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Improved biodiesel from palm oil using lipase immobilized calcium alginate and Irvingia gabonensis matrices
Beni-Suef University Journal of Basic and Applied Sciences volume 9, Article number: 59 (2020)
Lipase is an important microbial enzyme and biocatalyst in biodiesel production. The study investigated fuel properties of biodiesel produced from palm oil (PO) using lipase immobilized on Irvingia gabonensis and calcium alginate.
Biodiesel yield from PO using free and immobilized lipases was highest at 35 °C and pH 7, with product yield using calcium alginate-immobilized lipase, CAIL (94.42, 96.9%) higher than using Irvingia gabonensis-immobilized lipase, IGIL (92.54, 95.8%). Biodiesel produced using immobilized lipases had similar pour point, cloud point, and kinematic viscosity, and they possessed improved fuel properties compared to free lipase biodiesel in terms of densities at 15 °C and flash point. Pour points, flash point, and kinematic viscosity of biodiesel produced using CAIL and IGIL met American and European Standards but density at 15 °C and cloud points are below both standards. CAIL and IGIL biodiesel had similar fatty acid methyl ester (FAME) compounds and consisted more of unsaturated fatty acids (hexadecanoate, 9-octadecenoate, octadecanoate, dodecanoate, and 9,12-octadeca-dienoate) than obtained in biodiesel from free lipase. IGIL and CAIL were re-used in 8 and 12 cycles respectively, with > 90% biodiesel yield achieved in four and 11 cycles.
The study showed that lipase immobilized on Irvingia gabenensis and calcium alginate and used in biodiesel production retained high enzyme activity and biodiesel yield in repeated cycles.
Biodiesel is a renewable, non-toxic, environmentally friendly, biologically produced diesel fuel alternative to substitute petroleum-based diesel [1,2,3]. It is produced via enzymatic transesterification of several types of feedstocks such as edible plant oils and animal fats, and in recent times, non-edible oils are also being explored for biodiesel production . The enzymes directly involved in transesterification for biodiesel production are lipases.
Lipases are versatile, industrially important enzymes cheaply produced from microorganisms and serve as biocatalysts in several industrial purposes including food processing technology, fats and oil processing, pharmaceuticals, and biodegradation [5,6,7]. Microbial lipase is also important as the most common source for biodiesel production by transesterification due to their ease of fermentation and purification steps .
Shortcomings of free lipases include slow reaction rate and high cost, therefore, the need for immobilization, while immobilized lipases are advantageous in biodiesel production due to easy product separation and glycerol recovery, potential for continuous production with improved reusability, and reduced cost of lipase production [9,10,11,12].
Various techniques and support matrices have been used in lipase immobilization for biodiesel production. This includes different forms of silica and silica gel, amberlite, sepabeads, sepharose, and resin supports formulated using adsorption, cross-linking and entrapment methods, etc. [13, 14].
Entrapment is a physical restriction of enzymes within a confined space or network . This immobilization technique permits movement of both substrate and product through the matrix and is applicable to a wide range of carriers and lipases for biodiesel production . Different hydrogels have been used as entrapment for lipase [17, 18], while the use of alginates is widely reported in recent times [15, 19,20,21].
Irvingia gabonensis seeds are thickening agents known to possess hydrocolloid property and have been prepared as immobilization matrix for both microbial cells and lipase [22, 23], therefore, the need to determine its possible use as matrix for lipase immobilization in biodiesel production.
In this study, we investigated comparative biodiesel production from palm oil using lipase entrapped in calcium alginate and Irvingia gabonensis matrices based on fuel properties.
Lipase-producing Aspergillus niger strain from previous study  was obtained from a Culture Collection Center. Palm oil (PO) was obtained locally from an oil palm industry, and all chemicals were of analar grade.
Lipase production and purification assay
Aspergillus niger spores produced lipase on rice bran medium via solid-state fermentation as previously described , and enzyme activity was assayed . Sodium phosphate buffer (50 mM; pH 8) was mixed with fermented medium in an orbital shaker operated for 2 h at 150 rpm and 28 °C to obtain crude enzyme. The crude lipase presentin filtrate was further purified by mixing with olive oil emulsion, mixture incubated at 50 °C for 30 min and reaction stopped with absolute ethanol. According to Eq. (1), one unit (U) of lipase activity is the amount of enzyme released from the emulsion substrate 1 μmole of fatty acid per milliliter per minute under specific assay condition.
