Bioresource Technology xxx (2015) xxx–xxx
Contents lists available at ScienceDirect
Bioresource Technology journal homepage: www.elsevier.com/locate/biortech
Lipid extraction and esterification for microalgae-based biodiesel production using pyrite (FeS2) Yeong Hwan Seo a, Mina Sung a, You-Kwan Oh b, Jong-In Han a,⇑ a b
Department of Civil and Environmental Engineering, KAIST, 373-1, Guseong-dong, Yuseong-gu, Daejeon 305-701, Republic of Korea Clean Fuel Department, Korea Institute of Energy Research, 152 Gajeong-ro, Yuseong-gu, Daejeon 305-343, Republic of Korea
h i g h l i g h t s Pyrite can be used for lipid extraction as well as esterification processes. Microalgae cells can be effectively destroyed by the Fenton reaction using pyrite. Free fatty acids can be converted to biodiesel by pyrite under acidic condition. Two-step esterification process can solve the problem of alkaline catalyst.
a r t i c l e
i n f o
Article history: Received 14 January 2015 Received in revised form 19 February 2015 Accepted 20 February 2015 Available online xxxx Keywords: Pyrite Ferric chloride Fenton-like reaction Lipid extraction Two-step esterification
a b s t r a c t In this study, pyrite (FeS2) was used for lipid extraction as well as esterification processes for microalgaebased biodiesel production. An iron-mediated oxidation reaction, Fenton-like reaction, produced an expected degree of lipid extraction, but pyrite was less effective than FeCl3 commercial powder. That low efficiency was improved by using oxidized pyrite, which showed an equivalent lipid extraction efficiency to FeCl3, about 90%, when 20 mM of catalyst was used. Oxidized pyrite was also employed in the esterification step, and converted free fatty acids to fatty acid methyl esters under acidic conditions; thus, the fatal problem of saponification during esterification with alkaline catalysts was avoided, and esterification efficiency over 90% was obtained. This study clearly showed that pyrite could be utilized as a cheap catalyst in the lipid extraction and esterification steps for microalgae-based biodiesel production. Ó 2015 Elsevier Ltd. All rights reserved.
1. Introduction The world is in great need of alternative energy sources because of serious problems in energy supply, the environment and security involving current fossil fuels (Sung et al., 2014). Biodiesel is viewed as a green energy substitute for transportation because it has renewable, biodegradable and environment friendly properties. A potentially ideal source for biodiesel fuel can be found in the lipids produced by microalgae. These unique microbes can be cultivated 2–10 times faster than any terrestrial crop, which is the current source of biodiesel fuel, and accumulate oil up to 75% in their cells (Sayre et al., 2013). What is better, they grow via photosynthesis using sunlight as energy and assimilate CO2, the most problematic global warming gas, as their sole carbon source. Despite these numerous advantages, biodiesel production from microalgae has a long way to go before being commercialized due to its high energy and chemical costs. Considering the entire fuel⇑ Corresponding author. Tel.: +82 42 350 3629; fax: +82 42 350 3610. E-mail address: [email protected] (J.-I. Han).
making process, the lipid extraction and esterification processes are the main problems in this regard. The current approach for lipid extraction, especially from wet biomass, inevitably involves the removal of a great deal of water, which requires heating and pressurizing the biomass; this single step is responsible for up to 50% of the total energy consumed (Lardon et al., 2009). Esterification, in which the extracted lipids are converted into the final product of fatty acid methyl esters (FAMEs), uses a large amount of chemical catalyst and accounts for 30% of the total production cost (Tran et al., 2012). Acid catalysts are generally employed for this conversion, instead of the far more potent alkaline catalysts, because the substantial amounts of fatty acids (FFAs) in microalgae oil turn into soaps under alkaline conditions (Sendzikiene et al., 2004; Lopez et al., 2005). Our previous study proved that ferric chloride (FeCl3) enables lipids to be extracted from wet state biomass through a Fenton-like reaction and at the same time avoids the alkaline-induced saponification problem through the pre-conversion of FFAs (Seo et al., 2015). This iron-based method, needing only a minimum amount of FeCl3 and H2O2, is believed to reduce the overall process
http://dx.doi.org/10.1016/j.biortech.2015.02.083 0960-8524/Ó 2015 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Seo, Y.H., et al. Lipid extraction and esterification for microalgae-based biodiesel production using pyrite (FeS2). Bioresour. Technol. (2015), http://dx.doi.org/10.1016/j.biortech.2015.02.083
2
Y.H. Seo et al. / Bioresource Technology xxx (2015) xxx–xxx
cost to a substantial degree. To take full advantage of its potential, however, a cheaper source of iron must be found. Pyrite (FeS2), one of the most abundant iron minerals found in the earth’s crust, is a promising iron compound for the application. Natural weathering of pyrite is estimated to occur at the rate of 36 Mt/yr (Garrels et al., 1973), involving 0.02 mol of electrons per square meter of land-surface area (Wehrli, 1990). In fact, pyrite has been successfully used as a low cost iron source for the Fenton-mediated oxidation of contaminants such as trichloroethylene (TCE) and organic components including nucleic acid (Bhar et al., 2013; Cohn et al., 2006). The aim of this study therefore is to investigate the possibility of employing pyrite in the lipid extraction and esterification steps as a FeCl3 replacement. Furthermore, the oxidized form of pyrite, which is known to have higher activity than the raw mineral, has also been used (Hao et al., 2014).
