 Advances in Technology Innovation, vol. 3, no. 1, 2018, pp. 17 - 25 Prediction Primary Available Blend Biodiesel of Waste Oil from Aurantiochytrium sp. for General Diesel Engines Shu-Yao Tsai 1 , Hsiang-Yu Lin 2 , Guan-Yi Lu 1 , Chun-Ping Lin 1,* 1 Department of Health and Nutrition Biotechnology, Asia University, Taichung, Taiwa n, ROC. 2 Department of Neonatology, Children’s Hospital, China Medical University Hospital, Taichung, Taiwan, ROC. Received 05 June 2017; received in revised form 27 July 2017; accept ed 11 August 2017 Abstract Chemical and enzyme transesterification were compared by discussing preliminary transesterification of waste oil of Aurantiochytrium sp., which was then used in transesterification for the primary available blend biodiesel for a general diesel engine in this study. We made progress on the winterized characteristics of the waste oil’s biodiesel of Aurantiochytrium sp. and its biodiesel, which included the reactivity parameters and properties. This approach led to the development of a novel idea for the evaluation of kinetic parameters of winterization, along with obtaining the suitable operation and storage conditions of biodiesel. Therefore, the waste oil of Aurantiochytrium sp. could be developed for biodiesel production and successfully made into a suitable blend diesel. Overall, we acquired the best condition of mixtures and the highly mixed rate of petrodiesel: biodiesel = 80 : 20 (activation energy of winterization 21.32 kJ/mol; onset temperature of winterization -4.15 °C; heat of combustion 43.15 MJ/kg; kinematic viscosity 3.51 mm 2 /s; flash point 67.5 °C), which was an appropriate blend biodiesel from the waste oil’s biodiesel of Aurantiochytrium sp. Keywords: waste oil, Aurantiochytrium sp., biodiesel, winterization, blend biodiesel 1. Introduction This study focused on Aurantiochytrium sp., which is a type of microalgae, the oil of which is refined for docosahexaenoic acid (DHA) for incorporating into health food or food additives [1]. More than 20 mass% of the solid waste of Aurantiochytrium sp. is discarded as rubbish under current operating procedures in food factories. In this study, we implemented the preliminary transesterification of solid waste of Aurantiochytrium sp., to determine the advantages and disadvantages of chemical and enzyme transesterification. Chemical [1-7] and enzyme [8-15] transesterification were compared to discuss the preliminary transesterification of waste oil of Aurantiochytrium sp., which could then be beneficially used in the development of transesterification for an available mixture of biodiesel. The main stages o f chemical transesterification are as follows [1-7]: (a) saponification fixes the fatty acids, which are used for enhancing the yield of the transesterification reaction and removing impurities, glycerol, and free fatty acid; (b) reduction reaction, which reverts the fatty acids for convenient converting to biodiesel; and (c) acid-catalysed reaction, which makes the fatty acids and methanol for fully transesterifying into the fatty acid methyl ester. The main stages of enzyme transesterification are as follows: (a) triglyceride hydrolysis by hydrolytic enzymes is * Corresponding author. E-m ail address: cp.lin@ asia.edu.tw Tel.: +886-4-23323456; Fax: +886-4-23321206 Advances in Technology Innovation, vol. 3, no. 1, 2018, pp. 17 - 25 Copyright © TAETI 18 used for enhancing the yield of the transesterification reaction and removing impurities, glycerol, and free fatty acid [ 14]; and (b) immobilized enzyme reacts with the fatty acid , which reverts the fatty acids for converting to biodiesel [15]. We also developed the winterized characteristics of the waste oil’s biodiesel of Aurantiochytrium sp. and its biodiesel, which included the reactivity parameters and properties [16-18]. Differential