Optimization Of The Media Components By One Factor At A Time Methodology To Enhance Lipase Production By Bacillus Substilis KUBT4.

3601

Optimization Of The Media Components By One

Factor At A Time Methodology To Enhance

Lipase Production By

Bacillus Substilis

KUBT4.

Rubeen Dadakalandar Nadaf and Dr. Shivasharana Chandrabanda Thimmappa

Abstract: Lipases are an important hydrolysing enzyme with various applications and industrial potential. The maintenance of microorganisms in high oxygen concentrated liquid nutrient medium is possible by submerged fermentation. The present study focuses on the optimum production of lipase enzyme from Bacillus Substilis KUBT4 through submerged fermentation. The presence of 1% sucrose and 1% tryptone in the medium enhanced lipase production when compared with other carbon and nitrogen sources. The enzyme production by this strain expressed slightly alkaline. It has optimal activity at pH 7.5 and temperature at 36ºC where it is stable at pH 6.5-8.5 and temperature at 32ºC to 38ºC. Furthermore, 10% of inoculum size for 48 hrs of incubation period with the agitation of 150rpm increased the lipase production.

Key words: Lipase, submerged fermentation, Bacillus Substilis KUBT4, carbon source, nitrogen source, temperature, pH —————————— ◆ ——————————

1.

I

Lipases are triacylglycerol acylhydrolases that catabolise triacylglycerol to glycerol and free fatty acids [1]. Lipases are distributed extensively in animals, plants and microbes [2, 3]. In biotechnology, microbial lipase play a major role because of their stability at different temperatures and pH due to which gradually lipases are gaining attention in industries like detergent, dairy, fat, oleochemical, organic synthesis, biodiesel [4, 5], production of cosmetics, pharmaceuticals and paper manufacture [6]. Behaviour of lipase can be altered by amount of water content in the reaction mixture [7]. As of their incredible features, such as stability in organic solvents, wide range of substrate specificity, chemo-selectivity, stereoslectivity, regioselectivity and independent from cofactors, they are used in biotechnological applications, including food processing, chemical industry and biomedical sciences [8, 9, 10, 11]. Bacterial lipases have more stability than other lipases, commercially they have gained importance mainly due to ease in cultivation and higher yield [10], and are produced through submerged fermentation [12] since culture conditions can be easily controlled and maintained [13]. In literature various culture conditions which stimulated or suppressed the bacterial lipases production have been described. Normally, carbon and nitrogen sources, lipids, and salts influence the production of bacterial lipases [14]. Furthermore, production yield is affected by physio-chemical factors like temperature, pH and agitation [1]. Hence in this paper, the production of lipase from Bacillus Substilis KUBT4 through submerged fermentation (SmF) has been optimized.

2.

M

2.1. Microorganism: Organism screening for lipase-producing microbes on Tributyrin nutrient agar plates resulted in isolation of 59 Bacterial Strains. Isolates showing positive reaction were maintained on nutrient Agar plate at 37 °C and stored at 4°C. The best Lipase producing isolate was identified by 16S ribosomal RNA gene sequence deposited in Gen Bank data base and identified as Bacillus subtilis KUBT4.(Gen Bank accession number MH114029). This isolate is used here for the optimization study.

2.2. Lipase production media: The production medium used in this study contains; Tryptone 10 (g/l), glucose 10(g/l), Olive oil 15 (ml/l), CaCl2.2H2O 2 (g/l), MgSO4.7H2O 1 (g/l), 1% stock solution of FeCl3 4(g/l), pH 7. 24 hours old cultures were suspended in 5ml of sterile deionised water and used as the inoculum for pre culture to obtain an initial cell density to adjust the turbidity of 0.5 McFarland standards. The medium (100 ml) was dispensed into 250ml conical flask and autoclaved at 121ºC for 15min. the suspended culture was inoculated into media containing conical flask under sterile condition and incubated on rotary shaker at 100rpm at 37ºC temperature for 48hrs. The culture was centrifuged at 10000rpm for 20 minutes at 4ºC. The clear supernatant used as a source of crude enzyme for determining lipase activity.

2.3. Lipase assay: Lipase activity was measured spectrophotometrically using p-nitrophenyl phosphate [pNPP] (Sigma-Aldrich) as substrate. The assay was performed according to Kim et al. [14] with some modifications. The reaction mixture contained solution A (50 mM Tris-HCl buffer pH 8, 0.1% gum arabic, and 0.2% sodium deoxycholate) and solution B (10 mM pNPP in isopropanol). For the reaction, 100 μL of samples were added to 850 μL of solution A. The reaction was initiated by the addition of 50 μL of solution B and incubated at 35°C for 5 min. The reaction was stopped by the addition of 500 μL of 3M of HCl. After centrifugation, 500 μL of clear supernatant was transferred to a cuvette and 1 mL of 2M of NaOH was added. The mixture was read at 410 nm by UV-Vis 1800 Spectrophotometer (Hitachi, Japan) against a free enzyme mixture as blank. One lipase unit

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• Rubeen Dadakalandar Nadaf is currently pursuing doctorate program in Biotechnology in Karnatak University, Dharwad, Karnataka, India, PH-+917019103717. E-mail: [email protected],

is defined as the amount of enzyme required to liberate 1 mmol of p-nitrophenol per minute.

