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Wet in situ transesterification of microalgae using ethyl acetate as a co-solvent and reactant
Accepted Manuscript Wet in-situ transesterification of microalgae using ethyl acetate as a co-solvent and reactant Jeongseok Park, Bora Kim, Yong K. Chang, Jae W. Lee PII: DOI: Reference:
S0960-8524(17)30047-0 http://dx.doi.org/10.1016/j.biortech.2017.01.027 BITE 17522
To appear in:
Bioresource Technology
Received Date: Revised Date: Accepted Date:
15 November 2016 11 January 2017 12 January 2017
Please cite this article as: Park, J., Kim, B., Chang, Y.K., Lee, J.W., Wet in-situ transesterification of microalgae using ethyl acetate as a co-solvent and reactant, Bioresource Technology (2017), doi: http://dx.doi.org/10.1016/ j.biortech.2017.01.027
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Wet in-situ transesterification of microalgae using ethyl acetate as a co-solvent and reactant
Jeongseok Parka, Bora Kima, Yong K. Changa,b, Jae W. Leea,*
a
Department of Chemical and Biomolecular Engineering, KAIST, 291 Daehak-ro, Yuseong-
gu, Daejeon, Republic of Korea, 305-701 b
ABC Biomass R&D Center, KAIST, 291 Daehak-ro, Yuseong-gu, Daejeon, Republic of
Korea, 305-701
* Corresponding author Prof. Jae W. Lee Tel: +82-42-350-3940 Fax: +82-42-350-3910 E-mail: [email protected]
1
Abstract This study addresses wet in-situ transesterification of microalgae for the production of biodiesel by introducing ethyl acetate as both reactant and co-solvent. Ethyl acetate and acid catalyst are mixed with wet microalgae in one pot and the mixture is heated for simultaneous lipid extraction and transesterification. As a single reactant and co-solvent, ethyl acetate can provide higher FAEE yield and more saccharification of carbohydrates than the case of binary ethanol and chloroform as a reactant and a co-solvent. The optimal yield was 97.8 wt.% at 114 oC and 4.06 M catalyst with 6.67 ml EtOAC/g dried algae based on experimental results and response surface methodology (RSM). This wet in-situ transesterification of microalgae using ethyl acetate doesn’t require an additional co-solvent and it also promises more economic benefit as combining extraction and transesterification in a single process.
Keyword Biodiesel; Microalgae; Wet in-situ transesterification; Ethyl acetate; Response surface methodology
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1. Introduction Recently, microalgae have been considered as a renewable bio-feedstock for the commercial production of biofuels. Unlike oil crops, microalgae can grow fast and retain a high level of lipid productivity (Liao et al., 2012). They can multiply within 18 to 24 h (Sheehan et al., 1998) and their oil content can reach 80 wt.% of dry biomass at certain conditions (Spolaore et al., 2006). Also microalgae have no competition about an agricultural land to harvest them with other edible crops (Xu & Mi, 2011).
The lipid (triglyceride or fatty acid) oil exists inside a thick cell wall in microalgae (Siaut et al., 2011) and the microalgal cell wall has a complex and rigid structure which is mainly composed of polysaccharide such as cellulose and alginates (Scholz et al., 2014). Therefore, the disruption of the cell wall is needed to extract the lipid and it can be conducted with a physical method such as bead milling or a chemical method like acid pretreatment (Kim et al., 2013; Sathish & Sims, 2012). The extracted lipid oil is transesterified with an acyl acceptor, generating microalgal biodiesel that is the mixture of fatty acid alkyl esters (FAAEs) under catalyst (Lotero et al., 2005). Traditionally, methanol is used as an acyl acceptor due to its low cost and a mixture of fatty acid methyl ester (FAME) and glycerol are produced from transesterification (Encinar et al., 2007; Velasquez-Orta et al., 2013). Recently, alkyl acetate was suggested as a new acyl acceptor to eliminate the glycerol waste (Miesiac et al., 2013; Ranganathan et al., 2008). In case of alkyl acetate, glycerol triacetate is produced as a by-product and it can be directly added into diesel as a fuel additive or used as ingredients for cosmetics and gasoline-antiknock (Casas et al., 2010; Liao et al., 2010).
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Several researchers have proposed in-situ transesterification that is combining extraction and transesterification into a single process to increase the economic feasibility of process(El-Enin et al., 2013; Hidalgo et al., 2014; Kartika et al., 2013; Koberg et al., 2011; Patil et al., 2013). De-watering and harvesting of microalgae are performed for the downstreaming process after growing microalgae in pond, however, water still occupies most of reaction medium and only 10~20 % of microalgae is widely dispersed in water after dewatering (Mata et al., 2010). Since methanol is a polar organic solvent and miscible with water, water in a reaction medium inhibits both extraction and transesterification of lipid. Drying the cell can relieve the water inhibition but it is not desirable because the drying takes up to 77% of overall energy consumption of micro-algal biodiesel production (Lardon et al., 2009). Alternatively, a co-solvent can be added to separate reaction phase from water, extract more lipid and facilitate the mass transfer of reactant (Zhang et al., 2015). In a previous study, chloroform showed the best performance as a co-solvent of wet in-situ transesterification of microalgae (Im et al., 2014). Nonetheless, it is a toxic chemical and finding alternative of chloroform is also necessary for environmentally-benign in-situ transesterification.
This study firstly demonstrated wet in-situ transesterification of microalgae for lipid conversion to biodiesel and saccharification of the cell wall by utilizing only ethyl acetate as a single reactant and solvent. It can not only replace the existing toxic chloroform solvent but also play as a reactant itself for the transesterification with lipid. It was our conjecture that ethyl acetate is miscible with lipid and facilitates the mass transfer of lipid to ethanol that is generated as one of hydrolysis products of ethyl acetate under acidic conditions. The performance of ethyl acetate was compared with that of the pair of reactant ethanol and co4
solvent chloroform for the FAEE yield, the chemical product yield, and saccharification of carbohydrates in the cell. Extensive experiments were conducted by varying the reaction temperature, acid catalyst concentration and amount of ethyl acetate. Response surface methodology (RSM) was applied to find the optimal condition of maximizing the FAEE yield with the verification of experimental data.
