Hydrolysis of Oils in Saudi Arabia PDF

Hydrolysis of Oils in the Wadi Hanifah River in Saudi Arabia by Free and Immobilized Staphylococcus aureus ALA1 Lipase Abir Ben Bacha, a,b Islem Abid,c Imededdine Nehdi,d,e and Habib Horchanif a Biochemistry Department, Science College, King Saud University, P.O. Box 22452, Riyadh, 11495, Saudi Arabia b Laboratory of Plant Biotechnology Applied to Crop Improvement, Faculty of Science of Sfax, University of Sfax, Sfax, 3038, Tunisia c Botany and Microbiology Department, Science College, King Saud University, P.O. Box 22452, Riyadh, 11495, Saudi Arabia d King Saud University, College of Science, Chemistry Department, Riyadh, 1145, Saudi Arabia e El Manar Preparatory Institute for Engineering Studies (IPEIEM), Chemistry Department, Tunis El Manar University, Tunis, 2092, Tunisia f Science Department, College of Rivière-Du-Loup, Rivière-Du-Loup, Québec, G5R1E2, Canada Published online 00 Month 2018 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/ep.13000 This study describes the biochemical properties of Staphylo- offers the chance to overcome the problems associated with coccus aureus lipase immobilized by physical adsorption to lipase treatment of wastewater. The advantages of immobilized assess its role in the biodegradation of oils in the Wadi Hani- enzymes include mechanical stability, controlled reactions, fah River. After optimization of the immobilization conditions, and multiple usages. Various immobilization methods have the recovered enzyme activity was 95% with appreciable been reported ranging from reversible physical adsorption and increase in stability. The immobilized and free lipase retained ionic linkages to the irreversible stable covalent bonds, that is, 70% and only 10% of the initial activity after a 60-min incu- chemical engineering modiﬁcation, surface binding, and gel bation at 80 C, respectively. More than ~40% residual activity entrapment [7–10]. Among them, adsorption remains the most remained after 48 h of incubation at pH5 to 11. The immobi- simple and cost-effective method. However, the choice of the lized enzyme retained 100% of the initial activity when used support is an important factor inﬂuencing the main hurdle for for four cycles and 42% of initial activity when stored at widespread commercialization and the enzymatic reactions. 25 C for 120 days. Enzyme stability was enhanced in the Calcium carbonate (CaCO3) is chemically nonreactive and is presence of inactivating agents including β-mercaptoethanol, nontoxic. CaCO3 has proven to be an adequate adsorbent that SDS, EDTA, and Co2+. The bioremediation potential of lipid- allows elevated dispersion of crude Rhizopus oryzae and Can- rich wastewater by the immobilized and free lipases was dida rugosa lipases and retention of enzymatic activity [11,12]. explored by analyses of chemical oxygen demand and lipid Interestingly, enzyme readsorption is often possible by pH content. Both free and immobilized lipases efﬁciently hydro- modiﬁcation followed by binding of novel active lipase. lyzed all oils tested and organic matter present in the waste- Some characteristics of immobilized enzymes differ from the water. Overall, the results indicate the potential of same characteristics of soluble enzymes [14,15]. immobilized S. aureus lipase in biological wastewater treat- Biological treatment of wastewater has been used for more ment and offer new options for several industrial applications. than a century to reduce anthropogenic harm to the environ- © 2018 American Institute of Chemical Engineers Environ Prog, 2018 ment. Lipids (oils, fats, and greases) are the major problematic Keywords: bioremediation, wastewater, TOC, COD contaminants in the biological processes used for wastewater treatment. Oils kill many forms of aquatic life by forming a INTRODUCTION layer on the water surface due to their hydrophobic nature, Lipases are enzymes or biocatalysts that catalyze the hydro- which decreases the rate of oxygen transfer from air into the lysis of insoluble triglycerides at the lipid–water interface to water. Additionally, water drainage lines may be blocked fatty