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Optimization of phospholipase [A.sub.1] ([PLA.sub.1]) production from a soil isolate Bacillus subtilis subsp. inaquosorum RG1 via solid state fermentation.

Phospholipases are one of key enzymes in oil refinery industry and play a crucial role in degumming of edible oils. Physical refining using water removes only hydratable phospholipids so there is great demand for the environment friendly technique that can also remove the non-hydratable phospholipids [1] from edible oils. Various chemical processes are also used by the industries to remove phospholipids that includes acid degumming but enzymatic degumming using phospholipases is more advantageous and effective [2] because these enzymes can easily convert non-hydratable phospholipids to the hydratable phospholipids and can be further eliminated by centrifugation [3].

Soil is a major reservoir for a variety of microorganisms [4-6] and can be used for exploiting isolates possessing commercially important enzymes like [PLA.sub.1] Lyso-phospholipids, the product formed by action of [PLA.sub.1] on phospholipids are also in great demand as emulsifying agent, cosmetic agent as well as drug delivery agent. Various [PLA.sub.1] has been purified from various mammalian systems but to obtain high titers of [PLA.sub.1] microbial sources are still in demand [7].

Phospholipases are basically classified into four groups depending on the site of ester bond attacked i.e. phospholipase A (PLA), phospholipase B (PLB), phospholipase C(PLC) and phospholipase D (PLD) [8]. Phospholipases A is further categorized into Phospholipases [A.sub.1] ([PLA.sub.1]) and Phospholipases [A.sub.2] ([PLA.sub.2]). Numerous [PLA.sub.1] have been reported from a variety of microorganism that includes [PLA.sub.1] from Serritia liquifaciens [9-11], Fusarium sp. [12], Aspergillus oryzae [13-15], Streptomyces [16]. Out of which only a few enzymes are available commercially viz. [PLA.sub.1] from Novozymes (Fusarium [PLA.sub.1]) and Genecor (Streptomyces [PLA.sub.1]).

Fermentation process is the way out for producing high volume of enzymes. Solid state fermentation is an efficient method for enzyme production with less cost and reduced risk of contamination [17-19]. For solid state fermentation, agro-industrial waste can be used as a substrate. Keeping that in mind, the present study aims for production of extracellular [PLA.sub.1] by using defatted rice bran as fermentation substrate.


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 37[degrees]C for 24h. Isolates producing clear zone indicate lipase positive culture.

Qualitative assay for [PLA.sub.1]

Lipase positive cultures were patched on egg-yolk (5% v/v) agar plate and incubated at 37[degrees]C for 72h. Clear zone indicates phospholipase A positive cultures [20]. Hydrolysis capacity (HC) ratio was measured by dividing the clearing zone diameter by colony growth diameter [21] The glycerol stock of phospholipase A (PLA) positive cultures were preserved at -80[degrees]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.

[PLA.sub.1] 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.D.sub.600] reached 0.8 and was added (1% v/v) to the production medium followed by incubation at 37[degrees]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 [PLA.sub.1] activity assay was carried out with the supernatant.

Quantitative assay for [PLA.sub.1] activity

The quantitation of [PLA.sub.1] activity was done by using 'Enzchek[R] Phospholipase [A.sub.1] assay kit' (Molecular Probes Inc. USA) that uses [PLA.sub.1] 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 50[micro]l lipid-mix {30[micro]l of 10mM of DOPC (Dioleoylphosphatidylcholine), 30[micro]l of 10mM DOPG (Dioleoylphosphatidylglycerol) and 30[micro]l of 2mM [PLA.sub.1] substrate} slowly and steadily to the 5 ml of reaction buffer (1X) placed on a magnetic stirrer. The reaction was started by adding 50[micro]L of substrate-liposome mix to each well of 96-well plate already containing controls and samples. This was followed by incubation at 25[degrees]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[R] Ultra unit as provided in Enzchek[R] Phospholipase [A.sub.1] assay kit.

Identification by 16S rRNA gene sequencing

Two isolates with maximum [PLA.sub.1] 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[TM] 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 models [22].

[PLA.sub.1] production by solid state fermentation

The identified and selected isolate with maximum extracellular [PLA.sub.1] 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.D.sub.600] 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[degrees]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 [PLA.sub.1] 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 [PLA.sub.1] 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[degrees]C for 24 to 120 h.

Incubation temperature

The optimum temperature was determined by incubating the same medium at different temperatures (25-45[degrees]C) 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[degrees]C, for 48h.

Oil source

Different oil sources (0.5% v/v) were used as inducer for PLAt 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[degrees]C 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[degrees]C 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[degrees]C for 48h under static conditions.


