In situ transesterification of Chlorella sp. microalgae using LiOH-pumice catalyst

Accepted Manuscript Title: In situ transesterification of Chlorella sp. microalgae using LiOH-pumice catalyst Authors: Mark Daniel G. de Luna, Lorenzo Miguel T. Doliente, Alexander L. Ido, Tsair-Wang Chung PII: DOI: Reference:

S2213-3437(17)30196-3 http://dx.doi.org/doi:10.1016/j.jece.2017.05.006 JECE 1609

To appear in: Received date: Revised date: Accepted date:

5-3-2017 1-5-2017 3-5-2017

Please cite this article as: Mark Daniel G.de Luna, Lorenzo Miguel T.Doliente, Alexander L.Ido, Tsair-Wang Chung, In situ transesterification of Chlorella sp.microalgae using LiOH-pumice catalyst, Journal of Environmental Chemical Engineeringhttp://dx.doi.org/10.1016/j.jece.2017.05.006 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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In situ transesterification of Chlorella sp. microalgae using LiOH-pumice catalyst Mark Daniel G. de Lunaa,b,*, Lorenzo Miguel T. Dolienteb, Alexander L. Idoc, Tsair-Wang Chungd a

Department of Chemical Engineering, University of the Philippines, Diliman, Quezon City 1101,

Philippines,

Tel:

+632-981800,

Fax:

+632-9296640,

Email:

[email protected];

[email protected] b

Energy Engineering Program, National Graduate School of Engineering, University of the

Philippines Diliman, Quezon City 1101, Philippines c

College of Engineering and Technology, University of Science and Technology of Southern

Philippines, Claveria 9004, Philippines, Tel: +639173078639 d

Department of Chemical Engineering, Chung Yuan Christian University, Chungli, Taoyuan

32023 Taiwan. Tel: +886-3-2652500, Fax: +886-3-2652599, E-mail: [email protected]

Graphical abstract

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Highlights 

LiOH-pumice was synthesized by HCl treatment and LiOH wet impregnation.



LiOH-pumice was applied in the in situ transesterification of Chlorella sp.



Effects of catalyst, temperature, time, and methanol on FAME yield were evaluated.



47% highest FAME yield using LiOH-pumice at 80°C, 3 h, and 20 wt% catalyst.



% FAME yield by LiOH-pumice is comparable to those obtained from earlier researches.

Abstract Microalgae has gained substantial attention as a promising feedstock for producing biodiesel. In situ transesterification using a heterogeneous catalyst renders the conversion process more advantageous as lipid extraction and transesterification occurs simultaneously. In this study, LiOH-pumice catalyst was prepared via acid treatment and wet impregnation. X-ray diffraction analysis validated the successful LiOH impregnation to the pumice material while scanning electron microscopy and Brunauer–Emmett–Teller surface area analyses revealed that the LiOHpumice catalyst had a spherical and porous morphology and a high surface area. Moreover, the applicability of the LiOH-pumice catalyst for the in situ transesterification of Chlorella sp. was investigated by evaluating the effects of catalyst dosage, reaction temperature and time, and methanol-to-biomass ratio on the % fatty acid methyl ester (FAME) yield. The highest % FAME yield of 47% was obtained at 20 wt.% catalyst, 80°C reaction temperature, 3 h reaction time, and 12 mL g-1 methanol-to-biomass ratio. Overall, the results of this study shows that LiOH-pumice is a promising catalyst for the production of microalgae-based biodiesel via in situ transesterification.

Keywords: microalgae; Chlorella sp.; heterogeneous catalyst; LiOH; pumice; in-situ transesterification