Lipase was immobilized on both calcium alginate and Irvingia gabonensis as previously described [22, 25]. For immobilization on alginate, lipase was mixed with 2% v/v sodium alginate solution in equal ratio, and the mixture added drop wise into 0.2 M calcium chloride solution with continuous shaking at room temperature to form lipase-encapsulated calcium alginate beads. Lipase was immobilized on defatted Irvingia gabonensis seed powder of adequate gel strength (10% w/v) activated with 2.5% (w/v) glutaraldehyde solution. Beads were formed by adding enzyme-gel mixture drop wise into ethanolic-formaldehyde solution. Beads were washed severally with excess deionizing water and finally with sodium phosphate buffer (pH 7.5), dried, and their lipase activity assayed.
Optimization of environmental parameters on lipase immobilization
The effect of temperature on biodiesel production from palm oil using lipase immobilized on calcium alginate and Irvingia gabonensis was determined by carrying out transesterification between 25 and 45 °C at 5 °C intervals. Similarly, the acidity/alkalinity effect was investigated at pH range 5-9 with 0.5 pH changes considered.
Enzymatic production of biodiesel (fatty acid methyl ester) by immobilized lipase
Biodiesel production proceeded using the optimized transesterification reaction procedure previously described .
Transesterification reaction was carried out in a 250-ml conical flask on rotary orbital shaker as previously described . Briefly, immobilized lipase (2.5%) was added to 40 mL of PO and the mixture was supplemented with methanol (5 ml) in a starting molar ratio 1:1 to the oil, and reaction was completed with stepwise addition to methanol for a final 3:1 molar ratio. Reaction mixture was left for 48 h after which it was separated overnight using a separating funnel. Following separation, two layers were formed: the upper layer being the biodiesel product while glycerin settled at the bottom layer of the separating funnel.
The fatty acid methyl ester, FAME, yield, or biodiesel yield (% wt), relative to the amount of experimental oil used was calculated by comparing the weight of the upper layer biodiesel to the weight of the crude oil used as described in Eq. (2).
Characterization of biodiesel
Quality of biodiesel produced from palm kernel using lipase immobilized on calcium alginate and I. gabonensis beads was determined via fatty acid methyl ester (FAME) properties. This was analyzed as previously described  using gas chromatography system (Agilent Technologies 7890A model). Fuel properties determined included flash point (FC), cloud point (CP), pour point (PP), kinematic viscosity, and density at 15 °C of transesterified PO. These were compared with American (American Standard of Testing material, ASTM 6751–3) and European (European Union Standard, EN 14214) fuel standards. All properties were determined in triplicate experiments.
Effect of reusability of immobilized beads on yield in repeated batch cycles
The effect of reusability of immobilized beads on yield in repeated batch production of biodiesel was carried out. Immobilized lipase (2.5% v/v) beads were added to palm oil for esterification as previously described. After each batch of biodiesel production, beads were washed in excess distilled water before re-use.
Effect of temperature and pH on biodiesel production by immobilized lipase
Comparison of the effect of temperature on biodiesel production free lipase and lipase immobilized on both calcium alginate and Irvingia gabonensis was presented in Fig. 1. Biodiesel yield was optimum at 35 °C for free and immobilized lipases, while the highest yield was obtained when calcium alginate-immobilized lipase was used for esterification (94.42%) compared to free lipase (94.1%) and Irvingia gabonensis-immobilized lipase (92.54%). However, over 90% of biodiesel yield was produced when esterification proceeded with immobilized lipase at 40-45 °C while biodiesel yield between 73 and 81% was produced at similar temperatures by free lipase.
The effect of acidity or alkalinity on biodiesel production by free and immobilized lipases is described in Fig. 2. The highest biodiesel yield was produced at neutral pH 7 using free and immobilized lipases, and biodiesel yield was higher for immobilized lipases than free lipase. Furthermore, lipase immobilized on calcium alginate produced the highest biodiesel yield (96.9%) than Irvingia gabonensis-immobilized lipase (95.8%) and free lipase (93.14%). Similarly, over 90% of biodiesel yield was produced at pH 5-6 by immobilized lipases compared to free lipase (< 90%).
Fuel properties free and immobilized lipase
Using American and European biodiesel standards, the quality of fuel based on physicochemical fuel properties produced by immobilized lipases compared to free lipase are described in Table 1.