2.4. Nutritional parameters effects on lipase production: 2.4.1. Carbon source: The effect of carbon source on lipase production was estimated by using different carbon source like Glucose, sucrose, maltose, xylose and fructose in 1% concentration. 2.4.2. Nitrogen source: To test the effect of nitrogen sources on lipase production, different nitrogen sources such as yeast extract, soybean meal, sodium nitrate, peptone and tryptone were used. They were individually tested by replacing the tryptone present in the basal medium at the concentration of 10 g/L.

2.5. Physical Parameters effects on lipase production: 2.5.1. Optimum pH: To determine optimum pH of the media for maximum lipase production different ranges of pH (5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5 and 10) was used. Enzyme activity in the supernatant were estimated.

2.5.2. Optimum Temperature: studies on the effect of temperature were done by maintaining incubation at different temperature viz. 28ºC, 30ºC, 32ºC, 34ºC, 36ºC, 38ºC, 40ºC, 42ºC and 44ºC. Later activity of lipase were analysed.

2.5.3. Inoculum size: the optimum inoculum size was determined by using inoculum volume (%v/v) viz. 2.5 ml, 5ml, 7.5ml, 10ml, 12.5ml and 15ml respectively of 24 hrs old cell suspension of Bacillus Substilis KUBT4 inoculated in 100ml fermentation medium taken in 250 ml of conical flask. Activity of lipase in the supernatant were estimated.

2.5.4. Incubation time: In this investigation incubation time for lipase production were optimised by carrying out fermentation for different time intervals viz. 0hrs, 8hrs, 16hrs, 24hrs, 32hrs, 40hrs, 48hrs, 56hrs, 64hrs, 72,hrs, 80hrs, 88hrs and 96hrs respectively under the same fermentation conditions. Further lipase activities of the cell separated broth were measured.

2.5.5. Agitation: In the studies effect of agitation speed for optimum lipase production were carried out using different agitation speed viz. 110rpm, 120rpm, 130rpm, 140rpm, 150rpm, 160rpm, 170rpm, 180rpm, 190rpm and 200rpm under the same medium composition. Enzyme activity of the supernatant were estimated.

3.

RESULTS

AND

DISCUSSION

The production of enzyme and growth of the organisms by distinct process parameters have great impact i.e. carbon sources, nitrogen sources, pH, temperature, inoculum size and incubation time etc [12]. Components of media were optimized by one factor analysis.

3.1. Carbon source: among the carbon sources, it was found that 1% sucrose was the best carbon source for lipase production (43.32 ± 0.22 U/ml) followed by glucose (38.28 ± 0.12 U/ml), fructose (35.41± 0.28 U/ml), maltose (12.83 ± 0.06 U/ml) and xylose (11 ± 0.02 U/ml) (Figure 1) by Bacillus Substilis KUBT4. Sucrose as a carbon source showed higher lipase activity. Sucrose triggers the gene expression which leads to high lipase production

[15]. Earlier sucrose has been reported as the best carbon source for lipase production [16].

Graph 1: Effect of different Carbon sources on lipase production.

3.2. Nitrogen source: among the different Nitrogen sources, Tryptone (48.24 ± 0.22 U/ml) enhanced lipase production followed by peptone (43.37±0.12 U/ml), yeast extract (25.07 ± 0.09 U/ml), soyabean meal (20.66 ± 0.09 U/ml) and sodium nitrate (12.97 ± 0.05 U/ml) by Bacillus Substilis KUBT4. (Figure 2). Organic nitrogen sources are complex and plays an important role in promoting lipase production by Bacillus Substilis KUBT4.

Graph 2: Effect of 1% Nitrogen sources on lipase production.

Similar findings were reported for Bacillus sp.strain 42 [17]. In contrast, some research found that Pseudomonas species G6 and Burkholderia cepacia were not affected significantly by organic nitrogen sources for lipase production [18, 19].

3.3. pH: It was observed from the results that the bacterium is capable of producing lipase from initial pH 5.0 to pH 10.0. The enzyme production varied considerably from 1.06 to 42.34 U/ml. The bacteria Bacillus sustilis KUBT4 has optimum lipase production at pH 7.5 (42.34 ± 0.23 U/ml). (Figure 3). However it was noted that the lipase production was declined with increase in pH 7.0 to pH 10.0 but was able to produce lipase towards alkalotolerant nature. Factors like pH and

38.28 43.32

12.83 11 35.41

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1% Carbon source

Gl uco se

25.07 20.666666 67

12.97

43.373333 33

48.246666 67

0 10 20 30 40 50 60

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1% Nitrogen sources

3603 temperature have a high influence on processes such as

enzymatic reactions and permeability of cell membrane [20]. Bacterial lipases show maximum activity in alkaline pH [1]. In the present study results are consistent with the review of Gupta et al. (2004) [1].Variation of pH may change the bacterial metabolite production [21, 22] and lipase production [23]. The examined isolate here has good source of enzyme and it is slightly alkaline nature which is appropriate for stability and performance of biological treatments such as bioremediation of water, sewage treatment, detergent formulations and leather processing [24].