scanning calorimetry (DSC) was used to obtain the winterization temperature and the other detailed enthalpy of the exothermicity of the biodiesel (B100) and the various propor tion of biodiesel for mixing with petrodiesel (B0). The parameters and reactivity propertie s could be applied to the designs of the operation, application, and transportation safety conditions [1, 16-17]. It is an important project for developing high quality available blend biodiesel from the waste oil of Aurantiochytrium sp. An available blend of biodiesel has the appropriate heat of combustion, kinematic viscosity [16], and storage safety [18]. It is an important performance indicator of biodiesel for mixing with B0, which is concerned with effective fuel for diesel engines. Here, the heat of combustion, kinematic viscosity, and storage safety of blend biodiesel were tested by DSC [16-17], flash point tester [18], viscometer [19-20], and oxygen bomb calorimeter [21-23], and these were also compared to the various proportions of B100 for mixing with B0. Then, the best condition of mixtures was obtained as an available blend diesel of waste oil’s biodiesel of Aurantiochytrium sp. 2. Experimental and Method 2.1. Samples The waste oil of Aurantiochytrium sp., which was supplied directly from VEDAN Enterprise Corp. in Taiwan, was stored frozen at –20 °C. The original fatty acid profile of waste oil was conducted by gas chromatography (GC) analysis [ 24]. 2.2. Transesterification First, the fixed fatty acid composition was saponified with the three eq uivalents of sodium hydroxide under ca. 90 °C for three hours. Then, the impurities, the free fatty acids, glycerol, and microalgae cell wall were removed to form the waste oil soap. The oil soap was included in the further transesterification. We then obt ained a high purity fatty acid for the next step of the reduction reaction. Second, the reduction reaction was mixed with sulphuric acid to form fatty acids under 85 °C for three ho urs. Finally, the acid-catalysed reactions were mixed with dil. sulphuric acid (less than 0.02 mass% ratio of fatty acid) and methanol (fatty acids: methanol = 1:5, an amount of methanol more than five -times that of the fatty acids is better) under reflux overnight [1]. For preparation of the 0.1 M of disodium hydrogen phosphate-sodium dihydrogen phosphate buffer solution, the buffer solution was maintained at pH 7. Candida rugose is a type of hydrolytic enzyme of lipase, which showed the preferred results of the hydrolysis reaction under a pH 7 buffer solution [8-15]. A 0.1 mass % of C. rugose was mixed with solid waste oil of Aurantiochytrium sp. (triglycerides), and then the mixture was conducted for 260 rpm and stirring for 12 hours under isothermal 35 °C. From the above results, we removed the solid product and the oil layer of the upper layer (fatty acids), and then mixed this with C. antarctica (lipase acrylic resin of immobilized enzyme) (less than 2 mass% ratio of fatty acid) [15], methanol (fatty acids : methanol = 1:1), and isooctane (fatty acids: isooctane = 2:1) using a reflux apparatus under the isothermal conditions of 35 °C for 260 rpm and stirred for 16 hours . 2.3. Gas chromatography (GC) analysis Gas chromatography (GC) analysis Fatty acid methyl esters (FAMEs) were analysed by gas chromatograph Agilent 6890N (Agilent Technologies, USA) equipped with a flame ionization detector (GC-FID) and a Restek Rt-2340 NB Cap. column (105 m × Advances in Technology Innovation, vol. 3, no. 1, 2018, pp. 17 - 25 Copyright © TAETI 19 0.25 mm × 0.20 µm). The oven temperature was initially set at 170 °C and then programmed to 250 °C using helium as the carrie r gas, at a flow rate of 1.1 mL/min and held for 30 min. The injector and detector temperatures were 250 °C. The split ratio was 1:80. Fatty acids were identified by comparing the retention time of FAME peaks with Supelco 37 FAME mixture standards (Sigma) [1, 16, 24]. 