2. Materials and methods 2.1. Chemicals and reagents Nannochloropsis gaditana was purchased from AlgaSpring (Netherlands). For acyl acceptors, extra pure grade (99.5%) ethanol and ethyl acetate were purchased from Daejeong (South Korea). 95 wt.% of H2SO4 was purchased from JUNSEI(Japan) and used as an acid catalyst. For comparing the effect of acid catalysts, 35 wt.% HCl and 70 wt.% HNO3 were purchased from OCI (South Korea) and Sigma-Aldrich (USA), respectively. For the gas chromatography (GC) analysis, ethyl heptadecanoate (C17:0 FAEE) was purchased from Sigma-Aldrich (USA) as a standard reagent. For the HPLC analysis, glucose was purchased from Daejeong (South Korea) as a standard reagent.
2.2. Maximum lipid yield using lipid extraction and transesterification To measure the maximum amount of lipid which can be converted into FAEE in N.gaditana, the experiment was conducted based on the Folch’s method (Folch et al., 1957). 10mg dried microalgae was mixed with a 2ml mixture of ethanol and chloroform (1/2 v/v) in the test tube and agitated for 10 min. Then 1ml ethanol and 0.3ml H2SO4 were added into the 5
tube and tube was heated at 100 oC for 30 min. 1ml chloroform including 0.5 mg ethyl heptadecanoate was added as a standard reagent for the GC/MS analysis.
2.3. Wet in-situ transesterification of microalgae 0.75g wet algae (80 wt. % moisture), ethyl acetate and acid catalyst were mixed in a 14mL teflon-sealed tube (Daihan, South Korea). The test tube was heated for 2 hours under several conditions that vary the reaction temperature range (95~155oC), the amount of ethyl acetate (1ml~3ml) and H2SO4 (0.05ml ~ 0.3ml) based on the previous study (Im et al., 2015; Kim et al., 2015a). To compare the performance of ethyl acetate (EtOAC) system with ethanol (EtOH) and chloroform system, 2/1 (v/v) ethanol and chloroform system was selected as a reference group. In addition, nitric acid and hydrochloric acid were assessed to compare the catalyst effect on the FAEE yield with the case of H2SO4. After the reaction, the tube was cooled down at room temperature and sodium hydroxide solution was added to neutralize the reaction medium. 1ml ethyl acetate (for the EtOAC system) or chloroform (for the EtOH/Chloroform system) including 0.5 mg ethyl heptadecanoate was added as a standard reagent for the GC/MS analysis and the test tube was centrifuged at 3700rpm for 10 minutes to separate the phases. FAEE yield was calculated based on equation (1).
FAEE yield = × 100 (%)
(1)
2.4 Chemical analysis For the organic phase, the GC/MS analysis was conducted for 1 µl of the EtOAC layer. The MS analysis was conducted using Clarus 600 (USA) equipped with ELITE-5 column (30.0m X 0.32mm X 0.25µm) followed by the GC analysis using Agilent 7890b 6
(USA) equipped with HP-5 column (30.0m X 0.32mm X 0.26µm). The sample was injected to the FID detector at 280oC and helium was provided as a carrier gas at a flow rate of 2.1ml/min. The initial oven temperature was 50 oC and the oven was heated at a rate of 25 o
C/min up to 175 oC, followed by a heating rate of 4oC/min up to 240 oC. Then the
temperature was maintained for 15min.
The chemical contents were measured by calculating the ratio between standard and target chemicals. The FAEE yield is considered about C14 ~ C22 which is in the range of biodiesel (Ramirez-Verduzco et al., 2012). To analyze the sugar and acetic acid in aqueous phase, the HPLC analysis using ultimate 3000 (USA) equipped with Aminex HPX-87H column (300mm × 7.8mm, Bio-Rad, USA) was conducted. Total carbohydrate concentrations were measured via the phenol-sulfuric acid method with UV-1800 (Shimadzu, Japan).
2.5. Optimization of wet in-situ transesterification Response surface methodology (RSM) was used to optimize the FAEE yield with Design-Expert software 7.0 (Stat-Ease Inc., U.S.A). Based on the previous research (Hidalgo et al., 2013), the FAEE yield response is assumed to be affected by the acid concentration (X1), the reaction temperature (X2) and the amount of ethyl acetate (X3) whose ranges were set based on the experimental data. Using Box-Behnken design (Box & Behnken, 1960), the quadratic model was built with the empirical data and the optimization runs were evaluated by considering the economic constraints (X1, X2, and X3) of maximizing the FAEE yield.
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3. Results and Discussion 3.1. Comparison of ethanol and ethyl acetate systems The saponifiable lipid content of N. gaditana was 120.48 mg/g dry algae determined by the Folch’s method (section 2.2.). In the absence of co-solvent, either ethanol or ethyl acetate was used for wet in-situ transesterification. The variation of the FAEE yield and the generation of acetic acid over the reaction time were shown in Fig. 1. The acetic acid amount was measured in both aqueous phase by HPLC and organic phase by GC. In the case of ethyl acetate, neither ethanol nor chloroform was introduced to the reaction medium at all. With an acid catalyst in a heating condition, ethyl acetate is hydrolyzed into ethanol and acetic acid (Harned & Pfanstiel, 1922). Then the ethanol/ethyl acetate (EtOH/EtOAC) binary system is naturally created for in-situ transesterification. At the early stage of the reaction, the FAEE generation in the ethyl acetate case was lower than in the ethanol case as shown in Fig. 1 and the main product at the initial 30 min was acetic acid. This indicates that at the early stage, the generation of acetic acid is closely related to the hydrolysis of ethyl acetate. However, the acetic acid generation was negligible when only ethanol is used.