sulfuric acid (Seo and Han, 2014). Our previous work revealed that the lipid extraction was best at a H2O2 concentration of 0.5% and temperature of 90 °C (Seo et al., 2015); and therefore both values were adopted and maintained throughout. Extraction time was varied from 30 min to 3 h. After lipid extraction, the lipid suspension was used for esterification, and the lipid extraction efficiency was measured separately. To measure the lipid extraction efficiency, 5 mL of chloroform was added into the vial, and the mixture was shaken at 100 rpm for 1 h. A chloroform layer with lipid was separated using centrifugation at 1500 rpm for 5 min. Lipid was recovered from the chloroform using an evaporator (EZ2 plus, Geneva, UK), and weighed to estimate lipid extraction efficiency (Seo et al., 2015). The estimation was based on the final cell density (1.7 g/L) and the initial lipid content (35%) of the Chlorella species.
2. Methods
Lipid extraction efficiency ð%Þ
2.1. Microalgae and culture condition A locally isolated freshwater microalgae species Chlorella sp. KR-1 was obtained from the Korea Institute of Energy Research (KIER), and the cultivation conditions employed in this study were the same as other previous studies, using a Pyrex bubble-column reactor (length: 1180 mm, diameter: 85 mm, working volume: 6 L) equipped with 12 fluorescent lamps at the front and right/left sides (light intensity: 80 lmol/m2/s). The reported final cell density and lipid content was 1.7 g/L and 35% lipid content, respectively (Lee et al., 2013). The lipid was analyzed using the conventional method with chloroform: methanol (2:1, v/v) (Folch et al., 1957). 2.2. Pyrite stock solution preparation and oxidation Pyrite mineral was purchased from Sukrim Science (Daejeon, Korea) and milled to powder using a zirconia ball, according to Bae et al., 2013. The pyrite powder was washed using ultra-sonication in ethanol for 5 min, and oven dried (100 °C, 1 days). The pyrite was then stored in a closed glass vial. The oxidation of pyrite was performed in a Pyrex glass vial (40 mL). 1 g of the pyrite powder was dissolved in 10 mL of distilled water, and stirred in contact with air (200 rpm). During the oxidation process, pH was monitored, and dissolved Fe was also measured using the total FerroVer kit (Hach, USA). For comparison, a raw pyrite solution, where 1 g of pyrite was suspended in 10 mL of distilled water, was also prepared right before each experiment.
Esterification efficiency ð%Þ ¼
¼
Weighed lipid after the Fenton-like reaction Lipid content in 100 mL algae solution ð170 mg 35%Þ 100 ð1Þ
2.4. Two-step esterification using pyrite and alkaline catalyst The 10 mL lipid suspension was converted to FAMEs using a two-step esterification process. Esterification was performed at 90 °C, a temperature reported to be optimal for the process (Wang et al., 2007). As a kind of pretreatment, FFAs were first converted to FAMEs by the addition of 10 mL methanol, which was found to be an adequate concentration amount (Seo et al., 2015). The effects of reaction time, ranging from 30 min to 3 h, on the degree of FFAs conversion were investigated. After the solution was cooled to room temperature, alkaline treatment to convert triglycerides (TGs) into FAMEs was performed; pH was adjusted to a reported optimal value of 12 using sodium hydroxide (Seo et al., 2015). The temperature increased the same as the acid treatment, and reaction time was varied from 30 min to 2 h. After the FAME conversion, 5 mL of chloroform was added, and the mixture was shaken at 100 rpm for one hour and centrifuged at 1500 rpm for 5 min to separate the oil from methanol and cell residue. The organic phase of 1 mL was filtered using a 0.45 lm filter paper (Whatman, USA) and analyzed by gas chromatography (GC). Esterification efficiency was calculated based on the amount of extracted lipid.
