Hermansyah, H., Maresya, A., Putri, D.N., Sahlan, M., Meyer, M., 2018. Production of Dry Extract Lipase from Pseudomonas Aeruginosa by the Submerged Fermentation Method in Palm Oil Mill Effluent. International Journal of Technology. Volume 9(2), pp. 325-334
|Heri Hermansyah||Chemical Engineering Department, Universitas Indonesia, Depok, Indonesia|
|Ambar Maresya||Chemical Engineering Department, Universitas Indonesia, Depok, Indonesia|
|Dwini Normayulisa Putri||Chemical Engineering Department, Universitas Indonesia, Depok, Indonesia|
|Muhamad Sahlan||Universitas Indonesia|
|Michel Meyer||Université de Toulouse|
Palm Oil Mill Effluent (POME) is an agro-industrial waste product with high availability and which contains high quantities of organic compounds that are necessary for microbial growth. Cultures of Pseudomonas aeruginosa were grown in POME to produce lipase using the submerged fermentation method. The objective of this study is to obtain the optimum value of lipase activity produced by the cultures of Pseudomonas aeruginosa using POME as the substrate through the submerged fermentation method and to obtain the dry extract of lipase. In the study, the one factor at a time (OFAT) method was applied, which allowed observation of the effect of inoculum and additional nutrient concentrations, such as Ca2+ ion, olive oil, peptone and Tween 80, on the activity of lipase. These factors were investigated in shake flask fermentation at 30°C over 96 hours. The activity unit of lipase was determined by the titrimetric reaction of olive oil hydrolysis using crude lipase. The optimum value of the lipase activity unit (1.327 U/mL) was gained when 3% (v/v) of inoculum, 4 mM of Ca2+ ion, 0.4% (v/v) of olive oil, 0.9% (m/v) of peptone, and 0.9% of Tween 80 were added into the medium. Crude lipase was then dried using a spray dryer. Subsequently, 15.643 g of dry extract lipase was obtained from 500 mL of cell free supernatant. In further research, the lipase activity assay would be better achieved using the p-nitrophenyl palmitate hydrolysis method and examined by a spectrophotometer.
Activity unit; Lipase; Palm oil mill effluent; Submerged fermentation
Lipase is an enzyme which catalyzes the hydrolysis reaction of triglycerides into fatty acids and several types of glycerol, including di-acylglycerol and mono-acylglycerol (Mahadik et al., 2004). Recently, lipase has attracted significant interest for commercial production, as it has several advantages, including the fact that no cofactor is required, it is active in the interfaces of organic compounds, and facilitates a wide range of substrates (Hernández-Rodríguez et al., 2009). Therefore, lipase has many practical applications in industry, including as a biocatalyst for biodiesel synthesis, in bio-detergents, fine chemicals, waste water treatment, food additives, pharmaceuticals, leather processing and biomedical assays (Salihu et al., 2012).
At present, the utilization of extracellular enzymatic activity in several microorganisms has become a preferred approach because of its potential as a biological source of industrially economic enzymes (Abd-Elhalem et al., 2015). There are a number of lipases produced by microorganisms, such as fungi including Candida, Geotrichum and Rhizopus, and bacteria including Bacillus, Pseudomonas, Burkholderia, Staphylococcus and Streptomyces (Treichel et al., 2010). Among these strains, the Pseudomonas lipase is the most important, since it has better stability, selectivity, and a wide range of substrate specificity (Bose & Keharia, 2013). Furthermore, lipase from Pseudomonas aeruginosa has been extensively manufactured and used in organosynthetic reactions (Karadzic et al., 2006).
