International Journal of Current Biotechnology

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1 Doles Pereppadan Ephraim, Sarita Ganapathy Bhat and Chandrasekaran Muthuswamy, Lipase production by Immobilized marine Bacillus smithii BTMS11 and its potential application in waste water treatment, Int.J.Curr.Biotechnol., 2014, 2(12):1-8. International Journal of Current Biotechnology ISSN: Journal Homepage : Lipase production by Immobilized marine Bacillus smithii BTMS11 and its potential application in waste water treatment Doles Pereppadan Ephraim 1, Sarita Ganapathy Bhat 1* and Chandrasekaran Muthuswamy 1, 2 1 Department of Biotechnology, Cochin University of Science and Technology, Cochin , India. 2 Department of Botany and Microbiology, College of Science, King Saud University, Riyadh, Kingdom of Saudi Arabia. A R T I C L E I N F O A B S T R A C T Article History: Received 20 November 2014 Received in revised form 25 November 2014 Accepted 01 December 2014 Available online 08 December 2014 Key words: Marine lipase, Bacillus smithii, Optimization, Immobilization, Waste water treatment. Introduction Lipases (triacylglycerol acylhydrolase, E.C ) are enzymes widely distributed among animals, plants, and microorganisms that catalyze the reversible hydrolysis of glycerol ester bonds and, therefore, also the synthesis of glycerol esters (Treichel, et al., 2010). There is a large potential for lipases in industrial applications such as additives in foods, pharmaceuticals medical assay, cosmetics, leather, dairy industry fine chemicals, detergents, paper manufacture and waste-water treatment (Hasan et al., 2006). Lipases are produced by many microorganisms, including bacteria, fungi, yeasts and actinomycetes, although Candida, Pseudomonas, Rhizomucor and Rhizopus sp. stand out now a days as sources of most commercially available enzyme preparations (Arpigny and Jaeger, 1999; Sharma et al., 2001). Microbial lipases are preferred owing to their multifaceted applications in industries, easy production on cheap media components, easy downstream processing and unlimited supply due to renewable nature. Among the various microorganisms recognized as source of lipases, bacterial source are considered as the best source of extracellular lipase for large scale production by industries. Therefore, attempts to isolate microorganisms that facilitate the discovery of novel *Corresponding author. address: saritagbhat@gmail.com The main aim of the present study was the optimization of immobilization conditions for Bacillus smithii BTMS11 lipase production and its application on waste water treatment. Media optimization of lipase production by immobilized bacterial cells was carried out by one factor at a time method. Prior to that optimization of immobilization conditions were done. It was found that optimal concentration of immobilizing support material, sodium alginate concentration 3% (w/v), CaCl 2 concentration 0.2 (M), curing time of beads that could promote maximal production of lipase enzyme 8h, optimum activation time 24h, retention time 24 h and drop height for perfect shape 5 cm, respectively. It is also found that an agitation speed (rpm) of 150, temperature of 30 º C, ph 8, inoculum concentration of 20%, inoculum age of 24 h, incubation time of 18 h, glucose concentration of 1%, nitrogen source (Soyabean meal) of 1%, sodium chloride concentration of 1%, calcium chloride 0.2 %, magnesium sulphate concentration 2%, sesame oil concentration (Inducer) 1% were the best suited optimum for maximal enzyme activity. Results of the study indicated scope for application of this marine bacterial lipase in effluent water treatment. lipases always gain attention. Among bacterial lipases being exploited, those from Bacillus display properties that make them promising candidates for biotechnological applications. B. subtilis, B. pumilus, B. licheniformis and B. alcalophilus are among common bacterial lipase producers. Lipolytic enzymes secreted by Bacillus sp. are of substantial biotechnological interest and many have, therefore, been identified, cloned, and characterized (Lindsay et al., 2000). It was reported that Bacillus sp. were efficiently utilize complex nutrient sources. However, the literature on the development of a medium composed of natural nutrients such as vegetable oils for high lipolytic activity of isolated Bacillus species are limited. To the best of our knowledge, all the lipases reported are from terrestrial origin, and the marine environment that holds the vast diversity of flora and fauna remains under explored with respect to industrial enzymes. Immobilization of enzymes can offer several advantages including its reuse, ease in application of both batch and continuous systems, possibility of better control reactions, ease in removal from the reaction medium and improved stability (Betigeri et al., 2002). Entrapment, one of the immobilization techniques, can be defined as physical restriction of enzyme within a polycationic polymer by the addition of multi-valent counter-ions is a simple and common method of enzyme entrapment. Alginates are one of the most frequently used polymers 1 Int.J.Curr.Biotechnol. Volume 2; Issue 12; December, 2014

