Introduction

The demand for microbial industrial enzymes has attracted much interest owing to their novel & multifold applications in a wide variety of processes. Lipases are triacylglycerol hydrolases that catalyse the hydrolysis of triacylglycerol to glycerol and fatty acids.[7]Lipases have cosmic presence in nature such as soil, industrial effluents, oil contaminated areas etc and originated mostly from plants, animals, fungi, yeast and bacteria. Microbial lipases attracted more attention due to its easy isolation, ease of genetic manipulation, high yield possible, systematic amount due to absence of seasonal variations and quick growth of micro-organisms or low-priced media. Bacterial lipases are glycoproteins in nature but some extracellular lipases are lipoprotein. [9]Microbial lipases have high biotechnological applications. It becomes important biocatalyst in various industrial sectors such as dairy and food industries for cheese ripening, flavour enhancement and hydrolysis of milk fat, and lipolysis of cream and butter fat. Other applications include paper, pharmaceutical, cosmetics, detergent, leather, single cell protein production of fine chemicals, waste water treatment, bakery products, and biofuels industries.[9]The Major factor for the expression of lipase activity has always been reported as the carbon source, since lipases are inducible enzymes. These Enzymes are generally produced in the presence of a lipid such as oil or any other inducer, such as triacylglycerols, fatty acid, hydrolysable esters, tweens, bile salts and glycerols. It is well established that lipase production in microorganism is greatly influenced by media components. The present investigation is aimed at optimization of growth conditions and other parameters which have been pre-directed to play a significant role in enhancing the production of lipase enzyme. For this various parameters of nutritional and environmental factors were tested and growth and lipase activity were measured. [11]Various methods of lipase assay have been classified as; Titrimetry, Interfacial tensiometry, Spectroscopy, Chromatography, Immunochemistry and Conductimetry of these methods titrimetry is the simplest method which was used in our studies also.[7]

The purpose of the present study was to screen and identify potential lipase producing bacteria from various soil samples and optimize the production of lipase and partial purification of lipase.

Material And Mathods

Collection of soil samples

For the present study 11 Samples were collected from oil and fat contaminated soils from Area of Adajan, Jahangirpura and Olpad Of Surat,Gujarat. The soil samples were taken in appropriately labelled pre-sterilized bottles with the help of sterile spatula from the depth of 0.5 to 1.0 cm surface and subsurface. [4]

Table 1: Sampling sites for samples

Sample no Sample code Site
1 JA Garage soil from Jahangirpura ,suart
2 RA Milk waste soil from Rander, Surat
3 VS Milk waste soil from Olpad
4 HA Oil spilled soil from Hazira,Surat
5 AD Garage soil from Adajan, Surat
6 MS Milk waste soil from palanpur, Surat
7 TA Milk waste soil from talad, Olpad
8 PA Oil soil from palanpur, Surat
9 GS Garage soil from Surat
10 UD Oil Mill soil,Udhana
11 FS Motor Soil from farm,Olpad

Isolation of Lipase Producing Bacteria

Samples of soil were diluted serially from 10-1 up to 10-6 in sterile distilled water, each dilution were cultured on nutrient agar plates by Spread plate method to obtain isolated colonies after 24 hours of incubation. [7]

Screening of the Isolates for Lipase Activity

Pure bacterial isolates were screened for lipase production by Bacterial colonies were streaked on Tributyrin agar Plate [7], Phenol red agar Plate[6] and Tween 80 agar Plate[3] and incubated at 37˚C for 48 hours.

Identification of lipase producing Bacteria

The Morphological and Colony characteristics were studied using Nutrient agar Plate. The Physiological characteristics of all the obtained isolates were studied. The Biochemical characteristics (Indole, Catalase, Voges-Proskauer, Methyl Red, Citrate, Hydrogen sulphide production, nitrate Reduction, Oxidase, Gelatine hydrolysis test,) and sugar .fermentation tests were also carried out using standard reference Biochemical tests for identification of medical bacteria by jean E. Mac Faddin.[5]

Selection of Best Isolates for Lipase Production

The isolates capable of lipase production were further screened to isolate the best possible lipase producing bacteria based on agar well diffusion (Cup well method). Sterile Phenol Red Agar Plate with olive oil as a substrate use for the Agar well diffusion assay. These sterile agar plates were punched aseptically with sterile cup borer to obtain well of 4mm diameter. The isolates were grown in 10ml sterile nutrient broth for 24hrs and loaded with 50μl in each well separately and incubated at 37˚C in the incubator for 48 hrs. The developed yellow zones around the wells were measured (mm) and the data was used for further production of enzyme. [2]