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1. Introduction Declining petroleum reserves with associated critical issues, namely environmental and energy crisis have paved the way for the search of sustainable and renewable energy sources [1– 2]. Among these sources, microalgae (third generation feedstocks) appears to be a more attractive energy source owing to its greater oil content, higher oil yield, minimal land area requirement and superior biodiesel productivity compared to the first (food-based) and second generation feedstocks (non-food-based) [1,3,4]. Moreover, microalgae is compatible with food security, biodiversity and ecosystem, and is considered a technically viable biodiesel feedstock [5–7]. In recent years, microalgae to biodiesel fuel conversion route has been extensively explored. There is a shift from petroleum-based fuel to biodiesel because the latter is non-toxic, renewable, biodegradable, low or non-sulfur bearing, and cleaner-burning fuel with good lubricity and high flash point properties [7]. However, the production of biodiesel from microalgae on the commercial scale is still not economically viable, mainly due to the high cost associated with the present biomass production and fuel conversion routes [9–10]. Briefly, technical challenges need to be overcome for the biodiesel production to be economical and sustainable [10]. For instance, the conventional microalgae to biodiesel conversion route which involves lipid extraction and then conversion of lipids to fatty acid methyl esters (FAME) and glycerol [9] is costly and time consuming. In situ transesterification, an alternative to this process has a potential to reduce the process units and costs of the fuel conversion process [9] because lipid extraction and the conversion of lipid to biodiesel occurs simultaneously in a single reactor [11]. Another technical challenge in biodiesel production is the application of catalyst. Homogenous catalysts are difficult to separate from the product which leads to the increase of cost on product purification step and also results to the generation of more wastewater [12]. Heterogeneous catalysts, on the other hand, offers numerous advantages over homogeneous ones. Such catalysts are easy to recover, reusable, less corrosive, safe, more environmentally friendly and does not need neutralization when separated from the reaction system [13]. Hence, these are more preferred over homogeneous ones for biodiesel production. The catalytic activity and stability of heterogeneous catalysts are crucial aspects in heterogeneous catalysis. Puna et al. [14] evaluated the catalytic activity and stability of CaO heterogeneous catalyst impregnated with lithium nitrate, an alkaline metal salt, in the production of biodiesel from rapeseed oil. The CaO catalyst was impregnated with Li to improve the surface

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basicity of CaO. The authors reported similar catalytic activities for the fresh catalysts, having biodiesel with 93–98% FAME. The Li-impregnated catalysts, however, showed faster deactivation than the bare CaO catalysts because of Ca leaching promoted by the enhanced formation of calcium diglyceroxide on the Li-impregnated catalysts. In this paper, the feasibility of producing biodiesel from microalgae (Chlorella sp.) through in situ transesterification using synthesized LiOH-pumice catalyst and applying conventional heating on a hot plate was investigated. Pumice is an amorphous, porous volcanic rock which is composed mainly of SiO2 [15] characterized by high porosity, richness in silica, alumina, and natural zeolites making it a promising candidate as a catalyst base in reactions that require active support for the metallic function required in isomerization or hydrogenation reactions [16]. Besides, pumice is inexpensive natural porous materials [17]. With this, pumice offers interesting features to be used as catalyst support for heterogeneous catalysts in in situ transesterification. To date, no in situ transesterification of microalgae study using LiOH-pumice catalyst has been conducted. This study synthesized LiOH-pumice by HCl treatment and LiOH wet impregnation and its suitability as heterogeneous base catalyst was evaluated in the in situ transesterification of Chlorella sp. microalgae. In particular, the effects of reaction temperature, reaction time, catalyst amount, and methanol to biomass ratio on the % fatty acid methyl ester (FAME) yield were evaluated.

2. Experimental 2.1 Materials A culture of freshwater microalgae Chlorella sp. was obtained from the Department of Bioscience Technology, Chung Yuan Christian University, in Taoyuan City, Taiwan. Technical grade pumice stone granules (Panreac) was used as catalyst support material, having a 20-25% granules with particle size of < 2.4 mm and 70-80% granules with particle size between 2.4 and 4 mm. Chemicals and reagents used were analytical grade hydrochloric acid (35.5-36.5% HCl, Aencore), anhydrous lithium hydroxide (98% LiOH, Alfa Aesar), HPLC grade methanol (99.9% CH3OH, Aencore), n-hexane (95.0% C6H14, Seedchem), HPLC grade 2-propanol (99.9% (CH3)2CHOH, Echo), and methyl laurate (98.0% C13H26O2, TCI).