2.4. Differential scanning calorimetry (DSC) tests The temperature–programmed screening experiments were performed with DSC TA Q20-RCS90 (TA Instruments, USA). For the DSC analysis on the samples sealed in 20 μL aluminium pans, the lid was pressed onto the crucible using the pressure of a heavy mechanical force, and the seal tightened the crucible; the test cell was then sealed manually by a special tool equipped with TA’s DSC [1, 16]. Approximately 1.7 mg to 2.5 mg of the sample was used for obtaining the experimental data. The B0 and B100 of the dynamic tests of the scanning rate selected for the programmed temperature ramp we re 2, 4, 6, and 8 °C/min and B2-B50. The scanning rate selected for comparing the programmed temperature ramp was 4 °C/min for the range of temperatures cooling down from 30 °C to -40 °C for each for each winterization phase behaviour experiment. In all of studies with the DSC thermal analysis, high purity nitrogen was the purge gas, and the flow rate was 50 mL/min. 2.5. Diesel, biodiesel, and blend biodiesel winterization k inetic evaluation Here, the proto-kinetic equation was applied to evaluate the win terization of the phase transfer for the exothermic reaction as follows [17]: 1 2 0 (1 ) aE n nRT ir k e      (1) where Ea is the activation energy, k 0 is the pre-exponential factor, R is the ideal gas constant, α is the degree of conversion of a reaction or stage, and n1 and n2 are the reaction order of the phase transfer of the winterized exothermic reaction [17]. 2.6. Biodiesel and blend Biodiesel k inematic viscosity measurement The kinematic viscosity measurement is important for diesel engine fuel, and it is also an important indicator for biodiesel. To charge the sample into the viscometer, we placed the viscometer into the holder for fixing and inserted it into the consta nt temperature bath. The viscometer holder fit a Cannon-Fenske Routine viscometer 75 U534 (CANNON Instrument Company, USA) [16, 19]. We aligned the viscometer vertically in the bath by means of a small plumb bob in the tube. Approximately 10 minutes was allowed for the sample to come to the bath temperature of 40 °C. We repeated this process three times for each sample to measure the efflux time and check the run. 2.7. Biodiesel and blend Biodiesel heat of combustion measurement The heat of combustion analysis of the samples involved a Pa rr 1341 oxygen bomb calorimeter instrument (Parr Instrument Company, USA) [16, 21-22]. We did a heat of combustion analysis for all of the samples as follows. To prepare, the water temperature was approximately 1.5 °C below room temperature, for which it was not necessary to use exactly 2 kg, but the amount selected must be duplicated within ± 0.5 g for each experiment. We opened the filling connection control valve slowly and watched the gauge as the bomb pressure rose to the desired filling pressure (usually 30 bar, but never more than 40 bar). Then, we closed the control valve. Instead of weighing the bucket, it was filled from an automatic pipette or from any other volume tric device if the repeatability of the filling system was within +/ – 0.5 mL and the water temperature was held within a 1 ºC range. We Advances in Technology Innovation, vol. 3, no. 1, 2018, pp. 17 - 25 Copyright © TAETI 20 let the stirrer run for 5 minutes to reach equilibrium before starting a measured run [16, 21-22]. Approximately 0.5 g of all of the samples was used for acquiring the experimental data. At the end of this period, we recorded the time on the timer of the Parr 6775 digital thermometer and read the temperature. 