Scholarly article on topic 'Application of Factorial Design Methodology for Optimization of Transesterification Reaction of Microalgae Lipids'

Application of Factorial Design Methodology for Optimization of Transesterification Reaction of Microalgae Lipids Academic research paper on "Chemical sciences"

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Abstract of research paper on Chemical sciences, author of scientific article — Yu-Ru Li, Meei-Fang Shue, Yi-Chyun Hsu, Wen-Liang Lai, Jen-Jeng Chen

Abstract This study investigates the effects of alkali catalyst quantity, reaction temperature, reaction time, and acid catalyst quantity on biodiesel production via the transesterification of microalgae using two-level four-factor full factorial design. Under the experimental range considered, the most important factor for FAME yield is the base catalyst quantity. The FAME yield increases with increasing base catalyst quantity. The reaction time and acid catalyst quantity also have positive influences. There is an appreciable interaction between alkali catalyst quantity and the acid catalyst quantity, and thus the effects of these variables must be considered jointly. The best results for laboratory-scale biodiesel production via transesterification were obtained at a 1:65 weight ratio of dry microalgal biomass to alkali catalytic methanol (NaOH/MeOH, 2.5wt.%), with the acid catalysis process (HCl/MeOH, 5.8 vol.%), a 60oC reaction temperature, and a 30-min reaction time.

Academic research paper on topic "Application of Factorial Design Methodology for Optimization of Transesterification Reaction of Microalgae Lipids"

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Energy Procedia 52 (2014) 377 - 382

Application of factorial design methodology for optimization of transesterification reaction of microalgae lipids

Yu-Ru Li1, Meei-Fang Shue2, Yi-Chyun Hsu3, Wen-Liang Lai2, Jen-Jeng Chen2* 1 Graduate Institute of Bioresources, National Pingtung University of Science and Technology, Pingtung 91201, Taiwan 2Graduate Institute of Environmental Management, Tajen University, Pingtung 90741, Taiwan 3Department of Environmental Engineering, Kun Shan University, Tainan 71003, Taiwan

Abstract

This study investigates the effects of alkali catalyst quantity, reaction temperature, reaction time, and acid catalyst quantity on biodiesel production via the transesterification of microalgae using two-level four-factor full factorial design. Under the experimental range considered, the most important factor for FAME yield is the base catalyst quantity. The FAME yield increases with increasing base catalyst quantity. The reaction time and acid catalyst quantity also have positive influences. There is an appreciable interaction between alkali catalyst quantity and the acid catalyst quantity, and thus the effects of these variables must be considered jointly. The best results for laboratory-scale biodiesel production via transesterification were obtained at a 1:65 weight ratio of dry microalgal biomass to alkali catalytic methanol (NaOH/MeOH, 2.5 wt.%), with the acid catalysis process (HCl/MeOH, 5.8 vol.%), a 60 °C reaction temperature, and a 30-min reaction time.

Keywords: fatty acid methyl esters (FAMEs), microalgae, transesterification

Introduction

Biodiesel (fatty acid methyl esters, FAMEs) is a mixture derived from the esterification and transesterification of free fatty acids (FFAs) and triglycerides and is typically made from renewable biological resources such as vegetable oils, animal fats, or even used cooking oil (UCO). Biodiesel contains fewer sulfur compounds, has a high flash point (>130 °C), is non-toxic, and is highly biodegradable in water (98% biodegrades in just a few weeks). However, using vegetable oil for biodiesel production increases the price of edible oil [1]. Animal fats contain higher saturated fatty acids and normally exist in solid form at room temperature, which may cause problems in the biodiesel production process [2]. The quality of UCO varies because its physical and chemical properties depend on the contents of fresh cooking oil and UCO may contain many undesired impurities, such as water and FFAs [3].

Microalgae are another source of triglycerides. Microalgae not only have higher biomass production and faster growth than those of energy crops, producing 15300 times more oil for biodiesel production than

Corresponding Author: jerry@tajen.edu.tw

traditional oilseed crops on an area basis, but can also reduce the amount of global warming gases and consume other pollutants. Many microalgae are exceedingly rich in lipids and oils, which can be converted into biodiesel [4]. Microalgal oils suitable for making biodiesel are common [5].