The FAEE yield increased sharply in Fig. 1 when EtOAC is used because ethanol, acyl acceptor for FAEE formation, was produced from EtOAC hydrolysis and then the EtOH/EtOAC mixture system is built for the better algal lipid extraction than the single ethyl acetate or ethanol system (Lin et al., 2004).
The polarity of ethanol and ethyl acetate is 0.654 and 0.228, respectively (Reichardt 8
& Welton, 2011). More polar ethanol is suitable to extract phospholipids, whereas less polar solvent ethyl acetate can extract more neutral lipid (Lu et al., 2015). Therefore, the EtOH/EtOAC mixture system can extract more wide range of lipid than the sole ethanol system. Also using only ethanol is unfavorable in wet condition since ethanol is watermiscible so water inhibits the lipid extraction and conversion of FAEE (Park et al., 2016). Water-immiscible ethyl acetate can create the binary phase and minimize the water inhibition during the reaction. Also ethyl acetate itself can directly react with lipid to generate FAEE. Therefore, ethyl acetate as a single co-solvent and reactant showed better FAEE yield for wet in-situ transesterification.
3.2. Ethanol/chloroform and ethyl acetate systems for in-situ transesterification 3.2.1. FAEE yield comparison Sole ethanol is not effective for in-situ transesterification of wet algae due to water inhibition, therefore, a co-solvent is necessary for the higher yield. In the previous study, chloroform was chosen as a co-solvent (Kim et al., 2015a) since the polarity of chloroform is 0.259 and it is also water immiscible like ethyl acetate (Reichardt & Welton, 2011). Both binary ethanol/chloroform (EtOH/Chloroform) system and single ethyl acetate (EtOAC) system were assessed for dry and wet in-situ transesterification. In case of dry algae, there was no clear difference between EtOH/Chloroform and EtOAC systems and 100% FAEE yield was achieved for both cases (Fig. 2.a). However, the yield decreased for both systems in wet algal condition due to water inhibition. In wet algal condition, the EtOAC system showed higher FAEE yield than the EtOH/Chloroform system especially in the range of C14~C18 (Fig. 2.b). Because ethyl acetate is very selective for neutral lipids which is the target range of 9
biodiesel, whereas chloroform can extract broader range of lipids including phospholipid or glycolipid (Lu et al., 2015). Therefore, ethyl acetate is more suitable reactant/solvent for wet in-situ transesterification of microalgae than EtOH/Chloroform and can eliminate the usage of the toxic solvent of chloroform while the acetate itself is hydrolyzed to ethanol as a reactant.
3.2.2 Other chemicals Besides FAEE, triacetin and ethyl levulinate (EL) were detected in the organic phase by GC/MS. The generation of these chemicals was dependent on the moisture content of algae, the type of acyl acceptor (EtOAC or EtOH) and the amount of sulfuric acid (Table 1).
Triacetin is directly produced by transesterification between ethyl acetate and lipid triglyceride. It can be directly blended into biodiesel as an additive to adjust the cold property and the cetane number (Casas et al., 2010). Several studies already showed the generation of triacetin from the biodiesel production process using ethyl acetate, however, their input feedstock was dry oils such as rapseed oil and jatropha oil (Miesiac et al., 2013; Modi et al., 2007). Under the wet condition, triacetin is unstable and its yield is very low due to its acidic hydrolysis (Yamasaki, 1920) as shown in Table 1 while the triacetin yield is very high (231 mg/g dried algae) with the dry algae.
Ethyl levulinate (EL) can be used as a flavoring chemical or a biodiesel additive and it is derived from esterification of levulinic acid which is one of the products of cellulose 10
hydrolysis (Joshi et al., 2011; Leonard, 1956). Since N. gaditana has a cellulose-rich cell wall (Scholz et al., 2014), EL was generated during in-situ transesterification with EtOH/Chloroform by the saccharification of the cell wall (Alonso et al., 2013; Im et al., 2015). In this study, using EtOAC in wet in-situ transesterification also led to the generation of EL. The dry condition with 9.39M H2SO4 showed higher EL yield (Table 1) because water can induce the reverse reaction and EL decomposes into levulinic acid and ethanol. The total carbohydrate content was 128.2 mg/g dried algae measured by the phenol-sulfuric acid analysis. During wet in-situ transesterification with 1.56M H2SO4, more carbohydrates were saccharified using EtOAC (50.67%) than EtOH/Chloroform (43.12%) since less ethanol that dilutes the acid concentration exists in the water phase (Fig. 3). Thus, the ethyl acetate system has advantage of not only having higher FAEE yield but also more saccharification from wet in-situ transesterification of microalgae.
3.3. Effect of acid catalyst on the FAEE yield Transesterification is initiated by the protonation of carbon under a strong acid catalyst (Lotero et al., 2005). To figure out the effect of type of acid catalyst, each 3.12M of hydrochloric acid, nitric acid and sulfuric acid solution was added to the mixture of 13.3ml EtOAC/g dried algae at 125oC. As shown in Fig. 4, hydrochloric acid showed similar performance compared to sulfuric acid, therefore, it can be used as an alternative acid catalyst for sulfuric acid. Although nitric acid can oxidize other chemicals and decompose the chlorophyll which is an impurity of biodiesel (Lee et al., 2014), it showed low FAEE yields for wet in-situ transesterification with ethyl acetate. Due to the oxidation cleavage of FAEE, shorter chain (C6~C14) FAEEs were generated (Park et al., 2016). These shorter chain 11
FAEEs are not in the range of biodiesel, therefore, nitric acid is not a relevant acid catalyst for wet in-situ transesterification of microalgae.
