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Utilization of Waste Glycerol from Biodiesel Process as a Substrate for Mono-, Di-, and Triacylglycerol Production
Available online at www.sciencedirect.com
ScienceDirect Availableonline onlineatatwww.sciencedirect.com www.sciencedirect.com Available Energy Procedia 00 (2017) 000–000
ScienceDirect ScienceDirect
www.elsevier.com/locate/procedia
Energy (2017) 000–000 895–900 EnergyProcedia Procedia138 00 (2017) www.elsevier.com/locate/procedia
2017 International Conference on Alternative Energy in Developing Countries and Emerging Economies 2017 AEDCEE, 25‐26 May 2017, Bangkok, Thailand
15th International Symposium on District Heating and Cooling Utilization The of Waste Glycerol from Biodiesel Process as a Substrate for Mono-, Di-, andof Triacylglycerol Assessing the feasibility using the heatProduction demand-outdoor 2 3* temperature function1, for a long-term heat demand forecast Narisa Binhayeeding Sappasith Klomklaodistrict and Kanokporn Sangkharak a a b c I. Andrića,b,c *, A.ofPina , P. Ferrão J. Fournier ., B. Lacarrière , O. Le Correc Department Biotechnology, Faculty of, Science, Thaksin University, Phatthalung, Thailand 1
2 Department a
of Food Science and Technology, Faculty of Technology and Community Development, Thaksin University, Phatthalung, Thailand 3* IN+ Center for Innovation, Technology and PolicyFaculty Research - Instituto Superior Técnico, Phatthalung,, Av. Rovisco Pais 1, 1049-001 Lisbon, Portugal Department of Chemistry, of Science, Thaksin University, Thailand b Veolia Recherche & Innovation, 291 Avenue Dreyfous Daniel, 78520 Limay, France c Département Systèmes Énergétiques et Environnement - IMT Atlantique, 4 rue Alfred Kastler, 44300 Nantes, France
Abstract
Glycerol Abstract (or glycerin) is a major by-product in the biodiesel process. Ten percent of crude glycerol are created for every 100 pounds of biodiesel produced. As the utilization of biodiesel increasing, an excess of glycerol is being generated. However, the are purify of waste glycerolinisthe expensive, must search alternative methods District heating networks commonly addressed literature biodiesel as one of producers the most effective solutions for decreasing the for its utilization. This paper demonstrate the feasibility of producing mono-, diand triacylglycerol from waste greenhouse gas emissions from the building sector. These systems require high investments which are returned through the heat glycerol. was produced through glycerolysis reaction usingheat commercial fromcould Candida sp. sales. DueThe to acylglycerol the changed climate conditions and building renovation policies, demand inlipase the future decrease, Effects of various reaction parameters prolonging the investment return period. were evaluated. The optimum conditions for acylglycerol production were a The maintoscope this mole paper is to assess the100 feasibility using with the heat – outdoor temperature function for heat glycerol fattyofacid ratio of 6∶1, mg ofoflipase thedemand reaction temperature and time at 40°C anddemand 24 h, respectively. acylglycerol Layer Chromatography ether : diethyl of ether forecast. The Analysis district of of Alvalade, locatedbyinThin Lisbon (Portugal), was used as(TLC) a caseusing study.petroleum The district is consisted 665 buildings that vary in both construction and typology. Three weather scenarios (low,triacylglycerol medium, high) were and three district :methanol:acetic acid (90:7:2.5:0.5 v/v)period as mobile phase found that mono-, di- and produced scenarios were developed (shallow, intermediate, deep). To estimate the error, obtained lipase heat demand values were inrenovation high purity. Interestingly, the highest production of acylglycerol yeild from commercial and crude lipase compared withwhite resultsshrimp’s from a dynamic heat demand model, previously developed validated Attenuated by the authors. from pacific hepatopancreas were obtained at the same and conditions. Total Reflectance The results showed that whenInfrared only weather change is considered, the margin error could be acceptable for mono-, some applications (ATR)-Fourier Transform Spectroscopy (FTIR) proved that ofthe final product contains di- and (the error in annual demand was lower than 20% for all weather scenarios considered). However, after introducing renovation triacylglycerol.
scenarios, the error value increased up to 59.5% (depending on the weather and renovation scenarios combination considered). © 2017 The Authors. Elsevier Ltd. value slope Published coefficient increased ©The 2017 The of Authors. Published by by Elsevier on Ltd.average within the range of 3.8% up to 8% per decade, that corresponds to the Peer-review responsibility of the scientific committee of the the 2017 International Conference Energy in decrease in under the number of heating hours of 22-139h during heating season (depending on on theAlternative combination of weather and Peer-review under responsibility of the Organizing Committee of 2017 AEDCEE. Drenovation eveloping Countries EmergingOn Economies. scenarios and considered). the other hand, function intercept increased for 7.8-12.7% per decade (depending on the coupled scenarios). The values suggested could be Monoacylglycerol used to modify the function parameters for the scenarios considered, and Keywords: Glycerol, Glycerolysis, Hepatopancreas, Lipase, improve the accuracy of heat demand estimations. *© 2017 The Authors. Published by Elsevier Ltd. Corresponding Author: [email protected]
Corresponding Author: [email protected]
Peer-review under responsibility of the Scientific Committee of The 15th International Symposium on District Heating and Cooling.