FAME production ðFAME concentration analyzed by GC ðmg=mLÞÞ ðamount of chloroform ð5 mLÞÞ Extracted lipid ð170 mg 35% lipid extraction efficiencyÞ 100 ð2Þ
2.3. Lipid extraction using Fenton-like reaction
2.5. Analysis of pyrite and microalgal lipid
Lipid extraction was conducted using a microalgae suspension (17 g/L) harvested by centrifugation. A 10 mL algae solution was transferred to a glass vial (40 mL), to which pyrite was added with concentrations from 5 to 100 mM. FeCl3 was used as the control. The pH of the solution was adjusted to 3.0–3.5, which is known to be an optimal range for the Fenton-like reaction, using 1 M of
To analyze the pyrite characteristics, X-ray diffraction (XRD) and crystallinity analysis were performed by Williamson-Hall method (Mote et al., 2012). Results of the pyrite XRD showed good agreement with the Joint Committee on Powder Diffraction Standards (JCPDS) data files (JADE 9, Materials Data Inc.) (Fig. S1). The crystallinity indexes of crystallite size (CS), full width
Please cite this article in press as: Seo, Y.H., et al. Lipid extraction and esterification for microalgae-based biodiesel production using pyrite (FeS2). Bioresour. Technol. (2015), http://dx.doi.org/10.1016/j.biortech.2015.02.083
Y.H. Seo et al. / Bioresource Technology xxx (2015) xxx–xxx
at half maximum (FWHM), and crystallinity index (CI) of pyrite were determined to be 61.8 nm, 0.102°, and 99%, respectively. These indexes were calculated using the PDXL software (Rigaku, USA). Scanning Electron Microscope (SEM) images were used to investigate the uniformness of the microalgae cells’ surface, and to evaluate differences between the surfaces of the microalgae cells before and after the lipid extraction. SEM images were obtained using field emission Scanning Electron Microscopy (S-4800, Hitachi, Japan) at an acceleration voltage of 10 kV after Pt coating (SCD 050 platinum evaporator, Bal-tec, Germany). FAME content was analyzed by GC (Shimadzu, GC-2010, Japan) with a flame ionized detector (FID) and HP-INNOWAX capillary column (Agilent, USA, 30 m, 0.25 mm), and each fatty acid was identified based on the retention times and peak areas of FAME Mix, C8-C24 (Sigma Aldrich, 18918-1AMP, USA). All experiments and analyses were done in triplicate.
3. Results and discussion 3.1. Oxidation of pyrite solution The pyrite based Fenton-like reaction was enhanced by treating the pyrite in the presence of oxygen (Hao et al., 2014). The oxidative dissolution of pyrite allows for ferrous ions (Fe2+) and protons (H+) to be released into solution according to Eq. (3). Dissolved Fe2+ is active in the Fenton-like reaction (Eq. (4)), whereas raw pyrite, which has a mineral crust and cannot be dissolved in water, shows relatively low activity. þ FeS2 þ 7=2 O2 þ H2 O ! Fe2þ þ 2SO2 4 þ 2H
ð3Þ
Fe2þ þ H2 O2 ! Fe3þ þ HO þ OH
ð4Þ
Quantitative changes in the levels of pH and dissolved Fe were measured while the oxidation of pyrite was being performed (Fig. 1). The pH was found to drop immediately, from 5.7 to 3.0, with the addition of 1 g/10 mL pyrite. This was attributable to the rapid dissociation of H+ from the pyrite surface and its release into the suspension. After the initial drop, however, the pH remained unchanged, likely because pyrite oxidation no longer occurred. This reaction was limited mostly by the lack of dissolved oxygen in the solution, which was to some degree resolved by stirring. The pH remained at 3.0 without stirring, whereas stirring rendered a slow reduction in pH down to 1.5. The concentration of Fe ions, which are also a product of the pyrite oxidation, increased along with the decrease in pH (Fig. 1). Interestingly, even below the pH of 3.0, Fe concentration continued to increase, although the pH decrease slowed down. This phenomenon might be brought about at least in part by another product from the pyrite oxidation, i.e., sulfate ions (SO 4 ). The sulfate ion has a buffering capacity at pH 1.6–2.98, and hence, although a substantial amount of H+ is released from the pyrite, it ends up preventing a dramatic pH drop. In fact, this low-pH-range buffering capacity of SO 4 primarily released from pyrite may act as a pH controller in nature (Jha and Bose, 2005). After a certain point (pH 1.5), further and continuous stirring had a negligible effect on the oxidation, and the final Fe concentration was about 3700 mg/L (nearly 10% molar equivalent of Fe in 1 g/10 mL of pyrite). Since all reactions of crystalline-structured minerals occur only at the surface, the reaction rate decreases as crystallinity is increased (Wehrli, 1990). In light of the cubic, exceedingly dense, and almost complete structure of pyrite it was expected to have somewhat low reactivity. Even so, the amounts of dissolved Fe and H+ (3700 mg/L and pH 1.5) were
3
sufficient to be used as a stock solution source of iron for the ensuing Fenton-like reaction. 3.2. Lipid extraction using pyrite Lipids were extracted by way of Fenton-like reactions using both FeCl3 (control) and pyrite. There was a difference in lipid extraction efficiency between the FeCl3 and pyrite (Fig. 2). When the same amount (20 mM) of agent was used, non-pretreated pyrite took more than 2 h to obtain a lipid extraction efficiency over 90%, whereas only 1 h was needed with FeCl3. However, the pyrite extraction time was shortened to 1 h, equivalent to that of FeCl3, when the pre-oxidized pyrite was used. As mentioned above, the dissolved Fe concentration was speculated to be the controlling factor for this discrepancy: raw pyrite stock solution had 400 mg/L of iron ion while the oxidized pyrite solution had 3700 mg/L. To confirm the effect of Fe concentration in a systematic manner, various amounts of catalyst agent were tested with a fixed reaction time of 1 h (Fig. 3). For all the tested samples, there appeared to be an optimal catalyst concentration at around 20– 50 mM, above which the extraction efficiency was impaired. When the catalyst exceeded some critical level, hydroxyl radicals were not only produced but also quickly consumed according to Eqs. (3) and (4), resulting in a weakened oxidation potential (Malik, 2004).