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2.2 Microalgae preparation The nutrient source used in the culture was autoclaved for 1.5 h at 120°C and was completely dissolved via ultrasonic mixing for approximately 30 min. Nutrients and the microalgae strain (total volume of approximately 2 L) and the trace metal solution (approximately 40 mL) were then poured into a 40-L cultivation unit filled with 15 L de-ionized water. The culture was kept at 25°C under light condition of not more than 8000 lux using 7 x 28W Philips cool daylight lamps and was continuously supplied with air at a flowrate of 70 L/min. The microalgae cultivation duration lasted for 7 to 10 days then harvested through centrifugation. The resulting biomass were freeze-dried for 5 to 7 days, ground into powder form using a mortar and pestle, and then stored in a desiccator until the time of in situ transesterification. 2.3 Catalyst preparation The pumice as catalyst support was respectively acidified and impregnated with HCl and LiOH. Acid treatment was done by soaking the materials with 1 M HCl for 24 h (1 g pumice: 10 mL HCl). Acid treated material was repeatedly washed with de-ionized water until neutral pH and was dried in an oven (DOV40, Deng Yng) at 110°C for 3 h. The material was ground and those retained on 170 mesh was impregnated with aqueous solution of LiOH at a molar ratio of 2:1 (LiOH: pumice) in an Erlenmeyer flask. The mixture was heated in a water bath using a hot plate (PC-420D, Corning) at 90°C for 2 h under constant stirring and was then dried in an oven at 180°C for 3 to 5 h. The resulting impregnated pumice was calcined in a muffle furnace (CSF 1100, Carbolite Furnaces) at 500°C for 3 h.

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2.4 In situ transesterification The in situ transesterification experiments were conducted in a 100-mL round bottom flask. The round bottom flask was placed in a water bath heated by a hot plate (PC-420D, Corning) to maintain the reaction at the desired temperature. A magnetic stirrer was used for constant stirring at 500 rpm. The reaction temperature was monitored using a glass thermometer. Dried microalgae (0.3 g) was used in each transesterification run, and specified volumes of methanol as alcohol reactant and n-hexane (12 mL g-1 n-hexane-to-biomass ratio) as extraction solvent were added. Synthesized LiOH-pumice was used as heterogeneous base catalyst. The reaction flask was equipped with a reflux condenser to prevent the evaporation of methanol and n-hexane into the surrounding atmosphere, and thus maintain the volume of alcohol and extraction solvent in the reaction flask. After the reaction time has elapsed, the flask was removed from the water bath and allowed to cool down for 5 min. The microalgae debris and LiOH-pumice catalyst were separated from the reaction products by using a vacuum filter. The filtrate (reaction products) was then diluted to approximately 20 mL with n-hexane to facilitate separation into two layers. The upper layer, which contains n-hexane and FAME, was extracted and placed in a 50-mL beaker. The bottom later, which contains the reaction by-products, was discarded. In order to quantify the weight of the obtained FAME for % FAME yield determination, the n-hexane was allowed to evaporate in a fume hood. To hasten the process of evaporation, the beaker was placed in a water bath heated by a hot plate (PC-420D, Corning). When the n-hexane has completely evaporated, the obtained FAME was weighed. 2.5 Analytical methods The characteristics of untreated pumice and LiOH-pumice were evaluated using X-ray diffraction spectrometer (D8 Advance Eco, Bruker), Fourier transform infrared spectrometer (Nicolet 6700, Thermo Scientific), scanning electron microscope – energy dispersive X-ray spectrometer (SEM-EDX) and Brunauer–Emmett–Teller analyzer (ASAP 2020, Micromeritics). The lipid content of Chlorella sp. was determined using the static hexane lipid extraction method by Halim et. al [18]. The n-hexane was used as non-polar extraction solvent and 2-propanol was used as polar extraction solvent. The n-hexane and 2-propanol in 3/2 v/v single-phase mixture (n-hexane = 18 mL, 2-propanol = 12 mL) was added to 0.3 g of dried microalgae in a 100-mL round bottom flask. The flask was equipped with a reflux condenser to prevent the evaporation of the solvents into the surrounding atmosphere. The lipid extraction was conducted at 25°C for 3 h