Keywords: Heat demand; Forecast; Climate change 1876-6102 © 2017 The Authors. Published by Elsevier Ltd. Peer-review under responsibility of the Organizing Committee of 2017 AEDCEE.
1876-6102 © 2017 The Authors. Published by Elsevier Ltd. Peer-review under responsibility of the Scientific Committee of The 15th International Symposium on District Heating and Cooling. 1876-6102 © 2017 The Authors. Published by Elsevier Ltd. Peer-review under responsibility of the scientific committee of the 2017 International Conference on Alternative Energy in Developing Countries and Emerging Economies. 10.1016/j.egypro.2017.10.130
Narisa Binhayeeding et al. / Energy Procedia 138 (2017) 895–900 Author name / Energy Procedia 00 (2017) 000–000
896 2
1. Introduction Biodiesel is an alternative renewable fuel and can be produced from animal fats and vegetable oils. The typical process to produce biodiesel fuel is trasesterification [1]. Glycerol (also known as glycerin) is a major byproduct in the biodiesel manufacturing process. In general, for every 100 pounds of biodiesel produced, approximately 10 pounds of crude glycerol are created. As the biodiesel industry is rapidly expanding, a glut of crude glycerol is being created. Because this glycerol is expensive to purify for use in the food, pharmaceutical, or cosmetics industries, biodiesel producers must seek alternative methods for its disposal. However, crude glycerol could be used as a starting material for the production of some very valuable products such as mono-, di- and triacylglycerol by esterification reaction [2]. As consumers become more aware and concerned about the impact of the food they eat and the substances they commonly use on their health and general well- being, there is a growing interest in designer lipids. Among the lipid classes, surfactants, such as monoacylglycerol (monoglyceride, MAG), are desired by the food, cosmetic, pharmaceutical and chemical industries [3].They are manufactured by a chemical glycerolysis of triglycerides catalyzed by inorganic catalysts. The glycerolysis reaction has a high demand for thermal energy because it is conducted at elevated temperatures (200-250°C) to overcome the low solubility of glycerol in oil phases [4]. However, this process only yields 30-40% MAG [4] and has several drawbacks such as low yield, dark color and burnt taste. Lipases can be used as biocatalysts for the glycerolysis process and they have many advantages over the chemical process such as a mild reaction condition, high catalytic efficiency and stereoand positional specificities. Different lipase-catalyzed reactions have been reported, using organic solvents in monophasic, biphasic or solvent-free systems. Unfortunately, the cost of bacterial lipase is relatively high for industrial scale [5]. In this paper, some efforts have been made to explore the possibility to use a relatively cheap lipase from candida sp and crude lipase from Pacific white shrimp’s hepatopancreas. Production of tailor-made designer monoacylglycerols with targeted fatty acids therefore offers promising industrial opportunities. The complete steps for glycerolysis reaction are demonstrated in Figure 1. The monoacylglycerol are obtained through glycerolysis of triacylglycerol with glycerol excess; and glycerol esterification with fatty acids [6, 7].
Figure 1. The main reactions of glycerolysis of palm olein (R: palmitin or olein).