Isolation of cultures from soil samples

In total, 82 lipase positive isolates were screened on the basis of degradation of tributyrin in lipase plate assay. Out of 82, only 31 isolates were found to have PLA activity on the basis of formation clear zone on egg-yolk agar plate. The Gram staining and HC on egg-yolk agar plate of the selected 31 isolates is displayed in Table 1.

[PLA.sub.1] production by submerged fermentation

The PLA positive isolates were subjected to submerged fermentation. The supernatant activity of 31 isolates (U/ml) is displayed in Table 2.

Identification of bacteria by 16S rRNA sequencing

Two isolates with maximum PLAt activity S8-3 and S14-1 were selected for identification. The 16S rRNA gene sequence of the S8-3 displayed 99% identity with Bacillus subtilis subsp. inaquosorum KCTC 13429 and S14-1 strain showed 99.78% identity with Acinetobacter radioresistens DSM [6976.sup.T]. The S14-1 was not used for further studies due to its pathogenic nature. Nucleotide sequence obtained after sequencing of S8-3 was submitted in GENBANK under Accession no. KY088040 with strain name RG1. The Phylogenetic tree of both isolates was constructed by Neighbor joining method (Fig. 1) on the basis of sequence analysis.

Optimization of the cultural conditions

Bacillus subtilis subsp. inaquosorum RG1 was subjected to solid state fermentation for PLAj production. The optimization was carried out by classical approach by taking different factors in account including incubation temperature, incubation time, moisture content, different oils, different nitrogen and carbon sources (Figure 2 and Figure 3). Maximum PLAt activity of 51.55 U/gm was obtained after the incubation of 48h at 37[degrees]C with moisture content of 1: 1.5 using glucose and peptone as carbon and nitrogen source respectively with 0.5% olive oil as an inducer.

[PLA.sub.1] production (23.2 U/gm) was observed after incubation of 48h and after that there is a decrease in [PLA.sub.1] that may be due to decrease in viable number of cells [23] and depletion of nutrient availability [24].

Substrate to moisture ratio of 1:1.5 was found to be more suitable for [PLA.sub.1] production. The moisture content ratio plays a crucial role in the growth of microorganism ultimately affecting enzyme productions by solid state fermentation [25].

Temperature is a crucial factor for optimum enzymatic activity and overall metabolism [26]. In our study, optimum [PLA.sub.1] production was observed at 37[degrees]C. Our results are in accordance with previous reports wherein 37[degrees]C was the best temperature for maximum [PLA.sub.1] production in Pseudomonas gessardii and Trichosporon sp. [27].

In addition to physical factors, medium composition also affects the enzyme production as it play an important role in overall growth of microorganisms.

Various carbon and nitrogen source and different oils have different effects on growth of microorganisms and finally influenced enzyme production. Thus, these parameters need to be optimized for better production yields of the desired enzyme [24].

In our study, 0.5% of olive oil is observed to be the best inducer for [PLA.sub.1] production. Similar results were observed for lipase production from Bacillus sp. [28,29]. Among the carbon and nitrogen sources used, glucose and peptone were found to be the best carbon and nitrogen source respectively. In contrast, xylose and ammonium sulphate were found as good carbon and nitrogen source for [PLA.sub.1] in Serriatia sp. [11].

The use of [PLA.sub.1] has been restricted owing to its low stability and less availability. In addition, crystallographic and structural data for most of the [PLAs.sub.1] have not been obtained yet. So there is an increasing trend to isolate new microorganisms for [PLA.sub.1] production which can provide a platform to further improve these microbes to make them competent enough to be exploited at industrial scale.


In the present study, a [PLA.sub.1] producing microorganism was isolated from soil and identified as Bacillus subtilis subsp. inaquosorum. Maximum [PLA.sub.1] production of 51.55 U/gm was observed which is 2.15 folds more than unoptimized medium via solid state fermentation. This is the first report wherein [PLA.sub.1] was produced using one factor at a time approach. Moreover, the present study also provided a clean approach to successfully produce high titers of enzyme and will reduce burden of the environment by utilizing waste as substrate.


We would like to acknowledge Dr G.S. Prasad (IMTECH, Chandigarh) for identification of isolate and Dr. Deepak Sharma (IMTECH, Chandigarh) for their assistance in work.


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Gaganpreet Kaur * (1), Sanjeev Kumar Soni (2) and Rupinder Tewari (1)

(1) Department of Microbial Biotechnology, South Campus, New Hospital Building, Panjab University, Sector 25, Chandigarh--160 014, India.