Previous lipase production from Pseudomonas aeruginosa has been studied using Pseudomonas aeruginosa san-ai (Karadzic et al., 2006), Pseudomonas aeruginosa AAU2 (Bose & Keharia, 2013), and Pseudomonas aeruginosa LX1 (Ji et al., 2010). Lipase from Pseudomonas aeruginosa san-ai has been isolated and purified from the medium containing mineral cutting oil with pH 10, obtaining excellent properties of lipase, including stability and activity in organic solvents (Karadzic et al., 2006). Besides, lipase from Pseudomonas aeruginosa AAU2 has also been produced by the submerged fermentation method using Jatropha SeedCake (JSC) as the carbon source, obtaining solvent tolerant lipase with a 11.4-fold higher enzyme yield in the optimum condition (Bose & Keharia, 2013). Another organic solvent tolerant lipase has also been produced from Pseudomonas aeruginosa LX1 (Ji et al., 2010). From its activity in transesterification reactions, lipase from Pseudomonas aeruginosa LX1 is considered to have potential for biodiesel production.
However, in the production of microbial lipase, production costs are a problem due to the high cost of the substrates. In order to lower these costs, agro-industrial waste can be used as the fermentation substrates. The utilization of agro-industrial waste would be a solution to the problem of disposal (Bose & Keharia, 2013). Palm Oil Mill Effluent (POME) is an agro-industrial waste product with high availability in Indonesia. Organic content, such as carbohydrates, protein, nitrogenous compounds, lipids and minerals, are present in POME (Wu et al., 2009). These contents are suitable for microbial growth and lipase production. Since POME is available in liquid form, application of submerged fermentation is a suitable method. The utilization of POME as a medium has been made by Salihu using Candida cylindracea with the submerged fermentation method, giving satisfactory results (Salihu et al., 2011). In addition, POME was also used as the basal medium by Wu et al. (2006) for protease production from wild type Aspergillus niger with the submerged fermentation method.
Submerged fermentation is one of the fermentation methods that have been used by industry for example in enzyme production, due to the better control of the parameters that affect the yield of the product (Hansen et al., 2015); temperature, pH and agitation are well-established for scaling the processes for industrial production capacity (Hansen et al., 2015). Lipase produced from submerged fermentation can be dried to obtain dry extract lipase. In a previous study, dry extract lipase was obtained from submerged fermentation by Suci et al. (2018) using Bacillus subtilis as the source and waste cooking oil as the basal medium. Dry extract lipase is more durable than liquid lipase and can be stored for a long time (Utami et al., 2017).
2.1. Sample Collection
POME was collected in clean containers from a palm oil company at Bengkulu, Indonesia, and then stored at 4oC.
2.2. Inoculum Preparation and Microbial Culture
Pseudomonas aeruginosa B2290 was purchased from Indonesian Culture Collection (InaCC). The strain was then grown on Nutrient Agar (NA) plates at 30?35°C for 1 day. The isolate was maintained and preserved at 4°C. Preparation of the inoculum was made by suspending Pseudomonas aeruginosa from nutrient agar in 50 ml of sterile Luria Bertani broth using a sterile inoculating loop, and incubating it using a water shaker bath at 170 rpm and 30°C for 24 hours. The mixture was then used as the inoculum in further experiments.
2.3. Profile of Lipase Production
Inoculum of Pseudomonas aeruginosa was inoculated to an Erlenmeyer flask containing 100 ml of sterilized POME as the basal medium, and other nutrients including olive oil (0.2%, v/v), peptone (0.5%, w/v), CaCl2.2H2O (10mM) and Tween 80 (0.7%, v/v), The mixture was then incubated in a water shaker bath at 30oC. 5 ml aliquots were taken after 0, 12, 24, 36, 48, 60, 72, 84, 96 and 108 h of incubation in order to analyze the bacterial growth using a spectrophotometer (660 nm) and the lipase activity using lipase activity assay.
2.4. Optimization of the Medium for Lipase Production
The optimization of the medium was made using the one factor at a time (OFAT) method. In this way, the experimental factors were varied one at a time, while the other factors were kept constant. The medium for the lipase production was prepared using sterilized POME as the basal medium, which contained olive oil (0.1–0.2%, v/v), peptone (0.3–1.1%, w/v), CaCl2.2H2O (4.0–12.0 mM) and Tween 80 (0.3–1.1%, v/v). The inoculum (1.0–5.0%, v/v) was then added to the medium and incubated using a water shaker bath at 170 rpm and 30oC for 96 hours. After 96 hours of incubation, the culture was then centrifuged at room temperature, at 4000 rpm for 45 minutes, to obtain the cell-free supernatant. This was then filtered and assayed for the lipase activity calculation.