In the current work, we demonstrate the feasibility of producing mono-, di- and triglycerides from waste glycerol by a glycerolysis reaction with lipase catalyzed. 2. Materials and Methods 2.1. Materials The lipases from candida sp were obtained from Sigma-Aldrich and crude lipase from Pacific white shrimp’s hepatopancreas was provided by Assoc. Prof. Dr. Sappasith Klomklao (Thaksin Uiversity, Thailand). 2.2 Preparation of purified glycerol from crude glycerol Crude glycerol was obtained from a biodiesel lab (Thaksin University, Thailand) in which palm oil was used as raw material for biodiesel production through the transesterification process catalyzed by sodium hydroxide. Crude glycerol (1 kg) was acidified by the addition of 1.19 M H2SO4 to pH 2, left until phase separated into three distinct layers, with is a top layer of free fatty acids, a middle glycerol-rich layer and a bottom inorganic salt rich layer. The top layer was removed by slow decantation and the middle layer was then harvested and filtered. After that, the middle layer was neutralized with 12.5 M NaOH and evaporated at 105°C for 2 hours and followed by filtration. The enriched glycerol layer was then extracted with excess ethanol for 10 min. The glycerol-ethanol
Narisa Binhayeeding et al. / Energy Procedia 138 (2017) 895–900 Author name / Energy Procedia 00 (2017) 000–000
897 3
solution was harvested and then filtered. The obtained purified crude glycerol was evaporated to eliminate ethanol at 80°C for 20 min [8] and then was used in mono-, di-, and triacylglycerol production. 2.3. Optimum conditions for glycerolysis reaction A reaction mixture consisting of 20 g of palm oil; 15 g of purified crude glycerol and 0.1 g of Candida sp. lipase under an agitation speed of 250 rpm. Several reaction conditions were optimized including temperature, reaction time and molar ratio of glycerol to free fatty acids. The studied reaction temperatures were from 40°C to 60°C. Investigation of the reaction times were at 6, 12, 18, 24, 30, 36, 42 and 48 hours. The molar ratios of glycerol to fatty acids were 2:1, 4:1, 6:1,8:1, 10:1 and 12:1. Afterward, lipase from Pacific white shimp’s hapatopancreas was substituted and also studied. 2.4. Analytical Methods 2.4.1. Thin-Layer Chromatography The reaction product (MAG, DAG, TAG) was identified by Thin Layer Chromatography (TLC). Aliquots of the reaction mixtures were applied on a Silica gel 60 plate (Merck) and developed with a solvent mixture of petroleum ether :diethyl ether : methanol : acetic acid (90:7:2.5:0.5 v/v) as mobile phase. The TLC paper was allowed to stand until the solvent reach a level which must be just below the end line. Then, the TLC paper was dried and the bands of the different produced glycerides were detected under a UV lamp. In some cases, the bands did not clearly appear under the UV lamp. Accordingly, the TLC paper was exposed to an iodine vapour which allowed a direct visualization of the band by naked eye. 2.4.2 Chemical structures of mono-, di- and triacylglycerol Fourier transform infrared spectroscopy (FTIR) was used to investigate the chemical structures of mono-, di- and triacylglycerol, with a frequency range of 4000-400 cm-1 [9]. 2. Results and Discussion 3.1 Characteristics of the purified crude glycerol The original crude glycerol from biodiesel was dark liquid with a high pH (8.67±0.55). After the addition of H2SO4 to the original crude glycerol to pH 2, it automatically phase separated into three distinct layers, an inorganic salt layer on the bottom, a glycerol-rich layer in the middle and a free fatty acid layer on the top. This might be explained by the fact that the strong acid condition neutralized the alkaline catalyst, which precipitated out of the bottom layer, and also achieved the hydrolysis of insoluble free fatty acids at the top layers. After the glycerol-rich layer was neutralized with NaOH and extracted with ethanol, it obtained a high purity with a neutral pH (5.61 0.37), as shown in Table 1. Table 1 Characteristics of Crude Glycerol and Purified crude glycerol. Parameters pH Color
Crude glycerol 8.67±0.55 Dark brown
Purified crude glycerol 5.61±0.37 Light brown
3.2 Effects of temperature For the effect of temperature on mono-, di- and triacylglycerol production, the temperature was controlled at 40-60oC. When the temperature was controlled at 40oC, the mono-, di- and triacylglycerol production increased when increasing the temperature as shown in Figure 2a. This may be due to the fact that the rate of reaction increased as the temperature increased. Hence, it can be explained that candida sp. lipase and Pacific white shrimp’s hepatopancreas lipase was denatured by higher temperature.
Narisa Binhayeeding et al. / Energy Procedia 138 (2017) 895–900 Author name / Energy Procedia 00 (2017) 000–000
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3.3 Effects of time The effects or reaction time on glycerolysis were determined and the results are shown in Figure 2b. When increasing the reaction time, the mono-, di- and triacylglycerol production also increased. There fore, the reaction time at 24 h was used in further experimentations. 3.4 Effects of molar ratio on the esterification yield The third studied parameter was the optimum esterification molar ratio (glycerol:fatty acids). In this part, all experiments were carried out at the optimum obtained temperature (40°C). The esterification process was carried out at 2:1, 4:1, 6:1, 8:1, 10:1 and 12:1 (glycerol to fatty acids). As shown in the Figure 2c, a molar ratio at 6:1 of glycerol to palm oil gave the maximum yield of mono-, di- and triacylglycerol (50%). When the molar ratio of glycerol to palm oil was more than 6:1, the yield of mono-, di- and triacylglycerol slightly decreased. It may be explained since increasing the glycerol will decrease the amount of available substrate at the interface between oil and glycerol and, hence, decrease the mono-, di- and triacylglycerol yield.
(a) Temperature (oC)
(b) Time (h)
(c)
Molar ratio of substrate
Figure 2. Effect of temperature (a), time (b) and molar ratio of substrate (c) on mono-, di- and triacylglycerol production by candida sp. lipase and pacific white shrimp’s hepatopancreas lipase. The reaction mixture contained glycerol and palm oil. The amount of candida sp. lipase and pacific white shrimp’s hepatopancreas lipase used was 5% of original oil. The reaction was carried out at 40oC for 24 hours.