The acid catalyst concentration affects the hydrolysis of not only cellulosic cell wall, but also ethyl acetate. The hydrolysis conversion was calculated by measuring acetic acid in both organic and water phases after the reaction. First, a mixture of 0.6g water, 2ml ethyl acetate and sulfuric acid (1.56M, 5.47M and 9.39M) without algal cells was heated for 2 hours to see the effect of different H2SO4 concentrations on the EtOAC hydrolysis. The initial mole fraction is 0.381 and 0.619 for EtOAC and water (EtOAC = 2ml and water = 0.6 ml). After the hydrolysis with 1.56M H2SO4, the equilibrium mole fraction is 0.259, 0.497, 0.122 and 0.122 for EtOAC, water, acetic acid and EtOH, respectively. Still about 40 % (0.23 ml) of the initial water (0.6 ml) is left and the moisture content is around 60 wt. % in the microalgae if the hydrolysis of EtOAC occurs at the same extent as in the system without the algal cell. The moisture content can be reduced to 60 wt.% starting from 80 wt. % but the algal cell system is still wet. The wet (not dry) in-situ transesterification still occurs in the mixed system of wet algal cells, EtOH/EtOAC, acetic acid, and H2SO4. Since acetic acid that is also an acidic component (proton donor) is generated from the hydrolysis, the higher H2SO4 concentration inhibits the hydrolysis of EtOAC in Fig. 5.
For the wet in-situ transesterification which microalgae were added, EtOAC hydrolysis conversion was 75.45±0.88% for 1.56M H2SO4 and it decreased to 67.02 ±2.23% for 9.39M H2SO4. The overall hydrolysis extent was increased because ethanol, one of ethyl acetate hydrolysis products, is continuously consumed during transesterification and the 12
reaction goes forward direction. Also, distribution of acetic acid into water and organic phase was different with the amount of the acid catalyst. 92.8% of acetic acid was present in the aqueous phase for 1.56M H2SO4 and it decreased to 88.6% in the aqueous phase for 9.39M H2SO4. In a previous study, the high acid catalyst concentration showed high FAEE yields when ethanol is used (Im et al., 2015). However, the high acid (catalyst) amount hinders the hydrolysis of EtOAC. Therefore, an optimal acid catalyst concentration exists when ethyl acetate is employed as both reactant and solvent (Section 3.4).
3.4. Optimization of wet in-situ transesterification condition for the maximum FAEE yield Not only the acid catalyst concentration but also the amount of acyl acceptor and the reaction temperature are main factors that affect the FAEE yield in wet in-situ transesterification (Im et al., 2014; Kim et al., 2015a). To figure out the relation between these factors and the optimal condition for maximizing the FAEE yield, responses of the FAEE yield with varying wet in-situ transesterification conditions were modeled using Design-Expert 7.0. As shown in Table 2, analytical data are statistically calculated based on the following quadratic model: # = 93.24 + 5.53+, + 6.65+. − 12.32+0 − 25.09+, +. + 6.06+,+0 − 7.37+. +0 − 24.52+,. + 1.95+.. + 1.07+0. where Y, X1, X2, and X3 are the FAEE yield (%), the H2SO4 concentration (M), the reaction temperature (oC) and the amount of ethyl acetate (ml EtOAC/g dried algae), respectively.
ANOVA tests were conducted to determine the fitness of non-linear regression and 13
significance of each coefficient (Table 3). This quadratic model was statistically well-fitted since the R2 coefficient was 0.9971 and the F-value is 265.22. Furthermore, the insignificant lack of fit can show the adequacy of RSM for experimental results. All coefficients were significant (p < 0.05) and the interaction between the factors cannot be ignored. This correlation means that the acid catalyst concentration, the reaction temperature and the amount of ethyl acetate should be dealt together since they are not independent for each other. The optimal H2SO4 concentration is slightly changing depending on the EtOAC amount at a given temperature due to the balance between EtOAC hydrolysis and transesterification (Fig. 6a). Also higher reaction temperature allows higher conversions because both hydrolysis of ethyl acetate and transesterification are endothermic (Euranto et al., 1986; Xiao et al., 2010). Therefore, lower sulfuric acid catalyst concentration than 5.47M (middle point in the model) is possible to maintain at least 90% FAEE yield if the temperature is at least 107.3oC (Fig. 6b). Except for the fixed acid concentration case (Fig. 6c), the saddle-like graph means the several conditions can be optimal for the FAEE yield. With the acid concentration fixed (Fig. 6c), increasing ethanol that is a product of the EtOAC hydrolysis dilutes the acid catalyst in the aqueous phase and then the FAEE yield decreases.
The optimization run was conducted considering the maximization of the FAEE yield subject to the minimization of the acid catalyst amount, the reaction temperature and the amount of ethyl acetate. Based on these constraints, the optimal condition for wet in-situ transesterification was 4.06M H2SO4 and 6.67ml EtOAC/g dried algae at 113.6oC. Compared to one previous study with 6.12M H2SO4 and 42.55ml EtOH/g dried algae at 100 oC (Kim et al., 2015b), the amounts of reactant and acid catalyst were significantly reduced while the 14
similar FAEE yield was found with a slightly higher temperature. Therefore, ethyl acetate can provide more economic benefits for wet in-situ transesterification of microalgae allowing less acid catalyst usage. The experimental FAEE yield was 97.8 ± 4.57 % at that optimal condition and the quadratic model showed a good prediction capability (97.1% FAEE yield optimized by RSM).
4. Conclusions Ethyl acetate can replace the binary ethanol/chloroform system for wet in-situ transesterification of microalgae as both reactant and co-solvent. The acidic hydrolysis of ethyl acetate builds up the EtOH/EtOAC mixture allowing higher FAEE yield and more saccharification of carbohydrates of wet microalgae. Verified by both RSM and experiments, 117 mg FAEE/g dried algae (97.1% FAEE yield) was the optimal yield at 113.6oC and 4.06M of sulfuric acid catalyst with 6.67ml EtOAC/g dried algae. Using ethyl acetate in wet in-situ transesterification contributes not only the feasible biodiesel production eliminating additional
co-solvent
but
also
economical
transesterification process.
15
process
combining
extraction
and
Acknowledgement This work was supported by the Advanced Biomass R&D Center (ABC) as the Global Frontier Project funded by the Ministry of Science, ICT and Future Planning (ABC2011-0031348).