acids and glycerol. During the last decade, several by the aggregates produced by oil droplets and other particles, studies reported the treatment of oily wastes using lipases as which are especially prominent in wastewater. Oily wastewa- biocatalysts [2–6]. Most of these studies focused on the use of ter is mainly formed from municipal wastewaters, food proces- synthetically added fats for the wastewater pre-treatment. sing and metals, petroleum, textile, and cooling and heating Commercially available lipases are costly. Additionally, industries [17,18]. lipases can be used only once in solution, since they are gen- In the Kingdom of Saudi Arabia, some of the wastewater erally soluble and unstable. The use of immobilized enzymes treated in the main treatment plant, the Manfuha station (Al- has revolutionized the ﬁeld of enzyme biotechnology, and Riyadh), is used for the irrigation of landscaped plants, trees, and grass in municipal parks and for industrial purposes. The remaining wastewater is discharged into Wadi Hanifah. The © 2018 American Institute of Chemical Engineers Hanifah Valley and side valleys (tributaries) are located in an Environmental Progress & Sustainable Energy DOI 10.1002/ep 1 urban-industrial desert within the Kingdom of Saudi Arabia. In Lipase activity was expressed in the international unit (U), the city of Al-Riyadh, industrial areas, a tannery, the Manfuha which corresponds to 1 μmol of free fatty acid liberated per Sewage Treatment Plant, and agricultural activities inﬂuence minute. the quality of water ﬂowing in the channel. The water ﬂow through Wadi Hanifah occurs year-round, mainly due to Inﬂuence of Inhibitors and Activators human activity discharges and seasonal rain events. The suc- The inﬂuence of several compounds [β-mercaptoethanol, cessful restoration of Wadi Hanifah and its natural environ- phenylmethane sulfonyl ﬂuoride (PMSF), ethylenediaminete- ment would beneﬁt the region and serve as an example of the traacetic acid (EDTA), and sodium dodecyl sulfate (SDS)] potential of this comprehensive approach in areas featuring and,metal ions including Ba2+, Cd2+, Co2+, Cu2+, Fe2+, Mg2+, natural and human activities. Oil layer biodegradation using Mn2+, and Zn2+ possible inhibitors or activators of immobilized enzymatic treatment of wastewater that is reused for agricul- and free enzymes was assessed using the standard assay tural purposes in the governorate of Al-Riyadh has not been method described above. reported before. In a prior study, we optimized the conditions for Staphylo- Effects of Temperature and pH on Lipase Stability and coccus aureus lipase immobilized on CaCO3. Presently, the biochemical characterization of the free and the CaCO3- Activity immobilized lipase were investigated. The enzyme perfor- Lipolytic activity was checked at 60 C at various pHs mance in the hydrolysis of several oils and the effective bio- (8–13). The pH stability of immobilized and free enzymes was degradation of the oily Wadi Hanifah water was established. determined by incubating the lipase preparation at various pH values ranging from 2 to 13 for 48 h and at room temperature using the appropriate buffers. The remaining lipase activity MATERIALS AND METHODS was determined after centrifugation using the standard assay method. Each measurement was performed in triplicate. Production and Immobilization of S. aureus Lipase The optimum temperatures for the activities of the immobi- The lipolytic S. aureus ALA1 strain (Access number: KF lized and the free enzymes were determined by performing 678862) was isolated and produced from camel milk as previ- the lipase assay at pH 12 and at temperatures ranging from ously described. After 30 h of culture, cells were removed 25 to 75 C. Thermostability was determined by incubating the by centrifugation and the crude enzyme solution was precipi- lipase for 1 h at temperatures ranging from 30 to 90 C and tated by the addition of ammonium sulfate to 65% saturation. pH 12. The remaining enzyme activity was measured after cen- The precipitate was resuspended in 5 ml of a solution contain- trifugation of each preparation