In situ transesterification of highly wet microalgae using hydrochloric acid

Accepted Manuscript Short Communication In situ transesterification of highly wet microalgae using hydrochloric acid Bora Kim, Hanjin Im, Jae W. Lee PII: DOI: Reference:

S0960-8524(15)00278-3 http://dx.doi.org/10.1016/j.biortech.2015.02.092 BITE 14665

To appear in:

Bioresource Technology

Received Date: Revised Date: Accepted Date:

6 January 2015 23 February 2015 24 February 2015

Please cite this article as: Kim, B., Im, H., Lee, J.W., In situ transesterification of highly wet microalgae using hydrochloric acid, Bioresource Technology (2015), doi: http://dx.doi.org/10.1016/j.biortech.2015.02.092

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In situ transesterification of highly wet microalgae using hydrochloric acid

Bora Kima, Hanjin Ima, and Jae W. Leea,* a

Department of Chemical and Biomolecular Engineering, KAIST, 291 Daehak-ro, Yuseong-

gu, Daejeon, Republic of Korea, 305-701 *

Corresponding author

Prof. Jae W. Lee Tel: +82-42-350-3940 Fax: +82-42-350-3910 E-mail: [email protected]

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Abstract This study addresses in situ transesterification of highly wet microalgae with hydrochloric acid (HCl) as a catalyst. In situ transesterification was performed by heating the mixture of wet algal cells, HCl, methanol, and solvent in one pot, resulting in the fatty acid methyl ester (FAME) yield over 90 % at 95 oC. The effects of reaction variables of temperature, amounts of catalyst, reactant, and solvent, and type of solvents on the yield were investigated. Compared with the catalytic effect of H2SO4, in situ transesterification using HCl has benefits of being less affected by moisture levels that are as high as or above 80%, and requiring less amounts of catalyst and solvent. For an equimolar amount of catalyst, HCl showed 15 wt. % higher FAME yield than H2SO4. This in situ transesterification using HCl as a catalyst would help to realize a feasible way to produce biodiesel from wet microalgae. Keywords: In situ transesterification, Hydrochloric acid (HCl), Sulfuric acid (H2SO4), Biodiesel, Wet microalgae, High moisture content

1.

Introduction

Microalgae, a rising resource for biodiesel has been intensively investigated in a number of studies during past decades since they grow fast and store lots of lipids to be converted to biodiesel (Chisti, 2007; Halim et al., 2011). Despite many advantages of using microalgae as a potential resource for biodiesel, the cost of biodiesel production from microalgae hinders commercial uses due to the poor economic viability in the current production process involving cell harvest, oil extraction, transesterification, and purification. Among these processes, especially drying microalgae after harvesting them requires lots of energy to be consumed, which accounted for 20-30% of the total cost of biodiesel production (Mata et al., 2010). In situ transesterification is an efficient way to convert oil bearing biomass to biodiesel directly, hence, eliminating the extraction step which is required in the conventional 2

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method. Due to its simple process, various feedstock have been under the investigation using in situ transesterification including vegetable oil and microalgae. During in situ transesterification reaction, the importance of the presence of catalysts has been proved to be more critical for the improved yield of FAME (Atadashi et al., 2013). Therefore, studies on a wide variety of catalysts are actively ongoing including homogeneous catalysts (NaOH, KOH, H2SO4, HCl, etc.), enzymes, and solid catalysts (Zabeti et al., 2009). Due to the characteristic of algal cells that contain high percentage of free fatty acid whose presence causes saponification with alkaline catalysts, employing acid catalysts on biodiesel production from microalgae has been mainly studied. In addition, algal cells containing water also cause an adverse effect on FAME production when alkaline catalysts are used (Demirbas, 2009). Other acid catalysts such as BF3 and H3PO4 were also studied (Bharathiraja et al., 2014). The FAME yield using aforementioned acid catalysts was able to be reached nearly 100 wt. %, however, none of them has been reported to be comparable with a cost-effectiveness of H2SO4. Nonetheless, using H2SO4 for transesterification requires additional purification step of products and wastewater treatment, thereby increasing the cost of producing biodiesel. This work investigated the effectiveness of HCl as a catalyst since HCl has been rarely studied for in situ transesterification of wet microalgae to produce biodiesel. Then, the advantage of HCl as a catalyst for in situ transesterification of wet microalgae was addressed especially when the water contents of algal cells are high. Due to the high affinity of HCl with water, the performance of HCl may be unaffected by the moisture contents in algal cells (Su, 2013). As the conditional factors, the effect of HCl, co-solvent (methanol/chloroform and methanol/hexane), reaction temperature and moisture contents on the performance of in situ transesterification with highly saturated algal cells were elucidate. The effectiveness of HCl was demonstrated in achieving higher than 90% conversion of lipid to biodiesel even with smaller amounts of catalyst and solvent in comparison with the H2SO4 case. 3