2.8. Flash point measurement The flash point was determined by HFP 360 Pensky -Marten Flash Point Tester (Walter Herzog GmbH, Germany), which met the requirements of the ASTM D93B standard [23, 25], and it was used to estimate the flash points of the B0, B100, and the various proportions of B100 for mixing with B0. The ASTM D93 test method was applied to measure the flash points for each sample [23, 25]. The tester amalgamated control devices programmed the instrument to heat the sample at a specific designated rate within a temperature range close to the expected flash point. The flash point was automatically tested by using an ignit er at specified temperature test intervals. 3. Results and Discussion 3.1. The results of transesterification From the sequences of the saponification, reduction reaction, and acid -catalysed reactions, which were conducted for the full process of transesterification, and excluding the impurities, the free fatty acids, and glycerol, we successfully obtained the biodiesel of waste oil of Aurantiochytrium sp. [1, 16]. Fig. 1(a) shows the fatty acid profile of waste oil from GC analysis, which verified the fatty acid composition of the waste oil and confirmed that the amount of palmitic acid and DHA was more than 75 mass% of total fatty acids [1]. In addition, from Fig. 1(b), the C14-C24 of the fatty acids were more than 92 mass% of the total fatty acid methyl ester profile by chemical transesterification, but via the enzyme transesterification, they were only 60 mass%. (a) Fatty acid composition of waste oil (b) Fatty acid methyl ester composition of waste oil Fig. 1 Fatty acid and fatty acid methyl ester composition of wa ste oil of Aurantiochytrium sp Fig. 2 shows that the DSC tests programmed temperature ramps were 2, 4, 6, and 8 °C/min for the range of temperatures cooling from 30 to -40 °C for each B0 and B100 experiment, respectively. Moreover, Figs . 2 (a) and 2 (b) s how the onset temperature of ca. -6 °C and 20 °C for the winterization of B0 and B100, respectively. B100 was the onset temperature (ca. 20 °C) of winterization slightly below room temperature, which would clog the oil lines and affect the diesel engine pe rformance at the low ambient temperature. Advances in Technology Innovation, vol. 3, no. 1, 2018, pp. 17 - 25 Copyright © TAETI 21 (a) B0 for the range of temperatures cooling down from 30 to -40°C with scanning rates of 2, 4, 6, and 8 °C/min (b) B100 for the range of temperatures cooling down from 30 to -40°C with scanning rates of 2, 4, 6, and 8 °C/min Fig. 2 DSC thermal curves of heat flow versus temperature 3.2. Kinetic parameter of winterization Table 1 Comparisons of the B0 and B100 kinetic parameters for the evaluation with scanning rates of 2, 4, 6, and 8 °C/min Kinetic Parameter Sample Condition a 2 4 6 8 ln(k0)/ln(1/s) b B0 7.6886 7.0606 6.8166 8.0858 Ea c 26.7237 23.8218 22.5479 24.1341 n1 d 1.1179 1.1126 1.0977 1.1352 n2 e 1.0108 1.2552 1.1630 2.7679 ΔΗ f 5.1302 7.6498 7.0392 11.0410 ln(k 0)/ln(1/s) B100 4.1213 4.5116 4.4545 4.7674 Ea 14.8082 14.2822 14.2331 14.6001 n1 1.1501 1.1493 1.1126 1.1045 n2 2.0334 2.5039 2.0072 1.9112 ΔΗ 169.0000 179.6522 162.9779 168.3870 The B0 and B100’s kinetic parameters of winterization were evaluated as listed in Table 1. From Table 1, we compared the Ea and of winterization, B0 and B100 ca. 24 kJ/mol and 14 kJ/mol, respectively. We found that the simulation method of applying the proto-kinetic equation could be appropriately used in the exothermic reaction for winterization of waste oil’s biodiesel of Aurantiochytrium sp. Moreover, from Table 1, the results of the parameter evaluation for proto -kinetic simulation demonstrated that the model provided much more consistent results for B0 and B100. Therefore, while analysing the B0 and B100 kinetic parameters of winterization, we obtained a better model by proto -kinetic simulation in this study. Similar to the various proportions of B100 for mixing with B0 of B2-B50 blend biodiesel, Table 2 and Fig. 3 show that the DSC onset temperature, peak maximum temperature and enthalpy measured for the B2-B50 clearly discriminated the differences. Fig . 