Optimization of Various Physical and Chemical Parameters for Lipase Production by Bacillus coagulans

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1 American-Eurasian J. Agric. & Environ. Sci., 15 (5): , 2015 ISSN IDOSI Publications, 2015 DOI: /idosi.aejaes Optimization of Various Physical and Chemical Parameters for Lipase Production by Bacillus coagulans K. Larbi Daouadji, F.Z.I. Reffas, M.L. Benine and B. Abbouni Laboratory for Molecular Microbiology Proteomics and Health, Department of Biology, Faculty of Natural Science and Life, University of Djillali Liabès of Sidi Bel Abbés, Algeria Abstract: Six samples of spilled soils from olives oil processing factories, where lipids wastes are submitted to natural fermentation and three samples of the hot thermal water from Hammam Bouhanifia, Algeria were collected. A total of twenty four bacterial isolates belonging to a several bacterial species were screened for lipase production on lipid agar medium. Colonies with highest clear zone on the plate lipid agar medium were selected as potential lipase producing strains. The obtained results showed that the selected producer Lipase was identified as B. coagulans and the investigated various physical and chemical parameters for the optimization of lipase production indicated that the optimum temperature for lipase production was at 40 C (70 U/ml), which the enzyme production was affected and decreased after increase of temperature above 40 C to 60 C. Where, a maximum Lipase yield was achieved at ph 8 with inoculum level of 6 %. Furthermore, the conducted investigation for the optimization of inoculums concentration, indicated that the variation in the level of inoculum concentration from 1-10 % influence the lipase enzyme production, which the lipase activity varied from 66.5 to 80.0 U/ml. The optimum inoculums concentration for lipase production was 6% (80.0 U/ml). The results of the present study demonstrate that the B. coagulans is ideal candidate for extracellular lipase production at industrial level. Key words: Lipase Screening B. coagulans Production Optimization and Hot Thermal Water INTRODUCTION processing factories, dairies, soil contaminated with oil, oilseeds and decaying food compost heaps, coal tips and Lipases are glycerol ester hydrolases that act on hot springs [6, 7]. Microbial lipases have gained special acylglycerols to liberate fatty acids and glycerol. Lipases industrial attention due to their stability, selectivity and can hydrolyze long chain water-insoluble triglycerides broad substrate specificity [8-10]. Several extracellular into diglycerides, monoglycerides, glycerol and fatty lipases were isolated from many different bacterial acids [1, 2]. Lipases are ubiquitous enzymes which are species, including Bacillus13 and Pseudomonas [11, 12]. widely distributed in plants, animals and microbes [3]. Particular attention is focused on specific classes of Lipases have many applications especially in paper enzymes of species Pseudomonas, that are among the first industry, detergent, drugs, degradation of different studied and used in biotechnological production but also wastes, textile, food, pharmaceutical, leather, degumming because of their involvement in bacterial pathogenesis. of silk goods, manufacture of liquid glue, cosmetics, meat Lipases are found in a number of Pseudomonas [13, 14]. tenderization, cheese production, growth promoters etc Enzymes of Ps. aeruginosa, Ps. cepacia and Ps. [4, 5]. Lipases are produced by many microorganisms and fluorescens obtained in industrial conditions are used higher eukaryotes. Most commercially useful lipases are in organic synthesis, including catalysis of reactions in of microbial origin. Lipase producing microorganisms aqueous solutions [15, 16]. include bacteria, fungi, yeasts and actinomycetes. Lipase The microbial lipases are mostly extracellular and their producing microorganisms have been found in diverse production is greatly influenced by medium composition habitats such as industrial wastes, vegetable oil besides physicochemical factors such as temperature, Corresponding Author: Abbouni, Laboratory for Molecular microbiology proteomics and health, Department of Biology, Faculty of Natural Science and life, University of DjillaliLiabès of Sidi BelAbbés, Algeria. abbounibouziane@yahoo.de. 962

