonia consumption from ammonium sulfate which can cause reduction in pH, and this change in pH can diminish the oil degradation activity of bacteria [30].
Both isolates SI1 and SI7 showed positive results when tested for oil-degradation. Hence it is proved that the bacterial isolates obtained from the diesel contaminated soil were oildegrading bacteria.
30-40% of lipids are present in municipal waste water as compared to total chemical oxygen demand. Oily wastewaters were conventionally being treated by physical methods, which are currently being considered as inefficient especially if the fat is present in dispersed state. Fat, oil and grease can be degraded into miscible molecules by using biological treatment and thus applying microbes for removing oily wastes is considered the most efficient method. Thus, the usage of microbial lipases may solve the problem [31].

Conclusion
The present work was aimed to isolate and characterize lipolytic strains from various soil samples. Seven bacterial isolates were isolated on medium containing lipid as a sole source of carbon and zone of hydrolysis was observed. On screening, isolates having highest lipolytic activity were selected, and further studied. The methods of choice were selected based upon the purpose of study and the available laboratory equipment. The results of the research reported that isolates which were obtained from the diesel contamoinated soil and kitchen waste, can degrade the oil and lipids.Therefore, these strains can be applied for remediation of oily wastewater in biological treatment processes. as well as can be used as detergent additives for removal of oily stains. It is predictable that the findings of current study can be exploited for future studies, and the lipase producing bacteria isolated from diesel and kitchen wastes will help in future, as possible solution for combating environmental pollution arising due to industrial and household effluents and wastewater. The tremendous potential of lipases in degradation of oil will be helpful in future to develop new, cost-effective and environmentally friendly technologies for increased production, scaling up and purification of this useful enzyme.