using the standard assay ing 25 mM Tris–HCl, 2 mM benzamidine, and 50 mM NaCl method. (pH 8) and treated at 70 C for 15 min. Finally, the insoluble material was discarded by centrifugation (30 min, 12,000 rpm) and the resulting lipase solution was stored at 4 C for further Preliminary Characterization of Wadi Hanifah Water analysis. Water samples were collected from Wadi Hanifah located Lipase was immobilized on Celite 545, CaCO3, and silica in Al-Riyadh City, Kingdom of Saudi Arabia. Characteristic fea- gel as previously described. One milliliter of lipase solu- tures of total organic chloride (TOC), chemical oxygen tion (containing 3500 U) and 1 g of the support were mixed demand (COD), and lipid content were analyzed before and and incubated for 2 h at 4 C with intermittent stirring. Tripli- after treatment with free and immobilized S. aureus lipase. cate samples were used. Fractions were acquired at different Water treatment was performed by addition of 3500 U of times from 0 to 120 min. Each sample was centrifuged for the free or the immobilized lipase to 1 L of the water sample. 5 min at 8,000 rpm and the resulting supernatant was checked After rigorous shaking, the sample was divided into 20 fractions for enzyme activity and unbound protein. Then, the prepara- (50 mL each) and stirred at 200 rpm at room temperature. Sam- tions were ﬁltered, washed three times with double-distilled ples were withdrawn at regular intervals of 24 h for COD and water and dried in a vacuum desiccator for 8 h at room tem- TOC determinations according to American Public Health perature. The yield of the immobilized lipase activity was Association protocols. Simultaneously, a blank was run deﬁned as the ratio of the adsorbed lipase activity divided by with distilled water rather than sample. The lipid content was the total soluble lipase activity originally added to 1 g of the determined as previously described. Each measurement support. was performed in triplicate and data are presented as mean standard deviation. Protein Analysis, Lipase Activity, and Fatty Acid Proﬁle RESULTS AND DISCUSSION of Oils Protein quantiﬁcation was determined spectrometrically Immobilization of S. aureus Lipase using the Bradford method. The activities of the free and Physical adsorption is considered the cheapest and simplest the immobilized lipases were measured titrimetrically using a method to immobilize an enzyme, even though the adsorbed pH-stat and olive oil emulsion as a substrate under standard enzymes may detach from the support during use. Immo- conditions (pH 12, 60 C). For comparison, the hydrolysis bilization of S. aureus lipase was carried out on three solid efﬁciency of four commercial oils (sunﬂower, palm, coconut, support materials: CaCO3, Celite 545, and silica gel. The bind- and corn oils) by CaCO3-immobilized and free lipases were ing of lipase to the different carriers was evaluated by estimat- also investigated under the same conditions. ing the amount of enzyme that was adsorbed (Table 1). The fatty acid proﬁle of commercial oils was determined by CaCO3 adsorbed markedly more lipase (86%) than the Celite gas chromatography–mass spectrometry (GC–MS) using a 545 and silica gel supports (16% and 23%, respectively QP2010 Ultra chromatograph (Shimadzu, Japan) equipped (Table 1). This could be explained by the fact that the Celite with a model QP2010 mass spectrometer quadrupole detector. 545 and Silica gel surfaces are not physically inert towards A Rxi-5Sil MS column (30 m × 0.25 mm internal diameter, lipase, which could cause deactivation of the enzyme. A simi- 0.25 μm ﬁlm thickness) was used for the identiﬁcation and lar result was described for the immobilization of R. oryzae quantiﬁcation of each fatty acid. Fatty acid methyl esters lipase and the commercial C. rugosa type VII lipase on the (FAMEs) were prepared from the ﬁve oil samples as previously same supports [7,11]. Silica gel and Celite 545 were excluded described. from further analyses. 2 Environmental Progress & Sustainable Energy DOI 10.1002/ep Table 1. Adsorption of S. aureus lipase on different supports. 