Four main synthetic approaches have been used for biodiesel production, namely dilution, microemulsion, pyrolysis, and transesterification. The most commonly used method for converting microalgal oils to biodiesel is transesterification. The transesterification reaction is an important step in the overall process of biodiesel production, which is both energy- and cost-intensive. Transesterification can be catalyzed by both alkali and acid catalysts. NaOH, C^ONa, CH3OK, and KOH are the most commonly used as alkali catalysts [1]. For acid-catalyzed systems, sulfuric acid, HCl, BF3, H3PO4, and organic sulfonic acids have been used [6]. The alkali catalysis reaction is about 4,000 times faster than the acid catalysis reaction. However, the performance of alkali catalysts is strongly affected by the presence of FFAs in the feedstock.

Several factors influence the transesterification reaction, including alcohol quantity, reaction temperature, reaction time, and catalyst concentration. A transesterification reaction theoretically requires 3 mol of alcohol for 1 mol of triglyceride to produce 3 mol of fatty acid ester and 1 mol of glycerol. An excess of alcohol is used in biodiesel production to ensure that the oils or lipids are completely converted to esters. However, increasing the alcohol amount beyond the optimal ratio does not increase the FAME yield, but it increases the cost of alcohol recovery. D'Oca et al. [7] studied the extraction of lipids from microalgae Chlorella pyrenoidosa using Soxhlet extraction, magnetic stirring, and an ultrasonic bath with five solvents: a mixture of chloroform/methanol (2:1 v/v), methanol, chloroform, ethanol, and hexane. They found that chloroform/methanol (2:1 v/v) led to the highest lipid extraction, followed by methanol, chloroform, ethanol, and hexane. Sheng et al. [8] demonstrated that a mix of chloroform/methanol had the highest lipid recoveries for Synechocystis.

The catalyst concentration increases the conversion of triglycerides, increasing the FAME yield. This is because an insufficient amount of catalyst results in an incomplete conversion of the triglycerides into FAMEs. The FAME yield is highest when the catalyst (H2SO4) concentration is 2.0% (v/v), decreasing slightly with a further increase

1876-6102 © 2014 Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.Org/licenses/by-nc-nd/3.0/).

Selection and peer-review under responsibility of the Organizing Committee of 2013 AEDCEE doi: 10.1016/j.egypro.2014.07.089

in the catalyst concentration [9]. Patil et al. [10] reported that around 2.0 wt.% of catalyst (KOH/MeOH) resulted in the maximum FAME yield under microwave-assisted transesterification for dry algal biomass.

The conversion rate of FAMEs significantly depended on reaction time. The FAME yield is highest at a reaction time of < 90 min, and then remains relatively constant with a further increase in the reaction time [11]. Excess reaction time leads to a reduction in the FAME yield due to the backward reaction of transesterification, resulting in a loss of esters as well as causing more fatty acids to form soaps. The reaction temperature also influences the transesterification reaction. A higher reaction temperature decreases the viscosity of oils, resulting in an increased reaction rate and a shorter reaction time. However, if the reaction temperature is increased beyond the optimal level, the FAME yield decreases because a higher reaction temperature accelerates the saponification reaction of triglycerides and causes the extraction solvent to leak out via vaporization [12].

In the present study, an experimental design is implemented to examine the correlations among the factors that affect the FAME yield from microalgal biomass. The four variables considered are alkali catalyst quantity, reaction temperature, reaction time, and acid catalyst quantity. These variables have been shown to affect biodiesel production individually and through interaction effects. Preliminary studies that evaluated the extraction systems on composition of FAMEs were also conducted.

Materials and methods 2.1Microalgal culture

One of the dominant green microalgal species, Chodatella sp., was isolated from local source water and cultured in a medium according to the method presented by Norris et al. [13]. Axenic cultures of Chodatella sp. were grown in batch mode in a 5-L modified serum bottle containing 4-L of sterilized algal medium. CO2 (2% v/v) was supplied to the cultures every day. Cultures were harvested in the log growth phase after 7 days for experiments.