Biodiesel produced by immobilized lipases had improved fuel properties compared to that produced by free lipase in terms of its densities at 15 °C and flash point, while both had similar pour point, cloud point, and kinematic viscosity. Furthermore, over 10 °C increase in flash point was observed in biodiesel produced by immobilized lipases compared to free lipase, and a similar increase in density at 15 °C of biodiesel produced by immobilized lipases was also observed. Furthermore, the pour points, flash point, and kinematic viscosity of biodiesel produced by calcium alginate and Irvingia gabonensis-immobilized lipase met American and European standards, while the density at 15 °C and cloud points are below both standards.
Chromatogram of biodiesel product from palm oil esterification by calcium alginate and Irvingia gabonensis-immobilized lipase in terms of the fatty acid methyl esters (FAME) is described in Figs. 3 and 4, and the FAME produced is summarized in Table 2.Biodiesel product from both calcium alginate and Irvingia gabonensis-immobilized lipase had FAME compounds hexadecanoate (palmitic acid), 9-octadecenoate (oleic acid), octadecanoate (stearic acid), dodecanoate (lauric acid), and 9,12-octadeca-dienoate (linoleic acid) in common. However, 13-octadecanoate and 11-octadecanoate were present only in biodiesel produced with calcium alginate-immobilized lipase, while 8-octadecanoate and tetradecanoate were exclusive to biodiesel produced using Irvingia gabonensis-immobilized lipase. Furthermore, FAMEs including 13-octadecanoate, 11-octadecanoate, 8-octadecanoate, and tetradecanoate were present in biodiesel from immobilized lipases but not in biodiesel produced by free lipase.
Effect of bead reusability on immobilized lipase production of biodiesel
Re-usability of immobilized lipase in the production of biodiesel based on product yield is described in Fig. 5. Calcium alginate-immobilized lipase retained activity in twelve cycles, while Irvingia gabonensis-immobilized lipase was re-useable in eight cycles. Furthermore, over 50% of biodiesel yield was achieved in 11 cycles by calcium alginate-immobilized lipase while a similar yield was maintained in seven (7) cycles for Irvingia gabonensis-immobilized lipase. Also, > 90% yield was achieved in four (4) and eight (8) for Irvingia gabonensis- and calcium alginate-immobilized lipases respectively.
Temperature is an important factor in transesterification that can influence enzyme activity which affects reaction rate and FAME yield. Optimum temperature of 35 °C has previously been reported in biodiesel produced using alginate-immobilized lipase . It is however lower than previous studies that reported optimum biodiesel production at > 40 °C using lipase immobilized on calcium alginate and silica [29, 30]. Furthermore, optimum reaction at 35 °C indicates esterification would reduce energy costs, and prevent evaporation of alcohols. Decreased biodiesel yield above 35 °C in this study could be due to poor lipase stability at elevated temperatures.
Optimum biodiesel yield at pH 7 for both free and immobilized lipase indicates entrapment in alginate, and Irvingia gabonensis matrices did not influence the ionic property of the enzyme. Also, the optimum activity of lipase at neutral pH is similar to the report of biodiesel production by resin-immobilized lipase [31, 32].
Fuel properties determine the quality of produced fuel and influence several processes during usage. Pour point (PP) is the temperature at which the produced biodiesel starts melting from solid to liquid, and a lower PP temperature in biofuel produced via immobilized lipase indicates formation earlier melting of fuel compared to fuel from free lipase. In the same vein, cloud point (CP) is the temperature at which biodiesel begins to form cloud, and CP determined in the study is lower than previous reports for edible and inedible tallow methyl ester (15-16 °C) . This indicates produced biodiesel would be suitable for use in temperate regions, thereby reducing the possibility of clogging injectors, filters, and fuel pipelines . Kinematic viscosity influences the fuel injection process and atomization of fuel , and values determined in this study might be due to the presence of glycerol content in adequate concentration . Furthermore, similar kinematic viscosity of biodiesel produced using both free and immobilized lipases is also in agreement with the previous report . In addition, the density of biodiesel from immobilized lipases was higher than obtained from free lipase biodiesel, indicating an improved esterification process. Pour point of both biodiesels agrees with both American and European standards, while cloud point and flash point were higher than standards.
Unsaturated fatty acids are responsible for the FAME properties of the biodiesel product and their increased presence in biodiesel produced using immobilized lipases makes it a better biodiesel product . It also indicates improved esterification process due to immobilization which increased the surface area of enzyme-substrate interaction. Furthermore, minor differences in FAME compounds produced by the different immobilized enzymes could be due to the influence of matrix parameters.