120 100 Immobilization yield (%) Support Yield of immobilized lipase activity (%) CaCO3 86 2.3 80 Silica gel 23 1.5 Celite 545 16 0.7 60 The initial concentration of the enzyme solution was 3500 UT, 40 the support amount was 1 g, and the contact time of the enzy- matic solution-support was 30 min at 4 C. The activities of the 20 free and immobilized lipases were measured using olive oil emulsion as substrate at pH 12 and 60 C. Data are the means of triplicate determinations standard deviation. 0 0 1000 2000 3000 4000 5000 6000 7000 8000 Initial activity of soluble lipase (UT) Figure 2. Yields of immobilized S. aureus lipase using The kinetics of protein and lipase adsorption onto CaCO3 different initial activity of soluble lipase. The activity was revealed the absence of detectable unbound lipase, whereas measured using olive oil emulsion at pH 12 at 60 C. Data are the amount of lipase and protein was below saturation and means of triplicate determinations standard deviation. their adsorption to the support occurred rapidly (Figure 1A). The highest binding was reached within 40 and 60 min for lipase and protein, respectively. The ﬁndings indicated that Characterization of Immobilized and Free Lipase lipase is a relatively fast absorbing protein. However, a loss Storage Stability of enzymatic activity was observed after 50 min of incuba- The storage stability for immobilized enzymes is an advan- tion time with CaCO3 (Figure 1B), indicating denaturation of tage compared to the lack of stability during storage of free some of the immobilized enzyme with time. The maximum enzymes. Storage stability is thus an important index to binding of the enzyme to CaCO3 (95%) was achieved after evaluate the enzyme properties. As illustrated in Figure 3, the incubation for 40 min. This was consistent with prior CaCO3-immobilized lipase retained ~80 and 42% of its original descriptions for the immobilization of R. oryzae and activity when stored for 120 days at 4 and 25 C, respectively. C. rugosa lipases to CaCO3 and AmberliteXAD7, respec- In contrast, the residual activity of free lipase progressively tively [11,26]. decreased up to 35% over 120 days at 4 C, with no residual To determine the optimum immobilization conditions of enzyme activity observed following storage at 25 C for the the enzyme, the effect of the amount of lipase in aqueous same period (Figure 3). This extended stability could be solutions ranging from 1000 to 7000 U g−1 support was inves- explained by numerous linkages of the enzyme to its support, tigated (Figure 2). The immobilization yields gradually which would prevent intermolecular processes such as aggre- increased to reach a maximum value at 3500 TU as more gation and proteolysis, and facilitate the formation of a more lipase was loaded onto the CaCO3 support. Reduced activities rigid enzyme molecule with greater stability [33,34]. observed at low enzyme loading could be attributed to the loss of lipase conformation probably due to the maximum contact of the lipase molecules with the surface. However, Effect of Temperature and pH on Free and Immobilized Lipase for loadings more than 3500 U g−1 support, multilayer Stability and Activity adsorption may occur, which would likely inhibit or block The immobilization procedure and pH might modify the access to the active site of the enzyme. Thus, most of the sub- kinetic behavior of the enzyme and its original physicochemi- sequent experiments used a lipase loading of 3500 U g−1 cal properties. The effect of immobilization on the S. aureus CaCO3. These ﬁndings are in the range of those obtained by ALA1 activity was assessed. No change in the optimum pH other immobilization techniques, such as the covalent bind- (12) was evident for both the immobilized and aqueous lipase ing of enzyme on aldehyde-Lewatit , polyglutaraldehyde- activities (Figure 4A). A similar result was described for Bacil- activated olive pomace powder , chitosan [29,30], and lus lipase immobilized on HP-20 beads. However, the Toyopearl AF-amino-650 M resin. optimum pH (6.0) of the immobilized form of C. rugosa lipase (A) (B) 3.5 4000 Lipase Solution (UI/ml) Protein Solution (mg/ml) 