2.2Extraction methods and preparation of FAMEs

A stock culture of Chodatella sp. cells was collected by centrifugation at 5000 x g for 5 min. The precipitated microalgal cells were then washed and resuspended in deionized water in triplicate. Biomass was collected by centrifugation again and then dried in a freeze dryer.

2.2.1 Method A

Method A was carried out using a modified protocol based on that given by Lepage and Roy [14]. 0.1 g of dry biomass was mixed with 8 mL of a solvent system (acetyl chloride/methanol, 2:40 v/v) in Teflon-capped Pyrex tubes. The tubes were heated in a water bath at 100 °C for 1 h under pure nitrogen and darkness. The derivatized mixture was cooled to room temperature, and 1 mL of extraction solvent (hexane) was added. The tubes were then shaken and centrifuged. Two phases were produced;

the upper phase (hexane) was transferred to another tube. This operation was repeated twice to optimize sample lipid extraction. The collected organic phase containing FAMEs was purified by using 1 mL of saturated sodium chloride solution. The organic and aqueous phases were then separated. The upper extracted organic phase was recovered, dried over anhydrous Na2SO4, and analyzed using a gas chromatograph.

2.2.2 Method B

0.1 g of freeze-dried biomass was placed in 10-mL Teflon-capped Pyrex tubes and 8 mL of fresh reaction solution (hydrochloric acid/chloroform/methanol, 4:4:40 v/v/v) was added [15]. Biomass was suspended in this solution by vortex mixing and immediately placed at 90 °C for 1 h for transesterification. The tubes were removed from the water bath and cooled to room temperature. 3 mL of an extraction solvent (hexane/chloroform, 4:1 v/v) was then added to each tube. The FAMEs were extracted twice. The upper extracted organic supernatant was collected. All the supernatants were pooled together and analyzed by gas chromatography.

2.2.3 Method C

Freeze-dried biomass (0.1 g) was placed in 50-mL Teflon-capped Pyrex tubes and mixed with a premixed homogeneous solution of NaOH catalyst (2.5 wt.%) and methanol. Depending on the experimental design (Table I), 2.0 or 8.0 mL of alkali catalyst was added to the tubes. The reaction mixture was heated at 60 and 100 °C for 10 and 30 min, respectively, and the samples were well-mixed during heating. After the reaction was completed, the tubes were allowed to cool to room temperature. Then, 0 or 8 mL of acid catalyst (HCl:methanol, 5.8 vol.%) and 10 mL of BF3/methanol solution were added to the tubes in a water bath at 100 °C for 15 min. After the reaction was completed, the tubes were allowed to cool to room temperature again. Purification of the solution was achieved by using 2 mL of saturated sodium chloride solution. 4 mL of an extraction solvent (hexane) was then added to each tube. The upper solvent layer containing FAMEs was collected and analyzed by gas chromatography.

2.3 Gas chromatography analysis

FAME analysis was performed using an Agilent 7820A gas chromatograph with an autosampler, flame ionization detector, and a DB-23 Agilent column (length: 60 m, ID: 0.25 mm, film: 0.25 pm). Hydrogen was used as the carrier gas at a constant flow rate of 35 mL/min. The injector was held at 270 °C while the detector was kept at 280 °C. The oven was at 130 °C initially, ramped to 170 °C at 6.5 °C/min, then ramped again to 215 °C at 2.75 °C/min, and held at 215 °C for 12 min. The temperature was further increased to 230 °C at a heating rate of 40 °C/min and held for 3 min, giving a total heating time of 38.88 min. FAMEs were identified by comparing the retention times with those of standard fatty acids (SUPELCO, USA) and quantified by comparing them with the prepared calibration curves. Pentadecanoic acid (C15:0) was used as an internal standard.

2.4 Statistical analysis and experimental design

The effects of transesterification factors and modifications on the quantity of FAMEs obtained via Method C were analyzed by experimental design [16]. The experimental design consisted of four factors, namely alkali catalyst amount (NaOH:methanol 2.5 wt.%) (X1), reaction temperature (X2), reaction time (X3), and acid catalyst amount (HCl:methanol 5.8 vol.%.) (X4) at two levels. This 24 full factorial design (FFD) was used to determine the joint effects of several factors on a response. It was also used to determine the individual and cumulative effects of these variables and the mutual interactions between them. The complete design consisted of 16 runs (Table I), which were performed in duplicate to optimize the levels of selected variables. Data processing and calculations were carried out using a commercial statistical package, STATISTICA 9.0, to estimate the coefficients of the regression equation. The equations were validated using analysis of variance (ANOVA) to determine the significance of each term in the fitted equations and to estimate the goodness of fit in each case.