2 due to their mild gelling properties ease of formulation, biocompatibility, and acceptability as food additive and as oral drug delivery systems and non-toxicity (Won et al., 2005). Waste water generated from food manufacturing units, oil massage based treatment units in health resorts and hospitals, restaurants, hostel kitchen and municipal sewage that receive from domestic sewage containing oils and fats all pose serious challenge in safe and effective treatment of effluents and subsequent disposal into environment. In fact a large number of pretreatment systems are employed nowadays to remove fats and oil from the wastewater before the main treatment process which is often a biological treatment. However, there is a need for improving the existing wastewater treatment processes by complimenting with enzyme catalysts which could facilitate easy and rapid reduction of complex substances such as fats and oil. In this context, microbial lipases could have an impact on reducing the fats and oil contents in such effluents (Vulfson et al., 1994). In the present communication, we report the production of detergent compatible alkaline lipase by Bacillus smithii BTMS11 (Lailaja and Chandrasekaran, 2013) under immobilized conditions employing gel entrapment technique, and their potential for use in the treatment of waste water generated from a food producing commercial enterprise. Materials and methods Materials Sodium alginate, CaCl 2 anhydrous, Zobell s agar, Acetone, Polyvinlyl alcohol, Olive oil were purchased from Merck (India). All other chemicals used were of analytical grade. Microorganism and culture maintenance Microorganism Bacillus smithii strain BTMS11 isolated from the sediment of coastal areas of South India during an earlier investigation and available as stock culture in the Microbial Technology Laboratory, Department of Biotechnology, CUSAT, Cochin (Lailaja and Chandrasekaran, 2013) was used for the study. Growth media Marine Zobell s medium (HiMedia, India) was used for the maintenance and periodical subculturing of the bacteria. The same medium was used for preparation of inoculum. Inoculation Preparation For the preparation of inoculum, a loop full of cells from the freshly grown culture (Zobell s Marine Agar slant of B. smithii BTMS11 was transferred to a 10ml Zobell marine broth (ZB) and incubated at room temperature (28±2 C) on a rotary shaker. After 18 h of incubation this pre culture was again transferred to 100 ml of ZB in a 250 ml conical flask and incubated at room temperature on a rotary shaker at 180 rpm. After 18 h of incubation, cells were harvested by centrifugation at 10,000 rpm for 15 minutes at 4 o C. The sediment cell pellet was suspended in 20 ml of 0.8 % physiological saline. The prepared cell suspension was used as inoculum (10 x 10 8 CFU/ml) for immobilization studies. Enzyme Production Medium Lipase Production medium (Lailaja and Chandrasekaran, 2013) contained 1.5 % sesame oil as carbon source along with 0.2% soyabean meal, 0.5% glucose, 0.5% additional NaCl, 0.1mM CaCl 2 and 2mM MgSO 4. This medium was optimized for lipase production under submerged fermentation conditions by free cells. The prepared medium was inoculated with 3 % (v/v) of inoculum and incubated in an environmental shaker (Orbitek, Scigenics, India) at room temperature for the immobilization optimization studies. Bacterial Cell Immobilization into Calcium alginate beads Preparation of support material for immobilization Sodium alginate was used for preparation of immobilized cells employing gel entrapment technique. Initially 9 g of sodium alginate was slowly added to 300 ml of distilled water while being continuously stirred. The stirring was continued for a further 1-2 h in a magnetic stirrer until complete dissolution of sodium alginate solution. The prepared sodium alginate solution was then autoclaved at 121 o C for 20 minutes and used for preparation of beads. Preparation Under sterile conditions, the prepared cell slurry