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with constant stirring using a magnetic stirrer at 600 rpm. After extraction, the microalgae cell debris was separated from the lipid-solvent mixture by vacuum filtration. The filtrate was placed in a separating funnel and 15 mL of n-hexane and 15 mL of de-ionized water was added to induce biphasic layering. After settling, the solvent mixture partitioned into two distinct phases: a top dark-green hexane layer containing most of the extracted lipids and a bottom light-green aqueousisopropanolic layer containing most of the co-extracted non-lipid contaminants. The hexane phase was collected in a pre-weighed flask and heated to dryness by placing the flask in a water bath heated by a hot plate (PC-420D, Corning) under a fume hood to enable gravimetric quantification of the extracted lipid. The GC-FID analysis of the obtained FAME was performed in a GC-FID, HP 6890, Agilent with a capillary column (30.0 m x 250 μm x 0.25 μm nominal, J&W 122-7032 DB-WAX, Agilent). Obtained FAME was re-dissolved in a 10 mL n-hexane and subsequently added with 10 μL methyl laurate as internal standard. Approximately 1.5 mL of this solution was used in the analysis. The oven temperature was initially set at 50°C for 1 min. The temperature was raised to 200°C at a rate of 25°C min-1, followed by raising to 230°C at a rate of 3°C min-1 and held at this temperature for 3 min. The FID detector temperature was set at 150°C, and the injection volume of 1.0 μL was used for analysis. The % FAME yield was calculated by using Eq. 1: % 𝐹𝐴𝑀𝐸 𝑌𝑖𝑒𝑙𝑑 =

∑ 𝐴𝑀𝐸 𝐶𝐼𝑆𝑇𝐷 × 𝑉𝐼𝑆𝑇𝐷 × × 100 𝐴𝐼𝑆𝑇𝐷 𝑚

(1)

where: ΣAME = total peak area of fatty acid methyl ester AISTD = peak area of internal standard CISTD = concentration of internal standard, mg/mL VISTD = volume of internal standard, mL m = mass of the sample, mg 3. Results and Discussion 3.1 Catalyst characterization and lipid content The XRD analyses demonstrated that the untreated pumice was mainly composed of amorphous silica as shown in Fig. 1a. The presence of a peak in the diffraction spectrum of untreated pumice at 2θ = 31° is attributed to cristobalite (SiO2). This is consistent with the findings of Ismail et al. [19]. However, the peak corresponding to cristobalite disappears in the diffraction

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spectrum of LiOH-pumice (Figure 1b) and this is an indication that the material has lost crystallinity as a result of the high calcination temperature of 500°C during catalyst preparation. The loss of crystallinity of pumice after catalyst synthesis was also observed in the study of Borges and co-workers [17]. The appearance of peaks at 2θ = 22° and 24° and at 2θ = 28°, 34°, and 38° are respectively attributed to lithium disilicate (Li2Si2O5) and lithium metasilicate (Li2SiO3) indicating the successful impregnation of lithium onto the catalyst support.

The infrared (IR) spectra of untreated pumice shows bands present at 3394, 1018, and 779 cm-1 (Figure 2). The presence of a broad peak at 3394 cm-1 is attributed to O-H bonds, similar to the result of the study reported elsewhere [20]. The peaks present at 1018 cm-1 and 779 cm-1 are attributed to the Si-O bonds and O-H bending bonds, respectively [21]. LiOH-pumice appears to have a similar IR spectrum to that of untreated pumice except that its intensity is low in the fingerprint region (1500–600 cm-1). This disparity is due to the loss of crystallinity of LiOHpumice upon calcination at 500°C which was also reported in another study [16]. In particular, the IR spectrum of LiOH-pumice shows peaks present at 3672, 1458, 948, and 733 cm-1. The peak present at 3672 cm-1 is attributed to O-H stretching bonds while peaks present at 1458 cm-1 and 733 cm-1 are attributed to O-H bending bonds [21]. The peak situated at 948 cm-1 is attributed to Si-O bonds [21]. Lithium bonds cannot be identified in the IR spectrum of the LiOH-pumice because metal vibrations commonly occur in the far-infrared region of 400 – 100 cm-1 [22]. The SEM image of untreated pumice shown in Figure 3 reveals a glassy and crushed morphology similar to the findings in the literature reported elsewhere [23]. LiOH-pumice, however, has a spherical and porous morphology demonstrating its suitability as heterogeneous catalyst because this physical attribute minimizes the resistance to transport of reactants and products [24]. BET results confirmed the suitability of the LiOH-pumice catalyst with 2.30 m2 g-1 surface area, 12 times higher than 0.14 m2 g-1 of untreated pumice. The increase in surface area may be attributed to the acidification of pumice in the catalyst preparation step, wherein microporous structures were formed as structural cations were replaced by protons [16]. This assumption may be justified by the increase in micropore volume from 0.000023 cm3 g-1 for untreated pumice to 0.002032 cm3 g-1 for LiOH-pumice. The high surface area of LiOH-pumice relative to untreated pumice is favorable because a high catalyst surface area indicates the possibility of an optimized