2.5. Lipase Activity Assay
The lipase activity was assayed by alkali titration using olive oil as the substrate according to work done by Pinhiero et al (2008) with several modifications in the titration method. Olive oil (10% m/v) was prepared and emulsified with PVA (5% m/v) in a 50 mM sodium phosphate buffer with a pH of 7.5. A crude enzyme sample of 2 mL was added into 18 mL of the emulsion and then incubated in a shaker for 15 minutes at 37°C and 150 rpm. After the reaction was stopped, 20 mL of 95% ethanol solution (1:1 v/v) was added to extract the fatty acids. Thereafter, three drops of 1% phenolphthalein were added as the titration indicator. Titration with 0.05 M NaOH then occurred, until the colour of the sample solution turned light violet, in order to calculate the amount of fatty acids liberated. The blanks for titration were run in similar steps, but the sample was added after the addition of ethanol. The lipase activity assay was performed in duplicate.
A unit activity of lipase was defined as the amount of enzyme which liberated 1 ?mol of fatty acids per minute under the assay conditions. The protein concentration was estimated by following the Lowry method with BSA (fraction V) as the standard.
2.6. Production of Dry Extract Lipase
Scale-up production was conducted in a 30-L bioreactor with two six-flat-blade impellers. The bioreactor was filled with 20-L of POME as the basal medium, containing the optimum concentrations of olive oil, peptone, CaCl2.2H2O and Tween 80 obtained from the OFAT method. The reactor was sterilized in situ and inoculated with the optimum concentration of prepared Pseudomonas aeruginosa inoculum, as described above. The fermentation was run at 30°C for 96 hours. The fermentation temperature was maintained using a digital control system attached to the bioreactor.
The aliquots were taken after 96 hours and then centrifuged at room temperature, at 4000 rpm for 45 minutes. The cell-free supernatant was filtered and assayed for lipase activity. It was then collected and dried with a spray dryer at Center for Postharvest Research and Development (Balai Besar Penelitian dan Pengembangan Pascapanen) Bogor, with an inlet temperature of 150°C and outlet temperature of 80°C. Before being dried, skimmed milk powder (12%, w/v) was added to the cell-free supernatant. The dry extract lipase was then assayed for the lipase activity calculation.
The activity unit of lipase from Pseudomonas aeruginosa using POME as the basal medium reached a maximum value of 1.327 U/mL from the crude form. The optimum values of the lipase activity unit were gained when 3% (v/v) of inoculum, 0.9% (m/v) of peptone, 0.4% (v/v) of olive oil, 4 mM of Ca2+ ions, and 0.9% of Tween 80 were added into the medium and fermented for 96 hours. The dry extracellular lipase obtained had an activity unit of 28.5 U/g, with a specific activity of 2.417 U/g proteins. The utilization of POME as an alternative medium would result in a considerable reduction in the cost of lipase production. The values of the lipase activities would achieve better results if the experiment used the hydrolysis reaction of p-nitrophenyl palmitate and examination by spectrophotometer.
The authors are grateful for the research support provided by Ministry
of Research, Technology, and Higher Education through International Research
Collaboration Grant Program and for the publication support provided by the
United States Agency for International Development (USAID) through the
Sustainable Higher Education Research Alliance (SHERA) Program for Universitas
Indonesia’s Scientific Modeling, Application, Research and Training for
City-centered Innovation and Technology (SMART CITY) Project, Grant
#AID-497-A-1600004, Sub Grant #IIE-00000078-UI-1.