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2.

Methods

2.1 Chemicals and Strains H2SO4 (98 wt. %), HCl solution (35 wt. %), and methanol are extra pure grade chemicals whereas chloroform and hexane are guaranteed reagent grade and all were provided from Junsei Chemical. For a standard reagent to quantify FAME in gas chromatography (GC) analyses, methyl heptadecanoate from Fluka was used. N.Gaditana (Nannochloropsis Gaditana) was acquired from AlgaSpring located in Almere, Netherlands. N. Gaditana was selected due to its relatively high lipid productivity and growth rate under nearly any changeable growth conditions. Furthermore, above 84 % of the total lipids in N.Gaditana are the fatty acids containing C16 to C18, which classify themselves as a proper candidate to mimic petro-derived diesel. 2.2 In situ transesterification Our previous in situ transesterification method (Im et al., 2014) was modified in this work. First, in a 14 ml Teflon-sealed tube (Daihan, South Korea), 0.15 g of dry microalgae was well saturated with DI water and water in HCl to have moisture contents of 80 wt. % in total. After an adequate saturation time of 0.5 – 24 hours at room temperature, chloroform and methanol were added and evenly mixed. The tube cap was sealed tightly and then heated in a thermostat bath (WCH-8, Daihan, South Korea) set at a desired temperature for 2 hours. After the reaction, the tube was cooled down to room temperature for 30 minutes. Then chloroform containing a known amount of standard agent and DI water were added for phase separation. To accelerate this phase separation, the tube was centrifuged at 3700 rpm for 10 minutes then the FAME phase containing chloroform on the bottom of the tube was collected for GC analyses. Temperature, amounts of catalyst, reactant and solvents, moisture content in algal cells and moisture saturation time were varied to find optimal reaction conditions and 4

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all experiments were duplicated or quadruplicated to secure the reproducibility of measurements. 2.3 Maximum FAME yield using two steps of lipid extraction and transesterification To quantify the maximum amount of FAME that can be converted from lipids in N. gaditana, experiments were conducted based on Bligh and Dyer’s method (Dyer, 1959). Dry microalgae ranging from 10 to 20 mg in the tube was mixed with a 2 ml mixture of chloroform and methanol (2/1 v/v) and stirred for 5 minutes in order to allow lipids inside algal cells to be extracted. Then 1 ml of methanol and 0.3 ml of H2SO4 were added for transesterification reaction of lipids from lysed cells. The tightly sealed tube containing the mixture above was heated in a bath set at 100 °C for an hour followed by 30 minutes’ cooling at room temperature. In each tube, 1 ml chloroform containing 0.5 mg methyl heptadecanoate and 0.3 M of NaOH solution were added. The tube was centrifuged for 10 minutes at 3400 rpm for phase separation, then the organic phase was extracted for GC analyses. 2.4 FAME yield determined from GC analyses To quantify the amount of FAME in each sample, Agilent 7890b equipped with HP-5 column (30.0 m x 0.32 mm x 0.25 µm) was used. The FID detector was set at 280 °C, with a column flow of 2.1 ml minute-1 of helium as a carrier gas. The injected sample volume was 1 µL. The oven temperature began at 50 °C. It was increased to 175 °C at a rate of 25 °C minute-1, then to 240 °C at a rate of 4 °C minute-1 and held constant for another 15 minutes. The amount of FAME in algal cell was calculated by following equation: FAME contents g 

      !"  #$%&   '( $     '(

(1)

Based on the method described in section 2.3, the maximum amount of FAME from lipids in N. Gaditana was determined as 12.05 mg ± 0.59 FAME/100 mg dry cell. Thus, the FAME yield was calculated by using the following equation:

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FAME yield wt. % 

  #$%& 0  1    2" #$%& 3

100 (2)

2.5 Control experiment using H2SO4 H2SO4 has been widely employed for in situ transesterification since H2SO4 along with primary alcohols are suitable to conduct both direct esterification and transesterification reaction simultaneously, make one step process possible (Marchetti & Errazu, 2008). Thus to compare the catalytic performance of HCl in in situ transesterification with that of H2SO4 on the FAME yield, experiments using an equimolar amount of H2SO4 corresponding to the HCl was used considering the difference in molecular weight. For example, 0.3 ml of 35 wt. % HCl contains 0.126 g of HCl, equimolar amount of H2SO4 then be calculated to be 0.334 g, which has 2.7 times greater than the one of HCl.

3.

Results and discussion

3.1 Moisture contents on the FAME yield As a term ‘HCl solution’ implies, HCl used as a catalyst in this work is fully dissociated in water. Therefore, the effect of moisture in HCl must be investigated first to understand whether it is a factor affecting the FAME yield. After HCl ranging from 0.1 to 1.5 ml added to dried microalgae and saturated for 0.5 hour, 0.1 ml chloroform and 1.0 ml methanol were added and then the mixture sample in the tube was put in the thermostat bath for 2 hours at 95 o

C. With increasing HCl amounts added to the dried microalgae, the moisture contents move

up and the corresponding FAME yield from in situ transesterification decreases as shown in Table 1. The result suggests that the water in the HCl solution negatively affects the FAME yield as the amount of HCl solution increases. Then, DI water along with water in the HCl solution was added to dried algal cells in order to reproduce harvested microalgae where the moisture content of microalgae is ranged as 70~85 wt. %. Takisawa et al. (2013) reported

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that during in situ transesterification employing H2SO4, the moisture contents has a profoundly adverse effect on the FAME yield from either dried or wet microalgae. With HCl, however, the FAME yield with highly moisturized algal cells showed a bit different trend. With 1.5ml methanol and 0.1ml chloroform, the conversion of lipids to biodiesel can be achieved consistently around 90 wt. % even in high moisture contents of 70, 75 and 80 wt. % due to a large activity of HCl. Above 80 wt. %, however, the moisture contents become a primary factor on the FAME yield resulting sharp decrease in FAME yield of 78 wt. % when the moisture contents was 85wt. %. Therefore, the total moisture contents throughout the experiments are set up at 80 wt. % considering the water content in the HCl solution. 3.2 Determination of the amount of acid catalyst For a fixed moisture level in the sample (80 wt. %), the FAME yield with the increasing amount of HCl was analyzed. To maintain the same level of moisture for each HCl concentration, the amount of water added was calculated for different amounts of 35 wt. % HCl ranging from 0.1 ml to 0.7 ml. After half an hour for algal cell saturation with water, 1 ml of methanol and 0.5 ml of chloroform were added, and the tube was subjected to in situ transesterification at 95 oC. In general, the FAME yield increased in proportion to the increment of the amount of the catalyst. The conversion yield to FAME was 98.77 wt. % with 0.7 ml HCl used, which is 10 % higher than the result when the equimolar amount of H2SO4 was employed in the same condition. When 0.3 ml of HCl and the corresponding equimolar amount of H2SO4 were used, the FAME yields for both cases were almost identical as 80 wt. %; therefore, the amount of HCl was fixed at 0.3 ml for further study to compare the catalytic effect between HCl and H2SO4 by subjecting them to the changes in other reaction variables.