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rate of catalytic activity [25]. The porous structure, as confirmed by the micropore volume of LiOH-pumice, indicates the possibility of a high degree of dispersion of the active component [24]. Figure 4 shows the EDX spectra of (a) untreated pumice and (b) LiOH-pumice, and the corresponding elemental analysis. It revealed that the untreated pumice has 37.10% silicon, 6.71% aluminum, and 56.19% oxygen by weight, while elemental analysis of LiOH-pumice shows 29.75% silicon, 5% aluminum, and 65.25% oxygen by weight. Elemental lithium was not detected because EDX equipment is not capable detecting lithium. Nevertheless, result confirmed the presence of silicon and aluminum which are vital components of pumice as catalyst support in transesterification process. The mass of the extracted lipid from a 0.3 g of dried Chlorella sp. was found to be 0.0149 g. Thus, the lipid content of Chlorella sp. is 0.05 g (5% w/wDW) lipid/g dried microalgae. This value is within the range of 2.0–63.0% (w/wDW) lipid content of the various species of Chlorella reported in literature [8]. This suggests that the cultivation conditions employed for the Chlorella sp. was relatively effective. 3.2 Effects of transesterification parameters Figure 5a shows the effect of reaction temperature on the % FAME yield. The results show that the % FAME yield generally improved at higher reaction temperatures. Similar findings were reported elsewhere [26] and [9]. A significant increase in % FAME yield from 60 – 70°C (27 – 35%) was observed, however, the transesterification reaction seems to be only slightly affected by temperature in the 50 – 60°C range, having % FAME yields of 26% and 27%, respectively. Further increase in the reaction temperature from 70 – 80°C showed a decrease in the % FAME yield (35 – 33%) due to higher rates of methanol and n-hexane evaporation. The decrease of % FAME yield when reaction temperature was set above the boiling point of methanol (64.5°C) and hexane (68°C) is due to solvent loss and limitation of catalyst and solvent interaction [27]. In addition, the increase of reaction temperature beyond boiling point of solvents will result to a formation of large number of bubbles which inhibit the reaction on the three-phase interface of oil, methanol, and catalyst [28]. For the effect of reaction time on the % FAME yield (Figure 5b), reactions conducted in 1 h and 2 h did not show any conversion of triglycerides to fatty acid methyl esters. The reason for this is the cell wall of the microalgae was not easily broken down by n-hexane. N-hexane is