Narisa Binhayeeding et al. / Energy Procedia 138 (2017) 895–900 Author name / Energy Procedia 00 (2017) 000–000
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3.5 Esterification reaction using the optimized conditions In this part, the esterification reaction of fatty acids was performed under the obtained optimum conditions. A sample was taken for FTIR and TLC analysis to prove the composition of the obtained esterification products. Figure 3. shows the TLC paper plate of estrfied by candida sp. lipase and pacific white shrimp’s hepatopancreas lipase, respectively. Figure 4 shows the FTIR analysis. The results proved the existance of the carbonyl group (at wave number 1743.85 and 1744.09 of the ester formation [10]. The results showed the existence of the three main space of monoglyceride, diglyceride, and triglyceride.
Figure 3.TLC analysis of esterification reaction unsaturated fatty acids 1: esterification reaction of fatty acids with glycerol by candida sp lipase 2: esterification reaction of fatty acids with glycerol by pacific white shrimp’s hepatopancreas lipase.
(a)
(b)
Figure 4. FTIR analysis of esterification unsaturated fatty acids catalyzed by lipase from candida sp. (a) and pacific white shrimp’s hepatopancreas (b).
900 6
Narisa Binhayeeding et al. / Energy Procedia 138 (2017) 895–900 Author name / Energy Procedia 00 (2017) 000–000
4. Conclusion The production of mono-, di- and triacylglycerol from waste glycerol by using commercial lipase from candida sp and pacific white shrimp’s hepatopancreas lipase . The optimal conditions for mono-, di- and triacylglycerol production were a glycerol to fatty acid mole ratio of 6∶1, 100 mg of lipase with a reaction temperature and a time of 40°C and 24 h. The production of mono-, di- and triacylglycerol were produced with high purity. ACKNOWLEDGEMENTS The authors would like to thank the Research and Development Institute Thaksin University, the Graduate School Thaksin University and the Department of Chemistry, Faculty of Science, Thaksin University for their financial support. Finally, we would like to thank Mr. Christopher Joseph Forti (English adviser and English proofreader, Thaksin University) for useful comments and suggestions on the language and structure of our manuscript. References [1] Hayyan A, Mjalli FS, Hashim MA, Hayyan M, AlNashef IM. Conversion of free fatty acids in low grade crude palm oil to methyl esters for biodiesel production using chromosulfuric acid. Bulg Chem Commun 2013, 45,p. 394 – 399. [2] Isabel, D., Federico, M., Joaquın, P. and Enrique, S. Synthesis of MCM-41 materials dialkyl silane groups and their catalytic activity in functionalised with the esterification of glycerol with fatty acids. Applied Catalysis A: General.2003, 242,p. 161-169. [3] N. A. Mostafa , Ashraf Maher , Wael Abdelmoez. Production of mono-, di-, and triglycerides from waste fatty acids through esterification with glycerol Advances in Bioscience and Biotechnology, 2013,4,p. 900-907 [4] Sonntag, N. O. V. Glycerolysis of fats and methyl esters--status,review and critique. J. Am. Oil. Chem. Soc. 1982, 59, p.795A-802A [5] P. Pinyaphong, P. Sriburi, and S. Phutrakul. Synthesis of Monoacylglycerol from Glycerolysis of Crude Glycerol with Coconut Oil Catalyzed by Carica Papaya Lipase. International Journal of Chemical, Molecular, Nuclear, Materials and Metallurgical Engineering. 2012,6,p. 926-931 [6] Guner, F.S., Sirkecioglu, A., Yilmaz, S., Erciyes, A.T. and Erdem-Senatalar, A. Esterification of oleic acid with glycerol in the presence of sulfated iron oxide catalyst. Journal of the American Oil Chemists’ Society.1996, 73,p. 347-351. [7] Lampert D. Processes and Products of Interesterification. In: O’BRIEN R, FARR W, WAN P, editors. Introduction to Fats and Oils Technology. Champaign: AOCS Press; 1998, p.208-232. [8] S. Kongjao, S. Damronglerd and M. Hunsom. Purification of crude glycerol derived from waste used-oil methyl ester plant.2010,27,p. 944949. [9] Khare E, Chopra J, Arora NK. Screening for MCL-PHA-producing fluorescent pseudomonads and comparison of MCL-PHA production under iso-osmotic conditions induced by PEG and NaCl. Curr Microbiol 2014; 68, p. 457-62 [1] Michael J. Haas *, Andrew J. McAloon, Winnie C. Yee, Thomas A. Foglia. A process model to estimate biodiesel production costs. Bioresource Technology .2006,97,p.671–678 [10] Vlachos, N., Skopelitis, Y., Psaroudaki, M., Konstantini- dou, V., Chatzilazarou, A. and Tegou, E. Applica- tions of fourier transforminfrared spectroscopy to edible oils. Analytica Chimica Acta, 2006,573-574,p. 459-465.