was mixed with sodium alginate solution at ratio of 1:2 and mixed thoroughly to get cell alginate slurry. This was then extruded drop wise in to a 0.2M CaCl 2 solution, taken in a beaker, from a height of 5 cm using a burette. The entrapped calcium alginate beads were maintained in a solution of 0.2M calcium chloride solution for overnight for curing. After this beads were thoroughly washed with physiological saline 3-4 times and maintained at 4 o C until used. Activation of Immobilized cells Activation of the immobilized cells in calcium alginate beads was done by suspending 20 grams of the cells in a 50 ml solution of enzyme production medium taken a in a conical flask and incubation at 37 o C. Samples of the activation media were drawn at intervals of 6h, 12 h, 18 h and 24 h and checked for enzyme activity. Retention Time After activation the immobilized cells in beads were removed, washed with fresh enzyme production media and used for determination of optimal retention period. For optimization of retention time, 20 g of immobilized cell beads was weighed, transferred to 50 ml of enzyme production media taken in a 250 ml conical flask and incubated at room temperature. Samples were drawn at regular intervals of time (3 h, 6 h, 12 h, 15 h 18 h and 24 h) and were assayed for lipase activity. Lipase Assay Lipase assay was performed using olive oil as a substrate. The enzyme activity was determined by titration of the free fatty acids liberated from olive oil against standard alkali solution (Ota and Yamada, 1966). One unit of lipase activity is defined as the amount of enzyme that releases one micromole of free fatty acids per ml per minute under assay conditions. Lipase activity (U/mL/min) = (T-C) x Normality of NaOH x 100 Time of incubation Where (T-C) is the difference in titer value between test and control Protein Determination Protein analysis of the supernatants was determined as per the method proposed by Lowry et al (1951). Bovine Serum Albumin was used for preparation o f standard curve and computation of protein concentration in the samples. Volume 2; Issue 12; December, 2014 Int.J.Curr.Biotechnol. 2

3 Table 1: Parameters of waste water before and after treatment with immobilized B.smithii Parameters Effluent before treatment with immobilised beads with bacterial cells After Treatment with immobilised beads with bacterial cells Control (Beads without bacterial cells) BOD 1200 ± ± ± 1 ph 4.85 ± ± ± 0.05 Chloride 71 ± ± ± 0.15 DO 6.3 ± ± ± 0.05 TDS 396 ± ± ± 0.5 conductivity 660 ± ± ± 0.1 Figure 1: Optimization of support concentration (% w/v) Figure 2: Optimization of Calcium chloride concentration (M) Figure 3: Optimized beads after drop height experiment Figure 4: Optimization of curing time (h) 3 Int.J.Curr.Biotechnol. Volume 2; Issue 12; December, 2014

4 Specific Activity Specific activity of the sample was calculated by dividing the enzyme units with the protein content and was expressed as U/mg/min. Optimization of immobilization conditions Optimal concentrations of immobilizing support material, CaCl 2 concentration, and curing time of beads that could promote maximal production of lipase enzyme by immobilized cells in beads and drop height for obtaining perfect shape to cells under immobilized conditions were determined. Support concentrations The optimal support concentration required for the preparation of active and stable beads with immobilized viable cells for maximum enzyme production was determined using sodium alginate at different concentrations (1-5% w/v). Calcium chloride concentration The optimal concentration of calcium chloride required for the preparation of active and stable beads with immobilized viable cells for maximum enzyme production was determined by using calcium chloride at different molar concentrations ( M). Optimum curing time Optimum curing time for preparing stable beads with immobilized viable cells was determined by allowing the beads formed in optimum Calcium chloride concentrations to remain as such for varying periods of curing time (2-12 h). Later, the beads were washed with physiological saline and the optimal curing time was accessed by estimating lipase activity. Drop height The height at which the sodium alginate slurry containing bacterial cells needs to be dropped to get the prefect beads were determined by varying the heights (0.5-5 cm) of drop and checking the perfectness of beads. Optimization of bioprocess variables and media constituents Various bioprocess parameters and media constituents that influence lipase in calcium alginate beads were optimized for obtaining maximal