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therefore unable to immediately extract the algal lipids from inside the microalgae’s cell wall. When the reaction was conducted for 3 h, transesterification of the algal lipids took place, and produced 33% FAME yield. This improved formation of biodiesel at prolonged reaction time resulted from the favorable disruption of microalgal cell wall thereby enhancing triglyceride release and interaction with the reactant mixtures [29]. At 4 h reaction time, however, no increase in % FAME yield was observed and the % FAME yield (27%) obtained was even 6% lower than that obtained at 3 h. This result is attributed to lipid oxidation which is consistent with the findings reported in the study of Ma et al. (2014) [30]. The effects of catalyst amount on the % FAME yield shown in Figure 5c demonstrated an increase of % FAME yield from 33% to 47% as the catalyst amount was increased from 10 – 20%. However, at 25% catalyst amount, the % FAME yield decreased. These finding was also observed in the literature reported elsewhere [30] and [31]. The increase in % FAME yield from 10 – 20% is most likely due to the increase in availability of catalytic active sites [31] which allowed for higher reaction rates, and thus, higher ester yields. Too much catalyst amount, however, leads to aggregation of catalyst particles, resulting in mass transfer limitation, decrease in the interaction among the reactants, and lower FAME yields [31]. For the effect of methanol to biomass ratio on the % FAME yield (Figure 5d), it can be observed that the increase from 12 – 15 mL g-1 showed a significant increase of % FAME yield from 33% to 42%, respectively. Further increase in the amount of methanol showed a decrease in % FAME yield which is also observed in the work reported in literature [31] and [32]. The increase in the % FAME yield is mainly due to the more favored shift of the reaction equilibrium towards the product side as the amount of methanol increased [31]. Also, the increase in the % FAME yield may have been due to methanol acting not only as a reactant, but also a solvent in cell lysis [32]. On the other hand, the decrease in % FAME yield as the amount of methanol was further increased may be due to the dilution of the catalyst which could have affected its catalytic activity and thus slowed down or hindered the transesterification reaction [33]. Overall, the highest % FAME yield of 47% in this study is comparable to those obtained by previous studies [34] [35].

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4. Conclusions A novel pumice-based catalyst for transesterification reactions was synthesized in this study. The presence of lithium silicate peaks in the diffraction spectrum indicates the successful impregnation of lithium onto the catalyst support. The shape and structure of the LiOH-pumice signified its suitability as catalyst material. Its spherical shape minimizes the resistance to transport of reactants and products, and its porous structure indicates the possibility of a high degree of dispersion of the active component. The novel LiOH-pumice catalyst was also found effective in producing biodiesel from lyophilized Chlorella sp. via in situ transesterification reaction.

Acknowledgments The authors are thankful to the Ministry of Science and Technology, Taiwan (Contract Number MOST 104-2221-E-033-054) and the Department of Science and Technology, Philippines for providing financial support for this research endeavor.

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16



a

Intensity (a.u.)

Cristobalite (SiO2)

b





Li2Si2O5

Li2SiO3



 

0

10

20

30

40

50

60

70

80

2(deg)

Figure 1. X-ray diffraction spectra of (a) untreated pumice and (b) LiOH-pumice.

17 0.30

Si-O

Pumice LiOH-Pumice

Absorbance

0.25

0.20

O-H 0.15

Si-O O-H

0.10

O-H

O-H

O-H

0.05

0.00 4500

4000

3500

3000

2500

2000

1500

1000

500

0

-1

Wavenumber, cm

Figure 2. Infrared spectra of untreated pumice and LiOH-pumice

a

b

Figure 3. SEM images of (a) untreated pumice and (b) LiOH-pumice

18

a

b

Figure 4. EDX Spectra of (a) untreated pumice and (b) LiOH-pumice

19

[b]50

[a]50

Reaction conditions: 80°C, 10 wt.% (dry 40

30

20

Reaction conditions: 3 h, 10 wt.% (dry microalgae) catalyst, 12 mL g-1 methanol to

10

microalgae) catalyst, 12 mL g-1 methanol to biomass ratio, and 12 mL g-1 n-hexane to

FAME Yield, %

FAME Yield, %

40

30

biomass ratio

20

10

biomass ratio, and 12 mL g-1 n-hexane to biomass ratio

0

0 50

60

70

80

1

2

O

Temperature, C

40

40

FAME Yield, %

[d]50

FAME Yield, %

[c]50

30

20

Reaction conditions: 80°C, 3 h, 12 mL g-1 10

3

4

Time, h

M to biomass ratio, and 12 mL g-1 n-hexane

30

20

Reaction conditions: 80°C, 3 h, 10 wt.% (dry 10

to biomass ratio

microalgae) catalyst, and 12 mL g-1 n-hexane to biomass ratio

0

0 10

15

20

25

Catalyst Amount, wt% of dry microalgae

12

15

18

21

Methanol to Biomass Ratio, mL/g

Figure 5. Effect of (a) reaction temperature, (b) reaction time, (c) catalyst amount, and (d) methanol to biomass ratio on % FAME yield