ScienceDirect Availableonline onlineatatwww.sciencedirect.com www.sciencedirect.com Available Energy Procedia 00 (2017) 000–000
ScienceDirect ScienceDirect
www.elsevier.com/locate/procedia
Energy (2017) 000–000 895–900 EnergyProcedia Procedia138 00 (2017) www.elsevier.com/locate/procedia
2017 International Conference on Alternative Energy in Developing Countries and Emerging Economies 2017 AEDCEE, 25‐26 May 2017, Bangkok, Thailand
15th International Symposium on District Heating and Cooling Utilization The of Waste Glycerol from Biodiesel Process as a Substrate for Mono-, Di-, andof Triacylglycerol Assessing the feasibility using the heatProduction demand-outdoor 2 3* temperature function1, for a long-term heat demand forecast Narisa Binhayeeding Sappasith Klomklaodistrict and Kanokporn Sangkharak a a b c I. Andrića,b,c *, A.ofPina , P. Ferrão J. Fournier ., B. Lacarrière , O. Le Correc Department Biotechnology, Faculty of, Science, Thaksin University, Phatthalung, Thailand 1
2 Department a
of Food Science and Technology, Faculty of Technology and Community Development, Thaksin University, Phatthalung, Thailand 3* IN+ Center for Innovation, Technology and PolicyFaculty Research - Instituto Superior Técnico, Phatthalung,, Av. Rovisco Pais 1, 1049-001 Lisbon, Portugal Department of Chemistry, of Science, Thaksin University, Thailand b Veolia Recherche & Innovation, 291 Avenue Dreyfous Daniel, 78520 Limay, France c Département Systèmes Énergétiques et Environnement - IMT Atlantique, 4 rue Alfred Kastler, 44300 Nantes, France
Abstract
Glycerol Abstract (or glycerin) is a major by-product in the biodiesel process. Ten percent of crude glycerol are created for every 100 pounds of biodiesel produced. As the utilization of biodiesel increasing, an excess of glycerol is being generated. However, the are purify of waste glycerolinisthe expensive, must search alternative methods District heating networks commonly addressed literature biodiesel as one of producers the most effective solutions for decreasing the for its utilization. This paper demonstrate the feasibility of producing mono-, diand triacylglycerol from waste greenhouse gas emissions from the building sector. These systems require high investments which are returned through the heat glycerol. was produced through glycerolysis reaction usingheat commercial fromcould Candida sp. sales. DueThe to acylglycerol the changed climate conditions and building renovation policies, demand inlipase the future decrease, Effects of various reaction parameters prolonging the investment return period. were evaluated. The optimum conditions for acylglycerol production were a The maintoscope this mole paper is to assess the100 feasibility using with the heat – outdoor temperature function for heat glycerol fattyofacid ratio of 6∶1, mg ofoflipase thedemand reaction temperature and time at 40°C anddemand 24 h, respectively. acylglycerol Layer Chromatography ether : diethyl of ether forecast. The Analysis district of of Alvalade, locatedbyinThin Lisbon (Portugal), was used as(TLC) a caseusing study.petroleum The district is consisted 665 buildings that vary in both construction and typology. Three weather scenarios (low,triacylglycerol medium, high) were and three district :methanol:acetic acid (90:7:2.5:0.5 v/v)period as mobile phase found that mono-, di- and produced scenarios were developed (shallow, intermediate, deep). To estimate the error, obtained lipase heat demand values were inrenovation high purity. Interestingly, the highest production of acylglycerol yeild from commercial and crude lipase compared withwhite resultsshrimp’s from a dynamic heat demand model, previously developed validated Attenuated by the authors. from pacific hepatopancreas were obtained at the same and conditions. Total Reflectance The results showed that whenInfrared only weather change is considered, the margin error could be acceptable for mono-, some applications (ATR)-Fourier Transform Spectroscopy (FTIR) proved that ofthe final product contains di- and (the error in annual demand was lower than 20% for all weather scenarios considered). However, after introducing renovation triacylglycerol.
scenarios, the error value increased up to 59.5% (depending on the weather and renovation scenarios combination considered). © 2017 The Authors. Elsevier Ltd. value slope Published coefficient increased ©The 2017 The of Authors. Published by by Elsevier on Ltd.average within the range of 3.8% up to 8% per decade, that corresponds to the Peer-review responsibility of the scientific committee of the the 2017 International Conference Energy in decrease in under the number of heating hours of 22-139h during heating season (depending on on theAlternative combination of weather and Peer-review under responsibility of the Organizing Committee of 2017 AEDCEE. Drenovation eveloping Countries EmergingOn Economies. scenarios and considered). the other hand, function intercept increased for 7.8-12.7% per decade (depending on the coupled scenarios). The values suggested could be Monoacylglycerol used to modify the function parameters for the scenarios considered, and Keywords: Glycerol, Glycerolysis, Hepatopancreas, Lipase, improve the accuracy of heat demand estimations. *© 2017 The Authors. Published by Elsevier Ltd. Corresponding Author: [email protected]
Corresponding Author: [email protected]
Peer-review under responsibility of the Scientific Committee of The 15th International Symposium on District Heating and Cooling.