Graph 3: Effect of pH on lipase production.

3.4. Temperature: The results showed that the lipase is able to produce optimum temperature at 36ºC with high lipase activity (49.6 ± 0.25 U/ml) followed by 38ºC (46.4 ± 0.14 U/ml), 34ºC (35.27± 0.17 U/ml), 32ºC (20.60± 0.11 U/ml), 30ºC (15.05± 0.02 U/ml), 40ºC (12.48± 0.29 U/ml), 28ºC (8.74± 0.14 U/ml), 42ºC (4.55± 0.30 U/ml), 44ºC (1.13±0.02 U/ml) (Figure 4). At 36ºC temperature greater biomass concentration of lipase was attended. In this study, enzyme activity showed continuous increase with increase of temperature from 30ºC. Further increase of temperature above 38ºC showed decreased enzyme activity. Scientists reported that slight rise in temperature up to 38ºC add to enzyme activity [25, 26, 27]. The B.thuringiensis showed optimum temperature ranging from 30ºC to 35ºC [28].

Graph 4: Effect of Temperature on lipase production.

3.5. Inoculum size: The decrease in the size of inoculum reduced the enzyme activity. Among the tested inoculum size 10% proved to be the best with 39.25 ± 0.21 U/ml activity value.Throughout fermentation the inoculum size plays vital role in production of lipase. Bacterial cell population/ growth stage is dependent on amount of nutrient consumption [29]. Maximum lipase activity for Bacillus Substilis KUBT4 was obtained with 10% (v/v) inoculum after 48hrs of fermentation (Figure 5).

Graph 5: Effect of Inoculum size on lipase production.

3.6. Incubation time: the effect of incubation time on lipase production revealed that maximum lipase production 49.19 ± 0.23 U/ml for Bacillus Substilis KUBT4 was found to be at 48 hrs of incubation. The activity gradually decreased after 48 hrs (figure 6).Lipases are liberated during stationary or late logarithmic phase [30]. 5hrs to168hrs have been reported as optimum incubation time for different lipase producing microorganisms [31]. Lipases from SP5 Bacillus sp. [32] and Bacillus methylotrophicus PS3 [12] were shown to produce the highest level after 48hrs and 60hrs of fermentation respectively.

1.0653333332.19

5.153333333 30.61666667

39.31666667 42.34333333

25.33666667

20.25666667

10.39666667

5.18

1.133333333

0 5 10 15 20 25 30 35 40 45

5 5.5 6 6.5 7 7.5 8 8.5 9 9.5 10

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5 5.5 6 6.5 7 7.5

8.7615.06 20.59

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49.6246.38

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4.49 1.02 0

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28 30 32 34 36

10.606666 67

14.206666 67

25.113333 33

39.253333 33

24.003333 33 21.296666

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2.5 5 7.5 10 12.5 15

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Graph 6: Effect of Incubation time on lipase production.

3.7. Agitation: Agitation at 130rpm to 150rpm enhanced the lipase production. The optimum agitation speed for the production of lipase by the bacteria was 150 rpm (48.12 ± 0.74 U/ml). (Figure 7).The rate of agitation speed above 150 rpm led to decrease in the growth and enzyme production. The results suggest that agitation is needed for appropriate dispense of oil and medium along with bacterial culture for the lipase production [33, 21, 17].

Graph 7: Effect of Agitation on lipase production.

4.

CONCLUSION:

From the current research it can be concluded that oil spilled soil has an industrially beneficial source of bacteria that can be used for commercial importance. The study indicated that

Bacillus Substilis KUBT4 is a valuable source of lipase

isolated from oil spilled soil sample. The enzyme production was optimized with different nutritional, physical parameters and high yield produced using pilot scale fermenter. Sucrose

and Tryptone are good source for maximum enzyme production. It showed an optimal activity at pH 7.5 and 36ºC temperature. 10% of inoculum size for 48 hrs of incubation period with agitation of 150rpm enhanced the lipase production.

A

’

:

Rubeen Dadakalandar Nadaf has designed the work, conducted practical work, analysed and interpreted the data. Shivasharana Chandrabanda Thimmappa has drafted the work and critically reviewed the content.

ACKNOWLEDGEMENT

The authors are thankful to University Grant Commission for providing the fellowship grant under the scheme of Maulana Azad National Fellowship for Minority Students (F117.1/201617/MANF201517KAR57468) and authors also sincerely acknowledge, Post Graduate Department of studies in Biotechnology and Microbiology, Karnatak University, Dharwad.

CONFLICT

OF

INTEREST:

The authors declare that they have no conflict of interest.

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0 8 16 24 32 40 48

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