2 ph and dissolved oxygen. 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 oil or any other inducer, such as triacylglycerols, fatty acids, hydrolysable esters, tweens, bile salts and glycerol [17, 18]. However, nitrogen sources and essential micronutrients should also be carefully considered for growth and production optimization. The formation of opaque zones around the colonies is an indication of lipase production by the organisms. In the present study, due the importance of lipase enzyme, lipase producer strains has been screened, selected and optimized. MATERIALS AND METHODS Collection of Soil Sample: A spilled soil from olives oil processing factories, where lipids wastes submitted to natural fermentation and the hot thermal water were used were collected. The auger was used to make a depth of 30 cm using a grid or Zigzag sampling system. The soil sample for physiological analysis was collected with unused plastic bag sealed with heavy-duty rubber bounds. All samples were labeled with a permanent waterproof maker, while the microbiological analyses were collected using 200 ml capacity sterile glass sampling container. Isolation of Lipase Producers: The soil samples were collected from different olives oil processing factories and the hot thermal water was enriched by periodic sub culturing of samples in Nutrient Broth (NB) media. They were aseptically subjected to serial dilutions and plated on Nutrient Agar (NA) and incubated at 37 C for 24, 48 and 72 h. Acceptable plate counts for bacteria were between UFC/ml per plate. After incubation 200 predominant bacterial colonies were isolated and screened for lipase activity and then subjected to morphological, cultural and biochemical examinations. Screening and Isolation of Lipase Producing Bacteria: A total of 43 bacterial isolates were isolated from different sources including field soils, hot thermal water, effluents sources by serial dilution technique. The bacterial isolates were screened for lipase production on the lipid agar plates. The hydrolysis zone formed on lipid agar plates were visualized by flooding the plates with Gram s iodine solution. The lipolytic potential was estimated by using of the lipolytic Ratio (R/r) defined as the diameter of the hydrolyzation zone (R) divided by the diameter of the producing colony (r). Isolates having a higher lipolytic ratio of clearing zone to colony size were grown in liquid broth and the amount of lipase production was determined from cell free supernatant. The bacterial isolate that produced maximum lipase was selected and identified. The selected bacterial strain was maintained in nutrient agar slants. The bacterial strain was sub-cultured periodically after every 30 days and stored at 4 C. Preparation of Bacterial Inoculum: A volume of 50 ml nutrient broth supplemented with 1% soluble olive oils was taken in a 250 ml Erlenmeyer flask. The flask was sterilized in an autoclave at 15 lb pressure (121ºC) for 15 min. After cooling the medium, a loop full of bacteria from a 24 h old slant was aseptically transferred to the flask and kept at 37 C in a rotary shaker (150 rpm). After 24 h of incubation, 1 ml of this nutrient broth culture was used as the inoculum for 100 ml of production medium. Lipase Activity: Lipase activity was determined titrimetric by using olive oil hydrolysis [19]. Enzyme solution of 1 ml was added to the assay substrate containing 10 ml of 10% homogenized olive oil in 10% gum acacia, 2 ml of 0.6% CaCl2 solution and 5 ml of 0.2 mol/ l phosphate buffer, ph 7.2. The enzyme-substrate was incubated on orbital shaker at 125 rpm at 37 C for 1h [20]. 20 ml ethanol/acetone (1:1 v/v) was added to stop the reaction. Liberated fatty acids were titrated with 0.1 mol/lnaoh by using phenophtaline as indicator. The reaction mixture without the enzyme was titrated in the same way and used as blank. One lipase unit was defined as the amount of the enzyme that released one ìmol fatty acid per min under standard assay conditions [21]. Optimization of Process Parameters for Lipase Production: Various process parameters such as inoculum level, incubation period, ph, temperature, carbon sources and nitrogen sources were optimized. The strategy followed was to optimize each parameter, independent of the others and subsequently optimal conditions were employed in all experiments. Effect of the Medium ph-value: The effect of the initial ph-value of the used fermentation medium on the lipase production was performed by varying ph of the medium from 4 to 10 with one unit interval using 0.1 N Hcl and 0.1 N NaOH. An inoculum concentration of 10% was employed. The fermentation was carried out at 37 C for 72 h. After the incubation period, the optimum of the initial ph of the culture medium was determined by this step was fixed for subsequent experiments. 963

3 Effect of the Incubation Temperature: The effect of the temperature of the fermentation medium on the lipase production was performed by the varying incubation temperatures from 20 C to 60 C implicated in the of lipase production. An inoculum concentration of 10% was employed. After the incubation period, the optimum of the incubation temperature of the culture medium was determined by this step was fixed for subsequent experiments. Effect of Inoculum Concentration: The optimum inoculum concentration has been investigated for lipase enzyme production For this purpose, several inoculums concentration (1-10 %)were added to in 500 ml Erlenmeyer flasks containing 100 ml of the optimized liquid culture medium, incubated at 37 C for 72 hours on a rotary shaker (150 rpm) for lipase production. The contents of the flasks were harvested and assayed for lipase activity. The reached optimum inoculum level was used for further optimization studies. Effect of Carbon Source: The effect of carbon source on the lipase production was analyzed by replacing the olive oil with different carbon sources such as maltose, glucose, sucrose, starch, mannitol, Lactose, Fructose, Mannose, arabinose, galactose at a concentration of (1% w/v) were added into the production medium in 500 ml Erlenmeyer flasks containing 100 ml of liquid medium on a rotary shaker (150 rpm) and incubated at 36 C for 24 hours and the enzyme was assayed. Effect of Nitrogen Sources: The effect of nitrogen sources on the lipase production was studied by replacing the nitrogen source with organic nitrogen sources such as yeast extract, peptone, gelatin, casein, peptone and inorganic nitrogen sources ammonium nitrate, ammonium sulphate, ammonium dihydrogen phosphate, ammonium chloride, urea at a final concentration of 1 % (w/v) were added to the medium and incubated at 37 C for 24 hours in a rotary shaker (150 rpm). Lipase activity was determined in the cell free supernatant. RESULTS AND DISCUSSION Six samples of spilled soils from olives oil processing factories, where lipids wastes are submitted to natural fermentation and three samples of the hot thermal water from Hamman Bouhanifia, Algeria were collected. Table 1: Illustration of the lipase production of the isolates strains belonging to the genus of Bacillus Diameter of zone hydrolysis Isolate code Probable strains of lipase activity (mm) S1 Ps. aeruginosa 24 S2 Enterococcus 19 S3 B. coagulans 35 S4 St. aureus 24 S5 // 20 S6 B. coagulans 28 S7 // 27 S8 B. subtilis 17 S9 B. cereus 24 S10 Lactobacillus 19 S11 Pseudomonas 29 Totally, twenty four bacterial isolates belonging to a several bacterial species were screened for lipase production on lipid agar medium. Colonies with highest clear zone on the plate lipid agar medium were selected as potential lipase producing strains. Among the isolated strains, 21lipase positive strains were selected by measuring the diameter of zone of hydrolysis on the agar plates. Based on differences in enzyme production by clear zone on the plates, the highest lipase producer was selected for further investigation. The morphological study, the microscopic observation and biochemical characterization revealed the presence of Ps. aeruginosa (S1, S11), B. coagulans (S3. S6, S7), B. subtilis (S8), B. cereus (S9), S. aureus (S4, S5), Lactobacillus (S10) and Enterococcus (S2). All the isolated Bacillus were gram positive, rod-shaped, aerobic, catalase positive and spore forming as described by Menoncin et al.[22] and Bergey s manual of determinative bacteriology. The isolated lipase producer strains according to their obtained hydrolysis halos are presented in table 1, which B. coagulans (S3. S6, S7) had the highest halo (35, 29, 27 mm) respectively. Effect of the Incubation Temperatures: Temperature is a critical parameter that has to be controlled and it varies from organism to organism. Temperature influences secretion of extracellular enzymes by changing the physical properties of the cell membrane. Studies conducted for the optimization of temperature shows that the bacteria produces lipase in wide range of temperature from 20 C to 50 C.The lipase enzyme produced at different range of temperature was from 1 5 U/ml to 70.0 U/ml. The optimum temperature for lipase enzyme production was at 40 C (70 U/ml) and the enzyme production was affected and decreased after increase of 964

4 Fig. 1: The effect of the incubation temperature on the lipase production in the optimized culture medium of the selected producer strain B. coagulans, agitated at 75 rpm for 3 days. Fig. 3: The effect of the inoculum concentration on the lipase production in the optimized culture medium of the selected producer strain B. coagulans, agitated at 75 rpm for 3 days. producing lipase from the initial ph of medium from ph 5.0 to ph 9.0. The enzyme production varied considerably from 5.0 U/mlto40 U/ml (Figure 2). The bacteria B. coagulans has optimum lipase production at ph 8.0(40.0 U/ml). However, it was noted that the lipase production was declined with increase in ph from ph8.0 to ph 9.0.]