Abd-Elhalem, B.T., El-Sawy, M., Gamal, R.F., Abou-Taleb, K.A., 2015. Production of Amylases from Bacillus amyloliquefaciens under Submerged Fermentation using Some Agro-industrial By-products. Annals of Agricultural Sciences, Volume 60(2), pp. 193–202
Ali, C.H., Mbading, S.M., Liu, J.-F., Yang, S.-Z., Gu, J.-D., Mu, B.-Z., 2015. Significant Enhancement of Pseudomonas aeruginosa FW_SH-lipase Production using Response Surface Methodology and Analysis of its Hydrolysis Capability. Journal of the Taiwan Institute of Chemical Engineers, Volume 52, pp. 7–13
Bose, A., Keharia, H., 2013. Production, Characterization and Applications of Organic Solvent Tolerant Lipase by Pseudomonas aeruginosa AAU2. Biocatalysis and Agricultural Biotechnology, Volume 2(3), pp. 255–266
Cadirci, B.H., Yasa, I., 2010. An Organic Solvents Tolerant and Thermotolerant Lipase from Pseudomonas fluorescens P2. Journal of Molecular Catalysis B: Enzymatic, Volume 64(3–4), pp. 155–161
Dalmau, E., Montesinos, J.L., Lotti, M., Casas, C., 2000. Effect of Different Carbon Sources on Lipase Production by Candida rugosa. Enzyme and Microbial Technology, Volume 26(9–10), pp. 657–663
Gonçalves, F.A.G., Colen, G., Takahashi, J.A. 2014. Yarrowia lipolytica and Its Multiple Applications in the Biotechnological Industry. The Scientific World Journal, Volume 2014, pp. 1–14
Gullo, M., Verzelloni, E., Canonico, M., 2014. Aerobic Submerged Fermentation by Acetic Acid Bacteria for Vinegar Production: Process and Biotechnological Aspects. Process Biochemistry, Volume 49(10), pp. 1571–1579
Guragain, M., King, M.M., Franklin, M.J., 2016. The Pseudomonas aeruginosa PAO1 Two-component Regulator CarSR Regulates Calcium Homeostasis and Calcium-induced Virulence Factor Production through Its Regulatory Targets CarO and CarP. Journal of Bacteriology, Volume 198(6), pp. 951–963
Hansen, G.H., Lübeck, M., Frisvad, J.C., Lübeck, P.S., Andersen, B., 2015. Production of Cellulolytic Enzymes from Ascomycetes: Comparison of Solid State and Submerged Fermentation. Process Biochemistry, Volume 50(9), pp. 1327–1341
Hernández-Rodríguez, B., Córdova, J., Bárzana, E., Favela-Torres, E., 2009. Effects of Organic Solvents on Activity and Stability of Lipases Produced by Thermotolerant Fungi in Solid-state Fermentation. Journal of Molecular Catalyst B: Enzymatic. Volume 61(3–4), pp. 136–142
Ji, Q., Xiao, S., He, B., Liu, X., 2010. Purification and Characterization of an Organic Solvent-tolerant Lipase from Pseudomonas aeruginosa LX1 and its Application for Biodiesel Production. Journal of Molecular Catalysis B: Enzymatic, Volume 66(3–4), pp. 264–269
Karadzic, I., Masui, A., Zivkovic, L.I., Fujiwara, N., 2006. Purification and Characterization of an Alkaline Lipase from Pseudomonas aeruginosa Isolated from Putrid Mineral Cutting Oil as Component of Metalworking Fluid. Journal of Bioscience and Bioengineering, Volume 102(2), pp. 82–89
Li, C.Y., Cheng, C.Y., Chen, T.L., 2004. Fed-batch Production of Lipase by Acinetobacter radioresistens using Tween 80 as the Carbon Source. Biochemical Engineering Journal, Volume 19(1), pp. 25–31
Mahadik, N.D., Bastawde, K.B., Puntambekar, U.S., Khire, J.M., Gokhale, D.V., 2004. Production of Acidic Lipase by a Mutant of Aspergillus niger NCIM 1207 in Submerged Fermentation. Process Biochemistry, Volume 39(12), pp. 2031–2034