Fe2þ þ HO $ Fe3þ þ OH
ð5Þ
After the lipid extraction using oxidized pyrite, SEM images of cell surfaces before and after the lipid extraction were obtained (Fig. S2). There was a significant difference between the raw cells and lipid-extracted cells. The surfaces of the raw microalgae cells (A-1, A-2) appeared smooth and had no apparent holes, whereas the cells disrupted by the Fenton-like reaction (B-1, B-2) had many pores and a non-uniform surface. The images show that the Fenton-like reaction has the ability to disrupt the cell and extract lipids. The resulting lipid suspension, produced with an extraction efficiency of about 90%, was subjected to the subsequent esterification process. 3.3. Two-step esterification of lipids from microalgae The lipids extracted using pyrite and FeCl3 were put through a two-step esterification process; the first step of pyrite-catalyzed esterification of FFAs and the second step of alkaline-mediated transesterification of the remaining TGs. Each extracted lipid sample was prepared based on the optimal concentrations of agents: i.e., 1 h of the Fenton-like reaction using 20 mM of either oxidized pyrite or FeCl3, and 50 mM raw pyrite. It was found that the esterification was also improved by the surface oxidation of pyrite (Fig. 4), whereas the composition of the extracted lipid was not different (Table 1). When the reaction time in the second step was fixed at 30 min, raw pyrite was found to need more than 2 h to complete over 90% esterification, whereas only 1 h was needed with oxidized pyrite, and FeCl3. Worse yet, soap, which was the undesired byproduct that we wanted to avoid generating, was formed with raw pyrite in the upper layer at a reaction time of 1 h (data not shown). It is possible that the concentration of dissolved Fe ion produced with raw pyrite was too low to fully occupy all FFAs molecules, resulting in incomplete conversion and leaving some FFAs intact. To investigate the length of reaction time in the second transesterification step needed to achieve maximum esterification efficiency, samples that had been prepared for 1 h of reaction time in the first step were used (Fig. 5). All the tested samples showed
Please cite this article in press as: Seo, Y.H., et al. Lipid extraction and esterification for microalgae-based biodiesel production using pyrite (FeS2). Bioresour. Technol. (2015), http://dx.doi.org/10.1016/j.biortech.2015.02.083
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Fig. 1. (A) The pH drop in pyrite solution with or without stirring. (B) Change of Fe concentration in stirred pyrite solution.
Fig. 2. Lipid extraction using FeCl3, raw and oxidized pyrites. Catalyst concentration was fixed at 20 mM.
Fig. 3. Lipid extraction using FeCl3, raw and oxidized pyrites. Reaction time was fixed at 1 h.
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needed to reach pH 12, and this amount was 10 times less than the amount of acid catalyst used in the typical esterification process. In our two-step esterification process, cheap acid catalyst pyrites were used for pre-conversion of FFAs, which are the main problem in esterification using alkaline catalysts, and this step maximized the efficiency of the alkaline catalyst. 4. Conclusion
Fig. 4. Esterification using two-step process based on FeCl3, raw and oxidized pyrites. Reaction time of the second step (TG conversion) was fixed to 30 min.
Pyrite was utilized for the lipid extraction and esterification processes of microalgae-based biodiesel production, and over 90% FAME recovery was obtained. The lipid extraction efficiency as well as esterification efficiency was increased after pre-oxidation of the pyrite. The problem associated with the use of alkaline catalysts was also solved by this method, and the catalyst consumption was greatly reduced compared to an acid catalyst. This novel technology has strong potential and could be a breakthrough for the downstream processing in microalgae-based biodiesel production. Acknowledgements
Table 1 GC analysis after the two-step esterification using FeCl3, raw and oxidized pyrites. First and second reaction times were fixed to 1 h and 30 min, respectively. Retention time (min)
10.18 13.36 13.70
Fatty acid composition
C16:0 C18:1 C18:2 Others
Relative amount of total fatty acids (% w/w) FeCl3
Raw pyrite
Oxidized pyrite
50.1 ± 2.17 14.1 ± 0.19 12.2 ± 0.04 23.6 ± 0.12
49.9 ± 1.46 14.4 ± 0.23 12.5 ± 0.62 23.2 ± 0.42
50.5 ± 2.32 16.6 ± 1.18 10.5 ± 1.01 22.4 ± 1.85
This work was financially supported by the Advanced Biomass R&D Center (ABC) of Korea (Grant 2011-0031348) funded by the Ministry of Science, Korean Minister of Ministry of Land, Transport and Maritime Affairs (MLTM) as ‘‘U-city Master and Doctor Course Grant Program’’ and Technology of the Korean government of the Republic of Korea. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.biortech.2015.02. 083. References
Fig. 5. Esterification using two-step process based on FeCl3, raw and oxidized pyrites. Reaction time of first step (FFA conversion) was fixed to 1 h.