120 3 100 Lipase Solution (UI/ml) 3000 2.5 80 2 2000 60 1.5 1 40 1000 0.5 20 0 0 0 0 10 20 30 40 50 60 70 80 90 100 110 120 0 10 20 30 40 50 60 70 Incubation Time (min) Time (min) Figure 1. Adsorption kinetics of S. aureus lipase on CaCO3 (A) and yields of CaCO3 immobilized lipase using different incubation times (B). The activity was measured at the pH-stat on olive oil emulsion at pH 12 at 60 C. Bars indicate standard deviation. Environmental Progress & Sustainable Energy DOI 10.1002/ep 3 120 Immobilized 4°C substrate (Figure 4C). The maximum activity of both enzyme Immobilized 25°C forms was reached at 60 C. Interestingly, the decrease in the activity of the immobilized form was less compared to that of the free form above 60 C. At 78 C, only 10% of the activity of 100 Free 4°C Free 25°C free lipase was observed, compared to ~62% for the immobi- Residual lipase activity (%) 80 lized lipase. These results conﬁrmed that CaCO3 immobiliza- tion confers good heat resistance on S. aureus lipase. This phenomenon was also described for lipases of R. oryzae and 60 C. rugosa immobilized on CaCO3 [11,12]. However, in another study, the optimum temperature of immobilized Bacillus sp. lipase on HP-20 beads shifted from 60 to 65 C , while 40 Yang and Rhee observed a signiﬁcant change in optimum reaction temperature from 37 C (free lipase) to 50 C (immo- 20 bilized lipase). Likewise, Montero et al. reported that the optimal temperature of the C. rugosa lipase shifted slightly from 45 to 50 C when immobilized on Accurel. 0 Relatively high thermal stability of an enzyme is a desir- 0 10 20 40 50 60 90 120 able and attractive property for a variety of industrial applica- Days tions. To explore its potential applications, the tolerance of the immobilized lipase towards high temperatures was Figure 3. Activity retention (%) of free and immobilized checked. Free S. aureus lipase was only 10% active after a S. aureus lipases at 4 C and 25 C. Data are means of 1-h incubation at 80 C while the immobilized lipase exhib- triplicate determinations standard deviation. ited much better thermostability and retained 70% of its origi- nal activity at the same conditions (Figure 4D). Several studies reported a substantial loss of lipase activity at temper- on chitosan was slightly lower than that obtained for the aque- atures higher than 50 C. Ghamgui et al. observed that ous lipase (7.0). This pH shift towards an acidic value the free R. oryzae enzyme was totally inactivated upon incu- upon immobilization might be because of secondary interac- bation for 24 h at 50 C, while the immobilized lipase on tions between the matrix and the enzyme. CaCO3 retained 67% of its initial activity. Likewise, C. rugosa Conversely, the pH stability proﬁle shown in Figure 4B lipase immobilized on styrene-divinyl-benzene displayed a indicated that CaCO3 adsorption signiﬁcantly improved residual activity of 50% after heat treatment at 60 C for 1 h S. aureus lipase stability up to 58% in pHs ranging from 6.0 to while the free enzyme almost completely lost its activity 11.0 after incubation for 48 h (Figure 4B). under the same conditions. Here, we observed that the The effect of temperature on the activities of immobilized immobilization process tended to make S. aureus lipase more and free lipases was investigated using olive oil emulsion as stable. (A) (B) 100 Free Residual lipase activity (%) Immobilized Residual lipase activity (%) 80 80 60 60 40 40 Free Immobilized 20 20 0 0 6 8 10 12 14 0 5 10 15 pH pH (C) (D) 120 120 Residual lipase activity (%) Residual lipase activity (%) 100 100 80 80 60 60 40 40 Free Free 20 20 Immobilized Immobilized 0 0 0 20 40 60 80 0 20 40 60 80 100 Temperature (°C) Temperature (°C) Figure 4. pH effect on free and immobilized S. aureus lipases activity (A) and stability (B). Stability was analyzed after preincubating the pure enzyme for 48 h in different buffer solutions at various pH ranging from 2 to 13. Temperature effect on enzyme activity (C) and stability (D) of free and immobilized S. aureus lipases. For determining temperature stability, enzymes were preincubated at different temperatures for 1 h and the activity was measured under the standard conditions. All experiments were repeated at least three times. Bars indicate standard deviation. 