3 shows that there was an onset temperature from ca. -6 °C to 6 °C for the winterization of B2-B50 by DSC analyses. An important characteristic of a good blend biodiesel is the ability to avoid the winterization phenomenon under the low ambient temperature. Therefore, while at the ambient low temperature, a reliable blend biodiesel cannot be solidified in the pipeline and tank, wh ich could cause engine failure [1, 16]. In contrast to Tables 1 and 2, we observed the results of the proto -kinetic simulation for B0–B100, in which the kinetic parameters provided clear and specific results. The Ea values of B0-B100 were in the range of 23.82–14.28 kJ/mol. Moreover, Table 2 shows that the result was explicit: B0-B100 of Ea, along with the higher proportion of B100, became smaller. From the above results of the kinetic parameter evaluation of winterization, we obtained the highly proportio nal mixture of B100, which was easy for winterizing the blend biodiesel. Advances in Technology Innovation, vol. 3, no. 1, 2018, pp. 17 - 25 Copyright © TAETI 22 Table 2 Results of DSC test and proto-kinetic equation simulation from B2 to B50 kinetic parameters for the evaluation at a scanning rate of 4 °C/min Sample B2 B5 B8 B10 B20 B30 B50 Mass a 2.0 2.0 2.0 2.0 2.0 1.9 2.0 ExoTo b -6.22 -4.85 -5.71 -5.34 -4.15 -1.03 6.09 ExoTp c -7.31 -14.54 -14.50 -12.66 -7.67 -1.78 5.70 ExoΔH d 10.40 13.50 19.00 22.70 34.20 46.90 81.10 ln(k 0)/ln(1/s) e 7.9899 6.9724 6.5301 6.3565 6.6144 6.3312 5.2386 Ea f 26.8404 23.6787 23.0120 22.4650 21.3152 21.3428 18.7669 n1 g 1.0718 1.1073 1.0886 1.0935 1.1686 1.1335 1.1055 n2 h 0.6631 1.8153 0.9095 1.0190 1.6253 1.4865 2.2704 ΔΗ i 11.2995 18.4967 19.3829 23.1463 34.9629 47.6906 82.7576 Fig. 3 DSC thermal curves of heat flow vers us temperature for the B2-B100 for the range of temperatures cooling down from 30 to -40 °C with a scanning rate of 4 °C/min 3.3. The results of k inematic viscosity measurement From Fig. 4, we obtained the B0-B100 of the kinematic viscosity average valu es range from 3.14 mm 2 /s to 4.35 mm 2 /s. Generally, the biodiesel would be a fuel for a diesel engine. The kinematic viscosity range followed the ASTM D6751 and EN 14214 specification standards [23, 24], from 1.9 mm 2 /s to 6 mm 2 /s and from 3.5 mm 2 /s to 5.0 mm 2 /s, respectively. It was too thin for B2-B10 as a blend biodiesel for EN 14214 specification standards, and it could not provide the appropriate lubrication of diesel engines. We also observed that B20-B100 (3.51-4.35 mm 2 /s), whose values of kinematic viscosity were included in a suitable range, was also concerned with the effective fuel for diesel engine, such as the winterization, the heat of combustion, the f lash point, and the storage conditions of blend biodiesel (see Fig . 4). Then, the best conditions of the mixtures were obtained as an available blend biodiesel in this study. 3.4. Heat of combustion measurement We obtained the best condition of mixtures and the maximum proportion of biodiesel as a good blend biodiesel of waste oil’s biodiesel. In this study, for the heat of combustion of blend biodiesel, the basic value was B0 (43.98 kJ/g), and this value was used as a comparison benchmark for other diesels. From Fig. 4, we obtained the heat of combustion and showed that B100, B50, and B30 were les s than the others, which also showed that they were unsuitable for a blend biodiesel. From the comparisons of all samples heat of combustion, the mixed ratio of blend biodiesel smaller than B20 was acquired. This was the fuel that was suit able for use in compression ignition diesel engines in this study. Meanwhile, from Fig . 