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Table 1. Triacetin and ethyl levulinate (EL) yield (mg/ g dried algae) via wet in-situ transesterification with EtOAC or EtOH/Chloroform systems Ethyl acetate a
S0960-8524(17)30047-0 http://dx.doi.org/10.1016/j.biortech.2017.01.027 BITE 17522
To appear in:
Bioresource Technology
Received Date: Revised Date: Accepted Date:
15 November 2016 11 January 2017 12 January 2017
Please cite this article as: Park, J., Kim, B., Chang, Y.K., Lee, J.W., Wet in-situ transesterification of microalgae using ethyl acetate as a co-solvent and reactant, Bioresource Technology (2017), doi: http://dx.doi.org/10.1016/ j.biortech.2017.01.027
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Wet in-situ transesterification of microalgae using ethyl acetate as a co-solvent and reactant
Jeongseok Parka, Bora Kima, Yong K. Changa,b, Jae W. Leea,*
a
Department of Chemical and Biomolecular Engineering, KAIST, 291 Daehak-ro, Yuseong-
gu, Daejeon, Republic of Korea, 305-701 b
ABC Biomass R&D Center, KAIST, 291 Daehak-ro, Yuseong-gu, Daejeon, Republic of
Korea, 305-701
* Corresponding author Prof. Jae W. Lee Tel: +82-42-350-3940 Fax: +82-42-350-3910 E-mail: [email protected]
1
Abstract This study addresses wet in-situ transesterification of microalgae for the production of biodiesel by introducing ethyl acetate as both reactant and co-solvent. Ethyl acetate and acid catalyst are mixed with wet microalgae in one pot and the mixture is heated for simultaneous lipid extraction and transesterification. As a single reactant and co-solvent, ethyl acetate can provide higher FAEE yield and more saccharification of carbohydrates than the case of binary ethanol and chloroform as a reactant and a co-solvent. The optimal yield was 97.8 wt.% at 114 oC and 4.06 M catalyst with 6.67 ml EtOAC/g dried algae based on experimental results and response surface methodology (RSM). This wet in-situ transesterification of microalgae using ethyl acetate doesn’t require an additional co-solvent and it also promises more economic benefit as combining extraction and transesterification in a single process.
Keyword Biodiesel; Microalgae; Wet in-situ transesterification; Ethyl acetate; Response surface methodology
2
1. Introduction Recently, microalgae have been considered as a renewable bio-feedstock for the commercial production of biofuels. Unlike oil crops, microalgae can grow fast and retain a high level of lipid productivity (Liao et al., 2012). They can multiply within 18 to 24 h (Sheehan et al., 1998) and their oil content can reach 80 wt.% of dry biomass at certain conditions (Spolaore et al., 2006). Also microalgae have no competition about an agricultural land to harvest them with other edible crops (Xu & Mi, 2011).
The lipid (triglyceride or fatty acid) oil exists inside a thick cell wall in microalgae (Siaut et al., 2011) and the microalgal cell wall has a complex and rigid structure which is mainly composed of polysaccharide such as cellulose and alginates (Scholz et al., 2014). Therefore, the disruption of the cell wall is needed to extract the lipid and it can be conducted with a physical method such as bead milling or a chemical method like acid pretreatment (Kim et al., 2013; Sathish & Sims, 2012). The extracted lipid oil is transesterified with an acyl acceptor, generating microalgal biodiesel that is the mixture of fatty acid alkyl esters (FAAEs) under catalyst (Lotero et al., 2005). Traditionally, methanol is used as an acyl acceptor due to its low cost and a mixture of fatty acid methyl ester (FAME) and glycerol are produced from transesterification (Encinar et al., 2007; Velasquez-Orta et al., 2013). Recently, alkyl acetate was suggested as a new acyl acceptor to eliminate the glycerol waste (Miesiac et al., 2013; Ranganathan et al., 2008). In case of alkyl acetate, glycerol triacetate is produced as a by-product and it can be directly added into diesel as a fuel additive or used as ingredients for cosmetics and gasoline-antiknock (Casas et al., 2010; Liao et al., 2010).
3
Several researchers have proposed in-situ transesterification that is combining extraction and transesterification into a single process to increase the economic feasibility of process(El-Enin et al., 2013; Hidalgo et al., 2014; Kartika et al., 2013; Koberg et al., 2011; Patil et al., 2013). De-watering and harvesting of microalgae are performed for the downstreaming process after growing microalgae in pond, however, water still occupies most of reaction medium and only 10~20 % of microalgae is widely dispersed in water after dewatering (Mata et al., 2010). Since methanol is a polar organic solvent and miscible with water, water in a reaction medium inhibits both extraction and transesterification of lipid. Drying the cell can relieve the water inhibition but it is not desirable because the drying takes up to 77% of overall energy consumption of micro-algal biodiesel production (Lardon et al., 2009). Alternatively, a co-solvent can be added to separate reaction phase from water, extract more lipid and facilitate the mass transfer of reactant (Zhang et al., 2015). In a previous study, chloroform showed the best performance as a co-solvent of wet in-situ transesterification of microalgae (Im et al., 2014). Nonetheless, it is a toxic chemical and finding alternative of chloroform is also necessary for environmentally-benign in-situ transesterification.
This study firstly demonstrated wet in-situ transesterification of microalgae for lipid conversion to biodiesel and saccharification of the cell wall by utilizing only ethyl acetate as a single reactant and solvent. It can not only replace the existing toxic chloroform solvent but also play as a reactant itself for the transesterification with lipid. It was our conjecture that ethyl acetate is miscible with lipid and facilitates the mass transfer of lipid to ethanol that is generated as one of hydrolysis products of ethyl acetate under acidic conditions. The performance of ethyl acetate was compared with that of the pair of reactant ethanol and co4
solvent chloroform for the FAEE yield, the chemical product yield, and saccharification of carbohydrates in the cell. Extensive experiments were conducted by varying the reaction temperature, acid catalyst concentration and amount of ethyl acetate. Response surface methodology (RSM) was applied to find the optimal condition of maximizing the FAEE yield with the verification of experimental data.