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3.3 Methanol effect on the FAME Yield In this work, methanol was chosen considering the advantages of using methanol because of its inexpensive, high reactivity and ease of recovery after the reaction (Chongkhong et al., 2009). Due to the reversibility of transesterification reaction, an excess amount of methanol is required to achieve higher FAME yields if other conditions remain the same (Patil et al., 2011). When the amount of methanol varied from 0.1 to 2 ml with both amounts of HCl and chloroform fixed as 0.3 ml and 0.5 ml, respectively, the FAME yield is directly correlated to the amount of methanol added and the excessive methanol favors the formation of biodiesel. The yield was 38.39 wt. % when 0.1 ml methanol used, but 100.22 wt. % was obtained when 2 ml of methanol used. Cao et al. (2013) explains this direct correlation between methanol dosage and FAME yield since high water contents make the contact of lipids with methanol harder when a small amount of methanol is used, resulting in the low yield. 3.4 Chloroform effect on the FAME yield To see the effect of chloroform on the FAME yield, the amount of chloroform was varied from 0 to 2 ml while the following conditions were maintained; 95 °C, 0.3 ml of HCl, 80 wt. % cell moisture and 1ml methanol. Unlike the result of in situ transesterification using H2SO4 (Im et al., 2014), the yield of FAME seemed less affected by the amount of chloroform when HCl was used. There were not significant discrepancies among the FAME yields considering the degree of increment of chloroform. The FAME yield was 80.7 ± 4.0 wt. % with 0.1 ml of chloroform used while that was 85.7 ±1.7 wt. % with 2 ml of chloroform used. More experiments regarding the amount of chloroform were conducted to compare the catalytic effects of HCl and H2SO4. For the catalyst, 0.3 ml of HCl and the corresponding equimolar amount of H2SO4 were used. With no chloroform, the FAME yield using HCl was 72.35 wt. % while the yield using H2SO4 was as low as 53.52 wt. %. When HCl was used with 0.1 and 0.5 ml of chloroform, the yields were 80.7 and 80.3 wt. % where the results from the experiments 8

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using H2SO4 were 57.67 and 67.86 wt. %, respectively. The yields using H2SO4 showed steeper increment than the result of HCl as more chloroform was added. This proves the strong synergetic effect of chloroform and H2SO4. Besides, the yield from the experiment using HCl was higher than the yield from the H2SO4 case. Thus, the conversion of transesterification using HCl is less affected by the added chloroform while the H2SO4 case was more seriously affected. Also, the yield obtained without chloroform tells that HCl itself may involve in cell disruption more effectively than H2SO4 does, resulting more lipids to be released, thus higher yield of FAME. Thus, the results shown in Fig. 1a and b indicate the HCl case requires a smaller amount of solvent and may reduce the overall production cost. 3.5 Temperature effect on the FAME Yield Using a homogeneous acid catalyst for biodiesel production, achieving high FAME yields demands a harsh condition including high temperature (Lotero et al., 2005). Therefore, during in situ transesterification, the higher the reaction temperature, the more reaction can be driven. In our previous work using H2SO4 (Im et al., 2014), as the temperature goes up, the FAME yield greatly influenced from 49.1 wt. % to 90.6 wt. % when the temperature was at 65 and 95 ℃, respectively. To compare the temperature effect on the yield when HCl is used, the in situ transesterification experiment was conducted at 65, 75, 85 and 95 ℃ with the same condition as used in Section 3.4. The FAME yield from the experiments conducted at 85 and 95 ℃ were nearly the same, close to 80 wt. % in both cases. Nearly the same FAME yield was achieved regardless of the added chloroform 0.1 or 0.5 ml (max ±6.1 %). Here again, once a small amount of chloroform is added to the sample, the amount of chloroform makes no difference in the FAME yield. To recapitulate the result above, in situ transesterification using HCl does not require harsh conditions (i.e. reaction at high temperature) to achieve satisfactory FAME yields and less affected by temperature changes. Accordingly, using HCl

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can be applicable to the biodiesel production under the mild condition, also may contribute to reducing the overall cost. 3.6 Sensitivity analyses of reaction variables From all the experimental results stated in the previous sections, several variables were specified and investigated further for sensitivity analyses. The amount of methanol was narrowed down to two points, 1.5 ml and 1.75 ml, which cases provided the FAME yield higher than 90 wt. %. In the same sense, the amount of chloroform was also varied between 0.1 and 0.7 ml to find the point that yields the maximum FAME conversion while maintaining the minimum input of solvent. Nearly 90 wt. % of lipid conversion can be achieved with 1.5 ml methanol and 0.1 ml chloroform. An increase in methanol addition as 1.75 ml still obtained the same FAME yield as shown in Fig. 1b. It explains that the critical molar ratio of biomass to methanol for the high lipid conversion has reached when 1.5 ml methanol is added to 0.15 g dried based microalgae, which is 10 ml/g or 7.9 g/g. In addition, in situ transesterification of wet algal samples applying different saturation times was conducted to see whether the cells in each experiment were saturated evenly with a relatively short saturation time and whether the FAME yield is affected by the saturation time. All experiments throughout this work were based on the assumption that dried microalgae have been well saturated with water so that the results can be applied to the centrifuged wet microalgae, which has typically 65-90 % moisture contents. Applying the conditions found in the previous sensitivity analysis (1.5 ml methanol, 0.1 ml chloroform, 80 wt. % moisture contents, and 95℃), in situ transesterification was performed with samples that were saturated for 0.5, 5, 12 and 24 hours, respectively. The yields from all of the different saturated times were nearly the same regardless of the time applied for saturation, which were as high as 90 wt. %. Once the cells are evenly saturated, the saturation time seems to become no longer an influence on the FAME yield. 10