(2) Department of Microbiology, South Campus, Basic Medical Science (Block I), Panjab University, Sector 25, Chandigarh--160 014, India. 10.22207/JPAM.11.2.22

(Received: 20 February 2017; accepted: 15 April 2017)

* To whom all correspondence should be addressed. Tel.: +91-9988692556;


Caption: Fig. 1. Phylogenetic tree representing evolutionary relationship of RG1 and RG3: Neighbor-Joining method was used to infer evolutionary history. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) is shown next to the branches. The Kimura 2-parameter method was used to compute evolutionary distances and is in the units of the number of base substitutions per site. Evolutionary analyses were carried out using MEGA6. Pseudomonas aeruginosa JCM [5962.sup.T] (BAMA01000316) was taken as an out-group. NCBI Genbank accession numbers of 16S rRNA gene sequences are shown in parenthesis

Caption: Fig. 2. Effect of incubation conditions on [PLA.sub.1] production by Bacillus subtilis subsp. inaquosorum (a) incubation time (24h-120h) (b) incubation temperature (25-45[degrees]C) (c) moisture content (1:1-1:3.5) via solid state fermentation

Caption: Fig. 3. Effect of medium composition on [PLA.sub.1] production by Bacillus subtilis subsp. inaquosorum (a) oil variation (b) carbon sources (d) nitrogen sources
Table 1. Gram Character, Hydrolysis Capacity

S.No   Isolate   Gram Staining                             HC

1      S2-1      Isolated, thin, Gram positive rods        0.11
2      S3-1      Isolated, Gram positive rods              1.55
3      S3-2      Isolated, Gram positive rods              1.37
4      S6-3      Very small, Gram negative rods            1.36
5      S7-2      Small, Gram positive rods                 1.60
6      S7-5      Very small, isolated Gram negative rods   1.72
7      S7-6      Isolated, Gram negative rods              1.25
8      S8-1      Gram negative rods                        1.50
9      S8-3      Gram positive rods in chains              3.00
10     S9-1      Isolated, Gram positive thick rods        1.40
11     S9-2      Small, isolated, Gram negative rods       1.25
12     S10-2     Isolated, Gram positive rods              1.44
13     S10-7     Isolated, Gram positive rods              1.20
14     S11-4     Very small, Gram negative rods            1.18
15     S12-2     Small, Gram positive rods                 1.75
16     S12-3     Gram positive rods in chains              1.44
17     S13-1     Gram negative rods                        1.37
18     S14-1     Gram negative, cocco-bacilli,             1.90
19     S14-2     Gram positive cocci                       1.28
20     S14-3     Very small, Gram negative rods            1.44
21     S15-1     Gram negative cocci                       1.87
22     S15-3     Small, thin, Gram negative rods           1.76
23     S16-3     Gram negative rods                        1.50
24     S16-4     Very small, Gram positive cocci           1.75
25     S16-5     Gram negative cocci                       1.62
26     S17-4     Gram negative cocci                       1.05
27     S17-5     Gram positive cocci                       1.60
28     S18-1     Gram negative thin small rods             1.50
29     S20-2     Gram positive rods, isolated              1.36
30     S20-3     Gram positive rods, in chains             1.38
31     S20-4     Gram positive rods, in chains             1.50

Table 2. PLAj Activity (Submerged Conditions)

S.No.   Isolate Number   [PLA.sub.1] activity (U/ml)

1            S2-1                   0.614
2            S3-1                   0.133
3            S3-2                   0.005
4            S6-3                   0.125
5            S7-2                   0.014
6            S7-5                   0.472
7            S7-6                   0.087
8            S8-1                   0.394
9            S8-3                   1.250
10           S9-1                   0.839
11           S9-2                   0.064
12          S10-2                   0.013
13          S10-7                   0.023
14          S11-4                   0.019
15          S12-2                   0.021
16          S12-3                   0.241
17          S13-1                   0.404
18          S14-1                   1.170
19          S14-2                   0.071
20          S14-3                   0.513
21          S15-1                   0.016
22          S15-3                   0.263
23          S16-3                   0.271
24          S16-4                   0.910
25          S16-5                   0.097
26          S17-4                   0.018
27          S17-5                   0.078
28          S18-1                   0.315
29          S20-2                   0.043
30          S20-3                   0.228
31          S20-4                   0.195
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Author:Kaur, Gaganpreet; Soni, Sanjeev Kumar; Tewari, Rupinder
Publication:Journal of Pure and Applied Microbiology
Article Type:Report
Date:Jun 1, 2017
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