lipase production under immobilized conditions. Lipase at different levels of ph (5, 6, 7, 8, 9 and 10), incubation temperature (25, 30, 35 and 40 C), rate of agitation (0, 80, 100, 120 and 150 rpm), age of pre inoculum (6, 12, 18, 24,30,36,42 and 48 h) and initial inoculums concentrations (3, 5, 10, 20 and 30 %) were studied. Optimum incubation time was determined by incubating the production medium for a total period of 48 h and analyzing the samples at 8, 12, 18, 24 and 48 h. The effect of sodium chloride concentration (0.5, 1, 2 and 3%), and nitrogen source (soyabean meal) concentration (0.5, 1, 2 and 3%), glucose concentration (0.5, 1, 2 and 3%), calcium chloride concentration (0.1, 0.2 and 0.3mM), and MgSO 4 concentration (1, 2 and 3%) on lipase production by B. smithii BTMS11 under immobilized condition were also studied. Moreover, the effect of various lipase inducer sources was also studied by the addition of sesame oil at a level varying from 0.5 to 1.5 % (w/v) were also studied. Lipase Activity (U/mL/min), Protein concentration mg/ml and specific activity (U/mg/min), were determined for all the samples of the different experiments, which were conducted in triplicate. Treatment of kitchen effluent by immobilized B. smithii strain BTMS11 For the present study, waste water generated in the kitchen of a large scale commercial food catering unit located in Kochi, Kerala, India were used. All the major parameters that determine the overall quality of the wastewater/effluent that is being disposed were analyzed prior to the treatment with the lipase and after treatment with lipase producing B. smithii cells immobilized in calcium alginate beads. The parameters analyzed include biological oxygen demand (BOD), ph, chloride, total dissolved solids (TDS), dissolved oxygen (DO), and conductivity. To 50mL of effluent, 20g of calcium alginate beads containing lipase producing B. smithii were added. The experiments were conducted in triplicates in 250mL Erlenmeyer flask at room temperature in an environmental shaker (Orbitek, Scigenics, India) for 48h. The effluent without immobilized cells in beads was used as control. The efficiency of treatment was assessed in terms of changes in the values of various parameters analyzed. Results and Discussion Optimization of process parameters for immobilization of Bacillus smithii BTMS11 Support concentration Calcium alginate beads have been in used widely in various studies over the years ever since immobilization of cells was recognized for production of desirable products using microorganisms besides other applications. In the present study too it was noted that calcium alginate is an ideal choice for immobilizing B. smithii for lipase production. Results presented in Fig.1 indicated that immobilized cells prepared with 3% (w/v) concentration of sodium alginate produced relatively high level of enzyme activity and further increase in concentrations of sodium alginate led to decline in enzyme activity. The results indicate that 3% is the ideal concentration of sodium alginate for preparation of stable beads with immobilized B. smithii as in the case of several other microorganisms. Calcium chloride concentration Calcium chloride concentrations used for preparation of stable beads with immobilized cells influenced the lipase production by immobilized bacterial cells. The maximal enzyme activity was observed with a concentration of 0.2M of CaCl 2 indicating that it is the optimal concentration for preparing active and stable beads (Fig.2). Further increase in CaCl 2 concentrations showed a decreasing trend in enzyme activity. Drop Height From the studies it was inferred that drop height influence the perfect shape of the beads that subsequently affect stability and activity of the immobilized beads. From the data presented in Fig. 3 it was noted that at 2cm drop height the sodium alginates beads with entrapped cells attained prefect round shape and beads were stable unlike in the case of other heights tested. Optimization of Curing, Activation, & Retention periods From the results presented in Fig.4, it was observed that beads with immobilized cells cured for a period of 8 h could support maximal enzyme production and other periods of curing did not support enhanced lipase activity by immobilized cells. The results indicated that 8h of curing is optimal time for preparing stable and active Volume 2; Issue 12; December, 2014 Int.J.Curr.Biotechnol. 4