maximum transesterification efficiency in 30 min. This result was similar to a previous study using an alkaline catalyst for transesterification (Sendzikiene et al., 2004). The short reaction time is one distinctive advantage of using an alkaline catalyst for transesterification, along with the exceedingly high catalytic activity, about 4000 times as high as an acid catalyst. The relatively low amount of catalyst required for esterification was also another benefit (Lopez et al., 2005). For example, after the first esterification process using pyrite, less than 0.1% sodium hydroxide (w/v) was
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Contents lists available at ScienceDirect
Bioresource Technology journal homepage: www.elsevier.com/locate/biortech
Lipid extraction and esterification for microalgae-based biodiesel production using pyrite (FeS2) Yeong Hwan Seo a, Mina Sung a, You-Kwan Oh b, Jong-In Han a,⇑ a b
Department of Civil and Environmental Engineering, KAIST, 373-1, Guseong-dong, Yuseong-gu, Daejeon 305-701, Republic of Korea Clean Fuel Department, Korea Institute of Energy Research, 152 Gajeong-ro, Yuseong-gu, Daejeon 305-343, Republic of Korea
h i g h l i g h t s Pyrite can be used for lipid extraction as well as esterification processes. Microalgae cells can be effectively destroyed by the Fenton reaction using pyrite. Free fatty acids can be converted to biodiesel by pyrite under acidic condition. Two-step esterification process can solve the problem of alkaline catalyst.
a r t i c l e
i n f o
Article history: Received 14 January 2015 Received in revised form 19 February 2015 Accepted 20 February 2015 Available online xxxx Keywords: Pyrite Ferric chloride Fenton-like reaction Lipid extraction Two-step esterification
a b s t r a c t In this study, pyrite (FeS2) was used for lipid extraction as well as esterification processes for microalgaebased biodiesel production. An iron-mediated oxidation reaction, Fenton-like reaction, produced an expected degree of lipid extraction, but pyrite was less effective than FeCl3 commercial powder. That low efficiency was improved by using oxidized pyrite, which showed an equivalent lipid extraction efficiency to FeCl3, about 90%, when 20 mM of catalyst was used. Oxidized pyrite was also employed in the esterification step, and converted free fatty acids to fatty acid methyl esters under acidic conditions; thus, the fatal problem of saponification during esterification with alkaline catalysts was avoided, and esterification efficiency over 90% was obtained. This study clearly showed that pyrite could be utilized as a cheap catalyst in the lipid extraction and esterification steps for microalgae-based biodiesel production. Ó 2015 Elsevier Ltd. All rights reserved.
1. Introduction The world is in great need of alternative energy sources because of serious problems in energy supply, the environment and security involving current fossil fuels (Sung et al., 2014). Biodiesel is viewed as a green energy substitute for transportation because it has renewable, biodegradable and environment friendly properties. A potentially ideal source for biodiesel fuel can be found in the lipids produced by microalgae. These unique microbes can be cultivated 2–10 times faster than any terrestrial crop, which is the current source of biodiesel fuel, and accumulate oil up to 75% in their cells (Sayre et al., 2013). What is better, they grow via photosynthesis using sunlight as energy and assimilate CO2, the most problematic global warming gas, as their sole carbon source. Despite these numerous advantages, biodiesel production from microalgae has a long way to go before being commercialized due to its high energy and chemical costs. Considering the entire fuel⇑ Corresponding author. Tel.: +82 42 350 3629; fax: +82 42 350 3610. E-mail address: [email protected] (J.-I. Han).
making process, the lipid extraction and esterification processes are the main problems in this regard. The current approach for lipid extraction, especially from wet biomass, inevitably involves the removal of a great deal of water, which requires heating and pressurizing the biomass; this single step is responsible for up to 50% of the total energy consumed (Lardon et al., 2009). Esterification, in which the extracted lipids are converted into the final product of fatty acid methyl esters (FAMEs), uses a large amount of chemical catalyst and accounts for 30% of the total production cost (Tran et al., 2012). Acid catalysts are generally employed for this conversion, instead of the far more potent alkaline catalysts, because the substantial amounts of fatty acids (FFAs) in microalgae oil turn into soaps under alkaline conditions (Sendzikiene et al., 2004; Lopez et al., 2005). Our previous study proved that ferric chloride (FeCl3) enables lipids to be extracted from wet state biomass through a Fenton-like reaction and at the same time avoids the alkaline-induced saponification problem through the pre-conversion of FFAs (Seo et al., 2015). This iron-based method, needing only a minimum amount of FeCl3 and H2O2, is believed to reduce the overall process
http://dx.doi.org/10.1016/j.biortech.2015.02.083 0960-8524/Ó 2015 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Seo, Y.H., et al. Lipid extraction and esterification for microalgae-based biodiesel production using pyrite (FeS2). Bioresour. Technol. (2015), http://dx.doi.org/10.1016/j.biortech.2015.02.083
2
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cost to a substantial degree. To take full advantage of its potential, however, a cheaper source of iron must be found. Pyrite (FeS2), one of the most abundant iron minerals found in the earth’s crust, is a promising iron compound for the application. Natural weathering of pyrite is estimated to occur at the rate of 36 Mt/yr (Garrels et al., 1973), involving 0.02 mol of electrons per square meter of land-surface area (Wehrli, 1990). In fact, pyrite has been successfully used as a low cost iron source for the Fenton-mediated oxidation of contaminants such as trichloroethylene (TCE) and organic components including nucleic acid (Bhar et al., 2013; Cohn et al., 2006). The aim of this study therefore is to investigate the possibility of employing pyrite in the lipid extraction and esterification steps as a FeCl3 replacement. Furthermore, the oxidized form of pyrite, which is known to have higher activity than the raw mineral, has also been used (Hao et al., 2014).
