Isolation of microorganism from soil samples
Twenty soil samples were collected from various sites like Dhaba, Mandir, Temples and Motor -market in different regions of Chandigarh, Mohali and Shimla. One gm of soil was suspended in 10 ml followed by serial dilution. Isolates were plated on tributyrin (0.5 % v/v) agar plate and incubated at 37ºC for 24h. Isolates producing clear zone indicate lipase positive culture.

Qualitative assay for PLA₁
Lipase positive cultures were patched on egg-yolk (5% v/v) agar plate and incubated at 37°C for 72h. Clear zone indicates phospholipase A positive cultures²⁰. Hydrolysis capacity (HC) ratio was measured by dividing the clearing zone diameter by colony growth diameter²¹_.The glycerol stock of phospholipase A (PLA) positive cultures were preserved at -80°C. PLA positive cultures were selected for further studies. HC was calculated after 24h.

Gram staining characteristic
A loopful of overnight grown PLA positive culture in distilled water was put on a clean glass slide and a uniform smear was prepared. The smear was heat fixed by gently heating over the burner flame. After heat fixation the smear was stained with crystal violet complex for about one minute. The slide was gently washed under tap water. Next, smear was flooded with Gram’s iodine for about one minute and then slide was again washed gently with water. Decolourization of the smear was done by covering the smear with de-staining solution for 15-20 sec. Slide was again washed with water. Lastly, the smear was stained with safranin for about 40 sec followed by washing with water. Slide was blot dried and further examined under microscope (Quasmo, India) under 100X magnification.

PLA₁ production by selected isolates via submerged fermentation
PLA positive cultures were first subjected to submerged fermentation using Nutrient Broth (NB) supplemented with olive oil (0.5% v/v). The emulsion was formed with gum- acacia (1% w/v) in mixer grinder. An inoculum was prepared in NB until O.D₆₀₀ reached 0.8 and was added (1% v/v) to the production medium followed by incubation at 37°C in orbital shaker (Remi orbital shaker, India) with agitation speed 160 rpm for 24h. The medium was centrifuged at 10,000 rpm for 10minutes (Sigma Laborzentrifugen, Germany) and PLA₁ activity assay was carried out with the supernatant.

Quantitative assay for PLA₁ activity
The quantitation of PLA₁ activity was done by using ‘Enzchek^® Phospholipase A₁ assay kit’ (Molecular Probes Inc. USA) that uses PLA₁ dye-labeled glycerophosphoethanolamines with dye labeled acyl chain at the sn-1 position and dinitrophenyl quencher-modified head group. The standard curve for assay was prepared using lecitase provided in the kit.

Substrate-liposome mix was prepared by adding 50µl lipid-mix {30µl of 10mM of DOPC (Dioleoylphosphatidylcholine), 30µl of 10mM DOPG (Dioleoylphosphatidylglycerol) and 30µl of 2mM PLA₁ substrate} slowly and steadily to the 5 ml of reaction buffer (1X) placed on a magnetic stirrer.The reaction was started by adding 50µL of substrate-liposome mix to each well of 96-well plate already containing controls and samples. This was followed by incubation at 25°C for 30 minutes in dark and fluorescence was measured with excitation at 460nm and emission at 515 nm. One Unit (U) of the enzyme corresponds to one Lecitase^® Ultra unit as provided in Enzchek^® Phospholipase A₁ assay kit.

Identification by 16S rRNA gene sequencing
Two isolates with maximum PLA₁ activity were identified using 16S rRNA gene sequencing at IMTECH, Chandigarh, India. Genomic DNA was extracted from pure cultures using HiPurA^TM Bacteria Genomic DNA Miniprep Purification Spin Kit (Hi-Media, India) according to the manufacturer’s instructions. The amplification of 16S rRNA gene was achieved using the following primers: 27f (5’-AGA GTT TGA TCC TGG CTC AG-3’) and 1492r (3’-ACG GCT ACC TTG TTA CGA CTT-5’). Four sequencing primers and Big-Dye Terminator Cycle Sequencing Kit (Applied Biosystems, CA, USA) were used for the sequencing of the purified 16S rRNA gene.

Sequence assembly and phylogenetic analysis
DNA sequence assembling software SEQUENCHER™ 4.10.1 (Gene Codes Corporation, MI, USA) was used to assemble and analyse the sequence data obtained. In order to find out the similar sequences, the data was used to BLAST as query in nucleotide database of NCBI (http://www.ncbi.nlm.nib.gov/). Phylogenetic tree was constructed using Clustal W software by aligning all related and acquired sequences. Neighbour-joining method was used to measure the evolutionary distances and operated in MEGA 6 and the Kimura 2 parameter models²².

PLA₁ production by solid state fermentation
The identified and selected isolate with maximum extracellular PLA₁ activity in submerged conditions were subjected to solid state fermentation using defatted rice bran as substrate.

The olive oil emulsion (0.5% v/v) was formed in mixer grinder using 1% (w/v) gum acacia as emulsifying agent. An inoculum (1.5ml) was obtained in NB until O.D₆₀₀ reaches 0.8 and added to autoclaved defatted rice bran (5gm) with 6ml of olive oil emulsion maintaining surface to moisture content ratio of 1:1.5. The flasks were incubated at 37°C under static conditions for 48h.The content of the flask were suspended in 100ml of distilled water and kept at shaking 160rpm for 1h. Then centrifugation was carried out at 10,000rpm for 10minutes and supernatant was used for PLA₁ activity assay.

Classical approach for optimization in solid state fermentation
Different parameters like incubation time, incubation temperature, moisture conditions, oil sources, nitrogen and carbon sources were taken in account to optimize the culture conditions for maximum production of PLA₁ by solid state fermentation.

Incubation time
For optimization of incubation time, medium was prepared consisting of 5g defatted rice bran with 6 ml olive oil emulsion (0.5%, pH 7). The stirring of olive oil with water was carried out in a mixer grinder with 1% (w/v) gum acacia to prepare olive oil emulsion. The medium was sterilized and inoculum (1.5 ml) was added and incubated at 37^ºC for 24 to 120 h.

Incubation temperature
The optimum temperature was determined by incubating the same medium at different temperatures (25-45 ^ºC) for 48h.

Moisture content
The moisture content in the medium was optimized by incubating 5gm defatted rice bran and inoculums (1.5 ml) with following substrate to moisture ratio (rice bran : olive emulsion + inoculums): 1:1, 1:1.5; 1:2; 1:2.5; 1:3; 1:3.5 (pH 7) and medium was incubated at 37^ºC, for 48h.

Oil source
Different oil sources (0.5 % v/v) were used as inducer for PLA₁ production like sunflower oil, olive oil, soy-bean and groundnut oil. The moisture content of the medium was maintained at 1:1.5 by using 6ml of oil emulsion with 1.5ml of inoculums in 5gm defatted rice bran and incubated at 37^ºC for 48h.

Carbon source
The carbon source in the medium was optimized by taking different carbon sources (1% w/v) like glucose, sucrose, fructose, xylose, maltose in the same medium and incubated at 37^ºC for 48h.

Nitrogen source
The nitrogen source in the medium was optimized using 1% (w/v) of malt extract, yeast extract, peptone, urea and soybean meal in the medium and was incubated at 37^ºC for 48h under static conditions.