2. Materials and methods 2.1. Chemicals and reagents Nannochloropsis gaditana was purchased from AlgaSpring (Netherlands). For acyl acceptors, extra pure grade (99.5%) ethanol and ethyl acetate were purchased from Daejeong (South Korea). 95 wt.% of H2SO4 was purchased from JUNSEI(Japan) and used as an acid catalyst. For comparing the effect of acid catalysts, 35 wt.% HCl and 70 wt.% HNO3 were purchased from OCI (South Korea) and Sigma-Aldrich (USA), respectively. For the gas chromatography (GC) analysis, ethyl heptadecanoate (C17:0 FAEE) was purchased from Sigma-Aldrich (USA) as a standard reagent. For the HPLC analysis, glucose was purchased from Daejeong (South Korea) as a standard reagent.
2.2. Maximum lipid yield using lipid extraction and transesterification To measure the maximum amount of lipid which can be converted into FAEE in N.gaditana, the experiment was conducted based on the Folch’s method (Folch et al., 1957). 10mg dried microalgae was mixed with a 2ml mixture of ethanol and chloroform (1/2 v/v) in the test tube and agitated for 10 min. Then 1ml ethanol and 0.3ml H2SO4 were added into the 5
tube and tube was heated at 100 oC for 30 min. 1ml chloroform including 0.5 mg ethyl heptadecanoate was added as a standard reagent for the GC/MS analysis.
2.3. Wet in-situ transesterification of microalgae 0.75g wet algae (80 wt. % moisture), ethyl acetate and acid catalyst were mixed in a 14mL teflon-sealed tube (Daihan, South Korea). The test tube was heated for 2 hours under several conditions that vary the reaction temperature range (95~155oC), the amount of ethyl acetate (1ml~3ml) and H2SO4 (0.05ml ~ 0.3ml) based on the previous study (Im et al., 2015; Kim et al., 2015a). To compare the performance of ethyl acetate (EtOAC) system with ethanol (EtOH) and chloroform system, 2/1 (v/v) ethanol and chloroform system was selected as a reference group. In addition, nitric acid and hydrochloric acid were assessed to compare the catalyst effect on the FAEE yield with the case of H2SO4. After the reaction, the tube was cooled down at room temperature and sodium hydroxide solution was added to neutralize the reaction medium. 1ml ethyl acetate (for the EtOAC system) or chloroform (for the EtOH/Chloroform system) including 0.5 mg ethyl heptadecanoate was added as a standard reagent for the GC/MS analysis and the test tube was centrifuged at 3700rpm for 10 minutes to separate the phases. FAEE yield was calculated based on equation (1).
FAEE yield = × 100 (%)
(1)
2.4 Chemical analysis For the organic phase, the GC/MS analysis was conducted for 1 µl of the EtOAC layer. The MS analysis was conducted using Clarus 600 (USA) equipped with ELITE-5 column (30.0m X 0.32mm X 0.25µm) followed by the GC analysis using Agilent 7890b 6
(USA) equipped with HP-5 column (30.0m X 0.32mm X 0.26µm). The sample was injected to the FID detector at 280oC and helium was provided as a carrier gas at a flow rate of 2.1ml/min. The initial oven temperature was 50 oC and the oven was heated at a rate of 25 o
C/min up to 175 oC, followed by a heating rate of 4oC/min up to 240 oC. Then the
temperature was maintained for 15min.
The chemical contents were measured by calculating the ratio between standard and target chemicals. The FAEE yield is considered about C14 ~ C22 which is in the range of biodiesel (Ramirez-Verduzco et al., 2012). To analyze the sugar and acetic acid in aqueous phase, the HPLC analysis using ultimate 3000 (USA) equipped with Aminex HPX-87H column (300mm × 7.8mm, Bio-Rad, USA) was conducted. Total carbohydrate concentrations were measured via the phenol-sulfuric acid method with UV-1800 (Shimadzu, Japan).
2.5. Optimization of wet in-situ transesterification Response surface methodology (RSM) was used to optimize the FAEE yield with Design-Expert software 7.0 (Stat-Ease Inc., U.S.A). Based on the previous research (Hidalgo et al., 2013), the FAEE yield response is assumed to be affected by the acid concentration (X1), the reaction temperature (X2) and the amount of ethyl acetate (X3) whose ranges were set based on the experimental data. Using Box-Behnken design (Box & Behnken, 1960), the quadratic model was built with the empirical data and the optimization runs were evaluated by considering the economic constraints (X1, X2, and X3) of maximizing the FAEE yield.
7
3. Results and Discussion 3.1. Comparison of ethanol and ethyl acetate systems The saponifiable lipid content of N. gaditana was 120.48 mg/g dry algae determined by the Folch’s method (section 2.2.). In the absence of co-solvent, either ethanol or ethyl acetate was used for wet in-situ transesterification. The variation of the FAEE yield and the generation of acetic acid over the reaction time were shown in Fig. 1. The acetic acid amount was measured in both aqueous phase by HPLC and organic phase by GC. In the case of ethyl acetate, neither ethanol nor chloroform was introduced to the reaction medium at all. With an acid catalyst in a heating condition, ethyl acetate is hydrolyzed into ethanol and acetic acid (Harned & Pfanstiel, 1922). Then the ethanol/ethyl acetate (EtOH/EtOAC) binary system is naturally created for in-situ transesterification. At the early stage of the reaction, the FAEE generation in the ethyl acetate case was lower than in the ethanol case as shown in Fig. 1 and the main product at the initial 30 min was acetic acid. This indicates that at the early stage, the generation of acetic acid is closely related to the hydrolysis of ethyl acetate. However, the acetic acid generation was negligible when only ethanol is used.