Papagora, C., Roukas, T., Kotzekidou, P., 2013. Optimization of Extracellular Lipase Production by Debaryomyces hansenii Isolates from Dry-salted Olives using Response Surface Methodology. Food and Bioproducts Processing, Volume 91(4), pp. 413–420
Pinhiero, T.D., Menoncin, S., Domingues, N.M., Oliviera, D.D., Treichel, H., Di Luccio, M., Freire, D.M., 2008. Production and Partial Characterization of Lipase from Penicillium verrucosum Obtained by Submerged Fermentation of Conventional and Industrial Media. Ciência e Tecnologia de Alimentos, Volume 28(2), pp. 444–450
Rajendran, A., Thangavelu, V., 2012. Optimization and Modeling of Process Parameters for Lipase Production by Bacillus brevis. Food Bioprocess Technology, Volume 5(1), pp. 310–322
Reddy, L., Wee, Y., Yun, J., Ryu, H., 2008. Optimization of Alkaline Protease Production by Batch Culture of Bacillus sp. RKY3 through Plackett-Burman and Response Surface Methodological Approaches. Bioresource Technology, Volume 99(7), pp. 2242–2249
Salihu, A., Alam, M.Z., AbdulKarim, M.I., Salleh, H.M., 2011. Optimization of Lipase Production by Candida cylindracea in Palm Oil Mill Effluent based Medium using Statistical Experimental Design. Journal of Molecular Catalysis B: Enzymatic, Volume 69(1–2), pp. 66–73
Salihu, A., Alam, M.Z., AbdulKarim, M.I., Salleh, H.M., 2012. Lipase Production: An Insight in the Utilization of Renewable Agricultural Residues. Resources, Conservation and Recycling, Volume 58, pp. 36–44
Sharma, R., Chisti, Y., Banerjee, U.C., 2001. Production, Purification, Characterization, and Applications of Lipases. Biotechnology Advances, Volume 19(8), pp.627–662
Suci, M., Arbianti, R., Hermansyah, H., 2018. Lipase Production from Bacillus subtilis with Submerged Fermentation using Waste Cooking Oil. IOP Conference Series: Earth and Environmental Science, Volume 105, pp. 1–6
Treichel, H., Oliveira, D., Mazutti, M.A., Luccio, M.D., Oliveira, J.V., 2010. A Review on Microbial Lipases Production. Food and Bioprocess Technology, Volume 3(2), pp. 182–196
Utami, T.S., Hariyani, I., Gandhi, A., Hermansyah, H., 2017. Production of Dry Extract Extracellular Lipase from Aspergillus niger by Solid State Fermentation Method to Catalyze Biodiesel Synthesis. Energy Procedia, Volume 136, pp. 41–46
Vasiee, A., Behbahani, B.A., Yazdi, F.T., Moradi, S., 2016. Optimization of the Production Conditions of the Lipase Produced by Bacillus cereus from Rice Flour through Plackett-Burman Design (PBD) and Response Surface Methodology (RSM). Microbial Pathogenesis, Volume 101, pp. 36–43
Wu, T.Y., Mohammad, A.W., Jahim, J.Md., Anuar, N., 2006. Investigations on Protease Production by a Wild-type Aspergillus terreus Strain using Diluted Retentate of Pre-filtered Palm Oil Mill Effluent (POME) as Substrate. Enzyme and Microbial Technology, Volume 39(6), pp. 1223–1229
Wu, T.Y., Mohammad, A.W., Jahim, J.Md., Anuar, N., 2009. A Holistic Approach to Managing Palm Oil Mill Effluent (POME): Biotechnological Advances in the Sustainable Reuse of POME. Biotechnology Advances, Volume 27(1), pp. 40–52
Yates, G.T., Smotzer, T., 2007. On the Lag Phase and Initial Decline of Microbial Growth Curves. Journal of Theoretical Biology, Volume 244(3), pp. 511–517