Lipase Production

The isolate with higher zone production on phenol red agar plate was inoculated in the 15 ml of inoculum media (20 gm glucose, 10 gm yeast extract, 10 gm peptone, 10 gm CH3COONa.3H2O, 0.09 gm MgSO4, 0.03 gm MnSo4, 1.5 mg CuSO4.5H2O, 0.5 gm KCL, 5 ml Olive oil, 1000 ml Distilled water and pH 10.8). The inoculum flasks were then incubated at 37°C temperature for overnight on rotary shaker at 120 rpm. [8]

The composition of production medium used in this study was Peptone 0.2gm %,NH4 H2 PO4 0.1gm%, NaCl 0.25gm%,MgSO4.7H2O 0.04gm%, CaCl2.2H2O 0.04gm%, Olive oil 2ml, Tween 20- 2-3 drops. Submerged microbial cultures were incubated in 250 ml Erlenmeyer flasks containing 100 ml of liquid medium on a rotary shaker (120 rpm) and incubated at 37°C. After 96 hours of incubation, the culture was centrifuged at 10,000 rpm for 20 min at 4ºC and the cell free culture supernatant fluid was used as the sources of extracellular enzyme. The lipase activity in the supernatant was determined by the titrimetric method.[11]

Lipase Assay

Lipase activity was measured by titrimetric using olive oil as a substrate. One ml of the culture supernatant was added to the reaction mixture containing 2ml of Phosphate buffer with pH 7.0 and 1ml of olive oil and incubated at 37˚C for 60 min. The reaction was stopped and fatty acids were extracted by addition of 1.0 ml of acetone: ethanol solution (1:1). The amount of fatty acid liberated was estimated by titrating with 0.05M NaOH until pH 10.5 using phenophathelin as indicator. One unit of enzyme activity is defined as the amount of enzyme required to liberate 1 μmol of equivalent fatty acid under the standard assay conditions.[1]

Lipase activity (Units/ml) = Volume of alkali consumed × Strength of alkali × 1000/Volume of sample × Time in min

Result And Discussion

Isolation of Lipase Producing Bacteria

11 samples of different soil samples considered in study. 41 bacterial colonies obtained on nutrient agar plates which were subjected to qualitative screening on TBA for lipolytic strains. Out of 41 bacterial isolates only 20 isolates were lipase producer. A diversified lipolytic activity was observed ranges from a large clear zone (strong lipolytic activity) to small zone (weak lipolytic activity).

image 1

To test isolates as a lipase producer, streaked on Phenol Red containing Olive oil and tween 80 agar plate. production of yellow zone indicates lipase producer on Phenol red agar plate due to change in pH of the medium as the liberation of fatty acid. Tween have been the most widely used substrates for the detection of lipase producing microorganisms in agar media. Screening using tween agar plates shows precipitation around the lipase producing micro-organisms. The method is based on the precipitation as the calcium salt of the fatty acids released by hydrolysis of tweens. Liberated fatty acids bind with the calcium salt incorporated into the medium.

image 2

image 3

Identification of lipase producing Bacteria

The total Number of isolates counted and selected for further proceeded for their Morphological test. They all were Gram positive with different characteristics. Following Table 2 shows results of various biochemical characteristics of isolate

Table 2: Biochemical Characterization of isolates

Biochemical tests
Tests Carbohydrate
Isolate Indole MR VP Citrate H2S Production Nitrate Reduction Gelatine Catalase Oxidase Urea Glucose Fructose Lactose Xylose Probable
JA1 + + + + + + Staphylococcus spp.
JA2 + + + + + + Staphylococcus spp.
RA2 + + + + + + Lactobacillus spp.
RA3 + + + + + + Lactobacillus spp.
VS1 + + + + + Lactobacillus spp.
VS2 + + + + + + + Lactobacillus spp.
VS3 + + + + + + + Lactobacillus spp.
HA1 + + + + + + Staphylococcus spp.
HA2 + + + + + + Staphylococcus spp.
AD1 + + + + + + Staphylococcus spp.
AD2 + + + + + Staphylococcus spp.
MS1 + + + + + + Lactobacillus spp.
MS2 + + + + + + + Lactobacillus spp.
TA1 + + + + + + + Lactobacillus spp.
TA2 + + + + + + + Lactobacillus spp.
PA3 + + + + + Staphylococcus spp.
GS3 + + + + + Staphylococcus spp.
UD5 + + + + Staphylococcus spp.
FS1 + + + + + Staphylococcus spp.
FS2 + + + + + Staphylococcus spp.