Table I 24 full factorial experiment matrix.

X1 X2 X3 X4 Response

Run no. *Alkali catalyst amount (mL) Reaction temperature (°C) Reaction time (min) **Acid catalyst amount (mL) FAME yield (mg FAMEs/g dried microalgae)

1 2 60 10 0 31.7±0.53

2 8 60 10 0 32.6±0.18

3 2 100 10 0 30.0±1.03

4 8 100 10 0 33.8±0.71

5 2 60 30 0 33.7±1.36

6 8 60 30 0 36.7±0.29

7 2 100 30 0 27.9±0.93

8 8 100 30 0 37.8±0.22

9 2 60 10 8 28.4±1.58

10 8 60 10 8 39.9± 1.03

11 2 100 10 8 21.6±0.29

12 8 100 10 8 36.9±0.87

13 2 60 30 8 35.0±0.38

14 8 60 30 8 43.8±1.37

15 2 100 30 8 29.7±0.23

16 8 100 30 8 36.0±0.83

* NaOH:methanol (2.5 wt.%); ** HCl:methanol (5.8 vol.%)

Results and discussion 3.1 Catalyst type and FAME profile

A comparison of the yield of microalgae lipids to FAMEs through three extraction-transesterification methods is shown in Fig. 1. The FAME yield is expressed as the FAME weight relative to the dried microalgae weight. As shown in Fig. 1. biomass that reacted with methanol containing 5% acetyl chloride yielded 29.8 mg/g of FAMEs whereas the reaction with the alkali-acid-catalyzed transesterification process, 2.5% sodium hydroxide and 5.8% hydrochloric acid in methanol, resulted in a higher FAME yield of 41.9 mg/g. Method B with 1% hydrochloric acid and 1% chloroform in

methanol resulted in a FAME yield of 35.1 mg/g. Alkali-acid-catalyzed transesterification led to a higher FAME yield. These results reveal the importance of acid or/and alkali catalyst in accelerating the transesterification reaction.

The characteristics of the FAMEs obtained from the transesterification reaction are presented in Table II. The major FAMEs contained in the biodiesel were esters of palmitic acid (C16:0), oleic acid (C18:1), linoleic acid (C18:2), and linolenic acid (C18:3) from all of the methods, similar to those obtained from sunflower, palm, and corn [17]. No significant changes in the characteristics of the FAMEs were found between the three methods. The proportion of FAME unsaturation was greater than 60% of the total fatty acids. Table I also shows a low percentage of methyl esters with a carbon chain of >18 carbons. This guarantees a low viscosity for biodiesel.

Method A Acetyl chloride/MeOH (2:40 vol.)

Method C

NaOH/HCl/MeOH

m .к j d (1:2.3:40 wt.)

Method B

HCl/Chloroform/MeOH

(4:4:40 vol.)

Fig. 1. FAME yield of Chodatella sp. from three transesterification reactions.

Table II Fatty acid composition of Chodatella sp. obtained using three methods.

Composition (% wt)

Fatty acid Method Method Method

Caprylic (C8:0) N.D. N.D. N.D.

Capric (C10:0) N.D. N.D. N.D.

Laurie (C12:0) N.D. N.D. N.D.

Myristic (C14:0) N.D. N.D. N.D.

Palmitic (C16:0) 26.02 31.34 26.79

Palmitoleic (C16:1 cis) N.D. N.D. 5.16

Stearic (C18:0) 8.84 N.D. N.D.

Oleic (C18:1 cis) 17.94 20..51 19.43

Linoleic (C18:2 cis) 18.87 20.51 20.85

Linolenic (C18:3 cis) 28.34 27.65 27.76

Arachidic (C20:0) N.D. N.D. N.D.

Behenic (C22:0) N.D. N.D. N.D.

Erucic (C22:1 cis) N.D. N.D. N.D.