4 Environmental Progress & Sustainable Energy DOI 10.1002/ep Table 2. Effect of various metal ions and agents on the free and the immobilized S. aureus lipase activity. Residual activity (%)b a Substance Concentration (mM) Free lipase Immobilized lipase β-mercaptoethanol 5 5 0.7 40 0.7 EDTA 5 35.2 1.1 82 3.1 PMSF 2 31.5 2.4 61.5 1.7 SDS 5 46.7 3.2 86.5 2.5 Mg2+ 2 110 2.5 129 1.5 Zn2+ 2 110 2.7 136 3.1 Mn2+ 2 100 1.5 140 1.8 Cu2+ 2 100 2 135 2.7 Ba2+ 2 95 1.9 95 0.6 Cd2+ 2 60 3 98 0.5 Fe2+ 2 65 2.5 92 1.3 Co2+ 2 50 4.5 87 2.4 Values represent the mean of three replicates standard deviation. a Concentration in the preincubation mixture. b The activity is expressed as a percentage of the activity of untreated control. Effect of Inhibitors and Activators was reduced from 95% to 60% for β-mercaptoethanol, from 69% The effect of different metal ions and organic compounds as to 39% for PMSF, from 53% to 14% for SDS, from 65% to 18% possible inhibitors and activators on the CaCO3-immobilized for EDTA, and from 56% to 13% for Co2+. Furthermore, in con- and the free lipases was performed under the same conditions. trast to free enzyme, the addition of 2 mM Cd2+ or Fe2+ had no The free enzyme was almost totally inhibited by signiﬁcant effect on the immobilized lipase, whereas 2 mM Mg2 β-mercaptoethanol and to certain extent with EDTA, PMSF, and + , Zn2+, Mn2+, and Cu2+ immediately increased activity from SDS (Table 2). Additionally, only Cd2+, Co2+, and Fe2+ inhibited 29 to 40% compared to the control. more than 35% of the enzyme activity with Co2+ being the stron- gest inhibitor (50% inhibition). In contrast, lipase retained its full activity in the presence of Cu2+, Zn2+, and Mn2+ (Table 2). Inter- Hydrolysis Performance of Immobilized and Free Lipase estingly, CaCO3 adsorption conferred tolerance, compared to Hydrolysis of Commercial Oils free S. aureus lipase, against inhibition by β-mercaptoethanol, Five different types of substrates (coconut oil, corn seed PMSF, EDTA, SDS, and Co2+. Inhibition of immobilized enzyme oil, olive oil, palm oil, and sunﬂower oil) were used to Table 3. Fatty acid compositions (%) of ﬁve commercial edible oils. Fatty acid Common name Coconut Olive Sunﬂower Corn Palm C8 Caprylic 5.98 0.18 – – – 0.03 0.01 C10 Capric 5.55 0.12 – – 0.01 0.01 0.02 0.01 C12 Lauric 48.7 0.82 – – 0.02 0.01 0.20 0.01 C14 Myristic 19.8 0.13 0.01 0.02 0.07 0.02 0.06 0.02 0.86 0.07 C15 Pentadedecylic 0.01 0.01 0.01 0.01 0.02 0.01 0.02 0.01 0.04 0.01 C16 Palmitic 8.46 0.31 17.00 0.41 6.30 0.56 12.6 0.23 37.1 0.77 C17 Margaric 0.02 0.01 0.05 0.01 0.02 0.01 0.08 0.02 0.08 0.01 C18 Stearic 2.63 0.05 2.34 0.11 4.04 0.23 1.88 0.21 3.76 0.23 C20 Arachidic 0.06 0.02 0.38 0.05 0.26 0.04 0.38 0.04 0.35 0.05 C22 Behenic 0.02 0.01 0.12 0.04 0.65 0.07 – 0.06 0.01 C24 Lignoceric – 0.05 0.02 0.02 0.01 – – C16:1ω9 – – 0.08 0.02 0.02 0.01 0.03 0.01 0.03 0.01 C16:1ω7 Palmitoleic 0.01 0.01 2.15 .0.11 0.12 0.02 0.17 0.03 0.16 0.02 C17:1 ω7 – 0.01 0.01 0.08 0.02 0.11 0.02 0.12 0.02 0.03 0.01 C18:1 ω9 Oleic 6.78 0.21 56.66 1.36 35.14 0.13 25.4 0.25 45.6 0.41 C18:1 ω7 Cis -vaccenic 0.07 0.02 2.95 0.08 0.77 0.05 0.60 0.10 0.81 0.04 C20:1ω9 Gondoic 0.03 0.01 0.21 0.06 0.16 0.07 0.24 0.04 0.16 0.04 C22:1 ω9 Erucic 0.02 0.01 – – – – C18:2 ω6 Linoleic 1.73 0.09 17.17 0.47 52.06 0.88 57.5 0.86 10.4 0.25 C18:3 ω3 Linolenic 0.01 0.01 0.69 0.05 0.24 0.04 0.85 0.05 0.23 0.11 SFA – 91.23 19.95 11.38 15.05 42.50 MUFA – 6.91 62.13 36.32 26.56 46.79 PUFA – 1.74 17.86 52.30 58.35 10.63 SFA: saturated fatty acids, MUFA: monounsaturated fatty acids, PUFA: polyunsaturated fatty acids. Fatty acid methyl esters (FAMEs) were prepared from the ﬁve oil samples according to the laboratory protocol described by Nehdi et al.. Environmental Progress & Sustainable Energy DOI 10.1002/ep 5 140 not related to the chain length speciﬁcity of S. aureus lipase, Immobilized but were likely related to the accessibility of ester band to the 120 Free enzyme. Relative lipase activity (%) 100 Mojovic et al. and Ghamgui et al. described the improved catalytic activity of C. rugosa and R. oryzae lipases 80 using palm or olive oil as the substrate upon immobilization of the lipases to SGE-A2–94 copolymer or CaCO3, respectively. 