‘+’: Positive; ‘-‘: Negative

By studying Morphological and biochemical characteristics, 11 isolates of Staphylococcus spp. And 9 isolates of Lactobacillus spp. were obtained.

Agar well Diffusion Assay

Lipase producing isolates showed production of yellow zone of different diameter. Different zone size in mm given in Table.3. Among all the isolates, isolate no. AD2 obtained from Garage soil sample which was collected from adajan area of Surat showing zone of highest diameter, 27mm and isolate no. VS2 obtained from Milk waste soil from olpad showing zone of smallest diameter, 14mm. Zone with highest diameter can be further used for production and optimization of Lipase.

image 4

Table 3: Yellow zone production by Lipase producing bacteria

Isolate no. Zone size (in mm)
JA1 18mm
JA2 19mm
RA2 15mm
RA3 17mm
VS1 18mm
VS2 14mm
VS3 17mm
HA1 21mm
HA2 20mm
AD1 22mm
AD2 27mm
MS1 17mm
MS2 16mm
TA1 17mm
TA2 15mm
PA3 19mm
GS3 16mm
UD5 19mm
FS1 16mm
FS2 20mm

image 5

Production of Lipase

The isolate AD2, Staphylococcus spp., selected as higher lipase producer by agar well diffusion method and further use for production of enzyme. A time course of lipase production from this isolate showed that the lipase activity increased with time reaching a maximum at 4 days and decreased further. Lipase activity was measured by Titrimetric method using Olive oil as a substrate. Maximum activity of 4.27 U/ml obtained at 37°C for 96 hour at 120 rpm and pH 7.0.The activity of enzyme decrease after 96 hour as shown in figure:6.

image 6

Discussion

In present study, Oil and Fat contaminated soil used for isolation of lipase producing bacteria from area of Surat, Gujarat, India. lipolytic organisms were isolated and identified using preliminary and confirmatory tests. Total 41 isolates obtained by primrary screening on Nutrient agar plate and out of them 20 isolates grown in the selective medium like Tributyrin agar plate, Phenol red agar plate and Tween-80 agar plate similar to D.Kumar et al.(2012) and Li Pin Lee et al.(2015) were found to produce lipase. All the isolates were gram positive with 11 were identified as Staphylococcus spp. are similar to E. Sirisha et al.(2010) and remaining 9 were identified as Lactobacillus spp. are similar to Rashmi, B. S. et al. (2014).Their growth showed that they can use olive oil, Tributyrin and Tween 80 as carbon source and showed the lipase producing feasibility. All 20 isolates subjected to Agar well diffusion method for selection of maximum zone producing isolate for production lipase as similar to Bhavani M. et al (2012). Isolate no AD2obtained from garage site soil sample of Adajan area of Surat,Gujarat, India showing maximum lipolytic activity with zone size of 27mm which were further used for production. In recent study, Inoculum media used similar to Miral Patel et al. (2016) which was used for inoculation of Production media which was similar to S. Gaherwal et al.(2015).The maximum production of enzyme obtained after 96hr of incubation at 37°C and pH 7.0 in Rotary shaker at 120 rpm which is similar to Nadeem Ullah et al.(2015) except of production time of 48hr.Lipase activity was measured by titrimetric method using olive oil as a substrate as similar to A.kumar et al. (2012)

Conclusion

It may be concluded from the present study that the total 20 isolates obtained from oil and fat contaminated soil and isolate AD2, characterized as Staphylococcus spp. Obtained from Garage site soil from Adajan area of Suart,Gujarat,India with maximum zone production can be used as a potential bacterial source of lipase. In this study, Olive oil is used as a substrate for lipase production. Lipase activity was determined by titrimetric method with higher activity of 4.27 U/ml at 96 hours of production at 37°C and 120rpm at pH 7.0. The result obtained in the present study revealed the ability of Staphylococcus spp. to produce Lipase enzyme. It was concluded that Staphylococcus spp. could be used as a new potent microbial source of lipase. Further studies are recommended on the use of various other bacteria and other strains of Staphylococcus spp. for much better lipolytic activity.