Lignoceric (C24:0) N.D. N.D. N.D.

N.D.: not detected.

3.2Optimization of microalgal biodiesel production

The coded values of the test variables were used to optimize alkali catalyst amount (X1), reaction temperature (X2), reaction time (X3), and acid catalyst amount (X4). The experimental results of FAME yield in each case are presented in Table I. The results show individual effects of the combinations of the test variables, with significant variation between combinations. An analysis of these results shows that the highest FAME yield was obtained at a reaction temperature of 60 °C, a reaction time of 30 min, an alkali catalyst amount (2.5% sodium hydroxide) of 8 mL, and an acid catalyst amount (5.8% hydrochloric acid) of 8 mL (Run 14).

Table III shows the effects and interactions between the test variables. The results indicate that the FAME yield is mainly controlled by alkali catalyst amount. The FAME yield increased by 7.44 mg/g on average when the alkali catalyst amount was increased from 2 to 8 mL. Reaction temperature has a negative effect on the response; the FAME yield decreased by 3.51 mg/g when the reaction temperature was increased from 60 °C to 100 °C. Table IV shows the ANOVA results for the test variables. F-values with a very low probability (p < 0.05) indicate a very high significance for the corresponding coefficients. The effects of alkali catalyst amount and reaction temperature are both statistically significant in the range studied. The other variables and interactions showed a lower statistical significance.

The mathematical model representing the FAME yield as a function of the test variables in the experimental region is expressed by the following equation:

YFAMEs = 30.104 - 0.046X1 - 0.043X2 + 0.324X3 + 0.281X4 + 0.01296X1X2 - 0.007X1X3 + 0.127X1X4 -0.002X2X3 - 0.014X2X4 + 0.015X3X4, R = 0.962, adj. R = 0.862

The R values (0.962) indicate a good fit between the model and the experimental data. Adjusted R values derived from the sample size and from the number of terms in the model equation were used to correct the predicted R values. The differences between the predicted R values and the adjusted R values are small, and thus, they are in reasonable agreement. A positive term in the equations indicates a synergistic effect (i.e., increasing the variable increases the FAME yield), whereas a negative term indicates an antagonistic effect.

3.3 Influence of alkali catalyst concentration

Fig. 2. shows the interactive effects between alkali catalyst amount, reaction temperature, reaction time, and acid catalyst amount on FAME yield. Each response surface plot represents a number of combinations of two test variables with all other variables at zero levels. The FAME yield slightly increased with decreasing reaction temperature in the range of 60 °C to 100 °C and with increasing alkali catalyst amount in the range of 2 to 8 mL (Fig. 2A). The effect of alkali catalyst amount on FAME yield was more prominent than that of the reaction temperature. The increased alkali catalyst amount increa-

Table III Estimated effects from the 2 factorial experiment matrix. Effect on

Variables FAME yield (mg FAMEs/g dried microalgae) Standard error p-value

Mean 33.47 0.67 0.000

X1 7.44 1.35 0.003

X2 -3.51 1.35 0.048

X3 3.21 1.35 0.063

X4 0.89 1.35 0.539

X1X2 1.39 1.35 0.350

X1X3 -0.44 1.35 0.758

X1X4 3.04 1.35 0.074

X2X3 -0.94 1.35 0.517

X2X4 -2.21 1.35 0.161

X3X4 1.21 1.35 0.409

X1 = Alkali catalyst amount (NaOH:methanol 2.5 wt.%), X2 = Temperature (°C), X3 = Time (min), and X4 = Acid catalyst amount (HCl:methanol 5.8 vol.%)

Table IV Analysis of variance for FAME yield model.