The FAEE yield increased sharply in Fig. 1 when EtOAC is used because ethanol, acyl acceptor for FAEE formation, was produced from EtOAC hydrolysis and then the EtOH/EtOAC mixture system is built for the better algal lipid extraction than the single ethyl acetate or ethanol system (Lin et al., 2004).
The polarity of ethanol and ethyl acetate is 0.654 and 0.228, respectively (Reichardt 8
& Welton, 2011). More polar ethanol is suitable to extract phospholipids, whereas less polar solvent ethyl acetate can extract more neutral lipid (Lu et al., 2015). Therefore, the EtOH/EtOAC mixture system can extract more wide range of lipid than the sole ethanol system. Also using only ethanol is unfavorable in wet condition since ethanol is watermiscible so water inhibits the lipid extraction and conversion of FAEE (Park et al., 2016). Water-immiscible ethyl acetate can create the binary phase and minimize the water inhibition during the reaction. Also ethyl acetate itself can directly react with lipid to generate FAEE. Therefore, ethyl acetate as a single co-solvent and reactant showed better FAEE yield for wet in-situ transesterification.
3.2. Ethanol/chloroform and ethyl acetate systems for in-situ transesterification 3.2.1. FAEE yield comparison Sole ethanol is not effective for in-situ transesterification of wet algae due to water inhibition, therefore, a co-solvent is necessary for the higher yield. In the previous study, chloroform was chosen as a co-solvent (Kim et al., 2015a) since the polarity of chloroform is 0.259 and it is also water immiscible like ethyl acetate (Reichardt & Welton, 2011). Both binary ethanol/chloroform (EtOH/Chloroform) system and single ethyl acetate (EtOAC) system were assessed for dry and wet in-situ transesterification. In case of dry algae, there was no clear difference between EtOH/Chloroform and EtOAC systems and 100% FAEE yield was achieved for both cases (Fig. 2.a). However, the yield decreased for both systems in wet algal condition due to water inhibition. In wet algal condition, the EtOAC system showed higher FAEE yield than the EtOH/Chloroform system especially in the range of C14~C18 (Fig. 2.b). Because ethyl acetate is very selective for neutral lipids which is the target range of 9
biodiesel, whereas chloroform can extract broader range of lipids including phospholipid or glycolipid (Lu et al., 2015). Therefore, ethyl acetate is more suitable reactant/solvent for wet in-situ transesterification of microalgae than EtOH/Chloroform and can eliminate the usage of the toxic solvent of chloroform while the acetate itself is hydrolyzed to ethanol as a reactant.
3.2.2 Other chemicals Besides FAEE, triacetin and ethyl levulinate (EL) were detected in the organic phase by GC/MS. The generation of these chemicals was dependent on the moisture content of algae, the type of acyl acceptor (EtOAC or EtOH) and the amount of sulfuric acid (Table 1).
Triacetin is directly produced by transesterification between ethyl acetate and lipid triglyceride. It can be directly blended into biodiesel as an additive to adjust the cold property and the cetane number (Casas et al., 2010). Several studies already showed the generation of triacetin from the biodiesel production process using ethyl acetate, however, their input feedstock was dry oils such as rapseed oil and jatropha oil (Miesiac et al., 2013; Modi et al., 2007). Under the wet condition, triacetin is unstable and its yield is very low due to its acidic hydrolysis (Yamasaki, 1920) as shown in Table 1 while the triacetin yield is very high (231 mg/g dried algae) with the dry algae.
Ethyl levulinate (EL) can be used as a flavoring chemical or a biodiesel additive and it is derived from esterification of levulinic acid which is one of the products of cellulose 10
hydrolysis (Joshi et al., 2011; Leonard, 1956). Since N. gaditana has a cellulose-rich cell wall (Scholz et al., 2014), EL was generated during in-situ transesterification with EtOH/Chloroform by the saccharification of the cell wall (Alonso et al., 2013; Im et al., 2015). In this study, using EtOAC in wet in-situ transesterification also led to the generation of EL. The dry condition with 9.39M H2SO4 showed higher EL yield (Table 1) because water can induce the reverse reaction and EL decomposes into levulinic acid and ethanol. The total carbohydrate content was 128.2 mg/g dried algae measured by the phenol-sulfuric acid analysis. During wet in-situ transesterification with 1.56M H2SO4, more carbohydrates were saccharified using EtOAC (50.67%) than EtOH/Chloroform (43.12%) since less ethanol that dilutes the acid concentration exists in the water phase (Fig. 3). Thus, the ethyl acetate system has advantage of not only having higher FAEE yield but also more saccharification from wet in-situ transesterification of microalgae.
3.3. Effect of acid catalyst on the FAEE yield Transesterification is initiated by the protonation of carbon under a strong acid catalyst (Lotero et al., 2005). To figure out the effect of type of acid catalyst, each 3.12M of hydrochloric acid, nitric acid and sulfuric acid solution was added to the mixture of 13.3ml EtOAC/g dried algae at 125oC. As shown in Fig. 4, hydrochloric acid showed similar performance compared to sulfuric acid, therefore, it can be used as an alternative acid catalyst for sulfuric acid. Although nitric acid can oxidize other chemicals and decompose the chlorophyll which is an impurity of biodiesel (Lee et al., 2014), it showed low FAEE yields for wet in-situ transesterification with ethyl acetate. Due to the oxidation cleavage of FAEE, shorter chain (C6~C14) FAEEs were generated (Park et al., 2016). These shorter chain 11
FAEEs are not in the range of biodiesel, therefore, nitric acid is not a relevant acid catalyst for wet in-situ transesterification of microalgae.