5 Figure 5: Optimization of activation time (h) Figure 6: Optimization of retention time (h) Figure 7: Effect of ph on lipase production by immobilized cells Figure 8: Effect of Temperature ( º C) on lipase Figure 9: Effect of inoculum age(h) on lipase production by immobilized cells Figure 10: Effect of inoculum concentration (%) on lipase Figure 11: Effect of agitation speed (rpm) on lipase Figure 12: Effect of incubation time (h) on lipase 5 Int.J.Curr.Biotechnol. Volume 2; Issue 12; December, 2014

6 Figure 13: Effect of carbon source (glucose) on lipase Figure 14: Effect of sesame oil (enzyme inducer) on lipase Figure 15: Effect of nitrogen source (soyabean meal) on lipase Figure 16: Effect of sodium chloride on lipase Figure 17: Effect of calcium chloride on lipase Figure 18: Effect of magnesium sulphate on lipase Volume 2; Issue 12; December, 2014 Int.J.Curr.Biotechnol. 6

7 immobilized cells in beads for maximal enzyme production. After preparation of immobilized cells in alginate beads, optimal activation time required for maximal enzyme production was determined. Data presented in Fig.5 indicate that 24 h of activation is ideal for maximal enzyme production. Further increase in activation time led to decline in enzyme production. It was also noted that activation periods 6-12 h did not support enzyme production. After optimizing activation time, optimal retention period for obtaining maximal enzyme production by immobilized cells in beads was determined by subjecting the immobilized cells to different time intervals of retention and incubation in enzyme production medium. From the results presented in Fig. 6 it was noted that 24 h of retention time could support maximum enzyme activity compared to other levels of retention periods. Optimization of bioprocess variables that influence lipase production by immobilized viable cells Effect of ph on lipase The optimal ph for maximal production of lipase was found to be at ph 8. It shows the lipase activity maximum is at slightly alkaline condition (Fig.7). Further increase in ph decreased the activity. It may be noted that this bacteria was reported (Lailaja and Chandrasekaran, 2013) to produce detergent compatible alkaline lipase under submerged fermentation conditions and the present observations concur with the same. Effect of Temperature on lipase production by immobilized cells Effect of temperature on lipase production by immobilized cells found by keeping immobilized beads in production media at varying temperatures starting from 25 º C to 37 º C.It was found that 30 º C could promote maximal enzyme activity (Fig.8). At temperatures above 30 º C beads started dissolving into media. Effect of inoculum age and inoculum concentration on lipase It was found that incoulum age (h) that could support maximal production was found to be 24 h. At 6hr, 12 hr and 48 h there was no production of enzyme at all (Fig.9). Further, maximum lipase activity by immobilized cells was observed with 20% inoculum concentration. Inoculum concentrations above and below 20% did not support enhancement in enzyme activity and instead led to decrease in the enzyme activity (Fig.10). The higher level of inoculum concentration required for maximal enzyme activity may be attributed to the immobilized state of viable cells inside calcium alginate beads compared to their free state in submerged fermentation conditions. Effect of agitation speed (rpm) on lipase production by immobilized cells Effect of agitation speed on maximal enzyme activity was found by keeping the immobilized beads in production media at varying rpm. It was found that 150 rpm could promote maximal enzyme activity by immobilized cells in calcium alginate beads (Fig.11). Further increase in agitation speed showed a decreasing trend and finally dissolution of beads in the media due to shearing effects at high speed. Agitation enables easy permeation of enzyme production medium into the calcium alginate beads and subsequent availability of the same for the viable cells inside the beads for lipase production. At the same time higher agitation speeds would have exerted mechanical force that led to disruption of beads. Effect of incubation time on lipase production by immobilized cells Optimal incubation time directly influence the rate of enzyme production. In the present study it was observed that maximal enzyme in calcium alginate beads, under optimized bioprocess conditions, could be achieved only after incubation time for 18h (Fig.12). Optimization of media constituents From the results obtained for the studies on optimization of various enzyme production media constituents and presented in Figures 13 to18, it was found that maximal enzyme activity could be obtained with 1% carbon source (glucose), 1% nitrogen source (soya bean meal), 1 % sodium chloride concentration, 0.2 mm Calcium chloride concentration, 2mM magnesium sulphate and 1% sesame oil as lipase inducer. Treatment of commercial ready to eat food kitchen effluent by lipase produced by immobilized B. smithii BTMS11 Ready to eat food production has escalated in recent years owing to the change in life style of people and increase in tourism and travel. Consequently more and more commercial enterprises have proliferated that produce ready to eat food in large scale. In this process they generate more waste water rich in oils and fats along with food residues into the environment. This necessitates appropriate treatment of waste water before their disposal into environment, complying with environmental regulations. In this context, this experiment was attempted to evaluate the prospects of reducing oils and lipids in the waste water of a commercial ready to eat producing kitchen unit using lipase producing immobilized Bacteria. From the data obtained and presented in Table 1, it was observed that the lipase producing bacteria under immobilized conditions could accomplish reduction in values of parameters that determine the overall quality of waste water that is discharged. The average values of BOD, TDS and Chloride were considerably reduced in the treatment with immobilised B. smithii strain BTMS11 compared to the Control and increase in the parameters ph, dissolved oxygen (DO) and conductivity endorsing the lipid degradation capacity of marine B. smithii BTMS11 and its suitability in waste water treatment. Conclusion Based on the results of the present study, it is concluded that lipase produced by marine bacteria Bacillus smithii has potential for applications in oil and fat rich waste water generated in various commercial kitchen units besides applications in other areas where such biocatalyst is required. Enzyme catalysts are potential candidates for biotechnological solutions in effective waste and waste water management and the present studies endorse such an approach. References Arpigny JL, Jaeger KE (1999). Bacterial lipolytic enzymes: classification and properties. Biochem J 343: Betigeri, S.S., and Neau, S.H., (2002), Immobilization of lipase using hydrophilic polymers in the form of hydrogel beads, Biomaterials, Vol. 23, Gaur, R., Gupta, A. and Khare, S.K., Purification and characterization of lipase from solvent tolerant 7 Int.J.Curr.Biotechnol. Volume 2; Issue 12; December, 2014

8 Pseudomonas aeruginosa PseA, Process Biochemistry, Vol. 43, (2008), Hasan F, Shah AA, Hameed A (2006). Industrial applications of microbial lipases. Enzyme Microb Technol 39: Kumar S, Kikon K, Upadhyay A, Kanwar SS, Gupta R (2005). Production, purification and characterization of lipase from thermophilic and alkaliphilic Bacillus coagulans BTS-3. Protein Expr Purif 41:38 44 Lailaja VP, Chandrasekaran M, Detergent compatible alkaline lipase produced by marine Bacillus smithii BTMS 11, World J Microbiol Biotechnol (2013) 29: Lindsay D, Brozel VS, Mostert JF, von Holy A (2000) Physiology of dairy associated Bacillus sp. over a wide ph range. Int J Food Microbiol 54:49 62 Lowry OH, Rosenbrough NJ, Farr AL, Randal RJ (1951). Protein measurement with Folin phenol reagent. J Biol Chem 193: Macrae AR, Hammond RC (1985). Present and future applications of lipases. Biotechnol Genet Eng Rev 3: Ota, Y. and Yamada, K., Lipase from candida paralipolytica Part I. Anionic surfactants as the essential activator in the systems emulsified by polyvinyl alcohol, Agricultural and Biological Chemistry, Vol. 30, (1966), Sharma R, Chisti Y, Banerjee UC (2001). Production, purification, characterization, and applications of lipases. Biotechnol Adv19: Sharma R, Soni SK, Vohra RM, Jolly RS, Gupta LK, Gupta JK (2002). Production of extracellular alkaline lipase from a Bacillus sp. RSJ1 and its application in ester hydrolysis. Indian J Microbiol 42:49 54 Treichel, H. et al. (2010). A review on microbial lipases production. Food Bioprocess Technol. 3, Vulfson EN (1994). Industrial applications of lipases. In: Woolley P,Peterson SB (eds) Lipases-their structure, biochemistry and applications. Cambridge University Press, Cambridge, pp Wang Y, Srivastava KC, Shen GJ, Wang HY (1995). Thermostable alkaline lipase from a newly isolated thermophilic Bacillus,strain A30 1 (ATCC 53841). J Ferment Bioeng 79: Won, K., Kim, S., Kim, K.J., Park, W. and Moon, S.J., Optimization of lipase entrapment in Ca-alginate gel beads, Process Biochemistry, Vol. 40, (2005), Volume 2; Issue 12; December, 2014 Int.J.Curr.Biotechnol. 8