sulfuric acid (Seo and Han, 2014). Our previous work revealed that the lipid extraction was best at a H2O2 concentration of 0.5% and temperature of 90 °C (Seo et al., 2015); and therefore both values were adopted and maintained throughout. Extraction time was varied from 30 min to 3 h. After lipid extraction, the lipid suspension was used for esterification, and the lipid extraction efficiency was measured separately. To measure the lipid extraction efficiency, 5 mL of chloroform was added into the vial, and the mixture was shaken at 100 rpm for 1 h. A chloroform layer with lipid was separated using centrifugation at 1500 rpm for 5 min. Lipid was recovered from the chloroform using an evaporator (EZ2 plus, Geneva, UK), and weighed to estimate lipid extraction efficiency (Seo et al., 2015). The estimation was based on the final cell density (1.7 g/L) and the initial lipid content (35%) of the Chlorella species.
2. Methods
Lipid extraction efficiency ð%Þ
2.1. Microalgae and culture condition A locally isolated freshwater microalgae species Chlorella sp. KR-1 was obtained from the Korea Institute of Energy Research (KIER), and the cultivation conditions employed in this study were the same as other previous studies, using a Pyrex bubble-column reactor (length: 1180 mm, diameter: 85 mm, working volume: 6 L) equipped with 12 fluorescent lamps at the front and right/left sides (light intensity: 80 lmol/m2/s). The reported final cell density and lipid content was 1.7 g/L and 35% lipid content, respectively (Lee et al., 2013). The lipid was analyzed using the conventional method with chloroform: methanol (2:1, v/v) (Folch et al., 1957). 2.2. Pyrite stock solution preparation and oxidation Pyrite mineral was purchased from Sukrim Science (Daejeon, Korea) and milled to powder using a zirconia ball, according to Bae et al., 2013. The pyrite powder was washed using ultra-sonication in ethanol for 5 min, and oven dried (100 °C, 1 days). The pyrite was then stored in a closed glass vial. The oxidation of pyrite was performed in a Pyrex glass vial (40 mL). 1 g of the pyrite powder was dissolved in 10 mL of distilled water, and stirred in contact with air (200 rpm). During the oxidation process, pH was monitored, and dissolved Fe was also measured using the total FerroVer kit (Hach, USA). For comparison, a raw pyrite solution, where 1 g of pyrite was suspended in 10 mL of distilled water, was also prepared right before each experiment.
Esterification efficiency ð%Þ ¼
¼
Weighed lipid after the Fenton-like reaction Lipid content in 100 mL algae solution ð170 mg 35%Þ 100 ð1Þ
2.4. Two-step esterification using pyrite and alkaline catalyst The 10 mL lipid suspension was converted to FAMEs using a two-step esterification process. Esterification was performed at 90 °C, a temperature reported to be optimal for the process (Wang et al., 2007). As a kind of pretreatment, FFAs were first converted to FAMEs by the addition of 10 mL methanol, which was found to be an adequate concentration amount (Seo et al., 2015). The effects of reaction time, ranging from 30 min to 3 h, on the degree of FFAs conversion were investigated. After the solution was cooled to room temperature, alkaline treatment to convert triglycerides (TGs) into FAMEs was performed; pH was adjusted to a reported optimal value of 12 using sodium hydroxide (Seo et al., 2015). The temperature increased the same as the acid treatment, and reaction time was varied from 30 min to 2 h. After the FAME conversion, 5 mL of chloroform was added, and the mixture was shaken at 100 rpm for one hour and centrifuged at 1500 rpm for 5 min to separate the oil from methanol and cell residue. The organic phase of 1 mL was filtered using a 0.45 lm filter paper (Whatman, USA) and analyzed by gas chromatography (GC). Esterification efficiency was calculated based on the amount of extracted lipid.