. Sirisha et al. [30] has reported that a maximum lipase production was observed at ph 8.0 and temperature 37 C by S. aureus. Effect of the Inoculum Concentration: Further Studies conducted for the optimization of inoculums concentration, which indicated that the variation in the Fig. 2: The effect of the ph-value on the lipase level of inoculum concentration from 1-10 % influence production in the optimized culture medium of the the lipase enzyme production. The enzyme activity selected producer strain B. coagulans, agitated at varied from 66.5to 80.0 U/ml (Figure 3). The optimum 75 rpm for 3 days. inoculums concentration for lipase production was 6% (80.0 U/ml).YU et al. [31] have reported that the highest temperature above 40 C to 60 C. It was also noted that the lipase was produced when the inoculums concentration lipase enzyme production was deceased at temperature was 6% for Ps. Lip35. It is desirable to produce maximum 50 C (Figure 1). Similar result was reported that the enzyme activity with lower concentration of inoculums for maximum lipase production was at 37 C by Ps. industrial application. xinjiangensis [23,24]. It was also reported that the growth and lipase enzyme production of Ps.fluorescens was Effect of Carbon Source: Lipase production is influenced maximum at the temperature 36 C [25]. Kulkarni and by the type and concentration of carbon and nitrogen Kavitha et al. [26, 27] has reported that a maximum lipase sources, the culture ph, the growth and temperature. production was at 25 C for Ps. sp. Furthermore, Studies conducted on the effect of sugars supplied Benattouche et al. [28, 29] has reported that a maximum as additional carbon source have not enhanced the lipase production was at 35 C for Ps.aeruginosa31. lipase production when compared to control medium. The used Olive oils and almond oils have considerably Effect of the ph-value: The initial ph of the growth augmented the yields of lipase production (225, 200 U/ml) medium influences the rate of lipase production. It was respectively (Figure 4). While the other tested carbon inferred from the results that the bacteria is capable of source showed a middle level of lipase enzyme production 965

5 studied. Generally, microorganisms provide high yields of lipase when organic nitrogen sources are used, such as peptone and yeast extract, which have been used for lipase production by various thermophilic B. sp. and various Pseudomonas. Among the ten different organic nitrogen sources proteose peptone (168.7 U/ml) and peptone (132.5 U/ml) enhanced lipase production whereas lipase production was very low with casein, soya bean meal and soy peptone. Similar results were reported for organic nitrogen source by Ps. fluorescens NS2W41 [33]. Among the different nitrogen sources used peptone (2 g/l) was found to be the most suitable source for maximum lipase activity. Similarly inorganic nitrogen Fig. 4: The effect of the carbon source on the lipase sources ammonium sulphate (129 U/ml) and ammonium production in the optimized culture medium of the chloride (121.6U/ml) enhanced lipase production. selected producer strain B. coagulans, agitated at 75 rpm for 3 days. CONCLUSION In the present work, the optimum temperature for lipase enzyme production by B. coagulans was at 40 C (70 U/ml) and the enzyme production was affected and decreased after increase of temperature above 40 C to 60 C. While, the optimum lipase production was observed at ph 8.0 (40.0 U/ml). However, it was noted that the lipase production was declined with increase in ph from ph 8.0 to ph 9.0. Moreover, because of its pronounced thermal stability as well as preservation of activity and stability in organic solvents, this enzyme could be of significant biotechnological potential, particularly in organo synthetic reactions carried out at higher Fig. 5: The effect of the nitrogen source on the lipase temperatures. Future research will focus on purification production in the optimized culture medium of the structural characteristics of this enzyme. Moreover, selected producer strain B. coagulans, agitated at studies on sequencing, sub cloning and overexpression 75 rpm for 3 days. of these genes in E.coli may reveal the potential applications of this enzyme in future. compared to control medium. It was reported that starch was the best carbon source for lipase production by P. ACKNOWLEDGEMENT fluorescens. Reduction in the lipase production in the presence of sugars as carbon sources could be due to We are thankful to the department head of the catabolic repression by readily available carbon sources bacteriology laboratory of hospital of University of in the culture medium [32]. Sidi Bel Abbès Mr. Harrachi Abdelkader for providing us with the bacterial strains. We would like to thank Effect of Nitrogen Source: The used oils as additional Dr. Benattouche Zouaoui for your encouragement, your nitrogen source have been investigated for the lipase support and your useful discussion. Without the kind production. cooperation of the above mentioned people the work Among the 4 used nitrogen source yeast extract could not have been possible. (60 U/ml) and peptone of casein (40 U/ml) has considerably increased the lipase production (Figure 5). REFERENCES On the other hand, the investigated gelatin and casein caused a considerable reduction in enzyme production. 1. Gilham, D. and R. Lehner, Techniques to Furthermore, the effect of additional organic and inorganic measure lipase and esterase activity in vitro. nitrogen source 1 % (w/v) on the production of lipase was Methods. 36:

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