Reusability of immobilized lipase in over seven cycles and high biodiesel yield in 11 cycles using alginate-immobilized lipase agrees with the previous report of using Irvingia gabonensis as lipase immobilization matrix . Reduced efficiency of Irvingia gabonensis matrix in this study however disagrees with the report, and this could be attributed to matrix interference in esterification reaction and leakage of enzyme from the support matrix due to weak binding forces [23, 37].
The study showed that using lipase immobilized on Irvingia gabonensisand calcium alginate matrices resulted in better biofuel (FAMEs and physicochemical fuel properties) compared to free lipase biofuel due to possibly improved enzyme-substrate interaction. Also, calcium alginate matrix conferred high potential for lipase reuse and maintain high biodiesel yield, while reusability of Irvingia gabonensis matrix could be further improved. This study showed that with further improvement, the use of immobilized lipase in transesterification for biodiesel production could improve product properties and lead to cost-effectiveness in industrial usage due to continuous use.
Availability of data and materials
Calcium alginate-immobilized lipase
Fatty acid methyl esters
Irvingia gabonensis-immobilized lipase
American Standard of Testing Material
Wang Y, Liu J, Gerken H, Zhang C, Hu Q, Li Y (2014) Highly-efficient enzymatic conversion of crude algal oils into biodiesel. Bioresour Technol 172:143e149
Demirbas A (2003) Biodiesel fuels from vegetable oils via catalytic and non-catalytic supercritical alcohol transesterification and other methods: a survey. J Ener Conserv Manage 44(13):2093–2109
Vicente G, Martinez M, Aracil J (2007) Optimization of integrated biodiesel production. Part 1: a study of the biodiesel purity and yield. Biores Technol 98:1742–1733
Pinzi S, Leiva D, López-García I (2014) Redel-Macías MD and Dorado MP (2014) Latest trends in feedstocks for biodiesel production. Biofuel Bioprod Bioref 8:126–143
Gupta R, Gupta N, Rathi P (2004) Bacterial lipases: an overview of production, purification and biochemical properties. Appl Microbiol Biotech 64:763–781
Masse L, Kennedy KJ, Chou SP (2001) The effect of an enzymatic pretreatment on the hydrolysis and size reduction of fat particles in slaughterhouse wastewater. J Chem Technol Biotech 76:629–635
Takamoto T, Shirasaka H, Uyama H et al (2001) Lipase catalyzed hydrolytic degradation of polyurethane in organic solvent. Chem Lett 30:492–493
Al-Zuhair S (2007) Production of biodiesel: possibilities and challenges. Biofuel Bioprod Bioref 1:57–66
Mateo C, Palomo JM, Fernandez-Lorente G, Guisan JM, Fernandez-Lafuente R (2007) Improvement of enzyme activity, stability and selectivity via immobilization techniques. Enzyme Microb Technol 40:1451–1463
Zhao X, Qi F, Yuan C, Du W, Liu D (2015) Lipase-catalyzed process for biodiesel production: enzyme immobilization, process simulation and optimization. Renew Sustain Energy Rev 44:182–197
Poppe JK, Matte CR, Ruaro Peralba MDC, Fernandez-Lafuente R, Rodrigues RC, Zachia Ayub MA (2015) Optimization of ethyl ester production from olive and palm oils using mixtures of immobilized lipases. Appl Catal A 490:50–56
Adlercreutz P (2013) Immobilisation and application of lipases in organic media. Chem Soc Rev 42:6406–6436
Winayanuwattikun P, Kaewpiboon C, Piriyakananon K, Chulalaksananukul W, Yongvanich T, Svasti J (2011) Immobilized lipase from potential lipolytic microbes for catalyzing biodiesel production using palm oil as feedstock. Afr J Biotech 10(9):1666–1673
Sankaran R, Show PL, Chang J-S (2016) Biodiesel production using immobilized lipase: feasibility and challenges. Biofuel Bioprod Bioref 10:896–916
Ertan F, Yagar H, Balkan B (2007) Optimization of alpha-amylase immobilization in calcium alginate beads. Prep Biochem Biotechnol 37:195–204
Tan T, Lu J, Nie K, Deng L, Wang F (2010) Biodiesel production with immobilized lipase: a review. Biotechnol Adv 28:628–634
Betigeri SS, Neau SH (2002) Immobilization of lipase using hydrophilic polymers in the form of hydrogel beads. Biomaterial 23(17):3627–3636