Keywords: Heat demand; Forecast; Climate change 1876-6102 © 2017 The Authors. Published by Elsevier Ltd. Peer-review under responsibility of the Organizing Committee of 2017 AEDCEE.
1876-6102 © 2017 The Authors. Published by Elsevier Ltd. Peer-review under responsibility of the Scientific Committee of The 15th International Symposium on District Heating and Cooling. 1876-6102 © 2017 The Authors. Published by Elsevier Ltd. Peer-review under responsibility of the scientific committee of the 2017 International Conference on Alternative Energy in Developing Countries and Emerging Economies. 10.1016/j.egypro.2017.10.130
Narisa Binhayeeding et al. / Energy Procedia 138 (2017) 895–900 Author name / Energy Procedia 00 (2017) 000–000
896 2
1. Introduction Biodiesel is an alternative renewable fuel and can be produced from animal fats and vegetable oils. The typical process to produce biodiesel fuel is trasesterification [1]. Glycerol (also known as glycerin) is a major byproduct in the biodiesel manufacturing process. In general, for every 100 pounds of biodiesel produced, approximately 10 pounds of crude glycerol are created. As the biodiesel industry is rapidly expanding, a glut of crude glycerol is being created. Because this glycerol is expensive to purify for use in the food, pharmaceutical, or cosmetics industries, biodiesel producers must seek alternative methods for its disposal. However, crude glycerol could be used as a starting material for the production of some very valuable products such as mono-, di- and triacylglycerol by esterification reaction [2]. As consumers become more aware and concerned about the impact of the food they eat and the substances they commonly use on their health and general well- being, there is a growing interest in designer lipids. Among the lipid classes, surfactants, such as monoacylglycerol (monoglyceride, MAG), are desired by the food, cosmetic, pharmaceutical and chemical industries [3].They are manufactured by a chemical glycerolysis of triglycerides catalyzed by inorganic catalysts. The glycerolysis reaction has a high demand for thermal energy because it is conducted at elevated temperatures (200-250°C) to overcome the low solubility of glycerol in oil phases [4]. However, this process only yields 30-40% MAG [4] and has several drawbacks such as low yield, dark color and burnt taste. Lipases can be used as biocatalysts for the glycerolysis process and they have many advantages over the chemical process such as a mild reaction condition, high catalytic efficiency and stereoand positional specificities. Different lipase-catalyzed reactions have been reported, using organic solvents in monophasic, biphasic or solvent-free systems. Unfortunately, the cost of bacterial lipase is relatively high for industrial scale [5]. In this paper, some efforts have been made to explore the possibility to use a relatively cheap lipase from candida sp and crude lipase from Pacific white shrimp’s hepatopancreas. Production of tailor-made designer monoacylglycerols with targeted fatty acids therefore offers promising industrial opportunities. The complete steps for glycerolysis reaction are demonstrated in Figure 1. The monoacylglycerol are obtained through glycerolysis of triacylglycerol with glycerol excess; and glycerol esterification with fatty acids [6, 7].
Figure 1. The main reactions of glycerolysis of palm olein (R: palmitin or olein).