FAME production ðFAME concentration analyzed by GC ðmg=mLÞÞ ðamount of chloroform ð5 mLÞÞ Extracted lipid ð170 mg 35% lipid extraction efficiencyÞ 100 ð2Þ
2.3. Lipid extraction using Fenton-like reaction
2.5. Analysis of pyrite and microalgal lipid
Lipid extraction was conducted using a microalgae suspension (17 g/L) harvested by centrifugation. A 10 mL algae solution was transferred to a glass vial (40 mL), to which pyrite was added with concentrations from 5 to 100 mM. FeCl3 was used as the control. The pH of the solution was adjusted to 3.0–3.5, which is known to be an optimal range for the Fenton-like reaction, using 1 M of
To analyze the pyrite characteristics, X-ray diffraction (XRD) and crystallinity analysis were performed by Williamson-Hall method (Mote et al., 2012). Results of the pyrite XRD showed good agreement with the Joint Committee on Powder Diffraction Standards (JCPDS) data files (JADE 9, Materials Data Inc.) (Fig. S1). The crystallinity indexes of crystallite size (CS), full width
Please cite this article in press as: Seo, Y.H., et al. Lipid extraction and esterification for microalgae-based biodiesel production using pyrite (FeS2). Bioresour. Technol. (2015), http://dx.doi.org/10.1016/j.biortech.2015.02.083
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at half maximum (FWHM), and crystallinity index (CI) of pyrite were determined to be 61.8 nm, 0.102°, and 99%, respectively. These indexes were calculated using the PDXL software (Rigaku, USA). Scanning Electron Microscope (SEM) images were used to investigate the uniformness of the microalgae cells’ surface, and to evaluate differences between the surfaces of the microalgae cells before and after the lipid extraction. SEM images were obtained using field emission Scanning Electron Microscopy (S-4800, Hitachi, Japan) at an acceleration voltage of 10 kV after Pt coating (SCD 050 platinum evaporator, Bal-tec, Germany). FAME content was analyzed by GC (Shimadzu, GC-2010, Japan) with a flame ionized detector (FID) and HP-INNOWAX capillary column (Agilent, USA, 30 m, 0.25 mm), and each fatty acid was identified based on the retention times and peak areas of FAME Mix, C8-C24 (Sigma Aldrich, 18918-1AMP, USA). All experiments and analyses were done in triplicate.
3. Results and discussion 3.1. Oxidation of pyrite solution The pyrite based Fenton-like reaction was enhanced by treating the pyrite in the presence of oxygen (Hao et al., 2014). The oxidative dissolution of pyrite allows for ferrous ions (Fe2+) and protons (H+) to be released into solution according to Eq. (3). Dissolved Fe2+ is active in the Fenton-like reaction (Eq. (4)), whereas raw pyrite, which has a mineral crust and cannot be dissolved in water, shows relatively low activity. þ FeS2 þ 7=2 O2 þ H2 O ! Fe2þ þ 2SO2 4 þ 2H
ð3Þ
Fe2þ þ H2 O2 ! Fe3þ þ HO þ OH
ð4Þ
Quantitative changes in the levels of pH and dissolved Fe were measured while the oxidation of pyrite was being performed (Fig. 1). The pH was found to drop immediately, from 5.7 to 3.0, with the addition of 1 g/10 mL pyrite. This was attributable to the rapid dissociation of H+ from the pyrite surface and its release into the suspension. After the initial drop, however, the pH remained unchanged, likely because pyrite oxidation no longer occurred. This reaction was limited mostly by the lack of dissolved oxygen in the solution, which was to some degree resolved by stirring. The pH remained at 3.0 without stirring, whereas stirring rendered a slow reduction in pH down to 1.5. The concentration of Fe ions, which are also a product of the pyrite oxidation, increased along with the decrease in pH (Fig. 1). Interestingly, even below the pH of 3.0, Fe concentration continued to increase, although the pH decrease slowed down. This phenomenon might be brought about at least in part by another product from the pyrite oxidation, i.e., sulfate ions (SO 4 ). The sulfate ion has a buffering capacity at pH 1.6–2.98, and hence, although a substantial amount of H+ is released from the pyrite, it ends up preventing a dramatic pH drop. In fact, this low-pH-range buffering capacity of SO 4 primarily released from pyrite may act as a pH controller in nature (Jha and Bose, 2005). After a certain point (pH 1.5), further and continuous stirring had a negligible effect on the oxidation, and the final Fe concentration was about 3700 mg/L (nearly 10% molar equivalent of Fe in 1 g/10 mL of pyrite). Since all reactions of crystalline-structured minerals occur only at the surface, the reaction rate decreases as crystallinity is increased (Wehrli, 1990). In light of the cubic, exceedingly dense, and almost complete structure of pyrite it was expected to have somewhat low reactivity. Even so, the amounts of dissolved Fe and H+ (3700 mg/L and pH 1.5) were
3
sufficient to be used as a stock solution source of iron for the ensuing Fenton-like reaction. 3.2. Lipid extraction using pyrite Lipids were extracted by way of Fenton-like reactions using both FeCl3 (control) and pyrite. There was a difference in lipid extraction efficiency between the FeCl3 and pyrite (Fig. 2). When the same amount (20 mM) of agent was used, non-pretreated pyrite took more than 2 h to obtain a lipid extraction efficiency over 90%, whereas only 1 h was needed with FeCl3. However, the pyrite extraction time was shortened to 1 h, equivalent to that of FeCl3, when the pre-oxidized pyrite was used. As mentioned above, the dissolved Fe concentration was speculated to be the controlling factor for this discrepancy: raw pyrite stock solution had 400 mg/L of iron ion while the oxidized pyrite solution had 3700 mg/L. To confirm the effect of Fe concentration in a systematic manner, various amounts of catalyst agent were tested with a fixed reaction time of 1 h (Fig. 3). For all the tested samples, there appeared to be an optimal catalyst concentration at around 20– 50 mM, above which the extraction efficiency was impaired. When the catalyst exceeded some critical level, hydroxyl radicals were not only produced but also quickly consumed according to Eqs. (3) and (4), resulting in a weakened oxidation potential (Malik, 2004).