60 The immobilized lipase forms diverse interactions with the support through hydrophobic or hydrogen linkages stabilizing 40 the enzyme. 20 Bioremediation of Wadi Hanifah Water by Free and 0 Immobilized S. aureus Lipase Olive oil Palm oil Corn seed oil Sunflower oil Coconut oil A preliminary experiment used S. aureus ALA1 lipase to Figure 5. Effect of substrate type on free and CaCO3 treat Wadi Hanifah water. The general properties of the valley immobilized lipases activity. Hydrolysis reaction was carried water including TOC, COD, and lipid content were determined out at pH 12 at 60 C. Data are means of triplicate (Table 4). The initial TOC and COD content was 205 14 mg L−1 determinations standard deviation. and 1,950 67 mg L−1, respectively. After the treatment with the immobilized S. aureus lipase, the chloride content gradu- ally increased up to 835 35 mg L−1 on day 10 of incubation. The maximum reduction in COD (10 1.7 mg L−1) was observed on day 9 after the treatment with the CaCO3- determine the hydrolysis efﬁciency of CaCO3-immobilized immobilized S. aureus lipase. However, the TOC and COD and free lipases. The fatty acid proﬁles of commercial oils content of the treated water did not exceed 504 24 and (Table 3) revealed that coconut oil contained a high propor- 945 110 mg L−1, respectively, using the free lipase. The lipid tion of saturated fatty acid (91.23%) compared to palm oil content was also analyzed. The initial lipid content (42.5%), olive oil (19.95%), corn oil (15.05%), and sunﬂower (15,500 546 mg L−1) was decreased to 15 2.9 mg L−1 on oil (11.38%). Corn and sunﬂower oils contained the highest day 10 by the immobilized S. aureus lipase. In contrast, no sig- polyunsaturated fatty acid, speciﬁcally linoleic acid niﬁcant decrease (13,067 358 mg L−1) was recorded in the (57.5 0.86 and 52.06 0.88, respectively) (Table 3). The case of the free lipase (Table 4). Similar studies were carried hydrolysis activities of free and immobilized S. aureus lipases out on wastewaters hydrolyzed using free and immobilized for the tested commercial oil are presented in Figure 5. The lipases [6,13,41]. However, some studies were continued for two forms of lipase efﬁciently hydrolyzed all the oils, indicat- more than 10 days of incubation for the reduction of COD and ing the potential value of free or immobilized lipase in the lipid content. Other studies were performed with mixed biodegradation of oil-rich wastewater. There was no signiﬁ- cultures to facilitate the bioremediation process [42,43]. The cant difference in the activities of the immobilized and free collective results indicated the potential value of CaCO3- lipases using the same substrate, with the exception of corn immobilized S. aureus lipase as a biocatalyst of hydrolysis. seed oil. For the latter, the activity of immobilized lipase was greater (Figure 5). Among the substrates investigated, the highest activity of the free lipase was recorded when olive oil Repeated Use of Immobilized Lipase was used as the substrate. The CaCO3-immobilized lipase dis- Commercially lipases are generally expensive and tech- played the highest activity against corn seed oil followed by niques to extend their active life have been advantageously olive, coconut, sunﬂower, and palm oils. When the percent- developed. The main advantage of immobilization, especially ages of hydrolysis were compared with olive oil (100%), the for industrial and biotechnological applications, is the reduced activities were 78.2% for palm oil, 83.5% for sunﬂower oil, cost because the lipase can be used repeatedly. Pronounced 87% for coconut oil, and 125.76% for corn seed oil (Figure 5). stability could make the immobilized lipase more attractive The hydrolytic activities of free and immobilized lipases on and advantageous than the free form. However, we sunﬂower and coconut oil were very similar despite the dif- observed