4, we observed the B2-B20 (43.30-43.15 MJ/kg), whose values of heat of combustion were included in the suitable range, and this also showed that the highly mixed ratio of b lend biodiesel was B20 (43.15 MJ/kg) in this study. Advances in Technology Innovation, vol. 3, no. 1, 2018, pp. 17 - 25 Copyright © TAETI 23 Biodiesel (mass%) 0 20 40 60 80 100 F la sh p o in t (o C ) 40 60 80 100 120 140 160 180 200 220 K in e m a ti c v is c o si ty ( m m 2 /s ) 2.8 3.0 3.2 3.4 3.6 3.8 4.0 4.2 4.4 4.6 H e a t o f c o m b u st io n ( M J/ k g ) 38 39 40 41 42 43 44 Flash point Flash point regr. r 2 = 0.948, Y=1.26X+48.74 Kinematic viscosity Kinematic viscosity regr. r 2 = 0.968, Y=0.01X+3.20 Heat of combustion Heat of combustion regr. r 2 = 0.975, Y=-0.05X+43.75 Fig. 4 Heat of combustion, kinematic viscosity, and flash points of various proportions of B0 for mixing with B100 3.5. Flash point measurement Here, the results of the flash point tests of B0, B100, and the various proportions of B100 for mixing with B0 also followed the U.S. Department of Transportation (DOT 49 CFR 173.120) regulations [18]. From Fig. 4, we obtained the B0-B100 of the flash point temperature range from 45 °C to 184 °C, individually. From Fig. 4, we also observed the B2-B100 (63-184 °C) of flash points were all above 60.5 °C. This temperature of flash points exceeded the safety condition that was related to a suitable fuel fo r diesel engine. This study also showed that adding the waste oil’s biodiesel of Aurantiochytrium sp. could increase the storage and transport safety of diesel. The above transesterified results of fatty acid via GC analyses and DSC tests repeatedly corrobor ated the accuracy of the transesterification and the specific characteristic, proving that waste solid byproducts of Aurantiochytrium sp. oil formed biodiesel. Then, by conducting the DSC, bomb calorimeter, viscometer, and flash point tester, we compared the winterization, kinematic viscos ity, and heat of combustion of the various proportions of B100 for mixing with B0, which could establish the best conditions of the mixture B20 as an available blend biodiesel. 4. Conclusions The waste oil of Aurantiochytrium sp. can be developed for biodies el production and successfully made into a suitable blend biodiesel. We obtained the best conditions of mixtures and the highly mixed rate of B20 (Ea of winterization 21.32 kJ/mol at DSC scanning rate 4 °C/min; onset temperature of winterization -4.15 °C at DSC scanning rate 4 °C/min; heat of combustion 43.15 MJ/kg; kinematic viscosity 3.51 mm 2 /s; flash point 67.5 °C) as an appropriate blend biodiesel from the waste oil’s biodiesel of Aurantiochytrium sp. Thus, from a green energy perspective, this project addressed the problem of “will not be occupied” arable land, ease of management, and ease of extensive cultivation for the production of biomass. We will enhance the transesterification technology for the waste oil of Aurantiochytrium sp. to find improved energy efficiency, a less polluting method, and appropriate chemical and physical stabilities, which will be used in the mass production of green energy in futur e work. Advances in Technology Innovation, vol. 3, no. 1, 2018, pp. 17 - 25 Copyright © TAETI 24 Acknowledgements The authors are indebted to the donors of National Science Council (NSC), Taiwan, R.O.C. under the contract No s.: MOST 105-2221-E-468-001- & MOST 102-2221-E-468-001-MY2 and the Department of Medical Research, China Medical University Hospital, China Medical University, Taiwan, R.O.C. under the contract No.: DMR-100-165 for financial support. In addition, we are grateful to VEDAN Enterprise Corp. in Taiwan for providing the microalgae oil and its waste oil. References [1] S. Y. Tsai, H. Y. Lin, G. Y. Lu, and C. P. 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