Sum of Squares (SS)

Degrees of freedom (DF)

Mean Square (MS)

F-Ratio ^-Value

X1 221.27 1 221.27 30.50 0.003

X2 49.35 1 49.35 6.80 0.048

X3 41.28 1 41.28 5.69 0.063

X4 3.15 1 3.15 0.43 0.539

X1X2 7.70 1 7.70 1.06 0.350

X1X3 0.77 1 0.77 0.11 0.758

X1X4 36.91 1 36.91 5.09 0.074

X2X3 3.52 1 3.52 0.48 0.517

X2X4 19.58 1 19.58 2.70 0.161

X3X4 5.88 1 5.88 0.81 0.409

Error 36.28 5 7.26

Total SS 425.67 15

R2 = 91.48%, R2(adj) =74.43%

X1 = Alkali catalyst amount (NaOH:methanol 2.5 wt.%), X2 = Temperature (°C), X3 = Time (min), and X4 = Acid catalyst amount (HCl:methanol 5.8 vol.%)

sing the FAME yield is consistent with previous reports of transesterification reactions performed on microalgal biomass [9, 18]. Wahlen et al. [9] reported that an increase in reaction temperature from 60 °C to 80 °C significantly improved the FAME yield. In this study, when the reaction temperature was increased from 60 °C to 100 °C, the FAME yield did not improve at various alkali catalyst amounts. It is speculated that this may be due to the reaction temperature being beyond the boiling point of methanol, leading to the loss of alkali catalyst methanol. A reaction temperature of 60 °C led to the highest FAME yield, reaction was more beneficial than that of reaction temperature of 100 °C for energy consumption and operation cost in whole biodiesel conversion system.

Fig. 2B. shows the interactive effect of alkali catalyst amount and reaction time on FAME yield. A high FAME yield was observed at a high alkali catalyst amount and a long reaction time. Similar to the findings of Wahlen et al. and Ehimen et al. [9, 18], the FAME yield increased with increasing reaction time at various alkali catalyst amounts. With a long reaction time (30 min), increasing the miscibility of the reacting species at various alkali catalyst amounts was favorable for the FAME yield.

Factor

Fig. 2. Interactive effects of alkali catalyst amount, reaction time, reaction temperature, and acid catalyst amount on the FAME yield obtained using FFD.

The combined effect of alkali catalyst amount and acid catalyst amount was analyzed. Fig. 2C. shows that the FAME yield increased with increasing alkali catalyst amount and slightly increased with increasing acid catalyst amount. The variable with the largest effect on FAME yield was alkali catalyst amount. The maximum yield of FAMEs was found at a high alkali catalyst amount and acid catalyst amount. This is expected as a sufficient amount of alkali catalyst resulted in a complete conversion of triglycerides into fatty acid esters. Varying the catalyst amount greatly affected the FAME yield, which is consistent with results of acid-catalyzed in situ transesterification of microalgal Chlorella [9].

3.4Effect of alkali catalyst concentration

In addition to reaction time, reaction temperature, and concentration of reactants, an important variable in the efficient conversion of lipids to FAMEs is the concentration of the catalyst. Fig. 3. shows the effect of alkali catalyst concentration on FAME yield. All reactions were performed by Method C (60 °C, 30 min, HCl:methanol 5.8 vol.%) with varying concentrations of NaOH in methanol. There was an optimum alkali catalyst concentration. At an alkali catalyst concentration of 2.5 wt.%, the maximum FAME yield (37.2 mg/g) was observed. The FAME yield increasing with increasing NaOH catalyst concentration is consistent with reports of alkali-catalyzed transesterification reactions conducted on frying oil or microalgal extracted oil [9, 11, 18].

0 -1-1-1-1-

0 5 10 15 20

Alkali catylst concentration, % Fig. 3. Effect of alkali catalyst concentration on FAME yield.

Conclusion

A two-level full factorial design with four variables was used to determine the effects of alkali catalyst quantity, reaction temperature, reaction time, and acid catalyst quantity on biodiesel production from microalgae. Within the experimental range examined, the most important factor for increasing the biodiesel production is alkali catalyst quantity. Increasing the alkali catalyst concentration beyond 2.5 wt.% did not increase FAME yield. Reaction temperature is the second most important factor. It has a negative influence on the yield

of FAMEs. The interactive effects of alkali catalyst amount and acid catalyst amount on the FAME yield were identified. It was observed that alkali-acid-catalyzed transesterification led to a higher FAME yield than that obtained without catalyzed and only acid catalyzed transesterification.

Acknowledgment

The authors would like to thank the National Science Council of Taiwan for financially supporting this research under grants NSC 97-2622-E-127-002-CC2 and NSC 98-2622-E-127-001-CC2.

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