In the current work, we demonstrate the feasibility of producing mono-, di- and triglycerides from waste glycerol by a glycerolysis reaction with lipase catalyzed. 2. Materials and Methods 2.1. Materials The lipases from candida sp were obtained from Sigma-Aldrich and crude lipase from Pacific white shrimp’s hepatopancreas was provided by Assoc. Prof. Dr. Sappasith Klomklao (Thaksin Uiversity, Thailand). 2.2 Preparation of purified glycerol from crude glycerol Crude glycerol was obtained from a biodiesel lab (Thaksin University, Thailand) in which palm oil was used as raw material for biodiesel production through the transesterification process catalyzed by sodium hydroxide. Crude glycerol (1 kg) was acidified by the addition of 1.19 M H2SO4 to pH 2, left until phase separated into three distinct layers, with is a top layer of free fatty acids, a middle glycerol-rich layer and a bottom inorganic salt rich layer. The top layer was removed by slow decantation and the middle layer was then harvested and filtered. After that, the middle layer was neutralized with 12.5 M NaOH and evaporated at 105°C for 2 hours and followed by filtration. The enriched glycerol layer was then extracted with excess ethanol for 10 min. The glycerol-ethanol
Narisa Binhayeeding et al. / Energy Procedia 138 (2017) 895–900 Author name / Energy Procedia 00 (2017) 000–000
897 3
solution was harvested and then filtered. The obtained purified crude glycerol was evaporated to eliminate ethanol at 80°C for 20 min [8] and then was used in mono-, di-, and triacylglycerol production. 2.3. Optimum conditions for glycerolysis reaction A reaction mixture consisting of 20 g of palm oil; 15 g of purified crude glycerol and 0.1 g of Candida sp. lipase under an agitation speed of 250 rpm. Several reaction conditions were optimized including temperature, reaction time and molar ratio of glycerol to free fatty acids. The studied reaction temperatures were from 40°C to 60°C. Investigation of the reaction times were at 6, 12, 18, 24, 30, 36, 42 and 48 hours. The molar ratios of glycerol to fatty acids were 2:1, 4:1, 6:1,8:1, 10:1 and 12:1. Afterward, lipase from Pacific white shimp’s hapatopancreas was substituted and also studied. 2.4. Analytical Methods 2.4.1. Thin-Layer Chromatography The reaction product (MAG, DAG, TAG) was identified by Thin Layer Chromatography (TLC). Aliquots of the reaction mixtures were applied on a Silica gel 60 plate (Merck) and developed with a solvent mixture of petroleum ether :diethyl ether : methanol : acetic acid (90:7:2.5:0.5 v/v) as mobile phase. The TLC paper was allowed to stand until the solvent reach a level which must be just below the end line. Then, the TLC paper was dried and the bands of the different produced glycerides were detected under a UV lamp. In some cases, the bands did not clearly appear under the UV lamp. Accordingly, the TLC paper was exposed to an iodine vapour which allowed a direct visualization of the band by naked eye. 2.4.2 Chemical structures of mono-, di- and triacylglycerol Fourier transform infrared spectroscopy (FTIR) was used to investigate the chemical structures of mono-, di- and triacylglycerol, with a frequency range of 4000-400 cm-1 [9]. 2. Results and Discussion 3.1 Characteristics of the purified crude glycerol The original crude glycerol from biodiesel was dark liquid with a high pH (8.67±0.55). After the addition of H2SO4 to the original crude glycerol to pH 2, it automatically phase separated into three distinct layers, an inorganic salt layer on the bottom, a glycerol-rich layer in the middle and a free fatty acid layer on the top. This might be explained by the fact that the strong acid condition neutralized the alkaline catalyst, which precipitated out of the bottom layer, and also achieved the hydrolysis of insoluble free fatty acids at the top layers. After the glycerol-rich layer was neutralized with NaOH and extracted with ethanol, it obtained a high purity with a neutral pH (5.61 0.37), as shown in Table 1. Table 1 Characteristics of Crude Glycerol and Purified crude glycerol. Parameters pH Color
Crude glycerol 8.67±0.55 Dark brown
Purified crude glycerol 5.61±0.37 Light brown
3.2 Effects of temperature For the effect of temperature on mono-, di- and triacylglycerol production, the temperature was controlled at 40-60oC. When the temperature was controlled at 40oC, the mono-, di- and triacylglycerol production increased when increasing the temperature as shown in Figure 2a. This may be due to the fact that the rate of reaction increased as the temperature increased. Hence, it can be explained that candida sp. lipase and Pacific white shrimp’s hepatopancreas lipase was denatured by higher temperature.
Narisa Binhayeeding et al. / Energy Procedia 138 (2017) 895–900 Author name / Energy Procedia 00 (2017) 000–000
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3.3 Effects of time The effects or reaction time on glycerolysis were determined and the results are shown in Figure 2b. When increasing the reaction time, the mono-, di- and triacylglycerol production also increased. There fore, the reaction time at 24 h was used in further experimentations. 3.4 Effects of molar ratio on the esterification yield The third studied parameter was the optimum esterification molar ratio (glycerol:fatty acids). In this part, all experiments were carried out at the optimum obtained temperature (40°C). The esterification process was carried out at 2:1, 4:1, 6:1, 8:1, 10:1 and 12:1 (glycerol to fatty acids). As shown in the Figure 2c, a molar ratio at 6:1 of glycerol to palm oil gave the maximum yield of mono-, di- and triacylglycerol (50%). When the molar ratio of glycerol to palm oil was more than 6:1, the yield of mono-, di- and triacylglycerol slightly decreased. It may be explained since increasing the glycerol will decrease the amount of available substrate at the interface between oil and glycerol and, hence, decrease the mono-, di- and triacylglycerol yield.
(a) Temperature (oC)
(b) Time (h)
(c)
Molar ratio of substrate
Figure 2. Effect of temperature (a), time (b) and molar ratio of substrate (c) on mono-, di- and triacylglycerol production by candida sp. lipase and pacific white shrimp’s hepatopancreas lipase. The reaction mixture contained glycerol and palm oil. The amount of candida sp. lipase and pacific white shrimp’s hepatopancreas lipase used was 5% of original oil. The reaction was carried out at 40oC for 24 hours.