that the repetitive use of the immobilized lipase led ference in the fatty acid proﬁles (Table 3). Sunﬂower oil is to its deactivation. The enzyme was recovered after each reac- mainly composed of long chain fatty acid (linoleic acid, tion and reused for subsequent hydrolysis cycles. After each 52.06% 0.88%) and coconut oil is enriched in short chain cycle and before its reuse, the lipase was ﬁltered out, rinsed, fatty acid (lauric acid, 48.7% 0.82%). These ﬁndings were and ﬁnally dried. Although the activity of the immobilized Table 4. Bioremediation of Wadi Hanifah water by free and immobilized S. aureus ALA1 lipase. Lipid content (mg L−1) Total organic chloride (mg L−1) COD (mg L−1) Incubation time (days) Free Immobilized Free Immobilized Free Immobilized 0 15,500 546 15,500 546 205 14 205 14 1,950 76 1,950 76 1 15,200 678 10,750 257 234 11 295 21 1,805 87 1,450 120 2 14,8569 679 7,465 421 287 23 378 32 1,725 54 1,146 45 3 14,195 435 3,008 237 321 13 467 17.9 1,543 35 795 79 4 13,864 389 1,587 176 350 9.7 585 25 1,439 65 354 53 5 13,784 542 692 79 387 13.5 645 32 1,354 38 120 23 6 13,550 356 304 45 413 27.9 695 43 1,207 87 79 15 7 13,278 567 105 21 434 36 754 30 1,142 45 47 5.8 8 13,143 786 57 7 470 25 780 51 1,087 53 25 3.5 9 13,073 563 25 3.5 489 15.7 820 32 1,006 29 10 1.7 10 13,067 358 15 2.9 504 24 835 35 954 110 11 1.2 6 Environmental Progress & Sustainable Energy DOI 10.1002/ep 120 by lipolytic fungi as a bioaugmentation application, Envi- ronmental Technology, 11, 1–11. 3. Nanayakkara, C.M., Witharana, A. (2015). In S. Singh & Residual lipase activity (%) 100 K. Srivastava (Eds.), Handbook of research on uncovering 80 new methods for ecosystem management through biore- mediation (pp. 222–254), Pennsylvania (USA): IGI Global. 60 4. Azhdarpoor, A., Mortazavi, B., & Moussavi, G. (2014). Oily wastewaters treatment using Pseudomonas sp. isolated 40 from the compost fertilizer, J Environ Health Sci Eng, 12, 77. 20 5. Pintor, A.M., Vilar, V.J., Botelho, C.M., & Boaventura, R.A. (2016). Oil and grease removal from wastewaters: Sorption treatment as an alternative to state-of-the-art technologies, 0 0 5 10 15 20 A critical review. Chem Eng J, 297, 229–255. No. of cycle 6. Masse, L., Masse, D.I., & Kennedy, K.J. (2003). Effect of hydrolysis pretreatment on fat degradation during anaero- Figure 6. Effect of repeated use of immobilized S. aureus bic digestion of slaughterhouse wastewater, Process Bio- lipase on residual activity. Data are means of triplicate chemistry, 38, 1365–1372. determinations standard deviation. 7. Jeganathan, J., Bassi, A., & Nakhla, G. (2006). Pre-treatment of high oil and grease pet food industrial wastewaters using immobilized lipase hydrolyzation, Jour- enzyme began to decrease after ﬁve cycles, more than 55% of nal of Hazardous Materials, 137, 121–128. its original activity was retained even after 10 cycles, which is 8. Mohamad, N.R., Che Marzuki, N.H., Buang, N.A., comparable with the literature [4,11,43] (Figure 6). In contrast, Huyop, F., & Abdel Wahab, R. (2015). An overview of the activity loss was critical after 15 cycles showing that the technologies for immobilization of enzymes and surface immobilized enzyme activities decreased with continued use. analysis techniques for immobilized enzymes, Biotechnol- This is probably due to blockage of substrate/product and/or ogy Biotechnological Equipment, 29, 205–220. lipase leakage from the support material. 9. Selvam, K., & Vishnupriya, B. (2013). Partial puriﬁcation of lipase from Streptomyces variabilis NGP3 and its appli- CONCLUSION cation in bioremediation of waste water, International This study is the ﬁrst report of the evaluation of CaCO3- Journal of Pharmaceutical Sciences and Research, 4, immobilized lipase from S. aureus ALA1strain in the bio- 4281–4289. remediation of lipid-rich Wadi Hanifah water. The COD values 10. Minovska, V., Winkelhausen, E., & Kuzmanova, S. (2005). and lipid content of the treated water samples were decreased Lipase immobilized by different techniques on various from ~1,950 and 15,500 mg L−1, respectively, to

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