Fe2þ þ HO $ Fe3þ þ OH
ð5Þ
After the lipid extraction using oxidized pyrite, SEM images of cell surfaces before and after the lipid extraction were obtained (Fig. S2). There was a significant difference between the raw cells and lipid-extracted cells. The surfaces of the raw microalgae cells (A-1, A-2) appeared smooth and had no apparent holes, whereas the cells disrupted by the Fenton-like reaction (B-1, B-2) had many pores and a non-uniform surface. The images show that the Fenton-like reaction has the ability to disrupt the cell and extract lipids. The resulting lipid suspension, produced with an extraction efficiency of about 90%, was subjected to the subsequent esterification process. 3.3. Two-step esterification of lipids from microalgae The lipids extracted using pyrite and FeCl3 were put through a two-step esterification process; the first step of pyrite-catalyzed esterification of FFAs and the second step of alkaline-mediated transesterification of the remaining TGs. Each extracted lipid sample was prepared based on the optimal concentrations of agents: i.e., 1 h of the Fenton-like reaction using 20 mM of either oxidized pyrite or FeCl3, and 50 mM raw pyrite. It was found that the esterification was also improved by the surface oxidation of pyrite (Fig. 4), whereas the composition of the extracted lipid was not different (Table 1). When the reaction time in the second step was fixed at 30 min, raw pyrite was found to need more than 2 h to complete over 90% esterification, whereas only 1 h was needed with oxidized pyrite, and FeCl3. Worse yet, soap, which was the undesired byproduct that we wanted to avoid generating, was formed with raw pyrite in the upper layer at a reaction time of 1 h (data not shown). It is possible that the concentration of dissolved Fe ion produced with raw pyrite was too low to fully occupy all FFAs molecules, resulting in incomplete conversion and leaving some FFAs intact. To investigate the length of reaction time in the second transesterification step needed to achieve maximum esterification efficiency, samples that had been prepared for 1 h of reaction time in the first step were used (Fig. 5). All the tested samples showed
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Fig. 1. (A) The pH drop in pyrite solution with or without stirring. (B) Change of Fe concentration in stirred pyrite solution.
Fig. 2. Lipid extraction using FeCl3, raw and oxidized pyrites. Catalyst concentration was fixed at 20 mM.
Fig. 3. Lipid extraction using FeCl3, raw and oxidized pyrites. Reaction time was fixed at 1 h.
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needed to reach pH 12, and this amount was 10 times less than the amount of acid catalyst used in the typical esterification process. In our two-step esterification process, cheap acid catalyst pyrites were used for pre-conversion of FFAs, which are the main problem in esterification using alkaline catalysts, and this step maximized the efficiency of the alkaline catalyst. 4. Conclusion
Fig. 4. Esterification using two-step process based on FeCl3, raw and oxidized pyrites. Reaction time of the second step (TG conversion) was fixed to 30 min.
Pyrite was utilized for the lipid extraction and esterification processes of microalgae-based biodiesel production, and over 90% FAME recovery was obtained. The lipid extraction efficiency as well as esterification efficiency was increased after pre-oxidation of the pyrite. The problem associated with the use of alkaline catalysts was also solved by this method, and the catalyst consumption was greatly reduced compared to an acid catalyst. This novel technology has strong potential and could be a breakthrough for the downstream processing in microalgae-based biodiesel production. Acknowledgements
Table 1 GC analysis after the two-step esterification using FeCl3, raw and oxidized pyrites. First and second reaction times were fixed to 1 h and 30 min, respectively. Retention time (min)
10.18 13.36 13.70
Fatty acid composition
C16:0 C18:1 C18:2 Others
Relative amount of total fatty acids (% w/w) FeCl3
Raw pyrite
Oxidized pyrite
50.1 ± 2.17 14.1 ± 0.19 12.2 ± 0.04 23.6 ± 0.12
49.9 ± 1.46 14.4 ± 0.23 12.5 ± 0.62 23.2 ± 0.42
50.5 ± 2.32 16.6 ± 1.18 10.5 ± 1.01 22.4 ± 1.85
This work was financially supported by the Advanced Biomass R&D Center (ABC) of Korea (Grant 2011-0031348) funded by the Ministry of Science, Korean Minister of Ministry of Land, Transport and Maritime Affairs (MLTM) as ‘‘U-city Master and Doctor Course Grant Program’’ and Technology of the Korean government of the Republic of Korea. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.biortech.2015.02. 083. References
Fig. 5. Esterification using two-step process based on FeCl3, raw and oxidized pyrites. Reaction time of first step (FFA conversion) was fixed to 1 h.
maximum transesterification efficiency in 30 min. This result was similar to a previous study using an alkaline catalyst for transesterification (Sendzikiene et al., 2004). The short reaction time is one distinctive advantage of using an alkaline catalyst for transesterification, along with the exceedingly high catalytic activity, about 4000 times as high as an acid catalyst. The relatively low amount of catalyst required for esterification was also another benefit (Lopez et al., 2005). For example, after the first esterification process using pyrite, less than 0.1% sodium hydroxide (w/v) was
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