Narisa Binhayeeding et al. / Energy Procedia 138 (2017) 895–900 Author name / Energy Procedia 00 (2017) 000–000
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3.5 Esterification reaction using the optimized conditions In this part, the esterification reaction of fatty acids was performed under the obtained optimum conditions. A sample was taken for FTIR and TLC analysis to prove the composition of the obtained esterification products. Figure 3. shows the TLC paper plate of estrfied by candida sp. lipase and pacific white shrimp’s hepatopancreas lipase, respectively. Figure 4 shows the FTIR analysis. The results proved the existance of the carbonyl group (at wave number 1743.85 and 1744.09 of the ester formation [10]. The results showed the existence of the three main space of monoglyceride, diglyceride, and triglyceride.
Figure 3.TLC analysis of esterification reaction unsaturated fatty acids 1: esterification reaction of fatty acids with glycerol by candida sp lipase 2: esterification reaction of fatty acids with glycerol by pacific white shrimp’s hepatopancreas lipase.
(a)
(b)
Figure 4. FTIR analysis of esterification unsaturated fatty acids catalyzed by lipase from candida sp. (a) and pacific white shrimp’s hepatopancreas (b).
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Narisa Binhayeeding et al. / Energy Procedia 138 (2017) 895–900 Author name / Energy Procedia 00 (2017) 000–000
4. Conclusion The production of mono-, di- and triacylglycerol from waste glycerol by using commercial lipase from candida sp and pacific white shrimp’s hepatopancreas lipase . The optimal conditions for mono-, di- and triacylglycerol production were a glycerol to fatty acid mole ratio of 6∶1, 100 mg of lipase with a reaction temperature and a time of 40°C and 24 h. The production of mono-, di- and triacylglycerol were produced with high purity. ACKNOWLEDGEMENTS The authors would like to thank the Research and Development Institute Thaksin University, the Graduate School Thaksin University and the Department of Chemistry, Faculty of Science, Thaksin University for their financial support. Finally, we would like to thank Mr. Christopher Joseph Forti (English adviser and English proofreader, Thaksin University) for useful comments and suggestions on the language and structure of our manuscript. References [1] Hayyan A, Mjalli FS, Hashim MA, Hayyan M, AlNashef IM. Conversion of free fatty acids in low grade crude palm oil to methyl esters for biodiesel production using chromosulfuric acid. Bulg Chem Commun 2013, 45,p. 394 – 399. [2] Isabel, D., Federico, M., Joaquın, P. and Enrique, S. Synthesis of MCM-41 materials dialkyl silane groups and their catalytic activity in functionalised with the esterification of glycerol with fatty acids. Applied Catalysis A: General.2003, 242,p. 161-169. [3] N. A. Mostafa , Ashraf Maher , Wael Abdelmoez. Production of mono-, di-, and triglycerides from waste fatty acids through esterification with glycerol Advances in Bioscience and Biotechnology, 2013,4,p. 900-907 [4] Sonntag, N. O. V. Glycerolysis of fats and methyl esters--status,review and critique. J. Am. Oil. Chem. Soc. 1982, 59, p.795A-802A [5] P. Pinyaphong, P. Sriburi, and S. Phutrakul. Synthesis of Monoacylglycerol from Glycerolysis of Crude Glycerol with Coconut Oil Catalyzed by Carica Papaya Lipase. International Journal of Chemical, Molecular, Nuclear, Materials and Metallurgical Engineering. 2012,6,p. 926-931 [6] Guner, F.S., Sirkecioglu, A., Yilmaz, S., Erciyes, A.T. and Erdem-Senatalar, A. Esterification of oleic acid with glycerol in the presence of sulfated iron oxide catalyst. Journal of the American Oil Chemists’ Society.1996, 73,p. 347-351. [7] Lampert D. Processes and Products of Interesterification. In: O’BRIEN R, FARR W, WAN P, editors. Introduction to Fats and Oils Technology. Champaign: AOCS Press; 1998, p.208-232. [8] S. Kongjao, S. Damronglerd and M. Hunsom. Purification of crude glycerol derived from waste used-oil methyl ester plant.2010,27,p. 944949. [9] Khare E, Chopra J, Arora NK. Screening for MCL-PHA-producing fluorescent pseudomonads and comparison of MCL-PHA production under iso-osmotic conditions induced by PEG and NaCl. Curr Microbiol 2014; 68, p. 457-62 [1] Michael J. Haas *, Andrew J. McAloon, Winnie C. Yee, Thomas A. Foglia. A process model to estimate biodiesel production costs. Bioresource Technology .2006,97,p.671–678 [10] Vlachos, N., Skopelitis, Y., Psaroudaki, M., Konstantini- dou, V., Chatzilazarou, A. and Tegou, E. Applica- tions of fourier transforminfrared spectroscopy to edible oils. Analytica Chimica Acta, 2006,573-574,p. 459-465.