Stoytcheva M, Montero G, Toscano L, Gochev V, Valdez B (2011) The immobilized lipases in biodiesel production. In: Biodiesel – Feedstocks and Processing Technologies. IntechOpen, London, pp. 397–410
Nida J, Walvekar R, Shahbaz K, Abdullah N, Mohd Ghazi TI (2014) Biodiesel production from palm oil using lipase immobilized in ca-alginate gel and its application to an oscillatory flow reactor. 27th Symposium of Malaysian Chemical Engineers and 21st Regional Symposium on Chemical Engineering, pp 1–12
Zhao K, Di Q, Cao X, Wang M, Deng L, Wang F (2016) Production of biodiesel using immobilized lipase and the characterization of different co-immobilizing agents and immobilization methods. Sustainability 8:764
Matinja AI, Mohd Zain NA, Suhaimi MS, Alhassan AJ (2019) Optimization of biodiesel production from palm oil mill effluent using lipase immobilized in PVA-alginate-sulfate beads. Renew Ener 135:1178e1185
Osho MB, Popoola TOS, Kareem SO, Arowolo TA (2014) Transesterification of Jatropha seeds oil by vegetative sponge-immobilized lipase of Alternaria sp. MGGP 06 for fatty acid methyl ester production under optimized conditions. Petrol Technol Dev J 4(1):56–70
Adeogun AI, Omeike SO, Kareem SO (2016) Removal of Mn (II) from aqueous solution by Irvingia gabonensis immobilized Aspergillus sp. TU-GM14: Isothermal, kinetics and thermodynamic studies. J Environ Biotech Res 3(1):1–11
Kareem SO, Falokun EI, Balogun SA, Akinloye OA, Omeike SO (2017) Enzymatic biodiesel production from palm oil and palm kernel oil using free lipase. Egypt J Petrol 26:635–642
Riaz A, Qader S, Anwar A, Iqbal S (2009) Immobilization of a thermostable α-amylase on calcium alginate beads from Bacillus subtilis. Aust J Basic Appl Sci 3:2883
Taufiq-Yap YH, Abdullah NR, Basri M (2011) Biodiesel production via transesterification of palm oil using NaOH/Al2O3 catalysts. Sains Malay 40(6):587–594
Atabani AE, Silitonga AS, Badruddin IA, Mahila TMI, Masjuki HH, Mekhilef S (2012) A comprehensive review on biodiesel as alternative energy resource and its characteristics. Renew Sustain Energy Rev 16(4):2070–2093
Surendhiran D, Vijay M (2014) Biodiesel production from Nannochloropsis oculata oil using immobilized lipase with methyl acetate as an acyl acceptor. Biotechnol An Indian J 9(8):327–334
Kuan I-C, Lee C-C, Tsai B-H, Lee S-L, Lee W-T, Yu C-Y (2013) Optimizing the production of biodiesel using lipase entrapped in biomimetic silica. Energies 6:2052–2064
Garba A, Sallau AB, Ibrahim S, Abarshi MM, Muhammad A, Galadima MS, Babangida S (2018) Biodiesel production by lipase mediated transesterification of acacia nilotica seed oil. Nig J Basic Appl Sci 26(1):23–30
Egwim EC, Adesina AA, Oyewole OA, Okoliegbe IN (2012) Optimization of lipase immobilized on chitosan beads for biodiesel production. Glob Res J Microbiol 2(2):103–112
Palanisamy K, Kuppamuthu K, Jeyaseelan A (2015) Bacillus sp. Ps35 lipase-immobilization on styrene-divinyl benzene resin and application in fatty acid methyl ester synthesis. Iran. J Biotech 13(3):e1225
Vyas AP, Verma JL, Subrahmanyam N (2009) A review on FAME production processes. Fuel 89:1–9
Demirbas A (2008) Relationships derived from physical properties of vegetable oil and biodiesel fuels. Fuel 87:1743–1748
Encinar JM, González JF, Rodríguez-Reinares A (2005) Biodiesel from used frying oil: variables affecting the yields and characteristics of the biodiesel. Ind Eng Chem Res 44(15):5491–5499
Giakoumis E (2013) A statistical investigation of biodiesel physical and chemical properties, and their correlation with the degree of unsaturation. Renew Energy 50:858–878
Jaiswal N, Prakash O (2011) Immobilization of soybean 훼-amylase on gelatin and its application as a detergent additive. Asian J Biochem 6(4):337–346
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Kareem, S.O., Falokun, E.I., Balogun, S.A. et al. Improved biodiesel from palm oil using lipase immobilized calcium alginate and Irvingia gabonensis matrices. Beni-Suef Univ J Basic Appl Sci 9, 59 (2020). https://doi.org/10.1186/s43088-020-00084-6
- Irvingia gabenensis
- Calcium alginate