The acid catalyst concentration affects the hydrolysis of not only cellulosic cell wall, but also ethyl acetate. The hydrolysis conversion was calculated by measuring acetic acid in both organic and water phases after the reaction. First, a mixture of 0.6g water, 2ml ethyl acetate and sulfuric acid (1.56M, 5.47M and 9.39M) without algal cells was heated for 2 hours to see the effect of different H2SO4 concentrations on the EtOAC hydrolysis. The initial mole fraction is 0.381 and 0.619 for EtOAC and water (EtOAC = 2ml and water = 0.6 ml). After the hydrolysis with 1.56M H2SO4, the equilibrium mole fraction is 0.259, 0.497, 0.122 and 0.122 for EtOAC, water, acetic acid and EtOH, respectively. Still about 40 % (0.23 ml) of the initial water (0.6 ml) is left and the moisture content is around 60 wt. % in the microalgae if the hydrolysis of EtOAC occurs at the same extent as in the system without the algal cell. The moisture content can be reduced to 60 wt.% starting from 80 wt. % but the algal cell system is still wet. The wet (not dry) in-situ transesterification still occurs in the mixed system of wet algal cells, EtOH/EtOAC, acetic acid, and H2SO4. Since acetic acid that is also an acidic component (proton donor) is generated from the hydrolysis, the higher H2SO4 concentration inhibits the hydrolysis of EtOAC in Fig. 5.
For the wet in-situ transesterification which microalgae were added, EtOAC hydrolysis conversion was 75.45±0.88% for 1.56M H2SO4 and it decreased to 67.02 ±2.23% for 9.39M H2SO4. The overall hydrolysis extent was increased because ethanol, one of ethyl acetate hydrolysis products, is continuously consumed during transesterification and the 12
reaction goes forward direction. Also, distribution of acetic acid into water and organic phase was different with the amount of the acid catalyst. 92.8% of acetic acid was present in the aqueous phase for 1.56M H2SO4 and it decreased to 88.6% in the aqueous phase for 9.39M H2SO4. In a previous study, the high acid catalyst concentration showed high FAEE yields when ethanol is used (Im et al., 2015). However, the high acid (catalyst) amount hinders the hydrolysis of EtOAC. Therefore, an optimal acid catalyst concentration exists when ethyl acetate is employed as both reactant and solvent (Section 3.4).
3.4. Optimization of wet in-situ transesterification condition for the maximum FAEE yield Not only the acid catalyst concentration but also the amount of acyl acceptor and the reaction temperature are main factors that affect the FAEE yield in wet in-situ transesterification (Im et al., 2014; Kim et al., 2015a). To figure out the relation between these factors and the optimal condition for maximizing the FAEE yield, responses of the FAEE yield with varying wet in-situ transesterification conditions were modeled using Design-Expert 7.0. As shown in Table 2, analytical data are statistically calculated based on the following quadratic model: # = 93.24 + 5.53+, + 6.65+. − 12.32+0 − 25.09+, +. + 6.06+,+0 − 7.37+. +0 − 24.52+,. + 1.95+.. + 1.07+0. where Y, X1, X2, and X3 are the FAEE yield (%), the H2SO4 concentration (M), the reaction temperature (oC) and the amount of ethyl acetate (ml EtOAC/g dried algae), respectively.
ANOVA tests were conducted to determine the fitness of non-linear regression and 13
significance of each coefficient (Table 3). This quadratic model was statistically well-fitted since the R2 coefficient was 0.9971 and the F-value is 265.22. Furthermore, the insignificant lack of fit can show the adequacy of RSM for experimental results. All coefficients were significant (p < 0.05) and the interaction between the factors cannot be ignored. This correlation means that the acid catalyst concentration, the reaction temperature and the amount of ethyl acetate should be dealt together since they are not independent for each other. The optimal H2SO4 concentration is slightly changing depending on the EtOAC amount at a given temperature due to the balance between EtOAC hydrolysis and transesterification (Fig. 6a). Also higher reaction temperature allows higher conversions because both hydrolysis of ethyl acetate and transesterification are endothermic (Euranto et al., 1986; Xiao et al., 2010). Therefore, lower sulfuric acid catalyst concentration than 5.47M (middle point in the model) is possible to maintain at least 90% FAEE yield if the temperature is at least 107.3oC (Fig. 6b). Except for the fixed acid concentration case (Fig. 6c), the saddle-like graph means the several conditions can be optimal for the FAEE yield. With the acid concentration fixed (Fig. 6c), increasing ethanol that is a product of the EtOAC hydrolysis dilutes the acid catalyst in the aqueous phase and then the FAEE yield decreases.
The optimization run was conducted considering the maximization of the FAEE yield subject to the minimization of the acid catalyst amount, the reaction temperature and the amount of ethyl acetate. Based on these constraints, the optimal condition for wet in-situ transesterification was 4.06M H2SO4 and 6.67ml EtOAC/g dried algae at 113.6oC. Compared to one previous study with 6.12M H2SO4 and 42.55ml EtOH/g dried algae at 100 oC (Kim et al., 2015b), the amounts of reactant and acid catalyst were significantly reduced while the 14
similar FAEE yield was found with a slightly higher temperature. Therefore, ethyl acetate can provide more economic benefits for wet in-situ transesterification of microalgae allowing less acid catalyst usage. The experimental FAEE yield was 97.8 ± 4.57 % at that optimal condition and the quadratic model showed a good prediction capability (97.1% FAEE yield optimized by RSM).
4. Conclusions Ethyl acetate can replace the binary ethanol/chloroform system for wet in-situ transesterification of microalgae as both reactant and co-solvent. The acidic hydrolysis of ethyl acetate builds up the EtOH/EtOAC mixture allowing higher FAEE yield and more saccharification of carbohydrates of wet microalgae. Verified by both RSM and experiments, 117 mg FAEE/g dried algae (97.1% FAEE yield) was the optimal yield at 113.6oC and 4.06M of sulfuric acid catalyst with 6.67ml EtOAC/g dried algae. Using ethyl acetate in wet in-situ transesterification contributes not only the feasible biodiesel production eliminating additional
co-solvent
but
also
economical
transesterification process.
15
process
combining
extraction
and
Acknowledgement This work was supported by the Advanced Biomass R&D Center (ABC) as the Global Frontier Project funded by the Ministry of Science, ICT and Future Planning (ABC2011-0031348).
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