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3.7 Effect of solvent on the FAME yield Although being reported to be less effective on the FAME yield than chloroform, hexane has a minimal affinity towards non-lipid contaminants due to its low polarity and therefore higher selectivity for neutral lipids with less toxicity (Halim et al., 2011). Hence, in biodiesel production, hexane is usually employed under the harsh condition such as supercritical fluid or microwave assisted transesterification where other effects of variables were minimized. In this work, in situ transesterification using hexane with the same condition was conducted to study the effect of solvent when HCl is used. At any point, the results using chloroform showed higher yield than the hexane case as shown in Fig. 1b. With the same condition applied, 0.1 ml chloroform results around 90 wt. % FAME yield whereas 0.1 ml hexane was only able to obtain 70 wt. %. The discrepancy in the FAME yield was even bigger as 100 and 60 wt. % for chloroform and hexane cases with 0.7 ml of each solvent used. This may be due to the vulnerability of hexane to the presence of water and cell wall barriers.

4.

Conclusion

The effectiveness of HCl for in situ transesterification was investigated. Compared to H2SO4, HCl provides more effective as a catalyst, resulting in higher FAME yields when algal cells are highly saturated with moisture. Over 90 wt. % lipid conversion to FAME was achieved even with 80 wt. % moisture using a smaller amount of solvent. Thus, in situ transesterification using HCl may provide economic and ecofriendly approach for biodiesel production. Furthermore, the recovery of HCl after the reaction would be easier than that of H2SO4 due to the low boiling point of HCl raising a possibility of further cost reduction. Acknowledgements: This work was supported by the Advanced Biomass R&D Center (ABC) of Global Frontier Project funded by the Ministry of Science, ICT and Future Planning (ABC-20110031354). 11

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Table 1. FAME yields of dried microalgae with different HCl dosages (Methanol: 1 ml, Chloroform: 0.5 ml) Actual moisture HCl (ml) FAME yield (wt. %) contents (%) 0.1 30 100.18 0.3 56.5 98.20 0.5 68.4 91.57 0.7 75.2 85.07 1 81.2 82.11 1.5 86.7 72.60

14

a 100

FAME yield (wt. %)

80

60

40

35 wt.% HCl, MeOH:1.0 ml 35 wt.% HCl, MeOH:1.5 ml H2SO4, MeOH:1.0 ml H2SO4, MeOH:1.5 ml

20

0 0.1

0.3

0.5

0.7

Chloroform (ml)

b 100

80

FAME yield (wt. %)

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60

40

Chloroform, MeOH:1.5ml Chloroform, MeOH:1.75ml Hexane, MeOH:1.5ml Hexane, MeOH:1.75ml

20

0 0.1

0.3

0.5

0.7

Chloroform or Hexane (ml)

Fig. 1. (a) Interaction between each acid catalyst and chloroform on the FAME yield using 0.3 ml HCl and the corresponding equimolar amount of H2SO4 and (b) effect of solvents on the FAME yield using 0.3 ml HCl only using wet microalgae with 80 wt. % moisture contents for in situ transesterification at 95°C. 15

Highlights  In situ transesterification of highly wet microalgae using hydrochloric acid.  FAME yield over 90 wt. % with less amount of solvent in HCl than in H2SO4 catalysts.  HCl outperforms H2SO4 for in situ transesterification of high moisture containing microalgae

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