Production of Dry Extract Lipase from Pseudomonas Aeruginosa by the Submerged Fermentation Method in Palm Oil Mill Effluent

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 4^oC.

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 30^oC. 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 30^oC 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.

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